A method for controlling peanut bacterial wilt 6 A demethylase AhALKBH15 gene and identification and application thereof

Abstract

An m 6 A demethylase gene, named AhALKBH15 gene, the RNA sequence of which is shown as SEQ ID NO: 1, and the amino acid sequence encoded by the AhALKBH15 gene is shown as SEQ ID NO: 2. An anti-Ralstonia solanacearum resistance gene AhCQ2G6Y, the RNA sequence of which is shown as SEQ ID NO: 3. The amino acid sequence of the AhCQ2G6Y gene is shown as SEQ ID NO: 4. The m 6 A demethylase AhALKBH15 can remove the m 6 A modification, resulting in m 6 A level is reduced, the resistance gene AhCQ2G6Y is up-regulated, and the up-regulation of AhCQ2G6Y expression promotes the resistance of peanuts to BW. The great potential of AhALKBH15 for peanut resistance breeding is found.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to an m 6 A demethylase AhALKBH15 gene and identification and application thereof. BACKGROUND

[0002] Peanut (Arachis hypogaea L.) is one of the most important oil and economic crops in the world. Bacterial wilt (BW) is a typical vascular disease that severely affects the yield and quality of peanuts and can cause more than 50% yield loss (Jiang et al., 2017). The pathogen of peanut bacterial wilt is the soil-borne bacterium Ralstonia solanacearum (R. solanacearum). Once R. solanacearum exists in the soil of a specific site, it is difficult to completely eradicate. Therefore, the most economical and effective method to prevent BW damage is to breed new varieties resistant to BW, which requires the excavation of disease-resistant genes. In the past few years, efforts have been made to identify quantitative trait loci (QTLs) that control BW resistance (Zhao et al., 2016; Wang et al., 2018). Some candidate resistance genes (R-genes) have been identified and characterized in major QTLs (Luo et al., 2020; Qi et al., 2022). For example, Zhang et al. (2017) and Zhuang et al. (2019) cloned AhRLK1 and AhRRS5 (homologs of Arabidopsis resistance genes RRS1-R and ERECTA) and demonstrated that heterologous overexpression of these two genes in tobacco can improve BW resistance. Although several BW-resistant QTLs have been detected in different peanut varieties, it is not yet known whether epigenetic modifications are related to disease resistance in peanut-Solanaceae plant interactions.

[0003] Epigenetic modifications are common and have been shown to regulate plant growth, fruit development, and responses to fungal and viral stress (Hu et al., 2022; Chinnusamy et al., 2009). There are multiple types of chemical modifications in DNA, RNA, and proteins, but RNA modifications are the most abundant (Sonavane et al., 2009; He et al., 2021). To date, more than 170 types of RNA modifications have been detected, distributed in coding and non-coding RNAs (Lorenz et al., 2017). The most common mRNA modifications are N1-methyladenosine (m1A), 5-methylcytosine (m5C), and N-methyladenosine (m6A) (Frye et al., 2016; Shi et al., 2020). m 6 A modification is the most common, accounting for more than 80% of all RNA modifications (Shen et al., m 6 A modification is the most common, accounting for more than 80% of all RNA modifications (Shen et al., m6 The A modification is very abundant, both in terms of the proportion of mRNAs it affects and the number of modifications appearing on each modified mRNA. For example, approximately 33% of human and mouse mRNAs contain the A modification. 6 A (Klungland et al., 2014; Cai et al., 2021). In viruses, those carrying m... 6 Each mRNA of A contains 1 to 15 modifications (Yue et al., 2019; Zheng et al., 2020), but this number is lower in Arabidopsis (0.7 to 1) (Luo et al., 2014).

[0004] m 6 The modification of A is reversible and dynamic. 6 Methyltransferases, demethyltransferases, and methylation-reading proteins recognize them and trigger specific biological functions (Kan et al., 2017; Shi et al., 2019; Yang et al., 2019). Unlike humans (Homosapiens), mice (Musmusculus), yeast, etc., m 6 A has been shown to be unevenly distributed in the 5′ untranslated region (UTR), exons, and 3′ UTR (Lietal, m in the 5′ UTR). 6 A modifications are mainly related to mRNA editing, degradation, and splicing, while m in the 3′UTR... 6 A modification is mainly related to mRNA structural stability, translation initiation, and poly-A binding (Fustin et al., 2013). In strawberries, m... 6 A modification enhances the mRNA stability of NCED5 and AREB1, promoting the translation efficiency of ABAR. 6 A-methyltransferases MTA and MTB have a positive regulatory effect on strawberry fruit ripening (Zhou et al., 2021). FTO-mediated m 6 A demethylation promotes chromatin opening and transcriptional activation, leading to the upregulation of approximately 11,000 genes in leaves and approximately 7,000 genes in roots, activating multiple pathways (Yu et al., 2021). In *A. thaliana*, higher m values ​​were observed in abi1 and bes1. 6 A, they have a negative regulatory effect on ABA signal transduction. This leads to ABI1 and BES1 mRNA transmitting signals through m 6 Degradation via an A-dependent mechanism leads to a decrease in their expression levels (Tangetal, 2022). 6A modification has been proved to exist widely in the plant kingdom and plays an indispensable role in the regulation of many pathways, especially in the response to biotic and abiotic stresses (Liu et al., 2020; Mao et al., 2021; Wang et al., 2022; Ren et al., 2022). For example, the coat protein (CP) of alfalfa mosaic virus (AMV) interacts with Arabidopsis m 6 A demethylase protein AtALKBH9B interacts with, which affects the resistance of Arabidopsis to AMV (Martínez et al., 2017). Zhang et al. (2021) found that m 6 The level of A modification increases after viral infection and presents dynamic changes during the process of rice-virus interaction. Guo et al. (2022) found that Malushupehensis gene MhYTP2 affects m 6 The modification of A, m 6 A regulates the stability of Malus domestica susceptible gene MdMLO19 during powdery mildew infection. However, m 6 The characteristics and functions of A modification in peanut resistance to powdery mildew have not been elucidated. SUMMARY

[0005] The technical problem to be solved by the present application is to determine a gene capable of resisting peanut bacterial wilt.

[0006] To solve the technical problem, the present application provides an m 6 A demethylase AhALKBH15 gene and its identification and application.

[0007] The purpose of the present application is achieved by the following technical solutions.

[0008] An m 6 A demethylase AhALKBH15 gene, wherein the RNA sequence of the AhALKBH15 gene is shown as SEQ ID NO: 1.

[0009] The amino acid sequence encoded by the AhALKBH15 gene is shown as SEQ ID NO: 2.

[0010] An AhCQ2G6Y gene with resistance to peanut bacterial wilt, wherein the RNA sequence of the AhCQ2G6Y gene is shown as SEQ ID NO: 5.

[0011] The amino acid sequence of the AhCQ2G6Y gene is shown as SEQ ID NO: 6.

[0012] A method for improving the resistance to peanut bacterial wilt, wherein the m 6 A demethylase AhALKBH15 can remove the m6 A modification, resulting in m 6 A level reduction, resistance gene AhCQ2G6Y up-regulation, up-regulation of AhCQ2G6Y expression promotes resistance of peanuts to BW.

[0013] A method for improving resistance to peanut bacterial wilt, by identifying m 6 A demethylase AhALKBH15 gene and AhCQ2G6Y gene, verify m 6 A demethylase AhALKBH15 can remove m 6 A modification, resulting in m 6 A level reduction, resistance gene AhCQ2G6Y up-regulation, up-regulation of AhCQ2G6Y expression promotes resistance of peanuts to BW, the steps are as follows:

[0014] (1) BW inoculation and bacterial counting method

[0015] Peanut varieties H108 and H107 were inoculated with Ralstonia solanacearum,

[0016] The treatment method is water culture method, when the peanut root grows to 4 cm, the seedling is moved to the incubator (light / dark cycle is 14 hours / 10 hours),

[0017] At the 3-leaf stage, 0.5 cm of the main root tip was cut and soaked in a bacterial solution with a concentration of 108 CFU ml -1 of R.solanacearum, water was used as a control, and bacterial detection was carried out in an incubator;

[0018] (2) m 6 A methylation sequencing and analysis

[0019] Cutadapt (Martin M., 2011) and perl scripts were used to remove reads containing adapter contamination, low-quality bases, and undetermined bases; fastp was used to verify sequence quality,

[0020] HISAT2 was used to map reads to the genome of Arachis hypogaea with default parameters;

[0021] The candidate peak region was expanded on the genome to obtain a modeling region of a certain length;

[0022] According to the unique alignment of all reads in the region, a Poisson distribution model was used for testing, and the p value of the candidate peak region was calculated;

[0023] The mapped reads of IP and input library were provided to the R software package;

[0024] m 6 The position of A appearance area;

[0025] (3) RNA-seq and data analysis

[0026] H108 and H107 plants were inoculated and infected with R. oryzae at 0, 1 and 7 dpi;

[0027] Total RNA was extracted;

[0028] The fragmented RNA was reverse transcribed to create the final cDNA library;

[0029] Differentially expressed genes (DEGs) were identified with log2(fold change) > 1 and statistical significance (p < 0.05) by R package;

[0030] Gene ontology (GO, http: / / geneontology.org / ) and Kyoto Encyclopedia of Genes and Genomes (KEGG, https: / / www.kegg.jp / ) pathway enrichment analysis were performed;

[0031] (4) qRT-PCR and MeRIP-qPCR analysis

[0032] Real-time quantitative PCR (qRT-PCR) was used to determine the expression of peanut genes infected with R. oryzae and induced by plant hormones, and the roots, stems and leaves of H108 and H107 plants infected with R. oryzae were collected at 0, 0.5, 1.0 and 7.0 dpi, and the three-leaf stage H108 and H107 were treated with SA, MeJA and ABA, and the control was treated with sterile water;

