2个葡萄GID(Gibberellin Insensitive Dwarf)基因的克隆与表达

Cloning and expression analysis of two grape GID (Gibberellin insensitive dwarf) genes

为探讨外源GA介导的无核葡萄果实膨大的机理,本试验根据葡萄基因组序列设计特异性引物,以葡萄果实cDNA一链为模板,利用聚合酶链式反应(PCR)克隆了2个假定的葡萄GID基因。测序比对结果表明其中一个属于GID1ac家族,根据序列相似性,命名为VvGID1-3,另一个属于GID2 F-box基因家族,命名为VvGID2。在烟草中分别超表达这2个基因,结果表明:VvGID1-3和VvGID2转基因植株叶圆片在MS培养基上生长7d后面积都约为对照的140%;VvGID1-3和VvGID2转基因烟草种子的萌发率降低,萌发过程对0.5和1μmol/L的GA3处理表现超敏特征;内源激素水平测定结果显示VvGID1-3转基因烟草中的GA和ABA含量低于对照,VvGID2转基因植株中IAA含量高于对照;在葡萄胚性愈伤组织中分别表达VvGID1-3-GFP和VvGID2-GFP,信号均定位于细胞核中;对VvGID1-3和VvGID2的启动子进行克隆,pVvGID1-3::GUS和pVvGID2::GUS转基因烟草的染色结果表明,信号在幼嫩和快速生长的组织中最强,在成叶中主要在叶脉中表达,在不定根中的表达量很低。

In order to reveal the mechanism of seedless grape enlargement mediated by exogenous gibberellin （GA） application,specific primers were designed according to the grape genome sequence.The first strand cDNA of grape berry was served as the template.Two grape GID genes were cloned by PCR.Full sequence alignment showed that one isolate belongs to GID1ac family,and named as VvGID1-3 with close sequence similarity.The other was assigned to GID2 F-box gene family,named as VvGID2.The two genes were over-expressed in tobacco.The result showed that the area of VvGID1-3 and VvGID2 over-expressing leaf disc were about 140% of the control after 7 days growth on MS medium.Seed germination ratio of the transgenic lines was remarkably lower than the wild typeand demonstrated hypersensitivity to 0.5 and 1 μmol/L GA3 treatment during germination.Endogenous hormone level assay revealed lower GA and ABA content in VvGID1-3 transgenic lines and higher IAA content in VvGID2 lines.Expressing VvGID1-3-GFP and VvGID2-GFP in grape embryogenic callus,fluorescent signals were localized in the nucleus.Promoters of VvGID1-3 and VvGID2 were cloned.The pVvGID1-3：：GUS and pVvGID2：：GUS transgenic tobacco showed that blue stain was mainly in the young and fast growing tissues.In mature leaves,the expression was highlighted in the veins.And very limited signal was found in adventitious roots.