| Cucurbita ficifolia is one of the characteristic germplasm crops cultivated in Yunnan that has strong resistant abilities to wilt caused by Fusarium oxysporum,besides it has tolerant abilities to many abiotic stress,such as coldness,drought and barren soil,the exploration and utilization of its advantageous gene resources is of great significance in disease resistant breeding of Cucurbita crops.Cucurbita ficifolia has been widely used as grafting stock to control the occurrence and damage of wilt,Fusarium wilt is one of the main fungal diseases in the Cucurbitaceae planting area worldwide which remarkably affects quality and yield.It is commonly regarded that breeding new disease resistant cultivar is the most economic,effective and environment-friendly method to prevent wilt among other measures such as crop rotation,rafting,and application of fungicides.The host plants have developed and formed the complicated disease-resistance mechanism in the process of coevolution and adaptive interaction with the pathogens.Yet the previously published papers indicated that the molecular mechanism of wilt resistance involved multiple metabolic processes,multiple signal transduction pathways and multiple gene regulation networks.In general,the response mechanism of Curcubita wilt fungi infection and the molecular mechanism of host-pathogen interaction have not been fully clarified.In this study,using throughput sequencing technology,the molecular mechanism of C.ficifolia responding to F.oxysporum infection was explored and analyzed at the level of transcriptome and proteome.It is not only helpful to reveal the metabolic pathway and molecular regulation network in response to F.oxysporum infection,but also explore the molecular interaction mechanism between Cucurbita crops and Fusarium pathogen as well.The establishing transcriptomes and proteomics database would contribute to screening the key resistant genes,and offer advantageous genetic resources for genetic engineering breeding of Curcubita corps,thus it might be of great significance for Fusarium wilt-resistance cultivation and breeding in practice.In view of the situations,nucleotide binding site（NBS）type of disease-resistance genes homogenous were isolated from the genome of C.ficifolia firstly,and the high expression period of resistance genes was determined based on the Q-PCR analysis and wilt symptom performance.Secondly,wilt resistance level of three C.ficifolia varieties（the white peel,the stripe peel and the green peel）evaluated by indoors inoculation with F.oxysporum f.sp.Cucumerinum,expression of HQRGA2（Genebank ID:MG946756）were detected by Q-PCR among three varieties infected.Thirdly,high throughput RNA-Seq and iTRAQ technologies were used to establish differential expression gene and protein profiling in the resistant variety of green peel.Finally,the key NBS type resistant genes were selected and their expression characteristics were detected by Q-PCR,and the VIGS vectors of the selected NBS type gene were constructed and gene functions were confirmed initially.The main results showed that:1.The degenerated primers were designed according to the conserved regions of NBS type genes and consequently 8 resistance gene analogous（RGAs）were isolated and sequenced from C.ficifolia genome.The sequence named HQRGA2（Genbank ID:MG946756）that belongs to CC-NBS-LRR type gene,had got P-loop,Kinase-2,kinase-3a,and GLPL region,and another conservative structure domain NB-ARC.Furthermore,nucleotide similarity analysis indicated the identity of HQRGA2 varied from 87.0%to 99.0%compared with disease resistance genes from Cucurbita crops,yet98.0%and 97.0%of nucleotides of HQRGA2 were shown to have similarity comparable to that of resistant genes SQRGA-13 and SQRGA-8 from C.moschata respectively.By amino acid homology contrast of HQRGA2 with the known NBS type disease resistant genes,the homologous percentages were from 16.6%to 96.6%,among them,the gene SQRGA-13 from has got the highest percentage of 96.6%.Yet the neighbor-joining tree based on alignment of amino acid sequences indicated that HQRGA2 would probability be a new type of disease resistant gene fragment.By Q-PCR analysis,the expressions of HQRGA2 were all induced by F.oxysporum,of which the resistant variety Green peel exhibited the most rapid response to the F.oxysporum infection with the higher and more sustainable expression of HQRGA2.The green peel variety also had HQRGA2 expression feature with rising firstly and reducing finally,and the susceptible variety White peel had several rising-declining trend,moderate resistant Stripe peel variety declining-rising trend.With the regard of wilt symptom,the sampling time of transcriptomes and proteome were 48 hours and 96hours after inoculation with F.oxysporum at the stages of wilt inoculation and expansion.2.The wilt-resistant variety of green peel were sequenced by high throughput RNA-Seq after inoculation with F.oxysporum using root-cutting and root-irrigating method,after cutting and irrigating with water for 48 hours was used as control.The Clean Reads are assembled by Trinity to obtain the longest Unigene.In succession,the assembled Unigenes are contrasted on the database of NR,SWISSPROT,KOG,GO and KEGG,and the annotation results of unigenes in each library are obtained.By FPKM method,the expression of each unigene were calculated and differential expression