Application of AhPUGN1.1 in regulation and control of peanut nodulation

Abstract

The invention belongs to the technical field of plant gene engineering, and particularly relates to application of AhPUGN1.1 in regulation and control of peanut nodulation, the nucleotide sequence of the AhPUGN1.1 is shown as SEQ ID NO.1, the invention finds that the nodulation number is increased after overexpression of the AhPUGN1.1, and the nodulation number is reduced after knockout of the AhPUGN1.1, so that the application of the AhPUGN1.1 in regulation and control of peanut nodulation is provided.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to the application of AhPUGN1.1 in regulating peanut nodulation. Background Art

[0002] Peanuts (Arachis hypogaea L.) are the world's fourth-largest oilseed crop, after soybeans, rapeseed, and sunflower. Peanuts boast the highest average oil yield per acre among major oilseed crops, making them a powerful source of edible oils in my country. Beyond providing vegetable oil, peanuts, like other legumes, also provide essential protein for our daily lives.

[0003] Nodulation and nitrogen fixation provide approximately 55-70% of the nitrogen source for peanuts, accounting for the majority of their life cycle. Nodulation is a biological process unique to most legumes and a few non-legumes. The bacteroids in mature nodules fix nitrogen from the air into a nitrogen source that the plant can utilize, while the host plant provides the rhizobia with the carbon source they need for survival. This mutually beneficial host-rhizobium symbiosis ensures the high nitrogen requirements of the nodulating plant. Peanut is a representative species of the "Dalbergioid" family of legumes. Understanding the mechanisms of peanut nodule development and growth, and improving the nitrogen fixation efficiency of peanut nodules through breeding or genetic engineering, as well as cultivating peanut varieties with high nitrogen fixation efficiency, are key approaches to improving the competitiveness of my country's peanut industry.

[0004] The nodulation process in legumes can be summarized as follows: Under low nitrogen conditions, flavonoids secreted by legumes induce rhizobia to produce nodulation factors, which are recognized by host plant receptors. This initiates the infection process, with calcium ion oscillations, root hair deformation, and infection line formation as key hallmarks of successful rhizobium infection. Key transcription factors such as NSP1, NSP2, and NIN regulate the expression of key genes in the nodulation process, such as ENOD11 (Early Nodulin 11), initiating rhizobium infection, infection line formation, and nodule development.

[0005] Unlike other legumes, which invade through "infection threads," peanut nodulation primarily occurs through "crack-entry," where rhizobia enter the cortex directly. Nodules develop through endosymbiotic proliferation within infected cortical cells. Research on peanut nodulation is scarce, and no genes involved in nodulation regulation have been isolated and identified in peanut. Summary of the Invention

[0006] To solve the above problems, the present invention provides the use of AhPUGN1.1 in regulating peanut nodulation.

[0007]

[0008] Preferably, the regulation is to increase the number of peanut nodules by overexpressing AhPUGN1.1;

[0009] Inhibiting the expression of AhPUGN1.1 can reduce the number of peanut nodules.

[0010] Preferably, the step of overexpressing the AhPUGN1.1 is: connecting the CDS sequence of AhPUGN1.1 to a plant overexpression vector via a restriction endonuclease.

[0011] Preferably, the plant overexpression vector is a plant overexpression vector comprising a 35S promoter or an Ubi promoter.

[0012] Preferably, the plant overexpression vector is a pCAMBIA 1300 vector.

[0013] Preferably, the method of inhibiting AhPUGN1.1 is any one of RNA interference and gene editing.

[0014] Preferably, the method for inhibiting AhPUGN1.1 by RNA interference is as follows: AhPUGN1.1 is linked to pK7WG2D by the Gateway recombination system to achieve interference of AhPUGN1.1;

[0015] Preferably, the method for inhibiting AhPUGN1.1 by gene editing is: connecting the target sites shown in SEQ ID NO.16 to SEQ ID NO.17 to the pKSE401 gene editing vector to achieve the knockout of AhPUGN1.1.

