Ri质粒介导大豆花叶病毒外壳蛋白基因转化大豆的研究

STUDY ON TRANSFERRING SOYBEANS MOSAIC VRIOUS COATPROTEIN (SMV-CP) GENE INTO SOYBEANS BY RI-PLASMID

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.

The PES plasmid (T-DNA carrying SMV-CP gene) in the E. coli JM109 and was intro-duced into Agrobacterium rhizogenes (PRiA4b) with Ri-plasmid by tri-perental mating and wastranferred into cuiltived soybeans Hefen25, Heinong33 et al. by means of binary vecotors. Thehairy roots were inducted from cotyledonary node. Hypocotyl and whole plants by in Jection ofbacteria transferred. The analysis of paper electrophpresis showed that alkaliod found in the 25%hairy roots and 30% regenerated plants. The cullus of formation of hairy roots were inductedform hypocotylars. The cullus butting, adventition, leaf-like and embryoid were obtained fromimmature embryos and adventitiou were produced form organogeninsis. PCR and southern blot-ting proved that the SMV-CP gene have been introduced and interjected into genome of soybeans.