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

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

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。

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.

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