| Kiwifruit is one of the outstanding materials for fruit ripening and softening research.Postharvest softening is an essential process for commercial mature fruit to reach the edible firmness,however,the undesired rapid softening would reduce the storability and transportability.In order to improve the understanding on the basis of kiwifruit postharvest softening,the present research used Actinidia delisiosa'Hayward'(HWD)and Actinidia chinesis 'Hongyang'(HY)as the materials,employed ethylene(ETH)and 1-Methylcyclopropene(1-MCP)treatments to manipulate fruit postharvest softening,analyzed the dynamics of pectin and its related enzymes:Polygalacturonase(PG)and ?-galactosidase(?-Gal),performed a genome-wide genes isolation of PG and ?-Gal,characterized the key candidate genes for fruit softening,and also conducted a preliminary investigation on transcription factors that contribute to AdPG1 regulation.The main results are as follows:1.Analysis on cell wall polysaccharides.The content of water soluble pectin polysaccharide increased gradually,while the content of EDTA soluble pectin polysaccharide,Na2CO3 soluble pectin polysaccharide,KOH soluble hemicellulosic polysaccharides and CWM-residue polysaccharide decreased gradually during postharvest ripening.ETH and 1-MCP treatments can significantly accelerate and inhibit such changes of cell wall polysaccharides.These results once again indicated the association of pectin degradation and fruit softening.2.Analysis on pectin degradation enzymes(PG and ?-Gal).The results showed that PG and ?-Gal activity also has similar changs in 'HWD' and 'HY'.The activities of PG and ?-Gal graduately increased during fruit ripening,which were significantly enhanced by ETH.In 'HWD',activities of PG and ?-Gal in 1-MCP treated fruit remain at basal level at all examinated stages.Thus,PG and ?-Gal may contribute to pection degradation.3.Gene isolation and expression analysis.The reported coding genes for PG and?-Gal were very limited.Based on genome database and RNA-seq,13 novel AdPG genes were isolated and were designed as AdPG1-13.Phylogenetic tree indicated that AdPG1 was clustered with previously reported CkPGC.Meanwhile,9 AdGal genes were isolated and AdGal3 is a full-length ORF for previous reported fragment of a Gal gene.Gene expression analysis indicated multiple genes that associated with fruit ripening.Among the PG genes,CkPGC and AdPG1 showed positive responses to ETH and were negatively regulated by 1-MCP.Also,these two PG genes were also most abundant members.Correlation analysis further supported CkPGC and AdPG1 may correlated with both'HWD' and 'HY' softening.Unlike the PG genes,Gal genes showed differences between to cultivars.In 'HWD',AdGal1 showed the highest correlation with ?-Gal activity and most abundant.Although AdGal 1 was also most abundant member in 'HY' fruit,however,its correlation(R2)to ?-Gal activity was only 0.418.4.Yeast one hybrid library screening with AdPG1 promoter.Transcription factors AdBPCl,AdBPC2 and AdBPC3 were observed,with the promotor region of AdPGl,by using yeast one hybrid screen.After library screening,AdBPC2 and AdPBC3 were further verified with physical interaction on AdPG1 promoter,with individual yeast one hybrid assays.Dual luciferase assay in Nicotiana benthamiana leaves showed that AdBPC2 and AdBPC3 could repress the activities of the promoter of AdPG1.These results indicated the potential transcription regulation on pectin degradation related genes,however the mechanisms and roles of these transcription factors require further exploration. |
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