大豆启动功能片段的克隆及在转化甘草中启动β-葡糖苷酸酶基因的表达

Cloning of Soybean promoter Fragments and Expression in Transformed Glycyrrhiza uralensis Fisch

将大豆DNA的HindⅢ和BamHⅠ酶切片段连接到pBⅠ101载体缺启动子的GUS基因上游,构建了含有不同DNA片段的重组质粒,转化大肠杆菌C600,获得了具Km抗性的转化子。通过三亲交配将上述重组质粒转移至发根农杆菌R1000(PRiA4b)中,用注射法,将各含重组质粒的发根农杆菌菌液感染甘草外植体,在含cb的无激素MS培养基上诱发毛状根并再生成植株。转化甘草具Km抗性,有甘露碱和农杆碱存在。经组织化学法检测,在转化株C2和C13的毛根、茎、叶中有靛蓝色沉淀物产生。证明大豆启动功能片段在转化甘草中启动了GUS基因表达。重组质粒的酶切分析证明C2和C13大豆DNA插入片段均约为0.8kb。DNA分子杂交也证明C2、C13 DNA与大豆DNA和转化株DNA有同源性。

DNA fragments, BamHI and HindⅢ double digested, from soybean have been ligated ro the upstream of promoter-less GUS gene of pB1101 vector. The recombinant plasmids containing different DNA fragments of soybean were constructed, and transformed E. colt C600. Km-resistant transformants were obtained. The recombinant plasmids were transfered into A. rhizo-genes R1000(pRiA4b) by triparental mating method and the transferants were used to infect the embryo axis, stems and other explants from G. uralensis Fisch by injection. Hairy roots appeared from cultures on hormone-free MS medium with Img/ml cb and regenerated into plants. The transformed G. uralensis Fisch had resistance to Km and contained mannopine and agro-pine. In histochemical assay, blue precipitates were found in leaves, stems and hairy roots of transformed plants C13 and C2. It showed that soybean promoter had initiated GUS gene 10 express. Analysis of digesting recombinant plasmids had indicated that inserted fragments of toybean DNA in C2 and C13 were 0.8kb approximately. DNA-DNA hybridization confirmed that the DNA in recombinant plasmids is homology with both the DNA in soybean and transformed G. uralensis plant.