Genetically Engineering Rice For Increased Lysine

Plants are the primary source of all proteins consumed by humans and livestock. However, most plant proteins are nutritionally unbalanced, because they are deficient in certain essential amino acids. In general, cereal proteins are low in lysine and tryptophan while legume and most vegetable proteins are deficient in methionine and cysteine. Rice (Oryza sativa L.), 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 rice to over-express a gene encoding the lysine-rich protein (LRP) from winged bean. LRP contains 10.7 mol% lysine; its expression in the endosperm of transgenic rice should raise the content of lysine in rice grains. The research work includes three complementary parts: 1) isolation and analysis of the expression of promoters for rice seed storage proteins; 2) expression of LRP in transgenic rice seeds; and 3) expression of glutelin and LRP fusion protein in transgenic rice grains.For Part 1, three endosperm-specific promoters, namely, the glutelin Gt1 (also known as GlnA-2) and GluB-1 and the prolamin RP5 promoters, were isolated from theChinese rice varieties by PCR technique. These promoters, together with the rice albumin RAG1 and maize Ubi promoter, were fused transcriptionally to the GUS coding sequences. All these GUS chimeric genes were introduced into the same rice variety by /Igroforcterwm-mediated transformation. Results from GUS activity in transgenic rice plants showed that all the tested promoters could drive GUS expression in the endosperm of transgenic rice plants, but the expression level differed. The GUS activity driven by the promoter of rice glutelin or prolamin was significantly stronger than that by the promoter of rice RAG1 or CaMV 35S. The maize Ubi promoter also directed high GUS activity in the endosperm of transgenic rice plants. Analysis of the GUS activity in various tissues of transgenic rice plants showed that the control of endosperm-specific expression by glutelin promoters were more stringent than that by the prolamin RP5 promoter. In the case of RAG1 promoter, high GUS activity was detected in the stem and leaf of transgenic rice plants, indicating that the expression of RAG1 promoter is not endosperm-specific. It was interesting to find that the transcription of GUS chimeric gene could be enhanced after inserting the RP5 signal peptide coding sequence between the RP5 promoter and the GUS coding sequence; however, there was no or very low GUS activity in the endosperm of transgenic rice plants. Based on these results, the promoters of rice glutelin and prolamin are suitable for driving foreign proteins express in the rice endosperm.For Part II, the 477-bp cDNA coding sequence of LRP was fused to the 5' regulatory sequence of the rice glutelin Gtl gene or prolamin RP5 gene with or without the signal peptide coding sequence. These chimeric genes were all transferred into a Chinese elite rice variety Wuxiangjin 9 via Agrobacterium. Many transgenic rice plants were regenerated. Northern and Immunoblot analysis showed that the LRP was highly expressed in the grains of transgenic rice plants. Stable accumulation of the 18-kDa LRP in transgenic rice seeds was demonstrated by Tricine SDS-PAGE as well as Western blot, with the highest expression amounting to 12% of the total salt-soluble seed protein. No significant difference in LRP expression, at both mRNA and protein levels, was obse...