微生物學(xué)報(bào)Acta Microbiologica Sinica 48(6)：733~738; 4 June 2008
基金項(xiàng)目: “863 計(jì)劃” (2006AA10A212); 國(guó)家自然科學(xué)基金(30571252)
任羽1，劉華梅1，宋福平1,，黃大昉2,，張杰1*
*通訊作者。Tel: +86-10-62815921; E-mail: [email protected]
作者簡(jiǎn)介: 任羽(1979. ), 女, 北京懷柔人, 博士研究生, 主要從事蘇云金芽孢桿菌殺蟲(chóng)晶體蛋白分子設(shè)計(jì)研究,。E-mail: [email protected]
（1 中國(guó)農(nóng)業(yè)科學(xué)院植物保護(hù)研究所 植物病蟲(chóng)害生物學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室北京 100094）
（2 中國(guó)農(nóng)業(yè)科學(xué)院生物技術(shù)研究所,，北京100081）
摘要：蘇云金芽孢桿菌殺蟲(chóng)晶體蛋白Cry1Ca7 對(duì)重要的農(nóng)業(yè)害蟲(chóng)甜菜夜蛾具有較高毒力?！灸康摹勘疚牡难芯磕康氖峭ㄟ^(guò)定點(diǎn)突變的方法獲得毒力發(fā)生改變的毒蛋白,，為下一步研究工作提供有價(jià)值的實(shí)驗(yàn)材料?！痉椒ā坷弥丿B引物PCR 技術(shù)對(duì)cry1Ca7 基因進(jìn)行定點(diǎn)突變,，獲得了10 種突變基因，通過(guò)生物活性測(cè)定的方法確定了各突變基因表達(dá)產(chǎn)物對(duì)甜菜夜蛾的殺蟲(chóng)活性,?！窘Y(jié)果】活性降低的突變毒蛋白有G138S，T221D,，T221R,，N251S，439GGT440,，N306R,，W376F，R522E 和 R570G,，其中,，位于DomainⅡ內(nèi)的突變的活性依次是439GGT440<N306R<W376F；位于DomainⅢ內(nèi)的突變R522E< R570G,，二者的活性較Cry1Ca7 也有明顯降低,；只有位于結(jié)構(gòu)域Ⅰ中的R148G，產(chǎn)生了活性提高的突變毒蛋白,，其毒性較Cry1Ca7 提高了6 倍,，而同樣位于DomainⅠ內(nèi)的突變T221D<T221R<G138S<N251S,，尤其是T221D 活性完全喪失。研究結(jié)果表明,，在結(jié)構(gòu)域Ⅰ中的突變更容易產(chǎn)生活性提高的突變蛋白,，而結(jié)構(gòu)域Ⅱ和Ⅲ較難獲得毒力提高的突變蛋白?！窘Y(jié)論】本項(xiàng)研究所獲得的這些活性發(fā)生不同變化的蛋白為揭示Cry 蛋白的殺蟲(chóng)機(jī)理提供了基礎(chǔ)材料,，而活性提高的誘變基因及其表達(dá)產(chǎn)物將可作為新的殺蟲(chóng)資源，用于抗蟲(chóng)遺傳工程菌和轉(zhuǎn)基因植物的構(gòu)建,。
關(guān)鍵詞：蘇云金芽孢桿菌,；殺蟲(chóng)晶體蛋白；定點(diǎn)突變,；cry1Ca7 基因,；甜菜夜蛾
中圖分類(lèi)號(hào)：Q933 文獻(xiàn)標(biāo)識(shí)碼：A 文章編號(hào)：0001-6209 (2008) 06-0733-06
Effect of Cry1Ca7 protein modified by site-directed mutagenesis on inhibiting Spodoptera exigua Hübner
Yu Ren1, Huamei Liu1, Fuping Song1, Dafang Huang2, Jie Zhang1*
(1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100094, China)
(2 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China)
Abstract: [Objective] To obtain the mutants with different toxicity from the wild-type Cry1Ca7. [Methods] Insecticidal crystal protein Cry1Ca7 from Bacillus thuringiensis which is highly toxic to Spodoptera exigua, an important agricultural pest in China, and we mutated this toxin by over-lapping extensive PCR method in different domains to obtain 11 chimeric mutants. [Results] The results of bioassays against Spodoptera exigua neonates showed that several conserved amino acid sites were crucial to insects. The pesticidal activities of most of mutated proteins were decreased, including Glycine138 Serine, Threonine221 Aspartic acid, Threonine221 Argine, Asparagine251 Serine, 439GlycineGlycineThreonine440, Asparagine306 Argine, Tryptophan376 Phenylalanine, Argine522 Glutamic acid and Argine570 Glycine. The activity of those mutated proteins in the DomainⅡ was 439GlycineGlycineThreonine 440 < Asparagine 306 Argine < Tryptophan 376 Phenylalanine. In the DomainⅢ, the mutant Argine522 Glutamic acid < Argine 570 Glycine, their toxicities reduced distinctly compared with Cry1Ca7. The toxicities of the mutant Argine148 Glycine in DomainⅠincreased six-fold, nevertheless the activities of the mutants Glycine138 Serine、Threonine221 Argine and Asparagine251 Serine mutant reduced totally, even the mutant of Threonine221 Aspartic acid was not toxic entirely. In [Conclusion] It is relatively easier to obtain mutant with higher toxicity in DomainⅠof Cry1Ca7 protein than these in both DomainⅡ andⅢ. We can use the improved mutant genes as the potential resources to construct novel engineering bacteria and transgenic plant, meanwhile, to perform the study of interaction mechanism between insects and Cry proteins.
Keywords: Bacillus thuringiensis; insecticidal crystal protein; Site-directed mutagenesis; cry1Ca7 gene; Spodoptera exigua
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蘇云金芽孢桿菌Cry1Ca7 蛋白定點(diǎn)突變對(duì)甜菜夜蛾殺蟲(chóng)活性的影響
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