滇重楼可溶性无机焦磷酸酶基因的克隆及原核表达研究

发布时间：2019-02-24 09:00

【摘要】：目的为了研究可溶性无机焦磷酸酶(soluble inorganic pyrophosphatase,s PPase)基因在滇重楼生长发育中的功能,对滇重楼的s PPase基因进行了克隆、序列分析和原核表达研究。方法根据滇重楼c DNA文库中筛选到的s PPase的EST序列,克隆了滇重楼可溶性无机焦磷酸酶基因Pps PPase的c DNA全长序列;运用生物信息学的方法对该序列进行相似性比较和同源性分析,预测编码蛋白,并对其进行各种理化性质分析;将Pps PPase插入到原核表达载体p EASY-E1上,并导入到大肠杆菌BL21(DE3)细胞中进行诱导表达。结果滇重楼中Pps PPase的开放阅读框ORF为621bp,编码206个氨基酸,与高粱中s PPase的氨基酸序列相似性最高为94%;Pps PPase编码的蛋白相对分子质量是23.4k Da,理论等电点p I为5.6,特异识别区域即位于98-104位之间即DNDPMDV。成功构建了原核表达载体p EASY-E1-Pps PPase,并导入到大肠杆菌BL21(DE3)细胞中,经SDS-PAGE分析表明Pps PPase在大肠杆菌中能够表达,当IPTG诱导2h时表达量较高。结论首次从滇重楼中克隆得到可溶性无机焦磷酸酶基因Pps PPase,为滇重楼生长发育分子机制的研究提供参考。
[Abstract]:Objective to study the function of soluble inorganic pyrophosphatase (soluble inorganic pyrophosphatase,s PPase) gene in the growth and development of Polygonum yunnanensis, s PPase gene was cloned, sequenced and prokaryotic expression was studied. Methods according to the EST sequence of s PPase selected from c DNA library, the full length of c DNA sequence of soluble inorganic pyrophosphatase gene Pps PPase was cloned. Bioinformatics was used to compare the similarity and homology of the sequence, to predict the encoded protein, and to analyze its physical and chemical properties. The Pps PPase was inserted into the prokaryotic expression vector p EASY-E1 and induced into BL21 (DE3) cells. Results the open reading frame (ORF) of Pps PPase was 621 BP, encoding 206 amino acids, with the highest similarity to s PPase in sorghum. The relative molecular weight of the protein encoded by Pps PPase is 23.4k Da,. The theoretical isoelectric point Pi is 5.6. The specific recognition region is located between the 98-104 positions, that is, DNDPMDV.. A prokaryotic expression vector, p EASY-E1-Pps PPase, was successfully constructed and introduced into Escherichia coli BL21 (DE3) cells. SDS-PAGE analysis showed that Pps PPase could be expressed in E. coli, and the expression level was higher when IPTG was induced for 2 h. Conclusion the soluble inorganic pyrophosphatase (Pps PPase,) gene was cloned for the first time from Rhizoma Dianophora, which provides a reference for the study of molecular mechanism of growth and development of Rhizoma Dianophora.
【作者单位】：云南中医学院中药学院;
【基金】：国家自然科学基金(No.81160502) 云南省自然科学基金(No.2011FZ153)
【分类号】：S567.239;Q943.2

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