| 摘要: |
| [目的]克隆并表达苏云金芽胞杆菌(Bacillus thuringiens, Bt)杀虫晶体蛋白cry1Ab22基因,为解决昆虫抗性问题提供有效途径,并为构建新型转基因工程菌和转基因植物提供基因材料.[方法]根据GenBank中cry1Ab型基因序列设计1对引物,并加入BamH Ⅰ和Pst Ⅰ酶切位点.用全长基因PCR产物粘端定向克隆的方法,从苏云金芽孢杆菌S249菌株中扩增到1个苏云金芽胞杆菌杀虫晶体蛋白cry1Ab型基因cry1Ab22,将其克隆到表达载体pMAL-c2X中,转化大肠杆菌TB1,进行诱导表达,并对表达产物的杀虫活性进行检测.[结果] cry1Ab22基因长度约为3.5 kb;其与已知的cry1Ab3型基因同源性最高,所编码的蛋白质存在5个氨基酸差异.cry1Ab22基因已在GenBank中登录,登录号为EU220269,并被Bt毒素基因国际命名委员会正式命名为cry1Ab22.SDS-PAGE电泳结果表明,cry1Ab22基因表达了170 ku左右的融合蛋白.cry1Ab22蛋白对小菜蛾(Plutella xylostella)的LC50为225.1 μg/mL.[结论]S249菌株含有新型的cry1Ab基因,且该基因表达的蛋白具有较强的杀虫活性. |
| 关键词: 苏云金芽胞杆菌 杀虫晶体蛋白基因cry1Ab22 生物杀虫活性 |
| DOI： |
| 分类号:S482.398 |
| 基金项目:国家科技攻关项目，西北农林科技大学研究生教育创新计划? |
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| Cloning and expression of ICP cry1Ab22 gene of Bacillus thuringiens |
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WU Jin-yuan FENG Ji-nian ZHANG Xing
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| Abstract: |
| 【Objective】 Cloning and expression of ICP cry1Ab22 gene of Bacillus thuringiens not only give a way to resolve insect resistance,but also provide the genetic material for constructing the new transgenic project fungus and transgenic plant.【Method】 According to cry1Ab gene sequences of 5′-terminal and 3′-terminal,a pair of primers for full-length cry1Ab gene on the GenBank was designed.The cry1Ab type gene was cloned into the E.coli expression vector pMAL-c2X,which was then transformed into E.coli TB1,induction expression for Bacillus thuringiens and detected the activity for expression product.【Result】 PCR was performed to produce a cry1Ab type gene which was designated cry1Ab22 in GenBank (Accession No.EU220269).The length of gene was about 3.5 kb,and the comparison showed the highest homology with the cry1Ab3 and 5 amino acid differences existed in Encoded protein of cry1Ab22 .The 170 ku fusion protein could be identified obviously by SDS-PAGE. Bioassay showed that the LC50 of cry1Ab22 against Plutella xylostella-larve with a spread method was 225.1 μg/mL.【Conclusion】 There is a novel cry1Ab type in strain S249 and cry1Ab22 protein has a stronger biological insecticidal activity. |
| Key words: Bacillus thuringiensis Insecticidal crystal proteins cry1Ab22 gene Biological activity |
