Ahwrky41 gene for enhancing stem strength of peanut and application thereof

Abstract

This invention belongs to the field of biological gene and peanut stalk strength technology, and specifically discloses a method for enhancing peanut stalk strength. AhWRKY41 This invention relates to genes and their applications. By using GWAS combined with bioinformatics to locate candidate genes, it ultimately identified genes in peanuts that can regulate stem strength. AhWRKY41 Genes; when AhWRKY41 After gene overexpression transformation of plants, compared with wild type, AhWRKY41 Gene overexpression lines exhibited higher stem strength, higher cellulose and lignin content, and increased expression of key genes in the lignin synthesis pathway; therefore, AhWRKY41 Gene overexpression can enhance the strength of plant stems.

Description

Technical Field

[0001] This invention relates to the fields of biological genes and peanut stalk strength technology, and in particular to a method for enhancing peanut stalk strength. AhWRKY41 Genes and their applications. Background Technology

[0002] Currently, research on improving crop stem strength mainly focuses on cultivation regulation and hormone regulation, such as improving plant morphology by controlling planting density, fertilization methods, or exogenous regulators. However, these methods are easily affected by environmental conditions, have poor stability and reproducibility, and cannot fundamentally solve the problem of insufficient stem strength. In contrast, identifying and utilizing key genes regulating stem development and cell wall formation at the molecular level is an important approach to achieving stable improvement of crop lodging resistance. In recent years, with the development of high-throughput sequencing technology and the increasing maturity of genome assembly technology, more and more plant genome sequences have been successfully resolved, which has greatly promoted the development of research in the field of plant science, and genome-wide association studies (GWAS) are being used more and more widely.

[0003] Transcription factors play a central regulatory role in plant growth, development, and stress responses. Among them, the WRKY transcription factor family is a plant-specific class of transcriptional regulators that are widely involved in biological processes such as plant growth and development, hormone signal transduction, cell wall synthesis, and secondary cell wall thickening. Previous studies have shown that some WRKY transcription factors can influence the mechanical strength of plant stems and lodging resistance by regulating the biosynthesis of cell wall components such as lignin, cellulose, and hemicellulose. Currently, there is a lack of systematic research on the application of related genes in enhancing peanut stem strength, and there are no publicly available reports of related genes being used in molecular breeding for peanut lodging resistance or in related biotechnological applications. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for enhancing the strength of peanut stalks. AhWRKY41 Genes and their applications.

[0005] To achieve the above objectives, the present invention is implemented according to the following technical solution: One of the objectives of this invention is to provide a method for enhancing the strength of peanut stalks. AhWRKY41 Genes, the ones mentioned AhWRKY41 The gene is located on chromosome 15 of the peanut plant, and its nucleotide sequence is shown in SEQ ID NO:1. AhWRKY41 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO:2.

[0006] The second objective of this invention is to provide a method for enhancing the strength of peanut stalks. AhWRKY41 The application of genes in breeding plants with enhanced stem strength will AhWRKY41Transforming plants by gene overexpression yields plants with enhanced stem strength.

[0007] Furthermore, the plants include peanuts, Arabidopsis thaliana, and tobacco.

[0008] Compared with existing technologies, this invention uses GWAS combined with bioinformatics to locate candidate genes, ultimately identifying genes in peanuts that can regulate stem strength. AhWRKY41 Genes; when AhWRKY41 After gene overexpression transformation of plants, compared with wild type, AhWRKY41 Gene overexpression lines exhibited higher stem strength, higher cellulose and lignin content, and increased expression of key genes in the lignin synthesis pathway; therefore, AhWRKY41 Gene overexpression can enhance the strength of plant stems. Attached Figure Description

[0009] Figure 1 The phenotypic data of peanut stalk strength under different environments are normally distributed: A, B, C, and D are the phenotypic data of stalk strength in E3, E4, E5, and BLUP environments, respectively.

[0010] Figure 2 The results of GWAS analysis of stem strength under different environments using TASSEL are shown below: A, B, C, and D are Manhattan plots for environments E3, E4, E5, and BLUP, respectively; E, F, G, and H are QQ plots for environments EE3, E4, E5, and BLUP, respectively.

[0011] Figure 3 The results of GWAS analysis of stem strength under different environments using the mrMLM method are shown: A, B, C, and D are Manhattan plots for environments E3, E4, E5, and BLUP, respectively.

[0012] Figure 4 The results of GWAS analysis of stem strength under different environments using the mrMLM method are shown: A, B, C, and D are QQ plots for E3, E4, E5, and BLUP environments, respectively.

