Using Expression Vector With Gt1 Gene Under The Rice Waxy Promoter Changed The Quantity And Distribution Of The Rice Seed Protein

In this paper,we reviewed the advance of rice seed storage proteins,Glutelin, accounting for about 80%of total protein in the endosperm,and prolamin were major storage proteins in rice seeds(Yamagata H,et al.,1982).Compared with prolamin, glutelin not only contained relatively more lysine content(Zhao ZS,et al.,1995) which is the first limit amino acid,but also is easy to be digested(Tanaka Y,et al., 1975a,b).The research showed that the most of the rice storage protein accumulated in the sub-aleurone layer,and the little grain of rice located in the central region of rice.Therefore,in the research of improving the protein of rice,even if the selected promoter was the endosperm-specific expression promoter which could significantly improve the nutritional quality of rice,the problem of the rice protein loss couldn't be overcome.So we had to not only choose the promoter which is specially expressed in the rice endosperm,but also have to choose the promoter which can guide the foreign gene uniformly distributed in the endosperm.In this study,232 varieties of indica rice derived from waxy 2.8kb gene(Waxy) promoter,and with the gus reporter gene and Gt1 gene from the NO.11 rice varieties construct pCAMBIA1301-pWaxy-Gt1 recombinant expression vector.Using the Agrobactium tumefaciens,the pCAMBIA1301-pWaxy-Gt1 plasmid were introduced into Chao2-10 rice mature embryo callus induction,56 independent transgenic calli differentiated and regenerated 79 plants.Using PCR analysis showed that 37 lines that regenerated 37 platns among 56 independent transgenic ones were transformation plants.The vector pCAMBIA1301-pWaxy-Gt1 T-DNA in the rice genome was confirmed through PCR-Southern blot analysis.Gt1 gene could normally transcript in the rice immature seeds through RT-PCR analysis.And have nothing to do with the expression of endogenesis waxy gene and Gt1gene.Then T₁ seeds that regenerated from transformed lines were screened by using gus gene.The study showed that the most of the transfromation plants were single copy.And the result of both the SDS-PAGE and Western bolt indicated that the expression of the glutelin from transformed seeds were higher than the control's,which means exogenous Gt1gene increased the rice proteins.Base on that,we chosed the methords of In situ Western hybridization to detect the glutelin distribution in transformed mature rice seeds, compared to Chao2-10 rice seeds,the glutelin distribution in transformed mature rice seeds were especially in the inner portion of the endosperm. This research got the high-protein rice variety and will lay the groundwork for furthern education to filter the rice variety that extremely expressed the foreign protein in the inner portion of the endosperm to improve the nutritional quality of rice.