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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 disease (MDM) is a worldwide disease mainly caused by sugarcane mosaic virus (SCMV) in China. In order to explore an efficient and safe anti-SCMV transgenic pathway, a non-marker SCMV antisense coat protein gene (cp) expression vector pACP was constructed. The vector and the anti-herbicide marker (bar) vector were introduced into Agrobacterium LBA4404 by freeze-thaw method, respectively, and then co-transformed into immature embryos of maize inbreds. Thirty-five regenerated shoots were obtained from resistant callus by herbicide gradient screening. PCR test showed that 26 of them were resistant to barley marker and 14 of them were infected with SCMV antisense cp gene. The 14 maize plants with the gene of interest were selfed and their seeds were grown in the field (T1 generation). The T1 seedlings of corn were inoculated with SCMV by artificial selection, and two lines with more than 70% disease-resistant plants were selected. ELISA test showed that the SCMV content of the resistant strain was extremely low and the resistance level was high and anti-resistant. PCR analysis showed that the disease resistance was the result of the antisense cp gene and two resistant strains with positive cp gene and negative marker gene were obtained.