[0013] Figure 5 The results of GWAS analysis of stem strength under different environments using the IIIVmrMLM method are shown: A, B, C, and D are Manhattan plots for environments E3, E4, E5, and BLUP, respectively.

[0014] Figure 6 The results of GWAS analysis of stem strength under different environments using the IIIVmrMLM method are shown: A, B, C, and D are QQ plots for E3, E4, E5, and BLUP environments, respectively.

[0015] Figure 7Phylogenetic analysis of the peanut WRKY gene family. Using previously reported factors affecting stem strength in peony... PIWRKY41a The protein sequences of the gene were constructed by combining the WRKY family protein sequences of Arabidopsis thaliana and peanut WRKY family protein sequences.

[0016] Figure 8 for AhWRKY41 Gene heatmap and haplotype analysis: A: LD block analysis of SNP locus 15_16868122 and AhWRKY41 gene; B: AhWRKY41 gene haplotypes and amino acid mutations; C: Box plot of haplotype differences at SNP locus 15_16868122; D: Box plot of haplotype differences in AhWRKY41 gene; The significance test method was one-way ANOVA test. (*** indicates P < 0.001, ** indicates P < 0.01).

[0017] Figure 9 The lignin and cellulose content of peanut varieties with different stem strengths.

[0018] Figure 10 for AhWRKY41 Subcellular localization of the gene-encoded AhWRKY41 protein.

[0019] Figure 11 for AhWRKY41 Gene expression pattern analysis: A is AhWRKY41 Gene expression levels in roots, stems, and leaves during seedling emergence; B represents... AhWRKY41 Gene expression levels in roots, stems, leaves, flowers, and fruit pedices during the flowering and pecking stages; C represents... AhWRKY41 The expression levels of the gene in roots, stems, leaves, and pegs during the fruit-filling maturity stage; LS1, LS2, HS1, and HS2 are 19 and 69, respectively, for varieties with low stem strength; HS1 and HS2 are 41 and 49, respectively, for varieties with high stem strength.

[0020] Figure 12 for AhWRKY41 Gene overexpression transformation of Arabidopsis thaliana: A and B are the phenotypes of Arabidopsis thaliana transformed with AhWRKY41 overexpression; C is the identification of Arabidopsis thaliana plants transformed with AhWRKY41 overexpression vector; D is the expression level of AhWRKY41 gene in Arabidopsis thaliana; E, F, and G are the stem strength, stem diameter, and main stem height of the overexpressing Arabidopsis thaliana, respectively; the significance test method is one-way ANOVA test.

[0021] Figure 13 for AhWRKY41 Gene silencing: A represents the phenotypes of WT, TRV, and TRV-Z; B represents leaf differences among WT, TRV, and TRV-Z; C represents... AhWRKY41Gene expression levels in stems and leaves of WT, TRV, and TRV-Z; D, E, and F represent stem strength, plant height, and stem diameter of WT, TRV, and TRV-Z, respectively (TRV-Z is equivalent to TRV-...). AhWRKY41 The significance test method was a one-way ANOVA test.

[0022] Figure 14 This describes the expression patterns of key genes in the lignin synthesis pathway.

[0023] Figure 15 This refers to the activity of enzymes that transcribe key genes. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0025] The materials used in the following examples consisted of a natural population of 122 known peanut varieties collected from various regions, as shown in Table 1. Arabidopsis thaliana was used to construct the overexpression lines; Nicotiana benthamiana was used for subcellular localization.

[0026] Table 1. 122 peanut varieties ; Chemical reagents: Lignin content assay kit (AKSU010U) and cellulose content assay kit (AKSU007C), Beijing Box Biotechnology Co., Ltd. DNA extraction kit (FastPure). ® Plant DNA Isolation Mini Kit, RNA Extraction Kit (FastPure) ® Universal Plant Total RNA Isolation Kit, Reverse Transcription Kit (HiScript) ® III. RT SuperMix for qPCR, RT-qPCR reagent ChamQ Universal SYBR qPCR Master Mix Kit, and gel extraction kit FastPure ® Gel DNA Extraction Mini Kit, Plasmid Extraction Kit FastPure ® Plasmid Mini Kit, ClonExpress cloning kit ®II. The One Step Cloning Kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.; restriction endonucleases BamHI, SpeHI, and KpnHI were purchased from TaKaRa Bio.

