Peanut bag gene family and application in early identification of peanut bacterial wilt

Abstract

The application provides a peanut BAG gene family and application in early identification of peanut bacterial wilt, and belongs to the technical field of plant genetic engineering. The peanut BAG gene family provided by the application comprises AhYSVF0U, AhTEF0AP, Ah37JSRQ and AhCF9SCE genes. Four pairs of primers for detecting the peanut BAG family are disclosed, and the expression of the peanut BAG family genes can be rapidly and accurately detected by a fluorescence quantitative PCR method, thereby laying a foundation for identification and prevention and treatment of peanut bacterial wilt.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of plant genetic engineering, and particularly relates to a peanut BAG gene family and application thereof in early identification of peanut bacterial wilt. BACKGROUND

[0002] The Bcl-2-associated anti-apoptotic gene (BAG) family is highly conserved in eukaryotes and is a key hub for stress signal transduction and cell survival. The C-terminal of the family protein contains a BAG domain that interacts with HSP70 / HSC70 molecular chaperones, serving as a cooperative chaperone to regulate protein folding and degradation. Plant BAG proteins have unique structural differentiation: group I members (such as AtBAG1-4) contain a ubiquitin-like domain involved in stress adaptation, and group II (such as AtBAG5-7) contains a calmodulin-binding IQ motif. Functional studies have shown that they are involved in heat tolerance (AtBAG7-WRKY29 interaction), salt tolerance (OsBAG4-DNA methylation complex), and pathogen defense (AtBAG6-mediated autophagy). The promoter cis-acting elements and stress-induced expression patterns confirm that BAG genes achieve cross-regulation of hormones through the ABA and ethylene pathways. Although the functions are clear in model plants, the study of the peanut BAG family is still in the blank, which limits the application of this regulatory network in resistance to bacterial wilt. Therefore, it is of great significance to further detect the peanut BAG gene family in the process of resisting bacterial wilt infection. SUMMARY

[0003] The purpose of the application is to provide a peanut BAG gene family and application thereof in early identification of peanut bacterial wilt, laying a foundation for the research on identification and prevention of peanut bacterial wilt.

[0004] The peanut BAG gene family and application thereof in early identification of peanut bacterial wilt, wherein the peanut BAG gene family comprises AhYSVF0U, AhTEF0AP, Ah37JSRQ and AhCF9SCE genes, the nucleotide sequence of the AhYSVF0U gene is shown as SEQ ID NO. 1, the nucleotide sequence of the AhTEF0AP gene is shown as SEQ ID NO. 2, the nucleotide sequence of the Ah37JSRQ gene is shown as SEQ ID NO. 3, and the nucleotide sequence of the AhCF9SCE gene is shown as SEQ ID NO. 4.

[0005] The application of the q-PCR primer set in early identification of peanut bacterial wilt comprises four pairs of primers, and the sequences are shown as SEQ ID NO. 5-12, wherein the primer pairs are used for amplifying the AhYSVF0U, AhTEF0AP, Ah37JSRQ and AhCF9SCE genes respectively.

[0006] The application of peanut BAG gene family in early identification of peanut bacterial wilt, 13 AhBAG genes are identified in peanut genome, which are significantly up-regulated under Ralstonia solanacearum infection and abscisic acid treatment, indicating that they may be involved in regulating the resistance of peanut to bacterial wilt.

[0007] The method for identifying peanut BAG genes comprises the following steps:

[0008] S1, using BLASTP and HMMER tools, taking Arabidopsis BAG protein sequence as a query sequence, performing alignment analysis on peanut Tifrunner v2 genome, setting PF02179 domain E value threshold <1x10 5 ;

[0009] S2, verifying the domain integrity of the candidate gene through NCBI conserved domain database;

[0010] S3, analyzing the physicochemical properties of the protein by using ExPASy ProtParam online tool, and predicting the subcellular localization by Wolf PSORT. The expression of peanut BAG gene family can be accurately detected by the fluorescence quantitative PCR method, and the early identification and prevention and treatment of peanut bacterial wilt can be completed.

[0011] The application has the beneficial effects that: through whole genome system identification and function analysis, the knowledge gap in this field is filled, the evolutionary relationship, structural differentiation and stress response expression dynamics of peanut BAG gene are clarified by combining informatics and experimental means, the research results not only reveal the BAG-mediated molecular mechanism of peanut stress adaptation, but also lay a foundation for developing molecular markers and genetic engineering strategies to improve the stress resistance of this important crop. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 Evolutionary analysis and structural characteristics of peanut BAG gene family.

