杏CCD1和CCD4启动子克隆及顺式作用元件分析

Cloning and cis-acting Element Analysis of CCD1 and CCD4 Promoter in Apricot

以‘轮台小白杏’杏果实为材料,根据转录组数据库中的CCD1和CCD4基因片段,通过基因组步移法克隆了两个启动子pPaCCD1和pPaCCD4,获得了长度分别为2 233和1 838 bp的启动子序列。利用PlantCare数据库对启动子序列的顺式作用元件进行预测分析,结果表明两个启动子均含有多个光响应、脱落酸和茉莉酸甲酯等共有顺式作用元件,此外,PaCCD1启动子中含有刺激诱导和种子特异性调控等响应元件,PaCCD4启动子中含有赤霉素、低温和增强转录水平等响应元件,这暗示PaCCD1和PaCCD4的表达可能受光信号、激素和胁迫等多种信号的共同调节。为进一步确定其启动子核心区,基于基因结构特征与顺式作用元件的分布情况,分别构建两个不同长度缺失启动子与GUS基因融合的表达载体并瞬时转化烟草叶片。GUS活性检测发现,在PaCCD1的启动子上游–2 174~–1 700 bp、–1 700~–1 200 bp、–1 200~–600 bp区间内均包含影响启动子活性的顺式作用元件;PaCCD4启动子在上游–1740~–1200bp和–1200~–600bp这两个区间内含有顺式作用元件,随着启动子片段的缺失,PaCCD1和PaCCD4的GUS活性逐渐减弱。PaCCD1的启动子核心区域在–1200~2174bp区段内,PaCCD4的启动子核心区域在–1 200~1 740 bp区段内。

The promoters of pPaCCD1 and pPaCCD4 were cloned by genomic walking method using the CCD1 and CCD4 gene fragments in the transcriptome database. Sequencing showed that the lengths of pPaCCD1 and pPaCCD4 promoter were 2 233 bp and 1 838 bp,respectively. The cis-acting elements of promoter were analyzed and predicted using PlantCare databases. The results showed that two promoters contain multiple cis-acting elements associated with light signal,abscisic acid,and methyl jasmonate. In addition,the PaCCD1 promoter contains gibberellin,low temperature and transcription enhancer elements. The results suggested that the expression of PaCCD1 and PaCCD4 may be regulated by light signaling,plant hormone and stress. To further define two promoters’ core regions,two promoter-reporter vectors were respectively constructed based on structural characters of promoters and distributions of controlling elements in promoter using the promoter deletion mutation,and the vectors were fused to GUS gene and then were transformed into tobacco leaves. The efficient cis-acting element of PaCCD1 was included in–2 174 to–1 700 bp,–1 700 to–1 200 bp,–1 200 to–600 bp,while the efficient cis-acting element of PaCCD4 was included in–1 740 to–1 200 bp,–1 200 to–600 bp.The GUS enzyme activity of PaCCD1 and PaCCD4 was gradually weakened with the deletion of the promoter fragment. These results revealed that the core regions of the PaCCD1 and Pa CCD4 promoter were within –1 200 to 2 174 bp and–1 200 to–1 740 bp,respectively.