| Peanut(Arachis hypogaea L.)is one of the important cash and vegetable oil crops in China.In recent years,drought is becoming one of the main abiotic stresses that affect the yield and quality of peanut with the global warming and frequent occurrence of extreme weather,and therefore it is vital to study peanut drought resistance.In this study,two peanut varieties,L422(drought-resistant)and L677(drought-sensitive)were treated under well-watered and severe drought stress during the pod formation stage.Candidate genes for drought tolerance in peanuts were screened and WRKY transcription factors were identified using transcriptome technology combined with weighted gene co-expression network analysis(WGCNA)and bioinformatics methods.The WRKY transcription factors play an extremely important role in plant stress resistance,but those involved in the regulation of peanut drought stress resistance are rarely reported.Based on transcriptome data,the Ah WRKY family members were identified and analyzed using bioinformatics methods.The function of AhWRKY75 was characterized via genetic transformation and physiological experiments.Interaction proteins of AhWRKY75 transcription factors were also screened using yeast two-hybrid.The main results were as follows:1.The phenotype and physiological changes of peanut varieties L422 and L677 with different drought tolerance under drought stress were analyzed.From the observation of phenotypic changes,the growth of both varieties was severely affected and wilted during the severe drought stress stage(5 d,7 d and 9 d)compared with the control group(well-watered),but L677 was more severely.The measurement of physiological parameters showed that the relative water content(RWC)of the leaves of the two genotypes were decreased significantly compared with the control group,and the decrease of L677 was more significant than that of L422.Leaf relative electrical conductivity(REC),malondialdehyde(MDA),peroxidase(POD)activity,soluble sugar(SS)and soluble protein(SP)content were all increased to some extent,among of which REC and MDA of L677 were higher than that of L422,while POD,soluble protein and soluble sugar were higher in L422 than that of in L677.The results of phenotypic changes and physiological measurements showed that the drought tolerance of L422 was better than that of L677.2.The candidate genes for drought tolerance were mined using transcriptome sequencing in peanuts.The comparative analysis of the transcriptomic data under peanut drought stress was performed using bioinformatics methods.The DEGs were strictly screened under the conditions of FC≥4 and FDR<0.01.DEGs for the drought-resistant variety L422 were respectively identified 3,586,6,730 and 8,054 at the three-time points for 5,7 and 9 d of drought treatment.DEGs for the sensitive variety L677 were2,731 at 5d,5,315 at 7d and 6,887 at 9d.The number of induced DEGs was greatly increased with the continuation of drought stress time in the two peanut varieties.By using WGCNA,combining with the water-controlled situation and physiological data of the samples,two modules,namely MM.darkred and MM.brown,which significantly positively correlated with drought stress,were identified in L422 and L677(P<0.05).Further analysis of genes expressed in both modules revealed that 2,336 common DEGs including 112 transcription factors,among which many members of the WRKY family were induced by drought,and were up-regulated and highly expressed.In addition,GO and KEGG enrichment analysis revealed that these DEGs were mainly enriched in biological processes related to drought,such as oxidoreductase activity,redox process,plant hormone signal transduction and glutathione metabolism.3.The members of the WRKY family of peanut cultivars at the genome-wide level were analyzed.Using published genomic data of peanut cultivars,a total of 158 members of the WRKY family were identified.Through sequence alignment and phylogenetic evolution analysis,the 158 members were divided into three groups(group Ⅰ,group Ⅱ,and group Ⅲ),and group Ⅱ members were further divided into five subgroups(Ⅱa,Ⅱb,Ⅱc,Ⅱd,and Ⅱe).Studies of conservative motifs and gene structure,combined with phylogenetic analysis,revealed a certain degree of conservation among members of the same group.In the analysis of duplication events during the evolution of the AhWRKY gene family,it is found that tandem and fragment duplication events might be contributing to the generation of gene families and the evolution process.Most of the AhWRKY genes could be induced to express under drought stress based on the results of transcriptome sequencing,including 48 differentially expressed genes.Combined with the transcriptome data of peanuts under drought stress,it was found that most of the AhWRKY genes could be induced to express.A total of 37 differentially expressed WRKY genes were identified in the two peanut varieties throughout severe drought stress,including 28 up-regulated and nine down-regulated WRKY genes.Twelve WRKY genes were also identified in the MM.darkred and MM.brown modules related to drought stress,all of which showed up-regulated expression.These results indicated that members of the WRKY gene family play an important role in peanut drought stress response.4.Functional analysis of AhWRKY75 gene was conducted.Combined with the transcriptome data related to drought stress in peanuts,it was found that AhWRKY75 was all up-regulated and highly expressed throughout the severe drought stress.Subcellular localization experiment proved that AhWRKY75 was localized in the nucleus.The T3 homozygous transgenic Arabidopsis lines of AhWRKY75 were obtained through genetic transformation.The result of qRT-PCR experiment showed that the expression of the target genes in the transgenic lines was significantly improved compared with the wild-type Arabidopsis,and the highest was more than 2000-fold.Under Mannitol simulated drought stress,it was also found that both wild-type and transgenic Arabidopsis lines could grow normally on 1/2MS medium without mannitol,while the growth of both was inhibited on medium supplemented with different concentrations of mannitol,including the shorter root length and the smaller leaves,and the wild type Arabidopsis was more severely inhibited than the transgenic lines.Treatment results under water-controlling of wild-type and transgenic lines showed that wild-type Arabidopsis thaliana wilted or even died after drought stress,and could not return to normal growth after rewatering,and the survival rate was significantly lower than that of transgenic lines.In addition,the RWC of wild-type and transgenic lines decreased after drought treatment,and the performance of wild-type decreased more obviously.The relative conductivity of the wild type was significantly higher than that of the transgenic lines.These results indicated that the drought tolerance of the transgenic lines was better than that of wild-type Arabidopsis,and the overexpression of Ah WRKY75 improved the drought tolerance of Arabidopsis.5.The proteins that interacted with AhWRKY75 were screened.The self-activation test of the AhWRKY75 transcription factor showed that there was no self-activation activity.Forty potential interacting proteins were screened via the yeast two-hybrid experiment.Three candidate proteins that may interact with AhWRKY75 were predicated by Blast comparison and functional annotation of these proteins,which were named F-box protein,transcription factor NF-YC9 and autophagy-related protein,respectively.In summary,by using transcriptomics,bioinformatics,genetic transformation,qRT-PCR,yeast two-hybrid and other research methods,we obtained a gene regulating peanut drought tolerance and characterized its function.These results will provide important gene resources and lay a theoretical foundation for the genetic dissection of the mechanism of peanut drought tolerance. |
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