Efek Faktor Transkripsi Myb Selama Pertumbuhan Nodul Pada Lotus Japonicus
ii
EFFECT OF MYB TRANSCRIPTION FACTOR DURING
NODULE DEVELOPMENT IN Lotus japonicus
SUPRIADI
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
DECLARATION OF THESIS AND INFORMATION SOURCES
OF INFORMATION AND PATENT
I, Supriadi, hereby stated that this thesis entitled Effect of MYB
Transcription Factor During Development in Lotus japonicus is true of my own
work under the supervisor advisory board and that it has not been submitted before
in any form to any university. The content of this thesis has been examined by the
advising advisory board and external examiner. Sources of information which is
derived or cited either from published or unpublished scientific paper from other
writers have mentioned in the script and listed in the references at the end part of
this thesis.
I hereby handed the copyright of my thesis to Bogor Agricultural University.
Bogor, April 2016
Supriadi
A154130201
ii
RINGKASAN
SUPRIADI. Efek Faktor Transkripsi MYB Selama Pertumbuhan Nodul pada Lotus
japonicus. Dibimbing oleh RAHAYU WIDYASTUTI, DWI ANDREAS
SANTOSA, dan MIKA NOMURA.
Dalam beberapa tahun terakhir, beberapa capaian untuk memahami tahap
dalam proses perkembangan nodul telah banyak dilakukan dengan pendekatan studi
molekuler. Beberapa gen telah diidentifikasi memainkan peran sangat penting baik
dalam proses awal nodulasi atau pun nodul organogenesis. Penelitian sebelumnya
menemukan bahwa 76 jenis gen teregulasi selama infeksi Mesorhizobium loti ke
tanaman Lotus japonicus . Salah satu gen teregulasi adalah faktor transkripsi MYB,
molekul yang mengikat pada situs AACG /TG pada DNA. Karena fungsi yang
tepat dari faktor transkripsi MYB dalam proses nodulasi masih kurang diketahui,
maka fungsi faktor transkripsi MYB selama proses nodulasi penting untuk diteliti.
Untuk mengetahui pengaruh faktor transkripsi MYB dalam proses
nodulasi Lotus japonicus, kami selanjutnya membuat akar transgenik dari dari
Lotus japonicus dengan MYB over ekspresi (MYB OX) dan MYB tersupresi oleh
RNAi (MYB RNAi). Kami mengukur jumlah nodul, diameter nodul dan panjang
akar sebagai ekspresi fenotip. Untuk mengetahui aktifitas mereduksi Asetilen dari
nodul menggunakan analisis kromatografi gas untuk mengetahui aktivitas fiksasi
nitrogen. Untuk mengetahui ekspresi gen yang terkait nodulasi, kami mengukur
ekspresi gen enod40, gen nin, dan gen sistein protease menggunakan QRT-PCR.
Hasil penelitian menunjukkan bahwa MYB yang dieskpresikan berlebih
secara signifikan meningkatkan jumlah nodul dari Lotus japonicus di awal minggu
pasca inokulasi dan peningkatan aktivitas fiksasi nitrogen (N2) pada nodul. Pada
penekanan dari MYB secara signifikan menurunkan jumlah nodul dan aktivitas
fiksasi N2 serta menginduksi nodul senesens lebih awal. Kami juga menemukan
bahwa MYB tidak memberi efek pada diameter nodul dan panjang akar pada Lotus
japonicus.
Data pada QRT-PCR menunjukkan bahwa tingkat ekspresi enod40
memungkingkan MYB mengikat pada enod40. Sebaliknya, tingkat ekspresi nin
menunjukkan bahwa MYB mungkin tidak mengendalikan ekspresi gen nin tapi
kami memprediksi MYB memiliki fungsi paralel dengan gen nin dalam proses
nodulasi. Kami mengusulkan bahwa faktor transkripsi MYB mengikat pada gen
enod40 dan diaktifkan selama masa awal nodulasi.
Keyword: MYB, faktor Transkripsi, Nodul, Lotus japonicus
SUMMARY
SUPRIADI. Effect of MYB Transcription factor During Nodule Development in
Lotus japonicus. Supervised by RAHAYU WIDYASTUTI, DWI ANDREAS
SANTOSA, and MIKA NOMURA
In the past years, some achievments toward understanding the initial stages
of nodule developmental process has been done using molecular study. Several
genes have been identified that play very important roles whether in the early
nodulation process or in nodule organogenesis. Previous research found that 76
genes upregulated during Mesorhizobium loti infection to Lotus japonicus plant.
One of upregulated gene is MYB transcription factor, a kind of molecule bind to
AACG/TG site in DNA. Due to the precise function of MYB transcription factor
in the rhizobial symbiosis is still poorly unknown, we investigated function of MYB
transcription factor during nodulation.
To know the effect of MYB transcription factor in nodulation process of
Lotus japonicus we further created the transgenic hairy roots of Lotus japonicus
with MYB overexpression (MYB OX) and suppression by RNAi (MYB RNAi).
We measured the nodule number, nodule diameter and root lenght as phenotype
expression. In additional Acetylene reduction activity of nodules has been assayed
using gas cromatografh analysis to know nitrogen (N2) fixation activity. To know
the expression of genes related nodulation we measured expression of enod40 gene,
nin gene, and Cystein Protease gene using qRT-PCR.
The result revealed that the overexpression of MYB significantly increased
the nodule number of Lotus japonicus in early week post innoculation and
increased nitrogen (N2) fixation activity of nodule. Particularly the supression of
MYB has significantly decreased the nodule number and nitrogen fixation activity
and induce the scenesence nodule earlier. We also found that MYB did not give
effect to nodule diameter and root lenght of Lotus japonicus.
The qRT-PCR expression levels of enod40 suggested that MYB might
bind to enod40. In contras, the expression levels of nin suggested that MYB might
not a regulator of nin but we predicted MYB has a paralel function with nin in
nodulation process .We finally proposed that MYB transcription factor binds to
enod40 gene and activated during the early time of nodulation.
Keyword : MYB, transcription factor, nodule, Lotus japonicus
iv
© Copyright of Bogor Agricultural University, 2015
Protected by the laws
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prohibited. Citation is only for educational purpose, research, scientific writing,
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disadvantage on behalf of Bogor Agricultural University.
Any announcement and duplication of all contents or any part thereof without
permission from Bogor Agricultural University are strictly prohibited.
EFFECT OF MYB TRANSCRIPTION FACTOR DURING
DEVELOPMENT IN Lotus japonicus
SUPRIADI
A Thesis submitted for the Degree Programs
of Master of Science
in Soil and Environtmental Biotechnology
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
vi
External examiner : Dr Ir Iman Rusmana, Msi
viii
FOREWORD
First of all, I would humbly distinguish the Most Gracious Allah SWT, all
praises to Allah for the gifts and His blessing in completing this thesis with title
Effect of MYB Trancription Factor During Nodule Development in Lotus
japonicus. This thesis submitted for the Degree Programs of Master of Science in
Master of Science of Soil and Environtmental Biotechnology. I would like to
sincerely deliver my greatest gratitude to my advisor: Dr Rahayu Widyastuti, MSc,
Prof Dr Ir Dwi Andreas Santosa, MS, Prof Mika Nomura for their advices,
expertness, encouragement, and support. My sincere thanks also to Prof Shigeyuki
Tajima for all support.
My appreciations were also extended to Japan Student Services Organization
(JASSO) Scholarship for granting scholarship during the study and experiment in
Kagawa University and also for SUIJI-JDP (Six University Initiative JapanIndonesia Joint Degree Program) who allowed me to expand my knowledge and
experiences in Japan. This master thesis would not have been possible unless the
funding of Indonesian Government scholarship (Beasiswa Fresh Graduate).
I also addressed my gratitude to all staff and classmate in Soil and
Environtmental Biotechnologi (IPB) and my laboratorymate in Plant Nutrition
(Kagawa University) and I would like to take this moment to deeply express my
thankful feeling to my father and mother who have been praying, loving and
supporting as always. Finally, I hope this thesis can give information about
nodulation process in legume, specially the effect of MYB transcription factor
during those process.
Bogor, 26 May 2016
Supriadi
x
TABLE OF CONTENT
LIST OF TABLE
vi
LIST OF FIGURE
vi
INTRODUCTION
Objectives
1
1
LITERATURE REVIEWS
Characteristics of Lotus japonicus and Meshorhizobium loti
MYB transcription factor and myb 127
The formation process of nodule in Lotus japonicus
Producing transgenic hairy toot
2
2
2
3
3
MATERIAL AND METHODS
Bacterial strains and medium
Plant materials
Construction of MYB RNAi and MYBOX in L. japonicus
Acetylene reduction assay(ARA)
Quantitative real time PCR
Counting of bacteroid number
Staining of MYB promotor-GUS in L. japonicus
Microscope and nodule measurement
4
4
4
4
5
5
6
6
6
RESULT AND DISCUSSION
DNA amplification and electrophoresis results of PCR
Relative expression of transgenic hairy root
Phenotype expression of nodulation
Nitrogen (N2) fixation activity
Bacteroid number
GUS staining of MYB promoter-GUS
Quantitative real time PCR
8
8
9
9
12
14
14
15
CONCLUSIONS
17
RECOMENDATIONS
17
REFERENCES
18
APPENDIX
21
LIST OF TABLES
1 Calculation of construct electrophoresis results
8
LIST OF FIGURES
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Construction of MYB OX, MYB RNAi and controls
Transformation process of transgenic hairy root
Relative expression of myb in transgenic hairy roots
Nodule number per plant of MYB OX and GUS OX
Nodule number per plant of MYB RNAi and GUS RNAi
Root lenght of transgenic hairy roots
Nodule diameter of transgenic hairy roots
Acetylene reduction assay in OX hairy roots
Acetylene reduction assay in RNAi hairy roots
Bacteroid numbers of nodule in transgenic hairy roots
Blue staining in MYB promotor nodule and control
Relative expression of enod40 gene of transgenic hairy roots
Relative expression of nin gene of transgenic hairy roots
Relative expression of cyspro gene of transgenic hairy roots
5
7
8
9
9
10
11
12
13
13
14
15
16
17
INTRODUCTION
Excess fertilizer caused pollution. Emissions generated during the
application of synthetic fertilizer accounted for 13 % of agriculture greenhouse gas
emission in the world (FAO, 2014), it produced from fossil fuel with high economic
costs, pollution of the environment and its high energy costs. nitrogen fertilizer
which known as the biggest amount of syntetic fertilizer used could be reduced
without compromising crop yield using legume plants.
