| 方龙发1, 2, 王小奇2, 束长龙2, 宋福平2, 黄勃1, 张杰2*.Cry9Aa3蛋白对小菜蛾和亚洲玉米螟的最小活性区研究[J].植物保护,2015,41(2):80-85. |
| Cry9Aa3蛋白对小菜蛾和亚洲玉米螟的最小活性区研究 |
| The minimal active fragment of the Cry9Aa3 toxin against Plutella xylostella and Ostrinia furnacalis |
| 投稿时间：2014-03-23 修订日期：2014-04-11 |
| DOI： |
| 中文关键词: 苏云金芽胞杆菌 Cry9Aa3蛋白 最小活性区 小菜蛾 亚洲玉米螟 |
| 英文关键词:Bacillus thuringiensis Cry9Aa3 toxin minimal active fragment Plutella xylostella Ostrinia furnacalis |
| 基金项目:国家高技术研究发展计划“863”计划课题(2011AA10A203, 2013AA102701); 转基因生物新品种培育科技重大专项(2011ZX08009-003-001) |
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| 中文摘要: |
| 为了确定Cry9Aa3蛋白对亚洲玉米螟(Ostrinia furnacalis)和小菜蛾(Plutella xylostella)的最小活性区, 根据NCBI BLAST比对结果与SWISS-MODEL同源建模结果, 设计了12对截短引物, 以cry9Aa3全长序列为模板, 扩增出了12种不同截短片段, 与pEB载体重组后的阳性克隆, 转入大肠杆菌(Escherichia coli)菌株Rosetta(DE3)诱导表达, 提取蛋白。对小菜蛾和亚洲玉米螟的生物学活性测定, 发现截短蛋白Cry9Aa3-45～700、Cry9Aa3-1～654, 对小菜蛾和亚洲玉米螟有较高的活性, 其中截短蛋白Cry9Aa3-45～700对小菜蛾和亚洲玉米螟的LC50分别为5.27、7.56 μg/g, 截短蛋白Cry9Aa3-1～654对小菜蛾和亚洲玉米螟的LC50分别为7.76、7.79 μg/g, 与阳性对照无显著差异, 而截短蛋白Cry9Aa3-1～653和Cry9Aa3-46～700(LC50>200 μg/g)对幼虫几乎丧失生物学活性。这说明Cry9Aa3蛋白对小菜蛾和亚洲玉米螟的杀虫活性区相同, 最小活性区位于45I和654P之间。 |
| 英文摘要: |
| In order to identify the minimal active fragment of the Cry9Aa3 toxin, 12 pairs of primers were designed based on the structures predicted using SWISS-MODEL and BLAST. Twelve PCR products with different sizes were obtained using cry9Aa3 DNA as a template. Then they were cloned into pEB vectors separately. The positive clones were separately transformed into Escherichia coli strain Rosetta(DE3). The insecticidal activity was assayed using the full-length Cry9Aa3 toxin as a positive control against Plutella xylostella and Ostrinia furnacalis. The results showed that the truncated polypeptides Cry9Aa3-45—700 and Cry9Aa3-1—654 exhibited similar insecticidal activity as the full-length Cry9Aa3 protoxin. In contrast, the truncated toxin fragments Cry9Aa3-1—653 and Cry9Aa3-46—700 (LC50>200 μg/g) exhibited very low toxicity against the larvae. In conclusion, the minimal active fragment of the Cry9Aa3 toxin was located between the amino acid residues 45I and 654P. |
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