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以油用牡丹凤丹(Paeonia ostii T.Hong&J.X.Zhang)胚珠为试材,通过反转录PCR(reverse transcription PCR,RT-PCR)及RACE技术得到脂肪酸去饱和酶基因FAD2的c DNA全长,命名为PoFAD_2(Gen Bank注册号为KT153989)。该基因全长为1 695 bp,开放阅读框(open reading frame,ORF)为1 152bp,编码383个氨基酸。系统发育树结果表明PoFAD_2与芍药(P.lactiflora)FAD2具有最高的同源性,亲缘关系最近。此外,PoFAD_2编码的氨基酸序列中存在3个高度保守的组氨酸簇及4个跨膜区,且含有Delta12-FADs的特异位点,这些结果表明,该基因属于脂肪酸去饱和酶家族。实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)结果表明:PoFAD_2在凤丹各组织中的表达差异较大,在茎、萼片、雄蕊及花瓣中几乎不表达,在叶片中极微弱表达,表达量仅为胚珠中的1/73,在芽及雌蕊中低表达,表达量约为胚珠中的1/10~1/12,而在胚珠中高表达;种子发育进程中,PoFAD_2表达量呈现先急速上升之后迅速下降再回升的趋势,且于花后75 d出现表达量的最高峰。
Oil-based ovules of Paeonia ostii T. Hong & J.X. Zhang were used as materials to obtain full-length c DNA of fatty acid desaturase gene FAD2 by reverse transcription PCR (RT-PCR) and RACE techniques Long, named PoFAD_2 (Gen Bank registration number KT153989). The full length of this gene was 1 695 bp, and the open reading frame (ORF) was 1 152 bp, encoding 383 amino acids. The results of phylogenetic tree showed that PoFAD_2 has the highest homology with FAD2 of P.lactiflora and has the closest genetic relationship. In addition, there are three highly conserved histidine clusters and four transmembrane regions in the amino acid sequence encoded by PoFAD_2, and contain specific sites of Delta12-FADs. These results indicate that the gene belongs to the family of fatty acid desaturase. The results of real-time quantitative PCR (qRT-PCR) showed that the expression of PoFAD_2 in the tissues of P. phoenixensis varied greatly, almost not expressed in the stems, sepals, stamens and petals, and very weakly expressed in the leaves , And the expression level was only 1/73 in the ovule and low in the bud and pistil. The expression level was about 1/10 to 1/12 in the ovule and overexpressed in the ovule. In the process of seed development, the expression level of PoFAD_2 After the first rapid increase in the rapid decline and then rebound trend, and in 75 d after flowering the highest expression level.