丝裂原活化蛋白激酶基因CfMKK1调控果生炭疽菌的生长发育和致病力

MAPKK-encoding gene CfMKK1 in Colletotrichum fructicola is required for its growth and pathogenicity

【目的】由果生炭疽菌引起的炭疽病是油茶的主要病害,造成油茶产量下降。本文研究果生炭疽菌中丝裂原活化蛋白激酶CfMkk1的生物学功能,旨在为解析油茶炭疽病菌的致病机理提供依据。【方法】根据同源重组原理构建CfMKK1基因敲除载体片段,采用PEG介导法将载体导入原生质体中筛选获得突变体菌株DCfmkk1;PCR扩增果生炭疽菌含有启动子的CfMKK1基因回补片段,构建回补载体pYF11::CfMKK1;采用PEG介导法把回补载体转化至突变体的原生质体中,荧光筛选回补菌株ΔCfmkk1-C。测定野生型菌株、突变体菌株DCfmkk1及基因回补菌株ΔCfmkk1-C在营养生长、附着胞形成、胁迫应答和致病力等生物学表型。【结果】与野生型和回补菌株相比,CfMKK1基因敲除突变体ΔCfmkk1菌丝生长速率明显减缓;在含刚果红的PDA培养基上菌丝生长受到明显抑制,无法穿透玻璃纸,丧失了侵染寄主的能力;而且无法形成附着胞。【结论】研究结果表明CfMKK1基因参与调控油茶果生炭疽菌的生长发育、附着胞形成、致病力以及响应外界胁迫过程。

[Objective]Colletotrichum fructicola is an important pathogen that results in yield loss of Camellia oleifera.We studied the functions of the mitogen-activated protein kinase gene CfMKK1 in C.fructicola for analyzing the pathogenic mechanism of oil-tea tree anthracnose.[Methods]The homologous recombination method was used to construct the CfMKK1 gene-deleted fragment,which was transformed into the protoplasts generated by using PEG-mediated method to obtain the mutant strainΔCfmkk1.The PCR-amplified CfMKK1 gene-containing complement of the promoter of C.fructicola was taken to construct a complementary vector pYF11::CfMKK1,then the complementary vector was transformed into the mutant protoplasts by using PEG-mediated method to screen the complementary strainΔCfmkk1-C.The biological phenotypes of the wild-type strain CFLH16,the mutant strainΔCfmkk1,and the complementary strainΔCfmkk1-C were measured in vegetative growth,appressorium formation,stress response and pathogenicity.[Results]The mycelial growth rate ofΔCfmkk1 was significant slowed down compared to the wild-type strain CFLH16 and the complementary strainΔCfmkk1-C,more sensitive to Congo red,lost the ability to infect hosts and was unable to form appressoria.[Conclusion]CfMkk1 is involved in regulating the growth,response to external stress and appressorium formation of C.fructicola,affecting the pathogenicity.