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该研究旨在获得红花查尔酮合酶(chalcone synthase,CHS)基因全长片段,并在拟南芥中进行过表达,初步验证该基因的功能根据红花转录物测序结果中获得的中间序列,采用RT-PCR和cDNA末端快速扩增技术(rapid amplifi cation of cDNA ends,RACE)方法从红花花瓣中克隆到1个CHS基因的全长cDNA,命名为Ct CHS1,全长序列1 360 bp。生物信息学分析表明,该基因具有完整的开放阅读框(open reading frame,ORF),共1 113 bp,编码370个氨基酸。亚细胞定位预测结果显示,该基因编码的蛋白质定位于细胞质。结合其他物种的CHS基因构建系统树表明,Ct CHS1具有高度保守性,其与水母雪莲花的亲缘关系最近。荧光定量PCR(Real-time PCR)分析表明,Ct CHS1基因在吉红油姊妹系的衰落期和吉红一号的盛花期表达量最高。该研究成功构建了含有Ct CHS1基因的植物表达载体,并在拟南芥中进行过表达,获得了高黄酮含量的转基因拟南芥T2株系。结果表明,过表达红花CHS基因可以提高拟南芥中的黄酮含量,为后续该基因的功能验证奠定基础。
The aim of this study was to obtain the full-length fragment of the chalcone synthase (CHS) gene and overexpress it in Arabidopsis thaliana. The preliminary validation of the function of this gene was based on the results obtained in the sequencing of safflower transcripts Sequence and the full length cDNA of one CHS gene was cloned from safflower petals using RT-PCR and rapid amplification of cDNA ends (RACE) bp Bioinformatics analysis showed that the gene has a complete open reading frame (ORF) of 1 113 bp encoding 370 amino acids. The results of subcellular localization showed that the protein encoded by this gene localized in the cytoplasm. The phylogenetic tree based on the CHS genes of other species showed that Ct CHS1 is highly conserved and has the closest genetic relationship with A. septempunctata. Real-time PCR analysis showed that the Ct CHS1 gene had the highest expression in the decline period of Jiashi red sibling line and the flowering stage of Jihong-1. In this study, a plant expression vector containing Ct CHS1 gene was successfully constructed and overexpressed in Arabidopsis to obtain transgenic Arabidopsis T2 lines with high flavone content. The results showed that overexpression of safflower CHS gene can improve the content of flavonoids in Arabidopsis thaliana, and lay the foundation for the subsequent functional verification of this gene.