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

Salinity and alkalinity have been considered among most influential environmental constraints,which exert detrimental impact on crop/plant growth and production.Hence,it is important to generate 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,it is essential to understand the basic mechanisms of salt and alkaline tolerances via plenty of genes involved in stress responses and signaling networks for crop improvement.Wild soybean(Glycine soja)is distinguished from other legume crops due to its better adaptive nature and stress resistance capability which can serve as a vast source of stress resistance genes.Therefore,it is considered as an important material for mining and cloning of stress-resistant genes.Previously in our laboratory,many stress-related genes,including GsMIPS2 and GsSNAP33,induced by alkali stress conditions were obtained through transcriptome sequencing of wild soybean(G.soja 07256)roots by using fllumina sequencing technology.In this study,we functionally characterized GsMIPS2 gene under salt stress conditions via phenotype analysis at various growth stages and determined physiological parameters(MDA,chlorophyll content,electrolyte leakage),as well as expression level analysis of stress-related marker genes based on the comparative studies of mutant and overexpression plants.It was found that overexpression of GsMIPS2 confers enhanced salt tolerance in Arabidopsis thaliana via protection of photosystem,reduction in stress injuries and via up-regulation of other abiotic stress-responsive marker genes.Secondly,we also cloned the full-length sequence of GsSNAP33 by homology based cloning method and performed the bioinformatics analysis,GsSNAP33 expression pattern analysis,subcellular location analysis,and generated the transgenic Arabidopsis,lines for the functional analysis.This comprehensive research work will broaden our understanding towards the mechanism of plant tolerance under salt and alkali stress conditions.As these genes might have independent putative functions in salinity and alkalinity tolerance,therefore can serve as a new gene source for genetic engineering of stress-tolerant plant varieties as well as they might prove to be an important theoretical bases to elucidate plants stress tolerance mechanism.The brief summary of results is as follows:1.Isolation of GsSNAP33 and sequence analyses of GsMIPS2 and GsSNAP33 from Glycine soja.We isolated full-length cDNA of GsSNAP33 gene via homologous cloning method from Glycine soja.GsSNAP33 contains an open reading frame(ORF)of 903bp and codes a peptide sequence of 300 amino acids along with molecular mass of 33,31 Da.GsMIPS2 contains an open reading frame(ORF)of 1533bp coding a peptide sequence of 510 amino acids along with mass of 56,445 Da.Sequence analysis revealed presence of two conserved domains in GsSNAP33 protein.One is N-terminal domain of Qb/Qc subfamily members,while the 2nd one is SNARE domain of subgroup Qc.Similarly GsMIPS2 sequence analysis showed presence of four highly conserved motifs:GWGGNNG/LWTANTERY/NGSPQNTFVPG and SYNHLGNNDG.2.Expression pattern analysis of GsMIPS2 and GsSNAP33 genes and tissue specific analysis of GsSNAP33Real-time PCR experiment was performed to analyze the expression pattern of GsMIPS2 gene under salt stress conditions and induced levels of gene was observed,which reveals that GsMIPS2 gene may involve in salt stress signaling pathways.Similarly,expression level of GsSNAP33 gene was determined in roots and leaves of Glycine soja under 150mMNaCl,50mM NaHCO3,100?mol 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 alkali or 30%PEG stresses,implicating that GsSNAP33 is a salt-responsive gene.Furthermore,tissue specific analysis of GsSNAP33 gene showed higher abundance of GsSNAP33 transcripts in pods,roots and seeds respectively,while lower expression levels were observed in leaves,stems and flowers.3.Subcellular localization of GsSNAP33 proteinThe eGFP expression system was used to check out subcellular location of GsSNAP33 protein.pBSKII-GsSNAP33-eGFP vector was constructed and eGFP-fusion protein was transiently expressed in onion epidermal cells by biolistic bombardment method.GsSNAP33 protein was typically appeared 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 GsMIPS2 transgenic lines,atmips2 mutant and wild-type plants of Arabidopsis.We elucidated the positive function of GsMIPS2 gene under salt stress at germination,seedling and adult growth stages of transgenic plants.Our results showed that GsMIPS2 transgenic lines displayed increased tolerance as compared to WT and atmips2 mutant lines under salt stress conditions with significantly higher germination rates,increased root length and elevated levels of chlorophyll content but reduction in MDA(Malondialdehyde)content and electrolyte leakage.5.Expression pattern analysis of stress-related marker genes in WT,atmips2 mutant and GsMIPS2 transgenic plantsThe expression levels of some salt stress-responsive marker genes,including KINI,RD29A,RD29B,P5Cs and COR47 were found significantly up-regulated in GsMIPS2 over expression lines as compared to the wild-type and atmips2 mutant lines under salt stress treatment.These results might strengthen our conclusion that GsMIPS2 gene is a positive regulator of plant tolerance to salt stress.