| The rachis browning is the second most important problem causing the decline of the quality of table grape,and also the main obstacle to the development of new storage technology of table grapes.In order to improve the problem of rachis browning caused during postharvest storage,the main cultivation variety Seedless white grapes"Thompson Seedless"in Xinjiang was used as the material.After screening the suitable concentration by NO fumigation,RNA-seq was used to explore the main metabolic pathways and genes related to rachis browning.Candidate genes were identified according to the difference in NO response and gene function,and phenylpropane metabolic pathway was focused to explore the occurrence law and regulation mechanism.The purpose is to provide scientific basis and experimental data for the application of NO on grape storage.The main results are as follows:(1)The NO fumigation concentration was screened and optimized.The appropriate concentration of NO gas fumigation can delay the browning of grape rachis and maintain the quality of grape granule.However,the effect of NO was limited when the concentration was lower than 300?L·L-1,NO in the range of400?L·L-1?600?L·L-1 had obvious effect,and it was harmful when higher than 900?L·L-1.Combined with the analysis of physicochemical and microstructure quality,NO inhibited the rachis browning,effectively reducing the weight loss rate,shearing rate and rotting rate of grapes,and slowing down the decline of grape kernel hardness and sugar and acid.Especially,500?L·L-1NO significantly slowed down the increase of electrical conductivity,inhibited the degradation of chlorophyll and the accumulation of anthocyanin in grape rachis,especially delayed the degradation from chlorophyll a to chlorophyll b,and reduced the yellowing,but had no significant effect on the content of flavonoids.NO treatment not only reduced the number of cracks and cracking intensity on the rachis surface,but also benefited the maintenance of the morphology of the internal cells with the close arrangement and complete skeleton,so as to reduce the degree of local tissue depression.It slows down the consumption of inorganic substances in the xylem,thus delaying the breakdown of the cellular structure.Tissue staining analysis showed that NO maintained the volume of rachis epidermal cells,slowed down the cell wall thickening and cork formation,and inhibited the accumulation of brown matter in the epidermis.The results showed that 500?L·L-1NO was the appropriate concentration to delay rachis browning.(2)RNA-seq sequencing showed that the mRNA transcription of grape rachis during storage was significantly changed,and the effect of NO treatment on it was significant.There were 12869 genes expressed in rachis at different storage stages.At10 days after harvest,the up-regulated genes accounted for 72.35%of the total differential genes,and the down-regulated genes accounted for 27.65%of the total differential genes.The effect of NO treatment on the mRNA transcription of rachis was significant.Compared with harvest,759 differentially expressed genes were found in the treatment group and control group after 10 days of storage,among which62 differentially expressed genes were found.The results of qPCR for the top 32genes showed that the expression characteristics of 20 genes were prominent,among which the expression levels of genes PAL1,PAL3-5,PPO1-3,POD1,POD4-7 and transcription factors WRKY53,ERF003,MYB39 were significantly higher than that of genes PAL2,POD2-3 and transcription factors b HLH96,ERF095.The effects of NO treatment on the above genes were significant,especially in 5?25 d of cold storage and the first 2 d of shelf life.(3)Go,KEGG and protein enrichment indicated that phenylpropane metabolism pathway was closely related to rachis browning process,mainly involving PAL,PPO and POD family genes.RNA-seq data showed that 365 DEGs were involved in 50 metabolic pathways which were mainly distributed in the metabolic process,accounting for 81.10%(296)of the total DEGs.Moreover,phenylpropanol biosynthesis pathway was the main pathway,accounting for 11.82%(35).The second was phenylalanine metabolic pathway,accounting for 9.80%(29);It was followed by plant hormone signal transduction pathway and flavonoid synthesis pathway.The DEGs enriched in the first two items accounted for 21.62%of the total metabolic items(42 items),which because the main enrichment direction.In addition,the top three pathways were phenylpropanol biosynthesis pathway(KO00940),phenylalanine metabolism pathway(KO00360)and flavonoid biosynthesis pathway(KO00941).In combination with gene function,phenylpropane metabolic pathway related to rachis browning was selected to study the regulation mechanism,including9 candidate genes in this pathway,including VvPPO1-3,VvPAL1-3 and VvPOD1-3.(4)Correlation analysis showed that the changes of browning index and PPO activity in rachis were closely related to the changes of physicochemical quality and candidate genes,and the expression of different genes differed significantly.Browning index was significantly correlated with phenolic content,POD enzyme,VvPAL1 and VvPOD3,and was significantly correlated with water loss rate,PPO enzyme,VvPPO1 and VvPOD1.Meanwhile,PPO enzyme was significantly correlated with VvPOD1 and extremely significantly correlated with VvPPO1.The results showed that the expression of VvPPO1 was significantly higher than that of VvPPO2(7.05 times)and VvPPO3(5.56 times).VvPAL2 was significantly higher than VvPAL1(5.12 times)and VvPAL3(2.13 times).VvPOD3 was significantly higher than VvPOD1(4.35 times)and VvPOD2(21.81 times).Therefore,VvPPO1,VvPAL2 and VvPOD3 may be the genes with higher expression in grape rachis.(5)Transcriptional regulation studies showed that NO induced significant regulation of phenylpropane metabolism in rachis.The regulation effect was reflected in delaying water loss,reducing phenolic accumulation,inhibiting PPO and PAL activities,and inducing the increase of POD activity in rachis.The expression of VvPPO1,VvPAL2 and VvPAL3 was down-regulated,and the expression of VvPOD3was up-regulated.The expression profile of VvPPO1-3 showed that VvPPO1 was an important gene.NO treatment had significant inhibitory effect on VvPPO1(P<0.01),but had NO significant effect on VvPPO2 and VvPPO3.The results showed that VvPPO1 played an important role in the production and control of rachis browning,and might be a key gene related to rachis browning in the VvVPPO family.(6)Bioinformatics analysis and subcellular localization observation showed that VvPPO1 had a tyrosine domain and was able to travel on chloroplasts.The full length of VvPPO1 was 2010bp,containing 2007 bp ORF,and encoding 668 amino acid residues.The molecular formula was C3346H5215N909O987S23,the total number of atoms 10480,the molecular weight 74.71 k Da,and the theoretical PI 6.64.It is closely related to Vitis Vinifera"Shine Muscat"(BAO79387.1),and the similarity is greater than 99%.The sequence has been submitted to Gen Bank database,and the gene registration number is MN164611. |
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