深绿木霉中碳代谢抑制因子CRE1功能特性研究

Study on Functional Properties of Carbon Catabolite Repressor CRE1 in Trichoderma atroviride

木霉菌（Trichoderma spp.）是一种广泛存在于土壤及植物根系生境中的丝状真菌,具有拮抗植物病原真菌和促进植物生长的双重功效。碳代谢抑制子CRE1全局性调控细胞生长代谢过程,保障木霉在不同生境中的存活及拮抗病原菌特性。比较深绿木霉（T.atroviride）T23及其Cre1突变株（T23Δcre1）在不同培养基中的生长和代谢特性,结果表明：cre1基因沉默后,T23Δcre1较T23菌丝生长变慢,产孢滞后且降低一个数量级,cre1对菌丝生长和产孢的调控依赖于培养基组分。此外,cre1基因抑制几丁质酶、β-1,3-葡聚糖酶基因转录,区别性调控部分次级代谢合成的非核糖体肽合成酶NRPS和聚酮合成酶PKS基因的表达。综上,碳代谢因子CRE1作为一个多效性的转录调控因子,抑制细胞壁降解酶和代谢产物合成相关的基因表达,赋予木霉菌在环境中的适应性和竞争性,是深绿木霉T23生长的必要因子。

The genus Trichoderma comprises a large number of strains found in multitude of ecological niches.Some of them have been developed as biocontrol agents against plant diseases,which can antagonize plant pathogens and facilitate the growth of plants at the same time,showing an important value for biocontrol in agriculture.Carbon catabolite repression（CCR） is a wide-domain regulatory circuit,allowing the preferred assimilation of carbon sources of high nutritional value over others.CCR is mediated by the Cys2 His2 type transcription factor Cre A/CREI which has been cloned from numerous filamentous fungi.The latest studies have found that CRE1,the carbon catabolite repressor,regulates cell growth and metabolic procedures of Trichoderma spp.,which would be helpful for strains＇ antagonism against various pathogens and their own survival at different habitats.On basis of wide type strain T.atroviride 23,T23 and its cre1 knock out mutant strain T23 Δcre1 kept in laboratory,the phenotypic assays and function analysis of both strains have been carried out on different mediums to draw the following conclusions.Firstly,the cre1 gene in Trichoderma is essential for the growth and sporulation of T.atroviride.When cre1 is knocked out,the growth rate of T23 Δcre1 slows down,and its sporulation apparently lags behind that of T23 with the number of spores reduced an order of magnitude;Furthermore,the response of T23 Δcre1 to carbon sources and nitrogen sources are diffirent from those of T23.The results further indicate cre1-mediated regulation of growth and formation of sporulation depends on specific culture.Secondly,how CRE1 regulates the expression of some cell-wall degraditon enzymes,such as chitnase,β-1,3-glucanase,protease and cellulase,is detected.Results of enzyme activity assay on plate and q RT-PCR show that CRE1 represses the expression of the chitianse and β-1,3-glucanase obviously.In line with that,transcription activity of two enzyme activity encoding genes,chit42 and glucanase,respectively increase by 2 times and 4 times in T23Δcre1 compared with in T23.Enzyme activity comparison of cellulase and protease conducted on plate also indicates a higher activity level in T23Δcre1 rather than in T23.Finally,the regulation of CRE1 on the expression of several non-ribosomal peptides synthases（NRPSs） and polyketide synthases（PKSs）,which are found in charge of the synthesis of most valuable secondary metabolies for biocontrol,are deterimined.According to results of q RT-PCR,CRE1 plays a complicate role in the regulation on the transcription of NRPSs and PKSs.Among the three choosen NRPS encoding genes,the knock out of cre1 results in an increase of 8.72-fold expression of NRPS2,while NRPS1 expression is reduced to 1/2 and expression of NRPS3 is not significantly changed.Also,expression of PKS3 coding conidia pigment formation in T23Δcre1 increases by 3 times compared with that in T23,and PKS1 expression is detected being inhibited in T23.Thus,CRE1 indirectly affects the synthesis and secretion of Trichoderma secondary metabolites.In conclusion,carbon catabolite repressor CRE1 regulates mycelia growth,secondary metabolites biosynthesis,which is exactly of great importance in biocontrol procedure of T.atroviride.Those findings are pretty valuable for controlling synthesis and secretion of metabolites of T.atroviride and further to improve its biocontrol effects.