[0033] Total RNA was extracted according to the instructions of the plant RNA extraction kit, and then reverse transcription was performed using a reverse transcription kit;

[0034] qRT-PCR was performed on the obtained first-strand cDNA;

[0035] qRT-PCR specific primers for peanut genes were designed by the online website primerblast of NCBI;

[0036] Relative quantification (2 -ΔΔCT ) of candidate gene expression;

[0037] (5) m 6 A methylation enzyme and Rx_N (disease resistance protein) gene identification in peanut

[0038] The amino acid sequence of peanut was retrieved from PeanutBase, and the domains of peanut were predicted by HMMER3 website; the amino acid sequence was submitted to Pfam to determine the conserved domains;

[0039] The chromosome position map was drawn using MG2C software, and the protein domain was analyzed using MEME. Then, according to the sequence alignment results, a phylogenetic tree was constructed using MEGA7.0;

[0040] The cis-acting elements of the promoters of the selected genes were predicted and drawn using PlantCARE;

[0041] The construction of the plant vector and the transient transformation inserted the full-length coding sequence of the candidate gene between the 35S promoter and the yellow fluorescent protein (YFP) of the pCambi1300-YFP vector, and obtained the peanut expression vector;

[0042] The terminal primers of the full-length gene to be cloned were designed using Primer5.0 software;

[0043] The candidate gene fragment was amplified using Primer star Max DNA polymerase, cDNA and specific primers;

[0044] The purified PCR product was connected to the linearized pCambi1300-YFP vector;

[0045] The plasmid DNA was extracted;

[0046] The recombinant vector and the empty vector were transformed into Agrobacterium EHA105 by Agrobacterium-mediated method for transient overexpression of peanut candidate genes in tobacco and peanut tender leaves; the injected tobacco was cultured in a dark environment at 28℃ for 12 hours, and then cultured under light at 28℃ for 2 days;

[0047] The isolation and transformation of Arabidopsis protoplasts were observed and photographed by LSM710 laser scanning confocal microscope after being cultured in dark conditions at 23℃ for 24 hours;

[0048] LC-MS / MS quantitative analysis of mRNA m 6 A total of 200 ng of RNA sample was isolated from peanut plants, and the kit was treated according to the instructions with nuclease P1 and phosphodiesterase I;

[0049] The RNA was digested with 2 units and 0.01 units of enzyme in buffer (10 mM Tris-HCl pH 7, 20 mM sodium acetate, 2 mM ZnCl2) at 37℃ for 12 hours, and then digested with alkaline phosphatase at 37℃ for 1 hour;

[0050] The digested enzymes were removed with 10 kDa filter tubes; the RNA solution was diluted 10-fold, of which 10 μL was used for LC-MS / MS. Three independent biological replicates were set up for the experiment;

[0051] (6) mRNA stability assay

[0052] AhALKBH15-YFP and pCambia1300-YFP were transfected into H107 leaf protoplasts;

[0053] To the transfected peanut protoplasts, 30 μg ml -1 After 0.5 h incubation with actinomycin D, the peanut protoplasts were taken as the control at 0 h, and then samples were harvested every 3 h in triplicate;

[0054] The mRNA level of genes was detected by qRT-PCR,

[0055] Protein expression and purification The AhALKBH15 cDNA sequence was cloned into the pCold-GST vector backbone with a 6xHis-tag and a GST-tag added at the N-terminus;

[0056] AhALKBH15 and His-GST-tag were overexpressed in E. coli Rosetta2(DE3) cells;

[0057] The cells were lysed by ultrasonication;

[0058] The soluble proteins in the supernatant were purified and eluted;

[0059] The eluted protein fractions were desalted through a desalting column, and then the buffer was changed to storage buffer;

[0060] The purified protein was snap-frozen in liquid nitrogen and stored;

[0061] (7) m 6 A demethylation assay

[0062] In vitro m 6 The RNA demethylation assay was performed in 40 μL reactions containing 0.02-200 pmol of RNA fragments (including m 6A site (CCAUGAAGGm6ACUCACAGAA)), 5 μL AhALKBH15 protein and 2 μL SuperaseIn RNase Inhibitor, reaction buffer is 1×(10 mM KCl, 283 μM (NH4)2Fe(SO4)2, 50 mM MES, 300 μM 2-ketoglutarate and 2 mM l-ascorbic acid); AlkB is used as positive control, His-GST is used as negative control; the demethylation treatment reaction is carried out at 25°C for 3 hours, and then 4 μL 50 mM EDTA is added for quenching;

[0063] (8) m 6 Adotblot

[0064] After the demethylation treatment, the RNA dots are dotted on the nylon membrane and ultraviolet crosslinking is carried out;

[0065] The membrane is blocked with 5% defatted dry milk in 1×PBST at 25°C for 30 minutes, and then incubated with rabbit anti-m6A antibody in 1×PBST at 4°C overnight;

[0066] After large-area washing with 1×PBST, HRP-conjugated goat anti-rabbit IgG antibody in 1×PBST is used for incubation at 25°C for 1.5 hours;

[0067] The membrane is washed with 1×PBST, developed with ECL substrate, imaged with Touch imager, quantified with Touch viewer, and the results are normalized with Image J;

[0068] (9) Statistical analysis of the number of R. solanacearum colonies in leaves

[0069] pCambia1300-YFP, AhCQ2G6Y-YFP, AhCQ2G6Y-muta and Ah5DH5NX-YFP are transiently overexpressed in leaves for 3 days, and then R. solanacearum (concentration of 108 CFU ml -1 ) and ampicillin are inoculated in the leaves;

[0070] Statistical analysis of the number of R. solanacearum colonies in leaves is carried out;

[0071] Three replicates are prepared for each sample;

[0072] (10) Pathological characteristics of two peanut varieties to R. solanacearum infection

[0073] Pathological analysis is carried out on H108 and H107 inoculated with Ralstonia solanacearum;

[0074] To detect whether R. solanacearum effectively infected H108 and H107;

[0075] (11) Whole genome m 6 A methylation profiles

[0076] To explore m 6 A modification profiles in H108 and H107 plants infected by R. solanacearum;

[0077] To determine the differences that might be associated with peanut BW resistance;

[0078] (12) m 6 A modification differences between two peanut varieties;

[0079] To study how m 6 A modification affects the response of peanut to R. solanacearum infection;

[0080] To perform differential analysis of m 6 A modification between H108 and H107 infected by R. solanacearum;

[0081] (13) m 6 A methylation in mRNA transcripts might affect the expression and translation of target genes

[0082] At 0, 1 and 7 dpi, 16395, 8283 and 4504 genes in H108 were differentially m 6 A modified, respectively;

[0083] 5346 genes with different m 6 A modification were identified to exist only in R. solanacearum infected H108 samples; KEGG biochemical pathway enrichment analysis was performed on the genes;

[0084] (14) Whole genome identification of m 6 A modification related genes and their expression in peanut

[0085] Phylogenetic tree was constructed;

[0086] Expression profiles of peanut m 6 A related genes during R. solanacearum infection were studied;

[0087] Several types of cis-regulatory elements were found in the promoter regions of putative peanut m 6 A related genes;

[0088] Whether plant hormones would affect the expression of peanut m 6H108 and H107 seedlings were treated with SA, MeJA or ABA to modify the expression of genes;

[0089] The m 6 Genome-wide identification of A modification-related genes and their expression;

[0090] (15) m 6 Relationship between A modification and gene expression during R. solanacearum infection

[0091] LC-MS / MS was used to detect the mRNAs of H108 and H107 6 A total level;

[0092] Study how m 6 A modification affects the growth of peanuts and their response to biotic stress;

[0093] Study how m 6 A modification affects gene expression levels;

[0094] Determine whether m 6 A modification changed DEGs are related to the response of peanuts to Ralstonia infection, determine m 6 A modification changed DEGs are related to the response of peanuts to Ralstonia infection;

[0095] (16) Study of AhALKBH15-mediated m 6 A methylation in AhCQ2G6Y may improve the resistance of peanuts to Ralstonia infection Study the dynamics of m 6 A in H108 and H107 plants after inoculation with Ralstonia;

[0096] Pfam analysis showed that AhCQ2G6Y has a typical disease resistance protein structure;

[0097] The expression of Rx_N gene in H108 and H107 was detected;

[0098] Expression of AhCQ2G6Y-YFP and Ah5DH5NX-YFP fusion proteins in tobacco leaves;

[0099] Further study the function of AhCQ2G6Y, AhCQ2G6Y-muta and Ah5DH5NX in peanut BW resistance, transform tobacco leaves to transiently overexpress these three genes, then inoculate R. solanacearum;

[0100] Verify the inhibitory effect of AhCQ2G6Y on R. solanacearum infection, transiently overexpress the fusion protein AhCQ2G6Y-YFP in peanut leaves;

[0101] To verify the function of AhALKBH15 and AhALKBH18 as m6A demethylases in peanuts, recombinant glutathione S-transferase (GST)-AhALKBH15 and GST-AhALKBH18 proteins were expressed and purified from an Escherichia coli system; m 6 A modification was verified by MeRIP-qPCR, and it was found that the m 6 A modification level in AhCQ2G6Y was significantly reduced compared with the empty control. An m 6 A demethylase AhALKBH15 gene in peanut resistance to Ralstonia solanacearum.

