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本研究采用农杆菌介导法成功实现巴西橡胶树(Hevea Brasiliensis Muell.Arg)的遗传转化及植株再生。试验中以橡胶树未成熟花药的愈伤组织为转化材料,并通过与含有巴西橡胶树超氧物歧化酶基因(HbSOD)表达载体的根癌农杆菌进行共培养最终获得转基因植株。所用表达载体为CaMV 35S启动子控制下的组成型质粒表达载体,其中带有GUS(β—葡糖醛酸酶,uidA)报告基因,nptⅡ(新霉素磷酸转移酶)选择性标记基因,以及HbSOD(巴西橡胶树超氧物歧化酶)目的基因。为了进一步优化转化体系,试验中还就共培养时间,共培养基成分,以及卡那霉素浓度等因子对转化效果的影响进行了系统性的比较研究和分析。其结果显示,在卡那霉素浓度为300mg/L的培养基上存活的转化愈伤呈现出强烈的GUS阳性反应。之后,这些转化愈伤在含有2.0mg/L精胺及0.1mg/L脱落酸的MS2培养基上再生出体细胞胚胎。然后,成熟胚胎在附加0.2mg/L赤霉酸、0.2mg/L细胞分裂素,以及0.1mg/L吲哚-3-乙酸的MS4培养基上得以继续萌发并进一步发育成小植株。试验中所得转化频率为4%。转化植株在形态上与非转化植株相似。GUS组织学分析显示,GUS基因在转基因植株的胚胎及叶片中均得到表达。PCR(多聚酶链式反应)扩增及基因组Southern印迹杂交分析进一步证实了转基因植株基因组中uidA、nptⅡ及HbSOD基因的存在。
In this study, Agrobacterium-mediated transformation of Hevea Brasiliensis Muell.Arg was successfully carried out. In the experiment, the calli of immature anther of rubber tree were used as the transformation material, and the transgenic plants were finally obtained by co-culturing with Agrobacterium tumefaciens containing the expression vector of Superoxide dismutase gene (HbSOD) of the rubber tree. The expression vector used was a constitutive plasmid expression vector under the control of the CaMV 35S promoter with the GUS (beta-glucuronidase) reporter gene, the nptII (neomycin phosphotransferase) selectable marker gene, and HbSOD (Brazil rubber tree superoxide dismutase) gene of interest. In order to further optimize the transformation system, the effects of co-cultivation time, co-culture medium composition, and kanamycin concentration on transformation efficiency were also systematically compared and analyzed. The results showed that the transformed callus which survived on the medium with kanamycin concentration of 300 mg / L showed a strong GUS positive reaction. Afterwards, these transformed calluses regenerated somatic embryos on MS2 medium containing 2.0 mg / L spermine and 0.1 mg / L abscisic acid. Mature embryos were then germinated and further developed into plantlets on MS4 medium supplemented with 0.2 mg / L gibberellic acid, 0.2 mg / L cytokinin, and 0.1 mg / L indole-3-acetic acid. The resulting transformation frequency was 4%. Transformed plants are morphologically similar to non-transformed plants. GUS histological analysis showed that the GUS gene was expressed in embryos and leaves of transgenic plants. PCR (polymerase chain reaction) amplification and Southern blot analysis of the genome further confirmed the presence of uidA, nptII and HbSOD genes in the genome of transgenic plants.