[0016] A method for regulating peanut nodulation, comprising constructing a recombinant vector overexpressing the AhPUGN1.1 gene according to claim 1, transferring the recombinant vector into Agrobacterium rhizogenes, infecting peanuts with the Agrobacterium rhizogenes, obtaining positive peanut hairy root material after identification, and then inoculating rhizobia. The number of nodules in the obtained peanut chimeric plants is greater than the number of nodules in peanut chimeric plants that do not overexpress AhPUGN1.1.

[0017] A method for regulating peanut nodulation, comprising constructing a recombinant vector for inhibiting the AhPUGN1.1 gene according to claim 1, transferring the recombinant vector into Agrobacterium rhizogenes, infecting peanuts with Agrobacterium rhizogenes, obtaining positive peanut hairy root material after identification, and then inoculating rhizobia, so that the number of nodules in the obtained peanut chimeric plants is less than that in non-knockout and gene-edited AhPUGN1.1 peanut chimeric plants.

[0018] Compared with the existing technology, the beneficial effects of the present invention are:

[0019] The present invention found that the number of nodules increased after overexpression of AhPUGN1.1, and decreased after knockout of AhPUGN1.1, and therefore proposed the application of AhPUGN1.1 in regulating peanut nodulation.

[0020] Peanut belongs to the Dalbergoid subclass of the legume family. Its nodulation characteristics are different from those of other legumes. It is mainly infected through the "crack-entry" method. However, the signal recognition and signal transmission between the rhizobia and the host in this infection method are still blank. So far, apart from homologous cloning from model legumes, there have been no original reports on peanut nodulation and nitrogen fixation. The present invention obtained AhPUGN1.1, a key factor regulating peanut nodulation, for the first time through yeast two-hybrid screening in peanuts, and systematically studied its molecular mechanism in peanut nodulation, clarifying its specific role in regulating the occurrence and development of peanut root nodules. It provides important information and data for in-depth research in the field of genetic regulation of the unique rhizobium infection method of peanuts, and provides new ideas for molecular breeding to cultivate new peanut varieties with high nitrogen fixation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 3 shows the gene expression pattern and subcellular localization analysis of AhPUGN1.1. A shows the expression of AhPUGN1.1 in peanut roots at 0, 1, 3, 6, 12, and 24 hours after inoculation. B shows the expression of AhPUGN1.1 in roots, leaves, and nodules 14 days after inoculation. C to F show the expression pattern of proAhPUGN1.1::GUS by histochemical staining. C shows the nodule primordium, D and E show young nodules, and F shows a mature nodule. Bar = 1 mm, G, H are cross-sections of nodules expressing proAhPUGN1.1::GUS at 10 and 24 days after inoculation, scale bar = 200 μm, I is the subcellular localization of AhPUGN1.1-GFP transiently expressed in tobacco (N. benthamiana) leaves, GFP fluorescence was observed 2 days after transformation, scale bar = 70 μm, *P < 0.05; **P < 0.01; ***P < 0.001, ns indicates no statistically significant difference.

[0022] Figure 2Figure 5. AhPUGN1.1 expression in AhPUGN1.1-OE transgenic hairy roots. Figure 5. AhPUGN1.1 expression in AhPUGN1.1-OE transgenic hairy roots. Figure 5. AhPUGN1.1 expression in AhPUGN1.1-KD transgenic hairy roots. Figure 5. AhPUGN1.1 expression in AhPUGN1.1-KD transgenic hairy roots. Figure 5. AhPUGN1.1 expression in AhPUGN1.1-KD transgenic hairy roots. phenotype, scale bar = 5 mm, F is the number of nodules in empty vector and AhPUGN1.1-KD transgenic roots, G is the expression of AhPUGN1.1 in AhPUGN1.1-KD transgenic hairy roots, H is the nodule phenotype of vector control and AhPUGN1.1-KD roots, scale bar = 5 mm, I is the number of nodules in empty vector and AhPUGN1.1-KD transgenic roots, *P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no statistically significant difference.

