抗线虫基因表达载体构建与转化甘蔗研究初报

Construction of expression vector of nematode-resistant gene and transformation of sugarcane

试验研究利用德国引进的抗线虫基因Hs1pro 1构建表达载体并转化甘蔗 (SaccharumofficinarumL .) ,以获得抗线虫转基因植株。提取克隆载体P1832用NcoⅠ酶切、Klenow补平和SacⅠ酶切 ,回收基因片段 ;提取表达载体pBIL 1用KpnⅠ酶切、T4DNApolymerase补平和SacⅠ酶切 ,回收大片段 ;目的片段用T4DNAligase连接并转化E .coli,鉴定重组质粒 ;用基因枪轰击转化甘蔗品种“ROC”16获得 16株再生苗 ,其中 4株经PCR检测呈阳性 ,通过Southern杂交 ,证明Hs1pro 1基因已整合到其中

The expression vector of Hs1 pro-1 of nematode-resistant gene cloned by the Germany scientists was constructed and transformed into sugarcane to get the transgenic plants. The cloned vector P1832 was digested with restriction nuclease NcoⅠ, DNA polymerase I Large Fragment and SacⅠ in order. The short fragment was purified. Expression vector pBIL-1 was digested with KpnⅠ, blunted with T4 DNA polymerase and digested again with SacⅠ. The large fragment was isolated. The short fragment of P1832 was ligated with the large fragment of pBIL-1 by T4 DNA ligase and the reactant was transferred into E.coli. The recombinant plasmid was identified and transformed into sugarcane genotype“ROC”16 via particle bombardment. Sixteen kanamycin-resistant sugarcane seedlings were obtained and four seedlings were positive by PCR, of which three were proved to be transgenic plants by southern blot.