Duplex Construct Of Rice Pullulanase Gene And Its Transformation For The Gene Silencing

Starch is the major carbohydrate reserve in cereal grains. It constitutes the major source of calories in the human diet. Moreover, it is widely used as a raw material in industry, particularly in the food and medicine sectors. The function and end use of starch relies on its structure and property. According to our current understanding, starch biosynthesis occurs mainly through the action of four classes of enzymes: ADP-Glc pyrophosphorylase, starch synthase, starch-branching enzyme and debranching enzyme.The development of molecular biology techniques makes it possible to regulate the activity of key enzymes in starch biosynthesis and modify the structure and properties of starch by genetic engineering. The post-transcriptional gene silencing (PTGS) or RNA interference (RNAi) technique is a new and effective approach for this purpose.In the present study, the activity of a starch debranching enzyme of rice is down regulated by constructing and transforming an intron containing duplex plasmid into rice genome. This will facilate the understanding of its role in starch biosynthesis and the potent of altering the structure of amylopectin and starch properties by RNAi . The results are as following:1. An RNAi plasmid was generated that contains an duplex construct of a pullulanase gene fragment separated by an intron (duplex construct). It is confirmed by restriction enzymes digestion, sequencing and Southern blot.2. 29 resistant lines were obtained by Hygromycin selection. 28 of them were confirmed containing the duplex structure by PCR and Southern blot.3. Western blot with the protein from developing endosperms against the antibody of pullulanase showed that the duplex structure was expressed in the developing endosperm. Rice plants with decreased accumulation of pullulanase have been produced. Rice lines with varied pullulanase accumulation were obtained. 012 line has sharply decreased its accumulation of pullalanase to 10% of wildtype,suggesting the construction of duplex plasmid was effective.4. Rice lines where the Tos17 retrotransposon is inserted into pullulanase gene didn't show the knockout effect. The accumulation of pullulanase in the Tos17 insertion homozygous is in the same level with that of wildtype.This may be due to the fact that in the line available that the insertion position was in the region of an intron.