Genetic Transformation Of High Lysine Protein Gene Into Rice

Rice is the main food crop,it is a staple food for nearly one-half of the world's population. However, some storage proteins of gramineous plants such as maize and rice, lysine content is low. Lysine is the first limiting amino acid, seriously affect other amino acids digestion. Improve the lysine content of rice, can significantly improve the utilization of rice protein. So how to improve the lysine content of rice, has become the focus of concern.Traditional breeding has been tremendously successful in improving the lysine content of food and feed; however, this process is time-consuming and energy-consuming The possibilities are little.The development of genetic engineering technology, provides an effective means for improved nutritional quality of rice seed proteins, and some progress is made in the study. However, with the commercial production of transgenic plants, their safety issues aroused people's special attention. The use of selectable marker genes are the principal source of security issues.In this study, soybean high lysine genes, GsHLP2 and GsHLP8 the proportion by weight are 18.22% and 19.93%, With independent intellectual property rights transformated into rice, analysis of high lysine genes GsHLP2 and GsHLP8 transcription and lysine content, achieved the high-level expression of high lysine protein gene in rice,attempted to provide a approach to cultivate the transgenic rice with high lysine nutrition. Meanwhile, we will obtaine transgenic rice without selectable marker genes using of two T-DNA vector, with the reeombinationin of offspring, completely solved the security issues of selectable marker gene and what its product may lesd to. This study attempts to provide a new approach to cultivate the crops with high nutrition value and biosecurity.The major results were as follows:1. Cloning rice seed-specific promoter GluB-4Seed-specific promoter GluB-4 was cloned from rice by RT-PCR. The sequence of promoter GluB-4 was consistent with GenBank report.2. Construction of eight plant expression vectors with a high-lysine genes GsHLP2 and GsHLP8Eight two T-DNA plant expression vectors were constructed which regulated by constitutive Ubi or rice seed-specific promoter GluB-4, with and without enhancer P2, Bar gene for the selectable marker gene, cotainde high lysine protein genes of GsHLP2 or GsHLP8, and then were translated into Agrobacterium LBA4404 for rice transformation.3. Transformation and PCR identification of positive plantsThe plant expression vectors were transfered into rice "wuyoudao 1"by Agrobacterium Tumefaciens.10 resistant seedlings were obtained, which were transformed with LBA4404(pTTBUP2G8); 20 resistant seedlings were obtained, which were transformed with LBA4404(pTTBGG8); 12 resistant seedlings were obtained, which were transformed with LBA4404(pTTBGP2G8), PCR positive plants were 4,14 and 7 separately.4. Detection of high lysine gene GsHLP8 transcription in normal and induced plantsT0 generation plants of PCR positive transformed with pTTBUP2G8 were detected by Real-Time PCR, showed that pTTBUP2G8 were overexpressed in all lines.5. Lysine content analysis of transgenic plantsLysine content were detected from Real-Time PCR positive plants by Ninhydrin color method, showed that lysine content of the transgenic plants pTTBUP2G8 increased 110.51%.