反义Lox-3基因转化籼稻的遗传体系研究
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摘要
水稻是世界上最重要的粮食作物之一,全球接近一半的人口以其为主要的食物和能量来源,所以水稻的稳定增产保障了世界的粮食安全。然而,稻谷在储藏过程中由于陈化变质、虫害和霉变等引起大量损失,因此,开展水稻耐储藏特性的相关研究,培育耐储藏水稻新品种,意义重大。
本研究旨在通过农杆菌介导法,将反义lox-3基因导入籼型恢复系“航1号”中,期望从转基因植株中筛选出脂肪氧化酶低表达的株系,实现提高水稻耐储藏性的目的。围绕此目的,本研究主要得到以下结论:
1.通过对比“航1号”愈伤组织在6种不同的培养基上的生长情况,分析经过不同时间的培养,愈伤组织的增重情况,确定了其最适培养基。
2.利用农杆菌介导法,分别将载体pHG1PL, pHG4PL和pHG4P500L导入籼型恢复系“航1号”中,获得124株转基因植株,PCR结果表明,其中有96株检测到标记基因bar,初步说明反义lox-3基因已经整合到籼型恢复系“航1号”的基因组中。
3.从96株PCR检测为阳性的转基因植株中随机挑取5株进行Southern杂交和斑点杂交检测,结果4株检测到杂交信号,说明反义lox-3基因已经整合到籼型恢复系“航1号”的基因组中;而其中一株没有检测到杂交信号,说明反义lox-3基因没有整合到其基因组中,PCR结果可能为假阳性。
4.随机选取7株转基因植株,分别提取RNA,进行RT-PCR分析,结果表明7个样品均在mRNA水平上得到了表达,进一步说明反义lox-3基因已经整合到籼型恢复系“航1号”的基因组中。
5.分别利用常规转化法与快速转化法进行籼型恢复系“航1号”的遗传转化,对比两者的转化效率,发现快速转化法虽然缩短了转化流程,但是转化率却不及常规转化法。
6.对来自同一克隆的转反义lox-3基因的正常苗与白化苗的根、茎、叶进行显微结构解剖的对比研究,发现其根、茎、叶均存在差异。
Rice is one of the most important grain crops in the world. Almost half of the world’s population takes it as the main source of food and energy. So the stable increasing of rice yield ensures the world’s food supply. However there is a large loss caused by aging deterioration, pests and mildew during rice storage. Therefore, carrying out a series of related researches on rice storable characteristics and breeding new storable rice varieties, both are very important.
This study was designed to transform Cre/Lox-3 gene into indica restorer line“Hang No.1”, expecting to select lipoxygenase low-expression lines from transgenic plants, making preparation for breeding new varieties with high storage resistance, and achieving the purpose of improving rice resistance to storage. Aiming at this goal, this study obtained the following conclusions:
1. Analyzed the growth state of“Hang No.1”callus in six different media and their gain weight over different culture time, definited the optimum media for“Hang No.1”.
2. Plasmid pHG1PL, pHG4PL and pHG4P500L were transformed into indica restorer line“Hang No.1”respectively by Agrobacterium-mediated transformation, and 124 transgenic plants were obtained, the PCR results showed that the marker gene bar can be detected in 96 regenerated plants, preliminary indicating that the Cre/lox-3 gene had been integrated into indica restorer line“Hang No.1”.
3. Of altogether 96 PCR-positive transgenic plants, five ones were selected to perform Southern blot and dot blot analysis, and the result showed four transgenic plants detected the signal, indicating that the Cre/lox-3 gene had been integrated into their genomes; while one of them failed to detect any signal, indicating that the Cre/lox-3 gene had not been integrated into its genome, and the PCR result may be false positive.
4. Selected 7 transgenic plants at random, the result of RT-PCR analysis showed that all of them with expression on mRNA lever, further indicated that the Cre/lox-3 gene had been integrated into indica restorer line“Hang No.1.
5. Compared normal Agrobacterium-mediated transformation with early infection of agrobacterium, it was found that the transformation efficiency of the normal one was higher than the early infection, but it took much more time.
6. Compared the microstructure of the root, stem and leaf from the normal and albino seedlings which were differentiated from the same clone transformed with Cre/lox-3 gene. It was found that there were some differences between the root, stem and leaf.
