INTRODUCTION 1.1 Rationale Cloning Trehalose Synthase Gene And Its Expression In Arabidopsis thaliana

I. INTRODUCTION 1.1 Rationale

Trehalose is a disaccharide that is well known as a stress protecting agent for many organisms particularly microbes, insects, nematodes and some plants. It belongs to the unhydrobiotic group of organisms Crowe et al. 1982. The concentration of trehalose within the tissue of a certain organism correlates with the capability of the organism to face stresses such as osmotic and oxidative stresses Franco et al. 2000; Benaorouj 2002, heat stress Reinders 1999; Kandror 2003 and pressure Iwahashi et al. 2000 as Elbein 2003 suggested that trehalose is a multifunctional molecule. The presence of gene encoding trehalose synthesizing enzyme converted drought, cold, and salt plant resistance Garg et al. 2002; Jang et al. 2003, El-Bashiti 2003. The destructive effect of abiotic stresses often resulted from proteinenzyme denaturation, membrane disintegration, andor resulting ion balance disturbance within the cells. As a non- reducing sugar, trehalose has many unique features; it is very stable in a very wide range of conditions such as pH, temperature, and water status. This sugar further remains amorphous in completely dry conditions Hagen 1995. These unique features make trehalose is able to stabilize the surrounding compounds and structures and so minimize the destructive effect of stress; hence, it is also called as biological stabilizer Shinohara et al. 2002. There are three pathways of trehalose synthesis. The first is known as the OtsA-OtsB pathway. This pathway involves 2 steps of enzymatic reactions, using UDP-glucose and glucose-6-P to be condensed into trehalose-6-phosphate. This reaction is catalyzed by trehalose phosphate synthase TPS, encoded for example by TPS1 in yeast and Arabidopsis, or OtsA in E. coli. Trehalose phosphate phosphatase TPP that is encoded by OtsB in E.coli dephosphorylates trehalose-6-phosphate T6P, the next reaction of the pathway. The second pathway of trehalose synthase is known as TreY-TreZ. This pathway converts oligomers of glucose such as oligocyl maltose or glycogen to trehalose by converting the linkage of terminal glucose from an α -1-4 linkage into an α -1-1 linkage using the enzyme encoded by TreY, followed by cleaving the terminal disaccharide to release trehalose using the enzyme encoded by TreZ. This pathway can be found such as in Grifola frondosa. The third pathway of trehalose synthesis called TreS pathway, in which a single enzyme only is involved. The enzyme converts maltose directly to trehalose Smet et al. 2000. This enzyme is also capable to convert trehalose to maltose with equilibrium state at about 60 trehalose concentration Wei et al. 2004. Research on trehalose concerning stress resistance of many organisms has been done. Expression of either foreign OtsA or OtsB or both genes on plant caused stunted growth, such as on tobacco Romero et al 1997; Gidijn et al 1998; Almeida et al. 2005,. This event is resulted from accumulation of its intermediate product, trehalose-6-phosphate T6P that leads to feedback inhibition of trehalose phosphate phosphatase trehalose immediate enzyme synthesis. T6P accumulation depletes P inorganic that disturbs glycolysis influx and in turn disturbs energy conversion. The stronger effect on plant development, however, T6P induces starch immobilization leading to root starvation. In a certain level, however, T6P is needed for seedling growth at least it was proved in Arabidopsis Schluepmann et al. 2004. This problem has been solved by fusing both genes OtsA-OtsB into a molecule; hence, fused protein translated from the genes is obtained. Consequently, T6P resulted from TPS activity is directly converted into trehalose and T6P accumulation can be minimized. This work has been reported on rice contained a fused genes of OtsA-OtsB from E. coli . This rice transgenic showed more resistant to drought, salt and cold, as well as produced more yield than the control Garg et al. 2002; Jang et al. 2003. TreY-TreZ and TreS pathways, as mentioned above, do not produce T6P as intermediate product. Hence, questions rise, 1 whether introducing TreS that converts maltose to trehalose without producing T6P could overcome the problem resulted from T6Ptrehalose accumulation, 2 whether TreS can be used as a selectable marker and 3 whether TreS plant lines withstand various stresses e.g. drought, salt and pH. This research was addressed to answer those questions. When positive result is obtained, the gene will be transferred to crop plants. To be able to answer these questions, TreS from Thermobifida fusca and Mycobacterium tuberculosis were cloned separately, and were introduced into Arabidopsis thaliana.

1.2 Objectives for the research are:

1. To clone TreS gene from Thermobifida fusca and Mycobacterium tuberculosis. 2. To construct transgenic Arabidopsis expressing TreS gene 3. To evaluate whether TreS can be used as an alternative selectable marker in cloning strategy. 4. To charachterize the TreS expressing plants under stresses condition

1.3 Research Benefit

The result of this research would provide: 1. TreS gene inserted within pGemT and constructed within expression cassette 2. TreS expressing Arabidopsis thaliana as plant model for further research concerning on plant responses to stresses. 3. Information whether TreS can be used as an alternative of non toxic selectable marker in cloning strategy 4. Information about the expression of TreS gene from T. fusca on plant under stresses.

1.4. Hypothesis

1. Clones of TreS gene from T. fusca that has been characterized by Wei et al. 2004 would be available and could be used to develop transgenic plant for crop improvement. 2. Transgenic Arabidopsis which express trehalose synthas gene from Thermobifida fusca would be available 3. TreS can be used as an alternative among existed non toxic selectable markers in cloning strategy. 4. The present of the gene may improve plant tolerance to stresses.

II. LITERATURE CITATION