| The main factor limiting the commercial production of Carica papaya L. lies in the deterioration of fruit caused by rapid softening during postharvest storage and fresh-cut processing. In this paper, the key hydrolytic enzymeβ-GALs, which were responsible for the cell wall degradation and thus the fruit softening during postharvest ripening of papaya, were aimed at for the determination of relationship between its expression and fruit softening at the molecular level, on the advances of researches of postharvest physiology and genetic transformation of antisense ACS and ACO gene for papaya. The co-transformation transgenic papaya was obtained using co-cultivation of embryogenic calli with Agrobacterium tumefaciens harboring a RNAi-two-T-DNA plant expression vector driven by promoter characterized with fruit specificity, which facilitated the breeding of softening-resistant and marker-free transgenic papaya ripening normally and good for fresh-cut processing. The main results were as follows:1. Establishment of papaya embryogenesis system for genetic transformation. Explants of leaves and petioles of in vitro culture"zhanghong"papaya plantlets were inoculated on the modified MS media supplemented with 0.5 mg·L-1 KT and 1.0 mg·L-1 2, 4-D and 0.5 mg·L-1 BA and 0.1 mg·L-1 NAA and 400 mg·L-1 Glu and 30 g·L-1 sucrose and two kinds of the embryogenic calli were induced. Embryogenic calli of CⅡa type could be subcultured and multiplied and turn into CⅡb type, which were prone to forming somatic embryo. Two-step method for rooting showed the best rooting induction rate. Regenerated plantlets via somatic embryogenesis were firstly inoculated on the 1/2 MS media containing 0.5 mg·L-1 IBA and 1.0 g·L-1 AC and 30 g·L-1 sucrose in dark for 7 days, and then transferred onto 1/2 MS media containing 1.0 g·L-1 AC and 25μM·L-1 VB12 and 30 g·L-1 sucrose. After transplantation, the viability rate reached over 45%.2. Cloning and analysis of the first kind ofβ-Gal gene in papaya pulp. The total change tendency of expression level ofβ-Gal gene family during ripening and softening of papaya fruit was determined using a pair of degenerate primers, indicating the close relationship betweenβ-GALs and fruit softening. And the first kind ofβ-Gal gene cDNA with the highest expression abundance at the stage of 50% maturity, when the fruit pulp became rapid softening, was cloned. A 4501 bp genomic sequence coding for this gene was isolated, containing 17 introns and exons being one base different from the cDNA sequence. Bioinformatic analysis of this gene revealed that the protein belonged to 35 family of glycoside hydrolyase 42 superfamily, genetic relationship of which was closer with Arabidopsis thaliana and further with Persea americana and Picea sitchensis. Additionally, the predicted protein included a signal peptide located extracellularly, indicating the possible involvement of this enzyme in the degradation of cell wall matrix thus in the fruit softening.3. Isolation and preliminary characterization of the promoter of first kindβ-Gal gene. A 1143 bp of 5' regulated sequence of the first kindβ-Gal gene was isolated using inverse PCR. Online database prediction identified core promoter elements of TATA box and a predicted transcription start at -133 upstream of the start coden, motifs for responsiveness of phytohormone especially for ethylene, and stress, and for organ specificity, and enhancer region. Characterization of this putative promoter which, by driving GUS on pCAMBIA 1301 and using Agrobacterium tumefaciens co-cultivation with different organs, was wound-inducible and organ development-related, revealed that the GUS activity was the most in fruit pulp, moderate in embryo and root and none in other organs tested.4. Construction of plant expression vectors for the genetic transformation of papaya. Conserved region ofβ-Gal gene, which coded for a key enzyme ofβ-galactosidase involved in the cell wall degradation, with the highest expression abundance at the stage of rapid softening of papaya pulp was cloned. The RNAi intermediate expression vector of pKAN/RG was constructed containingβ-Gal genes in an inverted repeat orientation with the help of pKANNIBAL vector. hptⅡgene of the modified pCAMBIA 1300 vector was replaced by the hairpin structure of pKAN/RG, which resulted in the formation of intermediate expression vector of p1300-/MFRG. Single T-DNA region of p1300-/MFRG was isolated and incorporated into the pCAMBIA 2301 vector to produce the RNAi Two-T-DNA plant expression vector of p2301/TTRG. The transformation of p2301/TTRG into Agrobactrium tumefaciens EHA 105 was confirmed by restriction enzyme analysis and PCR assay. Embryogenic calli of papaya which showed kanamycin resistance and GUS positive were obtained via genetic transformation.5. Establishment and optimization of methods for the papaya genetic transformation mediated by Agrobacterium tumefaciens EHA 105 harboring plant expression vector p2301/TTRG. Methods of shaking in liquid media, infusion, pricking, and stem-infection for papaya genetic transformation were compared and optimized according to their efficiency of transformation or co-transformation. The first two methods both applied to the regeneration of transgenic papaya, with the optimized protocol that the concentration of 100μmol·L-1 AS for the preparation of Agrobacterium before diluting culture to OD 0.2 and the time of 20 min for infection and of 2 d for co-cultivation, for transformation; and the one consisting of 100μmol·L-1 AS and OD 0.2 and 20 min and 3 d, for co-transformation. However, the last two methods did not work well. Five regenerated transgenic plantlets were obtained, of which two were proved to be the result of co-transformation using PCR assay and southern blot, and a co-transformed one was induced to root and transplanted.6. Regenerated of BPTTRG-transformed plantlet and a preliminary study on the transformation of MFRG in papaya. CⅡb type of embryogenic calli were co-cultivated with Agrobacterium tumefaciens EHA 105 harboring plant expression vector p2301/BPTTRG and p1300-/MFRG, respectively, using infusion method. The BPTTRG transformation produced a co-transformed transgenic papaya showing positivity in GUS staining, PCR assay and southern blot. DNAs of the 198 regenerated plantlets from the MFRG transformation were extracted and divided into sixty-two multiple pools. Of two positive pools after PCR assay, none of individual plantlet from either pool was PCR positive, possibly indicating the chimeric phenomenon occurred during the co-transformation of papaya without selection pressure of antibiotics.In summary, the two-T-DNA co-transformed transgenic papaya with a RNAi-β-Gal structure introduced into genome was obtained. For the subsequent work, it is promising to breed a softening-resistant and marker-free papaya through sexual hybridization of the co-transformed transgenic plants, for the issue of safety of transgenic food.
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