北美鹅掌楸LtAGO1基因的克隆、表达及其启动子分析

Cloning, expression and promoter analysis of LtAGO1 from Liriodendron tulipifera

关键词:  北美鹅掌楸, AGO1, 叶极性, GUS, 组织表达

Key words:   Liriodendron tulipifera, AGO1, leaf polarity, GUS, tissue expression

摘要:

为深入探究叶原基分化成叶器官的形态建成机制,该研究以北美鹅掌楸为材料,采用RT-PCR和RACE克隆技术获得LtAGO1的cDNA全长和启动子序列并预测其功能,通过RT-qPCR分析LtAGO1在鹅掌楸属中的组织表达模式。同时,经抗性筛选和DNA鉴定获得ProAGO1 ∷ GUS的转基因拟南芥株系,并进一步对T2代阳性植株进行表型和GUS组织化学染色分析。结果表明:(1) LtAGO1基因包含3 300 bp的开放阅读框,编码1 100个氨基酸,分子量为122.14 kD,理论等电点(pI)为9.36。(2)氨基酸序列分析显示LtAGO1含Gly-rich-AGO1和Piwi两个典型的AGO基因结构域,同源性分析显示LtAGO1蛋白与沉水樟AGO1蛋白(RWR84608.1)亲缘关系最近。(3)组织表达特异性分析显示LtAGO1在北美鹅掌楸不同组织间的相对表达量为雄蕊>花芽>花瓣>花萼>叶片>雌蕊>叶芽>茎,LtAGO1在北美鹅掌楸叶片不同发育阶段的相对表达量为叶芽萌动期>幼叶期>衰老期>成熟期,AGO1在鹅掌楸属叶缘的表达量高于叶片的其他部位且北美鹅掌楸叶凹陷部位的表达量高于叶尖部位。(4)获得叶中-侧轴向和基-顶轴向的极性缺失、叶缘锯齿、重瓣花型的转化株系,GUS组织染色显示ProAGO1启动GUS基因在叶芽顶端稳定表达且在新分化的叶柄上表达较强,在成熟期的茎、叶、花和果的维管束中均特异表达。LtAGO1启动子的GUS活性强度为叶顶芽>花>维管束,这与实时定量PCR结果相一致。综上认为,LtAGO1基因在顶端分生组织特异表达且受到多种途径的调控而参与到叶和花器官的发育进程中。该研究结果为进一步了解北美鹅掌楸LtAGO1基因的基本功能及其调控叶形发育机制提供了理论基础。

Abstract:

To study the morphogenesis mechanism of leaf primordium differentiation, we used Liriodendron tulipifera to clone 2 001 bp upstream region of LtAGO1 CDS as the promoter by RT-PCR and RACE technology and predicted its function, and we also used real-time PCR to investigate expression patterns in Liriodendron, obtained the transgenic Arabidopsis thaliana of ProAGO1∷GUS by resistance screening and DNA dentification, and then monitored phenotype and GUS histochemical staining. The results were as follows:(1) The LtAGO1 gene included an open reading frame for 3 300 bp, encoding 1 100 amino acid, the molecular weight was 122.14 kD and theoretical isoelectric point(pI)was 9.36.(2)Amino acid sequence analysis showed that LtAGO1 consisted of Gly-rich-AGO1 and Piwi conserved domains of AGO family. Phylogenetic trees revealed that LtAGO1 was closed to Cinnamomum micranthum(RWR84608.1)in evolutional relationship.(3)The specific tissue expression analysis demonstrated that the expression order was stamen>floral bud>petal>calyx>leaf>pistil>leaf bud>stem among tissues, the expression order was leaf bud sprouting stage >young leaf stage>senescence stage >mature stage among stages, it was highly expressed in the leaf margin of Liriodendron L., and LtAGO1 gene expression in leaf tooth sinus was higher than that in leaf tooth tip of Liriodendron tulipifera.(4)The transgenic strains leaf polarity of the medio-lateral and proximo-distal axis were absent with serrated leaf margin and double petal flower. It was found that GUS staining was stably detected at the tip of leaf bud of transgenic seedlings, and higher GUS activity was observed at newly differentiated petioles. ProAGO1 promoter drove GUS gene to accumulate specifically in the vascular bundle of Arabidopsis thaliana leaf, flower, pod and stem, and GUS activity intensity order was leaf tip bud> flower>vascular bundle among tissues, which was in accordance with the real-time PCR results. Therefore, the LtAGO1 gene is predominantly expressed in apical meristem and is regulated by various pathways during the development of leaf and flower. The results provide a theoretical basis for further functional research of the LtAGO1 gene in Liriodendron tulipifera and regulation mechanism of leaf shape development.