Peach Acc Synthase Gene Separation And Peach, Tomato In Vitro Conversion System

Peach (Prunus persica L. Batsch), a favorite and famous fruit, well-received by people. But peach unique maturity characteristic resulted in bad preservation and freshness. The maturity of fruit was closely related to ethylene biosynthesis. ACC synthase and ACC oxidase are two key enzymes during the course. Juicy peach cv. Xinbaihua was used as materials to carry out isolation of ACC synthase gene ripening-related and establishment of peach and tomato in vitro culture transformation system which laid down a solid foundation for a prolonged shelf life using genetic engineering technology.Method of genomic DNA extraction was improved from such respects as enhancing concentration of CTAB up to 3%, adding of 3% PVP and prolonging water bath time to 1.5 h. Using this method, high quality genomic DNA solution was acquired, containing less polysaccharides, without browning and fit for further molecular operation.Two genomic DNA fragments were obtained by PCR method using two pairs of primers (P₁, P₂, P₃ and P₄) which are specific to peach cv. Hakuho ACC synthase gene cDNA ripening-related. The complete sequence (GenBank Accession: AY994054) assembly was analyzed by bioinformatics method. The total 2496 bp sequence comprised four exons and three introns. The length of codon sequence is 1479 bp. The coding region of peach ACC synthase was 98% and 84% identical to mume (Prunus mume) and pear (Pyrus communis). It encoded 492-amino-acid polypeptide (except the stop codon) with a predicted size of 55kD and a calculated pI of 8.26. The deduced protein contained the active site SLSKDMGLPGLR of ACC synthase.Tomato is the model plant studied on genetic transformation research field, as well as a climacteric fruit like peach. And the homology between these two ACC synthase genes was about 70%. So it is feasible to regard tomato as a receptor system to study characteristic and function of peach ACC synthase gene ripening-induced. It was necessary to establish in vitro culture system to study difference between transformation of peach and tomato with ACC synthase gene and its expression and regulation.In the study, a regeneration system of juicy peach was established. Explants were cultured on MS basal media supplemented with 6-BA (0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0mg/L) and IBA (0.0, 0.05, 0.1, 0.3, 0.5, 1.0, 2.0 mg/L) at different levels to compare their influence on axillary bud culture and shoot proliferation. The results showed that the multiple shoot induction was the most obvious on MS medium supplemented with 6-BA 2.0 mg/L. MS + 6-BA 1.0 mg/L was the most appropriate proliferation medium. MS + 6-BA 1.0 mg/L + IBA 0.5 mg/L was the most appropriate media for plantlet strengthening. Shoots cultured on half strength MS medium supplemented with 1 mg/L IBA and 2% sucrose for 7 days were transferred to MS + 6-BA 2.0 mg/L medium. Strong white roots were issued with the method of two-step induction. Then, regenerated plantlets were obtained from mature tissue.Meanwhile, tomato cv. Baoda 906 cotyledons and hypocotyls from axenic seedlings were cultured on MS basal media supplemented with 6-BA (0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/L) and NAA (0.0, 0.1, 0.3, 0.5 mg/L) at different levels to compare their influence on callus inducing and bud formation. The results showed that the most optimum medium for cotyledon differentiation was MS + 6-BA 1.0 mg/L + NAA 0.1 mg/L and the frequency was up to 93.5%. The most optimum medium for hypocotyl differentiation was MS + 6-BA 1.5 mg/L and the frequency was 55.6%. So cotyledon is the better explant for establishment of tomato transformation system. Strong buds, 1-2 cm long, were transferred on MS media to induce adventitious roots and the root rate up to 100% was obtained after 7 days. The reaction difference was compared between tomato cotyledons and hypocotyls on MS media supplement with Hyg (0, 5, 10, 15, 20, 30, 40, 50 mg/L) at different concentration. The optimal selected pressure of cotyledon and hypocotyl of Hyg was 20 mg/L and 40 mg/L respectively. Agrobacterium-mediated transformation was carried out using the system above-mentioned. Transformants were selected on the media with Hyg and PCR method was used to identify the transgenic plants.