苏云金芽孢杆菌<i>cry1Da5</i>基因的克隆与表达-《江苏农业科学》

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Title:: Cloning and expression of cry1Da5 gene from Bacillus thuringiensis strain

作者:: 关鹏¹; 秦培钢²; 代小娟¹; 宋金东¹; 1.渭南职业技术学院农学院，陕西渭南 714026； 2.新疆医科大学基础医学部，新疆乌鲁木齐 830011

Author(s):: Guan Peng; et al

关键词:: 苏云金芽孢杆菌; 简并引物; cry1Da5基因; 棉铃虫; 甜菜夜蛾; 杀虫活性

Keywords:: -

分类号:: S182

DOI:: -

文献标志码:: A

摘要:: 采用5对引物组合经2轮PCR扩增对苏云金芽孢杆菌QL75-2菌株中的cry基因进行鉴定，克隆得到1个cry1Da型基因片段。通过设计全长引物扩增得到该cry基因的全长序列，转入原核表达载体pET-28a中，在大肠杆菌BL21(DE3)中进行表达，最后对表达蛋白进行生物活性分析。序列分析结果表明，该cry1Da基因全长3 495 bp，推测其编码1 164个氨基酸，组成分子量约132.01 ku的蛋白质。该预测蛋白质与已知的Cry1Da3蛋白质具有最高的序列同源性(99.7%)，该基因被国际杀虫晶体蛋白基因命名委员会正式命名为cry1Da5。生物活性分析结果表明，Cry1Da5蛋白质对鳞翅目的甜菜夜蛾幼虫具有较强的杀虫活性，其LC₅₀为21.47 μg/mL，而对鳞翅目的棉铃虫幼虫无杀虫活性。因此，cry1Da5基因可作为一个新的cry基因资源在甜菜夜蛾的生物防治中应用。

Abstract:: -

参考文献/References:

［1］Priesnitz K U，Vaasen A，Gathmann A. Baseline susceptibility of different European lepidopteran and coleopteran pests to Bt proteins expressed in Bt maize：a systematic review［J］. Environmental Evidence，2016，5(1)：16-27.
［2］Soares-da-Silva J，Queirós S G，de Aguiar J S，et al. Molecular characterization of the gene profile of Bacillus thuringiensis Berliner isolated from Brazilian ecosystems and showing pathogenic activity against mosquito larvae of medical importance［J］. Acta Trop，2017，176：197-205.
［3］Kim H S，Noh S，Park Y. Enhancement of Bacillus thuringiensis Cry1Ac and Cry1Ca toxicity against Spodoptera exigua (Hübner) by suppression of a chitin synthase B gene in midgut［J］. Journal of Asia-Pacific Entomology，2017，20(1)：199-205.
［4］Reinoso-Pozo Y，Rincón-Castro M C D，Ibarra J E. Significant increase in toxicity of the Cry1Ac1 protein of Bacillus thuringiensis by the exchange of two amino acids in its domain III［J］. Biological Control，2018，125：1-6.
［5］王杨，周子珊，刘永磊，等. 对甜菜夜蛾具有杀虫活性的Bt菌株cry1Ca基因研究［J］. 植物保护，2016，42(2)：19-25，37.
［6］Grossi-de-Sa M F，Quezado de Magalhaes M，Silva M S，et al. Susceptibility of Anthonomus grandis (cotton boll weevil) and Spodoptera frugiperda (fall armyworm) to a cry1Ia-type toxin from a Brazilian Bacillus thuringiensis strain［J］. Journal of Biochemistry and Molecular Biology，2007，40(5)：773-782.
［7］Hernández-Rodríguez C S，Hernández-Martínez P，Rie J V，et al. Shared Midgut binding sites for Cry1A.105，Cry1Aa，Cry1Ab，Cry1Ac and Cry1Fa proteins from Bacillus thuringiensis in two important corn pests，Ostrinia nubilalis and Spodoptera frugiperda［J］. PloS One，2013，8(7)：e68164.
［8］Hernández-Martínez P，Ferré J，Escriche B. Susceptibility of Spodoptera exigua to 9 toxins from Bacillus thuringiensis［J］. Journal of Invertebrate Pathology，2008，97(3)：245-250.
［9］BenFarhat-Touzri D，Saadaoui M，Abdelkefi-Mesrati L，et al. Histopathological effects and determination of the putative receptor of Bacillus thuringiensis Cry1Da toxin in Spodoptera littoralis midgut［J］. Journal of Invertebrate Pathology，2013，112(2)：142-145.
［10］BenFarhat-Touzri D，Driss F，Jemli S，et al. Molecular characterization of Cry1D-133 toxin from Bacillus thuringiensis strain HD133 and its toxicity against Spodoptera littoralis［J］. International Journal of Biological Macromolecules，2018，112：1-6.
［11］宋福平，张杰，黄大昉，等. 苏云金芽孢杆菌cry基因PCR-RFLP鉴定体系的建立［J］. 中国农业科学，1998，31(3)：13-18.
［12］Berón C M，Curatti L，Salerno G L. New strategy for identification of novel cry-type genes from Bacillus thuringiensis strains［J］. Applied And Environmental Microbiology，2005，71(2)：761-765.
［13］Reyes-Ramírez A，Ibarra J E. Plasmid patterns of Bacillus thuringiensis type strains［J］. Applied And Environmental Microbiology，2008，74(1)：125-129.
［14］Pilhofer M，Bauer A P，Schrallhammer M，et al. Characterization of bacterial operons consisting of two tubulins and a kinesin-like gene by the novel Two-Step Gene Walking method［J］. Nucleic Acids Research，2007，35(20)：e135.
［15］Schgger H，von Jaqow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kD［J］. Analytical Biochemistry，1987，166(2)：368-379.
［16］黄天培，束长龙，张杰，等. 苏云金芽孢杆菌基因cry1Da3及其应用：CN201310286693.0［P］. 2013-07-09.

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