玉米赤霉烯酮毒素降解酶基因的克隆及在毕赤酵母中的高效表达

Expression of Zearalenone Degrading Enzyme Gene from Gliocladium Roseum in Pichia Pastoris GS115

以粉红黏帚霉菌株总RNA为模板，采用RT—PCR技术克隆得到玉米赤霉烯酮降解酶基因cD—NA序列，插入pPIC9K载体后，转化毕赤酵母。经MD板筛选，得到His＋型重组子，进行甲醇诱导表达，并进行了降解玉米赤霉烯酮能力测定。克隆得到的cDNA序列全长795bp，连续编码编码264个氨基酸；阳性克隆子在甲醇诱导培养3～5d后，HPLC检测证实，经表达后的酶液能完全降解液体中玉米赤霉烯酮（ZEN）。

In the study, the total RNA of gliocladium roseum was used as a template, from which the cDNA ot ZEN degradation enzyme was cloned by the RT- PCR, then was linked to the pPIC9K plasmid to construct the expression vector and was lastly transformed into the Pichia pastoris. The His ＋ recombinants were screened through MD screening, then cultured and induced with methanol. As a result, the cloned full-length cDNA of the sequence was 795 bp with 264 continuously encoded amino acids. The positive clones were cultured in the induction in 3-5 days later, and it was confirmed by HPLC test that the ZEN degradation was complete in the methanol induced enzvme solution.