Molecular cloning, promoter analysis and functional characterization of APETALA 1-like gene from precocious trifoliate orange (Poncirus trifoliata L. Raf.)
Molecular cloning, promoter analysis and functional characterization of APETALA 1-like gene from precocious trifoliate orange (Poncirus trifoliata L. Raf.)
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早熟枳（Poncirus trifoliata L. Raf.）APETALA 1样基因的分子克隆、启动子分析及功能鉴定
DOI：
10.1016/j.scienta.2014.08.015
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发表时间：
2014-10-23
影响因子：
4.3
通讯作者：
Hu, Chun-Gen
中科院分区：
文献类型：
--
作者：
Sun, Lei-Ming;Zhang, Jin-Zhi;Hu, Chun-Gen
对于开花植物，开花转换是对内源和外源环境信号的响应。在这一过程中，APETALA家族成员的MADS-box转录因子起着重要的作用。为了更好地了解APETALA1(AP1)调控柑橘开花的分子机制，本研究从早花三叶橙(早熟三叶橙、大鼠)和正常三叶橙中分离到一个APETALA1同源基因(PtAP1)。结果表明，PtAP1基因序列在两个三叶橙品种间无明显差异。序列比对和系统发育分析表明，PtAP1属于euAP1分支。利用实时荧光定量聚合酶链式反应技术研究了早熟三叶橙中PtAP1基因的空间和/或时间特征。结果表明，PtAP1在几乎所有的生殖组织中都有表达，但在营养组织中仅有微弱表达。此外，PtAP1的表达模式与相变期的开花诱导和开花密切相关。PtAP1在拟南芥AP1-1突变体中的转录显示部分挽救了AP1的功能。PtAP1在野生型拟南芥中的过表达导致了早花和构型的改变。用染色体走步方法从两个不同基因型的三叶橙中获得了约1kb的PtAP1启动子序列，两个启动子序列中也没有核苷酸差异。同时，通过生物信息学分析，预测了PtAP1启动子中一些可能的顺式调控元件。为了检测PtAP1启动子的表达，将GUS报告基因在PtAP1启动子控制下转入野生型拟南芥。结果表明，PtAP1启动子具有较强的活性，具有发育阶段和组织特异性，可能是柑橘遗传改良的理想启动子。此外，酵母单杂交实验表明，PtAP1可能与一些尚未在柑橘中获得功能特性的开花蛋白相互作用。这些结果表明，PtAP1可能在早熟三叶橙开花过程中起重要作用。(C)2014爱思唯尔B.V.保留所有权利。
For flowering plants, flowering transition is induced in response to endogenous and extrinsic environmental signals. During this process, the members of the APETALA family from MADS-box transcription factors play an important role. Here, to better understand the molecular mechanism of APETALA1 (AP1) in the regulation of citrus flowering, an APETALA1-like homologue gene (PtAP1) was isolated from an early flowering trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Rat) and normal trifoliate orange. The results indicated that the sequence of PtAP1 was not any differences in two genotypes of trifoliate orange. Sequence alignment and phylogenetic analysis revealed that the PtAP1 fell in the euAP1 clade. Specific spatial and/or temporal patterns of PtAP1 were investigated in precocious trifoliate orange by real-time PCR. The results showed that PtAP1 was detected in almost all reproductive tissues, but only faint expression in vegetative tissues. Moreover, the expression pattern of PtAP1 was closely correlated with floral induction and flowering at phase change stage. Transcription of PtAP1 in Arabidopsis ap1-1 mutant showed partially rescued AP1 function. Over-expression of PtAP1 in wild-type Arabidopsis resulted in early flowering and changed architecture. About 1KB promoter sequence of PtAP1 was obtained by chromosome walking from two genotypes of trifoliate orange, there was also not nucleotide difference in the two-promoter sequence. Meanwhile, a number of putative cis-regulatory elements were predicted in PtAP1 promoter by bioinformatics analysis. To determine the expression of PtAP1 promoter, GUS reporter gene was transformed into wild-type Arabidopsis under the control of PtAP1 promoter. The results revealed that the PtAP1 promoter had strong activity and showed developmental phase and tissue specificity, which might be ideal for citrus genetic improvement. In addition, a yeast one-hybrid assay indicated that PtAP1 may be interacted with some flowering proteins, which have not yet been functionally characterized in citrus. These findings suggested that PtAP1 may play an important role during the early flowering of precocious trifoliate orange. (C) 2014 Elsevier B.V. All rights reserved.