| Flax is an important oil crop in the northwest of China.The resistance of the resistant flax cultivars is lost due to the long-term cultivation of same cultivars,and leads to the serious occurrence of fusarium wilt.Hence,it has become an important disease that influcences the yield of flax.Therefore,breeding and cultivating disease-resistant flax cultivars has become an important issue to be solved urgently.In this study,resistant cultivar Jinya（R-7）and susceptible cultivar Shanxihuang 29（S-29）were chosen as the research objects.The seedling roots were sampled at 0 h,0.5 h,2 h and 8 h after inoculated with Fusarium oxysporum f.sp.lini（Foln）.Based on RNA-Seq technology,the transcriptome data of flax seedling roots during the interaction were analyzed.At the same time,the stress resistant physiological indexes of resistant and susceptible flax cultivars in response to pathogen infection were taken as the breakthrough point,to carry out module analysis in conjunction with weighted gene co-expression network analysis（WGCNA）in order to reveal the resistance of flax at the genetic level and provide a theoretical basis for revealing the molecular mechanism of the interaction between resistant and susceptible flax varieties and Fusarium oxysporum f.sp.lini.The specific research results are as follows:1.After Foln inoculation of flax,the host phenotypic test verified that the resistant and susceptible flax varieties showed significant differences in pathogenicity to pathogens,and the microscopic morphological observation experiment showed that under Foln stress from 0.5 h to 8 h,the number of spores around the root of resistant and susceptible flax showed significant differences.It will lay a theoretical and practical foundation for further study on transcriptome analysis,physiological and biochemical responses and expression responses of disease-resistant flax cultivars to Foln stress.2.A large number of differentially expressed genes（DEGs）were found in the resistant and susceptible flax cultivars inoculated with Foln within 8 hour post inoculation（hpi）compared with the non inoculated ones.A large number of DEGs were found at 0.5 hpi and 2 hpi in S-29 and R-7,respectively;The DEGs mainly enriched oxidoreductase activity and redox process;The resistant cultivar had more genes for terpenoids synthesis during the interaction with Fusarium oxysporum,while the susceptible flax cultivar mainly had genes for disease course related proteins and cell material functions.Meanwhile,there were significant differences in defense response pathways in resistant and susceptible varieties.In S-29,the DEGs in MAPK signaling pathway,phytopathogen interaction pathway,phenylpropanoid biosynthesis,andα-linolenic acid metabolism were significantly up-regulated（P≦0.05）at o.5 hpi;In R-7,plant photosynthesis antenna protein,plant hormone signal transduction,and estrogen signal pathways DEGs significantly up-regulated expressed at 2 hpi.The results of this study showed that plant hormone signal transduction and secondary metabolites play an important role in flax resistance to Fusarium oxysporum infection.The expression levels of DEGs involved in plant-pathogen interaction pathways,plant hormone signaling transduction and MAPK signaling pathways in R-7 were higher than those in S-29;In R-7,six genes belonging to the transcription factors family MYC2 were continuously expressed,while 11 genes were continuously expressed in susceptible variety;CML,CDPK,RBOH,WRKY and HSPA were played important roles in the plant-pathogen interaction pathway,of which WRKY 22 and WRKY33 regulate disease resistance of resistant and susceptible flax cultivars;Hsp 90 gene family was positively regulated the disease resistance of R-7;the MAPK signal pathway were regulated by IRAK4 and MEK;JA metabolism was regulated by transcription factor MYC2 and TIFY,which further regulate plant hormone signal transduction;CH3,ETR and ERS of plant hormone signal transduction pathway regulate resistance of flax;Meanwhile,seven core genes were screened out,of which Lus10025000.g and Lus10026447.g were simultaneously regulate plant hormone signal transduction and MAPK signal pathway.Transcription factors play a positive role in regulating the resistance of flax to Foln,and transcription factors that overlap in each comparison group in flax may play a greater regulatory role,among which b HLH and MYB family in the family plays a positive regulatory role in the resistance response of R-7,while the AP2/ERF,NAC and WRKY families play a negative regulatory role in the resistance response of S-29;the gene interaction networks of five transcription factor families revelaed that Lus10007326.g in the transcription factors family WRKY,Lus10009263.g in the MYB family of transcription factors,Lus10015902.g and Lus10006951.g in the b HLH family of transcription factors were the core genes of each transcription factor family,regulating the expression of other genes in the gene interaction network.3.The physiological indexes of resistant and susceptible flax varieties were differently changes after inoculation with Foln.Among them,the activity changes of PAL,MDA and MP in S-29 were higher than those of R-7;The activities of SOD and POD in R-7 were higher than those of S-29;The activity of CAT was down-regulated in R-7,and the activity of O2- showed little change in resistant varieties.Foln inoculated S-29 increased the reducing sugar,while R-7 decreased the reducing sugar,the IAA oxidase and PPO activities in R-7 and S-29 were lower than the control value,and the activity of S-29 was lower.Vc oxidase activity increased rapidly in R-7,but almost unchanged in S-29.It was concluded that the reduction of reducing sugar and the increase of Vc oxidase activity could improve the disease resistance of plants,and the activities of IAA oxidase and polyphenol oxidase decreased after pathogen infection.4.Through WGCNA analysis,eight specific gene modules related to physiological and biochemical stress resistance were obtained.Among them,blue module and POD,purple module and CAT,pink module and MDA were highly correlated;and it was found that both the purple and the blue module were enriched in amino acid metabolism and MAPK signaling pathways,and the pink module was enriched in the plant-pathogen interaction pathway;based on the gene interaction network,six core genes :Lus10003190.g and Lus10040664.g,Lus10014306.g,Lus10005553.g,Lus10036122.g and Lus10037357.g were screened out from three highly related modules.5.After Foln inoculation,WRKY41 encoded by Lus10003984.g was screened as the resistance gene regulating flax by transcriptomic analysis,and the function of Lu WRKY41 gene transferring from flax to Arabidopsis was verified.In the experiment,it was found that overexpressing Lu WRKY41 Arabidopsis plants and wild-type plants showed obvious symptoms after Foln inoculation,and the physiological indexes of stress resistance of overexpressing Lu WRKY41 Arabidopsis plants were higher than that of wild-type plants,confirming that Lu WRKY41 plays a regulatory role in the disease resistance of transgenic Arabidopsis.In this study,the transient stress defense response mechanisms of flax to pathogen infection was verified based on the RNA-seq technology.Meanwhile,the DEGs related to resistance phenotypes were screened out based on WGCNA technique.Transgenic Arabidopsis thaliana further confirmed that Lu WRKY41 has the function of regulating Fusarium wilt resistance. |
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