Optimization Of Oligogalacturonides Preparation And Study Of Its Biological Mechanisms

Fruit softening is an inevitable physiological process from ripening to senescence in most fruits,is an important indicator affecting fruit quality,and is a critical area of postharvest physiological research.Fruit ripening and softening exacerbate mechanical damage incurred during shipping and handling and increases pathogen susceptibility.In this study,oligogalacturonides（OGs）were produced in the hydrolysis produced of apple pectin with three fungal polygalacturonases（Pectin lyase PL,oligogalacturonase PG,Botrytis cinerea secreted protein）.The enzymolysis products and their optimum extraction conditions were detected by the method of HPLC.Using the model plant tomato（Solanum lycopersicum）as the main research material to tackle the problem of rapid softening of fruit after harvest.Combined with RNA-Seq technology to investigate the function of their differential genes,explored the relationship between OGs and resistance to fruit softening,and relational resistance genes expression,to investigate the mechanism of fruit softening.Evaluated the molecular biological mechanisms of OGs in the quality regulation of fresh fruits and vegetables and immune regulation.Taking the perishable and hard-to-store berries fruits as the representative,the OGs were used in practical application tests to establish the foundation for the development and enhancement of post-harvest physiological and pathological deterioration control techniques for fresh fruit and vegetable products.The main findings are as follows:（1）The optimum conditions for the enzymatic hydrolysis of apple pectin by the three pectin-degrading enzymes were optimized.The optimum extraction condition of enzyme 1was:12h,50℃,and pH at 4;enzyme 2 was:3 h,30℃,and pH at 7;and enzyme 3 was:3 h,30℃,and pH at 5.The OGs obtained from the hydrolysis of pectin derived from citrus peel and apple pomace by pectinase PG were more effective in alleviating fruit softening,while the degree of esterification of pectin differed in their effectiveness in alleviating fruit softening.（2）Separation of OGs was using Bio-Gel P-2,analysis was combined with HPLC,MALDI-TOF-MS,and plant physiological analysis.The molecular weight of OGs in368.98,505.21,681.27,and 857.33 Da were found to have biological functions in regulating fruit softening.（3）Tomato fruit treated with OGs can maintain fruit firmness at（8.37±0.45 N）at 13d of storage which was the same as the fruit firmness level of 5 d in the control group.RT-qPCR revealed significant down-regulation of SlPG2,SlPL3,SlPL5,and other cell wall degradation relevant genes after OGs induction.Transcriptome analysis indicated the ethylene signaling pathway,MAPK signaling cascade,reactive oxygen species,and calmodulin contributed to the OGsmediated firmness response.（4）By comparing the softening effects of OGs and the ethylene receptor inhibitor1-MCP,it was found that OGs could delay the softening of the whole tomato fruit without significantly affecting the total soluble solids and titratable acidity content of the fruit during the ripening process.At the same time,it can increase the production of jasmonic acid after wounding treatment,slow down the release of ethylene during fruit storage,and prolong the shelf life by inhibiting the expression of the endopectinase encoding gene in the fruit.（5）The application of OGs in berries,such as grapes,cherry tomatoes,blueberries,strawberries,and wolfberries,found that the ability of OGs in reducing fruit softening was also significant.When stored on the 7th day,the firmness of grapes,cherry tomatoes,blueberries,strawberries,and wolfberries in the OGs treatment group were 38.20,38.83,37.48,69.87,and 49.44%higher than those in the control group,respectively.And effectively reduce the loss rate of fruit during storage,and maintain the stable change of fruit soluble solid content.Demonstrating that a low degree of polymerization OGs can effectively inhibit post-harvest physiological and pathological quality deterioration of fruit.（6）Using OGs to induce treatment of wolfberry fruit,it was found that OGs treatment could significantly inhibit the softening of the fruit,reduce the fruit loss rate,and maintain the soluble solid content of the fruit and other basic fruit quality indicators more stably.By increasing the activity of enzymes related to the reactive oxygen species scavenging system such as SOD,CAT,and POD during storage,the ROS in the system can be removed in time,resulting in a reduction in membrane lipid peroxidation,thereby reducing the accumulation of H₂O₂ and MDA.At the same time,OGs treatment enhanced the PAL activity during storage and promoted the synthesis of secondary metabolites.As well as the ability of plant tissues to accumulate callose allows OGs induction to better maintain the stability of fruit quality during storage and extend the shelf life of wolfberry fruit.