甘蓝型油菜BnCOP1基因编码区全长cDNA的克隆与功能研究

Cloning and Characterization of a Full-length cDNA Encoding Brassica napus Constitutively Photomorphogenic 1

通过分析拟南芥、豌豆、番茄和水稻的COP1(constitutively photomorphogenic 1)的cDNA序列,运用RT-PCR和改进的基因组步行(genome walking)技术相结合的方法,首次从甘蓝型油菜中克隆到油菜BnCOP1编码区cDNA的全长序列,其全长2 034 bp,编码677个氨基酸.同源性分析表明,其编码的氨基酸序列与拟南芥的同源性高达94%.对BnCOP1编码序列(cDNA)演绎出的氨基酸序列分析表明,其编码的蛋白包含有N端的环形锌指结合域(ring finger zinc bindingdomain,RING)、中间的卷曲螺旋形结构域(coiled-coil domain,coiled-coil),7个C端的WD-40重复序列(WD-40 repeats,WD-40)的功能域.半定量RT-PCR和实时荧光定量PCR分析该基因在油菜中的表达模式,结果显示,BnCOP1在甘蓝型油菜的各个组织器官中均有表达,其中在花中的表达明显高于在根、叶、茎、果荚及子叶和胚轴中,暗示该蛋白可能与开花途径相关.过表达BnCOP1的转基因拟南芥植株在高度、主茎的直径和叶片大小上都呈现出比野生型弱小的表型,表明BnCOP1抑制了拟南芥光形态建成从而影响了植物的生长发育.

By analyzing the cDNA sequence of COP1（constitutively photomorphogenic 1） from Arabidopsis thaliana,Pisum sativum,Lycopersicon esculentum and Oryza sativa,combining RT-PCR and modified genome walking,full-length cDNA in encoding region of COP1 with 2 034 bp was first cloned from Brassica napus,which codes a protein with 677 amino acids.Sequence homology analysis of amino acid deduced from the cDNA showed that BnCOP1 shared high homology（94%） with AtCOP1.Sequence analysis of deduced amino acid indicated that one ring,one coiled coil and seven WD40 domains were contained in BnCOP1.Via semi-quantitative RT-PCR and real-time quantitative PCR analysis we demonstrated that BnCOP1 gene expression was detected in all analyzed tissues,the relative expression in flower was significantly higher than that in root,leave,stem,silique,cotyledon andhyposotyl,indicating the possible role of BnCOP1 in the Brassica napus flower pathway.Importantly,the plants appeared slimmer phenotypes in plant height,stem diameter length and leaves size when BnCOP1 gene was overexpressed in Arabidopsis thaliana,which implied BnCOP1 may impair the growth and development of plants by inhibiting plant photomorphogenesis.