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通过对蜻蜓凤梨转录组数据的分析,我们获得一个跟拟南芥AP2同源片段,基于此设计引物,利用RACE技术首次从蜻蜓凤梨中克隆到完整的Af AP2-1序列,其c DNA全长2 185 bp,开放阅读框全长1 404 bp,编码467个氨基酸。保守结构域分析其含有两个AP2结构域,属于AP2亚家族。系统进化树分析发现该蛋白与拟南芥的AP2 RAP2.7亲缘关系较近。通过在CDS序列两端分别引入XbaⅠ和KpnⅠ限制性内切酶酶切位点,我们成功构建了Ca MV35S-Af AP2-1-GUS过表达载体。利用农杆菌介导法转化拟南芥植株后,再通过GUS染色和RNA水平鉴定发现Ca MV35S-Af AP2-1-GUS已转入拟南芥中并且能正常表达。本研究可为Af AP2-1的功能鉴定奠定基础,对深入研究凤梨科植物开花调控的分子机理具有重要意义。
Based on the analysis of the data of the dragonfly pineapple transcriptome, we obtained a homologous fragment of AP2 with Arabidopsis thaliana. Based on this, primers were designed and the complete Af AP2-1 sequence was cloned from the dragonfly pineapple by RACE technology for the first time. 2 185 bp. The full length of the open reading frame is 1 404 bp, encoding 467 amino acids. Conserved domain analysis contains two AP2 domains, belonging to the AP2 subfamily. Phylogenetic tree analysis showed that this protein has a close genetic relationship with AP2 RAP2.7 in Arabidopsis thaliana. We successfully constructed CaMV35S-Af AP2-1-GUS overexpression vector by introducing Xba I and Kpn I restriction sites at both ends of the CDS sequence respectively. After transformation of Arabidopsis plants by Agrobacterium-mediated transformation, CaMV35S-Af AP2-1-GUS was transformed into Arabidopsis and expressed normally by GUS staining and RNA level identification. This study can lay the foundation for the functional identification of Af AP2-1, and is of great significance for further study on the molecular mechanism of the flowering regulation of bromeliads.