论文部分内容阅读
以马蔺幼根总RNA为模板,通过RT-PCR方法扩增得到马蔺H+-PPase全长序列并克隆到p MD19-T载体,命名为Il VP。序列分析表明该基因的开放阅读框为2 316 bp,编码771个氨基酸,推测等电点为5.16,分子量为80.7 k D,所得到的序列与Gen Bank中注册的高等植物液泡膜H+-PPase的核苷酸序列相比,其同源性均达到70%以上,其氨基酸序列的同源性则达到79%以上。用SmaⅠ和SacⅠ分别对目的基因质粒和植物表达载体p BI121进行双酶切,在DNA连接酶的作用下进行定向连接,构建成植物重组质粒p BI121-35S-Il VP-Nos,再转入根癌农杆菌EHA105中,为该基因在烟草等植物中遗传转化和基因功能研究奠定基础。
The full length sequence of Iris H + -PPase was amplified by RT-PCR from the total RNA of Iris lactea and cloned into p MD19-T vector and named as Il VP. Sequence analysis showed that the open reading frame of this gene was 2 316 bp, encoding 771 amino acids. The deduced isoelectric point was 5.16 and the molecular weight was 80.7 kD. The obtained sequence was identical to the H + -PPase of higher plant tonoplast registered in Gen Bank Compared with the nucleotide sequence, the homology was more than 70%, and the homology of the amino acid sequence was more than 79%. The recombinant plasmid pBI121-35S-Il VP-Nos was subcloned into plasmid pBI121 and the plasmid pBI121 was digested with SmaI and SacI, respectively. In Agrobacterium tumefaciens EHA105, this gene lays the foundation for genetic transformation and gene function research in plants such as tobacco.