Study Of The Effects Of PULL And ISA3 Gene On Rice Grain Quality Using RNAi Approach

Starch is one of the main components of rice endosperm. Thus, the quality of rice is affected by starch quality and quatity. It is generally accepted that many enzymes are involved in the synthesis of starch, including starch debranching enzyme (DBE). DBE was found to be correlated closely with the formation of final starch structures. Then, further studies on functions of DBE are required in explaining starch synthesis mechanism. In plants, there are two types of starch debranching enzymes, isoamylase (ISA) and Pullulanase (PULL or limit dextrinase). Three types of ISA been has been characterized from rice, ISA1, ISA2 and ISA3. In order to study the fuctions of PULL and ISA3, their RNA interference structures were introduced into a japonica rice variety, Nipponbare, using agrobacterium-mediated transformation. And double mutant were selected from the offspring of hybrids of two type transgenic rice. In present study, we focus on 4 double mutant lines (PULL/ISA3-RNAi) and their parent lines,4 RNAi lines for PULL gene (PULL-RNAi) and 3 RNAi lines for ISA3 gene (ISA3-BNAi). The main results were as follows:1. Real-time PCR analysis showed that the expression of PULL and ISA3 gene was reduced significantly in developing seeds of transgenic lines. The expression level of double mutant is very similar to their parental transgenic lines. It indicates that both PULL and ISA3 RNA interference structures successfully inhibit target gene expression.2. No significant difference was detected among different transgenic lines of the total starch content,amylose content and water-soluble polysaccharide. These results indicated that the effects of lower expression of PULL and ISA3 genes on starch and water-soluble polysaccharide distribution in endosperm were not significant.3. THe RVA and DSC analysis was performed for study the characters of transgenic plants. The results turned out that some of RVA and DSC values of the transgenic were lower than the wild type.4. No significant differences were detected in starch crystal between transgenic rice starch using X-ray diffraction and total reflection infrared spectroscopy analysis.5. Amylopectin structure denoted by FACE and GPC results showed that both of PULL-RNAi and ISA3-RNAi containing fewer chains with 10<DP<20 compaired with the wild type. The double mutants, PULL/ISA3-RNAi have more chains with 10≤DP≤35 than their parental lines. Then, both PULL and ISA3 affect the fine structure of amylopectin and the two genes have similar functions during starch synthesis.6. Most of the agronomic traits of the trangenic lines containing RNA interference structure are comparable to the wild type.