绿色木霉纤维二糖水解酶基因cbhII的克隆及其在粟酒裂殖酵母中的表达

食品科学 ›› 2012, Vol. 33 ›› Issue (21): 164-168.

绿色木霉纤维二糖水解酶基因cbhII的克隆及其在粟酒裂殖酵母中的表达

付永平1，宋冰1,2，王丕武1,*，卢实1

1.吉林农业大学农学院 2.吉林市农业科学院

收稿日期:2012-03-20 修回日期:2012-09-13 出版日期:2012-11-15 发布日期:2012-11-09
通讯作者: 王丕武 E-mail:peiwuw@yahoo.com.cn
基金资助:
大豆查尔酮还原酶（CHR）在大豆苷元合成中的作用机理研究

Cloning of CellobiohydrolaseⅡ Gene from Trichoderma viride and Its Expression in Schizosaccharomyces pombe

Received:2012-03-20 Revised:2012-09-13 Online:2012-11-15 Published:2012-11-09

摘要/Abstract

摘要： 采用粟酒裂殖酵母诱导表达系统进行绿色木霉纤维二糖水解酶基因cbhⅡ的表达研究。通过PCR方法从绿色木霉基因组DNA中克隆到长度为1611bp并含有信号肽序列的cbhⅡ基因；将其插入到粟酒裂殖酵母诱导型表达载体pESP-2的Pnmt1启动子下游，得到重组质粒pESP-2-cbhⅡ；利用电转化方法将其转入粟酒裂殖酵母SP-Q01细胞中，通过对酵母转化子质粒DNA的PCR和双酶切鉴定，得到了含有cbhⅡ基因的酵母转化子，对转化子进行诱导培养后提取酵母总蛋白，进行SDS-PAGE电泳检测，得到一条约为81kD的目的蛋白条带；利用DNS法对酵母转化子上清液中的纤维二糖水解酶活性进行测定，结果表明，酶活力最大值为165U/mL。以上结果证明：cbhⅡ基因在Pnmt1启动子控制下在粟酒裂殖酵母中成功地获得表达，并且cbhⅡ自身的信号肽可以被粟酒裂殖酵母正确的识别，并将表达产物分泌至胞外。

关键词: 绿色木霉, 纤维素酶, cbhⅡ基因, 粟酒裂殖酵母, 酶活力

Abstract: In this study, cbh (cellobiohydrolase)Ⅱgene from Trichoderma viride, 1611 bp in length and containing a signal peptide sequence, was cloned by PCR and inserted into the nmt1 promoter (Pnmt1) in Schizosaccharomyces pombe expression vector to obtain a recombinant plasmid pESP-2-cbh Ⅱ. The recombinant plasmid was transformed into S. pombe SP-Q01 by electroporation. S. pombe transformants carrying cbh Ⅱgene were successfully obtained as demonstrated by PCR and double enzyme digestion. The transformants were inductively cultured and total proteins were extracted from them and detected by SDS-PAGE to contain a protein band of approximately 81 kD. The maximum cellobiohydrolase activity of the fermentation supernatant of the S. pombe transformants was measured by DNS method to be 165 U/mL. The above results indicated that cbh Ⅱ gene was successfully expressed in S. pombe, which was controlled by Pnmt1 promoter and that the signal peptide sequence of the cbh Ⅱ gene was correctly recognized by S. pombe to produce extracellular expression products.

Key words: Trichoderma viride, cellulase, cbhⅡ gene, Schizosaccharomyces pombe, cellubiohydrolase activity

中图分类号:

Q939.97

付永平1，宋?冰1,2，王丕武1,*，卢?实1. 绿色木霉纤维二糖水解酶基因cbhII的克隆及其在粟酒裂殖酵母中的表达[J]. 食品科学, 2012, 33(21): 164-168.

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