hrpZ_(Psg12)基因植物表达载体的构建及花粉管通道法转化玉米的初步研究

Construction of Plant Expression Vector with hrpZ_(Psg12) Gene and Transformation into Maize by Pollen Tube Pathway

利用hrpZPsg12转化玉米,以期得到对玉米病害具有广谱抗性的新种质材料,为抗病育种提供新的种质资源。以植物表达载体pCAMBIA3301为基础载体,采用PCR法从克隆载体pGM-hrpZPsg12克隆来自丁香假单胞菌大豆致病变种(P.syringae pv.glycinea)的hrp ZPsg12基因,两端分别引入XhoⅠ酶切位点,构建了1个具有卡那霉素和除草剂草丁膦抗性标记的植物表达载体pCAMBIA3301-hrpZPsg12,并通过热激法转入到大肠杆菌DH5α中。采用花粉管通道法将构建好的植物表达载体的重组质粒导入玉米优良自交系"综31"中。结果表明:对得到的575株转化植株经PCR检测有45株呈阳性,阳性转化率为7.83%。

The objective of this study is to obtain new maize germplasm materials of broad-spectrum resistance to maize diseases and provide new germplasm resources for maize resistance breeding by transferring hrpZPsg12 gene into good inhybridline Zong 31. HrpZPsg12 gene,from Pseudomonas syringae pv.glycinea,was cloned from cloning vector p GM-hrpZPsg12 by PCR method and transferred into the based plant expression vector p CAMBIA3301. The two ends of pCAMBIA3301 were introduced XhoⅠ restriction sites and recombined to get plant expression vector p CAMBIA3301-hrpZPsg12,then the p CAMBIA3301-hrp ZPsg12 was transformed into the Escherichia coli DH5α through heat shock method. The recombinant plasmids of p CAMBIA3301-hrp ZPsg12 were extracted from constructed DH5α and imported into maize by pollen tube pathway method. The results showed that 45 positive plants were detected from the obtained 575 transgenic plants by PCR,and the positive conversion rate was 7. 83%.