茶树CsC7蛋白异源表达及抗真菌活性分析

Analysis of Heterologous Expression and Antifungal Activity of Tea Plant Chitinase CsC7 Protein

几丁质酶是一种重要的植物病程相关蛋白,本实验室前期研究发现,不同抗性的茶树品种在接种茶饼病病原菌(坏损外担菌)后,茶树几丁质酶CsC7相对表达量显著提高。本研究在此基础上,以“梅占”为材料,同源克隆得到茶树几丁质酶CsC7基因序列,通过生物信息学分析对CsC7蛋白功能进行初步研究。进一步通过载体构建将CsC7转入毕赤酵母X-33表达蛋白,利用平板抑菌法对脆弱毛霉菌、白桦茸担子菌、尖孢镰刀菌、赤星菌、灰霉菌、厚孢镰刀菌、枯叶格孢腔菌等7种真菌进行抗菌活性分析。结果表明,纯化后的酵母异源表达蛋白CsC7对脆弱毛霉菌、白桦茸担子菌、厚孢镰刀菌、尖孢镰刀菌、灰霉菌有明显抑制活性,抑菌率分别为27.07%,21.90%,28.45%,39.21%和32.23%,而对赤星菌和枯叶格孢腔菌没有显著抑制作用。

Chitinase is an important plant-pathogen related protein.Preliminary research in the laboratory found that the relative expression level of tea plant chitinase CsC7 significantly increased when different resistant tea varieties were inoculated with the pathogenic fungus of tea cake disease(Exobasidium vexans).Based on this,the homologous sequence of tea plant chitinase CsC7 gene was cloned from the'Meizhan'material and preliminary research on the function of CsC7 protein through bioinformatics analysis was conducted.Furthermore,CsC7 was transferred into Pichia pastoris X-33 expression express proteins through vector construction,and the antibacterial activity of purified yeast heterologous expressed protein CsC7 against seven fungi,including Mucor fragilis,Inonotus obliquus,Fusarium oxysporum,Alternaria alternata,Botrytis cinerea,Fusarium chlamydosporum,and Pleospora herbarum,was analyzed by plate inhibition assay.The results showed that purified yeast heterologous expressed protein CsC7 had significant inhibitory activities against Mucor fragilis,Inonotus obliquus,Fusarium chlamydosporum,Fusarium oxysporum,and Botrytis cinerea,with inhibition rates of 27.07%,21.90%,28.45%,39.21%and 32.23%,respectively,while there was no significant inhibition effect on Alternaria alternata and Pleospora herbarum.