柑橘原生质体瞬时表达体系的建立及默科特橘橙转GFP种质的创造
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摘要
柑橘是世界第一大水果,通过遗传改良新技术创造优良柑橘品种是提升我国柑橘市场竞争力的重要措施。珠心胚干扰、性器官败育、童期长、遗传上高度杂合等生物学特征严重阻碍了柑橘常规育种的进程。近年来,不断完善的转基因技术为柑橘品种改良提供了一种更加快速有效的途径。
本研究以柑橘原生质体为转化受体,以绿色荧光蛋白基因(Green fluorescentprotein gene,GFP)作为报告基因,建立了原生质体电击转化体系,同时对影响电击转化效率的几个因素进行了探讨和分析。另外,以默科特橘橙(Citrus reticulata×C.sinensis‘Murcott')不同组织或器官为外植体进行农杆菌介导的GFP转化,获得了一批稳定表达的转基因材料,为细胞融合、细胞凋亡以及遗传转化相关机制等研究提供了便利。主要结果如下:
1.柑橘原生质体电击转化瞬时表达体系的优化。以广东蜜橘(C.reticulataBlanco‘Guangdong tangerine')愈伤组织原生质体作为电击转化受体转化mgfp5-ER,分析了电击缓冲液pH、电击前原生质体预处理等因素对瞬时表达效率的影响。结果表明:在直流脉冲强度为1250 V/cm,脉冲作用时间45μs,脉冲个数5个,脉冲间隔0.5 s,交变电场强度为50 V/cm,作用时间40μs时,瞬时表达率最高,达到10.3%;电击缓冲液pH为7时瞬时表达率最高;电击前对原生质体冰浴10 min,瞬时表达率无显著提高。将该体系与PEG(聚乙二醇)介导的遗传转化法进行比较,瞬时表达率差异不显著。
2.柑橘不同品种原生质体转化效率差异较大。国庆一号(C.unshiu‘GuoqingNo.1')和广东蜜橘愈伤组织原生质体较易转化,瞬时表达率分别为9.7%和10.9%;伏令夏橙(C.sinensis‘Valencia')叶肉原生质体较难转化,偶见GFP瞬时表达;暗柳橙(C.sinensis Osbeck‘Anliu')和默科特橘橙愈伤组织原生质体转化后未检测到GFP瞬时表达。
3.默科特橘橙不同外植体转化mgfp5-ER。以默科特橘橙胚性愈伤组织和试管实生苗的上胚轴切段、根轴联结体、子叶为外植体,利用根癌农杆菌介导法进行转化,获得了稳定表达mgfp5-ER的转基因胚性愈伤组织和转基因芽;转化效率因外植体而不同,其中以愈伤组织和上胚轴切段为外植体转化效率较高,分别为13.3%和12.3%;种子成熟度影响转化效率,以未成熟种子和成熟饱满的种子获得的试管实生苗进行转化,转化效率分别为8.0%和12.6%,二者差异显著。
4.默科特橘橙转基因愈伤组织和转基因芽的荧光和分子检测。本实验共获得46个转化子:17个仍处于愈伤组织团状态,29个转基因芽及芽点;利用激光共聚焦显微镜进行荧光检测,结果表明GFP在默科特橘橙抗性愈伤组织和抗性芽中均可稳定表达;对GFP表达呈阳性的抗性愈伤组织进行PCR检测,PCR阳性率达100%。
Citrus ranks the number one in the world fruit production. Creating high qualities potential citrus varieties via genetic improvement is the main solution of enhancing the competitiveness of China's citrus market. The conventional breeding is constrained by the complex biological characteristics, such as polyembryony, apomixis, long juvenility, high heterozygosity and parthenogenesis etc. In recent years, the improving transgenic technology has provided an efficient way to the improvement of citrus varieties.
