Study On The Induced Resistance And Gene Expression Of Blueberry To Leaf Spot With BABA

Blueberry leaf spot is one of the most important diseases in the blueberry production,which mainly affects the leaves.In recent years,with the increase of blueberry planting area,varieties and the diversification of planting methods,the occurrence of blueberry leaf spot disease is also significantly increased,the loss is increasingly serious,and the difficulty of prevention and treatment is gradually increased,which has become a serious obstacle in blueberry production.BABA is a kind of secondary metabolism non protein amino acid isolated from tomato roots exposed to the sun.It is a phytochemical inducer with broad spectrum of disease-resistant activity which can induce plant resistance to fungal or bacterial infection.In this study,BABA was used as inducer,the "brilliant" variety of blueberry in rabbit eyes was selected as experimental material,blueberry inoculated with leaf spot pathogen as experimental group,and blueberry inoculated with leaf spot pathogen uninduced was used as control group to construct transcriptome sequencing library.Determined the content of anthocyanin in different blueberry varieties and investigated the leaf spot disease rate of blueberry.Through the analysis of biological information,gene function annotation and difference analysis,the related genes of blueberry resistance to leaf spot were screened out from the library.The expression of 8 related genes in the process of resistance to the disease was studied by q RT-PCR,and the changes of resistance expression of these genes to leaf spot were explored after BABA induction.Two selected genes with high expression of disease resistance were cloned and their functions were preliminarily analyzed and verified.The main research results are as follows: 1.The expression of induced resistance of blueberry to leaf spot induced by BABA was confirmed.The results of disease resistance identification indicated that the disease resistance of blueberry to leaf spot was significantly increased after being treated with BABA leaf spray.The determination results of plant defense enzyme activity showed that the activities of PPO,POD,PAL,?-1,3-glucanase in blueberry leaves were higher than those of the control group.The experimental results showed that PPO,POD,PAL and ?-1,3-glucanase could be increased in blueberry leaves after treatment of BABA spray.These highly active plant defense enzymes are the biochemical mechanism which from the BABA induces the induced resistance of blueberry to leaf spot.2.Constructed the blueberry transcriptome sequencing library induced by BABA,comprehensively analyzed the gene expression changes of blueberry leaves after induction inoculation,and excavated the genes and transcription regulatory factors related to the disease resistance of blueberry leaf spot.We get 78507 transcripts were obtained by assembling the sequencing data,and the average length is 957.92 bp,of which the longest is 15693 bp,and the shortest is 301 bp.In the KEGG metabolic pathways the annotation to the environment of information processing and signal transduction genes has 2122,annotation to the organic system and environmental adaptation has 3108 genes,annotation to the metabolic genes with 7566,notes to the genes of genetic information processing has 2694,annotation to the cell has 1717 genes,3953 genes were annotated to disease.1115 transcripts were involved in the plant-pathogen interactions of annotated into the transcripts of diseases,accounting for 14.74% of the annotated metabolic genes.There are 35 genes involved in anthocyanin synthesis.3.Screened out the up-regulated genes with high expression resistance to blueberry leaf spot by comparative analysis differentially expressed libraries.The results showed that there were 900 up-regulated genes,531 down regulated genes and 1431 total genes.Among the 70 genes that were highly expressed in the differentially expressed library,no features or assumed proteins genes are 24,disease-resistant proteins associated with the pathogen of blueberry leaf spot genes are 11,and 2 genes encoded anthocyanin synthesis.For functional annotation and classification by Blast2 PHI,it was found that in All-unigene associated with blueberry leaf spot disease,there were 727 transcription factors in the first category.The disease resistance defense category is the second category,has 200;There were 45 genes related to cell cycle,effector proteins and pathogenic development.other genes are relatively to 7.Through comparative analysis we select 11 genes with resistance to leaf spot of blueberry from the PHI and DEG database of transcriptome library.q RT-PCR results showed that 8 genes with better amplification specificity were expressed in blueberry leaves induced by BABA,but not in controls.The result of disease resistance identification showed that the induced-treated blueberries had a disease resistance phenotype to leaf spot,while the untreated blueberries showed a susceptible phenotype to leaf spot.So these 8 genes are related to BABA-induced resistance to leaf spot in blueberries.4.The functions of comp126500?c0?seq1 and comp126556?c0?seq2 were preliminarily analyzed and verified.The results show that the gene comp126500?c0?seq1 encoded 1077 amino acids,and the phylogenetic tree showed that the gene comp126500?c0?seq1 was closely related to actinidia chinensis.It was predicted that there were two conserved domains of comp126500?c0?seq1 protein.The secondary and tertiary structure prediction indicated that the protein contained 38?