[0023] Figure 3 Figure 5 Characteristics of AhPUGN1.1-overexpressing, AhPUGN1.1-knockout, and AhPUGN1.1-gene-edited nodules are shown. Nodules of roots: A is AhPUGN1.1-OE, B is AhPUGN1.1-KD, and C is AhPUGN1.1-KO. Scale bar = 1 cm. Fresh weight of nodules of single transgenic roots: D is AhPUGN1.1-OE, E is AhPUGN1.1-KD, and F is AhPUGN1.1-KO. G is cross-section of toluidine blue-stained nodules from AhPUGN1.1-OE, AhPUGN1.1-KD, or control roots 14 days after inoculation. Scale bar = 200 μm. *p < 0.05, **p < 0.01, ***p < 0.001. ns indicates no statistically significant difference.

[0024] Figure 4Figure 3 shows marker genes related to nodule formation and differentially expressed genes that may be regulated by AhPUGN1.1. A shows the expression of nodule-related marker genes AhCCaMK, AhHK1, AhNIN, AhSymREM, AhEFD, AhENOD40, AhCYP735A, AhCerberus, AhNPI1, AhScarN, and AhSYMRK in the roots of AhPUGN1.1-OE and the control group 28 days after inoculation. B shows the expression of nodule-related genes AhCCaMK, AhHK1, AhNIN, AhSymREM1, AhEFD, AhENOD40, AhCYP735A, AhCerberus, AhNPI1, AhScarN, and AhSYMRK in the roots of AhPUGN1.1-KD and the control group 28 days after inoculation. The transcription levels of I1, AhScarN, and AhSYMRK were set to "1" in the control group. C, E to F are heat maps showing the expression patterns of related genes, among which C is the nodule-related gene in AhPUGN1.1-KO roots and the control group, E is the cytokinin signaling pathway, F is the calcium ion signaling interaction, and D is the expression level of nodule-related genes verified by RT-qPCR. At 28 days after inoculation, the transcription levels of MtN21, EIN3, ERF3, CDPK1, NRT2.2, NUP85, FER4, ARF2, ARF4, and NSP1 were set to "1" in the roots of the empty vector control. The expression abundance of these genes has been normalized to the internal reference gene AhActin. *p<0.05, **p<0.01, ***p<0.001; n=3.

[0025] Note: In the figure, OE is the abbreviation of AhPUGN1.1-OE, and KD is the abbreviation of AhPUGN1.1-KD. DETAILED DESCRIPTION

[0026] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0027] The present invention uses overexpression of AhPUGN1.1, knockout of AhPUGN1.1 and AhPUGN1.1 gene editing to perform functional studies, mainly analyzing the effects of these treatments on the number phenotype of peanut nodules.

[0028] Table 1 Primer sequences of the present invention

[0029]

[0030] Example 1

[0031] AhPUGN1.1 gene expression pattern and subcellular localization

[0032] To investigate genes involved in nitrogen fixation in peanut nodules, researchers conducted differential analysis of genes expressed in peanut using RNA sequencing. They found several genes primarily expressed in peanut nodules, revealing their potential roles in nodule formation. Among them, a gene called PUGN1.1 (peanut-specific nodule gene 1.1) was found only in the peanut genome, with no homologous genes found in other legumes, suggesting it may be a unique gene in the peanut genome.

[0033] 1. To study the expression pattern of AhPUGN1.1 during rhizobium infection and nodule development, the present invention used qPCR to detect the transcription level of AhPUGN1.1 in leaves, roots, and nodules after inoculation with peanut rhizobium CCBAU.05117. The primers used in qPCR are shown in SEQ ID NO.14 to SEQ ID NO.15.

[0034] Our results showed that AhPUGN1.1 was mainly expressed in roots with nodules, which was consistent with the transcriptome FPKM data ( Figure 1 A). Further analysis revealed that the expression of AhPUGN1.1 increased significantly 1 hour after inoculation and reached a peak at 12 hours, indicating that AhPUGN1.1 plays a key role in nodule formation ( Figure 1 B).

[0035] 2. The present invention used peanut genomic DNA as a template and cloned the promoter using primers shown in SEQ ID NO.4 to SEQ ID NO.5. The promoter was constructed into a β-glucuronidase (GUS) reporter gene vector proAhPUGN1.1::GUS to investigate the spatial expression pattern of AhPUGN1.1 in peanut.

