A Study Of Genetic Transformation Of Arabidopsis Thaliana With A Novel Gene Involved In ALDH From Chinese Wild Vitis Pseudoreticulata

We did the study of genetic transformation of Arabidopsis thaliana with a novel gene involved in ALDH from chinese wild Vitis pseudoreticulata.Main results were obtained as follows:1.Linked aldehydehyde dehydrogenase gene being amplified by PCR with the previous obtained recombinant plasmid pGEM-T-ALDH template and pENTR/D-Topo Vector to obtain entry clone vector pENTR/D-ALDH,and then performed LR reaction with pENTR/D-ALDH and plasmid pHellsgate8 to obtain the plant RNAi vector pHellsgate8-ALDH.2.A nucleotide sequence of plasmid pWR306 carrying promotor ED35s,Omega TMV translation enhancer and nopaline synthase terminator was specifically cloned into plasmid pCAMBIA 1300 to construct intermediate overexpression vector pWR-â…¡;the segment of aldehydehyde dehydrogenase gene from the recombinant plasmid pGEM-T-ALDH was specifically cloned into intermediate overexpression vector pWR-â…¡to construct the plant overexpression vector pWR-â…¡-ALDH.3.The two plant expression vectors were transformed into Arabidopsis thaliana by the Agrobacterium tumefaciens-mediated floral-dip method to obtain 20 kanamycin-resistant lines and 9 hygromycin-resistant lines.PCR analysis indicated that 7 of the 20 kanamycin-resistant lines were positive and 3 of the 9 hygromycin-resistant lines were positive.4.Real-Time PCR analysis of positive hygromycin-resistant lines confirmed by PCR indicated that ALDH gene's relatively expression in transgenic plant was much higher than the expression of wild plant.There were some phenotype differences between transgenic plants and wild plants,it indicated that the ALDH gene's expression have an impact on the Arabidopsis thaliana's growth.