[0027] Example 1: Enhancing the strength of peanut stalks AhWRKY41 Gene screening GWAS analysis was performed on peanut stem strength under E3, E4, E5, and BLUP environments. Using 122 peanut germplasm resources shown in Table 1, three GWAS methods were employed for localization: TASSEL, mrMLM, and 3VmrMLM.

[0028] Phenotypic data are shown in Table 1 and Figure 1 As shown, the stem strength variation ranges from 0.21 to 0.29, and the generalized heritability calculation shows a value of 0.79. The stem strength of the natural population approximately follows a normal distribution.

[0029] Table 1. Variation and heritability analysis of peanut stalk strength phenotypic data ; GWAS results as follows Figures 2-6 As shown in Table 2, candidate genes were screened within a 50kb interval above and below the identified SNP sites based on the LD decay distance. Through functional annotation, analysis of previously reported homologous genes, and haplotype analysis, two genes potentially related to stem strength were finally selected. Arahy.ZTD7SX and Arahy. ICU7TP .

[0030] Table 2 Candidate genes related to stem strength ; Meanwhile, based on existing technology reports on factors affecting stem strength in peony... PIWRKY41a The protein sequences of the genes, combined with the WRKY family protein sequences of Arabidopsis and peanut, were used to construct a phylogenetic tree using MEGA11 software. Figure 7 ).Discover Arahy.ZTD7SX and PIWRKY41a Since they are homologous genes, the gene was renamed AhWRKY41 We will conduct a functional analysis on it.

[0031] AhWRKY41 The gene is 1220 bp in length, with three exons and two introns, and the coding region is 1005 bp in length. There are two SNPs (15_16876297, 15_16876206) within this region, both located in the exon region (see [link to gene description]). Figure 8 (B) of the identified AhWRKY41 Analyze the gene loci, and at that locus and AhWRKY41 Linkage blocks can be formed both upstream and downstream of the gene (see...) Figure 8 A in the middle), for AhWRKY41 Haplotype analysis revealed that this gene has two haplotypes (see...) Figure 8 The B in the formula caused mutations in alanine (Ala) to threonine (Thr) and leucine (Leu) to proline (Pro), respectively. This resulted in the identification of... AhWRKY41 SNP sites and AhWRKY41 Perform haplotype difference analysis separately (see Figure 8 (C) The results showed that stem strength phenotypes differed at this site, with haplotype CT having a larger phenotypic value than CC. AhWRKY41 There are also genetic differences; the phenotypic value of CG is greater than that of TA.

[0032] AhWRKY41 The gene sequence is:

[0033] AhWRKY41 The amino acid sequence of the gene-encoded protein is as follows: MECEGSWEKSELKSELVQGMEVARKLKAELELESSADNRDLLVDKILSSYDKALLILRWSGSVSKPHTLHQSTKTSPPHSPLPHNHQQELKHSSQKRKMISKWVNQVKVNSENGFEGSHEDGYSWRKYGQKDILGSKYPRSYYRCTFRNTKDCWATKQVQRSEDNPNIF EITYKGSHTSKGKKNAAMSAKSPDIQEPQEHHTNFQNMLSVTAAKPGNEEMACSFTSPSTSSGCRTQENQNLPSILENNPFLGSLSQTHLLLPNTPESNYFPSPFCANELDGIYSNRCSEFDVSEIISAHTSATNPPIFDFDFSLDPLELGQNFRFNASRFSH* (SEQ ID NO:2).

[0034] The lignin and cellulose contents of peanut stems from high-stem-strength varieties HS1 and HS2 and low-stem-strength varieties LS1 and LS2 were determined. The lignin content was HS1 > HS2 > LS1 > LS2, with a significant difference between HS2 and the low-stem-strength varieties, and a highly significant difference between HS1 and the low-stem-strength varieties. The cellulose content was HS1 > HS2 > LS2 > LS1, with significant differences between both high-stem-strength and low-stem-strength varieties. Figure 9 ).

[0035] Example 2 AhWRKY41 Subcellular localization and expression patterns of genes 1. A subcellular localization recombinant vector was constructed using the pCAMBIA2300-35S-eGFP vector, and subcellular localization was performed on tobacco leaves. Tobacco leaves were infected by injection after being grown for 4-5 weeks under an environment of 14 h light / 10 h darkness, 25℃ temperature, and 70% relative humidity.

[0036] (1) Preparation of Agrobacterium bacterial culture and infection solution (see Table 3) The PCR-verified bacterial culture was added to YEB liquid medium containing Kan+ and rifampin and cultured overnight at 28°C and 220 rpm with shaking. The culture was then centrifuged at 6000 rpm for 10 min. The supernatant was discarded, and the cells were resuspended in MMA buffer until the OD600 value reached 0.6–0.8. The culture was then incubated at room temperature in the dark for 3 h.

