Studies On Transgenic Regulation Of The Expression Of Glutelin Gt1Gene In Rice

Rice (Oryza sativa L.) seeds accumulate large amounts of storage proteins (seed storage proteins, SSP) in endosperm during seed development. Glutelin account for about80%of the total proteins and deposit in protein body-II.Any change in glutelin content or composition will alter rice quality. Rice, one of the leading food crops and the staple food of over half the world’s population, is a very good and relatively cheap source of energy and protein. However, like other cereals, rice proteins are nutritionally incomplete due to their deficiency in threonine, tryptophan, especially lysine. Traditional breeding approach has been attempted to increase the lysine content in a few food crops, but so has not been successful in rice. Therefore, the development of a more efficient approach to enhance the lysine content of rice protein is of extreme importance. Recent advancements in molecular biotechnology offer new opportunities to improve the nutritional quality of rice grains.In this study, we attempt to genetically engineering of rice to over-express two genes Gtl and LRP from rice and winged bean, reseparately. The lysine-rich protein (LRP) from winged bean contains10.7%lysine; its expression in the endosperm of transgenic rice should raise the content of lysine in rice grains. The research work includes two parts:1) Over express the rice seed storage proteins gene Gtl and analysis of the rice seed storage proteins content;2) expression and molecule character analysis of LRP and LRP fusion protein in transgenic rice grains.Part Ⅰ, two endosperm-specific promoters, namely, the glutelin Gtl (also known as GluA-2) and the prolamin RP5promoters were fused transcriptionally to the glutelin Gtl coding sequences. All these Gtl chimeric genes were introduced into two rice varieties named9983and WY3by Agrobacterium-mediated transformation. Results from glutelin and its mRNA content in transgenic rice plants showed that all the tested promoters could drive Gt1expression in the endosperm of transgenic rice plants, but, Only we can test the exist of mRNA, the increase of glutelin content is not obvious. After remove the glutelin signal peptide or replaced by prolamin RP5signal peptide, the prolamin RP5promoter also can drive Gt1expression; however, there was no obvious improve of glutelin in the endosperm of transgenic rice plants,too.Based on these results, we think, glutelin families is very big which contain lots of members. Only improve one of this members is useless to improve glutelin content in rice. To eliminate the selectable marker gene form transgenic rice, Super-binary vector system, harboring both the Gtl chimeric gene and selectable marker gene but at different integration sites, were thus produced. The super-binary vector system through Agrobacterium were demonstrated also can drive goal gene and select gene into rice genomics. But the super-binary vector system is high-efficiency.Through segregation, several selectable marker-free transgenic rice plants were obtained in the first generation transgenic plants.Part Ⅱ, the477bp cDNA coding sequence of LRP was fused to the5’regulatory sequence of the rice glutelin Gt1gene. Besides a fusion protein approach was used, that is, LRP was fused with glutelin, the major storage proteins of rice grains. The LRP cDNA, after removal of its stop code, was inserted into the basic subunit coding sequence of glutelin Gt1structural gene, resulting one chimeric fusion genes:Gt1::LRP. These chimeric genes were all transferred into a Chinese elite rice variety Wuxiangjin9via Agrobacterium. Many transgenic rice plants were regenerated. After selection, we obtain two transgenic lines HL1and HL5without the selectable marker. RT-PCR showed that all of these two fusion genes could normally transcribe in the developing transgenic rice seeds. The stable expression and accumulation of the LRP in the endosperm of transgenic plants could be observed through Western blot analysis. The T-DNA regions in HL1and HL5are double copy, but one insert site. They were inserted in chromosome10and9, reseparately. These results sufficiently demonstrated that the expression of foreign proteins in rice grains can be significantly enhanced via fusion protein strategy and the endogenous rice glutelin could serve well as a fusion receptor.