Leguminous plants have capability of establishing nitrogen (N2) fixing
symbiosis with soil bacteria of family Rhizobiaceae. Under conditions of low soil
nitrogen, soil bacteria collectively referred as rhizobia infects the roots of legumes
and induce the formation of root nodules, while host plants are house and feed the
bacterial symbiont. Within the root nodule rhizobia provided reduced nitrogen (N2)
to the plants instead of the host plants provide carbohydrates for exchange
mutualism. This critical important symbiosis to fix nitrogen around 200 million tons
per year (Ferguson 2010). Understanding the mechanisms of symbiotic nitrogen
(N2) fixation between rhizobia and legume expects to decrease the synthetic
nitrogen fertilizer consumption.
During symbiosis process, rhizobia and leguminous plants recognize each
other through the exchange of chemical signal. Some advance of researchs said that
precise interaction between phytohormones and the other signaling compounds are
imperative for nodule organogenesis (Ferguson 2003). Important progress has been
achieved in the past years toward understanding the initial stages of this complex
developmental process. Several genes have been identified that play a role in the
perception of the bacterial Nod factors (NFs), lipo-chito-oligosaccharidic signals
essential for triggering the symbiotic genetic program in specific legume hosts,
further several of upregulated genes have been identified playing very important
roles whether in the early nodulation process or in nodule organogenesis. Some of
the genes like nin gene, enod gene were known clearly based on their function but
many genes was still poorly understood. It’s make the complex developmental of
the nodulation process still actively investigated to know about the acts of gene
during nodulation.
In previous research, 76 genes upregulated during Mesorhizobium loti
infection to Lotus japonicus plant. One of upregulated gene is MYB transcription
factor (El Yahyaoui et al. 2004), a molecule binds to AACG/TG site in DNA
specifically we found myb127 as the kind of this transciption factor. Due to the
precise function of MYB transcription factor in the rhizobia symbiosis is still
unknown, this research tried to investigate about function of MYB transcription
factor during nodule development in Lotus japonicus.
Objectives
The objectives of this study are to investigate the effect of MYB
transcription factor in nodule formation by studying the phenotype expression of
nodule, nitrogen (N2) fixation activity and relative expression of gene related
nodulation.
LITERATURE REVIEWS
2
Characteristics of Lotus japonicus and Mesorhizobium loti
Mesorhizobium loti MAFF303099 or Rhizobium loti is a Gram negative
bacteria that able to set determinant type globular nodule with Lotus japonicus to
fix nitrogen from the atmosphere. It’s also known as a bacterial symbiont with
several lotus species. The complete sequence of genome of M.loti have been
published by Kaneko et al.. (2000). The genome consisted of three circular
molecules, a single chromosome with 7,036,071 bp and two plasmids pMLa and
pMLb with 351, 911 bp and 208, 315 bp respectively.
Lotus japonicus is a wild legume of family Fabaceae. Members of this
family are very diverse more than 20,000 species. Fabaceaea family has
significant importance for agricultural and biological as many of others legume
species that rich sources of protein and oil and can also fix nitrogen (N2). L.
japonicus has become a model plant for genome studies in legumes, particularly in
reference to rhizobia and arbuscular mycorhizal symbiosis. The genome size that
known of 470 Mb, the size of plant smaller than other legumes and the abundant
of resulted seed for a short life cycle (2 to 3 months) were some reasons why this
plants were used to genomic study during these last years (Udvardi et al. 2005).
The data of whole genome sequence L. japonicus already been published by Sato
et al. (2008).
MYB Transcription Factor and myb127
Nod factor, the signal molecules carried by rhizobia like an
oligopolysaccharide can activate a signal pathway that resulted the transcription
activation of transcription factors. These regulatory proteins recognize and bind to
specific sequences of DNA which located in the promoter of genes, its controlling
the activity of gene as the response to environmental. In legumes, several
transcription factors have been identified by their capacity to bind to nodulin
promoters, but the most significant advances have aroused from genetic approaches
in nodule development. The first transcription factor cloned was NODULE
INCEPTION (NIN), the other genes such as Astray, Ethylene Response Factor
Required for Nodulation 1 (ERN1) and ERN2,5,6 RR1,7 CYCLOPS,8 Nodulation
Signaling Pathway 1 (NSP1) and NSP2, Nuclear Factor Y (NF-Y) A1 and NFYC112 GRAS (NSP1 and NSP2) and NF-Y (NF-YA and NF-YC) (Ripodas et al.
2014).
The MYB proteins family is a group of functionally diverse transcriptional
activators found with plants or animal. MYB proteins characterized by DNAbinding domain of more than 50 amino acids. It was known binds to a specific DNA
sequence for C/TAACG/TG in most organisms (James et al. 1997) MYB proteins
classified into three subfamilies (one, two or three) depending on the number of
adjacent which repeats in MYB domain. MYB transcription factors in plants
participate in the cell development, plant responses to environmental factors and
mediate the action of some hormones (Stracke et al. 2001).
In Lotus japonicus some function of MYB trancription factor have been
reported.Volve et al. 2012 has found that MYB transription factor upregulated in
Lotus japonicus root during mycorhizal process, acted as the control for root growth.
Kang et al. 2013, also reported MYB subfamily of IPN2 has an important action in
nodule organogenesis, in which the suppression of IPN2 by RNAi will significantly
decrease the nodule number of L.japonicus.
The myb127 has been upregulated during M.loti infection in L.japonicus.
myb127 gene was strongly upregulated after 6 days M.loti inoculation, this
expression measured with water and KNO3 as the control. The high expression of
MYB transcription factor is an early indicator of myb127 gene as a gene which
affected the nodulation.
The formation process of nodule in Lotus japonicus
The nodule formation process for each plant, generally has the same
pathway. The nodules formation has started from the signal that given by plant as a
host, flavonoid was known as the compound used to create an early essential signals
for nodule establishment. Its known as the early signal for establishing contact and
became the first step in infection leading to nodule development (Lateif et al. 2012).
The activated signaling of flavonoids by plants then activate the NodD
proteins that contributing to stimulate the Nod-gene. Nod factor signal continuously
trigger the nodule development after M.loti-rhizobia get to the root and form the
root curling, then made the infection thread in conditioning the root to be low
oxygen condition. The increase number of rhizobia as bacteroids in plants root then
differentiate as the nodule (Madsen et al. 2010).
In Lotus japonicus the activation of Nod factor (NF) will express the early
nodulin gene or ENODs, The activation of ENODs is associated with calcium
spiking that acts as the signal in linking NF perception to change of gene expression.
The early NF signaling pathway is NFR1/NFR5 that encode predicted sugarbinding receptor-like kinases furtherly known as SymRK.
Producing transgenic hairy root
Transgenic hairy roots have been used for more than 30 years. In recent
years transgenic hairy has used Agrobacterium-rhizogenes to mediate hairy root
production as a biotechnology tool in a variety of plant species to discover novel
biological insights. Metabolic enzyme function can be determined by gene
overexpression or RNA interference (RNAi) approaches using hairy root
transformation or transgenic hairy roots (Ron et al. 2014).
Nowadays, hairy roots transformation used gateway clonning technology.
The Gateway clonning system is a molecular biology method that enables
researchers to transfer DNA-fragments between plasmids using a proprietary set of
recombination sequences efficiently, the "Gateway att" sites, and two proprietary
enzyme mixes, called "LR clonase", and "BP clonase". Gateway clonning technique
allows transfer of DNA fragments between different clonning vectors while
maintaining the reading frame. Using Gateway, one can clone or subclone DNA
segments for functional analysis. The system requires the initial insertion of a DNA
fragment into a plasmid with two flanking recombination sequences called “attL1”
and “attL2”, to develop a “Gateway entry clone” (Maekawa et al. 2008)
MATERIALS AND METHODS
Bacterial strains and medium
4
M.loti, a Gram-negative nitrogen (N2) fixing bacterium wild type was used
in this research. M. loti lives in soil and establish a symbiosis with legume plants.
A tryptone yeast extract (TY) medium was used for cultivation (Beringer 1974).
The medium contained 5 g tryptone, 3 yeast extract, 1 g CaCl 2.H2O controlled on
pH 6.8. During cultivation M.loti was controled at 28oC.
Plant materials
Seeds of L.japonicus B-129 (GIFU) were scarified by sand paper to remove
the seed coats. 2 ml of sodium hypochlorite solution and 10 µl of tween-20 fill up
10 ml water was used as sterilization solution of seeds, shacked for 10 minutes and
followed by washing with milli-Q water for 3-4 times and final shacked for
overnight. One hundred seeds then cultured for each plate for 3 days dark and 2
days light, then infection with Agrobacterium-rhizogenesis LBA1334 which
carrying transcription factor of MYB overexpression, MYB suppression, GUS over
expression, and GUS suppression, then infected to the shoot which has cut the root
and transferred to the Jensen medium for 1 day dark and 4 days light. HRE medium
was used as medium to grow the transgenic hairy root for ten days. Transgenic hairy
roots were selected under the flourescent microscope, then transferred to the jar pot.
Vermiculite was put in the upper compartment of sterile double Magenta Jars, the
lower jar was supplied with ½ B&D nitrogen-free nutrient solution (Broughton and
Dilworth 1971). Six transgenic hairy root of Lotus japonicus plant were cultured
in each jar pot. The Jars were placed in a growth cumber cabinet (EYELA FLI2000; Tokyo Rikakikai Co., LTD., Japan) controlled at 24oC and exposed to 16
h/light and 8 h/dark. After 1 day plants were inoculated with 10 ml of M.loti, which
cultured in TY Medium for 3 days and adjusted to OD550 nm =1.0 (1 x 109 cells
ml-1). The phenotype expression of nodule measured at 2 and 4 weeks post
inoculation (wpi).
Construction of MYB RNAi and MYBOX in L. japonicus
For making MYB RNAi (chr4.CM0004.2240.r2.d) suppression by RNAi
method, a 289 bp fragment (position 544- 832) was amplified from the cDNA using
a set of primers containing attB recombination sites for the Gateway vector; FW
primer;
5’ACAAGTTTGTACAAAAAGCAGGCTGTTGGGAATGATGCCACTTT-3’; Rv
primer;5’ACCACTTTGTACAAGAAAGCTGGGTACGGTAATTGGTCCAAG
CAG-3’ For MYB overexpression (MYB OX), a 900 bp fragment was amplified
using the following primers: FW primer;5’-ACAAGTTTGTACAAAA
AGCAGGCTATGGGCAGGAAGTGCTCACA-3’; Rv primer; 5’- ACCACTTTG
TACAAGAAAGCTGGGTTTAAGTCACGGTAATTGGTC-3’ (the underline
indicates the attB recombination sites). The PCR products were subcloned into
pDNOR/Zeo (Invitrogen), and then transferred into a Gateway binary vector, pUBGWS-GFP for RNAi construction and pUB-GW-GFP for MYB overexpression
(MYB OX) (Maekawa et al.. 2008).
