草木樨状黄芪与霸王科间体细胞杂交、PA1基因的克隆及对苜蓿的转化
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摘要
本研究首先建立了草木樨状黄芪(Astragalus molilotoides pall. )和霸王(Zygophyllum xanthoxylum)两种植物的原生质体分离、培养和再生的实验体系。在此基础上，开展了这两种植物的科间体细胞杂交，建立了双亲生理互补的杂种细胞筛选体系，并获得了杂种愈伤组织。本研究的另一部分是从食荚大菜豌(Pisum Linn. cultivar shijiadacaiwan)克隆出一个富硫氨基酸且具有抗虫性的双功能蛋白基因——PA1基因，通过农杆菌介导法将该基因转入紫花苜蓿，并获得转基因再生植株。探讨了转基因苜蓿体胚发生的变异及PA1基因在转基因紫花苜蓿中的表达。取得的主要进展如下：
1．取暗培养4d的草木樨状黄芪植株上完全伸展的嫩叶，进行60min质壁分离处理后，通过酶法游离出大量有活力的原生质体。原生质体经持续分裂形成愈伤组织并再生成植株。比较了原生质体培养密度和外源激素组合对原生质体分裂和再生的影响。结果表明：原生质体以3.0×10~5／ml的植板密度，在附加2，4-D(2.0 mg／L)、6-BA(0.2 mg／L)、2％蔗糖、0.4 mol／L甘露醇和500 mg／L水解酪蛋白的DPD培养基中进行液体浅层培养，其细胞分裂频率可达到55.6％。在附加了2，4-D(1.0 mg／L)、6-BA(1.0 mg／L)和KT(0.5 mg／L)的MS培养基上，原生质体来源的愈伤组织分化植株的频率高达96％，其再生苗移栽土壤成活。
2．对霸王的子叶和下胚轴愈伤组织分离的原生质体进行了培养，并获得了愈伤组织。结果表明：愈伤组织游离的原生质体产量和活力均高于子叶；用暗培养条件下继代14 d的松软的淡黄色愈伤组织为材料，经2％纤维素酶(Cellulase OnozukaR-10)，1％半纤维素酶(Hemicellulase Sigma)，0.5％果胶酶(Pectinase Serva)的酶组合溶液处理13 h后，分离的原生质体产率为2.4×10~6个／g·FW，原生质体活力达到89％。液体浅层培养下，原生质体分裂的最适外源激素组合为2，4-D(2 mg／L)和6-BA(1.0 mg／L)，最高分裂频率达到72％。
3．用修改的PEG-高pH高钙法诱导原生质体融合，得到了草木樨状黄芪和霸王的科间体细胞杂种融合细胞。通过罗丹明6G预处理草木樨状黄芪原生质使其细胞质失活、UV-B辐照霸王原生质体使其细胞核失活，结果双亲原生质体都不能持续分裂，而杂种细胞通过生理互补可以持续分裂，从而建立了有广泛应用价值的杂种细胞筛选体系，获得了两个杂种克隆及两棵小苗。细胞学和分子鉴定证实了杂种的真实性。
4．从食荚大菜豆克隆出具有抗虫作用同时富硫氨基酸的双功能蛋白质基因——PA1基因，并构建了植物表达载体pCAMBIA1301-PA1。采用农杆菌介导法用此基因转化了紫花苜蓿，并对其转化体系进行优化，得到了转基因植株。PCR和Southern杂交检测表明，PA1基因和潮霉素抗性基因整合到了宿主细胞，SDS-PAGE分析表明该基因在叶片中有一定表达。
The plant regeneration systems were established from protoplasts of Astragalus molilotoides pall. and Zygophyllum xanthoxylum. The conditions of protoplast isolation and culture were optimized. Based on this, somatic hybridization between Astragalus molilotoides pall. and Zygophyllum xanthoxylum was carried out, and somatic hybrid cells were selected by use of physiological complementation of parent protoplasts pretreated with UV radiation and Rhodamine-6G respectively. The hybrid calli were produced. At the same time, a gene of a sulphur-rich and insect-toxic seed albumin—PA1 gene was cloned from Pisum Linn. (cultivar shijiadacaiwan) and transformed into alfalfa thought Agrobacterium tumefaciens method. The transgenic plants were regenerated. The embryoid variations of transgenic plants were examined and PA1 gene expression was characterized. The main results were as follows:
1. Plenty of viable protoplasts were isolated from the totally extend young leaves of Astragalus molilotoides pall. after precultured in "dark for 4 days and pretreated in plasmolysal solution for 60 min. Protoplasts were induced to undergo sustained divisions in DPD medium supplemented with 2.0 mg/L 2,4-D, 0.2 mg/L 6-BA, 2%(w/v)sucrose, 0.4 mol/L mannitol and 500 mg/L casein hydrolyasate at a plating density 3×10~5/ml. The division frequency was about 55.6%. The regenerated calli were cultured on agar solidifed MS medium plus 1.0 mg/L 2,4-D, 1.0 mg/L 6-BA and 0.5 mg/L KT. The plant differentiation frequency was over 96%. The regenerated plants was successfully transplanted in soil.
