Cloning,Expression And Functional Research Of Dehydrin Protein Gene In Ammmopiptanthus Nanus

In this study,Ammopiptanthus was used as the research material.The dehydrin gene and its promoter in Ammopiptanthus were cloned and analyzed.Real-time fluorescence quantitative technique was used to explore the expression characteristicsat of dehydrin gene under stress conditions including low temperature,salt,drought and exogenous hormone.And to observe the growth of the situation when expression vectors imported into Prokaryotes E.coli.Through varieties of molecular biology techniques,to research the function and mechanism of dehydrin genes in resistance to abiotic stress.The specific test results are as follows:1.The full-length cDNA of the dehydrin protein gene was cloned from Ammopiptanthus by homologous cloning and RACE technology,and named it as AnDHN.The cDNA has 1005 bp in length and contains an open reading frame of 515 bp,which encodes 181 amino acid residues.By the classification of dehydrin protein structure,determining the encoded protein belongs to YK dehydrin protein.2.The promoter sequence of 830 bp was finally cloned by chromosome walking technique.PlantCARE and PLACE analysis has predicted a variety of functional elements related to stress response,including multiple relational response elements with low temperature,high salinity,drought,and hormones.The response element also contains some tissue-specific elements,such as endosperm-specific expression elements.3.The qRT-PCR technique was used to analyze the expression level of AnDHN gene of Ammopiptanthus under different abiotic stress conditions.AnDHN was specifically expressed under low temperature,high salinity and drought stress,and the expression levels of roots,stems and leaves all have upgraded in different degrees.Under the induction of hormone incluing abscisic acid,auxin and methyl jasmonate,the expression level also has showed an upward trend,and the expression of AnDHN gene was mainly induced by abscisic acid.4.The vector pET32a-AnDHN expression vector has successfully constructed by gateway cloning technology,and the protein has purified by affinity chromatography.After control bacteria pET32 a and recombinant strain AnDHN were induced by IPTG at 0.5 mM for6 h,to determine the effection of AnDHN protein on E.coli under abiotic stress,by observing on the culture of plate found that the AnDHN protein has improved tolerance to E.coli under abiotic stress.