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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

孙晓彤1, 赵丹1, 闫晓平3, 郭巍1,2, 徐大庆3, 李少雅1, 李景2   

  1. 1. 河北农业大学植物保护学院/河北省农作物病虫害生物防治工程技术中心, 保定 071001;2. 北京农学院植物科学技术学院, 北京 102206;3. 河北农业大学生命科学学院, 保定 071001
  • 收稿日期:2014-09-28 修回日期:1900-01-01 出版日期:2015-08-08 发布日期:2015-08-08
  • 通讯作者: 郭巍,教授,博士生导师,E-mail:guowei@hebau.edu.cn

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

SUN Xiaotong1, ZHAO Dan1, YAN Xiaoping3, GUO Wei1,2, XU Daqing3, LI Shaoya1, LI Jing2   

  1. 1. College of Plant Protection, Agricultural University of Hebei/Biological Control Center of Plant Diseases and Plant Pests of Hebei Province, Baoding 071001, China;2. Plant Science and Technology College, Beijing University of Agriculture, Beijing 102206, China;3. College of Life Sciences, Agricultural University of Hebei, Baoding 071001, China
  • Received:2014-09-28 Revised:1900-01-01 Online:2015-08-08 Published:2015-08-08

摘要: 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pPIC9K多克隆位点,转化大肠杆菌JM109感受态细胞,得到重组质粒pPIC9K-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 kDa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/mL。利用 RT-PCR对该基因进行mRNA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SeCDA1蛋白,为进一步明确SeCDA1蛋白的生理功能提供条件,并为以SeCDA1蛋白为靶标更有效地防治甜菜夜蛾提供理论依据。

Abstract: 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 secda1 was further cloned into pPIC9K and transformed into E.coli JM109 competent cells. The recombinant plasmid was named pPIC9K-secda1. Then the recombinant plasmid was linearized and transformed into Pichia pastoris GS115 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 secda1 sequence during various developmental stages and in different tissues by RT-PCR showed that secda1 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.

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