The constuct was checked using PCR; the standard cycling conditions were
98 C for 2 minutes, 40 cycles of 95 oC for 10 sec, 55 oC for 15 sec, and 68 oC for 2
minutes of 25 cycle amplification .
o
Figure 1. Constructions of MYB OX, MYB RNAi and controls
Acetylene reduction assay (ARA)
Acetylene reduction assay was determined to know the nitrogen (N2)
fixation activity (Banba et al.. 2001), 2 wpi of nodule and 5 wpi of nodule taken to
measure the acetylene reduction activity. Standard vial was prepared by injecting
1 ml of ethylene (C2H4) to 70 ml vial, 0.1 ml of them then taken into 25 ml vial,
then 0.25 ml transferred into 25 ml second vial. 0.1 ml, 0.5 ml and 1 ml gas from
25 ml second vial then injected to the Shimadzu GC-8A gas-chromatography
(shimazu, Kyoto, Japan) to make the standardcurve. The flow rate of nitrogen
carrier gas was set at 50 ml min-1, range set at 101 , attenuation 64, the injector and
column temperatures at 110 and 70 respectively.
After the standard prepared, 10 nodules for each thairy roots were inserted
to each sample vial. Then, 2.6 ml of Acetylene (C2H2) gas injected to the sample
vial substituted 2.6 ml of air extracted. The vials were closed with a rubber doublestopper then incubated at 28oC for 30 minutes. 1 ml of gas samples analyzed by
using six time repetition for each transgenic hairy root construct. The amount of
nmol ethylene evolved per hour per plant then calculated.
6
Quantitative real time PCR
The Lotus japonicus which carrying the transgenic hairy root of MYB
overexpression, MYB suppression, GUS over expression, and GUS suppression
were growed in the chamber. Two weeks post inoculation (wpi) and 4 weeks post
inoculation were a precise time for RNA extraction. The total RNA has extracted
using the RNeasy plant mini kit by Qiagen, CA, USA. In making cDNA, 0.5 μg of
total RNA mixed with The diluted cDNA stored at -30 oC until it used. A final
concentration of 200 ng cDNA was mixed with 5 μl of specific primer mix. The
ubiquitin used as the internal reference for relative quantification (Ubi forward
ATGCAGATCTTCGTCAAGACCTTG,
ubi
Reverse,
ACCTCCCCTCAGACGAAG), MYB transcripts were amplified using primers 5′CTGGGAAAAGGTGACTGGAG-3′ and 5′-CCACTTGTGTTGGGGTTCTT-3′.
to check the MYB Expression of each construct and then mixed with Takara SYBR
Green premix ExtaqII (Takara, Shiga, Japan) the real time PCR (Thermal Cycler
Dice real time SystemII Takara, Japan) the standard cycling conditions were 95 oC
for 30 sec, 40 cycles of 95 oC for 5 sec, 60 oC for 30 sec, and dissociation
amplification one cycle of 95 oC for 15 sec, 60 oC for 30 sec, and 95 oC for 15 sec.
Counting of bacteroid number
Samples of MYB OX, MYB RNAi, GUS OX and GUS RNAi, nodules at 2
wpi inoculated with Ds Red M. loti, were isolated about 15-20 nodules for each
hairy roots construct. The isolated nodules then sterilized using sterilizes solution
which contained 10% sodium hypochlorite (concentration of 0.5%), 0.1% tween20,
for 3 minutes and followed by water sterilization and shaked for 5 minutes for three
times. The sterlized nodules then added with 2 ml sterilized water and grinded with
pestle in three mortar for each hairy roots construct ( 1 mortar 5 nodules), 1 ml of
suspension bacteroid in the mortar then transferred to eventube and dilute for
10,100, and 1000 dilution. The bacteroid number of 10 μl bacteroids solution that
appear the red color under the microscope then counted in glass slide using
microscope and calculated according the formula below :
Bacteroid numbers per nodule =
�
�
∗� �
*10x = 381.18,40 x=6051.27
�
�
/
�
/
�
�
�
Staining of MYB Promotor-GUS in L. japonicus
A fragment of 1.0 kb upstream of the start codon of MYB transcription factor
was amplified from genomic DNA of L. japonicus using PrimeSTAR HS DNA
polymerase (Takara, Japan) with a specific primers: forward primer, 5’
AAATCTAGAGAGACTATTGTTTTGGTCAG -3’; reverse primer, 5’TTTCCCGGGTTTCACTTGAACAAGATTTT -3’. The PCR fragment was
digested into XbaI and SmaI. The resulting fragment was ligated into pCAMBIA
fused to GUS gene (Mai et al.. 2006). The construct was then introduced into L.
japonicus using hairy root transformation and GUS staining by 5-bromo-4-chloro3-indolyl-β-D-glucuronide cyclohexylammonium salt (X-gluc) was performed as
described previously (Mai et al.. 2006).
Microscope and nodule measurement
The fluorescent microscope has used to differentiate the number of
transgenic hairy roots before transferred to the pot, the root of transgenic hairy root
appeared the green color of roots. Bright-field and fluorescent microscopy were
performed with Olympus, the images of transgenic hairy roots were taken using DP
Controller (Olympus), NIS Elements (Nikon) or Photoshop (Adobe Systems). The
phenotype expression of root nodule such as the number of nodule, nodule diameter,
and the root length were observed at 2 wpi and 4 wpi by using Vernier calipers.
Figure 2. Transformation process of transgenic hairy root
RESULT AND DISCUSSION
8
DNA amplification and electrophoresis results of PCR
The electrophoresis results of PCR product showed that 12 samples of
constructed DNA has successfully amplified, it proof by appearing DNA bands
under the electrophoresis gel (Figure 3).
Figure 3. PCR electrophoresis results of construct, 1, 2, 7, 8 (MYB RNAi),
3, 4, 9, 10 (GUS RNAi), 5, 11 (MYB OX), 6, 12 (GUS OX)
Samples number 1, 2, 3, 4, 5, 6 are cDNA sample while sample number 7,
8, 9, 10, 11, 12 are sample colony that taken from Agrobacterium transfered the
expression clone. The calculation of DNA length using excel program showed the
expecteded size and PCR product as result on Table 1. The length of DNA expected
size and PCR product mostly same indicated that construction process of each
construct has succesfully done.
Table 1. Calculation of construct electrophoresis results
Sample
DNA name
Expected
Size of PCR
name
size (bp)
product (bp)
1
876 MYB
1855
1809
RNAi
2
7171 MYB
996
992
RNAi
3
876 GUS
1595
1609
RNAi
4
7171 GUS
720
722
RNAi
5
7171
1522
1583
MYBOX
6
7171
715
722
GUSOX
7
876 MYB
1855
1870
RNAi
8
7171
996
1008
MYBRNAI
9
10
11
12
876 GUS
RNAi
7171 GUS
RNAi
7171 MYB
OX
7171
GUSOX
1595
1531
720
722
1522
1609
715
785
Relative expression of transgenic hairy root
The first step to investigate the effect of MYB transcription factor during
formation of nodule is measurement of myb gene expression of transgenic hairy
root. Transgenic hairy root showed a different expression (Figure 4). MYB OX
revealed expressed myb gene very high compared with GUS OX as the control,
MYB OX expressed myb gene 125% more than GUS OX. By contrast, MYB RNAi
expressed myb gene very low compared with GUS RNAi as the control, MYB
RNAi was suppressed 82% than GUS RNAi .The high expression of MYB gene in
MYB OX and the suppression of myb gene in MYB RNAi indicated that the
transgenic hairy root for each construct successfully planted.
40
35
myb
Relative Expression
30
25
20
15
10
5
0
GUS OX
MYB OX
GUS RNAi
MYB RNAi
Figure 4. Relative expression of myb in transgenic hairy roots
Phenotype expression of nodulation
To know the effect of MYB transciption factor for nodulation responses.
We investigated the total nodule number, the nodule diamater and main root length
used calipers as the measurement. The nodule number for each transgenic hairy root
constructs performed the difference nodule number. Figure 5 showed the nodule
number average for each transgenic hairy roots of more than 35 plants for each
transgenic hairy root construct.
10
35
GUS OX
Nodule number per plant
30
MYB OX
25
*
20
15
10
5
0
0
2
4
Week post inoculation (wpi)
Figure 5. Nodule number per plant of MYB OX and GUS OX
The data of nodule number showed MYB Overexpression (MYB OX)
produced more numerous nodules (16.4 nodules per plant, n=44) than GUS
Overexpression (GUS OX) nodules (11.8 nodules per plant, n=46) as the control of
2 weeks post inoculation (wpi)(Figure 5). The different of nodule number of both
construct showed statistically significant. The nodule number of 4 wpi showed both
of constructs (MYB OX and GUS OX) produced nodule number with no significant
different, 19.1 and 20.4 respectively. The significance of these data that confirmed
by using Student’s t test analysis for (P < 0.01) for 2 wpi but the differences were
no significant at 4 wpi. The different of nodule number that showed MYB OX has
higher nodule number might indicated the symbiotic interaction of MYB OX
formed earlier after M.loti inoculation but it no longer at 4 wpi.
30
*
nodule number per plant
GUS RNAi
25
*
MYB RNAi
20
15
10
5
0
0
2
4
Week post Inoculation (wpi)
Figure 6. Nodule number per plant of MYB RNAi and GUS RNAi
The number of nodules of MYB suppression (MYB RNAi) produced lower
number of nodules (9.1 nodules per plant, n=38) than GUS Suppression (GUS
RNAi) nodules (14.6 nodules per plant, n=40) of 2 wpi (Figure 6). The different of
nodule number of both construct showed statistically significant. The nodule
number of 4 wpi showed MYB RNAi and GUS RNAi produced nodule number
with signifant different, 15.6 and 10.7 respectively. The significance of these data
that confirmed by using Student’s t test analysis for (P < 0.01) for 2 wpi and
(P0.05). These data (figure
6) indicated that MYB transcription factor did not affected the root lenght, no
significant different found in measuring more than 35 plants for each constructs. As
the conclusion, we stated that the MYB transcription factor did not give effect to
root length of Lotus japonicus.
