The Application Of Gene Stacking And MIGS Technology In The Starch Biosynthesis In Rice

Rice (Oryza sativa) is one of the three main food crops in the world. More than half of the world's people regard it as the main grain. By improving the yield and the quality of rice to satisfy the diversified needs of human beings in production and life have vital practical value. The accumulation of starch in seeds plays an important role in yield.The synthesis of starch is usually accomplished by a variety of crucial enzymes in the organism. In order to regulate the expression of genes in starch synthesis pathway, we use miRNA induced gene silencing technology and Gene Stacking technology to study it on the starch composition and starch yield. The main research contents and results are as follows:1. Based on the previously established MIGS efficient silencing system which suit for nmonocotyledonous, a MIGS-OsGBSSI single gene vector was designed to silence granule-bound starch synthase. The OsGBSSI gene was inhibited on the transcription level by semi-quantitative RT-PCR. The Iodine staining experiment showed that the wild type was blue-black and the transgenic was reddish brown which close to glutinous rice' color. Under the observation of scanning electron microscope, the transgenic starch granules were round and arranged densely, and there were a little different from wild type in the morphology. Finally, the amylose was determined by dual wavelength method. The content of starch decreased from 24.53% to 16.38%, the silencing efficiency was reached 33.20, and the amylose content in wild type was markedly significantly different from the transgenic. These indicate that the application of' MIGS in starch s\ynthesis pathway was successfully realized.2. Based on the gene stacking technology, a suitable multi-genes co-expression system was established. We selected two important gene OsAGPL2 and OsAGPS2b that involved in starch synthesis to set up two single gene vectors (pZHY973? pZHY975)and a gene stacking vector pZHY976. The results showed that the transcription level of OsAGPL2 or OsAGPS2b in the endosperm was higher than that in the wild type during 20-25DAP of grain filling stage, but slightly lower than the gene stacking vector's single gene. Semi-quantitative RT-PCR analysis of tissue expression patterns showed that the OsAGPL2 and OsAGPS2b could be detected in the leaves and endosperm, and OsAGPL2 dcesn't expressed in roots. In addition, the soluble sugar and total starch content of rice endosperm were determined by anthrone-sulfuric method. The results showed that soluble sugar content of transgenic plant endosperm were higher than wild type, and the difference was extremely significant. Compared with the wild type, the total starch content of OsAGPL2 or OsAGPSb was increased slightly, while the gene stacking was the highest to 74.31%, which increased about 11.70%. In addition, the 1000 grain weight of all the transgenic mateirials were higher than wild type, and there were significant differences. It is shown that the gene stacking system have been established and applied successfully to starch synthesis pathway in rice.