-Spiraling,32 ?-folding,14 protein kinase phosphorylation sites and 6 glycosylation sites.The GO enrichment analysis indicated that the protein comp126500?c0?seq1 was involved in DNA repair,regulating RNA splicing,extracellular secretion and other biological processes.NCBI sequence comparison was performed on the amino acid sequences.The results showed that comp126500?c0?seq1 protein was mainly the disease-resistant protein of actinidia chinensis,poplar,oak and durian.Comp126556?c0?seq2 encoding 1167 amino acids,the phylogenetic tree showed that the gene comp126556?c0?seq2 was closely related to Wild mulberry silkworm and jute,three conservative structure domain were predicted,The secondary and tertiary structure prediction indicated that the protein contained 45 ?-Spiraling,16 ?-folding,13 protein kinase phosphorylation sites and 9 glycosylation sites.GO enrichment analysis showed that comp126556?c0?seq2 was involved in DNA repair,receptor recovery,and mediated transport.NCBI sequence comparison results showed that comp126556?c0?seq2 protein was mainly compared with other disease-resistant genes of citrus,Wild mulberry silkworm,potato,pepper and the Leu enriched protein of castor.Both proteins are known to be disease-resistant.The protein expression results showed that comp126500?c0?seq1 and comp126556?c0?seq2 were both expressed successfully.The results of subcellular localization showed that both comp126556?c0?seq2 and comp126500?c0?seq1 were may located in the cell membrane and nucleus.We transformed comp126500?c0?seq1 and comp126556?c0?seq2 into Arabidopsis for Q-PCR detection.p BWA(V)HSADR1-L2-Glosgfp(L2)and p BWA(V)HS-ADR1-L1-Glosgfp(L1)transgenic Arabidopsis and WT were inoculated with the pathogen of blueberry leaf spot.Resistance test results showed that L1 positive rate was 100% and L2 positive rate was 89%.Observations showed that the leaf surface of WT arabidopsis began to show diseased spots and showed a response to the pathogen after 7 days,while the transgenic arabidopsis L1 and L2 showed no disease.Q-PCR results showed that the expression of disease-resistant genes in transgenic arabidopsis L1 and L2 was higher than WT after inoculation,and the expression in WT was almost zero.The results showed that p BWA(V)HS-ADR1-L2-Glosgfp and p BWA(V)HS-ADR1-L1-Glosgfp enhanced the resistance of transgenic arabidopsis to the pathogen of blueberry leaf spot.Therefore,comp126500?c0?seq1 and comp126556?c0?seq2,which produced in the body of a blueberry by induction of BABA,are related genes with resistance to leaf spot disease.5.Anthocyanin content is one of the physiological factors of blueberry resistance to leaf spot.Transcriptome data analysis showed that among the transcripts annotated into the disease,35 genes were involved in anthocyanin synthesis,and among the 70 genes with high upregulation in the differential expression library,2 genes encoded anthocyanin synthesis.In order to study the relationship between anthocyanin content of different varieties of blueberry and occurrence of leaf spot disease,and further clarify the physiological mechanism of blueberry resistance to leaf spot,we determine the total anthocyanin content of 9 varieties of blueberry and investigate the leaf rate of blueberry leaf spot.The results showed that Aonier's total anthocyanin content is the highest,up to 6.5 mg/m L,the lowest content is Canlan,only 4.34 mg/m L.The anthocyanin content of Canlan variety induced by BABA was 5.16 mg/m L,it was 0.82 mg/m L higher than that before induction.Combined with the investigation of disease resistance,it was found that among the 9 blueberry varieties,Aonier had the lowest leaf disease rate of 4.64%,while Canlan had the highest incidence rate of 12.98%.According to the results,field investigation and correlation analysis,anthocyanins have certain resistance to blueberry leaf spot,and the content of anthocyanins is inversely correlated with the incidence of blueberry leaf spot.It can be seen that the induction of BABA can not only increase the content of anthocyanin in blueberry,but also improve the resistance to blueberry leaf spot.Therefore,anthocyanin content is also one of the physiological factors of blueberry resistance to leaf spot.