红花CHS基因的过表达提高了拟南芥黄酮含量

Over-Expression of Chalcone Synthase of Safflower Increases Flavonoid Content in Arabidopsis thaliana

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 object of this study is to isolate the c DNA of CHS of safflower and overexpress it in Aropidopsis thaliana to vetify its function briefly. In this study, the full-length c DNA sequence of chalcone synthase（CHS） gene named Ct CHS1 was cloned from flowers of Carthamus tinctorius L.（safflower） by RT-PCR and RACE techniques according to the sequences of transcriptome in safflower. The full-length c DNA of Ct CHS1 was 1 360 bp and included a whole open reading frame of 1 113 bp, encoding a polypeptide with 370 amino acids. Subcellular localization prediction showed that Ct CHS1 may locate in the cytoplasm. Safflower CHS gene is highly conservative according to the blasting and phylogenetic analysis. Real-time PCR results indicated that relative expression of CHS gene was highest in fade of JIHONGYOU var. and early flowering period of JIHONGYIHAO var.. Plant expression vector of Ct CHS1 was constructed and transformed into A. thaliana genome. The T3 plants were obtained and the flavonoids content in transgenetic A. thaliana is higher than that in wild type. This results showed that over-expression of Ct CHS1 could improve the flavonoids content and lay a foundation for the functional verification of Ct CHS1 flavone content is determined in leaves,