苹果α-法尼烯合成酶基因转化番茄研究
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摘要
α-法尼烯是一种存在于生物体中的倍半萜类物质,已知与植物的抗冷性、昆虫的诱导及冷藏苹果和梨果实重大生理病害-虎皮病的发生有关,是苹果果实中重要的香气成分之一。α-法尼烯的生物合成是通过类异戊二烯途径中的甲羟戊酸途径,α-法尼烯合成酶是调控α-法尼烯合成的关键酶,催化FPP转变为α-法尼烯。
本实验根据Genbank中注册的α-法尼烯合成酶基因全长序列设计特定引物,利用RT-PCR的方法扩增青香蕉苹果α-法尼烯合成酶基因的全长,构建表达载体,优化番茄(中蔬6号)的再生和遗传转化体系,利用农杆菌介导法转化番茄,初步获得转基因植株,并研究了生理特性。
主要结果如下:
1、构建了pBI-AFS正义表达载体,并转化番茄,得到转AFS基因的番茄。
2、以番茄子叶为材料,优化了番茄的再生及遗传转化体系,最适宜番茄子叶再生的培养基为MS+ZT2.0mg/L+ IAA0.5mg/L+蔗糖30.0g/L+琼脂6.0g/L,生根培养基为MS+IAA0.5 mg/L+蔗糖30.0g/L+琼脂6.0g/L,确定在再生过程中Kan选择压为50mg/L;抑菌过程中头孢霉素100mg/L为宜。
3、通过克隆青香蕉苹果的α-法尼烯合成酶基因(AFS)片段,构建pEASY E1-AFS-原核表达载体,转化大肠杆菌,制备抗体,进行了Western杂交分析,显示该基因在番茄中成功表达。
4、高温处理后转AFS基因番茄比野生型番茄具有较高的耐热性,高温胁迫条件下转基因番茄维持较高的光合能力。
5、通过克隆到α-法尼烯合成酶基因(AFS)全长构建了pBI121-AFS-GFP真核表达载体,原生质体中瞬时表达AFS-GFP融合蛋白,发现该酶定位于叶绿体。
6、GC-MS分析显示,转基因植株叶片中单萜和倍半萜含量明显增加,但没有发现α-法尼烯的释放。
α-Farnesene, an acyclic sesquiterene existed in organisms, has been proved to play an important role in codling moth,chilling injury and development of scald on cold-storage apples. As one of aroma related volatiles in apple fruit,α-farnesene appears to be predominantly through classical mevalonate pathway. 3-Hydroxy-3- methylglutaryl CoA reductase (HMGR), AFS is the final, rate-limiting enzyme that converts farnesyl diphosphate (FPP) toα- farnesene in the mevalonic acid (MVA) pathway of terpene biosynthesis.
Specific primers ac cording to the full sequence ofα-farnesene synthase gene were designed, sense eukaryotic vector was transformed. A highly efficient regeneration system and genetic transformation system of tomato were established,and physiological characteristic of transgenic plant was studied.The main results were as follow:
1. A recombinant plant expression vector pBI-AFS was constructed and was used to transform to Tomato.
2. Adventitious bud regeneration from the cotyledon of tomato and efficient gene transformation system was optimized.The results showed that the optimum medium for adventitious bud regeneration was MS+ZT2.0mg/L+IAA 0.5mg/L+sucrose30.0g/L+ agar 6.0g/L, MS+ IAA 0.5mg/L+sucrose30.0g/L+agar 6.0g/L for root regeneration.The suitabale concentration of kan is 50mg/L,and cef is 100mg/L.
3. A recombinant prokaryotic expression vector pEASY-E1-AFS was constructed by cloning the fragment of AFS and was transformed to E.Coli. Western blot analysis indicated that AFS gene expressed in tomato.
4. The transgenic plants ,with higher adaptability to high temperature than wild type, could keep running of photosynthesis in high stress.
5. Constructed pBI-AFS-GFP eukaryotic expression vector by the cloned the cDNA of AFS. Transient expression of AFS-GFP fusion protein in protoplast showed that AFS was localized in the chloroplast.
