人表皮生长因子基因的克隆、表达及其在植物中的遗传转化研究
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摘要
香蕉是我国南方一些省重要的水果，但目前香蕉产业存在许多问题，如香蕉品质差、病虫害严重等。基因工程方法可将有用的外源基因导入香蕉，从而促进香蕉优良品种的培育，提高香蕉的品质，增强市场的竞争力。同时，香蕉作为生物反应器具有它的优越性，用它来表达一些医药蛋白具有广阔的前景。
本研究采用PCR方法克隆了人表皮生长因子基因，构建了原核表达载体和植物表达载体，研究了原核表达的外源蛋白的生物学活性；用薄层培养方法优化了香蕉、芦荟的受体再生系统；首次通过农杆菌介导法将人表皮生长因子基因导入香蕉获得抗性芽；系统研究了影响根癌农杆菌介导的香蕉、芦荟遗传转化的因素。
研究工作获得如下的几个方面的结果：
1、用PCR方法克隆了人表皮生长因子基因。
2、构建人表皮生长因子基因的原核表达载体和植物表达载体。植物表达载体p1301hEGF以35S为真核表达启动子，报告基因和目的基因以Tnos为终止子，植物选择标记基因以35S PolyA加尾，构成嵌合基因。原核抗性为Kan~r。
3、用原核表达得到的融合蛋白(GST-hEGF)进行了小鼠试验，证实其具有很高的免疫原性；用纯化得到的hEGF进行细胞活性检测，表明其能促进细胞生长。
4、建立了以香蕉吸芽为材料，诱导低代香蕉丛生芽，并用此材料进行薄层切片培养，同时对薄层切片的芽分化条件进行了系统的研究。通过这种方法能得到较高的芽分化率。
5、尝试用基因枪(PDS-1000Hi)对薄层切片进行转化，但没有筛选到转化体。
6、初步建立了一个有效的以农杆菌介导的香蕉转化系统。对3个菌株(EHA105、LBA4404和AGL1)浸染力比较研究表明，采用菌株EHA105介导的转化效率高；优化了各种影响转化的因素，如乙酰丁香酮(AS)、蔗糖、重悬液pH值、共培养条件、菌株、共培养时间、工程菌液的制备等。
7、在国内外首次获得仅以农杆菌介导的香蕉转基因抗性芽，也是首次将人表皮生长因子转化到植物中。经过筛选获得抗性芽，并对其进行dot-blotting杂交检测及PCR-Southern分析，证明外源基因已经整合到香蕉基因组中。
8、以芦荟试管苗为材料，采用薄层切片培养方法诱导芽的分化，并对影响薄层切片芽分化的多种因子进行了研究，建立了芦荟遗传转化受体系统。这些研究目前国内外未见报道。
9、对影响农杆菌介导的芦荟遗传转化的一系列因素进行了研究，初步建立了以农杆菌介导的芦荟遗传转化体系。首次将人表皮生长因子转入芦荟，获得较高的GUS基因瞬时表达率。
Banana(Musa spp.) is one of the most important fruits in the south of China and there are many problems in banana industry at present such as the quality of fruits, the serious diseases and insect pests etc. Some useful foreign genes could be transfered into banana by genetic engineering to improve banana varieties and enhance the competition ability of banana production in market. Banana could be served as bio-reactor to produce some medical proteins which has broad perspectives.
In the present study, the human epidermal growth factor(hEGF) was cloned by polymerase chain reaction(PCR), and both plant and Escherichia coli expression vectors were constructed, and bio-activities of the protein expression by Escherichia coli were studied. Meanwhile, the regeneration system of both banana and aloe using the thin cell layer was optimized, and banana transformed with the gene hEGF for the first time, and the gene was expressed in transgenic banana buds. At the same time, the factors for both banana and aloe genetic transformation mediated by Agrobacterium tumefaciens were optimized. The results were as follows:
1. The human epidermal growth factor was cloned by PCR.
2. Two expression plasmids were constructed for transformation. In the plant plasmid plSOlhEGF, the CaMV35S promoter and nos terminator fragments were syncretized with reporter gene and targeted gene. The selectable maker gene terminated by polyA fragment for transformation of plants was hygromycin phosphotransferase gene which allowed selection with hygromycin, and the selectable marker gene for plasmids was neomycin phosphotrasferase gene, which allowed selection with kanamycin.
3. The fusion protein GST-hEGF expressed in Escherichia coli in the experiments with mice showed the recombinant protein had good immunogenecity and the purifid protein hEGF could stimulate the growth of Hela cells efficiently.
4. The regeneration system of banana using the thin cell layers with the materials from lowest generation was optimized.
5. The transformation of banana thin cell layers via particle bombardment was tested but no transgenic bud was obtained.
6. An efficient banana transformation system mediated by Agrobacterium tumefaciens was established. Among three Agrobacterium tumefaciens strains that is, EHA105, LBA4404 and AGL1, the banana thin cell layers were the most sensitive to EHA105, and various factors that affect the genetic transformation of banana via Agrobacterium-mediated such as acetosyringone(AS), sucrose, the pH value of resuspension solution, condition of co-culture, strains of Agrobacterium tumefacients, time of co-culture and the preparation of Agrobacterium tumefacients liquid using for transformation were optimized.
7. For the first time, banana was transformed with the gene hEGF mediated only by Agrobacterium tumefacients. The presence and integration of foreign DNA in the transgenic banana buds were confirmed by polymerase chain reaction(PCR), PCR-Southern blotting and genomic dot-blotting analysis.
8. The regeneration system of aloe also was optimized by using the thin cell layers with the materials from plantlets and many factors that influence the bud differentiation of aloe thin cell layers were optimized.
