Cloning And Function Study Of StProDH1 Gene In Potato

Proline distrbued in plant different tissues and organs,which responses to various intracellular metabolic disorders that caused by environmental stresses,proline as stress adjustment molecure,which plays a very crucial biological function in plant response to stress.To some degree,?1-Pyrroline-5-carboxylate synthetase(P5CS)and proline dehydrogenase(ProDH)that are governed the proline metabolism.Which are also the rate-limiting enzymes in proline synthesis and catabolism respectively.In order to study the role of potato proline dehydrogenase(StProDH1)in the proline metabolism,StProDH1 gene was cloned from potato,designing and building the artificial microRNA interference vector pCPB121-amiR-ProDH1 to silent the StProDH1 gene.The transgenic plants were obtained by the method of agrobacterium-mediated transformation.Using the qRT-PCR technique detected the expression level of proline metabolism related genes and measuring the proline content in different organs of potato,The following research results are mainly obtained:1.The StProDH1 gene was cloned from the cDNA of potato cultivar Favorita,sequencing results indicated that the ORF was 1503 bp,encoding 500 amino acids.Through analysis it's promoter,it has ACTCAT and other cis-acting elements.In the StProDH amino acid sequence of different plants,the residues of serine,threonine and tyrosine are relatively conservative.2.AmiR-ProDH1 precursor was obtained by overlapping PCR cloning technology,and build amiR-ProDH1 interference expression vector pCPB121-amiR-ProDH1 with the CaMV 35 s promoter driven,the transgenic potato plants were obtained by the agrobacterium-mediated transformation method.3.qRT-PCR analysis showed that StProDH1 gene and StP5 CS gene had tissue specific expression in potato.The expression level of StProDH1 gene in root,stem and leaf was leaf > root > stem,and the expression level of StP5 CS gene was leaf > root > stem.The proline content showed that the content of transgenic plants was higher than that in non-transgenic plants in the root,stem and leaf.4.Under PEG stress,the expression of StProDH1 gene was decreased with stress time prolonging,and the expression of StProDH1 gene was up-regulated when the stress was eliminated.This trend was consistent in transgenic plants and non-transgenic plants,but the expression of StPro DH1 gene in transgenic plants was relatively lower than that of non-transgenic plants.With stress time prolonging,the expression quantity of StP5 CS gene increased,and decreased when the stress was eliminated.This trend was consistent in transgenic plants and non-transgenic plants,but the expression of StP5 CS gene was relatively low in transgenic plants.5.Under the low dose of exogenous proline treatment,the expression of StProDH1 gene was up-regulated,and in transgenic plants was higher than that of non-transgenic plants.