| Sand pear (Pyrus pyrifolia Nakai) is an important fruit crop in China. However, the short shelf-life of this fruit is a serious problem and causes a high loss of yield and commercial value. Six full-length cDNAs encoding enzyme or protein genes related to fruit ripening and softening, such as ACC synthase (ACS), ACC oxidase (ACO), xyloglucan endotransglucosylase/hydrolase (XTH) and expansins (EXP) were isolated from mature fruit of sand pear using RT-PCR and RACE methods. The expression of these genes as above was detected by real-time fluorescent qRT-PCR during fruit growth and development. Three binary antisense or sense expression vectors of Ppy-ACS and Ppy-ACO were constructed for plant transformation. In addition, the effects of exogenous ethylene and1-MCP on expression of Ppy-ACS and Ppy-ACO in postharvest fruit were determined, and conserved microRNAs were detected on mature fruit by microarray. Their results were as follow.1. The full-length cDNA (GenBank accession no. EF566865) of Ppy-ACS is1,939bp, which includes109bp of the5’-UTR, an ORF of1,488bp, and342bp of the3’-UTR. The Ppy-ACS encodes a polypeptide be composed of495amino acid residues, which includes seven conserved regions and an active site consisting of twelve essential amino acid residues, i.e. SLSKDMGFPGLR. Two binary expression vectors of Ppy-ACS, antisense named as pPpy-ACS(-) and sense named as pPpy-ACS(+), were constructed by inserting the target fragment in pYPX145. The target genes are controlled by a double CaMV35S promoter. The pPpy-ACS(-/+) was successfully introduced into Agrobacterium tumefaciens strain EHA105.2. The Ppy-ACO full-length cDNA (EF451060) is1,225bp, which contains is945nucleotides for ORF,63bp of the5’-UTR and217bp of the3’-UTR. The deduced amino acid sequence of the Ppy-ACO is314residues and includes twelve conserved amino acid residues belonging to non-heme iron (Ⅱ) dependent family of oxygenases/oxidases and three residues essential for emzyme activation. A binary antisense expression vector with Ppy-ACO coding region was constructed by inserting the target fragment in reverse orientation in pYPX145. The expression of the antisense gene is controlled by a double CaMV35S promoter. The vector was successfully introduced into Agrobacterium tumefaciens strain LBA4404.3. Expression of Ppy-ACS and Ppy-ACO using real-time fluorescent qRT-PCR increased with fruit growth and development, and expression of the Ppy-ACO was obviously higher than that of the Ppy-ACS during fruit growth and development.1-MCP treatment obviously inhibited the Ppy-ACS expression in12days after harvest and down-regulated the Ppy-ACO expression throughout fruit storage period. However, exogenous ethyl ene significantly up-regulated the Ppy-ACS expression at early and later stages, and evidently promoted the Ppy-ACO expression all time after harvest, only reduced at the end of fruit storage.4. Xyloglucan endotransglucosylase/hydrolase gene (named as Ppy-XTH, EU432411) is1,248bp contained73nuleotides of the5’-UTR, an ORF consiting of885nucleotides, and290nucleotides of the3’-UTR. The deduced amino acid sequence of the Ppy-XTH is294amino acid residues, which includes a signal peptide composed of22amino acid residues in the N-timminal, and shares a conserved DEIDFEFLD motif that is likely to be the catalytic site for both xyloglucan endotransglycosylase (XET) and xyloglucan endohydrolase (XEH) activity. The result of cluster analysis of amino acid sequence for mature Ppy-XTH polypeptide with other plant such as Arabidopsis, tomato, apple and kiwifruit showed Ppy-XTH belongs to group1, which expressed in flower and fruit. The accumulation of the Ppy-XTH mRNA in fruit could be detected during the whole fruit growth and development, obviously increased from the beginning of fruit rapid growth at98days after full bloom (DAFB), steadily maintained high level from98DAFB to126DAFB, evidently decreased at the end of fruit growth phase (140DAFB).5. Three expansin gene cDNAs, designate as Ppy-EXP1(EF602031), Ppy-EXP2(FJ619347) and Ppy-EXP3(FJ619348), were cloned from mature fruit of sand pear. The full-length cDNA of Ppy-EXP1, Ppy-EXP2and Ppy-EXP3were1,369bp,1,107bp and1,136bp, respectively. The Ppy-EXP1, Ppy-EXP1and Ppy-EXP3ordinally contain an ORF of759bp,756bp and765bp, and encode a polypeptide consisting of252,251and254amino acid residues, respectively. They all contain a histidine (His-Phe-Asp, HFD) motif as conversed amino acid domain of expansin for most plants in the central region. A phylogenetic tree was generated from the deduced amino acid alignment of three Ppy-EXPs and26other expansin homologues. Ppy-EXP1and Ppy-EXP2aligned within group B and Ppy-EXP3belonged to group C. Accumulation of Ppy-EXP1and Ppy-EXP2in the developing fruit was low level during0-98DAFB, slightly increased at the end of growth phase, and the increments of the Ppy-EXP1are more than that of the Ppy-EXP2. Accumulation of the Ppy-EXP3was higher than that of the Ppy-EXP1and the Ppy-EXP1in the developing fruit, and increased markedly with fruit growth and development, and reached to the highest level at the end of growth phase (140DAFB).6. Conserved microRNAs were detected in mature fruit by microarray based on6,703microRNAs sequences from miRBase11.0version (http://microrna.sanger.ac.uk/). Six hundred and forty-three microRNAs were detected based on positive signal. Eleven high frequently expressed microRNAs (eight from plant, three from animal) were selected for target prediction. Seven microRNA targets belong to transcription factor families, and the left one is involved in microRNAs metabolism. Isopentenyl transferase gene was predicted as a target for miR-30, but the other two animal microRNAs, miR-124and miR-181, didn’t find their targets. |