[0013] Figure 2 Conserved domain and three-dimensional structure analysis of peanut BAG protein.

[0014] Figure 3 Genome evolution and functional regulation characteristics of peanut BAG gene.

[0015] Figure 4 Expression regulation verification of peanut BAG gene under biological stress and hormone treatment.

[0016] Figure 5 Peanut BAG protein interaction network and functional prediction analysis. DETAILED DESCRIPTION

[0017] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0018] I. Materials and Methods

[0019] The bacterial wilt-resistant variety H108 and the highly susceptible variety H107, both developed by Professor Yin Dongmei's team at Henan Agricultural University, were selected.

[0020] II. Screening and Bioinformatics Analysis of the AhBAG Gene Family

[0021] Using BLASTP and HMMER tools (https: / / www.ebi.ac.uk / Tools / hmmer / search / phmmer), with the Arabidopsis thaliana BAG protein sequence (TAIR) as the query sequence, we performed alignment analysis on the peanut Tifrunner v2 genome (PeanutBase, https: / / www.peanutbase.org / ), setting the E-value threshold for the PF02179 domain to <1×10⁻. 5 The integrity of candidate gene domains was verified using the NCBI Conserved Domain Database (CDD, https: / / www.ncbi.nlm.nih.gov / ). Protein physicochemical properties (molecular weight, isoelectric point pI) were analyzed using the ExPASy ProtParam online tool (https: / / web.expasy.org / protparam / ), and subcellular localization was predicted using Wolf PSORT (https: / / www.genscript.com / wolf-psort.html). Phylogenetic trees were then reconstructed using full-length protein sequences from peanut (13 genes), Arabidopsis (7 genes), tomato (10 genes), and rice (6 genes), with the bootstrap set to 1000. TBools software was used to analyze the number of introns and exons, as well as the visualization of protein domains and gene structures. The sequence analysis of the peanut BAG gene family is shown in Table 1.

[0022] Table 1 Sequence information of the peanut BAG gene family

[0023]

[0024] After testing, such as Figure 1 As shown in B, there are 13 BAG gene family members throughout the peanut genome, distributed on 11 different chromosomes.

[0025]

[0026]

[0027]

[0028]

[0029] III. Treatment of materials

[0030] Mature seeds were surface sterilized with 0.1% HgCl2for 2 min and then rinsed with sterile water for 3 times, and placed on moist filter paper in the dark at 25°C for germination until the radicle emerged (about 36 h). The germinated seeds were transplanted to sterilized vermiculite and cultured in a phytotron (37°C, 70% relative humidity, dark) until the 3-leaf stage.

[0031] R. solanacearum inoculation: Root drenching with 1 x 10 8 CFU / mL bacterial suspension. Hormone induction: 100 μM ABA, 2 mM SA or 50 μM MeJA were applied by foliar spraying, respectively. Each treatment was set with 3 biological replicates, and samples were taken before treatment (0 d), 1 day (1 dpi) and 7 days (7 dpi) after treatment, respectively, and stored at -80°C after quick freezing in liquid nitrogen for RNA extraction.

[0032] IV. Extraction and reverse transcription of RNA

[0033] The total RNA was extracted from the treated leaves using NanoDrop 2000 and Agilent 2100, and reverse transcribed into cDNA by PrimeScript™ RT Kit (TaKaRa), and amplified by SYBR Green I (TB Green Premix ExTaq II, TaKaRa) in CFX96 Touch™ system (Bio-Rad).

[0034] V. Real-time quantitative PCR verification

[0035] The primers of AhYSVF0U, AhTEF0AP, Ah37JSRQ and AhCF9SCE genes were designed by PrimerBlast in NCBI, ensuring the specificity of the primers and crossing the intron region as much as possible, and the primer sequences are shown in Table 2. The specific reaction conditions were as follows: 95°C, 30 s; 95°C, 5 s; 58°C, 30 s; 40 cycles. Each treatment was performed with three biological replicates and three parallel samples, and the relative expression was calculated by 2-ΔΔCt method, and the significance was evaluated by one-way ANOVA (SPSS 26.0).

