对暗黑鳃金龟成虫具有杀虫活性的Bt蛋白筛选

doi:10.16409/j.cnki.2095-039x.2018.04.008

中国生物防治学报 ›› 2018, Vol. 34 ›› Issue (4): 546-552.DOI: 10.16409/j.cnki.2095-039x.2018.04.008

对暗黑鳃金龟成虫具有杀虫活性的Bt蛋白筛选

黄世智^1,2, 束长龙², 刘春琴³, 丁学知¹, 张杰²

1. 湖南师范大学生命科学学院/微生物分子生物学湖南省重点实验室/淡水鱼类发育生物国家重点实验室, 长沙 410081;
2. 中国农业科学院植物保护研究所/植物病虫害生物学国家重点实验室, 北京 100193;
3. 沧州市农林科学院植物保护研究所, 沧州 061001

收稿日期:2018-04-19 出版日期:2018-08-08 发布日期:2018-07-30
通讯作者: 丁学知,教授,Email:dingxuezhi@hunnu.edu.cn;张杰,研究员,E-mail:jzhang@ippcaas.cn。
作者简介:黄世智,硕士研究生,E-mail:1263056340@qq.com
基金资助:
国家重点研发计划（2017YFD0201201）；转基因生物新品种培育重大专项（2014ZX0800913B）

Screening of Insecticidal Activity Protein against Holotrichia parallela Adults from Bacillus thuringiensis

HUANG Shizhi^1,2, SHU Changlong², LIU Chunqin³, DING Xuezhi¹, ZHANG Jie²

1. State Key Laboratory of Developmental Biology of Freshwater Fishes/Hunan Provincial Key Laboratory of Microbial Molecular Biology/College of Life Science, Hunan Normal University, Changsha 410081, China;
2. State Key Laboratory for Biology of Plant Diseases and Insect Pests/Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
3. Institute of Plant Protection, Cangzhou Academy of Agricultural and Forestry Sciences, Cangzhou 061001, China

Received:2018-04-19 Online:2018-08-08 Published:2018-07-30

摘要/Abstract

摘要： 暗黑鳃金龟Holotrichia parallela是一种重要的农业害虫，其成虫和幼虫均能对多种作物造成严重危害。为筛选对暗黑鳃金龟成虫具有毒杀活性的Bt蛋白，本研究首先对暗黑鳃金龟成虫的生活习性进行观察，用浸泡蛋白的榆树叶饲喂成虫，建立了Bt蛋白对暗黑鳃金龟成虫的生物学活性测定方法；制备了6种Bt Cry8类杀虫晶体全长蛋白和胰蛋白酶消化蛋白，对暗黑鳃金龟成虫进行了杀虫活性测定。初筛结果发现Cry8Ga1原毒素和消化蛋白均对暗黑鳃金龟成虫具有较高毒杀活性，其在浓度1.0 mg/mL时对暗黑鳃金龟成虫的7 d校正死亡率分别为100%和55%。为更进一步确定Cry8Ga1蛋白的杀虫活性，进行了致死中浓度测定，7 d生测结果显示，Cry8Ga1原毒素的LC₅₀为0.387 mg/mL （95%置信区间为0.108~0.746 mg/mL），Cry8Ga1蛋白经胰蛋白酶消化后的LC₅₀为0.702 mg/mL （95%置信区间为0.150~2.664 mg/mL）。取食Cry8Ga1原毒素蛋白及其消化蛋白后的暗黑鳃金龟成虫中肠细胞产生显著的病理学变化。本研究筛选到对暗黑鳃金龟成虫有毒杀作用的Bt蛋白，研究结果将为金龟子的生物防治开辟新的途径。

关键词: 苏云金芽胞杆菌, Cry8Ga1蛋白, 暗黑鳃金龟成虫, 杀虫活性

Abstract: Holotrichia parallela is an important agricultural pests, damaging several crops at both the adult and larval stages. To screen Bacillus thuringiensis protein of high insecticidal activity a bioassay method of the Bt protein against H. parallela adults was established by feeding on the adults with Ulmus pumila L leaves soaked in Bt proteins. Six Cry8 full-length crystal proteins and trypsin-digested proteins were prepared and used in the insecticidal activity bioassay. Cry8Ga1 protoxin and digested protein showed high insecticidal activity in the primary test, with 7-day corrected mortality rates of 100% and 55%, respectively, at the concentration of 1.0 mg/mL. Further, the median lethal concentration (LC₅₀) values were determined to be 0.387 mg/mL (95% CL 0.108-0.746) for Cry8Ga1 protoxin and 0.702 mg/mL (95% CL 0.150-2.664) for digested Cry8Ga1 protein. When fed with Cry8Ga1 protoxin and digested protein, H. parallela adults exhibited obvious pathological changes at the organelles in midgut cells. The Bt protein showing high insecticidal activity against H. parallela adults is a new approach for the biological control of Scarabaeoideas.

Key words: Bacillus thuringiensis, Cry8Ga1 protein, Holotrichia parallela adults, insecticidal activity

中图分类号:

S476.1

黄世智, 束长龙, 刘春琴, 丁学知, 张杰. 对暗黑鳃金龟成虫具有杀虫活性的Bt蛋白筛选[J]. 中国生物防治学报, 2018, 34(4): 546-552.

HUANG Shizhi, SHU Changlong, LIU Chunqin, DING Xuezhi, ZHANG Jie. Screening of Insecticidal Activity Protein against Holotrichia parallela Adults from Bacillus thuringiensis[J]. journal1, 2018, 34(4): 546-552.

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对暗黑鳃金龟成虫具有杀虫活性的Bt蛋白筛选

Screening of Insecticidal Activity Protein against Holotrichia parallela Adults from Bacillus thuringiensis

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