抗甘蔗花叶病毒的无标记反向重复转基因玉米

Inverted-Repeat Transgenic Maize Plants Resistant to Sugarcane Mosaic Virus

构建了无标记基因源自玉米矮花叶病的主要病原——甘蔗花叶病毒(sugarcane mosaic virus,SCMV)复制酶基因的反向重复序列表达载体,通过农杆菌介导法以该表达载体和除草剂标记基因表达载体共转化玉米自交系综3的幼胚,用除草剂梯度筛选,获得了可育的再生株。对T1和T2代群体作PCR和DNA点杂交,结合温室和田间人工接种病毒进行抗病性鉴定,获得了比较稳定的对SCMV高抗的无标记转基因玉米株系。

RNA silencing is a post-transcriptional gene-silencing phenomenon induced by double-stranded RNA （dsRNA）. In an attempt to generate dsRNA-mediated transgenic maize plants resistant to SCMV （sugarcane mosaic virus）, we cloned SCMV NIb gene-specific sequences and inserted it into the binary vector p3301 in the sense and antisense orientations （ named SCMVirNIb）, which could produce RNAs capable of duplex formation in plant cells. Maize immature embryos were co-cultured with Agrobacterium carrying two vectors, one marker-free vector harboring the SCMVirNIb and one vector harboring bar gene as the selective marker. Resistant calli were recovered by selection on medium containing Biolaphos. Among the regenerated plantlets from resistant calli, 14 plants have been certified to contain SCMVirNIb by PCR amplification and DNA dot blot. T1 plants derived from the 14 plants were challenged in greenhouse with SCMV inoculums and the percentages of resistant plants in 11 T1 lines were higher than 60%. One plant in the T1 lines was found to carry SCMVirNIb without bar gene by PCR assay. T2 plants derived from T1 SCMV resistant transgenic plants were challenged with SCMV inoculums in field. The percentages of resistant plants from 3 lines, including the line derived from the marker-free transgenic plant, were higher than 85 %. And non-transgenic control plants were all susceptive. Further molecular analysis confirmed that the resistant plants from the marker-free transgenic line contained SCMVirNIb but bar gene.