12
GUS OX
MYB OX
GUS RNAi
MYB RNAi
Root lenght (cm)
10
8
6
4
2
0
0
2
Weeks post inoculation (wpi)
Figure 7. Root lenght of transgenic hairy roots
4
12
In addition, we also made an observation about the nodule diameter to
collect more data about the phenotype effect of MYB transcription factor in nodule
development (Figure 8). At 4 wpi of nodule we found that MYB transcription factor
has no effect in nodule diameter or nodule size. After measuring more than 35 plants
for each transgenic hairy root construct we found nodule diameter of MYB OX,
GUS OX, MYB RNAi, and GUS RNAi were 0.099, 0.093, 0.089, 0.090 cm
respectively. Same as the root length, the statistical analysis of nodule diameter also
concluded no significant different of nodule diameter of each transgenic hairy roots
construct.
Nodule Diameter
0,14
Nodule Diameter (cm)
0,12
0,1
0,08
0,06
0,04
0,02
0
GUS OX
MYB OX
GUS RNAi
MYB BRAi
Figure 8. Nodule diameter of transgenic hairy roots
Nitrogen (N2) Fixation Activity
To make explanation detailed about the effect of MYB transcription factor
during nodulation we also measured the acetylene reduction assay during nodule
formation at 2 wpi and 5 wpi (Figure 9). The positive transgenic nodules of each
construct has assayed using acetylene (C2H2) reduction assay (ARA) after 0.5 hours
incubation. ARA can measure the nitrogen fixing activity indirectly. Because
nitrogenase does not convert nitrogen to ammonia when excess acetylene is
included. Therefore, acetylene can convert to ethylene by nitrogenase. Acetylene
reduction Assay (Figure 8) showed that the nitrogen fixation capacity of MYB OX
was significantly higher than GUS OX with a different of 14.17 nmol/h/nodule and
11.44 nmol/h/nodule at 2 wpi. The nitrogen fixation assay of both constructs were
increased significantly at 5 wpi that nitrogen fixation activity of MYB OX and GUS
OX reached 28.68 nmol/h/nodule and 19.12 nmol/h/nodule respectively.
Ethylene Production(nmol/h/nodule)
ARA
40
GUS OX
35
30
25
20
15
10
5
0
0
2
5
Weeks post Innoculatuon (wpi)
Figure 9. Acetylene reduction assay in OX hairy roots
Ethylene Production (nmol/h/nodule)
Different with overexpression hairy roots of MYB OX that has a significant
different of activity at 4 wpi , MYB RNAi performed a similiar nitrogen (N 2)
fixation assay data with GUS RNAi at 2 wpi (Figure 10). Activity of 2 wpi MYB
RNAi reduced acetylene of 10.75 nmol/h/nodule and GUS RNAi reduced
achetylene of 12.29 nmol/h/nodule respectively. Interestingly, at 5 wpi the
acetylene reduction assay of MYB RNAi was lower than GUS RNAi that are 18.56
nmol/h/nodule and 32.14 nmol/h/nodule respectively.
These result showed that the over expression of MYB has a significant
impact on nitrogen (N2) fixation per nodule at 5 wpi, interestingly the number of
nodule in previous data (Figure 5) showed the MYB OX at 4 wpi did not increase
the nodule number. By Contrast, the ARA of RNAi constructs at 5 wpi suggested
that the nitrogen (N2) fixation of MYB RNAi was lower than GUSRNAi. These
results also showed that the MYB deficiency or MYB RNAi affected not only in
decreasing the nodule number but also the nitrogen (N2) fixation activity of nodule
after 5 wpi.
ARA
40
GUS RNAI
35
30
25
20
15
10
5
0
0
2
Weeks post Innoculatuon (wpi)
Figure 10. Acetylene reduction assay in RNAi hairy roots
5
14
The MYB transcription factor effects during nitrogen (N2) fixation activity
probably because of some reasons that already found by some previous studies.
Banba et al. (2001) reported the nodules formed with R. etli CE3 cannot utilize
photosynthate as an energy source for nitrogen (N2) fixation thereby decreased the
fixation activity of nitrogen (N2) and exhibit senescence. On the other hand, Ott et
al. (2005) also reported that suppression of plant leghemoglobin gene by RNA
interference in L. japonicus resulted higher oxygen concentrations inside nodules,
lower bacterial nif and fix gene expression, created the absence of nitrogenase
activity. However, the future study is needed to know the precious mechanism of
MYB transcription factor in effecting nitrogen (N2) fixation activity of nodules.
Bacteroid number
To know the effectivity of rhizobia-plants interaction we counted the
bacteroid number within the nodule (Figure 11). The bacteroid number of nodule
has known effectively correlated with the increased nitrogen (N2) fixation
(Wadisirisuk and Weaper 1985). We counted the bacteroid number of MYB OX,
MYB RNAi, GUS OX and GUS RNAi nodules at 2 wpi inoculated with Ds Red
M.Loti. We found the number of bacteroids was significantly increased in MYB
OX with 2.40 x 107 bacteroids per nodule, while that we found GUS OX with 1.56
x 107 bacteroids per nodule (Figure 10 ). In RNAi transgenic hairy roots we found
the bacteroids number of 1.63 x 107 bacteroids per nodule and 1.71 x 107
bacteroids per nodule for MYB RNAi and GUS RNAi recpectively. This analyzed
data showed that MYB OX was significantly greater at 2 wpi than GUS OX, but
MYB RNAi did not effect in bacteroid numbers. These data indicated that MYB
OX has more rapid in forming symbiotic interaction to form nodule at 2 wpi.
4
Bacteroid number per nodule
*
x107 cells per nodule
3,5
3
2,5
2
1,5
1
0,5
0
GUS OX
MYB OX
GUS RNAi
MYB RNAi
Figure 11. Bacteroid number of transgenic hairy roots
GUS staining of MYB promoter-GUS
In transgenic hairy roots carrying a MYB promoter-GUS, GUS staining
(blue color) of roots marks confirming the transient expression of GUS (Figure 12).
The GUS staining showed the MYB promoter was active within the root and 12
days post inoculation (dpi) nodule. The GUS Expression within the nodule
expressed in nodule primordia site (Kang et al. 2013). Further the control
experiments with Plambia 134 vector did not give blue coloration.
0.5
mm
0.5
mm
Figure 12. Blue staining in MYB promotor nodule and control
Quantitative real time PCR
In order to check the correlation of MYB transcription factor compared
with the expression of genes in nodule we investigated the expression analysis
of enod40 gene and nin gene using qRT-PCR (Figure 13). The expression levels
corresponding to enod40 were higher in MYB Supressing nodules than in the GUS
OX as control. By parallel the expression of enod40 lower in MYB RNAi. These
results suggest that MYB may binds to enod40. By Contrast, the expression levels
of early nodule development marker genes, nin in MYB hairyroots (Figure 14).
revealed that nin decreased in MYB OX and increased in MYB RNAi compared to
their control GUS OX and GUS RNAi. These results suggest that MYB may not a
regulator of nin but we predicted MYB has a parallel function with nin in nodulation
process .
The expression pattern of MYB was similiar with enod40. The
overexpression of enod40 accelerated nodule formation and the suppression of
enod40 suppressed nodule formation (Charon et al. 1999). On the other hand The
GUS staining of MYB promoter-GUS activity (Figure 12) and enod40 stated by
Martirani et al. (1999) was also similiar in which it expressed in the vascular of
nodule. These results indicate that MYB might regulate the enod40expression.
Relative Expression
1000
800
enod40
600
400
200
0
GUS RNAi
MYB RNAi
GUS OX
MYB OX
Figure 13. Relative expression of enod40 gene of transgenic hairy roots
16
14
nin
Relative Exspresssion
12
10
8
6
4
2
0
GUS RNAi
MYB RNAi
GUS OX
MYB OX
Figure 14. Relative expression of nin gene of transgenic hairy roots
To ensure the nitrogen (N2) fixation correlated with nodule senescence we
measured the expression of cyspro gene, the gene marker for nodule senescence in
MYB hairy roots (Figure 15). The cyspro expression of MYB RNAi was
significantly high compared with GUS RNAi, contrastly the MYB OX expressed
low Cyspro gene. These data totally supported the nitrogen (N2) fixation result, in
which the MYB RNAi significantly reduced the nitrogen (N2) fixation activity at
5 wpi nodule. These results indicate that downregulation of MYB expression by
RNAi suppression has an effect to form senescence nodule earlier, and negative
effect to nirtogen (N2) fixation and nodulation.
Banba et al. (2001) reported that nodules that formed with inoculation
CE3/Rhizobium etli that inoculated appeared to be of the early-senescence type
because greater amounts of disintegrated membrane structures of nodules. Early
senescence formed by disintegration of symbiosome membranes, deterioratio and
aggregation of bacteroids, and degradation of the cytoplasmic structures of the
infected cells. Although our experiments did not reveal the mechanism of
senescence formed in L. japonicus nodule by MYB RNAi but we so far concluded
the role of MYB RNAi in forming senescence nodule. Moreover, the study about
the mechanism of senescence of MYB RNAi is an interesting issue to be solved in
the future research.
Relative Exspression
2
1,8
cysteine protease
1,6
1,4
1,2
1
0,8
0,6
0,4
0,2
0
GUS RNAi
MYB RNAi
GUS OX
MYB OX
Figure 15. Relative expression of cyspro gene of transgenic hairy roots
CONCLUSIONS
a.
b.
c.
d.
e.
f.
In this present study, we concluded that :
the Overexpression of MYB increased the nodule number of nodule during
early nodulation time but no longer in mature nodule.
MYB overexpression significantly increased the nitrogen (N2) fixation
activity and plant-rhizobia symbiosys.
The suppression of MYB (MYB RNAi) decreased the number of nodule
and indirectly decreased the nitrogen (N2) fixation activity and symbiosis
process of plant-rhizobia.
MYB transcription factor expressed in nodule and root.
As an early genetically study we proposed that MYB transcription factor
might bind to enod40 gene but not as transcription regulator of nin gene.
The lack of MYB gene formed early senecence in nodulation.
RECOMENDATIONS
It is needed to analysis the effect of MYB genetic expression during
nodulation more deeply. The histological study of MYB nodule also very important
to be investigated more detailed.
18
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APPENDIX
Appendix 1. Phenotype expression of transgenic hairy roots under GFP
microscope. The photographs were taken in the GFP mode (A, C, E, G) and in the
light field (B, D, F, H). Scale bars represent 2 mm, A and B are MYB OX in 2 wpi,
C and D are GUS OX in 2 wpi, E and F are GUS RNAi in 2 wpi, G and H are GUS
RNAi in 2 wpi.
