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.PDFVIP

164 2012, Vol.33, No.21 食品科学 ※生物工程 cbh 1 1,2 1,* 1 付永平 ，宋 冰 ，王丕武 ，卢 实 (1.吉林农业大学农学院，吉林 长春 130118 ；2.吉林市农业科学院，吉林 吉林 130118) 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cbh Ⅱ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cbh Ⅱ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-cbh Ⅱ；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cbh Ⅱ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：cbh Ⅱ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cbh Ⅱ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。 绿色木霉；纤维素酶；cbhⅡ基因；粟酒裂殖酵母；酶活力 Cloning of CellobiohydrolaseⅡ Gene from Trichoderma viride and Its Expression in Schizosaccharomyces pombe 1 1,2 1,* 1 FU Yong-ping ，SONG Bing ，WANG Pi-wu ，LU Shi (1. College of Agriculture, Jilin Agricultural University, Changchun 130118, China； 2. Jilin City Academy of Agricultural Sciences, Jilin 130118, China) 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 SDSto