甜菜夜蛾几丁质脱乙酰酶1(SeCDA1)在毕赤酵母中的表达与活性测定

Expression and Enzyme Activity of Chitin Deacetylase 1 from Spodoptera exigua in Pichia pastoris

利用RACE-PCR方法,扩增得到编码甜菜夜蛾几丁质脱乙酰基酶基因secda1。将secda1基因插入到载体p PIC9K多克隆位点,转化大肠杆菌JM109感受态细胞,得到重组质粒p PIC9K-secda1。重组质粒经线性化后,电击转化缺陷性毕赤酵母GS115感受态细胞,构建含有目的基因的重组酵母菌株。经甲醇诱导表达,Western blot分析后表明secda1基因在毕赤酵母GS115中成功表达80 k Da蛋白。脱乙酰基酶活性测定结果显示重组蛋白酶活力为1.35 U/m L。利用RT-PCR对该基因进行m RNA水平上的表达分析,结果表明secda1在幼虫取食期的头、表皮、中肠、马氏管、脂肪体及蛹期虫体均有表达;本研究利用毕赤酵母成功表达具有酶活力的重组Se CDA1蛋白,为进一步明确Se CDA1蛋白的生理功能提供条件,并为以Se CDA1蛋白为靶标更有效地防治甜菜夜蛾提供理论依据。

Using rapid amplication of cDNA ends （RACE-PCR）, a 1.5 kb cDNA encoding chitin deacetylase 1 protein SeCDA1 was cloned from Spodoptera exigua. The gene secdal was further cloned into pPIC9K and transformed into E.coli JM109 competent cells. The recombinant plasmid was named pPIC9K-secdal. Then the recombinant plasmid was linearized and transformed into Pichiapastoris GS 115 competent cells by electroporation Antibodies reacting to the recombinant SeCDA1 recognized a single protein by Western blot analysis after induction by methanol. The result indicated SeCDA1 was expressed in the yeast. The CDA activity of recombinant SeCDA1 is 1.35 U/mL. Transcription analysis on secdal sequence during various developmental stages and in different tissues by RT-PCR showed that secdal was expressed in head, midgut, integument, malpighian tubules, fat body and pupa. The expression of recombinant SeCDA1 laid the foundation for further investigation and physiological function of the SeCDA1 protein, and provided a theoretical basis for the biocontrol of Spodoptera exigua targeting CDA protein.