A
C
B
D
E
F
G
H
22
Appendix 2. Table of nodules number of 2 wpi transgenic hairy roots
GUS
MYB
GUS
MYB
No
OX
OX
RNAi
RNAi
1
11
9
12
3
2
23
8
22
7
3
3
6
10
0
4
13
17
11
6
5
4
23
17
5
6
17
10
15
3
7
10
12
11
9
8
20
25
5
13
9
9
13
10
7
10
19
5
11
13
11
16
16
18
15
12
5
13
EFFECT OF MYB TRANSCRIPTION FACTOR DURING
NODULE DEVELOPMENT IN Lotus japonicus
SUPRIADI
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
DECLARATION OF THESIS AND INFORMATION SOURCES
OF INFORMATION AND PATENT
I, Supriadi, hereby stated that this thesis entitled Effect of MYB
Transcription Factor During Development in Lotus japonicus is true of my own
work under the supervisor advisory board and that it has not been submitted before
in any form to any university. The content of this thesis has been examined by the
advising advisory board and external examiner. Sources of information which is
derived or cited either from published or unpublished scientific paper from other
writers have mentioned in the script and listed in the references at the end part of
this thesis.
I hereby handed the copyright of my thesis to Bogor Agricultural University.
Bogor, April 2016
Supriadi
A154130201
ii
RINGKASAN
SUPRIADI. Efek Faktor Transkripsi MYB Selama Pertumbuhan Nodul pada Lotus
japonicus. Dibimbing oleh RAHAYU WIDYASTUTI, DWI ANDREAS
SANTOSA, dan MIKA NOMURA.
Dalam beberapa tahun terakhir, beberapa capaian untuk memahami tahap
dalam proses perkembangan nodul telah banyak dilakukan dengan pendekatan studi
molekuler. Beberapa gen telah diidentifikasi memainkan peran sangat penting baik
dalam proses awal nodulasi atau pun nodul organogenesis. Penelitian sebelumnya
menemukan bahwa 76 jenis gen teregulasi selama infeksi Mesorhizobium loti ke
tanaman Lotus japonicus . Salah satu gen teregulasi adalah faktor transkripsi MYB,
molekul yang mengikat pada situs AACG /TG pada DNA. Karena fungsi yang
tepat dari faktor transkripsi MYB dalam proses nodulasi masih kurang diketahui,
maka fungsi faktor transkripsi MYB selama proses nodulasi penting untuk diteliti.
Untuk mengetahui pengaruh faktor transkripsi MYB dalam proses
nodulasi Lotus japonicus, kami selanjutnya membuat akar transgenik dari dari
Lotus japonicus dengan MYB over ekspresi (MYB OX) dan MYB tersupresi oleh
RNAi (MYB RNAi). Kami mengukur jumlah nodul, diameter nodul dan panjang
akar sebagai ekspresi fenotip. Untuk mengetahui aktifitas mereduksi Asetilen dari
nodul menggunakan analisis kromatografi gas untuk mengetahui aktivitas fiksasi
nitrogen. Untuk mengetahui ekspresi gen yang terkait nodulasi, kami mengukur
ekspresi gen enod40, gen nin, dan gen sistein protease menggunakan QRT-PCR.
Hasil penelitian menunjukkan bahwa MYB yang dieskpresikan berlebih
secara signifikan meningkatkan jumlah nodul dari Lotus japonicus di awal minggu
pasca inokulasi dan peningkatan aktivitas fiksasi nitrogen (N2) pada nodul. Pada
penekanan dari MYB secara signifikan menurunkan jumlah nodul dan aktivitas
fiksasi N2 serta menginduksi nodul senesens lebih awal. Kami juga menemukan
bahwa MYB tidak memberi efek pada diameter nodul dan panjang akar pada Lotus
japonicus.
Data pada QRT-PCR menunjukkan bahwa tingkat ekspresi enod40
memungkingkan MYB mengikat pada enod40. Sebaliknya, tingkat ekspresi nin
menunjukkan bahwa MYB mungkin tidak mengendalikan ekspresi gen nin tapi
kami memprediksi MYB memiliki fungsi paralel dengan gen nin dalam proses
nodulasi. Kami mengusulkan bahwa faktor transkripsi MYB mengikat pada gen
enod40 dan diaktifkan selama masa awal nodulasi.
Keyword: MYB, faktor Transkripsi, Nodul, Lotus japonicus
SUMMARY
SUPRIADI. Effect of MYB Transcription factor During Nodule Development in
Lotus japonicus. Supervised by RAHAYU WIDYASTUTI, DWI ANDREAS
SANTOSA, and MIKA NOMURA
In the past years, some achievments toward understanding the initial stages
of nodule developmental process has been done using molecular study. Several
genes have been identified that play very important roles whether in the early
nodulation process or in nodule organogenesis. Previous research found that 76
genes upregulated during Mesorhizobium loti infection to Lotus japonicus plant.
One of upregulated gene is MYB transcription factor, a kind of molecule bind to
AACG/TG site in DNA. Due to the precise function of MYB transcription factor
in the rhizobial symbiosis is still poorly unknown, we investigated function of MYB
transcription factor during nodulation.
To know the effect of MYB transcription factor in nodulation process of
Lotus japonicus we further created the transgenic hairy roots of Lotus japonicus
with MYB overexpression (MYB OX) and suppression by RNAi (MYB RNAi).
We measured the nodule number, nodule diameter and root lenght as phenotype
expression. In additional Acetylene reduction activity of nodules has been assayed
using gas cromatografh analysis to know nitrogen (N2) fixation activity. To know
the expression of genes related nodulation we measured expression of enod40 gene,
nin gene, and Cystein Protease gene using qRT-PCR.
The result revealed that the overexpression of MYB significantly increased
the nodule number of Lotus japonicus in early week post innoculation and
increased nitrogen (N2) fixation activity of nodule. Particularly the supression of
MYB has significantly decreased the nodule number and nitrogen fixation activity
and induce the scenesence nodule earlier. We also found that MYB did not give
effect to nodule diameter and root lenght of Lotus japonicus.
The qRT-PCR expression levels of enod40 suggested that MYB might
bind to enod40. In contras, the expression levels of nin suggested that MYB might
not a regulator of nin but we predicted MYB has a paralel function with nin in
nodulation process .We finally proposed that MYB transcription factor binds to
enod40 gene and activated during the early time of nodulation.
Keyword : MYB, transcription factor, nodule, Lotus japonicus
iv
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EFFECT OF MYB TRANSCRIPTION FACTOR DURING
DEVELOPMENT IN Lotus japonicus
SUPRIADI
A Thesis submitted for the Degree Programs
of Master of Science
in Soil and Environtmental Biotechnology
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
vi
External examiner : Dr Ir Iman Rusmana, Msi
viii
FOREWORD
First of all, I would humbly distinguish the Most Gracious Allah SWT, all
praises to Allah for the gifts and His blessing in completing this thesis with title
Effect of MYB Trancription Factor During Nodule Development in Lotus
japonicus. This thesis submitted for the Degree Programs of Master of Science in
Master of Science of Soil and Environtmental Biotechnology. I would like to
sincerely deliver my greatest gratitude to my advisor: Dr Rahayu Widyastuti, MSc,
Prof Dr Ir Dwi Andreas Santosa, MS, Prof Mika Nomura for their advices,
expertness, encouragement, and support. My sincere thanks also to Prof Shigeyuki
Tajima for all support.
My appreciations were also extended to Japan Student Services Organization
(JASSO) Scholarship for granting scholarship during the study and experiment in
Kagawa University and also for SUIJI-JDP (Six University Initiative JapanIndonesia Joint Degree Program) who allowed me to expand my knowledge and
experiences in Japan. This master thesis would not have been possible unless the
funding of Indonesian Government scholarship (Beasiswa Fresh Graduate).
I also addressed my gratitude to all staff and classmate in Soil and
Environtmental Biotechnologi (IPB) and my laboratorymate in Plant Nutrition
(Kagawa University) and I would like to take this moment to deeply express my
thankful feeling to my father and mother who have been praying, loving and
supporting as always. Finally, I hope this thesis can give information about
nodulation process in legume, specially the effect of MYB transcription factor
during those process.
Bogor, 26 May 2016
Supriadi
x
TABLE OF CONTENT
LIST OF TABLE
vi
LIST OF FIGURE
vi
INTRODUCTION
Objectives
1
1
LITERATURE REVIEWS
Characteristics of Lotus japonicus and Meshorhizobium loti
MYB transcription factor and myb 127
The formation process of nodule in Lotus japonicus
Producing transgenic hairy toot
2
2
2
3
3
MATERIAL AND METHODS
Bacterial strains and medium
Plant materials
Construction of MYB RNAi and MYBOX in L. japonicus
Acetylene reduction assay(ARA)
Quantitative real time PCR
Counting of bacteroid number
Staining of MYB promotor-GUS in L. japonicus
Microscope and nodule measurement
4
4
4
4
5
5
6
6
6
RESULT AND DISCUSSION
DNA amplification and electrophoresis results of PCR
Relative expression of transgenic hairy root
Phenotype expression of nodulation
Nitrogen (N2) fixation activity
Bacteroid number
GUS staining of MYB promoter-GUS
Quantitative real time PCR
8
8
9
9
12
14
14
15
CONCLUSIONS
17
RECOMENDATIONS
17
REFERENCES
18
APPENDIX
21
LIST OF TABLES
1 Calculation of construct electrophoresis results
8
LIST OF FIGURES
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Construction of MYB OX, MYB RNAi and controls
Transformation process of transgenic hairy root
Relative expression of myb in transgenic hairy roots
Nodule number per plant of MYB OX and GUS OX
Nodule number per plant of MYB RNAi and GUS RNAi
Root lenght of transgenic hairy roots
Nodule diameter of transgenic hairy roots
Acetylene reduction assay in OX hairy roots
Acetylene reduction assay in RNAi hairy roots
Bacteroid numbers of nodule in transgenic hairy roots
Blue staining in MYB promotor nodule and control
Relative expression of enod40 gene of transgenic hairy roots
Relative expression of nin gene of transgenic hairy roots
Relative expression of cyspro gene of transgenic hairy roots
5
7
8
9
9
10
11
12
13
13
14
15
16
17
INTRODUCTION
Excess fertilizer caused pollution. Emissions generated during the
application of synthetic fertilizer accounted for 13 % of agriculture greenhouse gas
emission in the world (FAO, 2014), it produced from fossil fuel with high economic
costs, pollution of the environment and its high energy costs. nitrogen fertilizer
which known as the biggest amount of syntetic fertilizer used could be reduced
without compromising crop yield using legume plants.