[0036] Table 2 Primer sequences of peanut BAG gene family

[0037]

[0038] VI. Statistical analysis

[0039] One-way ANOVA (SPSS 26.0) was used to assess significance for all data, with p<0.05 and p<0.01 indicating significance and highly significant, respectively. Data were expressed as mean ± standard deviation (SD).

[0040] VII. Results and analysis

[0041] 1. Bioinformatics analysis of peanut BAG gene family

[0042] The classification of peanut BAG transcription factors is shown in Fig. Figure 1 A, all peanut BAG proteins were divided into four distinct clades. Clade I contained two paralogous plant-specific members (AtBAG7 and SlBAG10), clade II contained 14 members with species-specific expansion in peanut (6 AhBAGs) and tomato (4 SlBAGs). Clade III showed cross-species conserved orthologous relationships, including two peanut members (Ah35MDCZ and Ah9JS9HJ), while clade IV exhibited the highest diversity, containing 14 members with significant expansion in peanut (5 AhBAGs) and Arabidopsis (3 AtBAGs).

[0043] The chromosomal distribution of peanut BAG gene family is shown in Fig. Figure 1 B, chromosomal localization showed that 13 AhBAG genes were unevenly distributed on 11 of the 20 chromosomes in peanut. Chromosomes 4 and 14 each contained two pairs of paralogous genes, possibly resulting from segmental duplication events. This dispersed genomic distribution pattern suggests that multiple replication mechanisms have collectively contributed to the expansion of the peanut BAG family.

[0044] The exon-intron structure of peanut BAG gene family is shown in Fig. Figure 1 C, clade II members had the simplest structure, with 62% (4 / 6) containing only a single exon (AhD7MIWJ, AhG65IU1, Ah4JG1SZ, and AhJ75SJQ), and one member (AhYSVF0U) had three exons. In contrast, clade IV genes exhibited a conserved four-exon structure (5 / 5 members), while clade III members showed moderate complexity (Ah35MDCZ / Ah9JS9HJ contained 4 exons). The negative correlation between phylogenetic divergence and structural complexity suggests that exon loss / gain events may have driven functional specialization during the evolution of the BAG family. The protein structure of peanut BAG family is shown in Fig. Figure 2As shown, plant BAG genes typically encode a BAG domain of 70-80 amino acid residues. Conserved motif analysis further elucidated the genetic variation in peanut BAG proteins. Ten motifs were identified among the 13 AhBAGs, primarily composed of hydrophobic residues (valine V, isoleucine I, leucine L) and charged residues (glutamate E, aspartate D, arginine R, lysine K). Figure 2 B, C). All AhBAGs contain motif 2 and motif 3, which are consistently present in phylogenetic clusters and may be key elements essential for BAG protein function. Class IV proteins contain eight motifs (motif 1-8), and members encoding complete BD and possessing UBQ all contain these eight common motifs, indicating a degree of conservation in peanut BAG protein motifs. Phylogenetic analysis showed that AhYSVF0U in group III has high amino acid sequence similarity to AtBAG6. Structural prediction indicated that the IQ-calmodulin binding motif consists of two small α-helices connected by hairpin loops, interacting with the first α-helix (triple antiparallel α-helix bundle) of the BAG domain. The IQ-calmodulin motif of AhYSVF0U contains two α-helices, contrasting with the single helix observed in AtBAG6. Figure 2 D).

[0045] 2. Analysis of Peanut BAG Genome Collinearity and Cis-Regulatory Elements

[0046] The evolutionary conservation of BAG genes among peanut species was studied using comparative collinearity analysis. Eleven AhBAG genes in cultivated peanut (A. hypogaea) showed orthologous connections in diploid wild species (A. duranensis and A. ipaensis) and tetraploid wild species (A. monticola), indicating that functional conservation was maintained during domestication. Figure 3 A). Notably, the seven AhBAG genes distributed across seven chromosomes exhibit collinearity with their Arabidopsis AtBAG homologs. For example, AhYSVF0U shows collinearity with AtBAG6, a key regulator of fungal resistance, suggesting its conserved function in stress adaptation. This evolutionary conservation underscores the potential retention of AhBAG gene functions in mediating stress responses.