2. The cotyledons and the calli of. Zygophyllum xanthoxylum were used as materials for protoplast preparation, and large amount of protoplasts were isolated from calli. The protoplast yield and viability from calli of Zygophyllum xanthoxylum were higher than those from the cotyledons; 2.4×10~6/g·FW of protoplast yield and 89% of viability were obtained from light yellow calli after transferred and cultured for 14 days in the dark. The enzyme solution for protoplast isolation was composed of 2% cellulose, 1% hemicellulase and 0.5% pectinase and the enzyme digestion was completed in 13 h. Using liquid thin layer culture method and the proper phytohormone combination, i.e. 2 mg/L 2,4-D and 1.0 mg/L 6-BA, the division frequency of protoplasts was up to 72%.
3. Protoplast fusion between Astragalus molilotoides pall. and Zygophyllum xanthoxylum was carried out using modified PEG-high pH, high Ca~(2+) method. The interfamilial somatic hybrid cells were selected based on physiological complementation between both parents which were pretreated respectively with UV radiation and Rhodamine-6G. The system for somatic hybrid cell selection might be extensively useful. Two calli of hybrid were obtained from this selected system and two shoots were produced. Cytology and molecular biology identification confirmed the hybrid nature of two calli.
4. A gene of a sulphur-rich and insect-toxic seed albumin—PA1 gene was cloned from Pisum Linn. (cultivar shijiadacaiwan) and the plant expression vector of pCAMBIA1301-PA1 was constructed. The vector was used to transform into alfalfa by Agrobacterium tumefaciens method. The conditions for transformation were optimized. Transgenic regenerated calli and plants were obtained. PCR and Southern blotting identification confirmed that PA1 gene and hygromycin-B phosphotransferase-resistant gene were integrated into the genome of alfalfa, and SDS-PAGE analysis indicated PA1 gene expressed in leaves of transgenic plants.
1．取暗培养4d的草木樨状黄芪植株上完全伸展的嫩叶，进行60min质壁分离处理后，通过酶法游离出大量有活力的原生质体。原生质体经持续分裂形成愈伤组织并再生成植株。比较了原生质体培养密度和外源激素组合对原生质体分裂和再生的影响。结果表明：原生质体以3.0×10~5／ml的植板密度，在附加2，4-D(2.0 mg／L)、6-BA(0.2 mg／L)、2％蔗糖、0.4 mol／L甘露醇和500 mg／L水解酪蛋白的DPD培养基中进行液体浅层培养，其细胞分裂频率可达到55.6％。在附加了2，4-D(1.0 mg／L)、6-BA(1.0 mg／L)和KT(0.5 mg／L)的MS培养基上，原生质体来源的愈伤组织分化植株的频率高达96％，其再生苗移栽土壤成活。
2．对霸王的子叶和下胚轴愈伤组织分离的原生质体进行了培养，并获得了愈伤组织。结果表明：愈伤组织游离的原生质体产量和活力均高于子叶；用暗培养条件下继代14 d的松软的淡黄色愈伤组织为材料，经2％纤维素酶(Cellulase OnozukaR-10)，1％半纤维素酶(Hemicellulase Sigma)，0.5％果胶酶(Pectinase Serva)的酶组合溶液处理13 h后，分离的原生质体产率为2.4×10~6个／g·FW，原生质体活力达到89％。液体浅层培养下，原生质体分裂的最适外源激素组合为2，4-D(2 mg／L)和6-BA(1.0 mg／L)，最高分裂频率达到72％。
3．用修改的PEG-高pH高钙法诱导原生质体融合，得到了草木樨状黄芪和霸王的科间体细胞杂种融合细胞。通过罗丹明6G预处理草木樨状黄芪原生质使其细胞质失活、UV-B辐照霸王原生质体使其细胞核失活，结果双亲原生质体都不能持续分裂，而杂种细胞通过生理互补可以持续分裂，从而建立了有广泛应用价值的杂种细胞筛选体系，获得了两个杂种克隆及两棵小苗。细胞学和分子鉴定证实了杂种的真实性。
4．从食荚大菜豆克隆出具有抗虫作用同时富硫氨基酸的双功能蛋白质基因——PA1基因，并构建了植物表达载体pCAMBIA1301-PA1。采用农杆菌介导法用此基因转化了紫花苜蓿，并对其转化体系进行优化，得到了转基因植株。PCR和Southern杂交检测表明，PA1基因和潮霉素抗性基因整合到了宿主细胞，SDS-PAGE分析表明该基因在叶片中有一定表达。
