IN SILICO STUDIES AND FUNCTIONAL VALIDATION OF SALT INDUCIBLE S6PDH GENE IN RICE

Abstract

Rice is the most important staple crop, accounts for over 20 percent of global calorie intake and feed more than 50% of the global population. It has been assumed that the demand for rice consumption will be increase in the coming decades, due to the population explosion and cropland reduction. Salinity (high amount of salts in the soil and water) is the most widespread problem after drought in rice growing countries and is considered as a serious constraint to increased rice production worldwide (Glenn et al., 1997). To overcome these limitations and improve rice production under salt stress conditions, it is important to understand the mechanism of salt response and improve salt stress tolerance in crops. ABSTRACT The identification of novel genes, determination of their expression patterns in response to salt stress, and an improved understanding of their functions in stress adaptation will provide us the basis of effective engineering strategies to improve stress tolerance in plants (Cushman and Bohnert, 2000). Nowadays, the availability of rice genome mapping, sequencing and advanced molecular tools paved the way for studying the gene functions. Previous studies have reported that sorbitol 6 phosphate dehydrogenase (S6PDH) gene have an important role in salt stress tolerance in Rosaceae and other families of plants (Yamaki 1980; Kanayama et al., 1992; Gao et al., 2001, Liang et al., 2012). However, the S6PDH gene has not been identified and characterized at molecular level in rice (Oryza sativa L.). Present study entitled “In silico studies and functional validation of salt inducible S6PDH gene in rice” was aimed to identify and to perform the structural and functional studies of S6PDH gene in rice using bioinformatics and modern molecular tools. The present thesis is divided into nine chapters and covers the extensive studies carried out on S6PDH gene and protein in rice including in silico analysis, expression analysis, cloning, expression, purification, characterization, in silico post translational modification studies and preparation and transformation of RNAi construct for OsS6PDH. The thesis starts with Chapter 1 introduction; which briefly outlines the significance of rice, magnitude of abiotic stress on rice production and highlight the effect of salt stress on plants. It discusses about the salt tolerance mechanism in plants with special reference to sorbitol and S6PDH gene. At the end of the chapter the role of bioinformatics and modern ii molecular tools in the identification and functional validation of gene has been discussed and specific objectives of the present study have been specified. Chapter 2 review of literature; starts with the brief introduction of rice, and progresses with the detailed review on abiotic stresses affecting the growth and production of plants, salinity stress with special reference to rice, effect of salt stress on plants, salt stress signaling in plants, mechanism and molecular level analysis of salt tolerance in plants, role of sugar alcohols (sorbitol and mannitol) in salt stress tolerance, characteristics of sorbitol-6- phosphate dehydrogenase (S6PDH) and approaches for structural and functional validation of gene. This chapter also describes the application of RNA interference for the functional validation of gene in plants. The basic importance of the present study has also been highlighted in this chapter in various sections. Chapter 3 describes the in silico analysis and homology modelling of sorbitol-6- phosphate dehydrogenase of rice (OsS6PDH). The homologous sequence of S6PDH gene was identified by homology search method. The screening of the OsS6PDH gene along with mRNA, cDNA and protein sequences available at NCBI site indicated that it is a fully active gene. The sequence analysis revealed that the gene has two cDNA sequences having the same size of open reading frame (960 bp), the only difference is a 168 nucleotides long stretch at 5’end which turns into 56 amino acids long extension on N-terminal of the protein and this extension does not match with any other sequence in database. Multiple sequence alignment and phylogenetic analysis indicated that the protein is the member of AKR superfamily as it clusters with the members of AKR2A subfamily. Positioning of catalytic tetrad and other conserved features within the active site suggested that the OsS6PDH utilizes the same chemistry as the other AKRs used for the reduction and oxidation of the carbonyl compounds. The results of docking experiments suggested