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本研究采用农杆菌侵染法成功实现玄参花叶病毒(FMV)34S启动子控制下的氧物歧化酶基因对巴西橡胶树(Hevea Brasiliensis Muell.Arg)的遗传转化。来自巴西橡胶无性系RRⅡ105未成熟花药的愈伤组织用含有双元质粒载体的根癌农杆菌菌株EHA 101进行转化。该双元载体带有用作选择标记的新霉素磷酸转移酶(nptⅡ)基因及β-葡糖醛酸酶(GUS)报告基因,同时含有带FMV 34S启动子的Mn-超氧物歧化酶(Mn-SOD)目的基因。试验中采用含4.5μM 2,4-D,2.2μM BAP,以及1.1μM NAA的MS培养基筛选并增殖抗卡那霉素的GUS阳性愈伤组织,并利用附加1.5μM BAP,1.4μM KIN,1.1μM NAA,以及2.0μM GA_3的MS培养基诱导高频体胚发生。用于胚胎成熟的培养基为附加1.2μM GA_3及1.5μM TDZ(噻苯隆)的改良MS培养基。试验结果显示,成熟的转化胚胎中有20%发育成小植株。这些小植株经健化之后被移植到营养袋中并置于温室中继续培育生长。最后,本研究通过用SOD及nptⅡ特异性引物作PCR扩增以及用nptⅡ特异性探针与分离自转化植株的DNA模板进行Southern杂交进一步证实了转化事件的发生。
In this study, Agrobacterium tumefaciens infection was used to successfully transform the Hemoglobin gene (Hevea Brasiliensis Muell.Arg) under the control of the 34S promoter of figurine mosaic virus (FMV). Callus from unripe anther of RRII105 from the rubbery clones of Brazil was transformed with Agrobacterium tumefaciens strain EHA 101 containing the binary plasmid vector. The binary vector carries the neomycin phosphotransferase (nptII) gene and the beta-glucuronidase (GUS) reporter gene as selectable markers, together with the Mn-superoxide dismutase with the FMV 34S promoter Mn-SOD) gene of interest. Anti-kanamycin-resistant GUS-positive callus was screened and propagated using MS media containing 4.5 μM 2,4-D, 2.2 μM BAP, and 1.1 μM NAA and supplemented with additional 1.5 μM BAP, 1.4 μM KIN, 1.1μM NAA, and 2.0μM GA_3 in MS medium induced high-frequency somatic embryogenesis. The medium used for embryo maturation is a modified MS medium supplemented with 1.2 μM GA_3 and 1.5 μM TDZ (thidiazuron). Test results show that 20% of mature transformed embryos develop into small plants. These plantlets were harvested and transplanted into nutrition bags and placed in a greenhouse to continue their growth. Finally, we confirmed the occurrence of transformation events by PCR amplification using SOD and nptII-specific primers and Southern hybridization with nptII-specific probes and DNA templates isolated from transformed plants.