Leguminous plants have capability of establishing nitrogen (N2) fixing
symbiosis with soil bacteria of family Rhizobiaceae. Under conditions of low soil
nitrogen, soil bacteria collectively referred as rhizobia infects the roots of legumes
and induce the formation of root nodules, while host plants are house and feed the
bacterial symbiont. Within the root nodule rhizobia provided reduced nitrogen (N2)
to the plants instead of the host plants provide carbohydrates for exchange
mutualism. This critical important symbiosis to fix nitrogen around 200 million tons
per year (Ferguson 2010). Understanding the mechanisms of symbiotic nitrogen
(N2) fixation between rhizobia and legume expects to decrease the synthetic
nitrogen fertilizer consumption.
During symbiosis process, rhizobia and leguminous plants recognize each
other through the exchange of chemical signal. Some advance of researchs said that
precise interaction between phytohormones and the other signaling compounds are
imperative for nodule organogenesis (Ferguson 2003). Important progress has been
achieved in the past years toward understanding the initial stages of this complex
developmental process. Several genes have been identified that play a role in the
perception of the bacterial Nod factors (NFs), lipo-chito-oligosaccharidic signals
essential for triggering the symbiotic genetic program in specific legume hosts,
further several of upregulated genes have been identified playing very important
roles whether in the early nodulation process or in nodule organogenesis. Some of
the genes like nin gene, enod gene were known clearly based on their function but
many genes was still poorly understood. It’s make the complex developmental of
the nodulation process still actively investigated to know about the acts of gene
during nodulation.
In previous research, 76 genes upregulated during Mesorhizobium loti
infection to Lotus japonicus plant. One of upregulated gene is MYB transcription
factor (El Yahyaoui et al. 2004), a molecule binds to AACG/TG site in DNA
specifically we found myb127 as the kind of this transciption factor. Due to the
precise function of MYB transcription factor in the rhizobia symbiosis is still
unknown, this research tried to investigate about function of MYB transcription
factor during nodule development in Lotus japonicus.
Objectives
The objectives of this study are to investigate the effect of MYB
transcription factor in nodule formation by studying the phenotype expression of
nodule, nitrogen (N2) fixation activity and relative expression of gene related
nodulation.
LITERATURE REVIEWS
2
Characteristics of Lotus japonicus and Mesorhizobium loti
Mesorhizobium loti MAFF303099 or Rhizobium loti is a Gram negative
bacteria that able to set determinant type globular nodule with Lotus japonicus to
fix nitrogen from the atmosphere. It’s also known as a bacterial symbiont with
several lotus species. The complete sequence of genome of M.loti have been
published by Kaneko et al.. (2000). The genome consisted of three circular
molecules, a single chromosome with 7,036,071 bp and two plasmids pMLa and
pMLb with 351, 911 bp and 208, 315 bp respectively.
Lotus japonicus is a wild legume of family Fabaceae. Members of this
family are very diverse more than 20,000 species. Fabaceaea family has
significant importance for agricultural and biological as many of others legume
species that rich sources of protein and oil and can also fix nitrogen (N2). L.
japonicus has become a model plant for genome studies in legumes, particularly in
reference to rhizobia and arbuscular mycorhizal symbiosis. The genome size that
known of 470 Mb, the size of plant smaller than other legumes and the abundant
of resulted seed for a short life cycle (2 to 3 months) were some reasons why this
plants were used to genomic study during these last years (Udvardi et al. 2005).
The data of whole genome sequence L. japonicus already been published by Sato
et al. (2008).
MYB Transcription Factor and myb127
Nod factor, the signal molecules carried by rhizobia like an
oligopolysaccharide can activate a signal pathway that resulted the transcription
activation of transcription factors. These regulatory proteins recognize and bind to
specific sequences of DNA which located in the promoter of genes, its controlling
the activity of gene as the response to environmental. In legumes, several
transcription factors have been identified by their capacity to bind to nodulin
promoters, but the most significant advances have aroused from genetic approaches
in nodule development. The first transcription factor cloned was NODULE
INCEPTION (NIN), the other genes such as Astray, Ethylene Response Factor
Required for Nodulation 1 (ERN1) and ERN2,5,6 RR1,7 CYCLOPS,8 Nodulation
Signaling Pathway 1 (NSP1) and NSP2, Nuclear Factor Y (NF-Y) A1 and NFYC112 GRAS (NSP1 and NSP2) and NF-Y (NF-YA and NF-YC) (Ripodas et al.
2014).
The MYB proteins family is a group of functionally diverse transcriptional
activators found with plants or animal. MYB proteins characterized by DNAbinding domain of more than 50 amino acids. It was known binds to a specific DNA
sequence for C/TAACG/TG in most organisms (James et al. 1997) MYB proteins
classified into three subfamilies (one, two or three) depending on the number of
adjacent which repeats in MYB domain. MYB transcription factors in plants
participate in the cell development, plant responses to environmental factors and
mediate the action of some hormones (Stracke et al. 2001).
In Lotus japonicus some function of MYB trancription factor have been
reported.Volve et al. 2012 has found that MYB transription factor upregulated in
Lotus japonicus root during mycorhizal process, acted as the control for root growth.
Kang et al. 2013, also reported MYB subfamily of IPN2 has an important action in
nodule organogenesis, in which the suppression of IPN2 by RNAi will significantly
decrease the nodule number of L.japonicus.
The myb127 has been upregulated during M.loti infection in L.japonicus.
myb127 gene was strongly upregulated after 6 days M.loti inoculation, this
expression measured with water and KNO3 as the control. The high expression of
MYB transcription factor is an early indicator of myb127 gene as a gene which
affected the nodulation.
The formation process of nodule in Lotus japonicus
The nodule formation process for each plant, generally has the same
pathway. The nodules formation has started from the signal that given by plant as a
host, flavonoid was known as the compound used to create an early essential signals
for nodule establishment. Its known as the early signal for establishing contact and
became the first step in infection leading to nodule development (Lateif et al. 2012).
The activated signaling of flavonoids by plants then activate the NodD
proteins that contributing to stimulate the Nod-gene. Nod factor signal continuously
trigger the nodule development after M.loti-rhizobia get to the root and form the
root curling, then made the infection thread in conditioning the root to be low
oxygen condition. The increase number of rhizobia as bacteroids in plants root then
differentiate as the nodule (Madsen et al. 2010).
In Lotus japonicus the activation of Nod factor (NF) will express the early
nodulin gene or ENODs, The activation of ENODs is associated with calcium
spiking that acts as the signal in linking NF perception to change of gene expression.
The early NF signaling pathway is NFR1/NFR5 that encode predicted sugarbinding receptor-like kinases furtherly known as SymRK.
Producing transgenic hairy root
Transgenic hairy roots have been used for more than 30 years. In recent
years transgenic hairy has used Agrobacterium-rhizogenes to mediate hairy root
production as a biotechnology tool in a variety of plant species to discover novel
biological insights. Metabolic enzyme function can be determined by gene
overexpression or RNA interference (RNAi) approaches using hairy root
transformation or transgenic hairy roots (Ron et al. 2014).
Nowadays, hairy roots transformation used gateway clonning technology.
The Gateway clonning system is a molecular biology method that enables
researchers to transfer DNA-fragments between plasmids using a proprietary set of
recombination sequences efficiently, the "Gateway att" sites, and two proprietary
enzyme mixes, called "LR clonase", and "BP clonase". Gateway clonning technique
allows transfer of DNA fragments between different clonning vectors while
maintaining the reading frame. Using Gateway, one can clone or subclone DNA
segments for functional analysis. The system requires the initial insertion of a DNA
fragment into a plasmid with two flanking recombination sequences called “attL1”
and “attL2”, to develop a “Gateway entry clone” (Maekawa et al. 2008)
MATERIALS AND METHODS
Bacterial strains and medium
4
M.loti, a Gram-negative nitrogen (N2) fixing bacterium wild type was used
in this research. M. loti lives in soil and establish a symbiosis with legume plants.
A tryptone yeast extract (TY) medium was used for cultivation (Beringer 1974).
The medium contained 5 g tryptone, 3 yeast extract, 1 g CaCl 2.H2O controlled on
pH 6.8. During cultivation M.loti was controled at 28oC.
Plant materials
Seeds of L.japonicus B-129 (GIFU) were scarified by sand paper to remove
the seed coats. 2 ml of sodium hypochlorite solution and 10 µl of tween-20 fill up
10 ml water was used as sterilization solution of seeds, shacked for 10 minutes and
followed by washing with milli-Q water for 3-4 times and final shacked for
overnight. One hundred seeds then cultured for each plate for 3 days dark and 2
days light, then infection with Agrobacterium-rhizogenesis LBA1334 which
carrying transcription factor of MYB overexpression, MYB suppression, GUS over
expression, and GUS suppression, then infected to the shoot which has cut the root
and transferred to the Jensen medium for 1 day dark and 4 days light. HRE medium
was used as medium to grow the transgenic hairy root for ten days. Transgenic hairy
roots were selected under the flourescent microscope, then transferred to the jar pot.
Vermiculite was put in the upper compartment of sterile double Magenta Jars, the
lower jar was supplied with ½ B&D nitrogen-free nutrient solution (Broughton and
Dilworth 1971). Six transgenic hairy root of Lotus japonicus plant were cultured
in each jar pot. The Jars were placed in a growth cumber cabinet (EYELA FLI2000; Tokyo Rikakikai Co., LTD., Japan) controlled at 24oC and exposed to 16
h/light and 8 h/dark. After 1 day plants were inoculated with 10 ml of M.loti, which
cultured in TY Medium for 3 days and adjusted to OD550 nm =1.0 (1 x 109 cells
ml-1). The phenotype expression of nodule measured at 2 and 4 weeks post
inoculation (wpi).
Construction of MYB RNAi and MYBOX in L. japonicus
For making MYB RNAi (chr4.CM0004.2240.r2.d) suppression by RNAi
method, a 289 bp fragment (position 544- 832) was amplified from the cDNA using
a set of primers containing attB recombination sites for the Gateway vector; FW
primer;
5’ACAAGTTTGTACAAAAAGCAGGCTGTTGGGAATGATGCCACTTT-3’; Rv
primer;5’ACCACTTTGTACAAGAAAGCTGGGTACGGTAATTGGTCCAAG
CAG-3’ For MYB overexpression (MYB OX), a 900 bp fragment was amplified
using the following primers: FW primer;5’-ACAAGTTTGTACAAAA
AGCAGGCTATGGGCAGGAAGTGCTCACA-3’; Rv primer; 5’- ACCACTTTG
TACAAGAAAGCTGGGTTTAAGTCACGGTAATTGGTC-3’ (the underline
indicates the attB recombination sites). The PCR products were subcloned into
pDNOR/Zeo (Invitrogen), and then transferred into a Gateway binary vector, pUBGWS-GFP for RNAi construction and pUB-GW-GFP for MYB overexpression
(MYB OX) (Maekawa et al.. 2008).
