Energy Metabolism Of Pitting Of Blueberry Fruits After Low-tempertature Storage And Control Of Pitting

Blueberry is a small berry,it grows in March and April each year and produces a large amount of fruit in July and August,it quickly decay after harvesting at room temperature for a week or so.In order to solve the problem between the blueberry collection time of concentrated and the long-term demand of the market,refrigeration is widely used in production to delay the decay process.The refrigerating blueberry fruits removed to the room temperature shelf life are vulnerable emerging the symptoms of pitting in the bottom of fruit.With the prolongation of fruit storage time,the symptoms of the pitting on are aggravated,which seriously affects the commercial quality of blueberry fruit.This problem has become a bottleneck in the development of blueberry production.Therefore,studying the pitting mechanism of low-temperature storage blueberry and exploring its regulation technology have important effects and significance for the development of blueberry industry.In this experiment,iTRAQ technology was used to compare the blueberry fruits of 0d and 6d after harvesting and 0d and 6d after 30 d low temperature storage,and the differential proteins were screened for bioinformatics analysis.In order to validate the iTRAQ results,12 differentially expressed proteins were screened for Western blot and 8 differentially expressed proteins were screened for real-time PCR,and analysis from the protein level and the transcriptome level.Based on energy metabolism related substance of blueberry fruit at different temperature storage conditions after different cold storage time were analyzed.The gene expression levels of key enzymes in glycolytic pathway,tricarboxylic acid cycle and oxidative phosphorylation pathway were studied,and the mechanism of low-temperature storage blueberry pitting was revealed based on energy metabolism.Finally,the blueberry fruits were treated with exogenous ATP and DNP,and their effects in the cold storage process were observed.The effects of two exogenous energy materials treatment on the refrigerated blueberry fruit pitting and its mechanism were investigated.Results are following above:iTRAQ analysis identified a total of 2041 proteins,of which 1411 proteins were quantified.Functional analysis and classification of identified proteins were performed by GO analysis and KEGG analysis.Among them,163 differentially expressed proteins were associated with the blueberry fruit pitting,82 protein expression levels were up-regulated,and81 protein expression levels were down-regulated,these include the areas of "synthesis and decomposition of amino acids","reactive oxygen metabolism","membrane lipid metabolism" and "energy metabolism" and multiple specific metabolic pathways,explain the result of the fruit pitting of blueberry fruit caused by many aspects and multiple factors.There were 322 differential proteins related to changes in the process of cold storage,154 protein expression levels were up-regulated,and 168 protein expression levels were down-regulated,among them,"calcium ion signaling pathway","photosynthesis" and "photosynthetic carbon sequestration" all showed abnormal changes,It can be seen that refrigeration has affected the pre-synthesis and ion transport process of energy metabolism.A total of 228 proteins were involved in the change in the shelf life of 30 days at room temperature,122 protein expression levels were up-regulated,and 106 protein expression levels were down-regulated,among them,"vitamin B6 metabolism" and "ribosomes" changed greatly,and the up-regulation of vitamin B6 metabolic pathway was related to the up-regulation of oxidative activity,which indicated the activity of active oxygen metabolism.In order to verify the results,differential proteins were picked up by Ratio>±1.2 and FDR<0.05,among them,12 significantly different proteins were selected for Western blot verification.Based on the above,8 proteins related to blueberry fruit depression were selected for real-time PCR,The expression levels of these genes are exactly the same as the relative results of the proteome.After refrigerating,the blueberry fruit was transferred to the normal shelf life,and the pitting incidence and decay incidence increased significantly,and the ATP content and EC value decreased significantly,and the AMP content increased significantly,complex enzyme activity of oxidative phosphorylation decreased significantly.This shows that the decline of energy metabolism inside the blueberry fruit and the deficiency of energy substances eventually lead to the occurrence of depression in the bottom of blueberry fruit.The expression of glucokinase(GK)gene and pyruvate kinase(PK)in the glycolytic pathway were significantly down-regulated,which affected the formation of acetyl-Co A and the normalization of the tricarboxylic acid cycle.The citrate synthase(CS)and isocitrate dehydrogenase(ICD)in the Krebs cycle showed a significant downward trend,which reduced the supply of NADH and electrons and affected the progress of oxidative phosphorylation.The expression levels of NADH dehydrogenase gene,SDH gene,CCR gene,CCO gene and ATP synthase(ATPase)gene in the oxidative phosphorylation pathway were all down-regulated,indicating that the energy in blueberry fruit cells was deficient.Exogenous ATP and DNP were used as control group to treat refrigerated blueberries,respectively.ATP treatment significantly reduced the depression rate and decay rate of blueberry fruit,DNP treatment accelerates the occurrence of depressions and decay.The ATP-treated blueberry fruit maintained a high ATP content and EC value during the shelf life at room temperature after refrigeration,and the DNP-treated blueberry fruit showed a significant downward trend,indicating that ATP treatment can maintain the higher energy level of the cells,it resists the invasion of external substances and maintains the normalphysiological metabolism of cells.The relative expression levels of blueberry fruit gene expression in the glycolytic pathway,the Krebs cycle and the oxidized phosphate pathway were relatively stable.The DNP-treated blueberry fruit was relatively stable in the glycolysis pathway,which was significantly lower than the ATP treatment group.However,the relative expression levels of key enzyme genes in the tricarboxylic acid cycle pathway and the oxidative phosphorylation pathway were significantly higher than those in the ATP-treated group.This indicates that the DNP-treated blueberry fruit does not have the function of responding to stress during the shelf life after refrigeration,which will greatly consume the nutrients in the blueberry fruit and accelerate the decline of the fruit.