[0037] Table 3 Subcellular localization infection resuspension formulation ; (2) Infection of tobacco and tobacco cultivation Select healthy, flat-leaved leaves of *Nicotiana benthamiana*. Gently injure the underside of the leaves with a syringe needle, draw up the infection solution, inject it through the wound, and mark the injection area with a marker. Inject empty vector as a control.

[0038] Infected tobacco plants were cultured in the dark for 12-24 hours, then transferred to normal conditions for 2-3 days. Leaf sections near the wounds were selected for examination using a laser confocal fluorescence microscope to observe the presence and location of fluorescence. Results are as follows: Figure 9 .

[0039] 2. Using the Guangdong-grown variety Yueyou 7 as experimental material, total RNA was extracted according to the manufacturer's kit instructions. The concentration and purity of RNA (A260 / A280 ratio) were detected using a spectrophotometer. Subsequently, the total RNA was reverse transcribed into cDNA according to the reverse transcription kit instructions for subsequent experiments.

[0040] The results are as follows Figure 10 As shown, AhWRKY41 The gene-encoded AhWRKY41 protein is located in the cell nucleus.

[0041] For quantitative real-time PCR (qRT-PCR), peanut tissue samples were collected from the field at different time stages and immediately stored in liquid nitrogen before being brought back. qPCR primers were designed based on the gene cDNA template using the NCBI online website and PRIMER 5 software (see Table 4), synthesized by Beijing Qingke Biotechnology Co., Ltd., and used for PCR amplification. Primer quality was assessed by agarose gel electrophoresis. A qRT-PCR kit was used to prepare the quantitative real-time PCR system, which was then placed in a CFX96 Touch quantitative real-time PCR instrument (Bio-Rad Laboratories, USA). The reaction program was as follows: Step 1, 95℃ pre-denaturation for 30 s; Step 2, 95℃ denaturation for 10 s, 60℃ annealing for 30 s, repeated 40 times; finally, the instrument's default melting curve acquisition program was used. Actin (LOC112783800, Arachis hypogaea actin-7) was used as an internal reference gene, and the relative expression level was calculated using the 2−ΔΔCt method.

[0042] Table 4 qRT-PCR primer design ; The results are as follows Figure 11 As shown, the expression levels at different times and locations reveal AhWRKY41 The gene expression is relatively stable during the flowering and pegging stage and is mainly highly expressed in the stem.

[0043] Example 3: Construction of overexpression lines Overexpression recombinant plasmids were constructed using the pCAMBIA2300-35S-eGFP vector.

[0044] 1. Transformation of Arabidopsis thaliana by flower soaking method (1) Arabidopsis thaliana culture: After vernalization, Arabidopsis thaliana seeds were sown in the soil (vermiculite: nutrient soil = 1:3), and then placed in an incubator at 28℃ for 1 month under 16 h light / 8 h darkness conditions. When the Arabidopsis thaliana produced more inflorescences, the transformation was carried out.

[0045] (2) Bacterial culture: pCAMBIA2300S-35S- AhWRKY41 Agrobacterium culture was cultured in 20 mL of LB liquid medium containing 25 mg / L Rif and 50 mg / L Kana at 28°C and 200 rpm for 12–16 h until the OD600 of the culture was 0.8–1.0.

[0046] (3) Washing: Centrifuge at 5000 rpm for 10 min and collect the bacterial cells.

[0047] (4) Agrobacterium infection: Pick samples containing pCAMBIA2300S-35S- AhWRKY41 Agrobacterium vector was resuspended in a solution to prepare an OD600 of 0.8-1.0. Silwet-77 was added to a concentration of 0.02%. During infection, the inflorescence was immersed in the Arabidopsis flower infusion for 1 min. After infection, the inflorescence was cultured in the dark for 1 day, then transferred to normal light for one week. Infection could be repeated once to increase the genetic transformation efficiency of Arabidopsis. Seeds were harvested after maturity.

[0048] 2. Identification of positive Arabidopsis thaliana plants (1) Arabidopsis thaliana culture: Arabidopsis thaliana seeds were evenly distributed on MS medium containing 50 mg / mL Rif and cultured in an incubator at 28℃ for 1 week under 16 h light / 8 h darkness. The surviving Arabidopsis thaliana were transplanted into soil (vermiculite: nutrient soil = 1:3) and cultured for 1 month.

