Construction Of Expression Vector With Gy7 Gene Under The Gt1 Promoter And Transformation Of Rice

Crop proteins are the major source of dietary protein consumed by over half of the world population, but most plant proteins are nutritionally incomplete due to their deficiency in several EAAs. It's hard to improve the content of EAA efficiently by conventional breeding. The advance of modern biotechnology has provided a new way to enhance the nutritional value of crop proteins. The research and development of improving nutritional quality of crops by genetic engineering were reviews in this paper. It'll lay the foundation for larger-scale studies and applications in this area.In our studies, 5.3kb Gt1 5'upstream sequence with the Gt1 promoter and its singnal peptide was sucessfully cloned from rice cultivar Koshihikari. It was inserted into the plasmid pCAMBIA1300, which was called pCAMBIA1300-5.3kbGt1.Then Lys-rich soybean Gy7 gene which had been cloned in our lab was inserted into pCAMBIA1300-5.3kbGt1.Meanwhile,resistance marker gene (hygromycin) and 35S promoter in pCAMBIA1300 were cut out. The expression vector constructed was called pCAMBIA1300-5.3kbGt1-Gy7.We hope that the nutritional quality of rice will be improved by expressing Gy7 gene.The two kinds of Agrobactium tumefaciens, which respectively carried the vector p1300-HWG (contains hygromycin gene and gus gene) and vector pCAMBIA1300-5.3kbGt1-Gy7 (only has Gy7 gene without marker gene), were mixed at the density ratio of 1:9. The T-DNAs of the two vectors p1300-HWG and pCAMBIA1300-5.3kbGt1-Gy7 were introduced into Chao2-10 by the co-transformation method of which is safety transgenic technique. 67 independent transgenic calli differentiated and regenerated 255 plants. Using PCR analysis showed that 9 lines that regenerated 23 platns among 67 independent transgenic ones were co-transformation plants, 13.43% co-transformation frequency. The vector pCAMBIA1300-5.3kbGt1-Gy7 T-DNA in the rice genome was confirmed through Southern blot analysis. Gy7 gene could normally transcript in the rice immature seeds through RT-PCR analysis. Then T1 seeds of 13 plants that regenerated from 6 co-transformed lines, which were without report and marker gene, were screened by using gus gene.Gy7 gene were confirmed in 5 GUS negative T1 transgenic lines from these 6 ones by PCR. The study can lay the groundwork for filtrating new rice materials which containing homozygotic target gene, nutritional quality of protein rich in lysine and without the marker gene and report gene.In order to compare the expression of Gy7 gene with that of its cDNA under 5.3kbGt1 promoter, RNA were extracted from immature seeds of soybean Nannong 87C-38.Then Gy7 cDNA were cloned by RT-PCR. After being compared the two sequences with Genbank (AF319776 and AF319777) by vector NTI software, their identities were 99% and 99.6%. After that,11S glycinin Gy7 cDNA was inserted into pCAMBIA1300-5.3kbGt1 and the new expression vector was called pCAMBIA1300-5.3kbGt1-Gy7 cDNA. The vector can be introduced into rice by Agrobacterium-mediated transformation in the future. It will lay the foundation of finding the proper gene to improve the nutritional quality of crops such as rice.It was also first time to introduce the superior Lys-rich soybean Gy7 gene into rice under the 5.3kbGt1 promoter, which was cloned from rice cultivar Koshihikari containing higher level of glutelin, to improve the quality of rice both at home and abroad. This research will lay the groundwork for improving the nutritional quality of rice.