Physicochemical Properties Of Starch And Enhance The Amylase Content Of Rice By Genetic Engineering

As one of the most important crops in China, the yield and quality of rice are greatly important to our country's food safety and economic development. In recent years, researches focus mainly on improving quality of rice in China. Rice starch is mainly composed of amylose and amylopectin, the quality of rice and its use are determined by the content and proportion of amylose. The proportion of rice amylose and amylopectin can be altered by regulating the expression of rice key enzyme genes in starch biosynthesis, thus we can breed new varieties of rice that conform to the production requirements. In order to improve amylose content of rice, we carried out several research, the main results were as follows:1. We measured the starch physicochemical properties of five rice varieties with different amylose content, the results showed that the gelatinization temperature(GT) and the starch granules were different between different amylose content rice varieties. Next, we used the quantitative real-time RT-PCR method to make an examination of the expression profiles of 10 rice genes related to starch synthesis in developing seeds (3,5,7 days after flowering), the results showed that Wx, RBE1 and RBE3 were high expressed in developing seeds there were obvious differences between different rice varieties with different AC. The expression level of Wx Gene was positively correlated with AC value in different varieties of rice, while expression of other genes hardly present correlation with AC value.2. Our laboratory got a Zhonghua 11 mutant population by EMS mutagenesis, we used Tilling method and gene sequencing to find a single-base mutation (G to A) on the fourth exon of BE ?a (RBE4), this single-base mutation made the serine (S) to the asparagine (N). The protein structure domain analysis of BE ?a gene showed that the mutation wasn't in the conservative function structure domain of the protein, and the Q-PCR of BE ?a gene showed there were no obvious difference between Zhonghua 11 and the mutant on the relative expression quantity of BE ?a gene. The analysis of starch physicochemical properties of the mutant showed that AC of the mutant was slightly increased compared to the wild type,the average AC was increased 4.97%, at the same time, the GT type of the mutant was changed from the medium type (70?74?) into the low type (?70?). Scanning electron microscope of the starch granules observed that starch granules of mutant was larger than that of wild type, and the outline of the starch granule was not clear and the size was inconformity, but the arrangement was more closely.3. According to the literature's data and our previous results, we found Wx, RBE1 and RBE3 are great significance in AC of rice grain. In this study, we amplified the complete cDNA of Wx gene, SBE gene fragment from rice cDNA and GluB1 gene from rice genomic DNA in the way of RT-PCR and PCR from immature seeds of IR36. The plant expression vector were constructed with over-expression of Wx gene and RNAi of RBE1, it was controlled by a rice specific promoter GluB1. Then we transfered the expression vector into indica cultivar IR36ae (amylose extender) by Agrobacterium-mediated transformation, and verified transgenic plants.