本研究旨在通过农杆菌介导法,将反义lox-3基因导入籼型恢复系“航1号”中,期望从转基因植株中筛选出脂肪氧化酶低表达的株系,实现提高水稻耐储藏性的目的。围绕此目的,本研究主要得到以下结论:
1.通过对比“航1号”愈伤组织在6种不同的培养基上的生长情况,分析经过不同时间的培养,愈伤组织的增重情况,确定了其最适培养基。
2.利用农杆菌介导法,分别将载体pHG1PL, pHG4PL和pHG4P500L导入籼型恢复系“航1号”中,获得124株转基因植株,PCR结果表明,其中有96株检测到标记基因bar,初步说明反义lox-3基因已经整合到籼型恢复系“航1号”的基因组中。
3.从96株PCR检测为阳性的转基因植株中随机挑取5株进行Southern杂交和斑点杂交检测,结果4株检测到杂交信号,说明反义lox-3基因已经整合到籼型恢复系“航1号”的基因组中;而其中一株没有检测到杂交信号,说明反义lox-3基因没有整合到其基因组中,PCR结果可能为假阳性。
4.随机选取7株转基因植株,分别提取RNA,进行RT-PCR分析,结果表明7个样品均在mRNA水平上得到了表达,进一步说明反义lox-3基因已经整合到籼型恢复系“航1号”的基因组中。
5.分别利用常规转化法与快速转化法进行籼型恢复系“航1号”的遗传转化,对比两者的转化效率,发现快速转化法虽然缩短了转化流程,但是转化率却不及常规转化法。
6.对来自同一克隆的转反义lox-3基因的正常苗与白化苗的根、茎、叶进行显微结构解剖的对比研究,发现其根、茎、叶均存在差异。
Rice is one of the most important grain crops in the world. Almost half of the world’s population takes it as the main source of food and energy. So the stable increasing of rice yield ensures the world’s food supply. However there is a large loss caused by aging deterioration, pests and mildew during rice storage. Therefore, carrying out a series of related researches on rice storable characteristics and breeding new storable rice varieties, both are very important.
This study was designed to transform Cre/Lox-3 gene into indica restorer line“Hang No.1”, expecting to select lipoxygenase low-expression lines from transgenic plants, making preparation for breeding new varieties with high storage resistance, and achieving the purpose of improving rice resistance to storage. Aiming at this goal, this study obtained the following conclusions:
1. Analyzed the growth state of“Hang No.1”callus in six different media and their gain weight over different culture time, definited the optimum media for“Hang No.1”.
2. Plasmid pHG1PL, pHG4PL and pHG4P500L were transformed into indica restorer line“Hang No.1”respectively by Agrobacterium-mediated transformation, and 124 transgenic plants were obtained, the PCR results showed that the marker gene bar can be detected in 96 regenerated plants, preliminary indicating that the Cre/lox-3 gene had been integrated into indica restorer line“Hang No.1”.
3. Of altogether 96 PCR-positive transgenic plants, five ones were selected to perform Southern blot and dot blot analysis, and the result showed four transgenic plants detected the signal, indicating that the Cre/lox-3 gene had been integrated into their genomes; while one of them failed to detect any signal, indicating that the Cre/lox-3 gene had not been integrated into its genome, and the PCR result may be false positive.
4. Selected 7 transgenic plants at random, the result of RT-PCR analysis showed that all of them with expression on mRNA lever, further indicated that the Cre/lox-3 gene had been integrated into indica restorer line“Hang No.1.
5. Compared normal Agrobacterium-mediated transformation with early infection of agrobacterium, it was found that the transformation efficiency of the normal one was higher than the early infection, but it took much more time.
6. Compared the microstructure of the root, stem and leaf from the normal and albino seedlings which were differentiated from the same clone transformed with Cre/lox-3 gene. It was found that there were some differences between the root, stem and leaf.
引文
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2.云昌杰,我国农村储粮问题研究,粮食储藏,1996,25(4):24-27.
3. Aibarasi, Ismailia, Yamashitah, et al., Changes in Rice Bran Lipids and Fatty Acids during Storage [J], Agricultural Biological Chemistry, 1986, 50: 6652 673.
4. Takano K, et al., Advances in Cereal Chemistry and Technology in Japan [J], Cereal Foods World, 1993, 38: 6952 698.
5.王海滨,植物的脂肪氧化酶,植物生理学通讯,1990,(2):63-67.
6. Goffman F D, Bergman C, et al., Hydrolytic degradation of triacyl Glyce roles and changes in fatty acid composition in rice bran during storage, Cereal Chem, 2003, 80(4): 459-461.
7. Kloeti, A. & Potrykus, et al., I. Rice improvement by genetic transformation, In Molecular Biology of Rice (ed. Shimamoto, K.) 283–301. (Springer-Verlag, Tokyo, Japan, 1999).
8. Shimamoto, K., Terada, R., Izawa, T. & Fujimoto H., Fertile transgenic rice plants regenerated from transformed protoplasts, Nature 338, 274–276 (1989).
9. Datta, S.K., Peterhans, A., Datta, K. & Potrykus, I, Genetically engineered fertile indica-rice recovered from protoplasts, Bio/Technol. 8, 736–740 (1990).
10. Christou, P., Ford, T.L. & Kofron, M., Production of transgenic rice (Oryza sativa L.) plants from agronomically important indica and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos, Bio/Technol. 9, 957–962 (1991).
11. Hiei, Y., Ohta, S., Komari, T. & Kumashiro, T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, Plant J. 6, 271–282 (1994).
12. Davey, M.R., et al., 1991. Plant Sci Lett, 18 :307-313.
13.沈圣泉,张仁华,舒庆尧等,水稻常用的遗传转化技术及应用现状,中国农学通报,2001,17[1]:37-40.
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