蓖麻Caleosin基因的克隆及功能验证
发布时间：2018-08-04 20:10
【摘要】：生物柴油是一种可再生的生物能源,可以作为化石燃油的替代品,在缓解能源危机和减少环境污染方面有重要作用,通过基因工程方法调控植物体中脂肪酸的合成可以提高植物中的含油量,从而为制造生物柴油提供更多的原材料。油体是植物细胞中主要的储油细胞器,而油体钙蛋白(Caleosin)作为油体结构的重要组成成分,与油体的形成、油体的数量、油体结构的稳定以及植物细胞中油脂的积累有密切的关系。烟草具有繁殖快速,生物量大,遗传转化体系成熟等优点,通过基因工程手段提高烟草叶片中的油脂含量可以用来提取油脂制备生物柴油,为此本研究在做了以下试验：1.通过逆转录PCR方法克隆了油料作物蓖麻的油体钙蛋白(Caleosin)基因,并对该基因在蓖麻不同组织器官以及不同发育时期种子中的表达情况进行了半定量表达分析,结果表明,蓖麻Caleosin基因在嫩叶、茎、幼根、开花后20d、30d、40d的种子中检测到了表达,在种子中的表达量要高于在其他组织中的表达量,并且主要集中在种子发育中后期(油脂积累期)表达,说明Caleosin基因的表达与油脂积累过程是同步的。2.将克隆的蓖麻Caleosin基因与植物表达载体pCAMBIA 1303连接,构建了含有蓖麻Caleosin基因的重组植物表达载体pCAMBIA-RcClo。利用农杆菌介导的叶盘法将重组表达载体转入烟草,获得了转基因烟草植株。3.对获得的转基因烟草植株的叶片进行脂肪酸含量测定,结果表明转基因烟草叶片的脂肪酸含量比野生型烟草叶片提高了8.1%。
[Abstract]:Biodiesel is a renewable bio-energy source that can be used as an alternative to fossil fuels and plays an important role in alleviating the energy crisis and reducing environmental pollution, Controlling the synthesis of fatty acids in plants by genetic engineering can increase the oil content in plants and provide more raw materials for the manufacture of biodiesel. Oil body is the main organelle of oil storage in plant cells. As an important component of oil body structure, (Caleosin) is closely related to the formation of oil body, the quantity of oil body, the stability of oil body structure and the accumulation of oil in plant cells. Tobacco has the advantages of rapid reproduction, large biomass and mature genetic transformation system. Increasing the oil content in tobacco leaves by genetic engineering can be used to extract oil to produce biodiesel. In this study, the following experiments were carried out: 1. The oil troponin (Caleosin) gene of oil crop castor was cloned by reverse transcription PCR. The expression of the gene in different tissues and organs of castor and seeds at different developmental stages was analyzed by semi-quantitative analysis. The expression of ricin Caleosin gene was detected in young leaves, stems, roots and seeds 20 days after anthesis. The expression level in seeds was higher than that in other tissues, and it was mainly expressed in the middle and late stage of seed development (oil accumulation stage). It is suggested that the expression of Caleosin gene is synchronized with the accumulation of oil. 2. The recombinant plant expression vector pCAMBIA-RcCloin containing castor plant Caleosin gene was constructed by ligating the cloned ricin Caleosin gene with plant expression vector pCAMBIA 1303. The recombinant expression vector was transformed into tobacco by Agrobacterium tumefaciens mediated leaf disc method, and transgenic tobacco plant. 3. 3 was obtained. The fatty acid content of transgenic tobacco leaves was measured. The results showed that the fatty acid content of transgenic tobacco leaves was 8.1% higher than that of wild tobacco leaves.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2;S565.6
本文编号：2165007
[Abstract]:Biodiesel is a renewable bio-energy source that can be used as an alternative to fossil fuels and plays an important role in alleviating the energy crisis and reducing environmental pollution, Controlling the synthesis of fatty acids in plants by genetic engineering can increase the oil content in plants and provide more raw materials for the manufacture of biodiesel. Oil body is the main organelle of oil storage in plant cells. As an important component of oil body structure, (Caleosin) is closely related to the formation of oil body, the quantity of oil body, the stability of oil body structure and the accumulation of oil in plant cells. Tobacco has the advantages of rapid reproduction, large biomass and mature genetic transformation system. Increasing the oil content in tobacco leaves by genetic engineering can be used to extract oil to produce biodiesel. In this study, the following experiments were carried out: 1. The oil troponin (Caleosin) gene of oil crop castor was cloned by reverse transcription PCR. The expression of the gene in different tissues and organs of castor and seeds at different developmental stages was analyzed by semi-quantitative analysis. The expression of ricin Caleosin gene was detected in young leaves, stems, roots and seeds 20 days after anthesis. The expression level in seeds was higher than that in other tissues, and it was mainly expressed in the middle and late stage of seed development (oil accumulation stage). It is suggested that the expression of Caleosin gene is synchronized with the accumulation of oil. 2. The recombinant plant expression vector pCAMBIA-RcCloin containing castor plant Caleosin gene was constructed by ligating the cloned ricin Caleosin gene with plant expression vector pCAMBIA 1303. The recombinant expression vector was transformed into tobacco by Agrobacterium tumefaciens mediated leaf disc method, and transgenic tobacco plant. 3. 3 was obtained. The fatty acid content of transgenic tobacco leaves was measured. The results showed that the fatty acid content of transgenic tobacco leaves was 8.1% higher than that of wild tobacco leaves.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2;S565.6
【相似文献】
相关硕士学位论文 前2条
1 李传宝;蓖麻Caleosin基因的克隆及功能验证[D];山西农业大学;2016年
2 丁勇;甘蓝型油菜caleosin基因克隆与表达[D];华中农业大学;2006年
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