Molecular Cloning Of Aldehyde Dehydrogenase (BADH) From Atriplex Canescens And The Transformation To Tobacco

Today, soil salinization is a golobel problem in agricultural production.It is the new important way to breed new salt-tolerant variety by agricultural biotechnology. Atriplex canescens for Chenopodiaceae Atriplex are perennial evergreen or evergreen-shrub, is resistant to drought, cold-resistant, resistant to barren, salt-tolerant and so on.now, it is widely used for pasture improvement and soil, water conservation.This study got salt-tolerant genes from betaine aldehyde Catalase gene (BADH) cDNA from Atriplex canescens, plant expression vector pBI121-BADH was constructed. It was transformed to tobacco of mode plants through Agrobaterium-mediated system, and PCR testing.The results of the experiment were as follows:1. Guanidine isothiocyanate method was used to extract and purificate RNA from Atriplex canescens leaves. Agarose gel electrophoresis showed a typical three RNA band 28S, 18S, 5S and reverse transcription into cDNA.2. According to BADH cDNA sequences of the same family, the same plant Atriplex triangularis published in GenBank, Design a pair of gene-specific primer by oligo6.0. The 5' were introduced BamHⅠand SacⅠr estriction sites, using PCR technology access to salt-tolerant genes from betaine aldehyde Catalase gene (BADH) cDNA, Connected with pGEM-T Easy Vectors transformate into E.coli DH5α, After PCR, enzyme test, sequenced of BADH clones is submitted to sequenct. Comparative analysis with Homologous sequences in GenBank, results showed that it is 95.92% as high as the gene known BADH nucleotide sequences.3. plasmid DNA of pGEM-BADH and pBI121 was double digested with restriction endonucleases SacⅠa nd BamHⅠto retrieve the BADH gene and the linear vector without Gus gene.connectivity, transformed, the recombinant plasmid was obtained. After indentification of PCR, BamHⅠand SacⅠd ouble-identification.It show that middle expression vector and plant expression vector of CaMV35S promoter-driven pBI121-BADH was successful constructed.4. The recombination plasmid pBI121-BADH was transferred into Agrobacterium tumefaciens EHA105 by freeze-thaw method.Access to works of EHA105-pBI121- BADH.It was transformed to tobacco of mode plants through Agrobaterium-mediated system.Transgenic plants resistance to kana were got, after PCR testing.which confirmed that the BADH were introduced into the tobacco genome.