【作者】 黄永红；

【导师】 赵长增； 陆璐；

【作者基本信息】 甘肃农业大学 ， 果树学， 2005， 硕士

【摘要】 甜瓜（Cucumis melo L.）在世界园艺业中占有重要地位,种植面积和产量在十大水果中居第9 位,中国甜瓜种植面积和产量均居世界第一。由于大量甜瓜在8 月份成熟,形成明显上市高峰期,此期正值高温季节,甜瓜在贮藏、运输过程中,极易软化、腐烂,给瓜农造成巨大经济损失,也影响了我国甜瓜产业的发展。乙烯在高等植物的生长、发育、成熟、衰老过程中起着重要作用,乙烯的产生与果实的软化、腐烂有密切关系。1979 年,Adman和Yang阐明了乙烯的生物合成途径,证明了ACC 合成酶和ACC 氧化酶是植物体内乙烯合成中的两个限速酶。降低跃变型果实ACC 合成酶和ACC 氧化酶基因的表达,进而减少乙烯的生成量,就可以延缓果实的后熟过程,延长果实的贮藏期。大多数甜瓜属于呼吸跃变型果实,传统的贮藏保鲜措施如冷藏,气调贮藏,只能提供一个良好的贮藏环境,减少贮藏环境中乙烯的存在,但难以从根本上解决果实采后软化问题。本研究拟通过反义RNA技术获得转反义ACC 氧化酶基因甜瓜植株,以期获得耐贮藏的甜瓜新种质。本项目主要从两个方面进行了研究,一是甜瓜ACC 氧化酶反义基因植物表达载体的构建并转化普通烟草;另一个是甜瓜高效遗传转化体系的建立及用ACC 氧化酶反义基因转化一个甜瓜栽培品种‘GT-1’,获得了转基因植株。用限制性内切酶从目的基因供体质粒pBI-aACO1 上切下大小约2.3kb 的目的基因,将其连接在受体质粒pCAMBIA2301 载体上,构建成含有GUS 基因的甜瓜ACC氧化酶反义基因植物表达载体pCB-aACO1。采用直接转化法将pCB-aACO1 导入根癌农杆菌菌株EHA105,并用新构建的工程菌对普通烟草进行了遗传转化。在Kanamycin 选择压力下获得的烟草转化不定芽和完整植株,经过gus 基因组织化学法检测以及PCR 方法鉴定,证实了该反义基因已导入烟草基因组中。此项研究为下一阶段用该反义基因转化甜瓜品种以改良甜瓜果实耐贮运性打下可靠基础。以甜瓜品种‘GT-1’的幼龄子叶作为根癌农杆菌介导转化的外植体,通过对gus基因表达的分析,研究此转化体系的最佳实验参数。实验结果表明,预培养时间、预培养后对外植体进行再创处理、感染时间、共培养时间、农杆菌工程菌的浓度等对转化效率都有一定的影响;但是根癌农杆菌诱导物AS 并不能大幅度提高转化效果。预培养2d,对外植体进行伤口再创处理,用OD₅₆₀=0.3 的农杆菌工程菌感染15-25min,共培养3-4d 的处理,gus 基因表达率达到最高。在上述研究的基础上,以甜瓜‘GT-1’的幼龄子叶作为根癌农杆菌介导转化的外植体,将子叶预培养2d 后,对外植体进行伤口再创处理,用OD560=0.30 的的农杆菌工程菌pCB-aACO1/A.t.EHA105 侵染15min,共培养4d,外植体的GUS 表达率最高达83%。在选择诱导培养基上前期（20d）的Kana+不定芽诱导率高达77.2%。后期更多还原

【Abstract】 Cucumis melo production is an important part of the horticulture industry of world for its planting area and yield is in the ninth place in the ten important fruits. Both area and output in China are in the first place; Due to the large amount of Cucumis melo harvest in August when the temperature is usualy high, Cucumis melo soften and rot easily in the course of store and transport, which lead to great economic loss to planters and restrict the development of the melon industry of our country. Ethylene plays an important role in growth, development, ripeness and senescence of plants and the product of ethylene is correlative closely with softening and rot of fruit. The biosynthetic pathway of ethylene was clarified by Adman andYang in 1979, which prooed that ACC synthase and ACC oxidase were two crucial enzymes that output of ethylene was restricted. Controling the expression of ACC synthase and ACC oxidase gene in the climacteric fruit and reducing output of ethylene in the fruit could slower ripening process and prolong shelf life. Most of Cucumis melo is typical climacteric fruits. The traditional measures to store and keep fruits freshly such as cryopreservation, chemical preservation just provide well environment for preservation and reduce the amount of ethylene of the environment. But it is hard to solve problems of fruit softening thoroughly. The aim of the study is to transfer the Cucumis melo GT-1 with a Cucumis melo ACO1 antisense gene and obtain transgenic plants to improve its quality for store and transport. In the paper, two items were mainly researched .One was the construction of a plant expression vector containing melon ACO1 antisense gene and its transformation to Nicotiana tabacum;The other was the establishment of a high frequency genetic transformation system of Cucumis melo and the transformation of Cucumis melo‘GT-1’with the plant expression vector newly constructed. The result showed the transgenetic plants were obtained. A donator plasmid pBI-aACO1 was digested with restriction endonucleases, then the acquired target genes was directionly ligated to recipient plasmid pCAMBIA2301 which was digested with the same restriction endonucleases, and finally a plant expression vector named pCB-aACO1 expressing melon ACO1 antisense gene and gus gene was constructed. Moreover, pCB-aACO1 was transferred into Agrobacterium tumefacien EHA105 by direct DNA transfer. And the transformation with the new engineering bacterium to Nicotiana tabacum was studied. The adventitious shoots and complete plants obtained under the selection pressure of Kanamycin were determined by gus gene with histochemical method and confirmed with PCR of genome of Nicotiana tabacum.The results showed that the target gene was transferred into the plants. Using the rate of gus gene expression, the optimal conditions for transformation of Cucumis melo cotyledons mediated by Agrobacterium tumefaciens was investigated. Histochemical GUS assay showed that transient expression rate was affected significantly by pre-culture time of explants, treatments of explants cuts, infection time of explants with bacterium suspension, co-culture time of the explants with bacterium suspension and concentration of the engineering bacterium used in the experiment, while influence of adding acetosyringone was not significant. That the explants were pre-cultured 2d, treated newly, infected in engineering bacterium the OD560 of which was 0.30 for 15-25 min, co-cultured 3-4d contributed to the highest level of transient expression. Thus a high frequency transformation system was established. The transformation of Cucumis melo cotyledons mediated by Agrobacterium tumefacience was investigated on the basis of the optimal system. The explants were pre-cultured for 2 days, treated newly, infected in the engineering bacterium the OD560 of which was 0.30 for 15min, and co-cultured for 4days, the transient expression rate of GUS was 83%. The induction rate of adventitious buds with Kana resistance was 77.2% on the selection induction medium at the first period (20d), and that was 37.8% at the late period (40d). The explants co-cultured with bacterium suspension, the adventitious buds induced on the selection mediums and the leave of the regeneration plants were identified using histochemical method and there were a large amount of blue staining on the materials, which showed that gus gene was expressed in the tissues at the lever of protein. The nptII gene, melon ACO1 antisense gene and the gus gene were orderly arranged in series on the plant expression vector pCB-aACO1,therefore ,the melon ACO1 antisense gene were transferrd inte the Cucumis melo plants with the Kana resistance.更多还原