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本研究通过农杆菌介导玉米茎尖遗传转化法将含有花粉特异启动子Zm13驱动的Gene-deletor外源基因清除系统、表达元件Ubi-Zm SDD1-3’UTR、Actin-Bt-NOS和Ubi-Bar::GUS-3’UTR的植物表达载体遗传转化玉米交51,经过除草剂初步筛选、GUS组织化学染色和PCR分子鉴定,获得31株转基因植株。研究表明转基因植株比野生型植株具有更强抗旱性、抗玉米螟能力和抗除草剂草丁膦的能力。抗旱性鉴定表明干旱处理后转基因植株的存活率比野生型高70%,同时,转基因植株的叶片保水性显著高于野生型植株。抗虫性试验表明转基因植株的叶片蛀孔少而小,对玉米螟具有明显抗性,还抑制了玉米螟的取食和发育。叶片离体和田间抗除草剂试验表明转基因植株比野生型能够抵抗更高的除草剂浓度,最高可抵抗150 mg/L草丁膦浓度。同时,在统计的20株转基因玉米植株的基因删除效率时发现部分植株可完全清除玉米中的外源基因,对于未完全清除的玉米植株还需要进一步研究。本研究创制了一种抗虫、抗除草剂、抗旱的复合性状转基因玉米新种质,可为培育多性状叠加的转基因玉米新品种奠定基础,同时结合“Gene-deletor”技术,为实现转基因生物安全提供技术支撑。
In this study, Gene-deletor exogenous gene-driven system, which contains pollen-specific promoter Zm13, was constructed by Agrobacterium tumefaciens mediated transformation of maize shoot tips. The expression components Ubi-Zm SDD1-3’UTR, Actin-Bt- The plant expression vector of Bar :: GUS-3’UTR was transformed into Zea mays L. 51. After preliminary screening of herbicides, GUS histochemical staining and PCR molecular identification, 31 transgenic plants were obtained. Studies have shown that transgenic plants have stronger drought resistance than the wild-type plants, resistance to corn borer and resistance to herbicide glufosinate. Drought resistance identification indicated that the survival rate of transgenic plants after drought treatment was 70% higher than that of wild type plants. Meanwhile, the leaf water retention of transgenic plants was significantly higher than that of wild type plants. Insect resistance test showed that the leaves of transgenic plants had fewer and fewer holes, which were obviously resistant to Ostrinia furnacalis and inhibited the feeding and development of Ostrinia furnacalis. Leaf in vitro and field herbicide resistance tests showed that transgenic plants were able to resist higher herbicide concentrations than wild type, up to 150 mg / L glufosinate. At the same time, we found that some plants could completely eliminate exogenous genes in maize while the efficiency of gene deletion of the 20 transgenic maize plants was further studied. In this study, a new germplasm of transgenic corn with insect-resistant, herbicide-resistance and drought-resistance was developed, which laid the foundation for cultivating new varieties of transgenic maize with multi-trait superimposition, combined with “Gene-deletor” technology Genetically modified organisms to provide technical support.