大豆PM2（LEA3）蛋白及其短肽PM2A酵母表达载体的构建.pdf

大豆 PM2(LEA3)蛋白及其短肽 PM2A 酵母表达载体的
构建
生命科学学院生物技术专业汪为茂
学号:2002302011
【摘要】LEA 蛋白(胚胎晚期富集蛋白)是与细胞耐盐保护作用相关的一类蛋白质。
本实验室已经利用大肠杆菌异源表达体系证明大豆 PM2 蛋白(LEA3)及其缺失多肽 PM2A
的耐盐功能。为进一步研究PM2 和 PM2A 在真核细胞中的功能,利用PCR 扩增大豆 PM2 基因
及片段 PM2A,经 BamHⅠ和 EcoRI 双酶切后,连接到酵母表达载体 pYES2。连接产物转化至
大肠杆菌 Top10。培养大肠杆菌重组子,提取重组质粒,经PCR、酶切鉴定以及 DNA 测序后,
将鉴定正确的重组质粒通过电转化方法转化酿酒酵母 INVSc1。菌落 PCR 结果显示重组质粒
pYES2-PM2 和 pYES2-PM2A 成功转入酵母中。利用半乳糖诱导酵母重组菌株 INV/PM2 和
INV/PM2A 表达外源多肽。提取酵母的热稳定性蛋白质进行 SDS-PAGE 电泳,结果显示
INV/PM2A 菌株有目的蛋白表达。以上实验为进一步鉴定 PM2 蛋白及其缺失多肽 PM2A 在酵
母中的耐盐功能奠定基础。
【关键词】LEA3 蛋白,酿酒酵母,22-氨基酸重复序列,大豆,缺失克隆,耐盐结构
域
【教师点评】大豆 PM2 蛋白是与植物抗逆保护作用相关的主要蛋白。目前对其抗逆保
护机制尚不清楚。本论文以大豆 PM2 蛋白为研究对象,采取多种分子生物学技术,将扩增
的 PM2 基因连接,转化到酵母中,并使其诱导表达。为进一步利用重组酵母研究其耐盐功
能提供材料。汪为茂同学积极上进,刻苦钻研,勤奋好学,有较好的专业知识基础,掌握
了多种现代分子生物学技术,得到了有创新性的研究结果。论文工作饱满,达到了毕业论
文的要求。
点评老师:郑易之教授
Construction of yeast expression vector containing soybean PM2
and its truncated sequence PM2A
【Abstract】LEA proteins (later embryogenesis abundant) are related with salt tolerance
in cells. Our lab has already provided the evidence that PM2, a LEA3 protein from soybean and
PM2A (a truncated sequence of PM2) confer salt tolerant in Escherichia coli. In order to research
the function of PM2 and PM2A in the eukaryotic cell, we amplified soybean gene PM2 and its
segment PM2A by PCR method. The gene sequence was digested with BamHⅠ/EcoRI, and
ligated into yeast expression vector pYES2. The ligation products were introduced into
Escherichia coli TOP10. it proved that the gene was cloned into pYES2 vector by the means of
PCR, digestion with BamHⅠ/EcoRI and DNA sequencing. The binant plasmid was
transformed into the hromyces cerevisiae INVSc1 by using the Electroporation methods.
PCR was finished using the transformed yeast cells as template, and the result showed that
binant plasmid pYES2-PM2 and pYES2-PM2A had been transformed into yeast
essfully. The yeast binant strains INV/PM2 and INV/PM2A were induced to
expressing target polypeptide b