布渣叶查耳酮合酶基因全长cDNA克隆及其表达模式分析  

Cloning and expression pattern analysis of chalcone synthase gene in Microcos paniculata

作　　者：岳春华 张初梅 林爽 黄奕辉 李坤平 YUE Chun-hua;ZHANG Chu-mei;LIN Shuang;HUANG Yi-hui;LI Kun-ping(School of Pharmacy,Guangdong Pharmaceutical University,Guangzhou 510006,China)

机构地区：[1]广东药科大学药学院,广东广州510006

出　　处：《中草药》2018年第17期4118-4124,共7页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金项目(31300273);广东省科技计划项目(2015A030302082)

摘　　要：目的克隆布渣叶查耳酮合酶基因(Mp CHS)全长c DNA,并对其在不同部位和不同生长阶段叶片中的表达模式进行分析。方法以布渣叶叶片总RNA逆转录合成c DNA为模板,根据其转录组数据设计特异引物序列,PCR扩增Mp CHS基因全长c DNA,经质粒连接、转化、扩培,挑选阳性克隆测序、分析并构建原核表达载体。同时,采用实时荧光定量PCR(RT-q PCR)对Mp CHS基因的表达模式进行分析。结果成功克隆得到Mp CHS基因全长c DNA(Gen Bank:KY472608)。生物信息学分析表明其开放阅读框为1 176 bp,编码含391个氨基酸的蛋白,其相对分子质量为42 700,理论等电点6.11,具有CHS家族蛋白3个保守的功能活性位点(165 C、304 H和337 N)和特征多肽标签序列RLMMYQQGCFAGGTVLR和GVLFGFGPGL。系统进化树分析发现Mp CHS与可可、陆地棉等木本植物的亲缘关系比较近。RT-q PCR结果表明,Mp CHS基因在不同部位均有表达,在叶片中的表达量随着生长过程逐步降低。结论首次克隆得到Mp CHS基因,并分析了Mp CHS基因在布渣叶不同部位和不同生长阶段叶片中的表达模式,为Mp CHS基因的原核表达和功能验证奠定了基础,也为进一步解析布渣叶黄酮类生物合成途径提供了参考。Objective To clone the full-length cDNA of Mp CHS and analyze its expression pattern in different parts of Microcos paniculata and different growth periods of M. paniculata leaves. Methods Based on the transcriptome data of M. paniculata, we designed specific primers for Mp CHS gene. The full-length cDNA of Mp CHS was amplified by PCR and the positive clones were then sequenced, analyzed, and constructed prokaryotic expression vector. The bioinformatics analysis of Mp CHS was also performed. Meanwhile, the m RNA expression of Mp CHS was detected using real-time quantitative PCR. Results The relative molecular mass was 42 700, and its theoretical isoelectric point was 6.11, with three conserved functional active sites（165 C, 304 H, and 337 N） of the CHS family proteins and the tag sequence of RLMMYQQGCFAGGTVLR and GVLFGFGPGL. Phylogenetic tree analysis showed that Mp CHS had close relationship with woody plants such as cocoa and upland cotton. We successfully cloned the full-length cDNA of Mp CHS（Gen Bank： KY472608）. It had an ORF of 1 176 bp which encoded a protein of 391 amino acid residues. RT-qPCR results showed that Mp CHS was expressed in all parts of M. paniculata and its expression in leaves was gradually decreased along with its development. Conclusion Mp CHS is cloned from M. paniculate for the first time, and the gene expression pattern of Mp CHS in different parts of M. paniculata and different growth periods of M. paniculata leaves was analyzed. This study facilitates the further purification and functional validation of Mp CHS protein and provides reference for further analysis of flavonoids biosynthesis pathway in M. paniculata.

关 键 词：布渣叶 查耳酮合酶 基因克隆 基因表达模式 原核表达载体 

分 类 号：R282.12[医药卫生—中药学]