The Cloning And Analysis Of Dehydrin Promoter Of Suaeda Salsa

In this study,the Suaeda salsa of Panjin red beach is used as experimental material,the type of the dehydratidin gene(SsDHN)promoter under salt stress is analyzed by real-time fluorescence quantitative PCR,then the promoter of SsDHN gene is cloned by FPNI-PCR.The cis-acting element of the promoter is predicted by bioinformatics analysis.A deletion fragment is constructed by 5 'deletion in the promoter sequence,and constructs the deletion fragment expression vector.The deletion of the fragment by Agrobacterium-mediated is transformed into tobacco leaves,and performs the transient expression analysis.The results are as follows:1.Analysis of SsDHN gene expression in Suaeda salsa under NaCl stress40 d Suaeda salsa seedlings are treated in different time under 300 mM NaCl stress,qRT-PCR analysis showed that the expression of SsDHN gene in 12 h is the highest,and the gene expression in leaves is higher than in root,the expression of SsDHN gene increased firstly and then decreased.The results show that the SsDHN promoter is induced the expression of SsDHN gene under salt stress promoter.2.Cloning and sequence analysis of SsDHN gene promoterAccording to the SsDHN cDNA sequence the full length of SsDHN gene is cloned.The full length of the SsDHN gene is 1253 bp,which contains a 560 bp intron,and the border at both ends is GT-AG.The 872 bp promoter is cloned by FPNI-PCR.The results showthat the sequence contained TATA-box and CAAT-box,and contains the cis-acting elements related to stress,such as ABRE,ARE and MRE,CCGTCC-box,CGTCA-motif,GC-motif,TCA-motif,TGACG-motif and MBSI.3.Construction of deletion expression vectorsThe PCR amplified 5 'end deletion fragments connect to the cloning vector pMD18-T,and named:pMD18T-F1,pMD18T-F2,pMD18T-F3,pMD18T-F4.The deletions of the sticky ends is obtained by double enzyme digestion and connect with the frame of pCAMBIA 1303 plasmid,we constructed 4 recombinant expression vectors named SsDHNp1-GUS,SsDHNp2-GUS,SsDHNp3-GUS,SsDHNp4-GUS,The recombinant expression vector are successfully constructed by PCR,double enzyme digestion and sequence alignment.4.Analysis of promotersBy Agrobacterium tumefaciens mediated deletion recombinant plasmid are transformed into tobacco leaf.GUS transient expression analysis shows that the promoter may exist in response to cis-acting element of NaCl,MeJA,ABA,SA stress.