[0102] Compared with the prior art, the technical effect of the present application is that the present application provides an m 6 A demethylase AhALKBH15 gene, the identification and characterization of the AhALKBH15 gene, and m 6 A demethylase AhALKBH15 can remove the m 6 A modification of AhCQ2G6Y, resulting in m 6 A level, the up-regulation of the resistance gene AhCQ2G6Y, and the up-regulation of AhCQ2G6Y expression promotes the resistance of peanuts to BW, and the great potential of AhALKBH15 for peanut resistance breeding is found. BRIEF DESCRIPTION OF DRAWINGS

[0103] Figure 1 The influence of m 6 A methylation of peanuts by Ralstonia solanacearum.(a) The enrichment distribution of m 6 A peak values in H108 and H107 seedlings in the 5' untranslated region (UTR), exons and 3' UTR at 0, 1 and 7 days after inoculation.(b) The number of m 6 A peak values in H108 and H107 seedlings in the 5' untranslated region (UTR), exons and 3' UTR at 0, 1 and 7 days after inoculation.(b) The number of m 6 A peak values in H108 and H107 seedlings in the 5' untranslated region (UTR), exons and 3' UTR at 0, 1 and 7 days after inoculation.(b) The number of m 6 A peak values in H108 and H107 seedlings in the 5' untranslated region (UTR), exons and 3' UTR at 0, 1 and 7 days after inoculation.(b) The number of m 6 A-URUAY motifs of the four main types (UGUAU, UAUAU, UGUAC and UAUAC).(f) The common m 6 A modification genes in H108 and H107 seedlings after inoculation with Ralstonia solanacearum and the control.

[0104] Figure 2 The influence of m6 m of A peak 6 Examples of A modified transcripts. During the process of inoculation of peanuts with R. japonica, m 6 A modified transcript contains one m6A peak and multiple m 6 A peaks. The red box indicates the position of m 6 A peak. The blue line indicates the m 6 A immunoprecipitation (IP)-qPCR amplified fragments.

[0105] Figure 3 The most significant methylation motifs of H108 and H107 at 0, 1, 7 dpi were determined using HOMER software.

[0106] Figure 4 Differential m 6 GO enrichment analysis of A modified genes. The differentially up-regulated (a, c, e) and down-regulated (b, d, f) m 6 GO enrichment analysis of A genes. Each plot shows the top 20 categories.

[0107] Figure 5 Differential m 6 KEGG enrichment analysis of A modified genes. The differentially up-regulated (a, c, e) and down-regulated (b, d, f) m 6 KEGG enrichment analysis of A genes. Each plot shows the top 20 categories.

[0108] Figure 6 m 6 Genome-wide family member gene structure and conserved motif analysis of m

[0109] Figure 7 m 6 Specific expression of m 6 Tissue expression of methylation enzyme (SAM, WRITERs, EASER and READER) related genes.

[0110] Figure 8 m 6 Three-dimensional structure of demethylase (AhALKBH18).

[0111] Figure 9 m6 Three classes of enzymes of methylation machinery and their expression analysis in response to R. solanacearum. qRT-PCR analysis of the relative expression levels of two “Donor” components (a), “Writer” components (b), and “Reader” components (c). All qRT-PCR assays were set up in triplicate. Error bars represent mean ± standard deviation (*, P < 0.05; **, P < 0.01; t-test).

[0112] Figure 10 is the expression analysis of AhSAM, AhMTA and AhECT genes. SA (3 mmol l 6 A Cis-acting elements in the promoter region (~2000 bp) of the related genes in response to hormone regulation and defense response. (a-d) represent the m 6 A Response elements in the promoter region of methylation-related genes. The darker the red, the more response elements.

[0113] Figure 11 is the expression analysis of AhSAM, AhMTA and AhECT genes. SA (3 mmol l -1 ), MeJA (100 mmol l -1 ) and ABA (10 μg ml -1 ) on peanut genes. Y-axis represents the relative expression amount; error bars represent the mean ± standard deviation of three biological replicates (n = 3, *, P < 0.05; **, P < 0.01; t-test).

[0114] Figure 12 is the whole genome expression and differentially expressed genes analysis of peanut after R. solanacearum infection. (a) Gene expression characteristics and distribution on 20 chromosomes of peanut (H108 and H107) before and after R. solanacearum infection. (b) The number of significantly up-regulated and down-regulated genes in each comparison group. (c) GO enrichment number of differentially expressed genes in biological process, cellular component and molecular function. (Fold change ≥ 2; P value < 0.05)

[0115] Figure 13 is a violin plot showing the m 6 A and non-m 6 A modified genes in peanut during R. solanacearum infection. 6 A The expression level of genes with m 6 A modification is significantly higher than that of non-m 6 A modified genes, two asterisks indicate extremely significant difference (**, P < 0.01; t-test).

[0116] Figure 14 is the cumulative distribution of m 6 A genes and non-m 6 A genes expression changes in H108 and H107.

[0117] Figure 15 Cumulative distribution plot and boxplot analysis of m 6 Relationship between A modification and gene expression. (a) m 6 Cumulative distribution function of log2 peak intensity of A modification genes. (b) Comparison between low (FPKM < 1), medium (1 < FPKM < 5) and high (FPKM > 5) expressed genes in three periods m 6 Boxplot of A level. P values of three periods were calculated using two-tailed unpaired t-test.

[0118] Figure 16 Differential expression of H108 and H107 at 1 and 7 dpi compared to their respective controls. m 6 Correlation of A peak with differentially expressed genes. Red dots represent m 6 Genes and their expression are up-regulated simultaneously; green dots represent m 6 Genes and their expression are down-regulated simultaneously; yellow dots represent m 6 Genes are down-regulated and their expression is up-regulated; blue dots represent m 6 Genes are up-regulated and their expression is down-regulated; gray dots represent genes with no difference between them (different m 6 A is 1.5-fold, different genes are 2-fold, P < 0.05).

[0119] Figure 17 Differential genes of each control group during the process of Ralstonia solanacearum infection m 6 Scatter plot of log2 fold change and differentially expressed genes. m 6 Joint analysis of RIP-seq and RNA-seq data showed that H108_7dpi vs H108_CK (Pearson R = 0.16, P = 0.00), H107_7dpi vs H107_CK (Pearson R = 0.09, P = 0.00) and H108_CK vs H107_CK (Pearson R = 0.17, P = 0.00) were positively correlated with each other. H108_1dpi vs H108_CK (Pearson R = -0.24, P = 0.00), H107_1dpi vs H107_CK (Pearson R = -0.24, P = 0.01) and H108_7dpi vs H107_7dpi (Pearson R = -0.41, P = 0.00) were negatively correlated. In addition, in H108_1dpi vs H107_1dpi (Pearson R = -0.02, P = 0.85), m 6 No correlation between methylation and mRNA expression.

[0120] Figure 18 is a box plot representing the differentially expressed m 6 Expression level of A peak. Peanut 5'UTR region m 6 The expression level of A peak was lower than 3'UTR and exon before inoculation with Ralstonia solanacearum and significantly higher after inoculation. In addition, the expression level of the exon region was significantly lower than that of the 5'UTR and 3'UTR, regardless of before or after inoculation. The asterisk indicates that there is a significant difference between the two (*, P < 0.05; **, P < 0.01; ***, P < 0.001; NS indicates no difference, t test).

[0121] Figure 19 is the difference of different positions compared with H107 m 6 Heatmap of A peak and GO functional enrichment analysis. In each comparison group, differentially expressed m 6 A peak is mainly located in the 3'UTR region, accounting for more than 60%. After inoculation with Ralstonia solanacearum, differentially expressed m 6 The distribution proportion of A peak in the 5'UTR and exon regions increased, and the distribution proportion in the 3'UTR region decreased (fold change ≥ 1.5; P value < 0.05). The green line segment represents the 5'UTR, the black line segment represents the exon, and the red line segment represents the 3'UTR. The number of genes in the GO function enrichment is represented by a circle.

[0122] Figure 20 is the collinearity circle diagram of 25 candidate genes in the disease resistance pathway after inoculation with Ralstonia solanacearum.

[0123] Figure 21 is the expression analysis of 15 combined genes at 0, 0.5, 1.0, and 7.0 dpi by qRT-PCR. The Y-axis is the relative expression; significant differences are evaluated by Mann-Whitney U test and indicated by asterisks; a single asterisk (*) represents P < 0.05, and a double asterisk (**) represents P < 0.01. The average value of three biological replicates, and the error bar represents SD.

[0124] Figure 22 is the expression level and correlation analysis of peanut RX-N family members after inoculation with Ralstonia solanacearum. (a) Heatmap representing the expression level of H108 and H107 at different time periods (0d, 1dpi, and 7dpi). (b, c) Represent the correlation of the expression level of RX-N family members, red represents positive correlation, and blue represents negative correlation.

[0125] Figure 23Subcellular localization of pCambia1300-YFP, AhCQ2G6Y-YFP, Ah5DH5NX-YFP and AhALKBH15-YFP in tobacco leaves. (a) The transient expression vectors were obtained by cloning the full-length CDS of AhCQ2G6Y, Ah5DH5NX and AhALKBH15 into pcambia1300-YFP vector, respectively. (b) By laser confocal observation, pCambia1300-YFP showed green fluorescence in the whole cell, fusion protein AhCQ2G6Y-YFP expressed in the nucleus, fusion protein Ah5DH5NX-YFP expressed in the cell membrane, and fusion protein AhALKBH15-YFP expressed in the nucleus, scale bar 20 pm. PM: plasma membrane; DAPI: 4',6-diamidino-2-phenylindole.

