Integration and expression of Xa21 in transgenic rice CX8621 / 生物工程学报

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.

Application of competitive PCR for screening selectable marker-free Xa21 transgenic rice / 生物工程学报

Polymerase chain reaction (PCR) is a simple, quick and highly sensitive method. However the accuracy of the conventional PCR assay was often affected by false positives and false negatives. In this study, a protocol competitive PCR was used to reduce the false results in screening for selectable marker-free (SMF) Xa2l transgenic rice plants. The competitive template of Xa21 was the endogenous Xa2l homologous sequence located on chromosome 11. The competitive template of the selectable marker gene, hygromycin phosphotransferase (hpt), was an additive DNA extracted from hpt transgenic Nipponbare (Oryza sativa L). Through competitive PCR analysis of transgenic T1 plants produced by double right border binary vector, false positive or false negative samples were effectively diminished, and genuine SMF Xa21 transgenic plants were obviously obtained. Comparing with the conventional non-competitive PCR, competitive PCR increased the accuracy for selecting SMF Xa21 transgenic plants. The results of bacterial blight (BB) resistance tests and hygromycin B resistance assay of SMF Xa21 transgenic plants testified the reliability of this method.