9. Aloe was transformed with the gene hEGF mediated by Agrobacterium tumefacients and obvious GUS gene transient expression were observed, and a genetic transformation system of aloe was preliminarily set up.
本研究采用PCR方法克隆了人表皮生长因子基因，构建了原核表达载体和植物表达载体，研究了原核表达的外源蛋白的生物学活性；用薄层培养方法优化了香蕉、芦荟的受体再生系统；首次通过农杆菌介导法将人表皮生长因子基因导入香蕉获得抗性芽；系统研究了影响根癌农杆菌介导的香蕉、芦荟遗传转化的因素。
研究工作获得如下的几个方面的结果：
1、用PCR方法克隆了人表皮生长因子基因。
2、构建人表皮生长因子基因的原核表达载体和植物表达载体。植物表达载体p1301hEGF以35S为真核表达启动子，报告基因和目的基因以Tnos为终止子，植物选择标记基因以35S PolyA加尾，构成嵌合基因。原核抗性为Kan~r。
3、用原核表达得到的融合蛋白(GST-hEGF)进行了小鼠试验，证实其具有很高的免疫原性；用纯化得到的hEGF进行细胞活性检测，表明其能促进细胞生长。
4、建立了以香蕉吸芽为材料，诱导低代香蕉丛生芽，并用此材料进行薄层切片培养，同时对薄层切片的芽分化条件进行了系统的研究。通过这种方法能得到较高的芽分化率。
5、尝试用基因枪(PDS-1000Hi)对薄层切片进行转化，但没有筛选到转化体。
6、初步建立了一个有效的以农杆菌介导的香蕉转化系统。对3个菌株(EHA105、LBA4404和AGL1)浸染力比较研究表明，采用菌株EHA105介导的转化效率高；优化了各种影响转化的因素，如乙酰丁香酮(AS)、蔗糖、重悬液pH值、共培养条件、菌株、共培养时间、工程菌液的制备等。
7、在国内外首次获得仅以农杆菌介导的香蕉转基因抗性芽，也是首次将人表皮生长因子转化到植物中。经过筛选获得抗性芽，并对其进行dot-blotting杂交检测及PCR-Southern分析，证明外源基因已经整合到香蕉基因组中。
8、以芦荟试管苗为材料，采用薄层切片培养方法诱导芽的分化，并对影响薄层切片芽分化的多种因子进行了研究，建立了芦荟遗传转化受体系统。这些研究目前国内外未见报道。
9、对影响农杆菌介导的芦荟遗传转化的一系列因素进行了研究，初步建立了以农杆菌介导的芦荟遗传转化体系。首次将人表皮生长因子转入芦荟，获得较高的GUS基因瞬时表达率。
Banana(Musa spp.) is one of the most important fruits in the south of China and there are many problems in banana industry at present such as the quality of fruits, the serious diseases and insect pests etc. Some useful foreign genes could be transfered into banana by genetic engineering to improve banana varieties and enhance the competition ability of banana production in market. Banana could be served as bio-reactor to produce some medical proteins which has broad perspectives.
In the present study, the human epidermal growth factor(hEGF) was cloned by polymerase chain reaction(PCR), and both plant and Escherichia coli expression vectors were constructed, and bio-activities of the protein expression by Escherichia coli were studied. Meanwhile, the regeneration system of both banana and aloe using the thin cell layer was optimized, and banana transformed with the gene hEGF for the first time, and the gene was expressed in transgenic banana buds. At the same time, the factors for both banana and aloe genetic transformation mediated by Agrobacterium tumefaciens were optimized. The results were as follows:
1. The human epidermal growth factor was cloned by PCR.
2. Two expression plasmids were constructed for transformation. In the plant plasmid plSOlhEGF, the CaMV35S promoter and nos terminator fragments were syncretized with reporter gene and targeted gene. The selectable maker gene terminated by polyA fragment for transformation of plants was hygromycin phosphotransferase gene which allowed selection with hygromycin, and the selectable marker gene for plasmids was neomycin phosphotrasferase gene, which allowed selection with kanamycin.
3. The fusion protein GST-hEGF expressed in Escherichia coli in the experiments with mice showed the recombinant protein had good immunogenecity and the purifid protein hEGF could stimulate the growth of Hela cells efficiently.
4. The regeneration system of banana using the thin cell layers with the materials from lowest generation was optimized.
5. The transformation of banana thin cell layers via particle bombardment was tested but no transgenic bud was obtained.
6. An efficient banana transformation system mediated by Agrobacterium tumefaciens was established. Among three Agrobacterium tumefaciens strains that is, EHA105, LBA4404 and AGL1, the banana thin cell layers were the most sensitive to EHA105, and various factors that affect the genetic transformation of banana via Agrobacterium-mediated such as acetosyringone(AS), sucrose, the pH value of resuspension solution, condition of co-culture, strains of Agrobacterium tumefacients, time of co-culture and the preparation of Agrobacterium tumefacients liquid using for transformation were optimized.
7. For the first time, banana was transformed with the gene hEGF mediated only by Agrobacterium tumefacients. The presence and integration of foreign DNA in the transgenic banana buds were confirmed by polymerase chain reaction(PCR), PCR-Southern blotting and genomic dot-blotting analysis.
8. The regeneration system of aloe also was optimized by using the thin cell layers with the materials from plantlets and many factors that influence the bud differentiation of aloe thin cell layers were optimized.
9. Aloe was transformed with the gene hEGF mediated by Agrobacterium tumefacients and obvious GUS gene transient expression were observed, and a genetic transformation system of aloe was preliminarily set up.
引文
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