[0126] Figure 24 Transient overexpression of genes during Ralstonia solanacearum infection. (a, b) Disease symptoms, mesophyll cell morphology, evaluation of mesophyll cell damage by trypan blue (TB) staining, and H2O2 accumulation by 3,3'-diaminobenzidine (DAB) staining in R. solanacearum infected tobacco leaves at 3 dpi after transient overexpression of pCambia1300-YFP (replicate 2, 3), AhCQ2G6Y-YFP (replicate 2, 3), AhCQ2G6Y-muta-YFP (replicate 2, 3), Ah5DH5NX-YFP (replicate 1, 2, 3). (c) Colony number of R. solanacearum in tobacco leaves after transient overexpression of pCambia1300-YFP, AhCQ2G6Y-YFP, AhCQ2G6Y-muta-YFP, Ah5DH5NX-YFP and mock control at 3 dpi. "Mock control" represents healthy leaves inoculated with sterile TTC broth; "pCambia1300-YFP" indicates leaves overexpressing empty vector pCambia300-YFP; "AhCQ2G6Y-YFP" indicates leaves overexpressing fusion protein AhCQ2G6GY-YFP; "AhCQ2G6Y mutant" indicates leaves overexpressing fusion protein AhCQ2G6G mutant; "Ah5DH5NX-YFP" indicates leaves overexpressing fusion protein Ah5DH5NX-YFP. Mean values of three biological replicates, data expressed as mean ± standard deviation (n = 9). One-way ANOVA and Tukey's multiple range test (P < 0.05) were used.

[0127] Figure 25Protein induction and purification of AhALKB15 (a) and AhALKB18 (b). Relative gray values of AhALKB15 (a), AhALKB18 (b) and dot blot (c). Error bars represent the SD of three biological replicates. One-way ANOVA and Tukey's multiple range test (P < 0.05) were applied.

[0128] Figure 26 Evaluation of mesophyll cell damage by Trypan blue (TB) staining after transient overexpression of AhALKB15-YFP and pCambia1300-YFP in H107 leaves.

[0129] Figure 27 Detection of endogenous hormones in H108 and H107 at different stages. Comparison of JA content differences. The abscissa is the grouping and the ordinate is the content, error bars represent the mean ± standard deviation of three biological replicates (n = 3, *, P < 0.05; **, P < 0.01; t test).

[0130] Figure 28 Pathological analysis of R. solanacearum infection in the main roots of plants H108 and H107. (a) Representative control and R. solanacearum infected H108 and H107 plants at 28 days post inoculation (dpi). (b) Survival curves of H108 and H107. (c) Stereomicroscopy images of H108 and H107 main root cross sections at 28 dpi. (d) Trypan blue (TB) staining cross sections of H108 and H107 main roots at 1 dpi and 7 dpi. (e) R. solanacearum induced vascular occlusion in H108 and H107. Red arrows indicate the short rod-shaped form of R. solanacearum. (f) Abundance of R. solanacearum (CFU g-1) in H108 and H107 main roots at 0, 1, 3, 5 and 7 dpi with 108 CFU ml-1 of R. solanacearum. -1 -1

[0131] Figure 29 Comparison of different modifications of m 6 A in H108 and H107 after infection with Ralstonia solanacearum (R. solanacearum). (a) Purification of m 6 ​​mRNA fragments of A were then sequenced together with un-inoculated input fragments. (b)m 6 Distribution of peaks in the H108 and H107 mRNA transcript regions. (c)m 6 Consensus sequence motifs within A peaks. (d) LC-MS / MS analysis showed the total m 6 Changes in methylation levels. Error bars represent mean ± standard deviation of three biological replicates (n = 3, *, P < 0.05; Student’s t-test). (e) Significantly different m 6 Distribution of modified genomic regions. Red and blue peaks represent m 6 Increases and decreases in modification levels. (f) Significantly different m 6 Distribution of peaks. (g) Venn diagram showing unique and overlapping different m 6 Modified genes. (h) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of 5346 m 6 Biochemical pathway enrichment of modified genes.

[0132] Figure 30 involved in regulating m 6 Modified genes. (a) m 6 Phylogenetic analysis of m 6 Expression of m 6 demethylases (AhALKBH15 and AhALKBH18, Figure 8 ) with homologs in other plants. Blue arrow indicates key functional residue (2OG binding residue). (d) Relative expression levels of two methylation “erasers” in peanut varieties H108 and H107 upon R. solanacearum infection. Error bars represent mean ± standard deviation of three biological replicates (n = 3, *, P < 0.05; **, P < 0.01; Student’s t-test). (e) Relative expression levels of two methylation “erasers” in H108 and H107 treated with plant hormones salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA). Error bars represent mean ± standard deviation of three biological replicates (n = 3, *, P < 0.05; **, P < 0.01; Student’s t-test) (f) Schematic representation of m 6 Modification pathways and m 6A modification genes.

[0133] Figure 31 is m 6 Relationship between A modification and gene expression during R. solanacearum infection in peanuts. (a) m 6 A modification genes and non-m 6 Number of A modification genes. Transcript abundance was measured as fragments per kilobase of transcript per million mapped reads (FPKM); genes were classified as lowly expressed (FPKM < 1), moderately expressed (1 ≤ FPKM ≤ 5), or highly expressed (FPKM > 5). (b) Different m 6 Correlation between A modification and different gene expression. Red dots (Hyper-up), highly methylated and upregulated genes; blue dots (Hyper-down), highly methylated and downregulated genes; yellow dots (Hypo-up), lowly methylated and upregulated genes; green dots (Hypo-down), lowly methylated and downregulated genes. (c) Venn diagram showing the different m 6 Unique and overlapping differentially expressed genes (DEGs) of A modification. (d) DEGs of different m 6 Gene ontology (GO) term enrichment of DEGs of A modification. (e) Heatmap showing the expression of 25 disease resistance-related genes in H108 and H107 at 0, 1, and 7 dpi.

[0134] Figure 32 Transient overexpression of AhCQ2G6Y during R. solanacearum infection in peanuts. (a) m 6A modification. (b) Phylogenetic tree showing the relationship between peanut AhRx_N genes and homologous genes from other plant species. The phylogenetic tree was constructed using the neighbor-joining method and 1000 bootstrap replicates. (c) Relative expression of AhCQ2G6Y and Ah5DH5NX in H108 and H107 plants at 0, 1 and 7 dpi under R. solanacearum action. Error bars represent the mean ± standard deviation of three biological replicates (n = 3, *, P < 0.05; **, P < 0.01; Student's t-test). Expression normalization was performed using AhACTIN7 as the internal control gene. (d-g) Tobacco leaves transiently overexpressing pCambia1300-YFP, AhCQ2G6Y-YFP or AhCQ2G6Y-muta-YFP, disease symptoms (d), mesophyll cell morphology (e), Trypan blue (TB) staining to assess mesophyll cell lesions (f) and 3,3'-diaminobenzidine (DAB) staining to assess H202 accumulation (g) at 3 dpi of R. solanacearum infection. (h) Subcellular localization of AhCQ2G6Y in Arabidopsis protoplasts. (i) Disease symptoms (top panel) and TB staining (bottom panel) of peanut leaves transiently overexpressing pCambia1300-YFP or AhCQ2G6Y-YFP at 3 dpi of R. solanacearum action. (j) Colony-forming units of R. solanacearum in peanut leaves transiently overexpressing pCambia1300-YFP or AhCQ2G6Y-YFP at 12 h post-inoculation and 1, 2 and 3 dpi of R. solanacearum inoculation. Error bars represent the mean ± standard deviation of three biological replicates (n = 3, *, P < 0.05; **, P < 0.01; Student's t-test).

[0135] Figure 33 AhCQ2G6Y m 6 A methylation can increase the resistance of peanut to R. solanacearum infection. (a) m 6 A dot blot verified that GST-AhALKBH15 and GST-AhALKBH18 m 6 A action. m 6 A dot blot showed that GST-AhALKBH15 m 6A. Demethylation. GST was used as a negative control, and AlkB as a positive control. (b) mRNA lifetime of AhCQ2G6Y. TI: transcriptional repression. Data are expressed as mean ± SD of three biological replicates (n = 3, p = 0.0184, 0.0154). (c) Relative expression level of AhALKBH15 and relative mRNA lifetime of AhALKBH15 after transient overexpression of AhALKBH15-YFP and pCambia1300-YFP in H107 leaves. 6 A enrichment (d) and AhCQ2G6Y expression levels (e) at 3 dpi after exposure to R. solanacearum. Data are presented as mean ± SD of three biological replicates (n = 3, *, P < 0.05; **, P < 0.01; Student's t-test). (f) m 6 A proposed model for regulating peanut resistance to bacterial wilt (BW) mediated by the demethylase AhALKBH15. Following inoculation with *R. solanacearum*, the level of the endogenous hormone jasmonic acid (JA) in peanuts rapidly increased. This upregulates m... 6 AhALKBH15 is a demethylase whose promoter contains a JA response element. Upregulation of AhALKBH15 reduces the m-reactive element on the disease resistance gene AhCQ2G6Y. 6 A modification reversed the inhibition of AhCQ2G6Y expression, thereby enhancing peanut BW resistance. Red and green arrows indicate significant upregulation and downregulation, respectively (p<0.05). Detailed Implementation

[0136] The present invention will be further described below with reference to specific embodiments.

