绿僵菌胞外蛋白酶Pr1C诱导飞蝗中肠免疫功能分析  

Immune analysis of Locust migratoria midgut induced by Metarhizium anisopliae extracellular protease Pr1C

作　　者：郭隆隆 李贝贝 李霜 杜桂林[3] 农向群[1,2] 涂雄兵 王广君[1,2] 张泽华 GUO Longlong;LI Beibei;LI Shuang;DU Guilin;NONG Xiangqun;TU Xiongbing;WANG Guangjun;ZHANG Zehua(State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China;Scientific Observation and Experimental Station of Pests in Xilin Gol Rangeland, Ministry of Agriculture and Rural Affairs, Xilinhot 026000, China;National Animal Husbandry Service, Beijing 100125, China)

机构地区：[1]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 [2]农业农村部锡林郭勒草原有害生物科学观测实验站,锡林浩特026000 [3]全国畜牧总站,北京100125

出　　处：《植物保护》2020年第2期12-17,37,共7页Plant Protection

基　　金：国家重点研发计划(2017YFD0200402);国家现代农业产业技术体系建设专项(CARS-34-07);中央级科研院所基本科研业务费专项(Y208YJ16,Y2019CG03,S2019XM20);牛顿基金国际合作项目(ST/N006712/1)。

摘　　要：为明确金龟子绿僵菌Metarhizium anisopliae胞外蛋白酶Pr1C在绿僵菌侵染飞蝗Locusta migratoria中肠中的作用,从绿僵菌IMI330189菌株转录组中获得并分析了Pr1C基因全长序列,设计引物对该基因进行了克隆和原核表达。将表达的Pr1C蛋白与绿僵菌IMI330189混合后饲喂处理,测定了混合物对飞蝗的毒力,并通过荧光定量PCR检测了飞蝗中肠免疫相关基因的表达情况。结果显示,该基因属于绿僵菌类枯草杆菌丝氨酸蛋白酶家族,全长为2126 bp,蛋白大小为71 kDa。Pr1C与绿僵菌IMI330189混合后可以显著提高对飞蝗的毒力。荧光定量结果表明,Defensin,Persephone,Tube,Relish,Dredd 5个基因在各处理中表达量均呈现升高趋势,其中Pr1C蛋白和绿僵菌混合处理的变化比其他处理组变化快,于第2天达到较高水平;绿僵菌处理变化较慢,于第6天达到最高水平。本研究表明,绿僵菌胞外蛋白酶Pr1C可显著提高绿僵菌毒力,促进绿僵菌侵染速率,为进一步研制和应用生物制剂防治飞蝗提供理论依据。In order to explore the role of Metarhizium anisopliae in the midgut infection of Locusta migratoria,the full-length sequence of Pr1C gene was obtained from the transcriptome sequences of M.anisopliae strain IMI330189 and primers were designed to clone and express the Pr1C gene.The obtained Pr1C protein was mixed with M.anisopliae IMI330189 spore and its toxicity to the locust was checked by using bait-feeding method.The expression level of intestinal immune-related genes in locusts was detected by fluorescence quantitative PCR.NCBI Blast results showed that Pr1C gene belonged to the serine protease family of M.anisopliae with a full length of 2126 bp,and its protein size was 71 kDa.The bioassay results showed that the mixture of Pr1C and M.anisopliae IMI330189 could significantly increase the virulence to L.migratoria.The expression level of five genes Defensin,Persephone,Tube,Relish and Dredd showed an increasing trend in all treatments,but the treatment with the mixture of Pr1C protein and metarhizium induced a faster change than other treatments and reached to the highest level on the second day.The treatment with M.anisopliae induced slow change in the expression of genes,and the highest level was observed on the sixth day.This study indicated that the extracellular protease Pr1C of M.anisopliae could significantly increase the virulence of M.anisopliae and its infection rate,providing a theoretical basis for the further development and application of biological preparations for controlling L.migratoria.

关 键 词：绿僵菌 胞外蛋白酶 飞蝗 毒力 

分 类 号：S476.12[农业科学—农业昆虫与害虫防治]