genes（DEGs）were screened and selected out,after that,GO and Pathway enrichment analysis of the DEGs at the same and different stage in the process of fungi infection were carried out,and the gene expression database were constructed based on transcriptome.The results indicated that:（1）A total of 62,169 Unigenes were obtained from C.ficifolia transcriptome by de novo analysis,among them the longest one was 15,877 bp,and the shortest was 301 bp,with an average length of 1,160.42 bp.After compared with NR,SWISSPROT,KOG,GO and KEGG database,47,521 unigenes（76.44%）were annotated.Among them,11,676 unigenes（29.40%）and 11,674 unigenes（29.39%）could be annotated in melon genome and cucumber genome respectively.In addition,14,648 unigenes（23.56%）had not been annotated,it was speculated that the unnoted unigenes may be new transcript and specific genes that differ from other Cucurbita crops.The transcriptome database established can provide referring information for related investigation of disease resistance mechanism of other Cucurbita crops.（2）Functional annotation and enrichment analysis of DEGs were carried out at different infection time,and verification of Illumina sequencing results is reliable by Q-PCR testing.The results showed that:（1）After Fusarium fungi infection,most of the unigenes induced were commonly reported,involving in almost all metabolism process of plant growth and development.Totally 25,020 unigenes were enriched into 25molecular functional families,of which 4,437 unigenes（17.73%）were enriched to general function prediction only that was the most abundant category.Secondly 2,744unigenes were enriched to posttranslational modification,protein turnover and molecular chaperones;2,368 Unigenes were enriched in signal transduction mechanisms,837 and 228 genes were enriched in secondary metabolites biosynthesis,transport and catabolism,and defense mechanisms respectively.（2）The number of DEGs were increasing with infection time prolonged,the number of DEGs were 939 and 2,021respectively at 48 hours and 96 hours including a great number of transcription factor and R gene.721 DEGs were co-differentially expressed at two time points,and the expression pattern of 355 genes were up-regulation and 366 genes were down-regulation;the number of DEGs that had the same expression trend was 444,of which 238 genes were constant up-regulation and 206 genes were constant down-regulation.（3）Pathway enrichment analysis showed that differentially expressed genes were involved in process of programmed cell death（Necroptosis）,peroxisome,thiaminemetabolism,starchandsucrosemetabolism,apoptosis,glycolysis/gluconeogenesis,lysozyme,cytochrome P450 metabolism,pyruvate metabolism,glutathione metabolism,ascorbic acid and aldarate metabolism,plant hormone signal transduction,etc.Besides the above metabolic pathways,P53,VEGF and TNF signal pathways were activated after 96 hours of infection.It would be indicated that F.oxysporum had stimulated various resistance pathways in C.ficifolia and DEGs involved in defense response and signal transduction,suggesting that the molecular mechanism response to F.oxysporum is regulated by a multi-gene network system.3.Based on transcriptome,the differential protein expression profiles of Green peel variety infected by F.oxysporum were studied using iTRAQ technique.The results showed that:（1）Totally 1,907 credible proteins were identified.A total of 567 differential expression proteins were detected,of which 60 proteins were enriched to 55 pathways in group CK-VS-48h by KEGG analysis,and 329 proteins in group CK-VS-96h that had 198 proteins to 83 pathways.Among them,4 unknown proteins were detected and their functions were deduced by NCBI on-line analysis:the up-regulation protein CL11145contig1 was GPI-anchored nonspecific lipid transfer protein,which involves in abiotic stress-resistance,another up-regulation protein CL9717contig1 is unknown protein,two down-regulation proteins CL29643contig1 and CL4168contig1 are presumptive CSA proteins,and their functions need to be idenificted.（2）Union and intersection analysis showed that there were 23 differentially expressed proteins in three groups,of which S-adenosylmethionine synthase（SAM1and SAM2）was the main resistance-related proteins.It was speculated that SAM-related genes also played an important role in the process of disease resistance.The interaction analysis between DEGs and proteins with KEGG pathway showed that there was significant difference in gene interaction after 48 h and 96 h infection.There were more up-regulated expression genes and proteins after 48 h infection than that of96 h.（3）The correlation analysis between differentially expressed genes and proteins showed that 11 and 39 differentially expressed proteins were associated with DEGs after 48 h and 96 h infection respectively.Differential expression genes,proteins and related regulating genes were enriched to 20 pathways by KEGG enrichment,which mainly involved in ribosome,phenylpropanoid biosynthesis,carbon fixation in photosynthetic organisms,peroxisome,glycosylate and dicarboxylate metabolism,glycolysis/gluconeogenesis,galactose metabolism,alanine,aspartic acid and glutamic acid metabolism and plant hormone signal transduction.The results provided a basis for identifying metabolic pathways and key resistance proteins that need to be further verified.4.Based on the pathway enrichment analysis of transcriptome and proteome after48 and 96 hours infection,it was preliminarily deduced that the resistance signal transduction network of C.ficifolia responding to Fusarium wilt infection,the proposed resistance signal transduction network laid a primary foundation for further mining of disease resistance genes in C.ficifolia.