[0036] GUS activity in the divided cortical regions ( Figure 1 C) Nodule primordium ( Figure 1 D) bulge ( Figure 1 E) and mature nodules ( Figure 1 F). Further paraffin section analysis showed that GUS expression was detected in both the infected and nitrogen-fixing areas of rhizomes at 10 and 24 days after inoculation (dpi). Figure 1G, H). By expressing AhPUGN1.1-GFP fusion protein in tobacco leaves using primers shown in SEQ ID NO.6 to SEQ ID NO.7 and detecting under the control of CaMV 35S promoter, the present invention determined the subcellular localization of AhPUGN1.1. The results showed that the AhPUGN1.1-GFP fusion protein was localized in the cell nucleus ( Figure 1 I).

[0037] These results suggest that AhPUGN1.1 may be a nodule-specific gene that regulates nodule development in peanut infected with Rhizobium.

[0038] In summary, the expression characteristics and spatial distribution of AhPUGN1.1 reveal its important role in peanut nodule development, suggesting that this gene may serve as a regulatory factor in nodule formation, providing important clues for further study of its function.

[0039] Example 2

[0040] AhPUGN1.1 positively regulates peanut nodulation

[0041] 1. The present invention verifies the role of AhPUGN1.1 in regulating nodule formation by overexpression (OE), RNA interference (KD) technology and gene editing (KO) methods of AhPUGN1.1.

[0042] The present invention uses the cDNA of peanut nodule AhPUGN1.1 as a template and clones the CDS of AhPUGN1.1 using SEQ ID NO.2 to SEQ ID NO.3.

[0043] The overexpression method is as follows:

[0044] Primers were designed based on the CDS sequence of AhPUGN1.1 and the restriction enzyme sites of the pCAMBIA 1300 vector sequence, as shown in SEQ ID NO. 12 and SEQ ID NO. 13 in Table 1. Using peanut nodule cDNA as a template, the product obtained by PCR amplification was recovered using the Sangon SanPrep column DNA gel recovery kit.

[0045] The pCAMBIA 1300 vector and gel-recovered PCR product were digested with BamHI endonuclease. After 1 hour of digestion, the vector and fragment were recovered and subjected to an enzyme ligation reaction. The ligation product was transformed into DH5α competent cells, and the plasmid from the positive colony PCR clone was designated AhPUGN1.1-OE. The constructed AhPUGN1.1-OE plasmid was transformed into K599 competent cells, and positive colonies were selected to prepare a transformation culture with an OD600 of 0.08-0.1. In this example, OD600 was 0.1.

[0046] Healthy, uncontaminated peanut seeds were germinated in vermiculite for 3 days. A cross-shaped incision was then made along the hypocotyl, 0.2 cm below the embryonic axis. The explants were then suspended in a transformation bacterial solution for 60 minutes. The infected explants were then co-cultured at 28°C in the dark for 3 days. The co-cultured explants were then transplanted to moist vermiculite and incubated normally at 28°C for 3 days, protected from light, until roots emerged. The moisture barrier was then slowly removed. Approximately 2 weeks after transplanting to the vermiculite, chimeric seedlings with transformed roots were selected using a LUYOR-3415RG dual-wavelength portable fluorescent protein observation lamp and inoculated with rhizobia.

[0047] The RNA interference method is as follows:

[0048] The cloned AhPUGN1.1 CDS was ligated into pDONR207 via BP reaction. Positive clones were selected for plasmid extraction and ligated into pK7WG2D via LR reaction. The product of the enzymatic ligation reaction was transformed into DH5α competent cells. The plasmid of the positive clone from colony PCR was used as the AhPUGN1.1-KD construct. Subsequent steps were the same as for the overexpression method.

