Cry1Ac毒素突变体的构建、表达及杀虫活性测定

袁晓曼,丁照鑫,张淑臣,刘京国

中国农学通报. 2017, 33(36): 139-144

中国农学通报 ›› 2017, Vol. 33 ›› Issue (36) : 139-144. DOI: 10.11924/j.issn.1000-6850.casb16110021
植物保护 农药

Cry1Ac毒素突变体的构建、表达及杀虫活性测定

  • 袁晓曼,丁照鑫,张淑臣,刘京国
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The Construction, Expression and Insecticidal Activity Determination of Cry1Ac Toxin Mutant

  • 袁晓曼,, and
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摘要

为了构建对敏感和类钙粘蛋白突变的抗性昆虫都具有高杀虫活性的毒素，将Cry1Ac毒素结构域I 中α1 和α2 之间插入一个α胰凝乳蛋白酶作用位点，并分析突变体毒素在苏云金芽孢杆菌中形成伴孢晶体、目标蛋白的表达情况，以及鉴定突变体毒素的杀虫活性。结果发现：4 个突变体蛋白都能在苏云金芽孢杆菌中表达产生约130 kDa 的目标蛋白，但不能形成有规则的伴孢晶体。这可能是由于突变体蛋白在Bt 生长的平稳期达到最高，在衰亡期开始被降解造成的。4 个突变体中，Cry1Acm4 在培养48 h 时产量最高，而且其对甜菜夜蛾杀虫活性比野生型高1.6 倍，但是其产量只有野生型毒素产量的十分之一。尽管由于Cry1Acm4 的表达量较低而使其实际应用的价值不大，但这种构建突变体的方法对研究高毒力的Cry1Ac毒素提供一种新的思路。

Abstract

The aim is to construct the toxin with high insecticidal activity for resistant insects with sensitive resistance and cadherin-like protein mutant. α-chymotrypsin site was inserted between α1 and α2 of domain I of Cry1Ac toxin, the expression of target protein and the formation of parasporal crystal from mutant toxin in Bt was analyzed, the insecticidal activity of mutant toxin was determined. The results showed that, the four mutant proteins could express about 130 kDa target protein in Bacillus thuringiensis, but these mutants did not form regular parasporal crystal. The reason might be that the mutant protein reached the top at the stationary stage of Bacillus thuringiensis, and then it was degraded at the beginning of the decline stage. In the four mutants, Cry1Acm4 had the highest yield at 48 h, and its insecticidal activity for Spodoptera exigua was 1.6 times higher than that of the wild type, but the yield was only one tenth of toxin yield of wild type. Although it had little value in application for Cry1Acm4 because of the low expression, the construction method provided a new approach for researching Cry1Ac toxin with high insecticidal activity.

关键词

苏云金芽孢杆菌;Cry1Ac毒素;突变体,杀虫活性

Key words

Bacillus thuringiensis; Cry1Ac toxin; mutant; insecticidal Activity

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袁晓曼,丁照鑫,张淑臣,刘京国. Cry1Ac毒素突变体的构建、表达及杀虫活性测定. 中国农学通报. 2017, 33(36): 139-144 https://doi.org/10.11924/j.issn.1000-6850.casb16110021
袁晓曼,, and. The Construction, Expression and Insecticidal Activity Determination of Cry1Ac Toxin Mutant. Chinese Agricultural Science Bulletin. 2017, 33(36): 139-144 https://doi.org/10.11924/j.issn.1000-6850.casb16110021

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