摘要
目的克隆青天葵黄酮类药效成分生物合成途径第一步关键酶——查尔酮合成酶(chalcone synthase,CHS)的编码基因,并分析其序列特征及编码蛋白的功能。方法在前期青天葵转录组测序获得的CHS基因序列片段的基础上,采用逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)和cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)从青天葵叶片中获取编码CHS的cDNA全长并克隆其编码区;利用生物信息学方法对其编码蛋白进行同源性比对和功能分析。结果克隆获得的青天葵CHS基因编码区长度为1173 bp,编码391个氨基酸。编码蛋白的氨基酸序列预测显示,青天葵CHS蛋白为酸性、亲水性蛋白,分子量约为96.9 kDa;不含有信号肽、转运肽和跨膜结构域,可能定位于细胞质中;具有CHS保守功能域,归属于CHS超家族,与文心兰等同科植物CHS蛋白的同源性可达87%。结论成功克隆得到青天葵查尔酮合成酶基因,为后续的基因功能鉴定和生物合成调控奠定了基础。
Objective To clone the gene encoding chalcone synthase which is the first-step key enzyme regulating the flavones biosynthesis,from endangered herbaceous Nervilia fordii,and analyze its sequence characteristics and functions of the coding protein. Methods Based on the CHS sequence fragment acquired from previous transcriptome sequencing, the full-length cDNA and coding region of CHS was cloned from N. fordii leaves using reverse transcriptional PCR（RT-PCR） and rapid amplification of cDNA ends（RACE） technologies; and then the phylogenetic analysis and function prediction of the encoding protein were performed by bioinformatics approaches.Results The length of CHS gene cloned from N. fordii was 1173 bp,which encoded 391 amino acids. Sequence analysis and prediction revealed that Nf CHS protein with a molecular weight of 96.9 kDa was acidic and hydrophilic,possessed none signal peptide,transit peptide and transmembrane region,which indicated the protein probably located in cytoplasm. Nf CHS protein contained a chalcone synthase conserved functional domain and was cataloged into chalcone synthase superfamily,shared an identity of 87 % with the CHSs from Orchidaceae species such as Oncidium. Conclusion A CHS encoding gene was successfully cloned from N. fordii,which provided firm foundation for its functional identification of flavones biosynthesis regulation in the plant.
作者
黄琼林
曾湘达
卓一南
何瑞
詹若挺
HUANG Qionglin;ZENG Xiangda;ZHUO Yinan;HE Rui;ZHAN Ruoting(1. Guangdong Medical University, Zhanjiang 524023 Guangdong, China; 2. Research Center of Chinese Medieinal Resource Science and Engineering, Guangzhou University of Chinese Medicine, Guangzhou 510006 Guangdong, China; 3. Key Laboratory of Chinese Medicinal Resource from Lingnan, Ministry of Education, Guangzhou 510006 Guangdong, Chin)
出处
《中药新药与临床药理》
CAS
CSCD
北大核心
2018年第2期205-210,共6页
Traditional Chinese Drug Research and Clinical Pharmacology
基金
广东省自然科学基金博士启动项目(2015A030310519)
广东省普通高校创新团队项目(2016KYTD02)
关键词
查尔酮合成酶
青天葵
克隆
序列分析
Chalcone synthase （CHS）
Nervilia fordii
cloning
sequence analysis
