Cloning And Functional Analysis Of AhP5CS Genes In Peanut

Peanut(Arachis hypogaea Linn.),as one of the important oil crops and cash crops in the world,is mainly planted in Asia,America and Africa.Peanut plays a vital role in economic development in China due to the fact that China is the largest producer,consumer and exporter in Asia.However,the limited irrigation conditions and the extreme climate events caused by global warming,especially frequent floods and dry weather,have seriously affected peanut production in China.Under drought conditions,plants mainly enrich osmotic adjustment substances to resist adversity.Proline(Pro)is one of the most widely distributed endogenous osmotic adjustment substances in plants.?1-pyrroline-5-carboxylic acid synthase(P5CS)is a rate-limiting enzyme in the proline synthesis pathway,which plays an important role in regulating the content of proline in plants.In order to study the role of P5 CS gene resistance to stress in peanut,with a view to providing a theoretical basis for resistance breeding,this study cloned three P5 CS genes and analyzed the expression of P5 CS genes in roots and leaves under drought stress.Through constructing eukaryotic expression vector and transforming Saccharomyces cerevisiae,and constructing plant over-expression vector and transforming Arabidopsis thaliana by flower dipping method,the expression profiles of P5 CS genes under stress were characterized.The results would be helpful to dissect the function of P5 CS gene in peanut.The main findings are as follows:(1)Based on the homologous sequence method,three P5 CS genes,named Ah P5CSA02,Ah P5CSA03 and Ah P5CSA04,respectively,were successfully cloned from peanut.Sequencing results showed that the length of c DNA fragments corresponding to Ah P5CSA02,Ah P5CSA03 and Ah P5CSA04,were 2259 bp,2151 bp,and 2268 bp,respectively.The number of encoded amino acids was estimated to be 752 aa,716 aa and 755 aa.Transmembrane structure analysis showed that none of the three potential proteins was transmembrane protein,and subcellular localization analysis revealed that they were localized in mitochondria.(2)Through multi-sequence alignment and analysis of conserved domains,P5 CS genes were found to be highly conserved in plants,and the predictive proteins contained some domains,including glutamate kinase(GK)domain,glutamyl phosphate reductase(GPR)domain,and ATP binding site,NADPH binding site and leucine domain.Phylogenetic analysis showed that Ah P5CSA02 and Ah P5CSA04 had the closest genetic relationship with Cicer arietinum and Medicago truncatula,both which belonged to the legume family,while Ah P5CSA03 had the closest genetic relationship with another legume family,Medicago sativa.(3)The spatial and temporal expression patterns of the three genes were alanlyzed using real-time fluorescent quantitative PCR technology,and the results showed that all three genes were constitutively expressed.Among them,the Ah P5CSA02 gene is highly expressed in roots,the Ah P5CSA03 gene is mainly expressed in stems,and the expression level of Ah P5CSA04 is highest in leaves.Under indoor simulated drought stress,the expression of Ah P5CSA02 and Ah P5CSA04 genes were obviously induced by osmotic stress,which implied that they were stress-inducible genes.Another gene,Ah P5CSA03 gene,was not induced by osmotic stressin that no significant difference of gene expression was found before and after stress treatment.(4)Three eukaryotic expression vectors,p YES2-Ah P5CSA02,p YES2-Ah P5CSA03 and p YES2-Ah P5CSA04,were constructed and transformed into Saccharomyces cerevisiae cells to analyze the growth of yeast cells under osmotic stress.Overexpression of the Ah P5CSA02 gene enhanced the resistance to salt stress and osmotic stress.And the yeast lines transformed with the Ah P5CSA03 gene failed to show significant resistance changes.Meanwhile,the yeast lines transformed with Ah P5CSA04 gene showed enhanced penetration of cells,but the adaptability to salt stress was not significantly improved.(5)Three over-expression vectors were constructed and transformed into Arabidopsis thaliana to analyze the constitutive expression of three genes.Drought stress experiments were carried out using T3 homozygous transgenic plants.Under osmotic stress,the germination rate of Arabidopsis thaliana transformed with Ah P5CSA02 gene was 55%(twice that of wild type),and the average root length was 2.5 times that of wild type,indicating that transgenic Ah P5CSA02 gene can improve the stress resistance of transgenic plants.The germination rate of wild-type and positive plants transfected with Ah P5CSA03 gene was about 25%,and the average root length of transformed plants with Ah P5CSA03 gene was not significantly different from that of wild-type,indicating that Ah P5CSA03 gene could not improve the resistance of plants.The average germination rate of the positive lines transfected with Ah P5CSA04 gene was 48%(1.9 times that of wild type)and the average root length was 1.7 times that of wild type,indicating that transgenic Ah P5CSA04 gene can improve the stress resistance of transgenic Arabidopsis.