苏云金芽孢杆菌CPB012菌株的杀虫功能基因鉴定及其对害虫的控制作用

doi:10.3864/j.issn.0578-1752.2015.06.08

摘要/Abstract

摘要： 【目的】分析并鉴定对马铃薯甲虫(Leptinotarsa decemlineata)具强致病性的苏云金芽孢杆菌(Bacillus thuringiensis)CPB012菌株的杀虫基因型，为充分而有效地利用此生防菌奠定基础。【方法】CPB012菌株在LB培养基上30℃培养2—3 d，用苯酚品红进行晶体染色，观察伴孢晶体形态。cry1—cry10杀虫基因的检测选用技术成熟的PCR-RFLP（polymerase chain reaction-restriction fragment length polymorphism）法，即对某一类基因（如cry1、cry2…）保守区序列设计通用引物，并引入限制性内切酶识别序列，将扩增产物用相应的限制性内切酶处理，得到的酶切图谱可以初步判断杀虫基因多态性。同时结合特异性PCR方法对二、三级分类基因（如cry1A、cry2Aa…）设计特异性引物进行扩增，将扩增产物测序比对。cry11—cry40和vip基因通过设计引物进行PCR检测。菌株在LB培养基上培养24、48、72和96 h分别提取晶体蛋白，结合晶体蛋白的SDS-PAGE图谱分析其表达的杀虫蛋白。室内分别测定CPB012对鳞翅目家蚕(Bombyx mori)、鞘翅目马铃薯甲虫和双翅目橘小实蝇(Bactrocera dorsalis)的杀虫效果；室内和田间测定CPB012与球孢白僵菌（Beauveria bassiana）MJ07菌株对马铃薯甲虫的生物协同防治效果。【结果】通过菌株形态和晶体形态的连续观察，发现CPB012能够产生方形和球形等晶体。基因型检测表明CPB012菌株含有cry1Aa、cry1Ia、cry2Aa、cry2Ab和vip3A，结合蛋白电泳图谱，其可能分别编码了约135、80、70、85—90 kD的大分子杀虫蛋白；同时可能还含有30和50 kD的两种新蛋白。室内生测试验显示，除马铃薯甲虫外，CPB012对家蚕和橘小实蝇也具有很强的毒杀作用，处理7 d 后死亡率分别为80.30%和76.72%。将CPB012与球孢白僵菌MJ07菌株按0.5﹕0.5剂量混合后室内接种，处理14 d后2龄马铃薯甲虫的累计死亡率达96.5%，极显著地(P<0.01)高于球孢白僵菌MJ07或苏云金芽孢杆菌CPB012单独处理的累计死亡率90.1%(MJ07)或83.3%（CPB012）。多重线性回归方程计算得出单独接种CPB012的LC₅₀为1.88×10⁷cfu/mL,MJ07的LC₅₀为5.46×10⁷cfu/mL，而二者混用后LC₅₀为1.10×10⁷cfu/mL，CTC（共毒系数）值为254，增效明显。在田间施用CPB012+MJ07混合剂(0.5﹕0.5)后对1龄和2龄马铃薯甲虫的防治效果分别为47.5%和43.8%, 显著高于MJ07、CPB012和Bt药剂对照的防治效果。【结论】苏云金芽孢杆菌CPB012的杀虫功能由cry1Aa、cry1Ia、cry2Aa、cry2Ab和vip3A等基因所决定，这些基因编码135、80、70、85-90 kD的杀虫毒蛋白。该菌的杀虫谱较广，对鞘翅目、鳞翅目和双翅目害虫都具有很强的生物毒性，其与球孢白僵菌MJ07混合施用可显著提高对马铃薯甲虫的防治效果。