（1）After the invasion of F.oxysporum,the downstream signal transduction was induced by fungal elicitor/PAMPs recognized by membrane-anchored receptor protein.（2）The activating of calcium channel and increasing of cytoplasmic calcium would trigger the NAPDH oxidase induction that resulted in the erupting of reactive oxygen species（ROS）and increasing of H2O2.（3）The rapid invading with root tip cutting affected the recognition of effectors of fungi,which resulted in the physical and chemical metabolic processes of lignin reduction and cell gap expansion,cell wall degradation,photosynthetic rate reduction,wax synthesis declining,etc.The physical defense didn’t play a role in resistance to fungi.（4）With the reproduction of fungi,C.Ficifolia recognized the fungal effector factors by specific pathogen receptor（PRRS）and NBS-LRR proteins,and then activated MAPK signal transduction pathways.（5）ABA pathway might protected host from fungi by increasing pyruvate,mediating stomatal closing,reducing transpiration and programmed cell death（PCD）associating with transcription factor MYB and NAC.（6）The jasmine acid（JA）pathway mainly promoted the related transcription factors or genes expressions by methyl jasmonate addition,it was inferred that cytochrome P450 and PCD involved in the later stage defense process.（7）Salicylic acid（SA）pathway acted as the main signal transduction pathway,stimulated the expression of PR-1 protein,accounted with transcription factors WRKY and BZIP,and thus opening the systemic defense process（SAR）,mobilizing thiamine,lysozyme,programmed cell death,cell apoptosis,phagosome and other ways to eliminate pathogens.（8）ROS produced was eliminated by ascorbic Acid-glutathione cycle（ASA-GSH）.（9）Peroxisome maintained the balance of ROS production and dispelling by catalase.5.As for the important effects of NBS type genes on resistance signal transduction,the deep mining of the key NBS type genes were carried out by the combination analysis of second-generation and third-generation transcriptome.Results showed that:（1）Using NB-ARC as the conserved domain,43 CfNBS（NBS-type gene from C.ficifolia）genes were identified,which belonged to six subgene families:TNL,CNL,TN,RPW8-N and N.2 and 13 CfNBS genes were classified to the typical TNL and CNL respectively.The phylogenetic tree analysis showed that CfNBS genes could be classified to four groups,whose gene evolution type was more abundant than other Cucurbita species despite of the less NBS type resistance genes.（2）43 CfNBS genes were contrasted in second-generation transcriptome,11differentially expressed CfNBS resistant genes were obtained,which were significantly enriched in defense response,glutathione transport,receptor serine/threonine kinase binding and other biological processes and molecular functions.Two genes（CL19588contig1 and CL21402 contig1）homologous to TMV resistance proteins had the most enrichment pathways,implying the two genes played an important role in the process of diseases-resistance.By Q-PCR detection,10 CfNBS key resistant genes were expressed at 48 or 96 hours after inoculation with F.oxysporum,and the expression trends were consistent with that of transcriptome data.Tissue specific expression analysis showed that CL19588Contig1,CL52011Contig1 and CL34065Contig1 gene had the highest expression quantity in pericarp,stem and root respectively.6.The full-length gene of HQRGA2 was cloned from the Unigene database of C.ficifolia transcriptome named as CfRFN2（Gene Bank ID:MK618462）,whose Unigene code was CL7398Contig1.The gene CfRFN2 had 4,303 bp,and had a complete coding frame with a length of 4,092 bp,encoding 1,363 amino acids.（1）CfRFN2 was annotated as a homologous gene with disease resistant gene of At4g27190-like transcript variant X1 from Arabidopsis thaliana.The nucleotide identity analysis of CfRFN2 varied from 87%to 98%compared with the disease resistance genes from Cucurbitaceae crops.It carried conservative NB-ARC structure and two LRR domain.The deduced signal peptide sequence of CfRFN2 had 19 to 20aminos.CfRFN2 protein had the closest genetic homology with RPS2 protein from C.pepo and C.moschata.（2）The 415 bp fragment of CfRFN2 was amplified from the cloning vector pEASY-CfRFN2-3 and linked into the vector pTRV2,the VIGS silencing vector pTRV2-CfRFN2 was finally constructed.The silencing vector and co-transformation vector pTRV1 were transformed into C.ficifolia seedlings by Agrobacterium-mediated method.After 28 days,Agrobacterium infected seedlings were used to inoculated with F.oxysporum as Treatment and the wild type seedlings inoculation with F.oxysporum as control.After 7 days inoculation,the wilt symptom of treated plants performed more serious than that of control.By Q-PCR analysis,the expression of CfRFN2 in treated plant after 48 h and 96 h inoculation with F.oxysporum were reduced 34.75%and98.27%respectively in contrast with that of control.It was preliminarily indicated that CfRFN2 gene had the function of resistance to Fusarium wilt. |
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