人参查尔酮合成酶基因PgCHS1的克隆与表达分析  

Cloning and Expression Analysis of Chalcone Synthase Gene PgCHS1 in Panax ginseng

作　　者：张变玲[1] 黄雪梅 刘心怡 谢彪 黄合庆 张儒[1] Zhang Bianling;Huang Xuemei;Liu Xinyi;Xie Biao;Huang Heqing;Zhang Ru(College of Chemistry and Chemical Engineering,Hunan Institute of Engineering,Xiangtan 411104,China)

机构地区：[1]湖南工程学院化学化工学院,湘潭411104

出　　处：《中国细胞生物学学报》2018年第12期2010-2017,共8页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:81874332);湖南省自然科学基金(批准号:2017JJ3048;2016JJ2037);湖南省教育厅基金(批准号:16C0394);中国博士后特别资助和面上基金(批准号:2017T100601;2016M590746);国家大学生创新训练项目(批准号:201811342007)资助的课题~~

摘　　要：该文探究了人参查尔酮合成酶(chalcone synthase, CHS)基因的表达模式与人参黄酮含量及抗胁迫之间的关系。作者从新鲜4年生人参根中提取总RNA,反转录合成cDNA,根据转录组测序结果,利用PCR扩增克隆人参CHS基因的cDNA全长,对其进行生物信息学分析,采用荧光定量PCR(qRT-PCR)分析人参根、茎、叶和胁迫条件下发根中CHS基因的表达水平,并测定其黄酮含量。克隆得到人参CHS,命名为PgCHS1,序列完整开放读码框(ORF)长度为1 182 bp,编码393个氨基酸。分析表明,该序列属于CHS家族基因,其具有查尔酮合成酶催化中心4个高度保守的残基Cys164、Phe215、His303、Asn336和形成活性中心构象所必需的13个关键残基Pro138、Gly163、Gly167、Leu214、Asp217、Gly262、Pro304、Gly305、Gly306、Gly335、Gly374、Pro375、Gly376。基因表达分析表明,Pg CHS1基因在叶片中的表达量最高,其次是茎、根和发根,并且受SA和MeJA的诱导。人参黄酮含量与基因表达水平高度一致。结果提示, PgCHS1基因参与人参黄酮的生物合成,从而保护人参免受外界的胁迫。To clone the chalcone synthase(CHS) gene and analyze the relationship of CHS expression profiles and flavonoids content, anti-stress in Panax ginseng, total RNA from fresh 4-years roots of P. ginseng was extracted and synthesized to cDNA. Primers were designed based on the CHS sequence of transcriptome of P. ginseng. The full open reading frame(ORF) of CHS gene was obtained by reverse transcription polymerase chain reaction(RT-PCR) and PCR products were sequenced. The sequence was analyzed by bioinformatics. The expression profiles of CHS gene were identified by qRT-PCR, and the content of the total flavonoids was assayed in the roots, stems, leaves and hairy roots of P. ginseng. The CHS gene was cloned and designated as PgCHS1. The full ORF of PgCHS1 has 1 182 bp and encode 393 amino acids. The sequence analysis indicated that absolute conservation of Cys164, Phe215, His303 and Asn336 occur in PgCHS1 sequence of the catalytic center. Moreover, PgCHS1 protein exhibits strong conservation of residues shaping the geometry of the active site(Pro138, Gly163, Gly167, Leu214, Asp217, Gly262, Pro304, Gly305, Gly306, Gly335, Gly374, Pro375 and Gly376). The Pg CHS1 gene expression level remained the highest in leaves, follow by stems, roots and hairy roots. Expression analysis of PgCHS1 in elicitor treatments showed that salicylic acid(SA) and methyl jasmonate(MeJA) obviously induced PgCHS1 expression. Total flavonoids content of P. ginseng also enhanced in response to SA and MeJA, which correlated with increased PgCHS1 expression. Induction of PgCHS1 by SA and MeJA suggested its involvement in production of flavonoids, providing protection from environmental stresses.

关 键 词：人参 查尔酮合成酶 基因表达 黄酮 生物合成 

分 类 号：S567.53[农业科学—中草药栽培]