[0049] The gene editing methods are as follows:

[0050] sgRNA sequences were predicted using http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR. Target sites with high off-target scores and approximately 200-400 bp intervals were selected. Target sequence 1: TATGGAGACGGATCAGGGA, denoted as SEQ ID NO. 16, and target sequence 2: TATGGAGACGGATCAGGGA, denoted as SEQ ID NO. 17. Primers were designed based on the target sites (see SEQ ID NOs. 8 to 11 in Table 1). Using pCBC-DT1T2 as a template, nested PCR was performed using the designed primers to integrate the target sequences into the gene editing element. The target sequences were then ligated into the pKSE401 vector via enzyme digestion and ligation. Subsequent procedures were similar to the overexpression method. Roots that tested positive were sequenced, and the knockout and mutation-induced genes were selected for phenotypic analysis and subsequent analysis.

[0051] Compared with the control group, the expression of AhPUGN1.1 was significantly increased in the roots of AhPUGN1.1-OE, while it was significantly decreased in the roots of AhPUGN1.1-KD ( Figure 2 A, D). At 28 days after inoculation (DAI), the present invention observed that AhPUGN1.1-OE transgenic roots formed more nodules, and the fresh weight of single nodules was significantly higher than that of the control group ( Figure 2 B~C, Figure 3A~D). At the same time, the number of nodules in AhPUGN1.1-KD transgenic roots was significantly reduced, and the fresh weight of a single nodule was also lower than that in the control group ( Figure 2 E~F, Figure 3 B-E). Compared with the control group, the infected area of ​​the AhPUGN1.1-KD group was reduced and showed more thin-walled cell layers, while the AhPUGN1.1-OE group showed the opposite characteristics ( Figure 3 G).

[0052] 2. To further confirm the role of AhPUGN1.1 in peanut nodulation, the present invention used the CRISPR-Cas9 system to knock out AhPUGN1.1 in the root system.

[0053] The results showed that AhPUGN1.1 knockout (AhPUGN1.1-KO) led to a significant decrease in the number of nodules, which was significantly different from the control group ( Figure 2 GI). Transgenic hairy roots containing the empty vector (CK) formed an average of 15 nodules after inoculation, while AhPUGN1.1-KO transgenic hairy roots formed an average of only 5 nodules 28 days after inoculation (dpi) ( Figure 3 C, F).

[0054] These results indicate that AhPUGN1.1 plays a positive regulatory role in peanut nodulation.

[0055] Example 3

[0056] AhPUGN1.1 regulates nodulation by modulating the nodulation factor signaling pathway and "crack invasion"-related genes

[0057] Nodule number is primarily regulated by the nodulation factor (NF) signaling pathway, so it is important to investigate whether AhPUGN1.1 promotes nodulation through the NF signaling pathway. To this end, the present study detected the transcription levels of genes associated with the NF pathway, including AhCCaMK, AhHK1, AhNIN, AhSymREM, AhEFD, and AhENOD40.

[0058] The results show that Figure 4 As shown in Figure 3, the transcription of AhCCaMK, AhHK1, AhNIN, AhSymREM1, AhEFD, and AhENOD40 was significantly upregulated in AhPUGN1.1-OE transgenic roots, but was significantly downregulated in AhPUGN1.1-KD roots ( Figure 4A to B). In addition, genes CYP735A, Cerberus, NPI1, ScarN, and SYMRK have been reported to be associated with the "crack entry" pattern in peanut nodulation. The present invention analyzed the expression patterns of these genes in AhPUGN1.1-OE transgenic roots and AhPUGN1.1-KD roots and found that the transcription of AhCYP735A, AhNPI1, AhScarN, and AhSYMRK was significantly increased in AhPUGN1.1-OE roots, but decreased in AhPUGN1.1-KD roots ( Figure 4 A~B).

[0059] These results indicate that AhPUGN1.1 is co-expressed with NF signaling markers and genes involved in "crack entry." Therefore, modulating AhPUGN1.1 expression will affect the expression of NF signaling markers and genes involved in "crack entry," suggesting that AhPUGN1.1 may regulate the nodulation process in legumes by modulating these two signaling pathways.

