砀山酥梨多酚氧化酶基因(PbPPO)的原核表达及优化研究

doi:10.3969/j.issn.1009-7791.2017.03.002

›› 2017, Vol. 46 ›› Issue (03): 209-214.DOI: 10.3969/j.issn.1009-7791.2017.03.002

• 植物生理生化与分子生物学 • 上一篇下一篇

砀山酥梨多酚氧化酶基因(PbPPO)的原核表达及优化研究

常艳,刘晓峰,金青,蔡永萍,聂凡,林毅

(1.安徽农业大学生命科学学院，安徽合肥230036；2.安徽省农业科学院园艺研究所，安徽合肥 230031)

收稿日期:2017-06-28 修回日期:2017-08-02 出版日期:2017-09-30 发布日期:2017-09-30
通讯作者: 蔡永萍
作者简介:常艳，硕士，实验师，从事植物分子生物学研究。
基金资助:
国家自然科学基金(No. 31640068)

Prokaryotic Expression and Its Optimization of Polyphenol Oxidase Gene (PbPPO) in Pyrus bretschneideri ‘Dangshan Su’

CHANG Yan,LIU Xiao-feng,JIN Qing,CAI Yong-ping,NIE Fan,LIN Yi

(1.School of Life Sciences, Anhui Agricultural University, Hefei 230036, Anhui China; 2.Horticultural Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, Anhui China)

Received:2017-06-28 Revised:2017-08-02 Online:2017-09-30 Published:2017-09-30
Contact: CAI Yong-ping

摘要/Abstract

摘要： 从砀山酥梨Pyrus bretschneideri ‘Dangshan Su’果实中克隆得到一条多酚氧化酶基因(PbPPO)的全长cDNA序列(GenBank登录号：JF809859)。通过生物信息学分析表明，PbPPO基因CDS区全长1782 bp，编码593个氨基酸，氨基酸序列由N端叶绿体转运肽、Cu结合区与C端疏水区三部分组成。为进一步研究PbPPO基因的功能，成功构建其原核表达载体pET-28a-PbPPO，并在大肠杆菌Rosetta菌株中成功诱导表达，经优化后显示，在28 ℃、1.0 mmol·L-1 IPTG诱导5 h的条件下，融合蛋白表达量最高。

关键词: 厚砀山酥梨, 木质素, 石细胞, 多酚氧化酶, 原核表达

Abstract: This study cloned the full-length cDNA sequence of a polyphenol oxidase gene (PbPPO) from Dangshan Su pear. The bioinformatics analysis showed that the full-length of PbPPO gene was 1782 bp, encoding 593 amino acids. The amino acid sequence consisted of three parts: N terminal chloroplast transport peptide, Cu binding region and C-terminal hydrophobic region. In order to further study the function of PbPPO gene, the prokaryotic expression vector pET-28a-PbPPO was successfully constructed, and the fusion protein was successfully expressed in Escherichia coli Rosetta. After optimization, it was proved that the fusion protein had the highest expression at 28 ℃ and 1.0 mmol·L-1 IPTG for 5 h. This study not only created the conditions for the study of PbPPO enzymatic properties, but also provided the scientific basis for the molecular regulation of stone cell content and the improvement of Dangshan Su pear quality.

Key words: Pyrus bretschneideri ‘Dangshan Su’, lignin, stone cells, PPO, prokaryotic expression

中图分类号:

S661.2

常艳,刘晓峰,金青,蔡永萍,聂凡,林毅. 砀山酥梨多酚氧化酶基因(PbPPO)的原核表达及优化研究[J]. , 2017, 46(03): 209-214.

CHANG Yan,LIU Xiao-feng,JIN Qing,CAI Yong-ping,NIE Fan,LIN Yi. Prokaryotic Expression and Its Optimization of Polyphenol Oxidase Gene (PbPPO) in Pyrus bretschneideri ‘Dangshan Su’[J]. , 2017, 46(03): 209-214.

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砀山酥梨多酚氧化酶基因(PbPPO)的原核表达及优化研究

Prokaryotic Expression and Its Optimization of Polyphenol Oxidase Gene (PbPPO) in Pyrus bretschneideri ‘Dangshan Su’

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