金龟子绿僵菌黏附素基因mad1的敲除及功能分析

闫多子 蔡霓 农向群 王广君 涂雄兵 张泽华

摘要 金龟子绿僵菌能够寄生昆虫，也能与植物共生。黏附素MAD1是绿僵菌与宿主互作初期的黏附因子。已知它在昆虫的侵染和致病中起重要作用，但与植物的互作机制报道甚少。为了研究MAD1在绿僵菌与植物共生中的作用，我们通过同源重组构建了金龟子绿僵菌mad1敲除株，并检测了敲除株的生长、产孢、孢子萌发及毒力等生物学特性，进一步利用qRT-PCR分析了MAD1在调节花生免疫响应中的作用。结果显示，与野生型菌株相比，敲除株产孢量降低了43.67%，分生孢子萌发中时为27.69 h，显著长于野生型菌株的13.43 h。敲除株对家蚕的半致死时间LT50为8.9 d，较野生型菌株的7.62 d显著延长。敲除株处理花生6 h后，花生免疫类基因CNGC1、PCD4，抗病基因SWEET10及转录因子WRKY41、MYB86的转录水平出现显著上调，而钙调素CML5、CML19的转录表达受到明显抑制。本研究证明了mad1是金龟子绿僵菌产孢、孢子萌发及毒力的正相关基因，并且MAD1在金龟子绿僵菌与植物相互作用初期，抑制植物抗性级联反应，减弱过敏反应，降低对微生物的抵御能力，同时增强共生信号的传导，这些作用有助于金龟子绿僵菌在花生根组织上定殖。

关键词 金龟子绿僵菌; MAD1; 基因敲除; 共生; 花生

中图分类号： S 476.12

文獻标识码： A

DOI： 10.16688/j.zwbh.2020629

Knockout and functional analysis of the adhesin gene mad1 in Metarhizium anisopliae

YAN Duozi1， CAI Ni1， NONG Xiangqun1*， WANG Guangjun1，2， TU Xiongbing1， ZHANG Zehua1

（1. State Key Laboratory for Biology of Plant Diseases and Insect Pests， Institute of Plant Protection，

Chinese Academy of Agricultural Sciences， Beijing 100193， China; 2. National Plant Protection

Xilinguole Observation and Experiment Station， Xilinhaote 026000， China）

Abstract

Metarhizium anisopliae is an entomopathogenic fungus and also an endophyte in plants. The adhesin MAD1 is the adhesion factor in the initial stage of interaction between M.anisopliae and insects or plant hosts. It is known that the adhesin is very important for infection and pathogenicity to insects， but its role in the interaction with plants is still unclear. Here， we aimed to explore the role of MAD1 in the symbiosis between M.anisopliae and plants. We constructed mad1 knockout strain by homologous recombination and tested the biological characteristics such as growth， sporulation， spore germination and virulence of the mad1 mutant. Furthermore， the effect of MAD1 on the peanut defense response was analyzed by qRT-PCR. The results showed that the spore production of the knockout strain reduced by 43.67%， and the spore semi-germination time was 27.69 h， which was significantly longer than 13.43 h of the wild type strain. The semi-lethal time LT50 of the knockout strain against the Bombyx mori was 8.9 d， which was significantly longer than 7.62 d of the wild type strain. In addition，  6 h after inoculating the knockout strain to peanut， the transcription levels of the immune genes CNGC1 and PCD4， disease resistance gene SWEET10 and transcription factors WRKY41 and MYB86 were significantly up-regulated， while the transcriptional expression of calmodulin CML5 and CML19 were significantly inhibited. This study suggested that mad1 was a positively correlated gene of sporulation， spore germination and virulence in M.anisopliae. Moreover， MAD1 inhibited the plant resistance cascade， weakened the allergic reactions and reduced the resistance to microorganisms， but synchronously enhanced the transduction of symbiotic signals at the initial stage of the interaction between M.anisopliae and plants. These effects were conducive to the colonization of M.anisopliae in peanut root tissues.

Key words

Metarhizium anisopliae; MAD1; gene knockout; symbiosis; peanut

绿僵菌Metarhizium作为一类昆虫病原真菌，经过漫长的适应性进化，形成了基物腐生、植物共生、昆虫寄生3种生活方式。由于具有广泛的杀虫能力，被用于防治多种农林害虫，如蝗虫、蛴螬等[1]。绿僵菌的致病机理相对复杂，有很多基因在绿僵菌对昆虫寄主的附着、定殖、穿透中发挥重要作用。……