反义外壳蛋白基因介导的抗SCMV转基因玉米研究

SCMV-resistant Transgenic Maize Mediated by Antisense cp Gene

玉米矮花叶病(MDM)是一种世界性病害,在我国主要由甘蔗花叶病毒(SCMV)所致。为探索一条高效、安全的抗SCMV转基因途径,构建了无标记基因的SCMV反义外壳蛋白基因(cp)表达载体pACP。通过冻融法将该载体与抗除草剂标记基因(bar)载体分别导入农杆菌LBA4404,然后共转化玉米自交系综3的幼胚。通过除草剂梯度筛选,从抗性愈伤组织分化获得了35株再生苗。PCR检测证明,其中26株带有抗除草剂标记基因(bar),14株带有SCMV反义cp基因。这14株带有目的基因的玉米植株自交,其种子在田间种植成株行(T1代)。玉米T1代幼苗人工接种SCMV,筛选出2个抗病株率高于70%的株行。ELISA检测表明,抗病株SCMV含量极低,抗性达高抗水平。PCR检测表明,抗病性是反义cp基因作用的结果,并且获得了2株cp基因阳性而标记基因阴性的抗病株。

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. Tile 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.