The Effects Of Simultaneously Antisense Gene Targeting Of Two Branching Enzymes On The Starch Physiochemical Properties In The Endosperm Of Transgenic Rice

Rice endosperm starch consists of amylose and amylopectin, and both their composition and structure, i.e. the ratio of amylose to amylopectin and the distribution of branching chain-length of amylopectin, largely affect not only the cooking and eating quality, the processing quality, and even the productivities of rice, but also the properties of starch applied in industrial production. For example, the high amylase content starch has been used widely in industrial fields, especially in food industry.Through the manipulation of the genes encoding the starch branching enzymes, scientists have bred selected several novel varieties with high amylose content (AC) in maize and potato, but up to now it wasn't achieved in rice. In the previous study, a few transgenic rice lines containing the antisense Sbe1 or Sbe3 gene have been generated in our laboratory. Thus, in present study, the introduced two antisense Sbe genes were combined into the same rice plants via conventional crossing with the aim to simultinously decrease the expression of both endosgenous Sbe genes for maxiumly increasing the apparent amylose content of starch in transgenic rice plants. The experiments were performed to test the inheritance and expression of both antisense Sbe genes in thesubsequent the physichemical properties of endosperm starch. The main results were showed as followings.1. Two hybrids, XB3-3/LB1-5 and XB3-2/LB1-2, produced by the crossing between the transgenic rice of Xieqingzhao containing the antisense Sbe3 gene (XB3) and that of Longtefu transferred by the antisense Sbel gene (LB1). After three generations of self-pollination, the transgenic rice plants containing both homozygous antisense Sbe genes were screened. But the results from AC analysis showed that the AC in the mature seeds of these homozygous transgenic lines were measured to be comparable with that of either the non-transformed wild types or the transgenic rice containing the single antisense Sbe gene.2. Another hybrid LB3-1/TB1-1 was generated from the crossing between the transgenic rice of Longtefu containing the antisense Sbe3 gene (LB3-1) and that of Teqing containing the antisense Sbel gene (TB1-1). The inheritance of both transgenes was carefully studied in the subsequent five selfing generations, but, unfortunately, no transgenic rice line with both homozygous antisense Sbe genes was selected. The mechanism was not known. Thus, only the offsprings containing both heterozygous antisense Sbe genes were selected for further study. Contrarily, among F3 to F5 generation, the AC in mature seeds of the selected transgenic offsprings carrying both heterozygous transgenes was measured to much higher than that of the un-transferred wild type or transgenic rice containing the single antisense Sbe gene, and the highest reached to 46.9% in one combined plants in F3 generation, while it was only 28.54% and 27.65% in the non-transformed parents Longtefu and Teqin, respectively. Therefore, these heterozygous transgenic lines were selected for further analysis.3. Several offsprings in F4 generation derived from the hybrid LB3-1/TB1-1 containing different combination of transgenes, such as only antisense Sbel, only antisense Sbe3, both of them, or neither of them were selected, and the expression of endogenous Sbe genes in these lines were analyzed by both Northern blot and immunoblot hybridization. The results showed that the expression of endogenous Sbe genes were much lower in immature seeds of the plants containing both antisense Sbe genes than these in the plants containing neither of them, whereas the expression level in the plants carrying the single antisense Sbel gene was comparable with that in the non-transfomed wild types.4. The starch gelatinization property and absorbance spectrum were much poorer in the endosperm of mature seeds of individual rice plant containing both antisense Sbe genes than that of either un-transferred wild type or transgenic rice containing only the single antisense Sbe gene, indicated that the starch in transgenic rice plant by simult...