[0137] n-6-methylladenosine (m 6 A) is the most abundant mRNA modification in eukaryotes and is an important regulator of gene expression and many other key biological processes. However, m 6 The characteristics and role of A in peanut resistance to 'BW' are still unclear. This study analyzed the effects of Ralstonia solanacearum (R. solanacearum) infection on resistant peanuts (H108) and susceptible peanuts (H107). 6 The dynamic changes of A. Throughout the transcriptome, we found that "URUAY" is a peanut m... 6 A highly conserved motif of A. Most differences m 6 A is located within the 3' UTR of the transcript, and less frequently in exons. RNA-seq and m 6 Integration analysis of the A-methyl group showed that m 6A was associated with gene expression related to R. solanacearum infection, and functional analysis showed that m6A-related genes were related to plant-pathogen interaction. Our experimental analysis showed that AhALKBH15 was an m 6 A demethylase, leading to m 6 A demethylase, leading to m -1 A demethylase, leading to m

[0138] I. BW inoculation and R. solanacearum counting method

[0139] Peanut varieties H108 and H107 are near-isogenic lines with similar genetic backgrounds, and show high resistance and high sensitivity to BW, respectively. Both lines were inoculated with R. solanacearum (strain number 180731-1) provided by the Institute of Plant Protection of Henan Academy of Agricultural Sciences. The treatment method followed the water culture method of (Lü Jianwei, 2012) (Girdthai et al. When the peanut roots grew to 4 cm, the seedlings were moved to the incubator (light / dark cycle of 14 hours / 10 hours). At the 3-leaf stage, 0.5 cm of the main root tip was cut and soaked in a bacterial solution with a concentration of 108 CFU ml -1 of R. solanacearum, with water as a control, and then cultured in the incubator according to the bacterial detection method of Chen et al. (2014).

[0140] II. m 6 A methylation sequencing and analysis

[0141] Cutadapt (Martin M., 2011) and perl scripts were used to remove reads containing adapter contamination, low-quality bases, and undetermined bases. Then fastp was used to verify sequence quality. We used HISAT2 (Daehwan et al., 2015) to map reads to the genome of Arachis hypogaea (Bertioli et al., 2019) with default parameters. The candidate peak region was extended on the genome to obtain a certain length of the modeling region. According to the unique alignment of all reads in this region, a Poisson distribution model was used for testing, and the p-value of the candidate peak region was calculated. By default, if the p-value is less than 0.05, any region with a p-value less than this value will be considered as a peak. The mapped reads of IP and input libraries were provided to the R software package exomePeak (Meng et al., 2014), which can identify m 6Peak A (Foldchange > 1.5; FDR < 0.05), in bed or bam format, can be visualized using the UCSC Genome Browser or IGV software. We predicted m using the online website (http: / / www.cuilab.cn / sramp / ). 6 The location where A appears in the area.

[0142] III. RNA-seq and Data Analysis

[0143] H108 and H107 plants were inoculated and infected with *Ralstonia solanacearum* at 0, 1, and 7 dpi. Total RNA was extracted using Trizol reagent (Invitrogen, California, USA) according to the manufacturer's instructions. The lysed RNA fragments were reverse transcribed to create the final cDNA library according to the mRNASeq sample preparation kit (Illumina, San Diego, USA), with an average insert size of 300 bp (±50 bp) for paired-end libraries. Differentially expressed genes (DEGs) were identified by log2 (reduced change) >1 and statistical significance (p < 0.05) using R software. Pathway enrichment analyses were performed using Gene Ontology (GO, http: / / geneontology.org / ) and Kyoto Genome Encyclopedia (KEGG, https: / / www.kegg.jp / ).

[0144] IV. qRT-PCR and MeRIP-qPCR Analysis

[0145] Real-time quantitative PCR (qRT-PCR) was used to determine the expression of peanut genes induced by Ralstonia solanacearum infection and plant hormones. Roots, stems, and leaves of H108 and H107 plants infected with Ralstonia solanacearum were collected at 0, 0.5, 1.0, and 7.0 dpi. The expression of genes was measured using 3 mmol / L salicylic acid (SA) and 100 mmol / L... -1 Methyl jasmonate (MeJA) and 10 μg / ml -1 Abscisic acid (ABA) was used to treat H108 and H107 at the three-leaf stage, with sterile water as a control. The hormone concentrations used were determined according to previous studies (Zhang et al., 2017). Furthermore, the detection methods for endogenous hormones in peanuts were based on studies by Li et al. (2016) and floková et al. (2014). Total RNA was extracted according to the instructions of a plant RNA extraction kit, followed by reverse transcription using a reverse transcription kit. The obtained first-strand cDNA was subjected to qRT-PCR according to the method of Kuang et al. (2018). Peanut gene-specific qRT-PCR primers were designed using the NCBI online website Primerblast (Table S3). Relative quantification of candidate gene expression (2...) -ΔΔCT) method (Ren et al., 2020).

[0146] Table S3 Primer sequences required in the experiments

[0147]

[0148]

[0149] V. m 6 The amino acid sequences of peanut were retrieved from PeanutBase, and the domains of peanut were predicted by HMMER3 website. The amino acid sequences were submitted to Pfam to determine the conserved domains. The chromosome position map was drawn using MG2C software, and the protein domains were analyzed using MEME. Then, the phylogenetic tree was constructed using MEGA7.0 according to the sequence alignment results. The cis-acting elements of the promoters of the selected genes were predicted and drawn using PlantCARE. The construction of plant vectors and transient transformation The full-length coding sequence of the candidate gene was inserted between the 35S promoter and the yellow fluorescent protein (YFP) of the pCambi1300-YFP vector to obtain the peanut expression vector. The terminal primers of the full-length gene to be cloned were designed using Primer5.0 software (Table S3). The candidate gene fragment was amplified using Primerstar Max DNA polymerase, cDNA, and specific primers. According to the instructions of the Seamless Cloning Kit, the purified PCR product was ligated into the linearized pCambi1300-YFP vector. The plasmid DNA was extracted according to the steps of the Endo-Free Plasmid Maxi Kit. The peanut candidate genes were transiently overexpressed in tobacco and peanut tender leaves by Agrobacterium-mediated method. The recombinant vector and empty vector were transformed into Agrobacterium EHA105. The injected tobacco was cultured in the dark at 28°C for 12 hours, and then in the light at 28°C for 2 days. The isolation and transformation of Arabidopsis protoplasts were performed according to the method of Yoo et al. (2007) with slight modifications. After incubation in the dark at 23°C for 24 hours, the fluorescence of protein fusion was observed and photographed by LSM710 laser scanning confocal microscope. Quantitative analysis of mRNAm 6A total of 200 ng of RNA sample was isolated from peanut plants and treated with nuclease P1 (Sigma-Aldrich, USA) and phosphodiesterase I (Sigma-Aldrich) according to the manufacturer's instructions. The RNA was digested with 2 units and 0.01 units of enzyme in buffer (10 mM Tris-HCl pH 7, 20 mM sodium acetate, 2 mM ZnCl2) for 12 h at 37 °C, followed by alkaline phosphatase for 1 h at 37 °C. The digested enzymes were removed using 10 kDa filter tubes (Amicon Ultra, USA). The RNA solution was diluted 10-fold, and 10 μL was used for LC-MS / MS. Three independent biological replicates were set up for the experiment.

[0150] VI. mRNA stability assay

[0151] AhALKBH15-YFP and pCambia1300-YFP were transfected into H107 leaf protoplasts and incubated at 25 °C for 24 h. 30 μg ml-1 of hygromycin was added to the transfected peanut protoplasts in ddH2O and incubated at 25 °C for 24 h. The protoplasts were then harvested and resuspended in 100 μL of 1x PBS and 10 μL was used for LC-MS / MS. -1Cultures were incubated with 0.5 μΜ of DNR (Biovision, 1036-50MG) for 0.5 h before harvesting the peanut protoplasts. The mRNA levels of the genes were detected by qRT-PCR every 3 h. Protein expression and purification The AhALKBH15 cDNA sequence was cloned into the pCold-GST vector backbone with a 6xHis-tag and a GST-tag added at the N-terminus. The AhALKBH15 and His-GST-tag were overexpressed in E. coli Rosetta2(DE3) cells. One liter of cell culture was incubated at 37 °C in LB medium containing 50 μΜ ampicillin for 3 h until the optical density at 600 nm (OD600) reached 0.5-0.6. The overexpression was induced with 0.5 mM isopropyl β-d-1 -thiogalactopyranoside (IPTG) and the cells were harvested after 4 h of incubation at 37 °C and resuspended in 80 ml of lysis buffer (50 mM Tris-HCl, 0.5 M NaCl, 10% glycerol, pH = 7.5, dissolved half tablet of protease inhibitor cocktail, Pierce) per liter of culture. The cells were then lysed with sonication and centrifuged at 12,000 r.p.m. for 40 min at 4 °C. The soluble proteins in the supernatant were purified with Ni-NTA Superflow Resins (Qiagen) and eluted with a 50 mM to 0.5 M gradient of imidazole buffer (containing 50 mM Tris-HCl, pH = 7.5, 0.2 M NaCl and 10% glycerol). The eluted protein fractions were passed through a desalting column (PD-10, GE Healthcare) and then the buffer was exchanged to storage buffer (50 mM Tris-HCl, 0.2 M NaCl, 10% glycerol, pH = 7.5). The purified proteins were snap-frozen in liquid nitrogen and stored at -80 °C.

[0152] Seven, m 6 A demethylation assay

[0153] In vitro m 6 The RNA demethylation assay was performed in 40 μΐ^reaction containing 0.02-200 pmol of RNA fragments (including m 6A site (CCAUGAAGGm6ACUCACAGAA)), 5 pL of AhALKBH15 protein, and 2 pL of SuperaseIn RNase Inhibitor, with a reaction buffer of 1 x (10 mM KCl, 283 pM (NH4)2Fe(SO4)2, 50 mM MES, 300 pM 2-ketoglutarate, and 2 mM l-ascorbic acid). AlkB was used as a positive control, and His-GST was used as a negative control. The demethylation treatment reaction was performed at 25 °C for 3 hours, followed by quenching with 4 pL of 50 mM EDTA.

