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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。
In order to study the function of soluble inorganic pyrophosphatase (s PPase) gene in the growth and development of Polygonum sibiricum, the s PPase gene was cloned, sequenced and prokaryotic expressed. Methods The full-length cDNA sequence of soluble PPase gene Pps PPase was cloned from the cDNA sequence of s PPase selected from c DNA library of Yunnan red reed house. The similarity of this sequence was analyzed by bioinformatics The Pps PPase was inserted into the prokaryotic expression vector pEASY-E1 and introduced into E. coli BL21 (DE3) cells for induction of expression. Results The open reading frame (ORF) of Pps PPase was 621bp, encoding 206 amino acids, and the highest homology with s PPase in sorghum was 94%. The relative molecular mass of Pps PPase encoded protein was 23.4 kDa. The electric point p I is 5.6, and the specific identification region is located between 98 and 104, that is, DNDPMDV. The prokaryotic expression vector pEASY-E1-Pps PPase was successfully constructed and introduced into E. coli BL21 (DE3) cells. SDS-PAGE analysis indicated that Pps PPase was expressed in E.coli. . Conclusion Pps PPase, a soluble inorganic pyrophosphatase gene, was cloned for the first time from Polygonum orientalis, providing a reference for molecular mechanism of growth and development of Polygonum orientalis.