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农杆菌介导的转基因技术在植物中已被广泛应用,而目的基因能否发挥功能受到多种因素的影响。前期,通过农杆菌介导的转化,实验室创制了无选择标记、无载体骨架残留的水稻转Xa21基因系CX8621。截止目前,CX8621已稳定遗传16代,依然保持着对水稻白叶枯病的优良抗性。在此基础上,本研究对外源基因Xa21在CX8621中的整合和表达情况进行了分析。首先,通过在转化载体p BXa21的左右边界与Xa21基因序列设计嵌套引物,确定Xa21被完整地整合到CX8621中。随后,利用改良的Tail-PCR方法体外克隆了整合位点的边界序列,明确了Xa21被整合在CX8621的2号染色体上。然后,通过RT-PCR分析了Xa21在CX8621中不同时期和不同组织的表达情况,结果表明Xa21在CX8621中能稳定表达,其表达量的变化与之前报道的抗病性反应吻合。此外,还制备了天然XA21蛋白的抗体,对CX8621不同时期、不同组织中XA21蛋白的表达量进行了检测,结果发现在种子中检测不到XA21蛋白。由此,通过对外源基因Xa21的整合和表达分析,为CX8621的转基因生物安全评价提供了部分科学依据。
Agrobacterium-mediated transgenic technology has been widely used in plants, and whether the target gene can function is affected by many factors. In the early stage, the Agrobacterium-mediated transformation created a non-selectable marker in the laboratory, and the Xa21 gene line CX8621 in rice without vector remains. Up to now, CX8621 has been stably inherited for 16 generations and still retains excellent resistance to bacterial leaf blight. On this basis, we analyzed the integration and expression of exogenous gene Xa21 in CX8621. First of all, it was confirmed that Xa21 was completely integrated into CX8621 by designing a nested primer on the left and right borders of the transformation vector pBXa21 and the Xa21 gene sequence. Subsequently, the boundary sequence of the integration site was cloned by the modified Tail-PCR method. It was confirmed that Xa21 was integrated on chromosome 2 of CX8621. Then, the expression of Xa21 in CX8621 cells in different periods and different tissues was analyzed by RT-PCR. The results showed that Xa21 was stably expressed in CX8621, and the change of Xa21 was consistent with the previously reported disease resistance. In addition, the natural XA21 protein antibody was also prepared. The expression of XA21 protein in different tissues and tissues of CX8621 cells was detected. As a result, XA21 protein was not detected in the seeds. Therefore, the integration and expression analysis of exogenous gene Xa21 provided some scientific evidences for the safety evaluation of CX8621 transgenic organisms.