转基因棉花突变体的鉴定和生理指标分析
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摘要
植物转基因技术是定向改良植物性状、高频诱发突变体产生的一条有效途径,对于作物品种的改良和新品种选育具有重要意义。本研究以导入转座活化结构的不同陆地棉转基因株系后代为材料,通过分子生物学手段对主要转基因株系进行了初步鉴定及其诱发突变的效应,并初步分析了相关突变材料在生理生化代谢方面的差异。其主要结果如下:
1.以筛选基因NPTⅡ基因为特异性引物时,在4105为受体的转基因株系中的扩增阳性率为26.1%,以1107为受体的转基因株系的扩增阳性率为27.2%。两种受体材料的转化效率比较接近;以Bar基因为引物时,转基因4105和1107的后代株系的扩增阳性率分别为28.1%和31.8%。通过分析可得出只含Bar基因的植株共122株,占被分析植株的比例为17.4%。说明Ds发生了转座,并与Ac因子产生了分离,有利于得到具有稳定遗传效应的突变材料。
2.对T3代部分转座活化的棉株进行Southern Blotting分析,初步证明外源基因已整合到棉花基因组中。
3.转基因棉花株系的主要生理指标的测定结果为:4105的L3株系在叶绿素含量和光合速率上与对照呈极显著差异,1107为受体的L9株系在蕾期与对照相比存在显著差异,在花铃期和吐絮期与对照的差异达到极显著水平,间接说明转座活化结构中的35S四联体增强子可影响插入位点附近的基因表达。此外,以4105为受体的转基因株系的叶绿素含量和光合速率均高于以1107为受体的转基因株系,说明转基因的受体不同产生的突变效应也存在差异。总体来看,4105转基因棉花株系中产生的晚熟类型的变异较多;而1107转基因株系中出现的早熟类型的变异较多。
4.通过对不同转基因株系叶片可溶性蛋白质进行SDS-PAGE,得到结果如下:转基因棉花突变体在某些蛋白亚基的表达量上高于对照,尤其是分子量大约为50kD的亚基。此外,在相同的环境条件下,转基因棉花突变系的SDS-PAGE图谱中还出现了一条大约42kD的蛋白质条带,而在对照中则不存在此条带,说明导入转座活化结构的棉花后代株系中可能存在由增强子引起的蛋白质的“超表达”情况,但与该亚基相关的蛋白质的功能还需进一步的实验验证。
5.对不同转基因株系叶片中POD同工酶进行PAGE,发现转基因棉株突变体酶谱与对照之间的差异。说明转座结构的插入能引起某些基因的“过量”表达,从而影响转基因棉花的相关生理代谢。
Plant transgenic technique is an efficient approach to improve plant characters directionally and to induce mutations in high frequency. It is significant for crop varieties' improving and breeding. The progenies of different transgenic land cotton lines, which were introduced transposon activation construct to cotton genome via Agrobacterium tumefeciens transformation, were used as materials in this research. The preliminary identification of the main strains of transgenic land cotton by molecular biology and the effects of the construct on mutation induced, and a preliminary analysis of physiological and biochemical differences of related transgenic mutants were studied. The main results are as follows:
1. Based on the specific primers of screening marker of NPTⅡgene, the positive rate of PCR amplified in the transgenic 4105 lines was 26.1%, and 27.2% in that of transgenic 1107 lines. So the transformation efficiency was almost the same in the two receptors;. Based on the specific primers of Bar gene, which linked with Ds element, the positive rates of PCR amplified in the progenies of transgenic 4105 and 1107 were 28.1% and 31.8% respectively. So we could calculate the ratio of transgenic plants only harbored Bar gene was 17.4%, the total number was 122. It indicated that Ds element has been dissociated from its original location, and inserted into the other chromosome, and separated with Ac in the progenies. It is easy to obtain the stabilized mutants from the events.
2. The results of Southern Blotting in T3 plants basically proved that the foreign T-DNA had been integrated into the cotton genome.
3. The results of physiological and biochemical indices of some transgenic lines indicated that the content of chlorophyll and photosynthesis rate of L3 line from transgenic 4105 were significantly higher than that of the control; the differences of chlorophyll content and photosynthesis rate between L9 which from transgenic 1107 and its control were found to be achieved a significant differences during the bud stage, and a most significant differences during the flowering and the boll opening stage. These physiological differences reflected that CaMV35S enhancer of the transposon activation construct might affect over-expression of some genes nearby the Ds inserted site. In addition, the chlorophyll content and photosynthesis rate of transgenic 4105 were higher than that of transgenic 1107, which suggested that the mutation effects varied with transgene receptors. Totally, we found more late-maturing mutations in the transgenic 4105 progenies and more early-maturing mutations in the transgenic 1107 lines.
4. The analysis of leaf soluble protein subunits with SDS-PAGE showed the overexpression of some proteins in the transgenic plants, especially a subunit of approximately 50kD molecular weight. Furthermore, a new 42kD subunit appeared in the transgenic cotton progenies, but not in the related control, which indicated that the over-expression of some genes induced by the enhancer in the transgenic lines caused related proteins synthesis, but the function of the protein related with the subunits is still to proved by the further experiment.