The plant regeneration systems were established from protoplasts of Astragalus molilotoides pall. and Zygophyllum xanthoxylum. The conditions of protoplast isolation and culture were optimized. Based on this, somatic hybridization between Astragalus molilotoides pall. and Zygophyllum xanthoxylum was carried out, and somatic hybrid cells were selected by use of physiological complementation of parent protoplasts pretreated with UV radiation and Rhodamine-6G respectively. The hybrid calli were produced. At the same time, a gene of a sulphur-rich and insect-toxic seed albumin—PA1 gene was cloned from Pisum Linn. (cultivar shijiadacaiwan) and transformed into alfalfa thought Agrobacterium tumefaciens method. The transgenic plants were regenerated. The embryoid variations of transgenic plants were examined and PA1 gene expression was characterized. The main results were as follows:
1. Plenty of viable protoplasts were isolated from the totally extend young leaves of Astragalus molilotoides pall. after precultured in "dark for 4 days and pretreated in plasmolysal solution for 60 min. Protoplasts were induced to undergo sustained divisions in DPD medium supplemented with 2.0 mg/L 2,4-D, 0.2 mg/L 6-BA, 2%(w/v)sucrose, 0.4 mol/L mannitol and 500 mg/L casein hydrolyasate at a plating density 3×10~5/ml. The division frequency was about 55.6%. The regenerated calli were cultured on agar solidifed MS medium plus 1.0 mg/L 2,4-D, 1.0 mg/L 6-BA and 0.5 mg/L KT. The plant differentiation frequency was over 96%. The regenerated plants was successfully transplanted in soil.
2. The cotyledons and the calli of. Zygophyllum xanthoxylum were used as materials for protoplast preparation, and large amount of protoplasts were isolated from calli. The protoplast yield and viability from calli of Zygophyllum xanthoxylum were higher than those from the cotyledons; 2.4×10~6/g·FW of protoplast yield and 89% of viability were obtained from light yellow calli after transferred and cultured for 14 days in the dark. The enzyme solution for protoplast isolation was composed of 2% cellulose, 1% hemicellulase and 0.5% pectinase and the enzyme digestion was completed in 13 h. Using liquid thin layer culture method and the proper phytohormone combination, i.e. 2 mg/L 2,4-D and 1.0 mg/L 6-BA, the division frequency of protoplasts was up to 72%.
3. Protoplast fusion between Astragalus molilotoides pall. and Zygophyllum xanthoxylum was carried out using modified PEG-high pH, high Ca~(2+) method. The interfamilial somatic hybrid cells were selected based on physiological complementation between both parents which were pretreated respectively with UV radiation and Rhodamine-6G. The system for somatic hybrid cell selection might be extensively useful. Two calli of hybrid were obtained from this selected system and two shoots were produced. Cytology and molecular biology identification confirmed the hybrid nature of two calli.
4. A gene of a sulphur-rich and insect-toxic seed albumin—PA1 gene was cloned from Pisum Linn. (cultivar shijiadacaiwan) and the plant expression vector of pCAMBIA1301-PA1 was constructed. The vector was used to transform into alfalfa by Agrobacterium tumefaciens method. The conditions for transformation were optimized. Transgenic regenerated calli and plants were obtained. PCR and Southern blotting identification confirmed that PA1 gene and hygromycin-B phosphotransferase-resistant gene were integrated into the genome of alfalfa, and SDS-PAGE analysis indicated PA1 gene expressed in leaves of transgenic plants.
引文
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