| China is a big producer of fruits,with apple planting area and output ranking first in the world.In recent years,the changes in apple cultivation area and yield in our country have gradually stabilized,and the improvement of fruit quality has become the core issue of apple industry development.Most of the early/mid-ripening apple fruits are prone to decrease in firmness/brittleness during post-harvest storage,which seriously reduces the fruit quality and storage tolerance.Therefore,studying the regulation mechanism of postharvest apple fruit softening is of great significance to for improving fruit quality,prolonging shelf life and breeding new varieties with high quality.In this thesis,the apple fruits of ’Qinguan’ cultivar with faster softening rate after harvest and ’Nagafu 2’ apple with slower softening rate were selected as materials,and 1methylcyclopropene(1-MCP)treatment was carried out at 20℃.The changes in fruit firmness,ethylene production and total soluble solid content during fruit storage were analyzed.We focused on the degradation of pectin during fruit softening,and analyzed the changes in the content of different types of pectin during apple softening.Three main pectin degradation related hydrolase,β-galactosidase(β-Gal),polygalacturonase(PG),pectin methylesterase(PE)activity,were analyzed.Based on the apple genome database and RNASeq data,β-Gal/PG/PE encoding genes were isolated and their expressions were analyzed,and important candidate members of apple pectin degradation were identified.By analyzing the differentially expressed transcription factors during apple softening,the candidate AP2/ERF and bHLH gene family members were isolated.The regulatory effects of ERF and bHLH members on genes related to pectin degradation were analyzed by using dual-luciferase system.The main research results are as follows:(1)The changes in fruit firmness and ethylene production during postharvest storage were analyzed.The fruit firmness of the two apple varieties decreased with the increase of ethylene production at the early stage of storage,and the softening rate of the fruit decreased during the late period of ethylene release peak.Both ethylene production and fruit softening were inhibited by 1-MCP treatment,and ethylene production and fruit softening rate were related to the variety.The ethylene release of the control group ’Qinguan’ was always higher than that of ’Nagafu 2’,and the peak value of ethylene production of ’Nagafu 2’ was 138.49μL·kg-1·h-1 at 28 d,while the peak value of ’Nagafu 2’ was 35.62 μL·kg-1·h-1 at the same period the former was 3.9 times that of the latter.The 1-MCP treatment significantly inhibited the ethylene production of postharvest fruits and kept it at a low level without the ethylene production peak.After 60 days of storage,the firmness of ’Qinguan’ decreased by 39.7%,while that of ’Nagafu 2’ decreased by 24.4%,indicating that the softening process of ’Qinguan’was faster than that of ’Nagafu 2’.There was no significant difference in the content of soluble solids between the two cultivars.(2)The content changes of different pectin types and the activity of β-Gal,PG and PE related to pectin degradation during the post-harvest apple softening process were analysed.The content of propectin(ASP)decreased,while the content of water-soluble pectin(WSP)increased gradually during storage.The content of ASP and WSP in fruit was highly correlated with the firmness of apple fruit after postharvest,which reached significant or extremely significant level.1-MCP treatment can effectively slow down the above process.The activities of β-Gal and PG increased significantly,while the activities of PE increased slightly,and 1-MCP treatment significantly inhibited the activities of β-Gal,PG and PE.The results showed that α-Gal,PG and PE were closely related to pectin degradation and fruit softening.(3)The corresponding encoding genes of β-Gal,PG and PE were cloned and their expressions were analyzed.Based on transcriptome data and apple genome database,combined with WGCNA(weighted gene co-expression network analysis),a total of five pectin degrading enzyme encoding genes(Mdβ-GAL101、Mdβ-GAL18、MdPG1、MdPgu1、MdPE6)and three expansion proteins(MdEXP-A8a、MdEXP-A8b、MdEXP-A4-like)were screened.Three bHLH transcription factors(MdIBH1、MdbHLH121、MdEGL1-like)and three AP2/ERF transcription factors(MdERF2、MdERF008、MdAP2-like)were screened and obtained,and clustering analysis was performed with bHLH family and ERF subfamily transcription factors reported in tomato.Gene expression analysis showed that all of these genes were inhibited by 1-MCP treatment to varying degrees.Moreover,the expression of these genes in the faster softening ’Qinguan’ was significantly higher than that in the slower softening ’Nagafu 2’,indicating that these genes are important candidate genes for the postharvest softening of apple fruit.(4)The regulatory effects of softening candidate AP2/ERF and bHLH transcription factors on the promoters of cell wall degradation genes were analyzed by dual luciferase assay.The results showed that MdbHLH 121 and MdAP2-like transcription factors could activate the promoter activity of Mdβ-GAL18,respectively.These results suggest that MdbHLH121 and MdAP2-like may be involved in the regulation of apple softening process,but the specific mechanism remains to be further analyzed. |
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