In this study, a procedure for efficient protoplast electroporation was established using green fluorescent protein gene (GFP) as reporter gene. Meanwhile, a number of factors affecting the efficiency of electroporation were also discussed. In addition, the transformation of GFP into different explants of Murcott Tangor by Agrobacterium was conducted. The transformants expressing GFP could be applied to further investigate the mechanism of cell fusion, apoptosis, and the related genetic transformation in citrus. The main results were as follows:
1. Optimizing citrus protoplast transient expression system. Some factors influencing the transformation of mgfp5-ER gene into Guangdong tangerine were analyzed, including the pH value of electroporation buffer, The results showed that the optimal transient expression rate reached 10.3% with the direct current at 1250 V/cm, pulse time for 45 us, pulse number at 5, pulse interval for 0.5 s, alternating electric field at 50 V/cm and for 40 us. When the electroporation buffer pH value was 7, transient expression efficiency reached maximum. The transient expression efficiency was not significantly improved by ice pre-treating the protoplast for 10 min before electroporation. Moreover, compared with transformation mediated by PEG (Polyethylene glycol), the transient expression efficiency of this system had no significant difference.
2. Comparison of protoplast transformation efficiencies in different citrus species. The result indicated that the transformation efficiencies of Guoqing No.1 and Guangdong tangerine were 9.7% and 10.9%, respectively. Protoplasts isolated from suspension culture callus were easier for transformation. Occasional transient expression of Valencia mesophyllic protoplast transformation could be observed. The transient expression in the callus protoplasts of Anliucheng and Murcott Tangor which were considered to be the most difficult for transformation has not been detected.
3. Transformation of mgfp5-ER genes with different Murcott Tangor explants. By making use of agrobacterium tumefaciens-mediated transformation method, Some transgenic callus and buds with stable GFP expression have been obtained by using embryonic callus, epicotyl segment, the trimmed etiolated shoot/root region seedling and cotyledon of test-tube seedlings of Murcott Tangeor as explants. The transformation efficiency was different among different explants, The results showed that higher transformation efficiencies with respectively 13.3% and 12.3% were obtained by using the callus and epicotyl segment as explants. The transformation efficiency was affected by the maturation level of seed, which were 8.0% and 12.6% by using the test-tube seedlings obtained from the immature seeds and mature seeds as explants respectively.
4. The fluorescence and molecular analysis of Murcott Tangor transformants. 46 transformants were obtaind. 17 of them were still in a state of callus stage, and 29 of them were transgenic buds and bud points. The results showed that the GFP gene could be stably expressed in the resistant callus and resistant buds of Murcott Tangor by using the laser scanning Confocal microscopy. PCR detection of the fluorescence callus team of Murcott orange showed the PCR-positive rate was 100%.
本研究以柑橘原生质体为转化受体,以绿色荧光蛋白基因(Green fluorescentprotein gene,GFP)作为报告基因,建立了原生质体电击转化体系,同时对影响电击转化效率的几个因素进行了探讨和分析。另外,以默科特橘橙(Citrus reticulata×C.sinensis‘Murcott')不同组织或器官为外植体进行农杆菌介导的GFP转化,获得了一批稳定表达的转基因材料,为细胞融合、细胞凋亡以及遗传转化相关机制等研究提供了便利。主要结果如下:
1.柑橘原生质体电击转化瞬时表达体系的优化。以广东蜜橘(C.reticulataBlanco‘Guangdong tangerine')愈伤组织原生质体作为电击转化受体转化mgfp5-ER,分析了电击缓冲液pH、电击前原生质体预处理等因素对瞬时表达效率的影响。结果表明:在直流脉冲强度为1250 V/cm,脉冲作用时间45μs,脉冲个数5个,脉冲间隔0.5 s,交变电场强度为50 V/cm,作用时间40μs时,瞬时表达率最高,达到10.3%;电击缓冲液pH为7时瞬时表达率最高;电击前对原生质体冰浴10 min,瞬时表达率无显著提高。将该体系与PEG(聚乙二醇)介导的遗传转化法进行比较,瞬时表达率差异不显著。
2.柑橘不同品种原生质体转化效率差异较大。国庆一号(C.unshiu‘GuoqingNo.1')和广东蜜橘愈伤组织原生质体较易转化,瞬时表达率分别为9.7%和10.9%;伏令夏橙(C.sinensis‘Valencia')叶肉原生质体较难转化,偶见GFP瞬时表达;暗柳橙(C.sinensis Osbeck‘Anliu')和默科特橘橙愈伤组织原生质体转化后未检测到GFP瞬时表达。
