Cloning Of Anti-apoptosis Genes-BAG, DAD And HSP70from Malus Hupehensis (Pamp) Rehd. And Their Response To The Stress Of Cadmium And Osmosis

Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang (Chinese name is Ping Yi Tian Cha,PYTC) was the unique resource in our country, which was used widely as a commonrootstock of apple and ornamental crabapple. In this thesis, the full-length cDNA of MhBAG,MhDAD, MhHSP70gene were isolated based on the materials of PYTC, the character andfunction of the gene sequence and the protein that it encoded was analyzed by bioinformatics.At the same time, their expression patterns and the change tendency of apoptosis rate undercadmium and osmosis sresses was studied. Otherwise, transgenic Arabidopsis thalianatransformed with pBI121vector of MhBAG, MhDAD gene was obtained by floral diptransformation.1) The full longth cDNA of MhBAG was obtained. According to the homologous sequencesfrom other plants, degenerate primers were designed to amplify specific DNA fragmentusing cDNA prepared from PYTC roots. MhBAG was1841bp in length, containing an openreading frame of1017bp and encoding338amino acids, and the accession number ofGeneBank was HQ639935.2) The full longth cDNA of MhDAD was obtained. According to the homologous sequencesfrom other plants, degenerate primers were designed to amplify specific DNA fragmentusing cDNA prepared from PYTC roots. MhDAD was546bp in length, containing an openreading frame of360bp and encoding119amino acids, and the accession number ofGeneBank was JN390964.3) The full longth cDNA of MhHSP70was obtained. According to the homologous sequencesfrom other plants, degenerate primers were designed to amplify specific DNA fragmentusing cDNA prepared from PYTC roots. MhHSP70was2307bp in length, containing anopen reading frame of1953bp and encoding650amino acids, and the accession number ofGeneBank was HQ876864. 4) RT-PCR results indicated that under CdSO4and NaCl stress, the expression of MhBAG,MhDAD both decreased gradually and the expression of MhHSP70increased by theincrease of CdSO4, NaCl concentration, and the apoptosis rate increased gradually.5) The pET-30a-MhBAG vector was successful constructed and optimizing expressed in E.coli,and the results showed that the molecular weight of MhBAG protein was37KD.6) The pBI121-MhBAG vector was successful constructed in this study and transformedArabidopsis thaliana col-0using the floral dip method via agrobacterium mediated.Harvest seeds were put on MS medium containing glufosinate in order to select theresistant seedlings. Genomic DNA of resistant seedlings was extracted for PCR detection.In the end,13transgenic seedlings were identified, which lay the foundation for the furtherstudy of the function of MhBAG.7) The pBI121-MhDAD vector was successful constructed in this study and transformedArabidopsis thaliana col-0using the floral dip method via agrobacterium mediated and theT0seeds had been harvest.