Gene Expression Analysis Of PWaxy :: Gtl Transgenic Rice

Among four major storage proteins in rice seeds, glutelin not only contains relatively more lysine, but also is the richest. So, we can enrich glutelin content to improve the nutritional quality of rice. Rice storage protein glutelin Gt1 usually locates in the outer layer of rice endosperm. In this study, waxy gene promoter was chosen to direct Gt1 gene expression in the central region of rice endosperm, which may be helpful to the research on transportation and distribution of rice storage proteins and rice nutritional quality. Together with genetic engineering, crossbreeding can also be used in improving rice quality.In the first part of the study, 10 transgenic plants which seeds all showed positive GUS were obtained by screening pCAMBIA1301-pWaxy-Gt1 transgenic seeds. On 4DAF and 18DAF, Gt1 gene and Waxy gene expression in the seeds of the 10 plants were examined by RT-PCR. The result showed that the expression level of Gt1 gene in some transgenic seeds increased and the expression of endogenous Waxy gene didn't influenced by transformation. Western bolt indicated that Waxy gene promoter can direct exogenous Gt1 gene expression on 4DAF, earlier than endogenous Gt1gene. Immunofluorescence microscopy revealed that more Gt1 protein were localized in the central region of rice endosperm. Then, we chose the method of In situ Western hybridization to detect Gt1 in mature rice seeds. Compared to Chao2-10 rice seeds, the glutelin distribution in transgenic seeds were especially in the inner portion of the endosperm .In the second part, by crossbreeding transgenic rice which beared anti-waxy gene and transgenic rice with pCAMBIA1301-pWaxy-Gt1, we generated 3 new type of rice group, totally 18 plants. The seeds of one hybrid plant was very different from other F2 seeds through GUS testing. SDS-PAGE showed that Wx protein in 6 mature F2seeds decreased sharply. The result of Western blot displayed wider band of Gt1 basic subunit. The expression of Gt1 in the central region was higher in transgenic rice seeds and didn't have fixed location.The seeds from the hybrid plant which showed different gus gene expression also displayed a distinct pattern.