| The main purpose of the melon breeding is to improve the storage and transportation duration properties of the melon, and become an urgent problem should be solved as early as possible in the practice. In recent years, a set of transgene plants was acquired by the using of genetic engineer for the controlling of ripening in melon. However, the great majority methods adopt the constitutive promoter during Construction of Expression Vector. The expression of the gene in the transgenic plant can keep in stable levels under the controlling of the constitutive promoter. The foreign source gene, which show high-efficient and continuous expression of non-specific in the receptor plant, not only causes the waste of the energy, and it also causes that the anticipant purpose can not achieve because of the low expressing amount passed or large amount of time to express the gene. Therefore, the choosing of inducing or specific expression can improve the gene expression greatly in the transgenic study. This also reduces the consumption of energy and material and increases the expression amount. In order to meet the various needings, the expression amount of foreign source gene and spacial dependent characteristic should be controlled.In this study, it is focus on the waste of the energy and the security of the antibiotic caused by the using of the constitutive promoter in the ACC oxidase in the transgenic melon. Based on the cloned CM-ACO1 promoter, following researches have been done:1. The strategy of the construction of the plant expressional vector: A dual-vector pBI101.2 and a plasmid pCM-ACO1-promoter were digested with restriction endonuclease Hindâ…¢and BamH I. And then, the pBI101.2 wirelike vector and the promoter fragment of 886bp were acquired. Becase the promoter was modified by the Hindâ…¢and BamH I, the acquired target genes fragment, digested by the restriction endonuclease, was matched to the wirelike vector. The promoter was added to the GUS gene through the appraisement such as the joining, transforming and screening. And finally, a plant expression vector named pCAPG101.2 with nptâ…¡gene was constructed.2. The GUS and promoter pCAPG101.2 were transferred into Agrobacterium tumefacien EHA105. The Agrobacterium tumefacien was transferred into the plants through the Kan resisting screening and PCR. The transference with the Agrobacterium tumefacien to Nicotiana tabacum were further confirmed the feasibility of the vector.3. Adopt optimized transformation system of the melon cultivar: the explant infected in the engineering bacterium the OD560 of which was 0.30 for 15min, and co-cultured for 3days; Choosing cultivated in MS +1mg/L 6 - BA + 500mg/L carb + 75mg/L Kan, extend cultivated in MS +0.05mg/L 6 - BA + 300mg/L carb + 75mg/L Kan and rhizogenic cultivated in 1/2MS + 200mg/L cef + 25mg/L Kan. 2000 explants were transformed, and 14 transformed plants, with Kana resisting and masculine PCR were acquired. Some of these plants were cultivated in the greenhouse and the situation of growing were observe.4.The rhizoma, stem, leaf, flower, fruits (young fruit and ripe fruit) of the acquired transgenic plant under various growth processes were dyed by X-Gluc. And the results indicated that only anther and ripe fruit was blue and other organizations were not detected the expression of GUS gene. This phenomenon proved that the CM-ACO1 promoter promote the expression of special characteristics of GUS gene. The controlling of the expression amount of the foreign source gene and the spatial expressions of the gene were established. |
