| Rice starch is made up of amylose and amylopectin. The composition and structure of starch, which is measured as the ratio of amylose to amylopectin and the amylopectin branch chain-length distributions affect the eating quality, cooking and processing quality of rice and even have close relation with the productivities of rice. And it is also the key factors determining the uses of starch in industrial applications. In the past several decades, the productivities of rice have been improved to a much higher level because of the dwarfing dreeding and the utility of hybrids in China and therefore the rice is relatively overmuch. The producing of much better quality rice for industrial use is of extreme importance to develop the productivity and trade of rice, and it is even important for the economic and social benefits of China, in which rice is the top one crops. With the develo'pment of food and other industry, more and more different types of rice is needed, and high amylose rice starch is especially needed. There are very few rice varieties with high or very-high amylose content, so the development of a more efficient approach to enhance the amylose content of rice is very important. Recent advancements in molecular genetics and biotechnology offer new opportunities to enhancing of rice amylose content.In this study, we attempt to genetically engineering rice through introducing antisense sbe genes into rice to reduce the expression of sbe genes and to increase the apparent amylose content of starch in transgenic rice plants. The research work includes three complementary parts:(l) study on the inheritance of high amylose content inrice;(2) cDNA cloning and sequence analysis of rice Sbel and Sbe3 genes;(3) analysis of amylose content of mature seeds in transgenic rice plants with antisense sbe genes. The main results were as follows:1. 84 crosses were made between 14 rice varieties with high amylose content(AC), the analysis of Fj seeds of each cross showed: AC of FI seeds varied among different crosses, AC of FI seeds of majority crosses were lower than the average of their parents, AC of three crosses were higher than their parents' average, but one cross were significantly lower than its' parents. These results suggested that there were distinct difference on the genetic bases of AC among different rice varieties.2. Sbel and Sbe3 gene were cloned from the template cDNA library, which was synthesized by improved RT-PCR technique from the mRNAs of the immature rice seeds ofjaponica rice Wuyunjing 7. DNA sequencing analysis showed that the size of the cloned Sbel and Sbe3 cDNAs were 2490 bp and 2481 bp, respectively, and carried their entire coding sequences. Comparison analysis indicated that the Sbe3 sequences were the same as the reported sequence and their identity was 100%. There were only four base-pairs difference, which resulted in two deduced amino acids alteration, between the cloned Sbel cDNA and the reported3. On the basis of the cloning of sbe 1 and sbe3 cDNAs, several fragments of sbe genes were cloned by PCR technique in order to construct antisense sbe chimeric genes and introduced into rice through Agrobacterium-mediated transformation. Several antisense sbe chimeric genes were constructed driven by the endosperm-specific promoters, namely, the Wx and Sbe and glutelin Gtl promoters. These chimeric genes were all transferred into one japonica rice Wuxiangjing 9 and three indica rice varieties Longtefu B, Teqing and Xieqingzao B via Agrobacterium. The expression of the Sbe genes and the amylose content of the seeds in transgenic rice plants were analyzed, the main results were following: (1) Southern analysis showed that the antisense She genes had been integrated into the genome of most of the transgenic rice plants with one or two copies, butthere were more than two copies of antisense Sbe genes in some transgenic rice plants. The antisense Sbe genes were stably transferred to TI generation and the segregation of TI transgenic rice plants was accord with the Mendel's m...
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