[0049] (2) DNA detection: DNA was extracted from the leaves of Arabidopsis thaliana plants using the FastPure Plant DNA Isolation Mini Kit (DC104) from Vazyme, and then detected by PCR. After the PCR reaction, agarose gel electrophoresis was performed.

[0050] (3) qRT-PCR analysis: RNA was extracted from the leaves of Arabidopsis thaliana plants, cDNA was prepared, and finally qRT-PCR analysis was performed. The relevant primers are shown in Table 4.

[0051] The results showed AhWRKY41 The gene was significantly expressed in transgenic plants, but its expression was not detected in wild-type plants (see [link to original text]). Figure 12 (D in the text). Phenotypic analysis indicates (see D). Figure 12 In plants overexpressing E, F, and G, the plant height, stem diameter, and stem strength were all higher than those in the control group.

[0052] Example 4: Construction of Silent Lines Gene silencing experiments were conducted using pTRV2 vector to construct gene silencing recombinant vectors. Agrobacterium was used to transiently transform the peanut variety "Yueyou 7". One week after peanut planting, the peanuts were infected by injection.

[0053] 1. Prepare Agrobacterium bacterial culture and infection solution (see Table 5) The PCR-verified bacterial culture was added to YEB liquid medium containing Kan+ and rifampicin and cultured overnight at 28°C with shaking at 220 rpm; centrifuged at 5000 rpm for 5 min. The bacterial culture consisted of: pTRV2 recombinant vector, pTRV2 empty vector, pTRV1 helper vector, and pTRV2-AhPDS positive control. The supernatant was discarded, and the bacteria were resuspended in MMA buffer until the OD600 value was approximately 0.8–1.0. The culture was then incubated at room temperature in the dark for 3–4 h.

[0054] Table 5 Formulation of VIGS experimental infection resuspension ; 2. Infection of peanuts and peanut cultivation Before infection, equal volumes of bacterial suspension were mixed according to three combinations: pTRV1 helper vector + pTRV2 recombinant vector, pTRV1 helper vector + pTRV2 empty vector, and pTRV1 helper vector + pTRV2-AhPDS positive control. The lower epidermis of peanut leaves was gently scratched with a needle, and then the upper epidermis at the scratched area was gently held down. The VIGS system was injected through the wound using a syringe without the needle, infecting at least 3 / 4 of the leaf. After 48 hours of dark incubation, the leaves were cultured under normal light, and changes in leaf phenotype were observed. The appearance of whitening in the positive control indicated the success of the VIGS system. Two weeks after infection, phenotypic identification and sampling were performed to detect the expression level of the target gene.

[0055] The results showed that the expression levels in stems and leaves of WT and TRV empty negative controls were much higher than those in silent plants (see [link to study]). Figure 13 (C) The stem strength, plant height, and stem diameter of plants under different treatments were measured. Silent plants showed lower stem diameter, main stem height, and stem strength than the WT and negative control (see C). Figure 13 (D, E, F in the text).

[0056] Example 5: Expression patterns and enzyme activity assays of key genes in the lignin synthesis pathway Expression patterns of key genes in the lignin synthesis pathway were determined using qRT-PCR, following the same procedure. AhWRKY41 Expression pattern determination. The enzyme activities of PAL, C4H, 4CL, C3H, and F5H were determined using the EILSA kit provided by Shanghai Enzyme-Linked Biotechnology Co., Ltd.

[0057] Gene expression pattern results showed that five of the nine key genes in the synthetic pathway exhibited differential expression. Figure 14 The activities of the enzymes transcribed from these five genes were measured, and the results showed that there were significant differences among the five enzymes. Figure 15 ).

[0058] In conclusion, when AhWRKY41 After gene overexpression transformation of plants, compared with wild type, AhWRKY41 The overexpressing gene lines had higher stem strength, higher cellulose and lignin content, and increased expression of key genes in the lignin synthesis pathway.

[0059] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for enhancing the strength of peanut stalks AhWRKY41 Genes are characterized by: The AhWRKY41 The gene is located on chromosome 15 of the peanut plant, and its nucleotide sequence is shown in SEQ ID NO:

1. AhWRKY41 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO:

2.

2. A method for enhancing the strength of peanut stalks as described in claim 1 AhWRKY41 The application of genes in breeding plants with enhanced stem strength is characterized by: Will AhWRKY41 Transforming plants by gene overexpression yields plants with enhanced stem strength.

3. The application according to claim 2, characterized in that: The plants mentioned include peanuts, Arabidopsis thaliana, and tobacco.

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