6. Monoterpenes, sesquiterpenes, and other volatiles were found in relatively high amounts in transgenic tomato leaves, but no farnesene was detected in the both samples.
本实验根据Genbank中注册的α-法尼烯合成酶基因全长序列设计特定引物,利用RT-PCR的方法扩增青香蕉苹果α-法尼烯合成酶基因的全长,构建表达载体,优化番茄(中蔬6号)的再生和遗传转化体系,利用农杆菌介导法转化番茄,初步获得转基因植株,并研究了生理特性。
主要结果如下:
1、构建了pBI-AFS正义表达载体,并转化番茄,得到转AFS基因的番茄。
2、以番茄子叶为材料,优化了番茄的再生及遗传转化体系,最适宜番茄子叶再生的培养基为MS+ZT2.0mg/L+ IAA0.5mg/L+蔗糖30.0g/L+琼脂6.0g/L,生根培养基为MS+IAA0.5 mg/L+蔗糖30.0g/L+琼脂6.0g/L,确定在再生过程中Kan选择压为50mg/L;抑菌过程中头孢霉素100mg/L为宜。
3、通过克隆青香蕉苹果的α-法尼烯合成酶基因(AFS)片段,构建pEASY E1-AFS-原核表达载体,转化大肠杆菌,制备抗体,进行了Western杂交分析,显示该基因在番茄中成功表达。
4、高温处理后转AFS基因番茄比野生型番茄具有较高的耐热性,高温胁迫条件下转基因番茄维持较高的光合能力。
5、通过克隆到α-法尼烯合成酶基因(AFS)全长构建了pBI121-AFS-GFP真核表达载体,原生质体中瞬时表达AFS-GFP融合蛋白,发现该酶定位于叶绿体。
6、GC-MS分析显示,转基因植株叶片中单萜和倍半萜含量明显增加,但没有发现α-法尼烯的释放。
α-Farnesene, an acyclic sesquiterene existed in organisms, has been proved to play an important role in codling moth,chilling injury and development of scald on cold-storage apples. As one of aroma related volatiles in apple fruit,α-farnesene appears to be predominantly through classical mevalonate pathway. 3-Hydroxy-3- methylglutaryl CoA reductase (HMGR), AFS is the final, rate-limiting enzyme that converts farnesyl diphosphate (FPP) toα- farnesene in the mevalonic acid (MVA) pathway of terpene biosynthesis.
Specific primers ac cording to the full sequence ofα-farnesene synthase gene were designed, sense eukaryotic vector was transformed. A highly efficient regeneration system and genetic transformation system of tomato were established,and physiological characteristic of transgenic plant was studied.The main results were as follow:
1. A recombinant plant expression vector pBI-AFS was constructed and was used to transform to Tomato.
2. Adventitious bud regeneration from the cotyledon of tomato and efficient gene transformation system was optimized.The results showed that the optimum medium for adventitious bud regeneration was MS+ZT2.0mg/L+IAA 0.5mg/L+sucrose30.0g/L+ agar 6.0g/L, MS+ IAA 0.5mg/L+sucrose30.0g/L+agar 6.0g/L for root regeneration.The suitabale concentration of kan is 50mg/L,and cef is 100mg/L.
3. A recombinant prokaryotic expression vector pEASY-E1-AFS was constructed by cloning the fragment of AFS and was transformed to E.Coli. Western blot analysis indicated that AFS gene expressed in tomato.
4. The transgenic plants ,with higher adaptability to high temperature than wild type, could keep running of photosynthesis in high stress.
5. Constructed pBI-AFS-GFP eukaryotic expression vector by the cloned the cDNA of AFS. Transient expression of AFS-GFP fusion protein in protoplast showed that AFS was localized in the chloroplast.
6. Monoterpenes, sesquiterpenes, and other volatiles were found in relatively high amounts in transgenic tomato leaves, but no farnesene was detected in the both samples.
引文
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