| 丹参果糖磷酸酶基因的克隆和功能研究 |
| 投稿时间：2012-12-19 修订日期：2013-10-29 点此下载全文 |
| 引用本文：谭何新,刘颖,张磊.丹参果糖磷酸酶基因的克隆和功能研究[J].药学实践杂志,2014,32(1):35~41,48 |
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| 基金项目:国家自然科学基金项目(31160059);上海市科委浦江人才项目(13PJ1411000). |
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| 中文摘要:丹参是著名的药用植物,在中国、日本、美国和欧洲国家被广泛地用于心脑血管系统疾病的治疗。笔者首次从丹参中克隆出了一个新的果糖磷酸酶基因,并将其命名为SmFBA,GenBank编号为FJ540907。SmFBA的cDNA全长含有1 390个核苷酸,包含一个完整的1 065 bp的开放阅读框,编码355个氨基酸残基。SmFBA基因全长含有3个外显子和2个内含子。生物信息学分析显示SmFBA蛋白预测的等电点为5.60,预测的分子量为37.78 ku,和其他植物物种中果糖磷酸酶具有很高的序列同源性。用Southern杂交技术显示SmFBA在丹参基因组中呈低拷贝。该基因在丹参根、茎、叶等器官都表达,根中表达量最高。体外重组表达的SmFBA在大肠杆菌中具有酶活性,并且能够提高大肠杆菌的耐盐性。这项研究进一步拓展了人们对高等植物糖酵解途径的认识。 |
| 中文关键词:丹参 果糖磷酸酶(FBA) cDNA末端快速克隆技术(RACE) 表达特征 耐盐 |
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| Cloning and molecular characterization of fructose-1, 6-bisphosphate aldolase gene from Salvia miltiorrhiza |
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| Abstract:Salvia miltiorrhiza (Bung), a famous medicinal plant, is widely used in China, Japan, America and European countries to treat various conditions, such as cardiovascular and cerebrovascular disease, due to their excellent medicinal values. A novel fructose-bisphosphate aldolase gene (designated as SmFBA, GenBank accession no. FJ540907) was cloned from S. miltiorrhiza for the first time. The full-length cDNA of SmFBA was 1 390 bp with an open reading frame (ORF) of 1 065 bp which encoding a protein of 355 amino acid residues. The deduced protein had isoelectric point (pI) of 5.60 and a calculated molecular weight of about 37.78 ku. The deduced amino acid sequence of SmFBA gene shared high homology and identity with other plant FBAs. The SmFBA genomic DNA sequence was also obtained, revealing SmFBA had three exons and two introns. Southern-blot analysis indicated that SmFBA was a low-copy gene in S. miltiorrhiza genomic. Real-time quantitative PCR analysis showed that SmFBA expressed constitutively in all tested organs, with the highest expression level in roots. In addition, the recombinant SmFBA protein has enzyme activity in E. coli and could improve the high-salinity stress tolerance of E. coli. The successful isolation of the SmFBA gene will be helpful for studying EMP pathway in the near future. |
| keywords:Salvia miltiorrhiza fructose-bisphosphate aldolase (FBA) rapid-amplification of cDNA ends (RACE) express pattern salt-tolerance |
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