《植物生理学报》 2009, 45(9): 869-874

丹参中硫堇基因SmTHI 的克隆和表达分析

曹晓燕, 王喆之*

陕西师范大学药用资源与天然药物化学教育部重点实验室, 西北濒危药材资源开发国家工程实验室, 西安710062

通信作者：王喆之；E-mail: zzwang@snnu.edu.cn；Tel: 029-85310260

摘　要：

丹参 EST 序列的 Blast 分析表明, 一条序列与硫堇(thionin, THI)基因有较高的同源性, 该序列长 575 bp, 包含 1 个长 366 bp 的开放阅读框(ORF), 编码 121 个氨基酸, 命名为 SmTHI, GenBank 登陆号为 DQ212984。在此基础上设计引物, 分别 从 cDNA 和 gDNA 水平上克隆到该基因的全编码区序列的结果表明, 该基因无内含子。序列分析表明, 该编码蛋白与大多 数植物的 THI 蛋白前体高度同源, 并符合植物硫堇类蛋白的序列模式和特征: C-C-X(5)-R-X(2)-[FY]-X(2)-C, N 端具 17 个 氨基酸的信号肽, 中间 46 个氨基酸为成熟 THI 部分, C 端的 58 个氨基酸为酸性多肽部分。成熟的 THI 蛋白带正电荷, 偏碱 性, 推测可能有抗病原微生物活性。实时定量 PCR 检测 SmTHI 在丹参不同组织部位的表达以及在黄瓜细菌性角斑病菌 (PSL)、NaCl 和水杨酸(SA)溶液诱导下的表达结果表明: SmTHI 在植物的根、茎和叶中均有不同程度的表达, 其表达丰度 为叶>茎>根; 在 PSL、NaCl 和 SA 溶液诱导下该基因的表达呈上调趋势。

关键词：丹参; 硫堇; 克隆; 实时定量PCR

收稿：2009-07-01   修定：2009-08-03

资助：国家“ 十一五” 科技支撑计划(2006BAI06A12-04)。

Cloning and Expression of SmTHI from Salvia miltiorrhiza Bge.

CAO Xiao-Yan, WANG Zhe-Zhi*

National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest of China, Key Laboratory of the Ministry of Education for Medicinal Resources and Natural Pharmaceutical Chemistry, Shaanxi Normal University, Xi’an 710062, China

Corresponding author: WANG Zhe-Zhi; E-mail: zzwang@snnu.edu.cn; Tel: 029-85310260

Abstract:

By blasting EST sequences of Salvia miltiorrhiza, one sequence named as SmTHI, was found to have high homology with thionin. The sequence of SmTHI was 575 bp, and contained a 366-bp open reading fram (ORF) encoding a 121-amino acid peptide (GenBank accession number: DQ212984). The full-length sequences were cloned from cDNA and gDNA on the base of the sequence, respectly, and no introns existed in the encoding region. The deduced amino acid sequence of SmTHI protein shared high identity with THI precursor in most plants. The protein precursor of SmTHI possessed a conserved signature sequence of “C-C-X(5)-R-X(2)-[FY]-X(2)-C” and could be divided into three distinct domains, including an amino-terminal (N-terminal) signal peptide, a mature THI, and a carboxy-terminal (C-terminal) acidic domain, composed of 17, 46 and 58 amino acid, respectively. The mature THI had positive charge and showed slightly high basic, which was presumed involving in antimicrobial activity. Real-time quantitative PCR showed that expression of SmTHI is the highest in leaf, followed by in stem, and lowest in root. The expression of SmTHI was obviously increased under the induction of Pseudomonas lachrymans, sodium chloride (NaCl) and salicylic acid (SA).

Key words: Salvia miltorrhiza Bge.; thionin; cloning; real-time quantitative PCR