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

摘要:

[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△Cfmkk1-C。测定野生型菌株、突变体菌株DCfmkk1及基因回补菌株△Cfmkk1-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△Cfmkk1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。

关键词:油茶;果生炭疽菌;丝裂原活化蛋白激酶;CfMkk1;致病力

Abstract:

[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 strain △Cfmkk1. 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 strain △Cfmkk1-C. The biological phenotypes of the wild-type strain CFLH16, the mutant strain △Cfmkk1, and the complementary strain △Cfmkk1-C were measured in vegetative growth, appressorium formation, stress response and pathogenicity. [Results] The mycelial growth rate of △Cfmkk1 was significant slowed down compared to the wild-type strain CFLH16 and the complementary strain △Cfmkk1-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.

Key words:Camellia oleifera;Colletotrichum fructicola;mitogen-activated protein kinases;CfMkk1;pathogenicity