米曲霉木聚糖酶基因的克隆及其在毕赤酵母中的表达

目的:构建米曲霉木聚糖酶基因的真核表达载体,并转化巴斯德毕赤酵母,进行分泌表达。方法:以米曲霉总RNA为模板,根据已知的米曲霉木聚糖酶基因序列设计引物,采用RT-PCR技术克隆木聚糖酶基因cDNA序列,将其与pPIC9K质粒连接构建表达载体后转化毕赤酵母,经MM/MD快慢斑筛选,得到Muts型重组子,进行甲醇诱导表达。结果:克隆得到的cDNA序列全长666 bp,连续编码221个氨基酸;阳性克隆子在诱导培养数天后,将菌液点于RBB-木聚糖平板上,产生了明显的透明圈,表明重组木聚糖酶在毕赤酵母中获得表达。结论:木聚糖酶基因的真核表达载体构建成功,并能够在毕赤酵母中表达。

Cloning and Expression in Pichia pastoris of the Xylanase Gene from Aspergillus oryzae

Objective：To construct the eukaryotic expression vector of the xylanase gene（XynG） from Aspergillus oryzae,and transform it into the Pichia pastoris for secretive expression.Methods：The total RNA of A.oryzae was used as the template,from which the cDNA of xylanase was cloned by the RT-PCR,and then it was linked to the pPIC9K plasmid to construct the expression vector,lastly it was transformed into the P.pastoris.The Muts re-combinants were screened through MM / MD quick-slow spots screening,then they were cultured and methanol in-duced.The culture supernatants were dotted on the flat plate containing RBB-xylan to select the xylanase produc-ing strain.Results：The total length of the cloned cDNA fragment was 666 bp,encoding 221 amino acids.The e-merging transparent circles indicated that the XynG transformed P.pastoris had expressed xylanase.Conclusion：The eukaryotic expression vector of the xylanase gene was constructed successfully,and it could express xylanase in P.pastoris.