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利用RT-PCR结合RACE技术,从川乌头(Aconitum carmichaeli Debx.)花朵中克隆到1个类黄酮-3′,5′-羟基化酶基因的cDNA全长序列,全长1720bp,包含1个编码506个氨基酸的开放阅读框,命名为Ac-F3′5′H(GenBank登录号:JN635708)。序列分析表明Ac-F3′5′H编码的氨基酸序列中包含有已确定的保守基序,包括CYP基序、I螺旋区和血红素结合区等。氨基酸序列比对显示Ac-F3′5′H与其它物种的F3′5′H有很高的序列相似性。以川乌头18SrRNA基因(FJ748878)为内参,通过半定量RT-PCR对Ac-F3′5′H的时空表达模式进行了分析,结果显示Ac-F3′5′H随花朵发育,表达量呈递增趋势,并且在正在开放的花朵中达到最高,而在根、茎、叶中不表达,推测该基因可能在调节川乌头蓝色花朵形成中发挥作用。
The full-length cDNA sequence of a flavonoid-3 ’, 5’-hydroxylase gene from Aconitum carmichaeli Debx. Was cloned by RT-PCR and RACE technology. The full-length cDNA sequence was 1720 bp in length and contained 1 A 506 amino acid open reading frame was designated Ac-F3’5’H (GenBank accession number: JN635708). Sequence analysis showed that the amino acid sequence encoded by Ac-F3’5’H contains the defined conserved motifs, including CYP motif, I helix region and heme binding region. Amino acid sequence alignment shows that Ac-F3’5’H has high sequence similarity to F3’5’H of other species. The spatiotemporal expression pattern of Ac-F3’5’H was analyzed by semi-quantitative RT-PCR using Chuanwutou 18SrRNA gene (FJ748878) as an internal control. The results showed that the expression of Ac-F3’5’H with flower development was Increasing trend, and reached the highest in the open flowers, but not in the roots, stems and leaves, suggesting that the gene may play a role in regulating the formation of Sichuan aconite blue flowers.