水稻转基因系CX8621中Xa21的整合和表达

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。

Integration and expression of Xa21 in transgenic rice CX8621

Agrobacterium tumefaciens-mediated transformation system has been widely applied. However, the function of target gene is affected by multiple factors. With this system, we obtained a transgenic rice line CX8621 carrying the bacterial blight resistance gene Xa21. In previous work, we have confirmed that it was selectable maker-free and vector backbone-free. And after 16 generations of breeding, it still maintained perfect resistance to bacterial blight disease. On this basis, we analyzed the integration and expression of Xa21 in CX8621 at the present study. First, based on the border sequences of plasmid pBXa21 and Xa21, we designed nested primers and assured the integrity of Xa21 in CX8621. Second, we cloned the flanking sequences and located Xa21 on chromosome 2 using improved Tail-PCR. Then we analyzed the expression pattern of Xa21 in several tissues and at different developmental stages by RT-PCR. The results show that Xa21 can be stably expressed in CX8621, agreeing well with the disease resistance response as reported previously. In addition, we detected the protein levels of XA21 in CX8621 with antibody of natural XA21 protein. Surprisingly, no XA21 protein was detected in the seeds of CX8621. Thus, the integration and expression analysis of Xa21 in CX8621 provided a part of scientific evidences for the safety assessment of genetically modified rice.