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利用RT-PCR方法从大豆根部组织获得Glyma03g34310.1开放阅读框(ORF)全长,经测序验证、Blast比对与同源性分析发现该序列编码的蛋白质与其他植物的TIP1;1蛋白具有较高的相似性,故命名为GmTIP1;1基因(GenBank登录号为AK285481),该基因ORF长753bp,编码1个包含250个氨基酸的蛋白,在ORF内部第381个核苷酸处含有1个94bp的内含子,符合↓GT--AG↓的剪接方式;系统进化树分析发现GmTIP1;1聚类到豆科植物分支,其他不同科的植物也有规律地聚到了不同分支,推测该蛋白氨基酸序列可以作为植物分类的依据之一;半定量RT-PCR结果表明该基因在大豆的不同器官、不同器官的不同发育阶段均具较高且同等的表达水平,暗示该基因在植物的整个发育进程中均具重要作用;在盐胁迫的不同时间点其表达量有下降的趋势,但仍然保持较高的表达水平;以pYES2为酵母表达载体,转化酿酒酵母INVSc1菌株,获得重组酵母INVSc1(pYES2-GmTIP1;1),转化菌株在盐胁迫下的存活率明显高于对照INVSc1(pYES2),而在干旱胁迫下则没有显著差异,表明该基因的表达能有效地提高酵母的耐盐性。
The full length of Glyma03g34310.1 open reading frame (ORF) was obtained from soybean root tissue by RT-PCR. The results of sequencing, Blast comparison and homology analysis showed that the protein encoded by this sequence was more potent than TIP1; 1 protein of other plants (GenBank accession number is AK285481). The gene ORF is 753bp in length and encodes a protein of 250 amino acids. It contains a 94 bp at the 381th nucleotide in the ORF The intron was in accordance with the splicing pattern of ↓ GT - AG ↓. Phylogenetic tree analysis showed that GmTIP1 clustered to the branch of legumes and the plants of other branches also clustered regularly to different branches, suggesting that the amino acid sequence The results of semi-quantitative RT-PCR showed that the gene had higher and equal expression level in different organs and organs of soybean at different developmental stages, which implied that the gene in plant during its whole development All of which had an important role in the process of salt stress. However, the expression levels remained at a high level at different time points of salt stress. The pYES2 yeast expression vector was transformed into INVSc1 strain of Saccharomyces cerevisiae to obtain recombinant The survival rate of the transformed strain INV INV-INVSc1 (pYES2-GmTIP1; 1) was significantly higher than that of the control INVSC1 (pYES2), while no significant difference was found under drought stress, indicating that the expression of this gene can effectively improve the yeast resistance Salt.