Construction Of A Plant Expression Vector With Melon ACO1 Antisense Gene And Transformation Of Cucumis Melo'GT-1'

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.