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本文用含有Ri质粒的发根农杆菌介导,克隆在大肠杆菌JM109中,中间载体质粒PES(具有大豆花叶病毒外壳蛋白基因),通过三亲杂交的方法,将质粒PES导入具Ri质粒的发根农杆菌(pRiA4b)中,用二元载体法转化黑龙江省常见的大豆品种,合丰25,黑农33等品种.用子叶节,胚轴,幼胚和整株感染转化子菌液,诱导出毛状根,经纸电泳检测有25%左右的毛状根含有冠瘿碱.感染的下胚轴,诱导的毛状根直接形成愈伤组织,从愈伤组织分化出芽,幼胚培养获得不定芽,叶状体和胚状体结构,由器官发生途径产生丛生芽.从下胚轴和子叶节获得丛生芽并再生植株,经纸电泳检测约30%再生植株含有冠瘿碱,PCR和DNA斑点杂交检测均证明大豆花叶病毒外壳蛋白基因,已导入并整合到大豆基因组中.
In this paper, Agrobacterium rhizogenes containing Ri plasmid was introduced into Escherichia coli JM109, and the intermediate vector plasmid PES (with soybean mosaic virus coat protein gene) was introduced into Pichia pastoris Agrobacterium rhizogenes (pRiA4b) were transformed into common soybean cultivars, Hefeng 25 and Heinong 33 cultivars in Heilongjiang Province by binary vector method.The transformant bacterial cells were infected with cotyledonary node, hypocotyl, immature embryo and whole plant, Induced hairy roots, paper electrophoresis detected by about 25% of the hairy roots containing opine .Infected hypocotyls, hairy roots induced direct formation of callus, budding from the callus differentiation, immature embryo culture The adventitious shoots, fronds and embryoid bodies were obtained, and clusters of shoots were generated from organogenesis pathways. Clustered shoots were regenerated from hypocotyls and cotyledons and regenerated from plants by electrophoresis. About 30% of regenerated plants contained opine, And DNA spot hybridization test all proved that the soybean mosaic virus coat protein gene has been introduced and integrated into the soybean genome.