华北农学报
華北農學報
화북농학보
ACTA AGRICULTURAE BOREALI-SINICA
2014年
1期
62-68
,共7页
海带%配子体%lhcf6%启动子%GFP
海帶%配子體%lhcf6%啟動子%GFP
해대%배자체%lhcf6%계동자%GFP
Saccharina japonica%Gametophytes%lhcf6 gene%Promoter%Green fluorescent protein
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。
根據海帶配子體光捕穫蛋白基因lhcf6的cDNA全長序列(GenBank登錄號：DQ250739)設計引物,利用基因組步移技術剋隆得到其在雌、雄配子體中的5′-側翼序列,長度分彆為932,901 bp。測序結果比對顯示二者相似度為33.3%,說明該基因在雌、雄配子體中具有不同的上遊調控區。網絡服務器PlantCARE和Softberry啟動子元件預測結果錶明,二者均含有TATA框、節律性相關元件MBS、光反應相關元件MNF1和GAG-motif,其中雌配子體lhcf6基因5′-側翼序列還特有LHC基因特徵元件AT-rich序列,雄配子體特有節律性相關元件Circadian元件,這些元件可能與該基因錶達受光質、光彊、節律性等因素的調控有關。將其與GFP報告基因一起構建融閤錶達載體pCAMBIA1304-Flhcf6和pCAMBIA1304-Mlhcf6,利用pCAMBIA1304作對照,通過電擊轉化法分彆轉化萊茵衣藻。激光共聚焦熒光顯微鏡檢測結果顯示,含pCAMBIA1304-Flhcf6和pCAMBIA1304-Mlhcf6的轉基因藻體中GFP基因均有錶達,說明剋隆得到的海帶雌、雄配子體lhcf6基因的5′-側翼序列具有啟動子功能。
근거해대배자체광포획단백기인lhcf6적cDNA전장서렬(GenBank등록호：DQ250739)설계인물,이용기인조보이기술극륭득도기재자、웅배자체중적5′-측익서렬,장도분별위932,901 bp。측서결과비대현시이자상사도위33.3%,설명해기인재자、웅배자체중구유불동적상유조공구。망락복무기PlantCARE화Softberry계동자원건예측결과표명,이자균함유TATA광、절률성상관원건MBS、광반응상관원건MNF1화GAG-motif,기중자배자체lhcf6기인5′-측익서렬환특유LHC기인특정원건AT-rich서렬,웅배자체특유절률성상관원건Circadian원건,저사원건가능여해기인표체수광질、광강、절률성등인소적조공유관。장기여GFP보고기인일기구건융합표체재체pCAMBIA1304-Flhcf6화pCAMBIA1304-Mlhcf6,이용pCAMBIA1304작대조,통과전격전화법분별전화래인의조。격광공취초형광현미경검측결과현시,함pCAMBIA1304-Flhcf6화pCAMBIA1304-Mlhcf6적전기인조체중GFP기인균유표체,설명극륭득도적해대자、웅배자체lhcf6기인적5′-측익서렬구유계동자공능。
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-ele-ments prediction by web server PlantCARE and Softberry showed both of them contained TATA box,circadian re-sponsive 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 fe-male 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 report-er 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. Flu-orescence signal was detected in cells containing pCAMBIA1304-Flhcf6 and pCAMBIA1304-Mlhcf6 indicating both the two cloned 5′-flanking sequences showed promoter activity.
