Technology Of Cultivation Of A Novel Safety Transgenic Rice Based On The Strategy Of Molecular Excision

Great progress was achieved using the transgenic technology, which has been constantly developed for more than thirty years. It has made great contributions on the security of world foods and opens up a broad future prospects. But the argument on the question of bio-safety, especially the misgivings about the food security, it badly hinders the development and widespread application of the transgenic rice. The research showed that the strategy of excising the foreign gene based on the site-specific recombination came to be an ideal method to resolve the problem on the bio-safety of the transgenic plants at the present time. A method of cultivation new safety transgenic rice using the regulation technology of endosperm-specific promoter combined with the high-efficient site-specific recombination system was established. The high-efficient and specific foreign gene excision was took place in the endosperm of the transgenic rice seeds which was obtained using the method. It made the special foreign gene to be excised effectively in the polished rice (the main edible part of the rice) derived from the transgenic rice. At the same time, there is no specific excision was took place in the embryo of the seeds. They maintained all the genetic characteristics of the transgenic rice and could effectively be inherited to their progeny. The main results of this paper are described as the following:1. Validation of the rice high-efficient endosperm-specific excision system by blocking and activatingTo validate the high-efficient endosperm-specific excision system be used in rice, a rice genetic expression vector of p1300--LF-AGCSGS was constructed. It contains the recombinase gene cre which controlled by the endosperm-specific promoter pGtl, the GUS reporter gene which controlled by the constitutive promoter and two directed efficient fusion recognition sites of loxPFRT. The two sites with some space sequences were placed between the constitutive promoter and the GUS reporter gene. The vector was transformed into the rice mediated by Agrobacterium tumefaciens and the transgenic rice plants were obtained after screening and differentiation. An assaying was carried out to detect and analyze the transgenic rice plants and their progeny. The results showed that an endosperm-specific excision has been complicated in the endosperm of the transgenic rice seeds. The characteristic of the specific excision could be inherited to their progeny steadily and the frequency of the specific excision could be up to 100%. These results showed that it is perfectly feasible for the high-efficient endosperm-specific excision system applying in rice.2. Method of cultivation the safety transgenic rice based on endosperm-specific foreign gene excisionBased on the experimental results of the effective validation of the high-efficient endosperm-specific excision system applying in rice, another rice genetic expression vector, p1300-LF-GCSAGS was constructed. The recombinase expression box and the GUS expression box were constructed between the two directed fusion recognition sites of loxPFRT within the vector, it was hoping to excise both of them by the site-specific recombinase system.62 transgenic rice plants were obtained after the vector p1300-LF-GCSAGS was transformed into the rice mediated by Agrobacterium tumefaciens. A preliminary GUS staining assay was performed on selfing seeds of To plants. It was suggested that all of the transgenic rice plants represented a high-efficient endosperm-specific excision. The further analysis showed that:(A) the characteristic of the endosperm-specific excision could be inherited to their progeny steadily, (B) the molecular mechanism of the excision are consistent with the expectation of the experiment, (C) the frequency of the endosperm-specific excision was up to 100% in the selected five pure lines of the transgenic rice which were integrated a single copy or low copies of transgenes, (D) and the average excision efficiency of line or clone was up to 86.46%. The above results suggest that it is a feasible and efficient method for cultivation new safety transgenic rice by endosperm-specific foreign gene excision. Including the endosperm of the obtained new safety transgenic rice seeds, a specific excision was took place. It made the special foreign gene to be excised effectively from the endosperm genome of the transgenic rice and easy to obtain the safety polished rice. At the same time, the whole seeds maintain all the good genetic characteristics of the transgenic rice and ensure the advantages of their progeny.3. Method of cultivation the safety transgenic rice by hybridizationThe prefer results were indicated that when the recombinase gene and the recognition sites of the high-efficient endosperm-specific excision system were placed in the male and female parents, it is possible to make them together by hybridization method and to obtain the same whole high-efficient endosperm-specific excision system in F1 plants. To validate the method, a vector p1300-GCMS containing only the recombinase controlled by the endosperm-specific promoter of PGtl and a vector p1300-LF-AGS containing the recognition sites were constructed. After transformation, the pure lines integrated with single copy of transgene were selected as male and female parents for hybridization, respectively. The results showed that we could obtain the efficient endosperm-specific excision in the progeny of the hybrid rice and the characteristic of the specific excision could be inherited to their progeny steadily.