MYB转录因子调控棘孢木霉Tas653抗杨树叶枯病的调控通路构建
The Construction of Regulatory pathway of MYB36 Transcription Factor from Trichoderma asperellum Tas653 Resisting to Poplar leaf blight
[目的]明确 MYB 转录因子在调控棘孢木霉(Tas653)抗杨树叶枯病菌链格孢菌(Aa1004)中的作用,揭示Tas653 抗Aa1004 的分子机制,为杨树叶枯病防治提供理论指导,从调控层面上揭示生防木霉抗病原真菌的分子机理。[方法]利用真菌转基因的方法,获得MYB36 基因过表达型(Ta-Oe)、敲除型(Ta-Kn)和抑制型(Ta-In)菌株;研究转基因菌株对Aa1004 直接和间接的生防作用;再通过酵母单/双杂交、EMSA、RNA-seq和ChIP等多种实验方法探究MYB36 直接和间接调控的生防基因,构建MYB36 调控Tas653抗杨树叶枯病菌Aa1004 的通路。[结果]通过真菌转基因技术共获得 20 株Ta-Oe,25 株Ta-Kn和 20 株Ta-In,对MYB36 基因定量和PCR检测确定Ta-Oe-8,Ta-In-1 和Ta-Kn-1 为MYB36 的过表达,抑制表达和敲除型菌株。与野生型相比,Ta-Kn菌株对Aa1004 的抑制率下降了 11.06%,Ta-kn菌株的SOD,POD和CAT酶活降低了 82.15 U/g,0.19 OD470/min/g和 1631.2 μmol/min/g且相关基因S50(SOD),C820(CAT)和P120(POD)酶活基因分别下调 3.43,8.1 和 2.43 倍;Ta-Oe菌株不仅对Aa1004 的重寄生性增强,同时发酵液会使其菌丝膨大变形甚至破裂。MYB36 还可以通过结合“ACAT”和“ATCG”元件直接调控转录因子,解毒和防卫基因等。其中,MYB36调控的转录因子中主要包括20个锌指和bZIP转录因子,其中bZIP61.5,Zn37.5,Zn58.0,Zn67.4,Zn83.7 被MYB36 直接调控;调控的解毒基因包括ABC转运蛋白和MFS超家族转运蛋白,其中ABC174.3,MFS47.5,MFS61.5 和MFS53.8 被MYB36 直接调控;调控的防卫基因包括蛋白激酶(PK),8 个细胞色素P450 和 13 个水解酶基因,其中PK50.1,5 个细胞色素P450 和 3 个水解酶基因被MYB36 直接调控。有趣的是,在我们的试验中还发现MYB36 还可以间接调控dUTP、tRNA合成和翻译后修饰等相关基因的表达。[结论]MYB转录因子不仅可以提高生防木霉的重寄生性,抗氧化能力等,还可以通过直接或间接调控锌指等转录因子,MFS 等解毒基因和细胞色素 P450 和水解酶等防卫基因提高棘孢木霉Tas653 对杨树叶枯病菌Aa1004 的防治作用。
[Objective]This study aims to clarify the role of MYB transcription factors in regulating the resistance of T.asperellum(Tas653)to poplar leaf blight(pathogen:Alternaria alternata Aa1004),reveal its molecular mechanism,provide theoretical guidance for disease prevention and control in poplar,and reveal the molecular mechanism of biocontrol Trichoderma against pathogenic fungi at the regulatory level.[Method]The MYB36 overexpression(Ta-Oe),knockout(Ta-Kn),and inhibitory(Ta-In)strains were obtained by using fungal transgenic methods.It measures the disease resistance of the MYB36 transgenic strain to study its direct and indirect biocontrol effects on Aa1004.Through various experimental methods such as yeast one/two hybridization,EMSA,RNA-seq,and ChIP,we explored the biocontrol genes directly and indirectly regulated by MYB36 and constructed a disease resistance pathway through which MYB36 regulates Tas653.[Result]Through fungal transformation technology,20 Ta-Oe,20 Ta-In,25 Ta-Kn,and 20 Ta-Flag strains have been obtained.Quantitative and PCR detection of the MYB36 gene confirmed that Ta-Oe-8,Ta-In-1,and Ta-Kn-1 were overexpressed,inhibited,and knockout strains of MYB36.Compared with the Wt,the inhibition rate of the Ta-Kn strain on Aa1004 decreased by 11.06%,the SOD,CAT,and POD activity decreased by 82.15 U/g,0.19 OD470/min/g,and 1631.2 μmol/min/g,and the enzyme activity-related genes S50(SOD),C820(CAT),and P120(POD)were down-regulated by 3.43,8.1,and 2.43 times,respectively.Ta-Oe strain not only enhances the hyperparasitism of Aa1004,but also its fermentation broth would cause Aa1004 hyphae to swell,deform,or even rupture.MYB36 can also directly regulate transcription factors,detoxify,and defense genes by binding to"ACAT"and"ATCG"elements.Among them,the transcription factors regulated by MYB36 mainly include 20 zinc fingers and bZIP transcription factors,among which bZIP61.5,Zn37.5,Zn58.0,Zn67.4,and Zn83.7 are directly regulated by MYB36;the detoxification genes regulated include ABC transporters and the Major Facilitator Superfamily(MFS),with ABC174.3,MFS47.5,MFS61.5,and MFS53.8 being directly regulated;the regulated defense genes include protein kinase(PK),8 cytochrome P450,and 13 hydrolase genes,with PK50.1,5 cytochrome P450,and 3 hydrolase genes being directly regulated.Interestingly,MYB36 can also regulate dUTP,tRNA synthesis,and post-translational modification genes to regulate expression of genes.[Conclusion]MYB transcription factors can not only enhance the parasitism and antioxidant capacity of biocontrol Trichoderma,but also enhance the control effect of T.asperellum Tas653 on poplar leaf blight pathogen Aa1004 by directly or indirectly regulating transcription factors such as zinc finger,detoxification genes such as MFS,and defense genes such as cytochrome P450 and hydrolases
刘斌;李昱霄;季世达;安一博;韩静;刘志华;
沈阳农业大学林学院 沈阳 110866;沈阳农业大学林学院 沈阳 110866;沈阳农业大学园艺学院 沈阳 110866;国家林业草原国家储备林工程技术研究中心 重庆市林业投资开发有限责任公司 重庆 401120;沈阳农业大学林学院 沈阳 110866;沈阳农业大学林学院 沈阳 110866;
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