来源于嗜热真菌Talaromyces leycettanus JCM12802的类膨胀素基因鉴定及功能探究  

Identification and Functional Exploration of the Expansin Gene from Thermophilic Talaromyces leycettanus JCM12802

作　　者：顾源 寻子琦 郑菲[1] 涂涛[1] 姚斌[1] 罗会颖[1] GU Yuan;XUN Zi-qi;ZHENG Fei;TU Tao;YAO Bin;LUO Hui-ying(Feed Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081;Soybean Research Institute,Key Laboratory of Soybean Biology in Chinese Ministry of Education,Northeast Agricultural University,Harbin 150030)

机构地区：[1]中国农业科学院饲料研究所,北京100081 [2]东北农业大学国家教育部大豆生物学重点实验室,哈尔滨150030

出　　处：《生物技术通报》2018年第10期172-181,共10页Biotechnology Bulletin

基　　金：中国农业科学院基本科研业务费专项(Y2017JC31)

摘　　要：挖掘新颖的类膨胀素基因,丰富类膨胀素基因资源,探究其功能,加深我们对类膨胀素及其作用机制的认识,促进其工业应用。通过RT-PCR的方法从嗜热真菌Talaromyces leycettanus JCM12802中克隆得到了一个1 196 bp的类膨胀素基因Tlexlx1,并与大多数真菌来源的膨胀素相比,Tl EXLX1缺少一个N端的CBM结构域。Tl EXLX1与来源于Penicilliopsis zonata的假定蛋白ASPZODRAFT_140583具有最高的序列相似性81%,与Penicillium digitatum Pd1来源的Expansin-like protein 1相似性为56%。同时构建野生型Tl EXLX1和含有Tl SWO1的N端CBM区的突变型CBM-Tl EXLX1重组质粒并在毕赤酵母中表达纯化,并对其基本性质进行分析。该基因含有1个内含子(83 bp),编码370个氨基酸和一个终止密码子。Tl EXLX1推导氨基酸序列包括一个22个氨基酸的N端信号肽序列,一个类GH45结构域和一个类膨胀素结构域。结果表明,重组蛋白Tl EXLX1和融合蛋白CBMTl EXLX1具有较高的葡聚糖酶活性(地衣多糖:7.2 U/mg和17.2 U/mg;大麦葡聚糖:4.4 U/mg和9.4 U/mg)和微弱的微晶纤维素水解活性。以地衣多糖为底物时,二者的最适作用温度和pH一致(60℃和6.0)。Tl EXLX1可以破坏微晶纤维素整齐光滑的表面结构,与商业纤维素酶有一定的协同效果。获得新型的类膨胀素蛋白,有一定的水解活性,在木质纤维素降解等工业中存在潜在的应用价值。This study aims to obtain novel expansin-like genes,enrich expansin-like genes resources,explore its function,deepen our understanding of expansin-like and its mechanism of action,and promote its industrial application.In this study,an expansin-like gene Tlexlx1,1 196 bp in length,was cloned from thermophilic Talaromyces leycettanus JCM12802 by RT-PCR.In comparison to most expansin derived from fungi,TlEXLX1 lacks a CBM domain at the N-terminus.Deduced TlEXLX1 has the highest sequence similarities of 81%with the hypothetical protein ASPZODRAFT_140583 from Penicilliopsis zonata and 56%with the expansin-like protein 1 from Penicillium digitatum Pd1.Recombinant plasmids harboring CBM-TlEXLX1 with the CBM domain of TlSWOl at the N-terminus and wild TlEXLX1were constructed,their gene products were expressed in Pichia pastoris,and the basic features of their products were analyzed.This gene contained 1 intron(83 bp)and encodes a polypepetide of 370 amino acids and a stop codon.Deduced TlEXLX1 consisted of a 22-residue signal peptide at the N-terminus,a GH45 domain and an expansin-like domain.The purified recombinant TlEXLX1 and fusion protein CBM-TlEXLX1 had significant glucanase activities of degrading lichenan(7.2 and 17.2 U/mg,and barleyβ-glucan(4.4 and 9.4 U/mg),but weak hydrolytic activity of microcrystalline cellulose.Using lichenan as the substrate,both enzymes showed similar temperature and pH optima(60°C and 6.0).Moreover,TlEXLX1 had capability of destroying the neat and smooth surface structure of microcrystalline cellulose,and exhibited synergistic effect with commercial cellulase.Conclusively,a novel expansin obtained has hydrolytic activity and has potential value in the lignocellulose degradation industry.

关 键 词：类膨胀素 TALAROMYCES leycettanus JCM12802 基因克隆 异源表达 CBM融合蛋白 

分 类 号：Q933[生物学—微生物学]