Transcriptomics And MiRNAs Analysis Of Peanut(Arachis Hypogaea L.)response To Pod Rot Pathogen Infection And Mining Of Resistance-related Genes

Peanut pod rot has become a major disease in peanut production,posing a serious threat to the yield and quality of peanut.In recent years,the disease has been increasing in the producing area of northern China.To date,researches on peanut pod rot mainly focus on the classification and identification of the known pathogenic fungi,whereas there are few reports on the separation and identification of pathogenic fungi in Laixi of Shandong province where pod rot had occurred several times,and few reports on the mechanism of peanut responding to pod rot.In this study,the rhizosphere soil of peanut pot rot was taken as material and the pathogenic fungi of pod rot in Laixi were separated from the soil and peanut by 454 sequencing technology;the seeds of peanuts before and after being infected by pod rot disease were taken as samples and the expression profile of the related genes of peanut to respond to pod rot and mi RNAs were constructed through the transcriptome and miRNAs sequencing technology.The key gene AhMKK4 was also screened for gene cloning and expression analysis.Through these studies,it is possible to purposefully separate the pathogenic fungi from soil as well as clone the key genes and miRNAs for the process of peanut responding to pod rot,providing the pathogeny material and theoretical basis for improving the disease resistance of peanut by genetic engineering.The main results of this study are as follow:(1)The pathogenic fungi were separated from the peanut pot rot soil in Laixi area by 454 sequencing.A total of 46,723 fungi ITSsequences were obtained and annotated into 1,706 OTUs(Operational Taxonomic Units)among which are 1,206 OTUs in control samples,974 OTUs in pod rot samples and 474 OTUs in both of them.The fungi OTUs can be classified to 6 phyla,24 classes and 272 genera.At the species level,the fungi with the highest abundance was Fusarium sp.OreYA.The RDA(Redundancy analysis)showed that the peanut variety,environmental factors and pathogenic fungi had a close relationship with each other.These results indicated that the fungi in the soil decreased after the peanut being infected by the pod rot and the change trend of soil fungi was affected by both peanut varieties and soil environment factors.The main pathogenic fungi were Fusarium in Laixi area.Four kinds of unkown fungi were isolated from the peanut rot soil,and one kind of Fusarium was isolated from the infected peanut seed.Peanut seedlings and separated tissues were inoculated with the Fusarium,which showed that both the living and separated tissues were infected,suggesting that the Fusarium may be the pathogenic fungi of peanut rot.(2)Obtain the expression profile of the genes related to peanut responding to pod rot through transcriptome sequencing.A total of 60,756 Unigene was obtained,among which are a total of 14,482 DEGs including 10,147 up-regulated genes and 4,334 down-regulated genes.The expression trends of the randomly selected 21 genes analyzed by qRT-PCR were consistent with the prediction of transcriptome,which indicated that the sequencing results were reliable.The DEGs was classified by GO analysis into 24 cell components,5 molecular functions and 23 biological processes.The enrichment analysis of KEGG(Kyoto Encyclopedia of Genes and Genomes)showed that 4,291 DEGs were annotated into 34 metabolic pathways,including 70 DEGs related to multiple plant hormones signal transduction pathway,50 DEGs related to plant-pathogen interaction pathway and 17 DEGs related to MAPK signal transduction pathway.These analyses indicate that there are a large number of DEGs in the process of peanut responding to pod rot,and it is speculated that the disease-resistant process is the result of the combined action by multiple genes.(3)Hormone signal transduction pathways and key genes involved in peanut responding to pod rot disease were screened.In the disease-resistant pathway mediated by salicylic acid,the expressions of the two NPR1 genes,and five TGA genes in the pod rot samples were down-regulated while the expression of PR gene in the two samples had few changes.In the disease-resistant pathway mediated by jasmonic acid,the expression of the two JAR1 genes was up-regulated in the control sample while the expression of JAZ was down-regulated in pod rot sample.In the disease-resistant pathway mediated by ethylene,the expression of the one ETR gene was up-regulated in the control sample while the expression of the two EIN3 and one EBF1-2 genes were down-regulated in the control sample.These results indicate that the jasmonic acid increased and then may degraded the JAZ,promoting the initial transcription of expression of the downstream transcription factors such as EIN3 and EBF1-2.In the signal pathway mediated by auxin,the expressions of the three GH3 genes were up-regulated in pod rot sample,and the expressions of the six IAA genes were up-regulated in the control sample.These results mean that peanut responding to pod rot disease has great relationship with endogenous hormone.Through the analysis of the expression profile of key genes,we speculate that peanut may resist the infection of pod rot pathogen through the JA/ET-mediated disease-resistant pathway.(4)The miRNAs expression profile under the process of peanut responding to pod rot were detected through small RNA sequencing.A total of 334 miRNAs including 27 up-regulated and 97 down-regulated mi RNA stressed by pod rot were detected.Most of the expression trends of the randomly selected 15 miRNAs analyzed by qRT-PCR were consistent with the prediction of sRNA sequencing,which indicated that the sequencing results were reliable.Target gene prediction was conducted on mi RNA and1,998 target genes and 2,646 target sites were predicted in 303 mi RNAs.Whereas 152 target genes were predicted in 119 differentially expressed miRNAs.This result indicated that different miRNAs were produced in the process of peanut responding to pod rot and showed different expression trends,the same miRNA might target multiple genes and have multiple target sites.(5)The correlation analysis between transcriptome and miRNAs sequencing under pod rot disease.14 target genes regulated by 10 miRNAs were all detected in transcriptome sequencing results.The negative regulation was also detected between 4 mi RNAs and its target genes.This indicated that miRNA was involved in the molecular regulation of peanut responding to pod rot by negatively regulating target genes.(6)Gene cloning and expression analysis were conducted on the screened key gene AhMKK4.The full length of the AhMKK4 sequence was 1,434 bp,including a 5' non coding region of 317 bp and a 3? non-coding region of 151 bp.The ORF has a full length of 966 bp,encoding a protein sequence with 322 amino acids.AhMKK4 belongs to the group D of the MAPKK gene family and its molecular weight was predicted as 36.74 kDa.Subcellular localization analysis showed that AhMKK4 was located in the nucleus and cytoplasm of plant cells.The qRT-PCR analysis showed that the expression of AhMKK4 in roots was higher than that in other tissues,revealing that AhMKK4 has tissue expression specificity.The expression of AhMKK4 was up-regulated by JA induction,and was down-regulated by SA induction.These results suggest that AhMKK4 may be involved in JA-mediated disease-resistant process in peanut root.(7)Transgenic function analysis of peanut AhMKK4.The gene constructed on the sense plant expression vector was transferred into Arabidopsis.Transformants were selected and further confirmed,indicating that AhMKK4 had been expressed successfully in the Arabidopsis.Over-expression transgenic Arabidopsis were obtained.The results of resistance analysis under abiotic stresses revealed that the transgenic plants have no tolerance to salt and osmotic stress,and its root growth was inhibited under ABA,JA and IAA stress.This will provide experimental materials for further research on transgenic function analysis of peanut gene function verification.