[0154] Eight, m 6 Adotblot

[0155] After demethylation treatment, the RNA dots were blotted on a nylon membrane (GE Healthcare) and UV cross-linked. The membrane was blocked with 5% skim dry milk in 1 x PBST at 25 °C for 30 minutes, then incubated with rabbit anti-m6A antibody (1:2500, abeam) in 1 x PBST at 4 °C overnight. After extensive washing with 1 x PBST, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG antibody (1:2000, abeam) in 1 x PBST at 25 °C for 1.5 hours. The membrane was washed with 1 x PBST, developed with ECL substrate (Thermo Fisher Scientific), imaged with a Touch Imager, quantified data with Touch viewer (e-BLOT Life Science Co., Ltd), and the results were normalized with Image J.

[0156] Nine, statistical analysis of the number of R. solanacearum colonies in leaves

[0157] pCambia1300-YFP, AhCQ2G6Y-YFP, AhCQ2G6Y-muta, and Ah5DH5NX-YFP were transiently overexpressed in leaves for 3 days, then inoculated with R. solanacearum (concentration of 108 CFU ml -1 ) and ampicillin in leaves. After 3 days of culture, the same weight of leaves was taken. The samples were washed with sterile water and transferred to 2-milliliter centrifuge tubes. After grinding, 10 milliliters of sterile water were added, and then left to stand at 4 °C for 30 minutes. Multiple dilutions were performed with a 10-fold gradient, and 0.1 milliliters of the solution were spread on 2,3,5-triphenyltetrazolium chloride (TTC) medium (with ampicillin added at a concentration of 50 pg ml-1). The culture dishes were placed in a dark incubator at 28 °C for 2 days. The colony counting method was referred to the method of Chen et al. (2014). Three replicates were prepared for each sample.

[0158] Results

[0159] X. Pathological features of R. solanacearum infection in two peanut varieties

[0160] To explore the physiological mechanisms behind the resistance of peanut to BW, we inoculated H108 and H107 with R. solanacearum. Twenty-eight days post-inoculation (dpi), H108 seedlings were still robust, while H107 seedlings were mostly wilted ( Figure 28 a). The survival rate of H108 was 90.15%, much higher than that of H107 (23.65%) ( Figure 28 b). In addition, cross sections of the main roots showed that the vascular tissue of H108 plants remained healthy, while that of H107 plants was dark brown and severely damaged ( Figure 28 c).

[0161] To detect whether R. solanacearum effectively infected H108 and H107, we stained cross sections of the main roots of the two peanut lines with trypan blue (TB). At 1 dpi, there were no obvious blockages in the vascular bundles of the roots of both lines. However, at 7 dpi, only occasional dark blue spots were found in the vascular bundle tissue of H108 roots, while a large number of blockages were found in the vascular bundles of H107 ( Figure 28 d). Further examination using a scanning electron microscope showed that, at 1 dpi, there were no R. solanacearum colonies in the vascular bundles of the roots of H108 and H107. Consistent with the TB staining results, at 7 dpi, a few R. solanacearum colonies were found in the vascular bundles of H108 roots, while a large number of R. solanacearum colonies were found in the vascular bundles of H107, blocking the vascular bundles ( Figure 28 e). This indicates that infection by R. solanacearum caused the death of H107 plants. Next, we quantified the number of bacteria in the main roots of the two varieties during 1 to 7 dpi. From 3 to 7 dpi, the number of bacterial colonies in the roots of H107 was significantly higher than that in H108 ( Figure 28 f). These results suggest that the difference in the ability of R. solanacearum to colonize the taproot may be the reason for the resistance of H108 to BW and the susceptibility of H107 to BW.

[0162] XI. Whole-genome m 6 A methylation profiling Next, we explored the m 6 A modification profiles in H108 and H107 plants infected with R. solanacearum to identify differences that might be associated with peanut BW resistance. We performed m 6 A sequencing in H107 and H108 seedlings at three stages (0, 1, and 7 dpi) Figure 29a). Each sample generated approximately 7.60-9.98 Gb of clean data (Table S1). Overall, 232,799 m 6 A peaks were identified across the genome. Based on the sequencing data, we mapped the m 6 A modifications in H108 and H107 during the infection process of R. solanacearum. We found that the m Figure 1 A peaks gradually decreased as the infection of R. solanacearum progressed. 6 A peaks gradually decreased as the infection of R. solanacearum progressed. Figure 1 b). The proportion of transcripts with single m 6 A peaks increased in H108 and significantly increased in H107 at 1 dpi. Overall, the proportion of transcripts with two or more m 6 A peaks decreased over time, although in H108, the proportion of transcripts with two m 6 A peaks increased after 1 dpi. Figure 1 c). We also found 24,229 genes in H108 and 23,038 genes in H107 that exhibited m 6 A modifications at three time points. We randomly selected several genes that exhibited m 6 A modifications and verified them using m 6 A immunoprecipitation (IP)-quantitative (q)PCR, which indicated that the m 6 A-seq data were reliable. Figure 2 ).

[0163] Table S1 Comparison of m 6 A reference genome read statistics

[0164]

[0165] In plants, m 6 A modifications exist in 5' UTR, exons, and 3' UTR. The m 6 A distribution pattern in peanut is consistent with that in Zea mays L. (Miao et al., 2022), Solanum lycopersicum L. (Zhou et al., 2019), and Arabidopsis thaliana (Song et al., 2021). Figure 29 b; Figure 1 d). Most peaks occurred in 3' UTR, fewer in exons, and the least in 5' UTR, which is consistent with previous research results. m 6 A modifications usually occur in conserved sequence motifs in animals and plants. From m 6In A-seq data, we found a "URUAY" motif, which was highly enriched in m 6 A peaks (76.4%) ( Figure 29 c、 Figure 3 ) where R represents A or G and Y represents G or U. Figure 29 c) where R represents A or G and Y represents G or U. Further analysis showed that m 6 A peaks matching the URUAY motif were significantly enriched in the 0, 1, and 7 dpi samples 6 A modification, which is a particularly highly conserved motif ( Figure 1 e、 Figure 3 ) in A-seq data. We identified 21,120 m 6 A peaks common to both pre- and post-inoculation samples. GO enrichment analysis showed that most of the common m 6 A modification genes were significantly enriched in terms of "oxidation-reduction", "protein phosphorylation", "defense response", and "response to abscisic acid" ( Figure 1 f). These results suggest that m 6 A modification is prevalent in both growth and pathogenic response of peanut.

[0166] Twelve, m 6 A modification differences between two peanut varieties

[0167] To investigate how m 6 A modification affects the response of peanut to R. solanacearum infection, we performed differential analysis of m 6 A modification between H108 and H107 infected with R. solanacearum. There were 19,059, 9,186, and 9,012 different m 6 A peaks at 0, 1, and 7 dpi, respectively. In addition, the number of up-regulated m 6 A peaks significantly decreased, while the number of down-regulated m 6 A peaks dramatically increased as R. solanacearum infection progressed ( Figure 29 e). This suggests that m 6 A modification is involved in the response of peanut to R. solanacearum infection. We also found that different m 6 A modifications exhibited different distributions in mRNA transcripts during peanut-R. solanacearum interactions ( Figure 29 f). In H108 plants, differential m 6A peaks were mainly located in the 3'UTR (71.92%), less in the exons (23.02%) and the least in the 5'UTR (5.06%). However, during R. solanacearum infection, the differential m 6 The percentage of A peaks increased greatly, while the differential m 6 A peaks decreased by 8.5-11.5%. These results further suggest that m 6 A modifications are related to the response of peanut to R. solanacearum infection.

[0168] Thirteen, m 6 The methylation of A in mRNA transcripts can affect the expression and translation of target genes. At 0, 1 and 7 dpi, 16395, 8283 and 4504 genes were differentially m 6 A modified in H108, respectively. Among these genes, 5346 genes were differentially m 6 A modified genes were identified as only present in the H108 samples infected with R. solanacearum Figure 29 g) KEGG biochemical pathway enrichment analysis was performed on these genes, and it was found that pathways such as "response to change in environment" and "signal transduction" were significantly enriched Figure 29 h) Compared with H107, the genes with increased m 6 A levels in H108 at 1 dpi, the pathways of "hormone transduction", "MAPK pathway" and "plant-pathogen interaction" were significantly enriched. In contrast, the genes with decreased m 6 A levels in H108 at 1 dpi, the pathways of "hormone transduction", "MAPK pathway" and "plant-pathogen interaction" were significantly enriched. In contrast, the genes with decreased m 6 A modifications were mainly enriched in the pathways of "phosphatidylinositol metabolism", "plant-pathogen interaction" and "phosphatidylinositol signaling system". Interestingly, the pathways of "plant-pathogen interaction" and "phosphatidylinositol signaling system" were enriched in H108 at both 1 dpi and 7 dpi. This suggests that these two pathways, especially "plant-pathogen interaction", may play an important role in the response of peanut to R. solanacearum infection Figure 4 and Figure 5 ). The results suggest that genes involved in these pathways may be closely related to the difference in BW resistance between H108 and H107.

[0169] Fourteen, Genome-wide identification of genes related to m 6 A modifications in peanut and their expression Plants encode several conserved proteins related to m 6 A modifications: m 6A methylase, demethylase and methylation reader proteins. Therefore, we utilized the known A. thaliana m 6 A modified proteins to identify homologous genes in peanut. A total of 52 candidate genes were identified, i.e., 9 SAMs, 6 writers, 18 erasers and 19 readers. We constructed a phylogenetic tree to show the relationship between these proteins and homologous genes from other plant species Figure 30 a, 30c; Figure 6 , Figure 7 Table S2). We investigated the expression profiles of peanut m 6 A related genes during R. solanacearum infection Figure 30 b). After inoculation with R. solanacearum, 2 SAM genes (AhSAM1 and AhSAM2) and 4 writer genes (AhMTA1, AhMTA2, AhMTA4 and AhMTA5) were strongly upregulated in H108 compared to H107. In addition, 4 eraser genes (AhALKBH2, AhALKBH14, AhALKBH15 and AhALKBH18) and 6 reader genes (AhECT6-AhECT10 and AhECT13) were also significantly upregulated in R. solanacearum-infected H108 compared to H107, as confirmed by qPCR Figure 30 d, 30e; Figure 9 ). The results indicated that peanut m 6 A methylation-related genes might be involved in peanut resistance to BW.

