【作者】 何晓兰；

【导师】 侯喜林；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2003， 硕士

【摘要】 根据甜菜碱醛脱氢酶（BADH）基因保守域设计一系列特异引物和兼并引物，利用RT-PCR技术从耐盐蔬菜三角叶滨藜中分离了两个基因序列BADH3和BADH5，并利用cRACE和3′-RACE技术获全长BADH3（GenBank登录号：AY256971）。测序结果表明，BADH3和BADH5，彼此核甘酸和推测的氨基酸序列同源性分别为81％和80％。表明三角叶滨藜BADH基因是一个多基因家族，至少存在两个成员。全长BADH3 cDNA核苷酸序列长1.815 kb，78～1580bp为一个开放阅读框架，推测的氨基酸序列全长500个氨基酸残基，分子量为54.7KD，pl为5.5。BADH3推测的氨基酸序列与藜科滨黎属植物和藜科其它属植物的同源性较高，同源性分别为93％-96％和84％-86％。与其它科植物的同源性相对低些为61％-79％。表明BADH3与其它植物的同源性基本符合种属划分标准。植物BADHs分子进化树分析表明BADH3和BADH5可能分别属藜科和苋科的两个分支成员，三角叶滨藜BADH基因家族可能在藜科和苋科植物分化之前己进化为至少两种差异较大的基因。 根据全长BADH3序列设计引物，通过RT-PCR技术从三角叶滨藜中扩增并克隆了该基因ORF，同时在5′端和3′端分别引入BamH1和SacI酶切位点。利用BamH1和Sacl酶切位点将ORF_BADH3连接到pCAMBIA2301的多克隆位点，并把CaMV35S启动子和nos终止子分别连接到ORF_BADH3的上游和下游的多克隆位点上，获重组植物表达质粒pCBAMATBADH68，转入根癌农杆菌LBA4404中，获LBA4404（pCBAMATBADH68）菌种。用农杆菌介导法把三角叶滨藜ORF_BADH3转入甘蓝型油菜中，获得15株具有卡那霉素抗性小植株。对其中5株进行了PCR鉴定，1个呈阳性，初步证明该基因已经插入油菜基因组中。 根据三角叶滨藜BADH3 5′端序列设计特异引物，用反向PCR技术从三角叶滨藜总DNA中扩增并克隆了该基因的5′侧翼区约855bp的片段。距起始密码子ATG 77bp处的T碱基为可能的转录起始位点。在-25bp～ -30bp、-260bp～ -265bp、-337bp～ -342bp以及-715bp～ -720bp含有推测的TATA-boX（TATAAA），在-389bp～ -393bp和-720bp～ -724bp含有推测的CAAT-box（CCAAT），以及在-1 12bp～ -1 16bp和-397bp～ -401bp含有推测的ABA作用元件CACGT。仅其-33bp～ -173bp区与中亚滨藜BADH基因启动子区高度同源，同源性为90％。该BADH3基因5′侧翼区与其它植物BADH基因的启动子无同源性。更多还原

【Abstract】 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.更多还原