that the enzyme can accommodate both the substrates (glucose-6-phosphate and mannose-6-phosphate) but the orientation of the substrates may differ depending on their stereochemistry leading to a different mode of enzyme mechanism. In Chapter 4, expression analysis and salt inducible property of OsS6PDH gene have been described. The in silico expression of the gene was analysed using publicly available resources and rice microarray data accessible through Rice Oligonucleotide Array Database iii (ROAD, http://www.ricearray.org/expression/meta_analysis.shtml) and RiceXpro version 3 (Rice gene expression profile data sets) using MSU ID LOC_Os02g03100. The results of in silico expression were validated by real time PCR (RT-PCR) analysis and sorbitol content was measured by high performance liquid chromatography (HPLC) method. The metaanalysis of the published microarray data and the cloning of the gene via RT-PCR strategy confirmed the actual expression of the gene. Induction of the gene expression in response to salt stress supported by GEO database and as determined by real time PCR, suggested that this gene is a salt-inducible gene i.e. expression of the gene is enhanced in the presence of salinity stress. The results of this study are in concordance with previous study in other plant that also suggests the role of S6PDH gene in salt tolerance (Teo et al., 2006). This chapter also describes the cloning, expression and purification of OsS6PDH protein and evaluation of its function. OsS6PDH sequence was cloned, expressed and purified successfully using Ni-NTA column and the purity was determined by running on SDS-PAGE. Along with the physico-chemical properties, kinetic properties of the purified recombinant protein were also determined. Purified recombinant OsS6PDH protein catalyzed glucose-6- phosphate reduction using NADPH as a cofactor (Km = 15.9 ± 0.2). The purified enzyme also catalyzed sorbitol-6-phosphate oxidation using NADP as a cofactor (Km = 7.21 ± 0.5) however the protein did not utilize mannose-6-phosphate as a substrate. As the reaction rate of glucose-6-phosphate reduction was found to be higher than sorbitol-6-phosphate oxdation, the reaction was considered to occurr in the direction of glucose-6-phosphate reduction. In Chapter 5, characterization of N-terminal extended sequence of S6PDH protein of rice has been described. As aforementioned in chapter 3, the OsS6PDH protein has an extended N-terminal sequence of 56 amino acids which did not match with any other sequence of the proteins in database. The extended sequence was found not to be involved in enzyme activity as the protein without this sequence was found to be fully active (chapter 4). To characterize the extended N-terminal sequence, cloning, expression, activity assays, structural analysis and in silico post translational modification analysis were performed. The activity assays and structural analysis of the homology model suggested that the extended sequence inhibits the active sites of the enzyme. The in silico post-translational modification analysis of the protein suggested the role of this additional sequence in signalling and was iv predicted to be a chloroplast signal peptide which is in accordance with the previous studies (Yamaki 1981, Liang et al., 2012). Chapter 6 describes the preparation and transformation of RNAi construct of S6PDH gene in rice. In present study, RNAi construct was prepared using Gateway cloning technique in pANDA vector. RNAi construct in pANDA was then transformed into embrogenic calli of rice through agrobacterium mediated transformation. Regenerated RNAi transformants were selected using Kanamycin (G418) resistance and transformation was confirmed by PCR (gus, hpt and npt-specific) amplification and real time PCR analysis. RNA interference of Os02g0123500 ledd to the reduction in sorbitol content as determined by HPLC analysis and also led to the reduction in sorbitol-6-phosphate dehydrogenase enzyme activity. This study supports the results of cloning and expression studies described in chapter 4 and confirms that the Os02g0123500 gene encodes sorbitol-6-phosphate dehydrogenase and has a role in sorbitol production in rice. In Chapter 7 complete work of the study has been briefly summarized and future prospects of this study have been discussed. Since rice is the model crop for the research on monocot, the derived information of the present study will be useful to initiate or explore future research in understanding the salt tolerance mechanisms in other related crops. In Chapter 8, the references used in course of present study have been enlisted.