Construction Of Rice LOX RNAi Vector And Research Of Storability Of Genetic Transformation Into Rice

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.We cloned maize ubiquitin promoter (constitutive promoter) rice seed embryo specific promoter(specific promoter), the first intron of maize ubiquitin from maize and rice genomic DNA and also obtained terminator NOS from plant eukargon expression vectors PBI121 by searching genebank database. The cloned Ubi sequence had 99.6% homology to the reported sequence. Others has 100% homology. We find high homology sequence by comparing many plants LOX gene. According to RT-PCR , a pair of LOX gene reverse complementary sequence was obtained from rice total RNA.The 35S promoter in plant eukargon expression vectors PBI121 was substituted by maize ubiquitin promoter . the GUS result indicate that the promoter has high reactivity.Two plant expression vectors RNAi expression vectors were constructed sucessfully by making use of plant eukargon expression vectors and the cloned fregments, and detected by Enzyme Digestion.â‘ p13UL, containing maize ubiquitin promoter, lox cDNA hairpin structure and hpt gene;â‘¡p13SL, containing rice seed embryo specific promotor lox cDNA hairpin structure and hpt gene.Calli was transformed with p13SL and p13UL via Agrobacterium respectively. All regenerated plant strain were zhonghua 16. we obtain 4 positive plants out of 14 transgenic plant which transformed with p13SL and 3 positive plants out of 6 transgenic plant which transformed with p13UL. This show both of the two vector had inserted into rice genome.Utilizing semi-quantitative RT-PCR, we analyzed the expression level of LOX gene after wound. The result shows that of LOX gene were increased in both controls groups and experimental groups, but the expression level in controls groups was obviously lower than that in experimental groups. This indicted that the lox cDNA hairpin structure inserted into rice genome play a role in RNA interference.