Functional Analysis Of GsMIPS2 And GsSNAP33 Genes Under Salt And Alkali Stress Conditions

Salinity and alkalinity have been considered among most influen tial environmental constraints, which exert detrimental impact on crop/plant growth and production. Hence, it is important to develop salt and alkaline tolerant crops. In general, salt and alkaline stresses mainly disrupt osmotic and ionic equilibrium of cell environment. It is known fact that stress signals first perceive at membranes via receptors and then further transduce into the cells for activation of stress-responsive genes to mediate stress tolerance. Therefore, understanding the basic mechanisms of salt and alkaline tolerances with plenty of genes involved in stress responses and signaling networks is essential for crop improvement.Wild soybean(Glycine soja) is distinguished by its better adaptive nature, stress resistance capability from other legume crops which can serve as a large source of stress resistance genes. Therefore, it is considered as an important material for mining and cloning stress-resistant genes. The transcriptome profiling of G. soja G07256 under alkali stress(Na HCO3) has been performed in previous study of our laboratory. Two genes Gs MIPS2 and Gs SNAP33 were selected as probably alkaline stress-responsive genes from the transcriptome data by bioinformatics methods. In this study, we functionally characterized Gs MIPS2 gene under salt stress conditions via phenotype analysis at various growth stages and determined physiological parameters(MDA, chlorophyll content, electrolyte leakage), and expression level analysis of stress-related marker genes based on the mutant and overexpression plants. It was found that overexpression of Gs MIPS2 confers enhanced salt tolerance in Arabidopsis thaliana via protection of photosystem, reducing stress injuries and up-regulation of other abiotic stress-responsive genes. We also cloned the full-length sequence of GsSNAP33 and performed the bioinformatics analysis, expression pattern analysis and subcellular location analysis, and generated the transgenic Arabidopsis for the function and mechanism analysis. This comprehensive research work will broa den our understanding to the mechanism of plant tolerance under salt and alkali stress conditions. As these genes have independent putative functions in salinity and alkalinity tolerance and can serve as a new gene source for genetic engineering of stress-tolerant plant varieties and can also prove to be an important theoretical bases to elucidate plants stress tolerance mechanism. The brief summary of results is as follows:1. Isolation of Gs SNAP33 and sequence analyses of Gs MIPS2 and Gs SNAP33 from Glycine sojaWe isolated full-length c DNA of Gs SNAP33 gene via homologous cloning method from Glycine soja. GsSNAP33 contains an open reading frame(ORF) of 903 bp and codes a peptide sequence of 300 amino acids along with molecular mass of 33,31 Da. Gs MIPS2 contains an open reading frame(ORF) of 1533 bp coding a peptide sequence of 510 amino acids along with mass of 56,445 Da. Sequence analysis revealed presence of two conserved motifs in Gs SNAP33 protein. One is N-terminal motif of Qb/Qc subfamily members, while the 2nd one is SNARE motif of subgroup Qc. Similarly Gs MIPS2 sequence analysis showed presence of four highly conserved motifs: GWGGNNG / LWTANTERY / NGSPQNTFVPG and SYNHLGNNDG.2. Expression pattern analysis of Gs MIPS2 and GsSNAP33 genes and tissue specific analysis of Gs SNAP33Real-time PCR experiment was performed to analyze the expression pattern of Gs MIPS2 gene under salt stress conditions and induced levels of gene was observed, which reveals that Gs MIPS2 gene may involve in salt stress signaling pathways. Similarly, expression level of Gs SNAP33 gene was determined in roots and leaves of Glycine soja under 150 m MNa Cl, 50 mM Na HCO3 alkali, 100μmol ABA and 30 % PEG stress conditions. GsSNAP33 gene expression levels were found significantly higher in roots than in leaves under these however, higher expression level was noted under salt and ABA treatment than those under alkali or 30% PEG stresses, implicating that Gs SNAP33 is an alkali and salt-responsive gene. Furthermore, tissue specific analysis of Gs SNAP33 gene showed higher abundance of Gs SNAP33 transcripts in pods, roots and seeds respectively, while lower expression levels were observed in leaves, stems and flowers.3. Subcellular localization of Gs SNAP33 proteinThe eGFP expression system was used to check out subcellular location of Gs SNAP33 protein. p BSKII-GsSNAP33-e GFP vector was constructed and e GFP-fusion protein was transiently expressed in onion epidermal cells by biolistic bombardment method. Gs SNAP33 protein was found to be localized in plasma membrane and can play an important role in signaling and trafficking of stress-related compounds.4. Overexpression of GsMIPS2 gene in Arabidopsis thaliana confers enhanced salt tolerance at various growth stagesA phenotype analysis was performed between Gs MIPS2 transgenic lines, atmips2 mutant and wild-type plants of Arabidopsis. We elucidated the positive function of Gs MIPS2 gene under salt stress at germination, seedling and adult growth stages of transgenic plants. Our results showed that Gs MIPS2 transgenic lines displayed increased tolerance as compared to WT and atmips2 mutant lines under salt stress conditions with increased germination rate, root length and increased levels of chlorophyll content but decreased in MDA content and electrolyte leakage.5. Expression pattern analysis of stress-related marker genes in WT and Gs MIPS2 transgenic plantsThe expression levels of some salt stress-responsive marker genes, including KINI, RD29 A, RD29 B, P5 Cs and COR47 were found significantly up-regulated in Gs MIPS2 over expression lines as compared to the wild-type and atmips2 mutant lines under salt stress treatment. These results might suggest that Gs MIPS2 gene is a positive regulator of plant tolerance to salt stress.