Construction And Transformation Of Met-rich Protein And Lys-rich Protein Genes Co-expressing Vector In Rice

There are more than 20 kinds of amino acids Constituting a human proteins. Human and animals are unable to synthesized eight kinds of essential amino acids by themselves, and therefore have to require these amino acids from outside. Lysine and methionine are the two kinds of essential amino acids. In the nutritional quality of rice, Lysine and methionine are two of the most important restricting amino acids. And the content of these two amino in rice is so low that other amino acids in rice could not be made full use. Therefore, enhancing the content of lysine and methionine in rice grain, you can improve the quality and utilization efficiency of rice grain.In this study, we attempt to genetically engineering rice to co-express a gene encoding the lysine-rich protein ( Lys ) from winged bean and another gene encoding the methionine-rich protein ( Met ) from sesame. Their expression in the endosperm of transgenic rice should rise the content of lysine and methionine in rice grain.1. According to sequences which have been reported, we design a pairs of specific amplification primers. We cloned the glutelin Gt1 promoter JAPAN Japonica rice varieties by PCR and the sequence of the promoter, 99% homologous with the original, was 1314 bp in length. Bioinformatics analysis showed that the cloned Gt1 promoter sequence contained essential promoter components including TATA box and CAAT box and other components such as GCN4 motif and AACA motif which are positive regulatory factors necessary for specific expression in endosperm, and had the ability in controlling gene specific expression in seed.2. Double-gene ( Lys and Met ) plant expression vector p1300-Gt1-Met-Lys was constructed, which contained hygromycin resistance gene and double-gene in the T-DNA segment. Met-rich gene was driven by rice endosperm specific Gt1 promoter and Lys-rich gene was driven by rice endosperm specific GluB-1 promoter.3. The plant expression vector was transformed to the embryonic callus tissue of Taijing 9 through Agrobacterium-mediated method. we obtained 4 transgenic plants, two of which were positive by PCR testing.