3.默科特橘橙不同外植体转化mgfp5-ER。以默科特橘橙胚性愈伤组织和试管实生苗的上胚轴切段、根轴联结体、子叶为外植体,利用根癌农杆菌介导法进行转化,获得了稳定表达mgfp5-ER的转基因胚性愈伤组织和转基因芽;转化效率因外植体而不同,其中以愈伤组织和上胚轴切段为外植体转化效率较高,分别为13.3%和12.3%;种子成熟度影响转化效率,以未成熟种子和成熟饱满的种子获得的试管实生苗进行转化,转化效率分别为8.0%和12.6%,二者差异显著。
4.默科特橘橙转基因愈伤组织和转基因芽的荧光和分子检测。本实验共获得46个转化子:17个仍处于愈伤组织团状态,29个转基因芽及芽点;利用激光共聚焦显微镜进行荧光检测,结果表明GFP在默科特橘橙抗性愈伤组织和抗性芽中均可稳定表达;对GFP表达呈阳性的抗性愈伤组织进行PCR检测,PCR阳性率达100%。
Citrus ranks the number one in the world fruit production. Creating high qualities potential citrus varieties via genetic improvement is the main solution of enhancing the competitiveness of China's citrus market. The conventional breeding is constrained by the complex biological characteristics, such as polyembryony, apomixis, long juvenility, high heterozygosity and parthenogenesis etc. In recent years, the improving transgenic technology has provided an efficient way to the improvement of citrus varieties.
In this study, a procedure for efficient protoplast electroporation was established using green fluorescent protein gene (GFP) as reporter gene. Meanwhile, a number of factors affecting the efficiency of electroporation were also discussed. In addition, the transformation of GFP into different explants of Murcott Tangor by Agrobacterium was conducted. The transformants expressing GFP could be applied to further investigate the mechanism of cell fusion, apoptosis, and the related genetic transformation in citrus. The main results were as follows:
1. Optimizing citrus protoplast transient expression system. Some factors influencing the transformation of mgfp5-ER gene into Guangdong tangerine were analyzed, including the pH value of electroporation buffer, The results showed that the optimal transient expression rate reached 10.3% with the direct current at 1250 V/cm, pulse time for 45 us, pulse number at 5, pulse interval for 0.5 s, alternating electric field at 50 V/cm and for 40 us. When the electroporation buffer pH value was 7, transient expression efficiency reached maximum. The transient expression efficiency was not significantly improved by ice pre-treating the protoplast for 10 min before electroporation. Moreover, compared with transformation mediated by PEG (Polyethylene glycol), the transient expression efficiency of this system had no significant difference.
2. Comparison of protoplast transformation efficiencies in different citrus species. The result indicated that the transformation efficiencies of Guoqing No.1 and Guangdong tangerine were 9.7% and 10.9%, respectively. Protoplasts isolated from suspension culture callus were easier for transformation. Occasional transient expression of Valencia mesophyllic protoplast transformation could be observed. The transient expression in the callus protoplasts of Anliucheng and Murcott Tangor which were considered to be the most difficult for transformation has not been detected.
3. Transformation of mgfp5-ER genes with different Murcott Tangor explants. By making use of agrobacterium tumefaciens-mediated transformation method, Some transgenic callus and buds with stable GFP expression have been obtained by using embryonic callus, epicotyl segment, the trimmed etiolated shoot/root region seedling and cotyledon of test-tube seedlings of Murcott Tangeor as explants. The transformation efficiency was different among different explants, The results showed that higher transformation efficiencies with respectively 13.3% and 12.3% were obtained by using the callus and epicotyl segment as explants. The transformation efficiency was affected by the maturation level of seed, which were 8.0% and 12.6% by using the test-tube seedlings obtained from the immature seeds and mature seeds as explants respectively.
4. The fluorescence and molecular analysis of Murcott Tangor transformants. 46 transformants were obtaind. 17 of them were still in a state of callus stage, and 29 of them were transgenic buds and bud points. The results showed that the GFP gene could be stably expressed in the resistant callus and resistant buds of Murcott Tangor by using the laser scanning Confocal microscopy. PCR detection of the fluorescence callus team of Murcott orange showed the PCR-positive rate was 100%.
引文
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