本氏烟Ⅰ型启动子的克隆及其转录起始位点分析

doi:10.13523/j.cb.20140105

中国生物工程杂志

2014, Vol. 34

Issue (1): 28-35 DOI: 10.13523/j.cb.20140105

研究报告

本氏烟Ⅰ型启动子的克隆及其转录起始位点分析

李志英^1,2, 牟红珍², 高丁梅², 丁国平², 马婷², 王盛^1,2

1. 西部特色生物资源保护与利用教育部重点实验室银川 750021;
2. 宁夏大学生命科学学院银川 750021

Cloning and Analysis of the RNA Polymerase i-Promoter of Nicotiana benthaminana

LI Zhi-ying^1,2, MU Hong-zhen², GAO Ding-mei², DING Guo-ping², MA Ting², WANG Sheng^1,2

1. Key Laboratory of Ministry of Education for Protection and Utilization of Special Biological Resources in the Western China, Yinchuan 750021, China;
2. School of Life Science, Ningxia University, Yinchuan 750021, China

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摘要： 启动子是转录水平上一个重要的调控元件，其决定着基因的表达模式和表达强度。Ⅰ型启动子具有高转录活性和种属间特异性等特点。如将其应用于植物RNA病毒载体表达系统，有利于提高表达系统的表达效率和生物安全性。本氏烟（Nicotiana benthaminana）是一种被广泛地应用于植物生物反应器和植物病理学的模式生物，但是现有核酸数据库中尚没有其Ⅰ型启动子的相关信息。因此，克隆本氏烟Ⅰ型启动子并分析其转录起始位点就具有重要的应用价值。通过半巢式PCR获得了514 bp的本氏烟Ⅰ型启动子序列（KC352713）；生物信息学分析初步预测其转录起始位点位于其核心序列TATA（G）TA（N）GGGGG中的第3位A处；通过植物RNA病毒表达载体和5’RACE技术在体内验证本氏烟Ⅰ型启动子转录起始位点与生物信息学预测结果一致。研究结果为深入研究Ⅰ型启动子和构建Ⅰ型启动子介导转录的植物RNA病毒载体表达系统奠定了基础。

关键词： 本氏烟; Ⅰ型启动子; 转录起始位点; 病毒载体

Abstract: Promoter is an important transcriptional regulatory element, which controls the levels and patterns of gene expression. RNA Pol Ⅰ is a cellular enzyme that is abundantly expressed in cells and transcribes rRNA precursor lacking a 5' cap, a 3' poly (A) tail and introns. Thus, viral RNA synthesized in cells transfected with Pol Ⅰ-driven plasmids containing viral genomic cDNA has precise sequences. This may increase the expression level and biological safety of the plant RNA-based expression system. In addition, Nicotiana benthaminana is a kind of model organism which widely used in plant bioreactor and plant pathology. However, there is no nucleic acid sequence data about its RNA Pol Ⅰ promoter for now. Therefore, Nicotiana benthaminana Pol Ⅰ promoter sequence we cloned and analysized its transcription initiation site (TIS). The 514 bp RNA pol Ⅰ promoter sequence (KC352713) was amplified by semi-net PCR form Nicotiana benthaminana and the third A residue in the core sequence TATA (G) TA (N) GGGGG was predicted to be the transcription initiation site (TIS) by comparison of sequences flanking RNA pol Ⅰ promoter TIS in silicon. The predicted result was conformed by the 5' RACE and sequence analysis. This result will be beneficial for the further research of both Pol Ⅰ promoter and Pol Ⅰ-based plant virus expression vectors.

Key words: Nicotiana benthaminana RNA polymerase Ⅰ promoter Transcription initiation site Viral vectors

收稿日期: 2013-10-21 出版日期: 2014-01-25

ZTFLH:

Q781

基金资助: 国家自然科学基金资助项目（31060023）

通讯作者: 王盛，E-mail：wang_s@nxu.edu.cn E-mail: wang_s@nxu.edu.cn

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引用本文:

李志英, 牟红珍, 高丁梅, 丁国平, 马婷, 王盛. 本氏烟Ⅰ型启动子的克隆及其转录起始位点分析[J]. 中国生物工程杂志, 2014, 34(1): 28-35.

LI Zhi-ying, MU Hong-zhen, GAO Ding-mei, DING Guo-ping, MA Ting, WANG Sheng. Cloning and Analysis of the RNA Polymerase i-Promoter of Nicotiana benthaminana. China Biotechnology, 2014, 34(1): 28-35.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140105 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I1/28

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