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茶叶科学 ›› 2012, Vol. 32 ›› Issue (3): 269-275.doi: 10.13305/j.cnki.jts.2012.03.012

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茶树PPO融合蛋白真核表达载体的构建与表达

王乃栋1,2, 张丽霞1,2*, 黄晓琴1,2, 韩晓阳1,2, 李智1,2   

  1. 1. 山东农业大学园艺科学与工程学院,山东 泰安 271018;
    2. 作物生物学国家重点实验室,山东 泰安 271018
  • 收稿日期:2011-11-15 修回日期:2012-02-06 出版日期:2012-06-15 发布日期:2019-09-05
  • 作者简介:王乃栋(1984– ),男,山东泰安人,博士,主要从事茶树生物技术研究工作。
  • 基金资助:
    高等学校博士学科点专项科研基金项目(20103702110002)、山东省自然科学基金面上项目(ZR2010CM026)

Construction and Expression of Fusion Protein Eukaryotic Expression Vector of Polyphenol Oxidase Gene of Tea Plant (Camellia sinensis)

WANG Nai-dong1,2, ZHANG Li-xia1,2*, HUANG Xiao-qin1,2, HAN Xiao-yang1,2, LI Zhi1,2   

  1. 1. College of horticulture science and engineering Shandong Agriculture University, Taian 271018, China;
    2. State key laboratory of crop biology, Taian 271018, China
  • Received:2011-11-15 Revised:2012-02-06 Online:2012-06-15 Published:2019-09-05

摘要: 以迎霜品种茶树基因组为模板,利用高保真聚合酶pfu,通过PCR方法扩增得到茶树PPO基因的编码区序列。通过设计酶切引物将其成功融合至真核表达载体pPICZa中,构建出茶多酚氧化酶的融合蛋白真核表达载体pPICZa-PPO,并将其转化进入巴斯德毕赤酵母(Pichia pastoris)GS115中,成功筛选出多个阳性转化子。对阳性转化子进行甲醇诱导,采用Western-Blotting方法在培养液中成功检测到诱导表达的目的蛋白。酶活检测结果也显示诱导产物具有较高的相对酶活性,能够正确发挥酶蛋白的生物功能。

关键词: 茶树, 多酚氧化酶, 融合蛋白真核表达载体, 巴斯德毕赤酵母, 诱导表达

Abstract: The polyphenol oxidase (PPO) cds sequence was amplified by PCR from the genome of Yingshuang tea plant with the high fidelity polymerase pfu. The cds sequence was recombinanted into the eukaryotic expression vector pPICZa with the designed primer, and the fusion protein eukaryotic expression vector of tea polyphenol oxidase “pPICZa-PPO” was succeeded to be constructed, and then it was transformed into Pichia pastoris GS115. Therefore, several positive converters were screened. After those positive converters were induced by methanol,the target protein was successfully detected in the culture medium by using Western-Blotting method. The detection result of enzyme activity showed that the induced products had a relatively high enzyme activity and can correctly play their biological activity.

Key words: Camellia sinensis, PPO, fusion protein eukaryotic expression vector, Pichia pastoris, inducible expression

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