摘要
【目的】对玉米大斑病菌(Setosphaeria turcica)漆酶基因Stlac2进行生物信息学分析,推测其蛋白功能,探究木质素降解过程中产生的小分子物质对Stlac2表达的影响,并对其进行克隆及原核表达,以便深入研究该基因在病菌生长、发育及致病过程中的作用。【方法】通过NCBI查询获得玉米大斑病菌EOA90070(Stlac2)基因的全序列,通过Clustal X与马尔尼菲青霉菌(Penicillium marneffei)PbrB(PMAA_082060)基因、烟曲霉(Aspergillus fumigatus)Abr2(AFUA_2G17530)基因、构巢曲菌(Aspergillus nidulans)YA(AN6635)基因等漆酶家族基因进行蛋白序列比对,查看Stlac2中的铜离子结合位点,利用在线软件SOPMA和ProtParam对其二级结构及理化性质进行检测,通过SWISS-MODEL对Stlac2蛋白质的三维结构进行预测,采用SAVES对三维结构进行评价。通过对ABTS的氧化试验确定木质素降解过程中产生的小分子物质对玉米大斑病菌漆酶产量的影响;利用RT-qPCR方法分析小分子物质对Stlac2表达规律的影响;采用原核表达系统,以pET-30a表达载体为框架,对其进行原核表达,并将表达蛋白纯化,以ABTS为底物,420 nm下检测漆酶活性。【结果】Stlac2具有典型的Cu离子结合位点,与漆酶典型的Cu离子结构域相似。其蛋白二级结构中α-螺旋、延伸链、β-转角、无规则卷曲所占比例分别为18.59%、25.63%、11.91%和43.86%,分子量为61.64 k D,等电点为5.00,平均疏水性为-0.37,表明其为亲水蛋白。当在PDA中添加0.01 g·L^(-1)香草醛、4-羟基苯甲醛、丁香醛、香草酸、4-羟基苯甲酸和香兰素时,玉米大斑病菌胞外漆酶的产量为香草酸>香兰素>4-羟基苯甲醛>4-羟基苯甲酸>丁香醛>对照。而在4-羟基苯甲醛和4-羟基苯甲酸存在时,玉米大斑病菌Stlac2的相对表达量明显升高,约为对照的5—8倍。利用原核表达系统,在67 k D处成功诱导表达出一条特异性蛋白条带,大量表达纯化后,漆酶活性为(40.7±0.3)U·L^(-1)。【结论】阐明了Stlac2的生物信息学特性,初步断定其为漆酶基因;4-羟基苯甲醛和4-羟基苯甲酸可显著提高Stlac2相对表达量,表明其在木质素降解过程中起到了一定的作用;该基因所编码的蛋白可通过pET-30a原核表达系统表达,体外漆酶活性可达(40.7±0.3)U·L^(-1),该研究结果为后期研究蛋白性质打下了基础。
【Objective】The objective of this study is to analyze the bioinformatics and infer the functions of Stlac2 in Setosphaeria turcica, research on the effects of substrates generated in the process of lignin degradation on the relative expression of Stlac2. For further study on the function of Stlac2 in the growth, development, and pathogenicity, the Stlac2 was successfully expressed by prokaryotic expression system. 【Method】The protein sequences of Stlac2 were obtained through NCBI and aligned with the known laccases such as Penicillium marneffei PbrB（PMAA_082060）, Aspergillus fumigatus Abr2（AFUA_2G17530）, and Aspergillus nidulans YA（AN6635） by Clustal X for the Cu ion binding sites. The secondary structure and biochemical properties were predicted online by online softwares SOPMA and Prot Param, and the three-dimensional structure was modelled by SWISS-MODEL, and analyzed by SAVES. The effects of lignin degradation substrates on the expression of extracellular laccase were measured by oxidazing ABTS and the effect on the relative expression of specific gene Stlac2 was analyzed by using RT-qPCR. Stlac2 was fused into the pET-30 a plasmid and expressed by prokaryotic expression system. The protein Stlac2 expressed by prokaryotic cell was extracted and the laccase activity was detected using ABTS as substrate at 420 nm.【Result】 Stlac2 had typical sites bonding with Cu ion. The proportion of α-helix, extended strand, β-turn and random coil were 18.59%, 25.63%, 11.91% and 43.86%, respectively. The protein molecular weight is 61.64 k D, pI is 5.00, grand average of hydropathicity is-0.37, indicating Stlac2 is a hydrophilic protein. The effect of lignin degradation substrates on the production of extracellular laccase was vanillic acid 〉 vanillin 〉 4-hydroxybenzaidehyde〉4-hydroxybenzoic acid〉syringaldehyde〉CK. The relative expression of Stlac2 was increased about 5-8 folds compared with CK under the substrates 4-hydroxybenzoic acid and 4-hydroxybenzaidehyde conditions. Stlac2 protein was successfully expressed by prokaryotic expression system and a specific protein band of 67 k D was induced. The laccase activity of this specific protein is（40.7±0.3） U·L^-1.【Conclusion】The biochemical properties of Stlac2 were analyzed systematically and predicted that Stlac2 is a laccase gene. 4-Hydroxybenzoic acid and 4-hydroxybenzaidehyde could increase the relative expression of Stlac2 significantly, indicating it plays a role in the process of lignin degradation. Stlac2 protein can be expressed by the prokaryotic expression systems of pET-30 a and the laccase activity is（40.7±0.3） U·L^-1, thus laid a foundation for further study.
出处
《中国农业科学》
CAS
CSCD
北大核心
2016年第21期4130-4139,共10页
Scientia Agricultura Sinica
基金
国家自然科学基金(31101402)
国家玉米产业技术体系(CARS-02-12)
河北省高等学校科学技术研究项目(ZD2014053
QN2014091)
