Research On The Mining And Regulation Mechanism Of Disease-resistant Genes In Postharvest Blueberry Fruit In Response To Alternaria Tenuissima Infection

Blueberry belongs to Vaccinium in Ericaceae.Its fruit are small blue berries rich in antioxidant substances.At present,they are currently mainly eaten fresh,with a sour and sweet taste that consumers love.Blueberry fruit are prone to mechanical damage during harvesting,storage and transportation,which provides favorable conditions for microorganism infection.These resulting in a large number of fruit decay.Alternaria tenuissima is the main pathogen that endanger blueberries in Liaoning Province.Currently,there are few reports on the response mechanism of blueberry fruit against A.tenuissima infection.In this paper,the responses of blueberry fruit to A.tenuissima were studied at the physiological,biochemical,and transcriptional levels,and then Kyoto Encyclopedia of Genes and Genomes（KEGG）metabolic pathways with significantly enriched differentially expressed genes were screened.And than the expression patterns of key structural genes and transcription factors in this pathway in blueberry fruit in response to A.tenuissima infection were explored.And the transient expression technology was used to further validate the regulatory role of candidate transcription factors in response to A.tenuissima infection.Based on this,the regulatory effects and mechanisms of exogenous burdock fructooligosaccharide（BFO）regulation treatment on disease resistance and senescence of blueberry fruit were analyzed.The main results are as follows:1.Physiological and biochemical studies on the response of blueberry fruit to A.tenuissima infection.After inoculation with A.tenuissima on blueberry fruit,it was found that at the 3 days post inoculation（dpi）,a small amount of gray white mycelium were produced at the pedicel and the interior browning occurred.With the extension of the inoculation time,on the 5 dpi,dense greenish-brown mycelium were produced at the pedicel and spread to the deep interior of the fruit.At the physiological and biochemical level,the infection of A.tenuissima induced an increase in the content of hydrogen peroxide（H₂O₂）in blueberry fruit in the early stages of inoculation,causing the occurrence of reactive oxygen species bursts,and enhanced the activities of superoxide dismutase（SOD）,catalase（CAT）,peroxidase（POD）,ascorbate peroxidase（APX）and disease-course related proteinsβ-1,3-glucanase（GLU）and chitinase（CHI）,and promoted the accumulation of soluble sugar and soluble protein in blueberry fruit.2.Transcriptomic studies on blueberry fruit response to A.tenuissima infection.Transcriptome sequencing and c DNA library construction were performed on blueberry fruit from sterile water control group and spore suspension treatment group using transcriptome sequencing technology.A total of 1567 differentially expressed genes were selected,including1310 up-regulated genes and 257 down-regulated genes.Gene Ontology（GO）functional enrichment analysis showed that the differentially expressed genes were mainly enriched in:catalytic activity,metabolic process,binding,cellular process,and single organism process;The results of enrichment analysis of KEGG metabolic pathway showed that phenylpropanoid biosynthesis,starch and sucrose metabolism,amino acid and nucleotide sugar metabolism,and MAPK signaling pathway-plant were significantly enriched,indicating that the infection of A.tenuissima initiated multiple disease resistance signal pathways in blueberry fruit.Among of them,the enrichment of phenylpropanoid biosynthesis is the most significant,while the biosynthesis of lignin is the main branch of phenylpropanoid biosynthesis to resist the infection of A.tenuissima.3.Functional research on key structural genes and transcription factors in lignin biosynthesis pathway.Focusing on the biosynthesis of lignin,the content of lignin in blueberry fruit A.tenuissima inoculated and related genes including VcPAL,Vc4CL-like7,VcCCR1,VcHCT,VcCSE-like,VcPOD51,VcC3,H,VcF5H,VcLAC14,and VcMYB14 were significantly up regulated,while VcALDH2C4 was significantly down regulated,thereby the synthesis of lignin was activated in blueberry fruit and its content was increased.Correlation analysis showed that lignin and VcMYB14 showed a highly significant positive correlation,with the largest correlation coefficient.The results of subcellular localization showed that the transcription factor VcMYB14 was localized in the nucleus.In addition,VcMYB14 was over-expressed and silenced in blueberry fruit by a grobacterium-mediated transient expression method.The results showed that over-expression of VcMYB14 could significantly increase the lignin content of inoculated blueberry fruit,reduce the diameter of diseased spots,and silence of VcMYB14 had no significant impact on the lignin content and the diameter of diseased spots.Furthermore,we speculated that the transcription factor VcMYB14 could mediate lignin biosynthesis through positive regulation to resist infection by A.tenuissima.4.Study on the induction of BFO on the disease resistance of blueberry fruit.0.5%BFO treatment as an activator induced gene expression of the VcMYB14,increased lignin content,and enhanced the resistance of blueberry fruit to A.tenuissima;BFO treatment caused an outbreak of H₂O₂in inoculated blueberry fruit,triggering an early defense response.At the same time,BFO treatment increased the antioxidant enzyme activity and gene expression of inoculated blueberry fruit,thereby maintaining the dynamic equilibrium of reactive oxygen species in blueberry fruit;In addition,BFO treatment also increased the activity and gene expression of GLU and CHI,further improving the disease resistance of blueberry fruit.5.Effect of BFO treatment on fruit quality and membrane lipid peroxidation of blueberry.0.5%BFO treatment delayed the increase of natural incidence rate and weight loss rate of blueberry fruit during normal temperature shelf life,as well as the decrease of firmness and soluble solids（SSC）,which maintained quality of blueberry fruit,and then extended the shelf life;At the same time,BFO treatment inhibited the accumulation the content of H₂O₂and malondialdehyde（MDA）by increasing the activity of antioxidant enzymes and gene expression in blueberry fruit,delaying the peroxidation of blueberry fruit;BFO treatment inhibited the activity and gene expression of key enzymes in membrane lipid peroxidation,including phospholipase D（PLD）,lipoxygenase（LOX）,and lipase,maintaining a high fatty acid unsaturated index and fatty acid unsaturation,thereby maintaining the integrity of the cell membrane and delaying the senescence of blueberry fruit.