Cloning And Genetic Transformation Of A Gene Which Encodes1-Aminocyclopropane-1-Carboxylate Oxidase (ACO) In Kiwifruit

With a rich nutrition, especially the high vitamin content, the kiwi fruit(Actinidia deliciosa) is known as "the king of the fruit" by people, and has a good market prospect. But the kiwi fruit is a kind of climacteric fruit, the fruit will be rotten rapidly under the bad storage environment and will lose their economic value. This is a difficult problem in the production and sales of kiwifruit. Ethylene will be synthesised rapidly at the stage of climacteric, and produce a series of physiological and biochemical changes in the fruits. Ethylene is important plant mature hormone, regulating of fruit mature at the aging process, reducing the synthesis of fruit internal vinyl, it’s the most effective way to extend the storage and preservation of fruit. In the control measures of ethylene biosynthesis endogenous, antisense RNA technology is one of the most effective ways, antisense RNA technology inhibit ethylene synthesis of speed limit of important enzyme ACC oxidase gene expression, will reduce the ethylene endogenous synthesis. In this study, Heywood kiwi fruit was used as the test materials, had achieved the following main results by cloning aco genes, building the RNAi express carriers and researching the experiment of genetic transformation.1. Having Cloned the ACO gene of Actinidia deliciosa successfully. According to the ACO genes sequence of other plants which Gene Bank accepted, the unique primers were synthened to amplify and to clone the full-length cDNA sequence of ACO gene from A.deliciosa. The full-length cDNA sequence contents1003bp of nucleatides, the CDS is960bp,and code the319amino acid residues. This cDNA sequence have a high homology with other ACO genes which had logged on gene bank. The top similarity up to94%. The amplified sequence has7conservative regions and have some others characteristics and biochemical characteristics of ACO gene, can be confirmed as the ACO gene(GenBank accession number:JQ062390)。.2. Constructed the antisense expression vector for the ACC oxidase gene of A.deliciosa ’Heywood’. The amplified ACO gene and the amplified pCAMBIA1300plasmid were digested respectively with the enzymes of Sma Ⅰ and Sal Ⅰ. The digested products were ligated with T4DNA ligation enzyme. And the antisense expression vector of ACO gene were successfully constructed. The ligated plasmid was transformed into EHA105Agrobacterium, which was then used for gene transformation to kiwifruit. To detect the constructed plasmid, the technology of PCR was used. The PCR results revealed that the antisense gene was successfully transformed into EHA105.3. Optimized the tissue culture system of A.deliciosa. The inductive culture medium are:MS+6-BA (10mg/L)+NAA(0.2mg/L)+TDZ(0.2mg/L); the subculture medium are:MS+6-BA (0.25mg/L)+NAA(1mg/L). Leaf vaccinated to the inductive medium,7days or so after the leaf vaccination, can see the veins of the leaf blades had formed start at swollen, callus began to grow, kiwi callus induction leaves rate was100%, it can be subcultured once after20days or so.4. Constructed the efficient and stable genetic regeneration systems The shoots and leaves from a superior individual plant,1year old with no pest diseases were selected. Callus shoot and rooting induction were influenced by different hormone combinations of culture medium YS:MS+6-BA (0.25mg/L)+NAA (1mg/L)+cephalosporins (500mg/L)+Carbencillin (400mg/L)+hygromycin (20mg/L), pH6.0. The Agrobacterium tumefaciens were cultured until its OD value to0.6, callus were immersed in A. tumefaciens media for30min, and were shanked every5min. The callus were cultured under25to26℃with a dark environment for3～4d. The RNAi gene was successfully transformed into the culture leaf in vitro of A.deliciosa, and detected the transformation of the plant after the appraisal by PCR. The PCR results showed that the antisense ACO gene had successfully transformed into the leaves, the rate of infected plant were71%. The result of Quantitative Real-Time Fluorescence PCR Technology shows that the ACC oxidase gene expression of regrowth leaves is19%of the wild type.5. Analyzed the rootstocks’effect after grafted.To study the rootstocks’effects, high resistance varieties, ordinary variaties, the kiwifruit of miliangyihao, hongyang and cuiyu were chosen as study varieties. The results showed that, compared with common varieties, it was significantly improved with the rootstock of high resistance varieties, in addition to hongang of vitamin C depressed after grafted outside. The high resistance varieties is an ideal meters of rootstock for miliangyihao and hongyang, but not so good for cuiyu.