杜仲EuFLC1基因克隆与生物信息学分析

以杜仲（Eucommia ulmoides Olive）雄花花芽为材料,依靠实验室构建的杜仲转录组库,采用cDNA末端快速扩增法克隆了3＇端包含完整开放阅读框的745bpcDNA序列。经3＇RACE产物和转录组库中5＇端序列拼接,获得全长为872bp的杜仲FLC的cDNA序列,编码86个氨基酸,命名为EuFLC1。生物信息学分析显示：EuFLC1蛋白分子量约为10.005 kD,理论等电点为10.47,为亲水性蛋白;存在3个可能的磷酸化位点、1个可能的核定位信号和1个可能的核输出信号;不存在跨膜螺旋和信号肽;蛋白二级结构中包含2个α螺旋和4个β折叠,无规卷曲连接α螺旋及β折叠;蛋白三级结构同源建模结果表明,EuFLC1蛋白包含2个互相垂直的α螺旋,α螺旋间有2个处于同一平面的β折叠,蛋白的N端和C端为无规卷曲并伸向整体结构的一侧;是含有MEF2_like型MADS-box结构域的MADS-Box基因。Blastp结果中,与EuFLC1同源性较高的序列有：葡萄的floweringlocus C、小粒种咖啡的MADS-box protein FLC subfamily、巨峰葡萄的flowering locus C-like MADS-box protein,因此认为EuFLC1是杜仲FLC基因。系统发育树表明杜仲与同属唇形类植物的小粒咖啡聚为一支,与APGⅢ分类系统的分类结果一致。该基因为首次从第三纪孑遗植物杜仲中克隆到的MADS-Box基因,为研究MADS-Box基因的演化的提供了一个重要材料,为研究杜仲开花时间的分子调控机制奠定了基础。

Molecular Cloning and Bioinformatics Analysis of Gene EuFLC1 in Eucommia ulmoides Olive

Based on the transcriptome library constructed from Eucommia ulmoides Olive by our laboratory, the gene FLC of Eucommia ulmoides was cloned by rapid amplification of cDNA ends（RACE） by using male flower buds of Eucommia ulmoides as experiment material and named as EuFLC1. The gene encodes a protein composed of86 amino acids. The predicted molecular mass and the isoelectric point（PI） of EuFLC1 are 10.005 kD and 10.47, respectively. Protein sequence analysis revealed the presence of three phosphorylation sites, one nuclear localization signal（NLS） and one nuclear export signal（NES）, and the absence of signal peptide sequence and transmembrane helix structure. The protein secondary structure of EuFLC1 has two α-helixs and four β-strands. The result of protein structure homology modeling shows that EuFLC1 has two mutually perpendicular α-helixs and two coplanarβ-strands between the α-helixs, and the N-terminal and C-terminal of EuFLC1 are randon coils extending toward the same side of the overall structure. With EuFLC1 containing a MEF2_like sequence, EuFLC1 belongs to the MADS-Box gene. According to Blastp analysis, EuFLC1 is more homologous to flowering locus C（Vitis vinifera）,MADS-box protein FLC subfamily（Coffea arabica） and flowering locus C-like MADS-box protein（Vitis labrusca×Vitis vinifera）. Phylogenetic tree shows that Eucommia ulmoides and Coffea arabica are in same branch, which is concordant with APG Ⅲ classification results that two trees are all members of the lamiids. EuFLC1 is a MADS-Box gene that was cloned for the first time from the tertiary relict plant Eucommia ulmoides, which may provide an important material for studying the evolution of MADS-Box gene and lay the foundation for the study of the molecular mechanism of EuFLC1 regulating flowering time in Eucommia ulmoides.