[0170] In the putative peanut m 6 A related genes, several types of cis-regulatory elements were found in the promoter regions. Among them, SA, MeJA and ABA response elements were included; MeJA response elements were the most prevalent Figure 30 e, 30f; Figure 10 ). To further investigate whether plant hormones would affect the expression of peanut m 6 A modification genes, H108 and H107 seedlings were treated with SA, MeJA or ABA Figure 11 ). Hormone treatment induced AhSAM1, AhSAM2, AhALKBH15 and AhALKBH18, but inhibited AhMTA2, AhMTA5, AhECT6 and AhECT7. Interestingly, the m 6 A peaks gradually decreased in both peanut varieties as R. solanacearum infection progressed, which might be due to m 6 A demethylase activity. Based on the above results, we speculate that AhALKBH15 and AhALKBH18 might play a key role in the resistance response of peanut to BW.

[0171] Table S2 Amino acid sequence information for plants

[0172]

[0173]

[0174]

[0175]

[0176] Fifteen, m 6 Relationship between A modification and gene expression during R. solanacearum infection

[0177] LC-MS / MS detected mRNAs of H108 and H107 6 A total levels, and the results showed that R. solanacearum infection caused a significant decrease in peanut mRNAs 6 A levels Figure 29 d) To investigate how the modification of m 6 A affects the growth of peanut and its response to biotic stress, we performed RNA-seq analysis on mRNA samples from H108 and H107 plants. Three independent biological replicates for each sample were consistent with each other, demonstrating the reliability of the data Figure 12 ). For each sample, we first divided genes into those with m 6 A modification or without m 6 A modification. Then, genes were divided into three categories according to transcript abundance in units of fragments per kilobase of transcript per million mapped reads (FPKM): low (FPKM < 1), medium (1 ≤ FPKM ≤ 5), and high (FPKM > 5) Figure 31 a). In un-inoculated H108 plants, 29,194 genes were m 6 A modified, most of which (86.7%) were expressed at medium or high levels. In contrast, in 21,317 genes that were not m 6 A modified, most (67.2%) were expressed at low levels. At 1 dpi and 7 dpi, m 6 A modified genes in H108 plants were still mainly expressed at medium and high transcript abundance, while most genes without m 6 A modification were expressed at low levels. Similar results were observed in H107 plants. These results indicated that the expression levels of m 6 A modified genes were generally much higher than those of genes without m 6 A peak Figure 13 , S14). In m 6In A-modified genes, the m gene with low expression 6 The total content of A was significantly higher than that of moderately and highly expressed genes. Figure 15 ).

[0178] To study m in more detail 6 How does A modification affect gene expression levels? We investigated different m... 6 Correlation analysis was performed on A-modified genes and different expression genes (DEGs). The differences between H108 and H107 compared to unvaccinated individuals were m 6 A showed a negative correlation with gene expression at 1 dpi and a positive correlation at 7 dpi. Figure 16 Compared to H107 plants, H108 plants had 815, 108, and 96 DEGs with different m values ​​at 0, 1, and 7 dpi, respectively. 6 A modification, in which 44% of genes, 56.5% of genes, and 52.1% of genes are in m 6 A shows a consistent trend at both the modification and expression levels. Figure 31 b; Figure 17 Further analysis showed that m associated with DEG 6 Peak A is primarily located in the 3′ UTR, suggesting that it may play an important regulatory role in gene expression levels during the peanut's response to BW. Interestingly, in both varieties, in the uninoculated state, peak A is present in the 5′ UTR. 6 The expression level of genes in peak A is much lower than that in the 3′ UTR with m 6 The expression level of genes in peak A was observed, but after inoculation with R. solanacearum, the expression level of genes with m in the 5′ UTR was significantly reduced. 6 Peak A showed high gene expression levels. Notably, at all three time points, m was present in either the 5′ UTR or the 3′ UTR. 6 The expression levels of genes in peak A were all higher than those in exons with m 6 The gene expression level in peak A is much higher. Figure 18 , Figure 19 This indicates that R. solanacearum infection has complex effects on gene expression levels and mRNA modification.

[0179] To determine m 6 Whether the altered DEGs (determined by A) are related to the peanut's response to Ralstonia solanacearum infection, we determined m 6 Are the DEGs modified by A related to the peanut's response to Ralstonia solanacearum infection? Figure 31 c). Surprisingly, only Ah7RJD9X (β-glucosidase) underwent different modifications and expression across the three time points, indicating that m 6A modification and gene expression occurred highly dynamic changes. However, after inoculation with R. solanacearum, 171 DEGs in H108 plants were found to be m 6 A modification occurred. GO analysis showed that these genes were significantly enriched in multiple terms, including "induction of programmed cell death", "defense response to fungus", "response to bacteria", and "disease resistance" ( Figure 31 d; Figure 20 ). These terms further indicated that m 6 A modification was involved in peanut disease resistance. Different m 6 A modification DEGs with "disease resistance" annotation were further studied, and the expression levels of 15 genes were confirmed by qPCR ( Figure 31 e; Figure 21 ). Eight genes (including AhCQ2G6Y, AhG5Y6Q1, AhW8IC6S, AhDGK5DJ, Ah3475Q7, AhMHUR4I, Ah25DMGV, and AhIZ1RGH) were significantly induced in both peanut varieties after R. solanacearum infection, while the other six genes were inhibited. In the process of R. solanacearum infection, 10 genes were enriched in m 6 A in H107 and H108 plants, including AhEXF4LS, AhYSVF0U, and AhX4AMHZ. In addition, 10 genes were deleted in m 6 A, including Ah25DMGV, AhIZ1RGH, and Ah5K9V6V. Most strikingly, there was one gene (AhCQ2G6Y) that was significantly m 6 A-enriched only in H107, with m 6 A modification levels ≥50-fold higher than in H108.

[0180] Sixteen, AhCQ2G6Y m 6 A methylation mediated by AhALKBH15 may improve peanut resistance to Solanaceae bacterial infection

[0181] We next studied the dynamics of m 6 A in AhCQ2G6Y after inoculation of H108 and H107 plants with R. solanacearum ( Figure 32 a). At 0 dpi, no obvious m 6 A peak was found in AhCQ2G6Y in both varieties. At 1 dpi, AhCQ2G6Y showed m 6 A enrichment in H108 plants, with a peak of 3.44, but no m 6 A in the corresponding H107 samples. However, at 7 dpi, m 6A modification sharply increased, with an enrichment value of 117, much higher than the peak of 2.36 at 7 dpi in H108. In addition, AhCQ2G6Y was significantly upregulated in both varieties infected by R. solanacearum, but the transcript abundance in H108 was much higher than that in H107. Therefore, we speculated that AhCQ2G6Y might be involved in the m 6 A mediated the response of peanuts to R. solanacearum infection.

[0182] Pfam analysis showed that AhCQ2G6Y had a typical structure of disease resistance protein. The phylogenetic tree showed the relationship between the 19 peanut Rx_N proteins and 24 disease resistance proteins of other plant species Figure 32 b) Rx_N proteins were divided into two subgroups: TNL and CNL families. In CNL, AhCQ2G6Y was clustered with a protein conferring resistance to potato late blight (Chen et al., 2018). Other peanut proteins were closely related to Arabidopsis downy mildew protein RPP13 (Bittner-Eddy et al., 2001) and soybean Fusarium wilt (Phytophthora sojae) resistance protein Rps1-k1 (Gao et al., 2008). We also detected the expression of Rx_N genes in H108 and H107 Figure 22 ) Overall, AhCQ2G6Y and its homologous gene Ah5DH5NX showed a positive response to R. solanacearum infection, with significantly increased expression in H108 and H107 plants upon R. solanacearum infection Figure 32 c) Expression of AhCQ2G6Y-YFP and Ah5DH5NX-YFP fusion proteins in tobacco leaves showed that AhCQ2G6Y and Ah5DH5NX were localized in the nucleus and cell membrane, respectively Figure 23 ) These data support the hypothesis that homologs AhCQ2G6Y and Ah5DH5NX might promote the resistance of peanut varieties H108 and H107 to BW. AhCQ2G6Y-muta is a mutation of base A (182 bp) in AhCQ2G6Y to base C. To further study the function of AhCQ2G6Y, AhCQ2G6Y-muta and Ah5DH5NX in peanut BW resistance, tobacco leaves were transformed to transiently overexpress these three genes (Zhao et al., 2022), and then inoculated with R. solanacearum Figure 32 d) At 3 dpi, tobacco leaves overexpressing AhCQ2G6Y-muta-YFP and Ah5DH5NX-YFP showed similar yellowing symptoms as those carrying the empty vector control Figure 24 a,b) In contrast, inoculated leaves overexpressing AhCQ2G6Y-YFP were comparable to those inoculated with growth medium Figure 32e) Cell death and leaf lesion were significantly reduced Figure 32 f) The bacterial content in leaves overexpressing AhCQ2G6Y-YFP was significantly lower than that in leaves overexpressing pCambia1300-YFP, AhCQ2G6Y-muta-YFP and Ah5DH5NX-YFP Figure 24 c) In addition, the accumulation of H02 in leaves overexpressing AhCQ2G6Y-YFP was reduced compared to leaves overexpressing AhCQ2G6Y-muta-YFP, Ah5DH5NX-YFP or empty vector control Figure 32 g) This indicates that overexpression of AhCQ2G6Y directly inhibits the infection and multiplication of R. solanacearum in tobacco leaves.