The constuct was checked using PCR; the standard cycling conditions were
98 C for 2 minutes, 40 cycles of 95 oC for 10 sec, 55 oC for 15 sec, and 68 oC for 2
minutes of 25 cycle amplification .
o
Figure 1. Constructions of MYB OX, MYB RNAi and controls
Acetylene reduction assay (ARA)
Acetylene reduction assay was determined to know the nitrogen (N2)
fixation activity (Banba et al.. 2001), 2 wpi of nodule and 5 wpi of nodule taken to
measure the acetylene reduction activity. Standard vial was prepared by injecting
1 ml of ethylene (C2H4) to 70 ml vial, 0.1 ml of them then taken into 25 ml vial,
then 0.25 ml transferred into 25 ml second vial. 0.1 ml, 0.5 ml and 1 ml gas from
25 ml second vial then injected to the Shimadzu GC-8A gas-chromatography
(shimazu, Kyoto, Japan) to make the standardcurve. The flow rate of nitrogen
carrier gas was set at 50 ml min-1, range set at 101 , attenuation 64, the injector and
column temperatures at 110 and 70 respectively.
After the standard prepared, 10 nodules for each thairy roots were inserted
to each sample vial. Then, 2.6 ml of Acetylene (C2H2) gas injected to the sample
vial substituted 2.6 ml of air extracted. The vials were closed with a rubber doublestopper then incubated at 28oC for 30 minutes. 1 ml of gas samples analyzed by
using six time repetition for each transgenic hairy root construct. The amount of
nmol ethylene evolved per hour per plant then calculated.
6
Quantitative real time PCR
The Lotus japonicus which carrying the transgenic hairy root of MYB
overexpression, MYB suppression, GUS over expression, and GUS suppression
were growed in the chamber. Two weeks post inoculation (wpi) and 4 weeks post
inoculation were a precise time for RNA extraction. The total RNA has extracted
using the RNeasy plant mini kit by Qiagen, CA, USA. In making cDNA, 0.5 μg of
total RNA mixed with The diluted cDNA stored at -30 oC until it used. A final
concentration of 200 ng cDNA was mixed with 5 μl of specific primer mix. The
ubiquitin used as the internal reference for relative quantification (Ubi forward
ATGCAGATCTTCGTCAAGACCTTG,
ubi
Reverse,
ACCTCCCCTCAGACGAAG), MYB transcripts were amplified using primers 5′CTGGGAAAAGGTGACTGGAG-3′ and 5′-CCACTTGTGTTGGGGTTCTT-3′.
to check the MYB Expression of each construct and then mixed with Takara SYBR
Green premix ExtaqII (Takara, Shiga, Japan) the real time PCR (Thermal Cycler
Dice real time SystemII Takara, Japan) the standard cycling conditions were 95 oC
for 30 sec, 40 cycles of 95 oC for 5 sec, 60 oC for 30 sec, and dissociation
amplification one cycle of 95 oC for 15 sec, 60 oC for 30 sec, and 95 oC for 15 sec.
Counting of bacteroid number
Samples of MYB OX, MYB RNAi, GUS OX and GUS RNAi, nodules at 2
wpi inoculated with Ds Red M. loti, were isolated about 15-20 nodules for each
hairy roots construct. The isolated nodules then sterilized using sterilizes solution
which contained 10% sodium hypochlorite (concentration of 0.5%), 0.1% tween20,
for 3 minutes and followed by water sterilization and shaked for 5 minutes for three
times. The sterlized nodules then added with 2 ml sterilized water and grinded with
pestle in three mortar for each hairy roots construct ( 1 mortar 5 nodules), 1 ml of
suspension bacteroid in the mortar then transferred to eventube and dilute for
10,100, and 1000 dilution. The bacteroid number of 10 μl bacteroids solution that
appear the red color under the microscope then counted in glass slide using
microscope and calculated according the formula below :
Bacteroid numbers per nodule =
�
�
∗� �
*10x = 381.18,40 x=6051.27
�
�
/
�
/
�
�
�
Staining of MYB Promotor-GUS in L. japonicus
A fragment of 1.0 kb upstream of the start codon of MYB transcription factor
was amplified from genomic DNA of L. japonicus using PrimeSTAR HS DNA
polymerase (Takara, Japan) with a specific primers: forward primer, 5’
AAATCTAGAGAGACTATTGTTTTGGTCAG -3’; reverse primer, 5’TTTCCCGGGTTTCACTTGAACAAGATTTT -3’. The PCR fragment was
digested into XbaI and SmaI. The resulting fragment was ligated into pCAMBIA
fused to GUS gene (Mai et al.. 2006). The construct was then introduced into L.
japonicus using hairy root transformation and GUS staining by 5-bromo-4-chloro3-indolyl-β-D-glucuronide cyclohexylammonium salt (X-gluc) was performed as
described previously (Mai et al.. 2006).
Microscope and nodule measurement
The fluorescent microscope has used to differentiate the number of
transgenic hairy roots before transferred to the pot, the root of transgenic hairy root
appeared the green color of roots. Bright-field and fluorescent microscopy were
performed with Olympus, the images of transgenic hairy roots were taken using DP
Controller (Olympus), NIS Elements (Nikon) or Photoshop (Adobe Systems). The
phenotype expression of root nodule such as the number of nodule, nodule diameter,
and the root length were observed at 2 wpi and 4 wpi by using Vernier calipers.
Figure 2. Transformation process of transgenic hairy root
RESULT AND DISCUSSION
8
DNA amplification and electrophoresis results of PCR
The electrophoresis results of PCR product showed that 12 samples of
constructed DNA has successfully amplified, it proof by appearing DNA bands
under the electrophoresis gel (Figure 3).
Figure 3. PCR electrophoresis results of construct, 1, 2, 7, 8 (MYB RNAi),
3, 4, 9, 10 (GUS RNAi), 5, 11 (MYB OX), 6, 12 (GUS OX)
Samples number 1, 2, 3, 4, 5, 6 are cDNA sample while sample number 7,
8, 9, 10, 11, 12 are sample colony that taken from Agrobacterium transfered the
expression clone. The calculation of DNA length using excel program showed the
expecteded size and PCR product as result on Table 1. The length of DNA expected
size and PCR product mostly same indicated that construction process of each
construct has succesfully done.
Table 1. Calculation of construct electrophoresis results
Sample
DNA name
Expected
Size of PCR
name
size (bp)
product (bp)
1
876 MYB
1855
1809
RNAi
2
7171 MYB
996
992
RNAi
3
876 GUS
1595
1609
RNAi
4
7171 GUS
720
722
RNAi
5
7171
1522
1583
MYBOX
6
7171
715
722
GUSOX
7
876 MYB
1855
1870
RNAi
8
7171
996
1008
MYBRNAI
9
10
11
12
876 GUS
RNAi
7171 GUS
RNAi
7171 MYB
OX
7171
GUSOX
1595
1531
720
722
1522
1609
715
785
Relative expression of transgenic hairy root
The first step to investigate the effect of MYB transcription factor during
formation of nodule is measurement of myb gene expression of transgenic hairy
root. Transgenic hairy root showed a different expression (Figure 4). MYB OX
revealed expressed myb gene very high compared with GUS OX as the control,
MYB OX expressed myb gene 125% more than GUS OX. By contrast, MYB RNAi
expressed myb gene very low compared with GUS RNAi as the control, MYB
RNAi was suppressed 82% than GUS RNAi .The high expression of MYB gene in
MYB OX and the suppression of myb gene in MYB RNAi indicated that the
transgenic hairy root for each construct successfully planted.
40
35
myb
Relative Expression
30
25
20
15
10
5
0
GUS OX
MYB OX
GUS RNAi
MYB RNAi
Figure 4. Relative expression of myb in transgenic hairy roots
Phenotype expression of nodulation
To know the effect of MYB transciption factor for nodulation responses.
We investigated the total nodule number, the nodule diamater and main root length
used calipers as the measurement. The nodule number for each transgenic hairy root
constructs performed the difference nodule number. Figure 5 showed the nodule
number average for each transgenic hairy roots of more than 35 plants for each
transgenic hairy root construct.
10
35
GUS OX
Nodule number per plant
30
MYB OX
25
*
20
15
10
5
0
0
2
4
Week post inoculation (wpi)
Figure 5. Nodule number per plant of MYB OX and GUS OX
The data of nodule number showed MYB Overexpression (MYB OX)
produced more numerous nodules (16.4 nodules per plant, n=44) than GUS
Overexpression (GUS OX) nodules (11.8 nodules per plant, n=46) as the control of
2 weeks post inoculation (wpi)(Figure 5). The different of nodule number of both
construct showed statistically significant. The nodule number of 4 wpi showed both
of constructs (MYB OX and GUS OX) produced nodule number with no significant
different, 19.1 and 20.4 respectively. The significance of these data that confirmed
by using Student’s t test analysis for (P < 0.01) for 2 wpi but the differences were
no significant at 4 wpi. The different of nodule number that showed MYB OX has
higher nodule number might indicated the symbiotic interaction of MYB OX
formed earlier after M.loti inoculation but it no longer at 4 wpi.
30
*
nodule number per plant
GUS RNAi
25
*
MYB RNAi
20
15
10
5
0
0
2
4
Week post Inoculation (wpi)
Figure 6. Nodule number per plant of MYB RNAi and GUS RNAi
The number of nodules of MYB suppression (MYB RNAi) produced lower
number of nodules (9.1 nodules per plant, n=38) than GUS Suppression (GUS
RNAi) nodules (14.6 nodules per plant, n=40) of 2 wpi (Figure 6). The different of
nodule number of both construct showed statistically significant. The nodule
number of 4 wpi showed MYB RNAi and GUS RNAi produced nodule number
with signifant different, 15.6 and 10.7 respectively. The significance of these data
that confirmed by using Student’s t test analysis for (P < 0.01) for 2 wpi and
(P0.05). These data (figure
6) indicated that MYB transcription factor did not affected the root lenght, no
significant different found in measuring more than 35 plants for each constructs. As
the conclusion, we stated that the MYB transcription factor did not give effect to
root length of Lotus japonicus.
12
GUS OX
MYB OX
GUS RNAi
MYB RNAi
Root lenght (cm)
10
8
6
4
2
0
0
2
Weeks post inoculation (wpi)
Figure 7. Root lenght of transgenic hairy roots
4
12
In addition, we also made an observation about the nodule diameter to
collect more data about the phenotype effect of MYB transcription factor in nodule
development (Figure 8). At 4 wpi of nodule we found that MYB transcription factor
has no effect in nodule diameter or nodule size. After measuring more than 35 plants
for each transgenic hairy root construct we found nodule diameter of MYB OX,
GUS OX, MYB RNAi, and GUS RNAi were 0.099, 0.093, 0.089, 0.090 cm
respectively. Same as the root length, the statistical analysis of nodule diameter also
concluded no significant different of nodule diameter of each transgenic hairy roots
construct.
