delta DNA序列介导的酿酒酵母整合表达载体的构建.docVIP

  delta DNA 序列介导的酿酒酵母整合表达载 体的构建# 闫凯，林蒋海，龚映雪，李晶博，肖文娟，刘泽寰** 5 10 （暨南大学生命科学技术学院分子生物研究中心，广州 510632） 摘要：通过 PCR 扩增得到酿酒酵母染色体上的 delta（δ）序列，在含有强启动子 PGK 及 G418 抗性的酵母多基因表达载体 pScIKP 的础上构建了以 δ 序列为整合位点的酿酒酵母整合型表 达载体 pδRCMB。为验证载体表达可行性与稳定性，将米曲霉 α-淀粉酶基因（amy）插入载 体，获得重组质粒 pδRCMB/amy。用电击穿孔转化法转化酿酒酵母 As2.489，得到酿酒酵母 重组菌株 As2.489/pδRCMB/amy。淀粉水解透明圈与 SDS蛋白电泳结果表明 α-淀粉 酶基因在酵母重组菌株中成功分泌表达，重组菌株传代 96 次后，其 α-淀粉酶仍能稳定分泌 表达，为研究其它外源基因在酿酒酵母中的稳定表达奠定了基础。 关键词：δ 序列；重叠 PCR；PGK 启动子；酿酒酵母；分泌表达 中图分类号：Q782 15 Construction of delta DNA-mediated stable expression vectors in Saccharomyces cerevisiae YAN Kai, LIN Jianghai, GONG Yingxue, LI Jingbo, XIAO Wenjuan, LIU Zehuan (Research Center for Molecular Biology, College of Life Science and Technology, Jinan 20 25 30 35 40 University, GuangZhou 510632) Abstract: The delta(δ) DNA fragment was amplified from the genomic DNA of Saccharomyces cerevisiae As2.489 using PCR. Then it was inserted into the expression vector pScIKP, to construct a new δ DNA-mediated integrated expression vector, designated as pδRCMB. To demonstrate its feasibility and stability, the amy gene from Aspergillus oryzae was inserted into the expression vector pδRCMB and a recombinant plasmid pδRCMB/amy was created. The recombinant plasmid was then transformed into the Saccharomyces cerevisiae As2.489 strain by electroporation and the recombinant yeast named As2.489/pδRCMB/amy was constructed. The activity of the α-amylase in the recombinant yeast was verified by starch transparent circle and SDS. The transformants showed stable expression level of α-amylase after at least 96 generations. This work will provide the foundation for other foreign genes to be stably expressed in S. cerevisiae. Keywords: δ sequence; overlap PCR; PGK promoter; Saccharomyces cerevisiae; secretion expression 0 引言 酿酒酵母是一种重要的工业微生物，由于其基因组学、分子生物学、生理学等方面背景 知识清楚，因此在工业上应用范围非常广。在发酵工业中，酿酒酵母主要用于生产生物乙醇、 啤酒、面包、药物类代谢物、酵母蛋白等。目前，在酿酒酵母所有应用当中，以酿酒酵母为 宿主，以基因工程和代谢工程为技术基础，通过引入外源基因诱导分泌表达异源目的蛋白具 有极大潜力[1-5]。 酿酒酵母表达系统较复杂，能够使用的载体类别非常有限。传统的载体有附