茶树黄金芽CsHIPP26.1蛋白螯合离子的筛选与鉴定

doi:10.13305/j.cnki.jts.2022.02.006

茶叶科学 ›› 2022, Vol. 42 ›› Issue (2): 179-186.doi: 10.13305/j.cnki.jts.2022.02.006

茶树黄金芽CsHIPP26.1蛋白螯合离子的筛选与鉴定

刘富浩^1,2, 范延艮^1,2, 王域^1,2, 孟凡月^1,2, 张丽霞^1,2,*

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:2021-12-06 修回日期:2022-02-18 出版日期:2022-04-15 发布日期:2022-04-15
通讯作者: * lxzhang@sdau.edu.cn
作者简介:刘富浩,男,硕士研究生,主要从事茶树分子生物学研究。
基金资助:
山东省“双一流”奖补资金项目（SYL2017YY03）、山东省现代农业产业技术体系创新团队项目（SDAIT-19-05）、鲁渝科技协作计划项目（2020LYXZ005）

Screening and Identification of Chaperone CsHIPP26.1 Chelating Ions in Tea Cultivar ‘Huangjinya’

LIU Fuhao^1,2, FAN Yangen^1,2, WANG Yu^1,2, MENG Fanyue^1,2, ZHANG Lixia^1,2,*

1. College of Horticulture Science and Engineering, Tai'an 271018, China;
2. State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an 271018, China

Received:2021-12-06 Revised:2022-02-18 Online:2022-04-15 Published:2022-04-15

摘要/Abstract

摘要： 重金属相关异戊二烯化植物蛋白（HIPPs）由于其独特的重金属结合域和异戊二烯序列的结构特点,成为一类重要的金属分子伴侣。为鉴定茶树（Camellia sinensis）黄金芽CsHIPP26.1蛋白的螯合离子,将pET-32a-CsHIPP26.1重组质粒和空载体分别转入大肠杆菌BL21,在分别添加4 mol·L^-1的单一金属离子（CuCl₂、ZnCl₂、MgCl₂、FeCl₃、CaCl₂）或5种金属离子混合液以及1 mmol·L^-1 IPTG诱导的LB液体中培养,观测大肠杆菌的生长情况,并用His-tag蛋白纯化磁珠纯化获得融合目的蛋白,经原子吸收分光光度计分析融合蛋白中金属离子含量,计算蛋白螯合的离子数目。结果表明,CsHIPP26.1蛋白仅与Zn²⁺和Cu²⁺螯合,且螯合Zn²⁺的能力显著强于Cu²⁺。根据其结合金属离子与目的蛋白质量摩尔比推测,CsHIPP26.1蛋白螯合Zn²⁺、Cu²⁺金属离子的最大数目分别为2和1。

关键词: 重金属异戊二烯植物蛋白, CsHIPP26.1, 金属离子, 茶树

Abstract: Heavy metal-associated isoprenylated plant proteins (HIPPs) is an important metallochaperones due to its unique heavy metal binding domains (HMA) and the structural characteristics of isoprenylation motif. In order to identify the chelating ions of CsHIPP26.1 protein in Camellia sinensis (L.) cv. ‘Huangjinya’, the pET-32a-CsHIPP26.1 recombinant plasmids and empty carriers were respectively transferred into E. coli BL21, and then were cultured in LB liquid culture medium with 4 mol·L^-1 single metal ions (CuCl₂, ZnCl₂, MgCl₂, FeCl₃, CaCl₂) or multiple metal ions and 1 mmol·L^-1 IPTG. The growth of E. coli in different ion media was observed, meanwhile the fusion target protein was obtained by His-tag protein purification magnetic bead. The contents of metal ions in fusion protein were analyzed by atomic absorption spectrophotometer, and the number of ions chelated by the protein was calculated. The results show that CsHIPP26.1 protein was only chelated with Zn²⁺ and Cu²⁺, and the chelating ability to Zn²⁺ was significantly higher than Cu²⁺. Based on the molar ratio of its bound metal ions to the target protein, the maximum number of Zn²⁺, Cu²⁺ chelated by CsHIPP26.1 protein was 2 and 1, respectively.

Key words: heavy metal-associated isoprenylated plant protein, CsHIPP26.1, metal ions, Camellia sinensis

中图分类号:

S571.1

刘富浩, 范延艮, 王域, 孟凡月, 张丽霞. 茶树黄金芽CsHIPP26.1蛋白螯合离子的筛选与鉴定[J]. 茶叶科学, 2022, 42(2): 179-186. doi: 10.13305/j.cnki.jts.2022.02.006.

LIU Fuhao, FAN Yangen, WANG Yu, MENG Fanyue, ZHANG Lixia. Screening and Identification of Chaperone CsHIPP26.1 Chelating Ions in Tea Cultivar ‘Huangjinya’[J]. Journal of Tea Science, 2022, 42(2): 179-186. doi: 10.13305/j.cnki.jts.2022.02.006.

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茶树黄金芽CsHIPP26.1蛋白螯合离子的筛选与鉴定

Screening and Identification of Chaperone CsHIPP26.1 Chelating Ions in Tea Cultivar ‘Huangjinya’

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