[0060] In summary, AhPUGN1.1 plays an important role in regulating nodulation factor signaling pathways and may further influence the nodulation process by regulating genes involved in "crack entry." By synergistically regulating these two signaling pathways, AhPUGN1.1 may play a key role in peanut nodulation, thereby promoting the symbiotic relationship between legumes and rhizobia. This discovery provides a new perspective for understanding the function of AhPUGN1.1 in legume growth and development, and may lay a theoretical foundation for improving the nodulation ability of peanut and other legume crops.

[0061] Example 4

[0062] Comparative transcriptome analysis reveals the positive regulatory role of AhPUGN1.1 in peanut nodulation

[0063] To explore the regulatory mechanism of AhPUGN1.1 in peanut nodulation, we screened for differentially expressed genes (DEGs) between rhizobium-treated and empty vector-transformed roots 10 days after treatment. DEGs related to nodulation, such as NQYF9R (MtN21), RX34PI (EIN3), 798WTR (ERF3), 961PRY (CDPK1), T69QN8 (NRT2.2), 2KYU87 (Nup85), B2GHU0 (FER4), 9IQ583 (ARF2), 849EC4 (ARF4), and PLG903 (NSP1), were all downregulated in AhPUGN1.1-KO roots (see Figure 2). Figure 4 C). RT-qPCR experiments further verified the expression changes of these genes in AhPUGN1.1-KO roots (see Figure 4D). In-depth analysis also revealed that AhPUGN1.1-KO affected the expression of CDPK, CCaMK, CNGC and RRs family members (see Figure 4 E, F), these genes play a key role in regulating the initiation and maturation of nodule formation in legumes, involving calcium and cytokinin signaling pathways. Therefore, the present invention speculates that AhPUGN1.1 may influence the nodulation process in peanut by regulating signaling pathways related to nodule initiation and nodule number.

[0064] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0065] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0066] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. The application of AhPUGN1.1 in regulating peanut nodulation is characterized by: The nucleotide sequence of AhPUGN1.1 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The regulation is to increase the number of peanut nodules by overexpressing AhPUGN1.1; Inhibiting the expression of AhPUGN1.1 can reduce the number of peanut nodules.

3. The use according to claim 2, characterized in that The step of overexpressing the AhPUGN1.1 is as follows: connecting the CDS sequence of AhPUGN1.1 to a plant overexpression vector via a restriction endonuclease.

4. The use according to claim 3, characterized in that The plant overexpression vector is a plant overexpression vector comprising a 35S promoter or an Ubi promoter.

5. The use according to claim 3, characterized in that The plant overexpression vector is a pCAMBIA 1300 vector.

6. The use according to claim 2, characterized in that The method of inhibiting AhPUGN1.1 is either RNA interference or gene editing.

7. The use according to claim 6, characterized in that The method for inhibiting AhPUGN1.1 by RNA interference is as follows: AhPUGN1.1 is connected to pK7WG2D through the Gateway recombination system to achieve interference of AhPUGN1.

1.

8. The use according to claim 6, characterized in that The method for inhibiting AhPUGN1.1 by gene editing is as follows: the target sites shown in SEQ ID NO.16 to SEQ ID NO.17 are connected to the pKSE401 gene editing vector to achieve the knockout of AhPUGN1.

1.

9. A method for regulating peanut nodulation, characterized in that: A recombinant vector overexpressing the AhPUGN1.1 gene according to claim 1 is constructed, the recombinant vector is transferred into Agrobacterium rhizogenes, peanuts are infected with Agrobacterium rhizogenes, positive peanut hairy root material is obtained after identification, and then rhizobia are inoculated. The number of nodules in the obtained peanut chimeric plants is greater than the number of nodules in the peanut chimeric plants that do not overexpress AhPUGN1.

1.

10. A method for regulating peanut nodulation, characterized in that: A recombinant vector for inhibiting the AhPUGN1.1 gene according to claim 1 is constructed, the recombinant vector is transferred into Agrobacterium rhizogenes, peanuts are infected with Agrobacterium rhizogenes, positive peanut hairy root material is obtained after identification, and then rhizobia are inoculated. The number of nodules of the obtained peanut chimeric plants is less than that of the non-knockout and gene-edited AhPUGN1.1 peanut chimeric plants.

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