浙江农业学报 ›› 2021, Vol. 33 ›› Issue (3): 470-478.DOI: 10.3969/j.issn.1004-1524.2021.03.12
赵秀平, 王双, 闫星伊, 段强, 张帅, 陈永胜, 李国瑞*()
收稿日期:
2020-06-19
出版日期:
2021-04-02
发布日期:
2021-03-25
通讯作者:
李国瑞
作者简介:
, 李国瑞, E-mail: degree1982@163.comZHAO Xiuping, WANG Shuang, YAN Xingyi, DUAN Qiang, ZHANG Shuai, CHEN Yongsheng, LI Guorui*()
Received:
2020-06-19
Online:
2021-04-02
Published:
2021-03-25
Contact:
LI Guorui
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。
中图分类号:
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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