Nodule Diameter
0,14
Nodule Diameter (cm)
0,12
0,1
0,08
0,06
0,04
0,02
0
GUS OX
MYB OX
GUS RNAi
MYB BRAi
Figure 8. Nodule diameter of transgenic hairy roots
Nitrogen (N2) Fixation Activity
To make explanation detailed about the effect of MYB transcription factor
during nodulation we also measured the acetylene reduction assay during nodule
formation at 2 wpi and 5 wpi (Figure 9). The positive transgenic nodules of each
construct has assayed using acetylene (C2H2) reduction assay (ARA) after 0.5 hours
incubation. ARA can measure the nitrogen fixing activity indirectly. Because
nitrogenase does not convert nitrogen to ammonia when excess acetylene is
included. Therefore, acetylene can convert to ethylene by nitrogenase. Acetylene
reduction Assay (Figure 8) showed that the nitrogen fixation capacity of MYB OX
was significantly higher than GUS OX with a different of 14.17 nmol/h/nodule and
11.44 nmol/h/nodule at 2 wpi. The nitrogen fixation assay of both constructs were
increased significantly at 5 wpi that nitrogen fixation activity of MYB OX and GUS
OX reached 28.68 nmol/h/nodule and 19.12 nmol/h/nodule respectively.
Ethylene Production(nmol/h/nodule)
ARA
40
GUS OX
35
30
25
20
15
10
5
0
0
2
5
Weeks post Innoculatuon (wpi)
Figure 9. Acetylene reduction assay in OX hairy roots
Ethylene Production (nmol/h/nodule)
Different with overexpression hairy roots of MYB OX that has a significant
different of activity at 4 wpi , MYB RNAi performed a similiar nitrogen (N 2)
fixation assay data with GUS RNAi at 2 wpi (Figure 10). Activity of 2 wpi MYB
RNAi reduced acetylene of 10.75 nmol/h/nodule and GUS RNAi reduced
achetylene of 12.29 nmol/h/nodule respectively. Interestingly, at 5 wpi the
acetylene reduction assay of MYB RNAi was lower than GUS RNAi that are 18.56
nmol/h/nodule and 32.14 nmol/h/nodule respectively.
These result showed that the over expression of MYB has a significant
impact on nitrogen (N2) fixation per nodule at 5 wpi, interestingly the number of
nodule in previous data (Figure 5) showed the MYB OX at 4 wpi did not increase
the nodule number. By Contrast, the ARA of RNAi constructs at 5 wpi suggested
that the nitrogen (N2) fixation of MYB RNAi was lower than GUSRNAi. These
results also showed that the MYB deficiency or MYB RNAi affected not only in
decreasing the nodule number but also the nitrogen (N2) fixation activity of nodule
after 5 wpi.
ARA
40
GUS RNAI
35
30
25
20
15
10
5
0
0
2
Weeks post Innoculatuon (wpi)
Figure 10. Acetylene reduction assay in RNAi hairy roots
5
14
The MYB transcription factor effects during nitrogen (N2) fixation activity
probably because of some reasons that already found by some previous studies.
Banba et al. (2001) reported the nodules formed with R. etli CE3 cannot utilize
photosynthate as an energy source for nitrogen (N2) fixation thereby decreased the
fixation activity of nitrogen (N2) and exhibit senescence. On the other hand, Ott et
al. (2005) also reported that suppression of plant leghemoglobin gene by RNA
interference in L. japonicus resulted higher oxygen concentrations inside nodules,
lower bacterial nif and fix gene expression, created the absence of nitrogenase
activity. However, the future study is needed to know the precious mechanism of
MYB transcription factor in effecting nitrogen (N2) fixation activity of nodules.
Bacteroid number
To know the effectivity of rhizobia-plants interaction we counted the
bacteroid number within the nodule (Figure 11). The bacteroid number of nodule
has known effectively correlated with the increased nitrogen (N2) fixation
(Wadisirisuk and Weaper 1985). We counted the bacteroid number of MYB OX,
MYB RNAi, GUS OX and GUS RNAi nodules at 2 wpi inoculated with Ds Red
M.Loti. We found the number of bacteroids was significantly increased in MYB
OX with 2.40 x 107 bacteroids per nodule, while that we found GUS OX with 1.56
x 107 bacteroids per nodule (Figure 10 ). In RNAi transgenic hairy roots we found
the bacteroids number of 1.63 x 107 bacteroids per nodule and 1.71 x 107
bacteroids per nodule for MYB RNAi and GUS RNAi recpectively. This analyzed
data showed that MYB OX was significantly greater at 2 wpi than GUS OX, but
MYB RNAi did not effect in bacteroid numbers. These data indicated that MYB
OX has more rapid in forming symbiotic interaction to form nodule at 2 wpi.
4
Bacteroid number per nodule
*
x107 cells per nodule
3,5
3
2,5
2
1,5
1
0,5
0
GUS OX
MYB OX
GUS RNAi
MYB RNAi
Figure 11. Bacteroid number of transgenic hairy roots
GUS staining of MYB promoter-GUS
In transgenic hairy roots carrying a MYB promoter-GUS, GUS staining
(blue color) of roots marks confirming the transient expression of GUS (Figure 12).
The GUS staining showed the MYB promoter was active within the root and 12
days post inoculation (dpi) nodule. The GUS Expression within the nodule
expressed in nodule primordia site (Kang et al. 2013). Further the control
experiments with Plambia 134 vector did not give blue coloration.
0.5
mm
0.5
mm
Figure 12. Blue staining in MYB promotor nodule and control
Quantitative real time PCR
In order to check the correlation of MYB transcription factor compared
with the expression of genes in nodule we investigated the expression analysis
of enod40 gene and nin gene using qRT-PCR (Figure 13). The expression levels
corresponding to enod40 were higher in MYB Supressing nodules than in the GUS
OX as control. By parallel the expression of enod40 lower in MYB RNAi. These
results suggest that MYB may binds to enod40. By Contrast, the expression levels
of early nodule development marker genes, nin in MYB hairyroots (Figure 14).
revealed that nin decreased in MYB OX and increased in MYB RNAi compared to
their control GUS OX and GUS RNAi. These results suggest that MYB may not a
regulator of nin but we predicted MYB has a parallel function with nin in nodulation
process .
The expression pattern of MYB was similiar with enod40. The
overexpression of enod40 accelerated nodule formation and the suppression of
enod40 suppressed nodule formation (Charon et al. 1999). On the other hand The
GUS staining of MYB promoter-GUS activity (Figure 12) and enod40 stated by
Martirani et al. (1999) was also similiar in which it expressed in the vascular of
nodule. These results indicate that MYB might regulate the enod40expression.
Relative Expression
1000
800
enod40
600
400
200
0
GUS RNAi
MYB RNAi
GUS OX
MYB OX
Figure 13. Relative expression of enod40 gene of transgenic hairy roots
16
14
nin
Relative Exspresssion
12
10
8
6
4
2
0
GUS RNAi
MYB RNAi
GUS OX
MYB OX
Figure 14. Relative expression of nin gene of transgenic hairy roots
To ensure the nitrogen (N2) fixation correlated with nodule senescence we
measured the expression of cyspro gene, the gene marker for nodule senescence in
MYB hairy roots (Figure 15). The cyspro expression of MYB RNAi was
significantly high compared with GUS RNAi, contrastly the MYB OX expressed
low Cyspro gene. These data totally supported the nitrogen (N2) fixation result, in
which the MYB RNAi significantly reduced the nitrogen (N2) fixation activity at
5 wpi nodule. These results indicate that downregulation of MYB expression by
RNAi suppression has an effect to form senescence nodule earlier, and negative
effect to nirtogen (N2) fixation and nodulation.
Banba et al. (2001) reported that nodules that formed with inoculation
CE3/Rhizobium etli that inoculated appeared to be of the early-senescence type
because greater amounts of disintegrated membrane structures of nodules. Early
senescence formed by disintegration of symbiosome membranes, deterioratio and
aggregation of bacteroids, and degradation of the cytoplasmic structures of the
infected cells. Although our experiments did not reveal the mechanism of
senescence formed in L. japonicus nodule by MYB RNAi but we so far concluded
the role of MYB RNAi in forming senescence nodule. Moreover, the study about
the mechanism of senescence of MYB RNAi is an interesting issue to be solved in
the future research.
Relative Exspression
2
1,8
cysteine protease
1,6
1,4
1,2
1
0,8
0,6
0,4
0,2
0
GUS RNAi
MYB RNAi
GUS OX
MYB OX
Figure 15. Relative expression of cyspro gene of transgenic hairy roots
CONCLUSIONS
a.
b.
c.
d.
e.
f.
In this present study, we concluded that :
the Overexpression of MYB increased the nodule number of nodule during
early nodulation time but no longer in mature nodule.
MYB overexpression significantly increased the nitrogen (N2) fixation
activity and plant-rhizobia symbiosys.
The suppression of MYB (MYB RNAi) decreased the number of nodule
and indirectly decreased the nitrogen (N2) fixation activity and symbiosis
process of plant-rhizobia.
MYB transcription factor expressed in nodule and root.
As an early genetically study we proposed that MYB transcription factor
might bind to enod40 gene but not as transcription regulator of nin gene.
The lack of MYB gene formed early senecence in nodulation.
RECOMENDATIONS
It is needed to analysis the effect of MYB genetic expression during
nodulation more deeply. The histological study of MYB nodule also very important
to be investigated more detailed.
18
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APPENDIX
Appendix 1. Phenotype expression of transgenic hairy roots under GFP
microscope. The photographs were taken in the GFP mode (A, C, E, G) and in the
light field (B, D, F, H). Scale bars represent 2 mm, A and B are MYB OX in 2 wpi,
C and D are GUS OX in 2 wpi, E and F are GUS RNAi in 2 wpi, G and H are GUS
RNAi in 2 wpi.
A
C
B
D
E
F
G
H
22
Appendix 2. Table of nodules number of 2 wpi transgenic hairy roots
GUS
MYB
GUS
MYB
No
OX
OX
RNAi
RNAi
1
11
9
12
3
2
23
8
22
7
3
3
6
10
0
4
13
17
11
6
5
4
23
17
5
6
17
10
15
3
7
10
12
11
9
8
20
25
5
13
9
9
13
10
7
10
19
5
11
13
11
16
16
18
15
12
5
13