调控油茶果生刺盘孢bZIP转录因子CfAp1的生物学功能

Biological Function bZIP-Type Transcription Factor CfAp1 in Colletotrichum fructicola

【目的】研究油茶炭疽病的主要流行致病菌果生刺盘孢菌b ZIP转录因子Cf Ap1的生物学功能,以期阐明果生刺盘孢菌致病的分子机制,为油茶炭疽病的防治提供理论依据。【方法】提取果生刺盘孢菌全基因组DNA,根据同源重组原理构建Cf AP1基因敲除载体片段,采用PEG介导法把该片段转化至果生刺盘孢菌野生型菌株的原生质体中,验证筛选突变体菌株;PCR扩增果生刺盘孢菌含有启动子的Cf AP1基因回补片段,构建回补载体p YF11::Cf AP1;采用PEG介导法把回补载体p YF11::Cf AP1转化至果生刺盘孢菌突变体菌株的原生质体中,获得回补菌株ΔCfap1/AP1。测定野生型菌株、突变体菌株ΔCfap1-8及基因回补菌株ΔCfap1/AP1在生长发育、附着胞形成、外界胁迫和致病力等生物学表型。【结果】果生刺盘孢菌中具有1个与灰色大角间座壳(稻瘟菌) b ZIP转录因子MoAp1直系同源的基因,命名为Cf AP1;该基因全长1 804 bp,编码566个氨基酸,该蛋白含有1个碱性亮氨酸链(b ZIP)结构域、2个PAP1结构域和2个未知功能的结构域;与野生型和回补菌株相比,突变体ΔCfap1-8生长速率没有明显影响,但气生菌丝显著减少;通过测量产孢量,发现突变体ΔCfap1-8的分生孢子显著减少;ΔCfap1-8在含2.5、5 mmol·L-1H2O2和0.7 mol·L-1Na Cl的PDA平板上菌丝生长受到明显抑制;致病力测试结果表明,果生刺盘孢菌基因敲除突变体ΔCfap1-8对无伤和有伤油茶叶片的致病力下降明显,与野生型和回补菌株的致病力差异显著;突变体分生孢子产生附着胞数量减少且膨压降低。【结论】转录因子Cf Ap1参与调控油茶果生刺盘孢菌的生长发育、产孢、致病力以及响应外界氧压胁迫和渗透压胁迫过程。

【Objectve】Colletotrichum fructicola is one of the major causal pathogens of Camellia oleifera.This experiment aims to study the biological functions of a leucine zipper(b ZIP)type transcription factor CfAp1 in order to elucidate the molecular mechanism of C.fructicola,providing theoretical basis for controlling oil-tea tree anthracnose.【Method】The whole genomic DNA of C.fructicola was extracted,and the CfAP1 gene knockout vector fragment was constructed based on the principle of homologous recombination.The fragment was transformed into the protoplasts of the wild type strain of C.fructicola by PEG-mediated method.Putative transformants were screened on hygromyc in media,and then verified by PCR amplification.Thus,a deletion mutant was obtained.PCR-amplifying CfAP1 gene-containing complement of the promoter of C.fructicola was used to construct a complement vector pYF11::CfAP1.PEG-mediated transformation of pYF11::CfAP1 was transformed into the protoplasts of theΔCfap1 mutant,and then the complement strainΔCfap1/AP1 was obtained.The biological phenotypes of wild-type strain,theΔCfap1-8 mutant and the gene complementationΔCfap1/AP1 were measured for growth and development,appressorium formation,external stress and pathogenicity.【Result】Our results showed that the transcription factor CfAp1 of C.fructicola had 566 amino acids with one bZIP domain,two PAP1 domains and three unknown function domains,encoded by a 1804 bp gene or thologous to MoAp1 of the rice blast fungus Magnaporthe oryzae.Compared with the wild-type strain and the gene complementationΔCfap1/AP1,the growth rate ofΔCfap1-8 mutant had no remarkable change,but the aerial hyphae decreased significantly.The conidia of theΔCfap1-8 mutant were remarkably reduced.Stress response assay showed that theΔCfap1-8 mutant was remarkably inhibited on the PDA plate containing 2.5 mmol·L-1,5 mmol·L-1 H2 O2 and 0.7 mol·L-1 Na Cl.The pathogenicity of theΔCfap1-8 mutant was remarkably decreased in virulence to C.oleifera.The appressorium pressure of the conidium was reduced,which may lead to a decrease in the pathogenicity of C.fructicola.【Conclusion】The study reveals that the transcription factor CfAp1 plays critical roles in growth and development,conidiation,appressorium formation,pathogenicity and response to oxidative stress and osmotic stress in C.fructicola.