聚合双反义分支酶基因对稻米淀粉理化特性的影响
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摘要
稻米淀粉主要由直链淀粉与支链淀粉两部分组成,两者的组成和结构与稻米食用品质、蒸煮加工品质、甚至于产量都有着密切的联系,同时也是决定其工业用途的最重要因素之一。直链淀粉由于其特殊的分子结构目前在食品等工业领域中的应用越来越广泛。目前,科学家们已经利用基因工程等技术,对控制支链淀粉合成的分支酶基因进行遗传操作,在玉米和马铃薯等作物中培育出了直链淀粉高达70%以上的新品种,然而在水稻中还没有相关报导。本研究是在已经分别获得转单个反义分支酶基因Sbe1和Sbe3水稻材料的基础上通过常规杂交技术,将两个反义分支酶基因聚合入同一水稻植株内,以期高效抑制水稻胚乳中Sbe基因的表达,达到抑制支链淀粉合成进而相对提高稻米直链淀粉含量的目的。本研究对两个反义分支酶基因在同一水稻植株内的遗传、表达及其对抑制内源分支酶基因表达和稻米淀粉理化特性等的影响作了较为详细的研究,主要结果如下:
1、利用籼稻协青早转反义Sbe3基因(XB3)和龙特甫转反义Sbe1基因(LB1)材料,分别组配了2个杂交组合XB3-3/LB1-5和XB3-2/LB1-2,经连续3次自交,并结合对导入基因的分子检测,从其自交后代中分别选育到了双纯合体材料。对成熟种子中直链淀粉含量的测定结果表明,聚合两个反义分支酶基因的双纯合体植株种子中的直链淀粉含量与未转化对照亲本、转单个反义Sbe基因亲本等比较并没有明显提高。
2、利用籼稻龙特甫转反义Sbe3基因(LB3-1)和特青转反义Sbe1基因(TB1-1)
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 the
subsequent 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
1、利用籼稻协青早转反义Sbe3基因(XB3)和龙特甫转反义Sbe1基因(LB1)材料,分别组配了2个杂交组合XB3-3/LB1-5和XB3-2/LB1-2,经连续3次自交,并结合对导入基因的分子检测,从其自交后代中分别选育到了双纯合体材料。对成熟种子中直链淀粉含量的测定结果表明,聚合两个反义分支酶基因的双纯合体植株种子中的直链淀粉含量与未转化对照亲本、转单个反义Sbe基因亲本等比较并没有明显提高。
2、利用籼稻龙特甫转反义Sbe3基因(LB3-1)和特青转反义Sbe1基因(TB1-1)
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 the
subsequent 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
引文
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