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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。
Using the male flower bud of Eucommia ulmoides Olive as materials, the 745bp cDNA sequence containing the complete open reading frame (ORF) at the 3 ’end was cloned by rapid amplification of cDNA ends. The 3’RACE product and transcriptome library 5 ’end sequence splicing, the full length of 872bp of Eucommia FLC cDNA sequence encoding 86 amino acids, named EuFLC1. Bioinformatics analysis showed that the molecular weight of EuFLC1 protein was about 10.005 kD and the theoretical isoelectric point was 10.47, which was a hydrophilic protein. There were three possible phosphorylation sites, one possible nuclear localization signal and one possible nuclear And the signal was exported. The transmembrane helix and signal peptide were absent. The protein secondary structure contained two α-helices and four β-sheets. The results showed that the tertiary structure of EuFLC1 The protein consists of two α-helices perpendicular to each other and two β-sheets in the same plane between α-helices. The N-terminal and C-terminal of the protein are randomly curled and extend to the side of the monolith. Domain MADS-Box gene. In the Blastp results, the sequences with high homology to EuFLC1 were: floweringlocus C of grape, MADS-box protein FLC subfamily of small-grain coffee, and flowering locus C-like MADS-box protein of Kyoho grape. Therefore, EuFLC1 is considered as Eucommia ulmoides FLC gene. Phylogenetic tree showed that the Eucommia ulmoides Olive and the same lip-shaped plants gathered in one small grain coffee, and APG Ⅲ classification system classification results. This gene is the first MADS-Box gene cloned from the Eucommia ulmoides plant, providing an important material for studying the evolution of the MADS-Box gene and laying a foundation for studying the molecular regulation mechanism of the flowering time of Eucommia ulmoides.