稻瘟病菌MGG-01005的表达纯化与生物信息学分析

doi:10.3969/j.issn.1004-1524.2021.03.12

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (3): 470-478.DOI: 10.3969/j.issn.1004-1524.2021.03.12

稻瘟病菌MGG-01005的表达纯化与生物信息学分析

赵秀平, 王双, 闫星伊, 段强, 张帅, 陈永胜, 李国瑞^*()

内蒙古民族大学生命科学与食品学院,内蒙古自治区高校蓖麻产业工程技术研究中心,内蒙古自治区蓖麻育种重点实验室,内蒙古自治区蓖麻产业协同创新中心,蓖麻产业技术创新内蒙古自治区工程研究中心,内蒙古通辽 028043

收稿日期:2020-06-19 出版日期:2021-03-25 发布日期:2021-03-25
作者简介:*, 李国瑞, E-mail: degree1982@163.com
赵秀平(1995—),女,黑龙江齐齐哈尔人,硕士研究生,主要从事分子育种研究。E-mail: 2247632876@qq.com
通讯作者: 李国瑞

Expression, purification and bioinformatics analysis of Magnaporthe oryzae MGG-01005

ZHAO Xiuping, WANG Shuang, YAN Xingyi, DUAN Qiang, ZHANG Shuai, CHEN Yongsheng, LI Guorui^*()

College of Life Science and Food Science, Inner Mongolia University for Nationalities, Inner Mongolia Industrial Engineering Research Center of Universities for Castor, Inner Mongolia Key Laboratory of Castor Breeding, Inner Mongolia Collaborative Innovation Center for Castor Industry, Inner Mongolia Engineering Research Center of Industrial technology Innovation of Castor, Tongliao 028043, China

Received:2020-06-19 Online:2021-03-25 Published:2021-03-25
Contact: LI Guorui

摘要/Abstract

摘要：

MGG-01005是与稻瘟病菌的菌丝生长有重要关系的基因,对其进行一般理化性质、结构域、功能位点预测等一系列生物信息学分析,构建了原核表达载体pETM13-MGG-01005,利用IPTG诱导重组蛋白表达,并通过镍离子亲和层析、阴离子交换层析以及分子筛层析进行蛋白纯化。结果显示,该蛋白相对分子质量约为16 471.49 u,编码153个氨基酸,含Tctex-1结构域,无跨膜结构和信号肽,无功能位点,存在磷酸活性位点,为不稳定亲水蛋白;该蛋白可被0.1 mmol·L ^-1 IPTG诱导表达,亲和层析的最适洗脱液组分为20 mmol·L ^-1 Tris-HCl,500 mmol·L ^-1 NaCl,80 mmol·L ^-1咪唑;阴离子交换层析表明该蛋白对低盐条件具耐受性;分子筛层析具有形态均一且对称性良好的构象,最大洗脱峰出峰位置对应的蛋白相对分子质量约为35 ku,表明该蛋白以二聚体形式存在。本研究最终得到大量高纯蛋白,以期为进一步探索该蛋白的功能以及稻瘟病菌的后续相关研究奠定理论与实践基础。

关键词: 稻瘟病菌, 原核表达, 纯化, 生物信息学分析

Abstract:

MGG-01005 is an important gene associated with mycelia growth of Magnaporthe oryzae.In this study, a series of bioinformatics analysis including general physical and chemical properties, structural domains, and functional site prediction were carried out to construct the prokaryotic expression vector PETM13-MGG-01005. IPTG was used to induce the expression of recombinant protein, and the protein was purified by nickel ion affinity chromatography, anion exchange chromatography and molecular sieve chromatography. The results showed that the molecular weight of the protein was about 16 471.49 u, which encoded 153 amino acids. It contained the domain of Tctex-1, had no transmembrane structure and signal peptide, had no functional site, and had the active site of phosphoric acid, and was an unstable hydrophilic protein. The protein could be induced to express by 0.1 mmol·L ^-1 IPTG. The optimal eluent of affinity chromatography was divided into 20 mmol·L ^-1 Tris-HCl, 500 mmol·L ^-1 NaCl, and 80 mmol·L ^-1 imidazole. Anion exchange chromatography showed that the protein was resistant to low salt conditions. The molecular sieve chromatography has the conformation of uniform morphology and good symmetry, and the molecular weight of the protein corresponding to the maximum elution peak is about 35 ku, indicating that the protein exists in the form of dimer. In this study, a large amount of high purity protein was obtained in order to lay a theoretical and practical foundation for further exploration of the function of this protein and subsequent related studies on Magnaporthe oryzae.

Key words: Magnaporthe oryzae, prokaryotic expression, purification, bioinformatics analysis

中图分类号:

S435.111.4 ⁺1

赵秀平, 王双, 闫星伊, 段强, 张帅, 陈永胜, 李国瑞. 稻瘟病菌MGG-01005的表达纯化与生物信息学分析[J]. 浙江农业学报, 2021, 33(3): 470-478.

ZHAO Xiuping, WANG Shuang, YAN Xingyi, DUAN Qiang, ZHANG Shuai, CHEN Yongsheng, LI Guorui. Expression, purification and bioinformatics analysis of Magnaporthe oryzae MGG-01005[J]. Acta Agriculturae Zhejiangensis, 2021, 33(3): 470-478.

图/表 10

图1 蛋白亲疏水性分析

Fig.1 Analysis of Protein Hydrophilicity and Hydrophobicity

图2 蛋白质保守结构域预测

Fig.2 Protein conserved domain mapping

图3 蛋白质的跨膜结构

Fig.3 Protein transmembrane structure diagram

图4 蛋白磷酸化位点预测

Fig.4 Prediction of protein phosphorylation sites

图5 蛋白二级结构预测

Fig.5 Protein secondary structure prediction

图6 蛋白三级结构预测

Fig.6 Protein tertiary structure prediction

图7 系统进化树

Fig.7 Phylogenetic tree

图8 pETM13-MGG-01005 亲合层析的SDS-PAGE检测 20~300依次表示洗脱液中咪唑浓度(20、40、60、80、100、200、300 mmol·L-1)。

Fig.8 Affinity chromatography (Ni-NTA) and SDS-PAGE analysis of pETM13-MGG-01005 20-300 represented the concentration of imidazole in the elution buffer(20, 40, 60, 80, 100, 200, 300 mmol·L-1), respectively.

图9 pETM13-MGG-01005离子交换层析(Resource Q)及SDS-PAGE检测 M,蛋白分子量标准;A-C,脱盐A-C液;X1-X2,穿出;A1-A4,洗脱液。

Fig.9 Ion-exchange column chromatography (Resource Q) and SDS-PAGE analysis of pETM13-MGG-01005 M, Protein marker; A-C, The liquid of desalt A-C; X1-X2, Protein exudates; A1-A4, Eluents.

图10 pETM13-MGG-01005分子筛分析(Superdex 75 10/300 GL)及SDS-PAGE检测 M,蛋白分子量标准;A1-A4,洗脱液。

Fig.10 Gel filtration (Superdex 75 10/300 GL) and SDS-PAGE analysis of pETM13-MGG-01005 M, Protein marker; A1-A4, Eluents.

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稻瘟病菌MGG-01005的表达纯化与生物信息学分析

Expression, purification and bioinformatics analysis of Magnaporthe oryzae MGG-01005

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