β-1,3-葡聚糖酶基因高效表达载体的构建及对小麦的转化

Construction of Expression Vector with BG2 Gene and Its Transformation in Wheat

利用PCR方法设计引物 ,进行特异扩增 ,在得到的BG2基因片段的两端引入可以与表达载体多克隆位点相匹配的酶切位点 ,将该基因插入高效表达载体质粒pATC94 0中 ,获得表达质粒pATCBG2。通过基因枪转化法 ,将pATCBG2转化优质小麦品种龙辐麦 10、龙辐麦 3号 ,获得抗卡那霉素 (Kanamycin)的再生植株 ,经PCR ,PCR Southern和Dot blotting检测 ,结果表明 ,有 5个转基因植株在以上各项检测中全为阳性 ,证明目的基因已整合入这些转基因小麦基因组中。田间接菌发病检测结果表明 ,转基因植株比对照的抗病性提高 1～ 2级。

β-1,3-glucanase( BG2 )is one of the pathogensis-related-proteins(PR).Study of these proteins and their related genes is one of the hot points in plant genetic engineering of disease resistance for a long time.In this research,specific primers were designed with the enzyme cleavage site of Spe Ⅰ in its forward one and Not Ⅰ site in the backward according to the BG2 gene sequence.Using this pair of primers, BG2 gene,which was contained in the plasmid of pRTL2,was amplified and confirmed by sequencing the amplified fragment inserted into T-easy vector.The positive clone containing BG2 gene was digested with the enzymes of Spe Ⅰ/ Not Ⅰ and then BG2 gene was inserted into the Xba Ⅰ/ Not Ⅰ sites of super expression binary vector pATC940.The reconstructed expression vector named as pATCBG2 was introduced into the wheat of Longfumai10 and Longfumai3 ( Triticum aestivum L.em.Thell) through the particle gun transformation method.The Kanamysin resistant (Km r) transformants were obtained.PCR,Dot-blotting and PCR-Southern hybridization analysis showed that the BG2 gene was integrated into the genome of wheat.Result of pathogen inoculation assay on the transgenic plants showed that the transgenic plants had a higher resistant disease score of 1～2 grade than the control.