摘要： 根据海带配子体光捕获蛋白基因lhcf6的cDNA全长序列(GenBank登录号:DQ250739)设计引物,利用基因组步移技术克隆得到其在雌、雄配子体中的5’-侧翼序列,长度分别为932,901 bp。测序结果比对显示二者相似度为33.3%,说明该基因在雌、雄配子体中具有不同的上游调控区。网络服务器PlantCARE和Softberry启动子元件预测结果表明,二者均含有TATA框、节律性相关元件MBS、光反应相关元件MNF1和GAG-motif,其中雌配子体lhcf6基因5’-侧翼序列还特有LHC基因特征元件AT-rich序列,雄配子体特有节律性相关元件Circadian元件,这些元件可能与该基因表达受光质、光强、节律性等因素的调控有关。将其与GFP报告基因一起构建融合表达载体pCAMBIA1304-Flhcf6和pCAMBIA1304-Mlhcf6,利用pCAMBIA1304作对照,通过电击转化法分别转化莱茵衣藻。激光共聚焦荧光显微镜检测结果显示,含pCAMBIA1304-Flhcf6和pCAMBIA1304-Mlhcf6的转基因藻体中GFP基因均有表达,说明克隆得到的海带雌、雄配子体lhcf6基因的5’-侧翼序列具有启动子功能。

关键词: 海带, 配子体, lhcf6, 启动子, GFP

Abstract: lhcf6 gene 5'-flanking sequences were isolated respectively from genomic DNA of female and male gametophytes of Saccharina japonica by nested PCR using genomic walking method. The length of the fragments were 932 bp in female and 901 bp in male. Sequence alignment showed a low similarity of 33. 3% between the two fragments,indicating this gene had different upstream regulatory region in female and male gametophytes. Cis-elements prediction by web server PlantCARE and Softberry showed both of them contained TATA box,circadian responsive element MBS,as well as light responsive elements MNF1 and GAG-motif. Otherwise,some elements were specific to female or male lhcf6,such as LHC gene promoter typical element AT-rich sequence was specific to female and circadian element circadian was specific to male. These elements might take part in the regulation of lhcf6 expression by light quality,light intensity and circadian. These two 5'-flanking sequences were fused with the reporter gene GFP to construct plant expression vectors,named pCAMBIA1304-Flhcf6 and pCAMBIA1304-Mlhcf6. The recombinant vectors as well as vector pCAMBIA1304 as positive control were transferred to the green alga Chlamydomonas reinhardtii by electroporation. GFP was observed under laser confocal fluorescence microscopy. Fluorescence signal was detected in cells containing pCAMBIA1304-Flhcf6 and pCAMBIA1304-Mlhcf6 indicating both the two cloned 5'-flanking sequences showed promoter activity.

Key words: Saccharana japonaca, Gametophytes, Lhcf6 gene, Promoter, Green fluorescent protein

中图分类号: 

• Q78