核盘菌角质酶的克隆、表达及活性分析  

Cloning, expression and activity analysis of cutinase from Sclerotinia sclerotiorum

作　　者：吕蕊花 史琳娜 张喜荣 冯昭 LÜ Ruihua;SHI Linna;ZHANG Xirong;FENG Zhao(College of Medical Technology,Shaanxi University of Chinese Medicine,Xianyang 712046,Shaanxi,China)

机构地区：[1]陕西中医药大学医学技术学院,陕西咸阳712046

出　　处：《生物工程学报》2022年第1期386-395,共10页Chinese Journal of Biotechnology

基　　金：陕西省自然科学基础研究计划(2021JQ-725);陕西中医药大学学科创新团队(2019-YS04);陕西省教育厅一般专项科研计划(21JK0595);陕西中医药大学科学研究计划(2017PY01)。

摘　　要：角质酶可降解脂肪族或芳香族聚酯,对聚对苯二甲酸乙二醇酯也具有较好的降解作用,但目前市面上角质酶产品非常稀缺,因此寻求一种高效表达的角质酶用于工程酶的开发非常有必要。本研究从核盘菌中克隆得到8个角质酶基因,利用PCR结合RT-PCR技术筛选出主效基因SsCut-52,利用大肠杆菌Escherichia coli BL21将SsCut-52进行异源表达并通过镍柱亲和层析的方法分离纯化,研究重组酶的特性及其致病性。SsCut-52全长为768 bp,N端有17个信号肽,进化树分析显示其氨基酸序列和葡萄孢属(Botrytis)角质酶同源性最高,和蜡盘菌属(Rutstroemia)角质酶亲缘关系也较近。SsCut-52在核盘菌侵染植物的过程中高效表达,在菌丝形成菌核的过程中表达量也高于其他角质酶基因。异源表达检测结果显示,角质酶以包涵体的形式存在,复性的SsCut-52在pH 6.0时活性最高,比活力达到3.45 U/mg,在pH为4.0–10.0时活性均较高。最适温度为20–30℃,当温度高于50℃时该酶的残余活性低于60%。植物叶片侵染结果表明,SsCut-52对核盘菌侵染板蓝根叶片具有一定的促进作用。Cutinase can degrade aliphatic and aromatic polyesters, as well as polyethylene terephthalate.Lack of commercially available cutinase calls for development of cost-effective production of efficient cutinase. In this study, eight cutinase genes were cloned from Sclerotinia sclerotiorum. The most active gene SsCut-52 was obtained by PCR combined with RT-PCR, expressed in Escherichia coli BL21 and purified by Ni-NTA affinity chromatography to study its characteristics and pathogenicity. Sscut-52 had a total length of 768 bp and 17 signal peptides at the N terminals. Phylogenetic analysis showed that its amino acid sequence had the highest homology with Botrytis keratinase cutinase and was closely related to Rutstroemia cutinase. Sscut-52 was highly expressed during the process of infecting plants by Sclerotinia sclerotiorum. Moreover, the expression level of Sscut-52 was higher than those of other cutinase genes in the process of sclerotia formation from mycelium. The heterologously expressed cutinase existed in the form of inclusion body. The renatured SsCut-52 was active at pH 4.0–10.0, and mostly active at pH 6.0, with a specific activity of 3.45 U/mg achieved. The optimum temperature of SsCut-52 was 20–30 °C, and less than 60% of the activity could be retained at temperatures higher than 50 °C. Plant leaf infection showed that SsCut-52 may promote the infection of Banlangen leaves by Sclerotinia sclerotiorum.

关 键 词：核盘菌 角质酶 硝基苯丁酸酯 原核表达 蛋白活性 

分 类 号：S432.44[农业科学—植物病理学]