Cloning Of BADH Genes From Atriplex Triangularis

In this study, BADH3 and BADH5, two partial BADH cDNAs were cloned from Alriplex Iriangularis through RT-PCR technique by specific primers designed on the basis of BADU gene on relative species. Comparison between BADH3 and BADH5 showed 81% identity for nucleotide sequences and 80% identity for deduced amino acid sequences, respectively. Moreover, a 1815bp full-length cDNA sequence of BADH3 (GeneBank accession No. AY256971) was obtained using nested PCR, cRACE and 3'-RACE technique. Further analyses on BADH3 indicated that an open reading frame (ORF) of 1500bp contained in this gene and predicted to encode a protein of 54.7KD with 500 amino acids. The blast results exhibited higher identity (93%-96%) to members of Alriplex genus than to those of other genus from Chenopodiaceae family (84%-86%), as well as other families (61%-79%). Phylogenetic analyses based on BADH in plant indicated that BADH3 and BADH5 likely belonged to the branch of Chenopodiaceae and Amaranlhaceae, respectively. Before Chenopodiaceae and Anunanthaceae developed, BADH gene family of Alriplex Iriangularis had possibly evolved into two genes at least.The ORF of BADH3 was amplified and cloned from Alriplex triungularis using RT-PCR technique by two specific primers based on BADH3 sequence with BamHl and SacI sites introduced. The ORF was cut by the two aforementioned enzymes and ligaled to the multiclone sites of pCAMBIA2301, then assembled CaMV35S promoter and nos terminator to the upstream and downstream of ORFBADH3, respectively. Resulting plasmid pCAMATBADH68 was introduced into Agrobacterium tumefaciens LBA4404. ORFbadm was transferred into Brassica napus by agrobacterium-mediated transformation. Fifteen kanamycin resistant transformants were selected. Among the five kan-resistant shoots being examined using PCR, one was positive. This showed that ORFBadH3 was introduced into genome of Brassica napus.A 855bp Alriplex triangularis DNA fragment upstream of the BADH3 gene was isolated with inverse PCR technique using two pairs of primers designed based on the sequence of BADH3 5 ' end, which had four predicted TATA motif (-25bp~ -30bp,-260bp~ -265bp, -337bp~ -342bp and -715bp~ -720bp) , two predicted CCAAT motif(-389bp~ -393bp and -720bp~ -724bp) and two predicted ABA response elements (-112bp~ -116bp and -397bp~ -401bp). The nucleotide sequences in -33bp~ -173bp showed 90% identity to the promoter of BADH gene from Alriplex ceturalasialica.