[0047] Cis-regulatory element analysis of the AhBAG promoter showed a significant enrichment of hormone response elements. Figure 3B). 10 genes contain ABRE (abscisic acid responsive element), of which AhYSVF0U and AhADAM8V contain 5 ABRE elements each, and 7 genes have CGTCA motif (methyl jasmonate responsive element). Ah37JSRQ uniquely carries three MeJA responsive elements, which is consistent with its rapid induction under MeJA treatment. In addition, four AhBAG promoters contain W-box elements, which play a key role in defense-related transcriptional reprogramming after pathogen-associated molecular pattern (PAMP) recognition. These findings suggest that AhBAG genes are transcriptionally regulated by ABA / JA signaling cascade and may be involved in pathogen defense.

[0048] 3. Expression analysis of peanut BAG gene family at different developmental stages

[0049] AhBAG genes showed distinct spatiotemporal expression patterns in different tissues Figure 3 C). AhYSVF0U was specifically highly expressed in root nodules and seeds (FPKM>50), which is consistent with its putative function in symbiotic nitrogen fixation and seed development. In contrast, six AhBAG genes showed constitutively low expression (FPKM<5), suggesting their stress-induced regulation. AhTEF0AP was only activated in floral tissues, indicating its specialized function in reproduction.

[0050] Under biotic and abiotic stresses, qRT-PCR revealed genotype- and time-dependent expression dynamics. In the resistant cultivar H108, AhYSVF0U was upregulated 5-fold in roots 7 days after P. solanacearum inoculation (p<0.01) Figure 4 A), consistent with its predicted function in limiting pathogen spread through programmed cell death (PCD). ABA treatment induced an 8-fold increase in AhYSVF0U expression in H108 leaves after 1 day, while MeJA rapidly activated AhTEF0AP and Ah37JSRQ (4-fold increase, Figure 4 B, D). Interestingly, salicylic acid (SA) suppressed AhYSVF0U expression in H108 Figure 4 C), highlighting the antagonistic role of SA with ABA / JA pathways in regulating BAG-mediated stress responses.

[0051] 4. Protein interaction network and functional association analysis

[0052] STRING-based protein interaction prediction revealed a multi-level regulatory network centered on AhYSVF0U Figure 5). Ah0J9HE3 is a HSP20 family chaperone involved in heat tolerance, suggesting that AhYSVF0U can stabilize stress-denatured proteins. AhTGXT8E is a xylanase inhibitor that fortifies the cell wall by neutralizing pathogen-derived xylanases. AhK7D5N2 is a calmodulin that transduces Ca2+ signals in immune responses via an IQ motif. This network positions AhYSVF0U as a multi-layered stress adaptation hub that integrates chaperone activity, cell wall fortification, and calcium signaling. Notably, the lack of direct interactions with canonical immune receptors suggests that AhBAGs act downstream of pathogen recognition.

[0053] The embodiments listed in the specification are only exemplary embodiments embodying the core technology of the present application, and serve to explain the technical principles and do not constitute a limitation on the scope of protection of the present application. For those skilled in the art, based on the technical solutions disclosed in the specification, the derived embodiments realized by equivalent replacement or technical adjustment all belong to the foreseeable technical extension range. Any creative changes, such as adaptive modifications, structural optimizations and functional expansions, made on the basis of the design concept of the present application, are considered to fall within the scope of patent protection of the present application.

Claims

1. An application of the peanut BAG gene family in the early identification of peanut bacterial wilt, characterized in that, The peanut BAG gene family includes the AhYSVF0U, AhTEF0AP, Ah37JSRQ, and AhCF9SCE genes. The nucleotide sequence of the AhYSVF0U gene is shown in SEQ ID NO.1, the nucleotide sequence of the AhTEF0AP gene is shown in SEQ ID NO.2, the nucleotide sequence of the Ah37JSRQ gene is shown in SEQ ID NO.3, and the nucleotide sequence of the AhCF9SCE gene is shown in SEQ ID NO.

4. Early identification of peanut bacterial wilt is achieved by detecting the expression levels of these genes.

2. The application of a q-PCR primer set in the early identification of peanut bacterial wilt, characterized in that, It includes 4 primer pairs, with sequences shown in SEQ ID NO.5~12, which are used to amplify the AhYSVF0U, AhTEF0AP, Ah37JSRQ and AhCF9SCE genes as described in claim 1.

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