关键词: 苏云金芽孢杆菌CPB012, 功能基因, 杀虫晶体蛋白, 马铃薯甲虫, 球孢白僵菌MJ07, 生物防治

Abstract: 【Objective】The objective of this study is to identify the functional genes, with their expressed proteins, and insecticidal spectrum of Bacillus thuringiensis strain CPB012. This will be important for effective application of CPB012 in pest control in the future.【Method】CPB012 was cultured at 30℃ on LB plates for two to three days and the bacterial cells were stained with phenol magenta for microscopy observation of the protein crystals. cry1-cry10 were identified with the polymerase chain reaction-restriction frame length polymorphism (PCR-RFLP) technique, namely, a universal primer pair for a group of genes was designed and used to amplify the genes with PCR. The amplified genes were then catalyzed with restriction enzymes and thus the polymorphism of the insecticidal genes were decided. On the other hand, specific primers were designed for identifying sub-group or genes (such as cry1A, cry2Aa. etc.) in groups cry1-cry10 via PCR amplification and then these genes were sequenced and compared. cry1-cry40 and vip groups were identified with PCR through the design of primers. The crystal proteins were extracted, respectively, from the bacterial cells cultured on LB plates for 24, 47, 72 and 96 hours and SDS-PAGE chromatographic fingerprint analysis was applied to demonstrate different insecticidal proteins. The lethality of Bombyx mori (Lepidoptera), Leptinotarsa decemlineata (Cleoptera) and Bactrocera dorsalis (Diptera) by CPB012 was measured in the laboratory, the cooperative effects of CPB012 and MJ07 strains of Beauveria bassiana were tested under both laboratory and field conditions. 【Result】Continuous microscopic observations found that CPB012 produced spherical and cubic protein crystals in blastosporic cells. Gene type analysis showed that B. thuringiensis CPB012 contained cry1Aa, cry1Ia, cry2Aa, cry2Ab and vip3A genes. These genes were shown via SDS-PAGE chromatographic fingerprint to code crystal proteins of about 135, 80, 70, and 85-90 kD, respectively. Also, the bacterium was demonstrated to contain two new crystal proteins which were 30 and 50 kD. Laboratory tests showed that CPB012 infected B. mori, B. dorsalis and L. decemlineata caused more than 76% deaths of each pest. After a mixture of B. thuringiensis CPB012 and B. bassiana MJ07 (mixed at 0.5: 0.5) was applied in a laboratory test, the cumulative mortality rate of 2nd-instar L. decemlineata larvae was 96.5%, which was significantly higher than that of individual CPB012 (83.3%) and that of MJ07 (90.1%). The LC₅₀ calculated from the multiple regression equation was 1.10×10⁷cfu/mL and the CTC was 254, which indicated the combined use of the two biocontrol agents increased pest control efficacy markedly. Under the local field condition, the mixture of CPB012+MJ07 treatment also improved the pest control effect markedly; the cumulative mortality of the 1st- and 2nd-instar larvae were 47.5% and 43.8%, respectively, which were significantly higher than those of separate CPB012, MJ07 and commercial Bt preparation.【Conclusion】The insecticidal function of B. thuringiensis CPB012 was decided by cry1Aa, cry1Ia, cry2Aa, cry2Ab and vip3A and these genes encoded insecticidal proteins of 135, 80, 70, and 85-90 kD. This bacterium had wide spectrum of insecticidal activity which was shown by high death rates of B. mori, B. dorsalis and L. decemlineata induced by CPB012 treatment. The insecticidal efficacy was significantly improved by combined application of CPB012 of B. thuringiensis and MJ07 of B. bassiana. Therefore, CPB012 is thought of as a wide-spectrum biocontrol agent and will be of potential significance and great value in developing effective bio-insecticide.

Key words: Bacillus thuringiensis CPB012, functional genes, insecticidal crystal proteins, Leptinotarsa decemlineata, Beauveria bassiana MJ07, biological control

陆慧慧，林志强，谭万忠，罗华东，鲜菲，毕朝位，余洋，杨宇衡. 苏云金芽孢杆菌CPB012菌株的杀虫功能基因鉴定及其对害虫的控制作用[J]. 中国农业科学, 2015, 48(6): 1112-1121.

LU Hui-hui, LIN Zhi-qiang, TAN Wan-zhong, LUO Hua-dong, XIAN Fei, BI Chao-wei, YU Yang, YANG Yu-heng. Insecticidal Protein Genes of Bacillus thuringiensis Strain CPB012 and Its Effects in Controlling Different Insect Pests[J]. Scientia Agricultura Sinica, 2015, 48(6): 1112-1121.

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