| Chinese flowering cabbage is an important leafy vegetables,which has widely cultivated area and great economic benefits,it plays an important role in the balanced supply of vegetables in south C hina area and the foreign exchange earning.But the high temperature and humidity climate in south China area are conducive to the propagation and spread of pathogens,and anthracnose cauased by Colletotrichum higginsianum is one of the most destructive diseases which seriously affecting the quality and yield of it,and it will cause 30%~40% loss of production when it gets seriusly.The objective of this research was to study the interactions of C hinese flowering cabbage and Colletotrichum higginsianum and to screen some differentially expressed genes(DEGs)related to host plant resistence,discussing the possibly molecular mechanisms of disease resistence,which have great significance to the prevention of anthracnose and the molecular breeding of disease resistance.In this research,we identified the disease resistence of 41 varieties of C h inese flowering cabbage and used RNA-seq technology to analyze the transcriptome before and after infected by Colletotrichum higginsianum in resistant cultivar and susceptible one.The main results are as following:1 ? Disease resistence could be differentia ted by spraying inoculation with Colletotrichum higginsianum,the concentration of the spore suspension and the moisturizing time after inoculation were determined by the different temperatures.The results showed that the disease index was between 35.045 and 64.643 among 41 kinds of Chinese flowering cabbage infected by Colletotrichum higginsianum,and that except for middle resistant and susceptible cultivars,there were no high susceptible cultivars as well as high resistant cultivars were found,of whic h 33 cultivars of disease-resistant and 8 cultivars of susceptible cultivars.According to the survey of pathogen inoculation as well as the disease index and the identification of cultivars,combined with the growth and characteristics of plants,the C hixin NO.2(disease index 35.045,hereinafter referred to as resistant cultivar)which is disease resistant and Youlv 701(disease index 63.265,hereinafter referred to as susceptible cultivar)which is disease susceptible were selected.These two C hinese following cabbages were used to transcriptomic analysis;2?Samples of C hixin NO.2(resistence)and Youlv 701(susceptible)inoculated with sterile water(control)and Colletotrichum higginsianumfor 48 h were used to analyze the transcriptome by RNA-seq,the sequencing reads were then mapped to the reference genome sequence of C hinese cabbage.After the functional annotation of the genes,the GO(Gene O ntology)and KEGG(K yoto Encyclopedia of Genes and Genomes)Pathway enrichment analysis were carried out,the results are as follows:(1)After infected by Colletotrichum higginsianum,there are 664 DEGs were up-regulated and 847 were down-regulated of 1511 DEGs in resistant cultivar,and there are 16 DEGs were up-regulated and 52 DEGs were down-regulated of 68 DEGs in susceptible one,and there are only 17 DEGs that are common to both varieties;indicating that there were more biological processes involved in regulating the resistance of C hinese flowering cabbage in resistant cultivar;(2)Through the GO enrichment analysis,the 3123 DEGs were significantly enriched in 28 GO term in resistant cultivar,including biological processes and catalytic activity and so on,but there have no significantly enriched GO term in susceptible cultivar;through the KEGG Pathway enrichme nt analysis,Photosynthesis-antenna proteins and Glutathione metabolism pathways were significantly enriched in resistant cultivar,and only Ascorbate and aldarate metabolism pathway was significantly enriched in susceptible one;(3)Screening the disease related genes in DEGs,found that WRKY15,WRKY18,WRKY33,WRK Y40,WRKY53,WRKY60 and ATAF1,AN AC042,ANAC055,ANAC072(RD26)transcription factors were up-regulated,and MYB,b ZIP,b HLH,ERF transcription factors were also differentially expressed in resistant cultivar,but there only two transcription factors were differentially expressed in susceptible cultivar,ANAC029 was down-regulated and MYB111 was up-regulated;these results showed that transcription factors play an important role in regulating the disease resistance in response to pathogen;(4)Infection of pathogens can strengthen the cell wall,genes relating with lignin synthesis were up-regulated in resistant cultivar,including cytochrome P450,cinnamoyl-Co A reductase family,cinnamate-4-hydroxylase,caffeoyl-Co A O-methyltransferase,O-methyltransferase 1 and cinnamyl-alcohol dehydrogenase family,but they are not differentially expressed in susceptible cultivar;indicating that resistant cultivar were maythrough strengthening the cell wall to preve ntion the pathogen in the process of pathogen infection of C hinese flowering cabbage;(5)Plant hormones are involved in responses to pathogens infection,related genes of SA pathway,including PAD4,PR5,TIR-NBS-LRR were up-regulated,genes involving in JA pathway were also up-regulated,including PR3,PR4,JAZ1,JAZ2,JAZ5,acyl-Co A oxidase 4,allene-oxide cyclase,lipoxygenase and zinc finger(CCCH-type)family protein,in addition to these genes,1-aminocyclopropane-1-carboxylate synthase,ERF1 and C EJ1 involved in ET and JA/ET pathways were also up-regulated,which is contrast to this,in susceptible cultivar,JAZ9,zinc finger(CCCH-type)family protein and ethylene-responsive element-binding protein were all down-regulated;these results demonstrated that SA,JA and ET were closely related with protecting the plant from pathogen and JA/ET pathway maywas the mainly signal regulating pathway;(6)Infection of pathogens can induce ROS burst in plants and then inhibit the growth of pathogens,but the excess of ROS will cause damage to cell structure,in this paper,in resistant cultivar,genes involved in ROS detoxifying,including C AT,SOD,POD,GPX7,GR,GST,were all up-regulated,and PR15/PR16 were down-regulated which could generate ROS,but there have no genes related to antioxidation were differentially expressed in susceptible cultivar;these results indicated that ROS may involved in regulating the defence response to pathogens;(7)A total of 30 DEGs were randomly selected for validation through quantitative Real-Time PC R(q RT-PCR)and the results were consistent with RNA-seq,indicating that the results of RN A-seq were reliable.In conclusion,after C hinese flowering cabbage responses to Colletotrichum higginsianum infection,there will be a variety of different pathways involved in the disease resistance to form a complex regulatory network,in which transcription factors,plant hormones and ROS may play a major regulatory role in disease resistance of Colletotrichum higginsianum in Chinese flowering cabbage. |
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