基于RNAi和蛋白质组学研究胶孢炭疽菌CgCDC2基因的功能

Research on the function of CgCDC2 gene in Colletotrichum gloeosporioides with RNAi and TMT-based proteomic analysis

【目的】CDC2是调控细胞周期的主要因子之一,对植物病原菌的生长发育有着重要作用,但目前还未有胶孢炭疽菌CDC2蛋白生物学功能方面的研究。本试验的目的是获得胶孢炭疽菌(Colletotrichum gloeosporioides)CgCDC2的RNAi突变体菌株,并分析CgCDC2基因的功能。【方法】利用RNAi技术和PEG介导法获得CgCDC2的RNAi突变体菌株,通过表型分析、TMT蛋白质组学分析和酶活性测定来确定该基因的生物学功能。【结果】通过PCR扩增获得CgCDC2基因,编码一个326个氨基酸的蛋白,其RNAi突变体菌株与野生型相比,生长速率减缓、分生孢子产量显著下降、对H2O2敏感性增强,PG、PL蛋白质含量、基因相对表达量和酶活性均显著降低,致病力减弱。【结论】CgCDC2参与调控胶孢炭疽菌的生长,分生孢子产量,氧化应激反应以及PG、PL的酶活性,从而降低病原菌的致病力。

【Objective】CDC2 is one of the main factors in cell cycle regulation,which plays an important role in the growth and development of plant pathogens.However,there are few studies on the biological function of CDC2 in Colletotrichum gloeosporioides.The purpose of this study was to obtain and analyze the CgCDC2 RNAi mutants of Colletotrichum gloeosporioides and provide some information about the function of CgCDC2.【Methods】The CgCDC2 gene was cloned by RT-PCR.RNA interference and the CgCDC2 RNAi mutants were obtain by using PEG-mediated protoplast transformation system.PCR amplification,agarose gel electrophoresis,along with qRT-PCR were used to determine whether the transformant obtained were RNAi mutants.The biological phenotypes including colony growth rate,spore yield and sensitivity to stress factors were tested to understand the phenotypic differences between mutants and wild-type strain.TMT-based proteomic analysis and enzyme activity assay were performed in order to analyze the differential proteins in the mutants and the wild-type strain.The pathogenicity of the mutants and the wild-type strain were determined by inoculating them on the mango.The diameter of the lesion was observed and measured every other day.【Results】The CgCDC2 gene encoded a protein containing 326 amino acids.It was found that through sequence alignment analysis the protein sequence of CgCDC2 was highly conserved.Compared with other plant pathogenic fungi,CgCDC2 was highly homologous.RNAi mutants were obtained by constructing RNAi expression vector and PEG-mediated protoplast transformation system.After the mutants and the wild-type strain inoculated on PDA and Czapek plates for several days,the colony growth rate of the mutants was found lower than that of the wild-type strain by observing and measuring the diameter of lesion.The collected conidia were counted by the hemocytometer and it was found that the conidia yield of mutants significantly decreased compared with the wild-type strain,it was about 1/8 of that of the wild-type strain.The sensitivity test of the mutants and the wild type strain to stress factors showed that both of them were not inhibited on the medium containing 0.7 mol·mL^-1 NaCl or 100μg·mL^-1 Congo Red.However,the growth of the mutants was inhibited to varying degrees,while the growth of the wild-type strain was not inhibited on the medium containing 5 mmol·mL-1 H2O2.TMT-based proteomic analysis showed that 5420 proteins were identified both in the mutants and the wild-type strain.In pSilent-1:CgCDC2-1 vs WT,86 significant differential proteins were identified,of which 47 were up-regulated and 39 were down-regulated.In pSilent-1:CgCDC2-2 vs WT,73 significant differential proteins were identified,of which 37 were up-regulated and 36 were down-regulated.GO enrichment analysis showed that in pSilent-1:CgCDC2-1 vs WT,the biological processes involved in differential proteins were mainly concentrated in the carbohydrate metabolic process,the regulation of transcription from RNA polymerase III promoter,nucleosome assembly and amino acid transmembrane transport.The molecular function were mainly concentrated in lyase activity,substrate-specific transmembrane transporter activity.The cellular components were mainly concentrated in chromatin.In pSilent-1:CgCDC2-2 vs WT,the biological processes involved in differential proteins were mainly concentrated in the aromatic amino acid family metabolic process.The molecular functions were mainly concentrated on oxidoreductase activity,carbon-sulfur lyase activity and antioxidant activity.The expression levels of PG and PL genes in the mutants the wildtype strain were determined by qRT-PCR.The results showed that the expression levels of PG and PL genes were significantly down-regulated in the mutants compared with the wild-type strain.The results of enzyme activity assay showed that the enzyme activities of PG and PL in mutants were significantly lower than those of the wild-type strain,which was consistent with the results of TMT-based proteomic analysis.The result of pathogenicity test showed that after inoculating mango for 5 days,the diameter of lesion caused by mutants was significantly smaller than that of the wild-type strain.This indicated that the pathogenicity of the mutants was weakened.【Conclusion】Sequence alignment results showed that the CgCDC2 protein was highly conserved.The biological phenotypic determination of the mutants indicated that the CgCDC2 gene was involved in the regulation of the colony growth rate,conidia yield,oxidative stress response,the expression levels of PG and PL genes,the expression levels of PG and PL proteins and enzyme activities in Colletotrichum gloeosporioides,thereby reducing the pathogenicity.