版纳龙竹CONSTANS同源基因的克隆与序列分析

Cloning and Sequence Analysis of CONSTANS Homologous Gene from Dendrocalamus xishuangbannaensis

以版纳龙竹基因组DNA为模板，采用前人基于水稻CO同源基因Hd1序列的保守区所设计的特异引物COS1和COA1，运用PCR方法扩增出一条1520bp的DNA片段，并克隆到pGEM—T载体。测序和序列分析结果显示：该片段含有1个590bp的内含子，编码区930bp共编码310个氨基酸；该基因被命名为DxCO1，其DNA序列在GenBank中的注册号为GQ358925。在GenBank中进行同源性检索的结果显示：其核苷酸序列与其它禾本科植物CO同源基因的氨基酸序列同源性高达81％～91％；推测的DxC01蛋白质序列与其它种子植物CO同源基因蛋白质序列的系统发育分析结果显示：DxC01与小麦Hdl—like等5个基因聚成了一个强烈支持的分支；另外，在该片段推测的蛋白质序列的氨基端含有一个类似锌指蛋白的B．box（Cx2Cx8Cx7Cx2Cx4Hx8H）结构域，羧基端含有一个CCT（CO，CO—like，TOC1）结构域。序列和结构的高度同源性表明：DxCO1是版纳龙竹的1个CO-like基因，可能对其开花调控有着重要作用。

CONSTANS （CO） is one of the key genes controlling flowering time of the flowering plants in Photoperiod Pathways. In current study, two primers, COS1 and COA1, based on the conserved region of Hdl sequences from CO-like gene of rice （ Oryza sativa）, were employed to amplify a 1 520 bp fragment by polymerase chain reaction （PCR） with genomic DNA of Dendrocalamus xishuangbannaensis D. Z. Li ＆ H. Q. Yang. The fragment was cloned into a pGEM-T vector and then sequenced. Result of the 1 520 bp fragment cloned into a pGEM-T vector produced a fragment of gene, i.e. DxCO1, with accession number GQ358925 in GenBank. The DNA sequence analysis indicated that DxCO1 included one intron of 590 bp and two exons encoding putative 310 amino acids. Using homology analyses of DNA sequence, DxCO1 was homologous to the other six CO-like genes from Gramineae plants with 81% - 91% supports, respectively. Phylogenetic analysis, based on protein sequences of DxCO1 and the known CO-like genes of seed plant in GenBank, showed that DxCO1 and other five CO-like genes from Gramineae plants clustered into a monophyly with bootstrap value of 92%. Meanwhile, there was a zinc-finger B- box （Cx2Cx8Cx7Cx2Cx4Hx8H） domain near amino terminus and a CCT （ CO, CO-like, TOC1） domain near carboxy terminus in putative amino acid sequence of DxCO1. These results suggested that DxCO1 belonged to CO-like gene family, and it might have the same function of controlling flowering time like other CO homologous genes.