Study On Improving The Storable Character Of Rice Grains By Using Genetic Engineering

Food security, which is related to the political stability and economic development of a country or even the whole world, is a strategic problem for the whole world. Rice is still one of the primary staple foods in China and the number of people living on rice is about 8 billion, which is 65 percent of the total population. Foodstuff reserve is the key ring of food security. The losing that was due to deterioration of rice grains during storage is about 3% of the total yield and it wasted several billion yuan per year. Rice grains begin deterioration normally after about one year storage and this will faster in the region with higher temperature and humidity. In order to minimize the losing of rice grains, they are replaced in batch periodically in storage period and this made a big waste of labor force, materials and money.Many research works in recent years indicated that the deterioration of rice grains during storage was caused by lipid peroxidation and lipoxygenase (LOX) was the key enzyme of this reaction, which can be broken up by loss or silence of lox gene. A local variety Daw Dam, which lost lox gene, showed better storable character after a series of storage experiments. Unfortunately, this variety could not be used for rice production due to its bad agronomic characters. This might due to the loss of the lox gene also affected the metabolism of the whole plant. In order to produce the rice with better storage and agronomic characters, antisense RNA, RNA interference technology and endosperm specific promoter were adopted, in our works, to induce the silence of endogenic lox gene in rice endosperm specifically.Considering the target organ of this study was rice grain, which mainly used as food, we used the manA gene from E. coli encoding phosphomannose isomerase (PMI) instead of antibiotic or herbicide markers, which provoked much public concern, as a selectable marker. Mannose selection system was expected to set up in rice transformation and pave the way for future study.Main results are as follows.1) Three binary expression vectors were constructed: 1) p13W4H, containing loxcDNA hairpin structure controlled by endosperm specific promoter (ESP) and hpt gene; 2) p13WSAP, containing antisense lox cDNA controlled by ESP and manA gene; 3) p13W8HP, containing lox cDNA hairpin structure controlled by ESP and manA gene.2) Japonica rice Ishikari-shiroge and T162 were transformed with p13W4H via Agrobacterium. 158 and 56 independent lines were regenerated respectively. There are 149 (94.3%) of Ishikari-shiroge regenerated shoots and 55 (98.2%) of T162 regenerated shoots were PCR positive. Southern blot and genetic analyses showed that the plants contained the foreign genes and the genes were transmitted to the progeny in Mendelian fashion. The immature embryos transformation frequency in this experiment was 13.49% and the mature embryos' was 11.96%. In contrast with the immature embryos transformation frequency (30-40%) normally got in our lab, the frequency we got was relatively low and we made a discussion here.3) Ishikari-shiroge was transformed with p!3W8AP and p!3W8HP via Agrobacterium. 13 and 37 independent lines were regenerated respectively after selected on media containing different concentrations of mannose. The highest transformation frequency (6.9%) was produced on medium containing 15g/l mannose with 5g/l sucrose. Molecular and genetic analyses showed that the plants contained the foreign genes and the genes were transmitted to the progeny in Mendelian fashion. The results indicated that the mannose selection system, which was devoid of the disadvantages of antibiotic or herbicide selection, could be used for rice Agrobacterium-mediaied immature embryo transformation.4) The Tl generation seeds of 9 lines transformed with p13W4H, 2 lines with p13W8AP and 4 lines with p13W8HP were detected with artificial aging method and also LOX activities were measured respectively. With these results, a series of comparison were made with the results of hygromycinB o...