关键词: 玉米, 抗SCMV, 反义外壳蛋白基因, 无标记基因表达载体

Abstract:

Maize Dwarf Mosaic (MDM) disease is one of the most widely distributed and economically important virus diseases of maize in many countries. The disease is caused mainly by sugarcane mosaic virus (SCMV) in China. The resistance to the disease in maize containing virus-derived transgenes by the expression of functional coat protein gene(cp) from maize dwarf mosaic virus (MDMV) or SCMV has been reported and transgenic plants showed modest resistance to related viruses. But protein expression or mRNA transcription may pose problems with acceptance because of possible interactions with other viruses. And in these studies, selectable marker genes conferring antibiotic or herbicide resistance were used for the efficient transformation. The selectable marker genes have caused public concerns on the biosafety.
In an attempt to generate safe marker-free transgenic maize plants resistant to SCMV, we inserted SCMV cp gene in antisense orientation into 3’ end of a maize ubiquitin promoter and constructed a marker-free expression vector pACP that only harbors an antisense SCMV-cp gene. Maize immature embryos were co-transformed with the binary vector pACP and a vector harboring bar gene as the selective marker, using Agrobacterium inoculation procedure. Resistant calli were recovered by selection on medium containing Biolaphos. Among 35 regenerated plantlets from resistant calli, 14 plants were certified to contain antisense SCMV-cp gene by PCR amplification. T₁ lines derived from the 14 transgenic plants were challenged with SCMV inocula in field and the percentages of resistant plants in two lines were higher than 70%. Two SCMV-resistant transgenic plants in one T₁ line were found to be marker free by PCR assay. The results demonstrated that stable expression of antisense SCMV-cp gene in transgenic maize plants results in resistance to SCMV, which provides a new useful and effective alternative to combat SCMV because it does not require the expression of a functional viral gene product and avoid possible interactions with other viruses. In addition, generation of selectable marker-free transgenic plants avoids responds to public concerns on the biosafety of selectable markers and will support multiple transformation cycles for transgene pyramiding. The results further confirmed that co-transformation is a comparatively simple and available system for obtaining marker-free plants of a cross-pollinating propagated crop.

Key words: Maize, Resistance to SCMV, Anti-sense RNA, Expression vector without marker gene