[0183] To verify the inhibitory effect of AhCQ2G6Y on R. solanacearum infection, the fusion protein AhCQ2G6Y-YFP was transiently overexpressed in peanut leaves Figure 32 h) As expected, peanut leaves overexpressing AhCQ2G6Y-YFP developed leaf lesions, but the rate of cell death was reduced Figure 32 i) As the infection of R. solanacearum progressed, the number of bacterial colonies on peanut leaves overexpressing AhCQ2G6YFP or empty vector control gradually increased. Notably, there was no difference in the number of bacterial colonies between the two at 12 hours post inoculation (hpi). However, more R. solanacearum colonies were observed on peanut leaves expressing empty vector control than on peanut leaves overexpressing AhCQ2G6YFP Figure 32 j).

[0184] To verify the function of AhALKBH15 and AhALKBH18 (the gene sequence of AhALKBH18 is shown as SEQ ID NO: 3, and the amino acid sequence is shown as SEQ ID NO: 4) as m 6 To verify the function of AhALKBH15 and AhALKBH18 (the gene sequence of AhALKBH18 is shown as SEQ ID NO: 3, and the amino acid sequence is shown as SEQ ID NO: 4) as m 6 To verify the function of AhALKBH15 and AhALKBH18 (the gene sequence of AhALKBH18 is shown as SEQ ID NO: 3, and the amino acid sequence is shown as SEQ ID NO: 4) as m 6 To verify the function of AhALKBH15 and AhALKBH18 (the gene sequence of AhALKBH18 is shown as SEQ ID NO: 3, and the amino acid sequence is shown as SEQ ID NO: 4) as m 6 To verify the function of AhALKBH15 and AhALKBH18 (the gene sequence of AhALKBH18 is shown as SEQ ID NO: 3, and the amino acid sequence is shown as SEQ ID NO: 4) as m 6A signal was significantly reduced, while m 6 A signal remained unchanged, indicating that GST-AhALKBH15 had m 6 A demethylase activity Figure 33 a; Figure 25 ). Transfection of AhALKBH15-yellow fluorescent protein (YFP) and pCambia1300-YFP into H107 leaf protoplasts found that m 6 Transient overexpression of m Figure 33 b) AhALKBH15-YFP fusion protein and control in H107 leaves, qPCR confirmed that the expression level of demethylase fusion protein AhALKBH15-YFP was significantly higher than that of empty control Figure 33 c). TB staining showed that overexpression of AhALKBH15-YFP in peanut leaves could reduce cell death and lesion development Figure 26 ). In addition, we detected the m 6 A modification of the candidate gene AhCQ2G6Y by MeRIP-qPCR, and found that compared with the empty control, the m 6 A modification level in AhCQ2G6Y was significantly reduced Figure 33 d), while its expression level was significantly increased Figure 33 e). In addition, we detected the content of endogenous hormone JA in H108 and H107 plants inoculated with R. solanacearum for 1 day, respectively. We found that there was a significant difference between the two, with a fold difference of more than 3 times. It is speculated that JA may affect the expression of demethylase AhALKBH15 Figure 33 f; Figure 27 ). This result shows that AhALKBH15 is a functional m 6 A demethylase in peanut, which can directly remove m 6 A in AhCQ2G6Y, and then enhance the resistance to BW mediated by AhCQ2G6Y by reducing the m 6 A modification level of disease resistance protein gene AhCQ2G6Y.

[0185] Seventeen, discussion

[0186] During the long-term co-evolution of plants and pathogens, the plant immune system has gradually developed the ability to distinguish self from non-self, transmit signals to the nucleus, regulate gene expression, and initiate defense responses to external invaders (Mark et al., 2001; Dou et al., 2012). With the detection of epigenetic markers such as m 6With the development of the technology of A modification), more and more species began to study these markers. In the past, m 6 A modification was mainly studied in the context of its important role in plant growth and development (Li et al., 2014; Shen et al., 2016; Wei et al., 2018). For example, the methylation of m 6 A modification has been shown to cause phenotypic differentiation in plants, such as defective leaves, hairy branches, over-proliferation of vegetative branches, and even embryonic death. However, m 6 A modification may also play a key role in biological stress response. Over 57.42% of m 6 A modification is distributed in the 3'UTR, which is consistent with the results of corn (Cai et al., 2021; Hu et al., 2021), m 6 A peak is mainly related to the stability, editing, degradation and splicing of mRNA, while the peak occurring in the 3'UTR is mainly related to the structural stability of mRNA (Machnicka et al., 2012; Fustin et al., 2013). In addition, m 6 A modification is positively correlated with gene expression, while m 6 A peak may have an inhibitory effect (Arribas-Hernandez et al., 2018). After R. solanacearum infection, m 6 The proportion of m 6 A peak increased sharply in the BW-resistant peanut variety H108, but the proportion in the 3'UTR decreased in both varieties. Other studies investigating plant infection have shown that contact with pathogens affects the distribution of m 6 A peak in peanut-specific genomes URUAY. We found that m 6 A peak was significantly enriched after R. solanacearum infection, similar to the results of other plant-specific genomes in Arabidopsis, corn and tomato (Miao et al., 2020). Overall, m 6 A modification increased after inoculation with P. solanacearum, and m 6 A modification is highly correlated with gene expression.

[0187] m 6 The regulatory mechanism of m 6 A is catalytically active when the substrate is m 6A lacks catalytic activity (Zhang et al., 2016; Mauer et al., 2017). Utilizing known m from other species... 6 Based on the sequences of A-regulated genes, we identified 18 homologs of the AlkB family in peanuts. Figure 6 c). AhALKBH15 has an alanine-rich region and a unique spiral structure at its N-terminus. The disease resistance gene AhCQ2G6Y is a member of the RX_N family, possesses a typical conserved CC domain, and responds to Ralstonia solanacearum infection. Furthermore, overexpression of AhCQ2G6Y inhibits the reproduction of R. solanacearum in peanuts. Although at 7 dpi, AhCQ2G6Y showed increased activity at H108 m 6 The enrichment level in A is lower than that in H107, but the gene is significantly upregulated in H108. 6 Transient overexpression of the demethylase AhALKBH15 reduces the m of AhCQ2G6Y. 6 The A modification level was increased, and the stability of AhCQ2G6Y mRNA was enhanced. We further demonstrated that m 6 The demethylase AhALKBH15 can remove the m-type of AhCQ2G6Y. 6 A modification, which in turn affects the abundance of the AhCQ2G6Y transcript and peanut BW resistance ( Figure 33 f).

[0188] In summary, we have mapped the first complete transcriptome of peanut genome. 6 A modified spectrum was used to analyze the m process during infection in resistant and susceptible peanut varieties. 6 The dynamic changes modified by A. Through the integration of m 6 A-modification and transcriptome analysis revealed that AhALKBH15 is a m 6 A demethylase, which can remove m from AhCQ2G6Y 6 A, while AhCQ2G6Y has a typical RxN (disease-resistant protein) terminal structure, and in m 6 A plays a key role in peanut BW resistance mediated by A. These findings not only validate a strong candidate gene for breeding BW-resistant peanuts, but also provide a wealth of data that is expected to facilitate future breeding efforts.

[0189] In this study, we analyzed m in two peanut varieties 6 The response of A modifications and transcriptional changes to R. solanacearum infection. We describe the dynamics of m in peanut. 6 A-methyl group and its association with gene expression. m 6 Multi-omics analysis and experimental data from A indicate that m 6A demethylase AhALKBH15 can remove the m 6 A modification, thereby enhancing BW resistance in peanuts. Our research results provide new insights that help to understand the role of epigenetic modifications in mediating the interaction between plants and biomass in peanuts.

[0190] These findings demonstrate the great potential of AhALKBH15 for peanut resistance breeding. Our identification and characterization of the AhALKBH15 gene provide a basis for a deeper understanding of the resistance mechanism of peanuts to BW, and provide information for future research to improve the genetic methods of peanut resistance to BW.

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[0253] Zhou L, Tian S, Qin G. 2019. RNA methylomes reveal the m6A-mediated regulation of DNA demethylase gene SlDML2 in tomato fruit ripening. Genome Biology 20: 1-23.

[0254] Zhou L, Tang R, Li X, Tian S, Li B, Qin G. 2021. N6-methyladenosine RNA modification regulates strawberry fruit ripening in an ABA-dependent manner. Genome Biology 22: 1-32.

[0255] Zhuang WJ, Zhang C, Chen H, Zhuang RR, Chen YT, Deng Y, Cai TC, Wang SY, Liu QZ, Tang RH, et al. 2019.

[0256] Overexpression of the peanut CLAVATA1-like leucine-rich repeat receptor-like kinase AhRLK1 confers increased resistance to bacterial wilt in tobacco. Journal of Experimental Botany 70: 5407-5421.

Claims

1. A method of increasing resistance to peanut bacterial wilt, characterized by, m 6 A demethylase AhALKBH15 removes m 6 A modification, resulting in m 6 A levels, the resistance gene AhCQ2G6Y is up-regulated, and the up-regulation of AhCQ2G6Y expression promotes the resistance of peanuts to bacterial wilt; The RNA sequence of the AhALKBH15 gene is shown as SEQ ID NO:

1. The RNA sequence of the AhALKBH15 gene is shown as SEQ ID NO:

1.

2. An m 6 A demethylase AhALKBH15 gene in the application of peanut anti-rhizome disease genetic engineering, the RNA sequence of the AhALKBH15 gene is shown as SEQ ID NO: 1.

Images