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为深入研究夏菊的春化机理,利用多聚酶链式反应(PCR)结合5′RACE、3′RACE技术克隆夏菊品种‘优香’[Chrysanthemum morflorium(Ramat.)Kitam.‘Yuuka’]开花抑制基因Cm FLC-like1的c DNA全长序列,获得其全长为945 bp,开放阅读框(ORF)为636 bp,编码1条211个氨基酸残基的多肽,且具有典型的MADS结构域。同源性分析表明,Cm FLC-like1与葡萄(Vitis vinifera)Vv FLC和龙眼(Dimocarpus longan)Dl FLC的同源性最高,分别为55%和52%。进化树聚类分析表明,Cm FLC-like1蛋白与拟南芥和核桃的FLC遗传距离最近。亚细胞定位表明,Cm FLC-like1基因定位在核上。在酵母体系中发现Cm FLC-like1没有转录激活活性,拟南芥原生质体转化发现具有转录抑制活性。菊花植株营养生长期不同组织器官的RT-PCR表明Cm FLC-like1在叶片中表达量最高,茎和茎尖中次之,根中最少。低温(4℃)可抑制Cm FLC-like1表达,且处理时间越长,抑制表达越明显。
In order to further study the vernalization mechanism of summer chrysanthemum, the chrysanthemum variety Chrysanthemum morflorium (Ramat.) Kitam. ’Yuuka’ was cloned by polymerase chain reaction (PCR) combined with 5 ’RACE and 3’RACE techniques The full-length cDNA sequence of Cm FLC-like1 was 945 bp in length and 636 bp in open reading frame (ORF). It encoded a polypeptide of 211 amino acid residues with typical MADS domain. Homology analysis showed that Cm FLC-like1 had the highest homology with Dl FLC of Vitis vinifera Vv FLC and Dimocarpus longan, 55% and 52% respectively. Phylogenetic tree clustering analysis showed that the FLC genetic distance between Cm FLC-like1 protein and Arabidopsis thaliana and walnut was the closest. Subcellular localization showed that the Cm FLC-like1 gene is located on the nucleus. Cm FLC-like1 was found not to have transcriptional activation activity in yeast and Arabidopsis protoplast transformation was found to have transcriptional inhibitory activity. The RT-PCR of different tissues and organs of chrysanthemum plants during vegetative growth indicated that Cm FLC-like1 had the highest expression in leaves, followed by the stems and shoot tips, and the least in roots. Low temperature (4 ℃) can inhibit the expression of Cm FLC-like1, and the longer the treatment, the more obvious the inhibition of expression.