5. The results of the POD isozymes in the different transgenic cotton lines by PAGE showed the different pattern of the POD isozymes compared with that of the control. It also suggested that some genes might be activated by the enhancer followed the insertion of Ds nearby, and therefore influenced the related metabolism in the transgenic cotton plants.
1.以筛选基因NPTⅡ基因为特异性引物时,在4105为受体的转基因株系中的扩增阳性率为26.1%,以1107为受体的转基因株系的扩增阳性率为27.2%。两种受体材料的转化效率比较接近;以Bar基因为引物时,转基因4105和1107的后代株系的扩增阳性率分别为28.1%和31.8%。通过分析可得出只含Bar基因的植株共122株,占被分析植株的比例为17.4%。说明Ds发生了转座,并与Ac因子产生了分离,有利于得到具有稳定遗传效应的突变材料。
2.对T3代部分转座活化的棉株进行Southern Blotting分析,初步证明外源基因已整合到棉花基因组中。
3.转基因棉花株系的主要生理指标的测定结果为:4105的L3株系在叶绿素含量和光合速率上与对照呈极显著差异,1107为受体的L9株系在蕾期与对照相比存在显著差异,在花铃期和吐絮期与对照的差异达到极显著水平,间接说明转座活化结构中的35S四联体增强子可影响插入位点附近的基因表达。此外,以4105为受体的转基因株系的叶绿素含量和光合速率均高于以1107为受体的转基因株系,说明转基因的受体不同产生的突变效应也存在差异。总体来看,4105转基因棉花株系中产生的晚熟类型的变异较多;而1107转基因株系中出现的早熟类型的变异较多。
4.通过对不同转基因株系叶片可溶性蛋白质进行SDS-PAGE,得到结果如下:转基因棉花突变体在某些蛋白亚基的表达量上高于对照,尤其是分子量大约为50kD的亚基。此外,在相同的环境条件下,转基因棉花突变系的SDS-PAGE图谱中还出现了一条大约42kD的蛋白质条带,而在对照中则不存在此条带,说明导入转座活化结构的棉花后代株系中可能存在由增强子引起的蛋白质的“超表达”情况,但与该亚基相关的蛋白质的功能还需进一步的实验验证。
5.对不同转基因株系叶片中POD同工酶进行PAGE,发现转基因棉株突变体酶谱与对照之间的差异。说明转座结构的插入能引起某些基因的“过量”表达,从而影响转基因棉花的相关生理代谢。
Plant transgenic technique is an efficient approach to improve plant characters directionally and to induce mutations in high frequency. It is significant for crop varieties' improving and breeding. The progenies of different transgenic land cotton lines, which were introduced transposon activation construct to cotton genome via Agrobacterium tumefeciens transformation, were used as materials in this research. The preliminary identification of the main strains of transgenic land cotton by molecular biology and the effects of the construct on mutation induced, and a preliminary analysis of physiological and biochemical differences of related transgenic mutants were studied. The main results are as follows:
1. Based on the specific primers of screening marker of NPTⅡgene, the positive rate of PCR amplified in the transgenic 4105 lines was 26.1%, and 27.2% in that of transgenic 1107 lines. So the transformation efficiency was almost the same in the two receptors;. Based on the specific primers of Bar gene, which linked with Ds element, the positive rates of PCR amplified in the progenies of transgenic 4105 and 1107 were 28.1% and 31.8% respectively. So we could calculate the ratio of transgenic plants only harbored Bar gene was 17.4%, the total number was 122. It indicated that Ds element has been dissociated from its original location, and inserted into the other chromosome, and separated with Ac in the progenies. It is easy to obtain the stabilized mutants from the events.
2. The results of Southern Blotting in T3 plants basically proved that the foreign T-DNA had been integrated into the cotton genome.
3. The results of physiological and biochemical indices of some transgenic lines indicated that the content of chlorophyll and photosynthesis rate of L3 line from transgenic 4105 were significantly higher than that of the control; the differences of chlorophyll content and photosynthesis rate between L9 which from transgenic 1107 and its control were found to be achieved a significant differences during the bud stage, and a most significant differences during the flowering and the boll opening stage. These physiological differences reflected that CaMV35S enhancer of the transposon activation construct might affect over-expression of some genes nearby the Ds inserted site. In addition, the chlorophyll content and photosynthesis rate of transgenic 4105 were higher than that of transgenic 1107, which suggested that the mutation effects varied with transgene receptors. Totally, we found more late-maturing mutations in the transgenic 4105 progenies and more early-maturing mutations in the transgenic 1107 lines.
4. The analysis of leaf soluble protein subunits with SDS-PAGE showed the overexpression of some proteins in the transgenic plants, especially a subunit of approximately 50kD molecular weight. Furthermore, a new 42kD subunit appeared in the transgenic cotton progenies, but not in the related control, which indicated that the over-expression of some genes induced by the enhancer in the transgenic lines caused related proteins synthesis, but the function of the protein related with the subunits is still to proved by the further experiment.
5. The results of the POD isozymes in the different transgenic cotton lines by PAGE showed the different pattern of the POD isozymes compared with that of the control. It also suggested that some genes might be activated by the enhancer followed the insertion of Ds nearby, and therefore influenced the related metabolism in the transgenic cotton plants.
引文
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