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[摘要]
目的 克隆鱼腥草查耳酮合成酶1(CHS1)基因并分析其蛋白质序列。方法 根据已经克隆的植物CHS基因的保守序列设计一对引物,以鱼腥草总RNA为模板,采用RT-PCR和SON-PCR的方法快速获得CHS基因序列并连接到pMD18-T Simple载体上,阳性克隆经PCR检测后进行测序。结果 得到一段1 188 bp的序列,序列分析表明,该片段编码395个氨基酸,与其他高等植物CHS基因氨基酸序列同源性在62.3%以上,生物信息学分析表明,该蛋白含有CHS家族的特征多肽序列“RLMMYQQGCFAGGTVLR”和“GVLFGFGPGL”,不含信号肽序列。相对荧光定量PCR分析表明,CHS1基因在花中的表达量最高,其次是地上茎,再次是地下茎,叶片中表达量最低。结论 首次从鱼腥草中克隆了CHS1基因,为有效利用该基因奠定了基础。
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[Abstract]
Objective To clone and sequence the chalcone synthase1 (CHS1) gene from the leaves of Houttuynia cordata. Methods The cloning primers were designed on the basis of the conserved sequences of the cloned CHS gene in other plants. The total RNA was extracted from the leaves of H. cordata. The sequence of CHS gene was cloned by reverse transcription polymerase chain reaction (RT-PCR) and SON-PCR techiniques, and then the gene was ligated with pMD18-T Simple vector. The positive clone was sequenced after the identification of clone by PCR. Results We cloned a fragment of 1 188 bp. The analysis of sequencing indicated that the fragment encoded 395 amino acids and shared the sequence homology of more than 62.3% with CHS gene sequences from other higher plants. Bioinformatic analysis indicated that the CHS amino acids had no signal peptide sequences, but possessed the CHS family’s characteristic sequences, RLMMYQQGCFAGGTVLR and GVLFGFGPGL. Relative real-time PCR analysis indicated that CHS1 showed the highest transcript abundance in the flowers, moderate levels in the stems, lower levels in the rhizomes, and the lowest levels in the leaves. Conclusion It is the first report that a novel CHS gene is cloned from H. cordata, which lays a foundation for the effective use of CHS1 gene.
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[基金项目]
国家自然科学基金资助项目(30870230);湖南省高校创新平台开放基金项目(12K132);湖南省科技计划重点项目(2013FJ6090)