中国水仙<i>NtCCHC</i>锌指蛋白基因的克隆与原核表达

邹晖; 李海明; 王伟英; 林江波

doi:10.19303/j.issn.1008-0384.2019.08.004

中国水仙NtCCHC锌指蛋白基因的克隆与原核表达

doi: 10.19303/j.issn.1008-0384.2019.08.004

福建省农业科学院亚热带农业研究所, 福建漳州 363005

基金项目:

福建省财政专项——福建省农业科学院科技创新团队建设项目 STIT2017-2-11

详细信息

作者简介:
邹晖(1982—), 男, 研究实习员, 主要从事园艺作物研究(E-mail:21880338@qq.com)

通讯作者:
林江波(1976—), 男, 硕士, 副研究员, 主要从事农业生物技术研究(E-mail:linjianbo1976@yahoo.com.cn)

中图分类号: S682
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- 被引次数: 0
出版历程
- 收稿日期: 2019-03-07
- 修回日期: 2019-07-18
- 刊出日期: 2019-08-01

Cloning and Prokaryotic Expression of NtCCHC in Narcissus tazetta var. chinensis

Subtropical Agriculture Research Institute, Fujian Academy of Agricultural Sciences, Zhangzhou, Fujian 363005, China

摘要

摘要: 目的利用建立的多效唑诱导中国水仙的SSH文库，克隆中国水仙NtCCHC锌指基因的cDNA序列，进行原核表达，为深入研究NtCCHC锌指基因在中国水仙中的功能奠定基础。方法采用Trizol法提取多效唑处理后的中国水仙叶片总RNA，反转录成cDNA后，根据已知的CCHC锌指蛋白基因序列设计引物进行基因克隆和同源性分析，并构建原核表达载体进行诱导表达。结果克隆一段810bp的cDNA编码区序列，编码269个氨基酸，同源性分析表明与多个物种的CCHC基因存在着较高的同源性，命名为NtCCHC。该基因能成功在pGEX-4T-3原核表达载体上实现诱导表达，系统进化树分析表明：NtCCHC与中国莲遗传距离最近。结论克隆了中国水仙NtCCHC基因序列，并成功诱导表达。
- 中国水仙 /
- 锌指蛋白 /
- 基因克隆 /
- 蛋白表达
Abstract: Objective Based on the information in the SSH Library on Narcissus tazetta var.chinensis, the zinc finger gene was cloned and prokaryotic expressed for future studies. Method Total RNA of N.tazetta var. chinensis from the paclobutrazol-treated leaves was extracted using the Trizol method. After reverse transcription into cDNA, gene cloning and homology analysis were carried out on it with primers designed according to the known sequence of CCHC. A prokaryotic expression vector was constructed to induce expression. Result The 810 bp sequence encoding 269 amino acids was cloned. The cloned gene showed high homologies with the CCHC genes of many species and was named NtCCHC, which could be successfully induced and expressed in pGEX-4T-3. The phylogenetic tree constructed by MEGA 6.0 indicated that the closest genetic distance of NtCCHC lied with Nelunbo nucifera. Conclusion This study successfully cloned and expressed the NtCCHC gene sequence of N. chinensis.
- Narcissus tazetta var.chinensis /
- zinc finger protein /
- gene cloning /
- protein expression

HTML全文

图 1 中国水仙NtCCHC PCR电泳检测

注：M为Marker；1、2为NtCCHC基因。

Figure 1. PCR electrophoresis of NtCCHC in N.tazetta var.

Note:M:Marker; 1, 2:NtCCHC.

下载: 全尺寸图片幻灯片

图 2 NtCCHC基因核苷酸序列及推导的氨基酸序列

Figure 2. DNA and deduced amino acid sequences of NtCCHC

下载: 全尺寸图片幻灯片

图 3 中国水仙NtCCHC基因的系统进化树的构建

Figure 3. Construction of phylogenetic tree for NtCCHC in N.tazetta var.

下载: 全尺寸图片幻灯片

图 4 pGEX-NtCCHC的酶切鉴定

注：M为Marker; 1为酶切pGEX-NtCCHC。

Figure 4. Enzyme digestion identification of pGEX-NtCCHC

Note:M:Marker; 1:restriction enzyme pGEX-NtCCHC

下载: 全尺寸图片幻灯片

图 5 NtCCHC基因诱导表达蛋白SDS-PAGE鉴定

注：泳道M为标准蛋白分子量; 泳道1为诱导前；2为诱导后。

Figure 5. SDS-PAGE identification of NtCCHC-induced expression protein

Note: M:protein marker; 1:Before induction; 2:After induction.

下载: 全尺寸图片幻灯片

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