Application of ahpgip1 gene in regulating peanut nodule number

Abstract

The application provides application of an AhPGIP1 gene in regulating peanut nodule number, and belongs to the technical field of genetic engineering. The AhPGIP1 gene negatively regulates peanut nodule number; the amino acid sequence of the protein encoded by the AhPGIP1 gene is shown in SEQ ID NO. 2. The application identifies an AhPGIP1 gene that negatively regulates peanut nodule number. Research shows that the nodule number on peanut hairy roots of a peanut strain overexpressing the AhPGIP1 gene is significantly reduced, and interference with the AhPGIP1 gene significantly increases the nodule number on peanut hairy roots, proving that AhPGIP1 negatively regulates the nodule number of peanuts. The application can provide an effective theoretical basis and gene resources for accelerating the cultivation of peanut high-efficiency nitrogen fixation varieties through molecular technical means.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the AhPGIP1 gene in regulating the number of peanut nodules. Background Technology

[0002] peanut( Arachis hypogaea Peanuts (L.) are an important economic and oilseed crop in my country. However, their production relies heavily on nitrogen fertilizer, which is unsustainable and often results in increased yield without increased nitrogen input. Therefore, reducing reliance on nitrogen fertilizer in peanut production is crucial for its green and sustainable development.

[0003] Nodule fixation is the primary means of nitrogen supply in peanuts, contributing up to 50% to nitrogen accumulation. Among the nitrogen-fixing factors, the number of nodules is one of the key factors limiting the nitrogen-fixing capacity of peanut nodules. Therefore, identifying genes that regulate peanut nodule phenotypes and elucidating their mechanisms of action can provide a theoretical basis for precision and intelligent peanut breeding, improving peanut nodule nitrogen-fixing capacity, reducing nitrogen while increasing efficiency, and achieving sustainable agricultural development. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the AhPGIP1 gene in regulating the number of peanut nodules.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of the AhPGIP1 gene in regulating peanut nodule number, wherein the AhPGIP1 gene negatively regulates peanut nodule number; the amino acid sequence of the protein encoded by the AhPGIP1 gene is shown in SEQ ID NO.2.

[0006] Preferably, the nucleotide sequence of the AhPGIP1 gene is shown in SEQ ID NO.1.

[0007] This invention provides a method for increasing the number of peanut nodules by interfering with the expression of the AhPGIP1 gene in peanuts, wherein the amino acid sequence of the protein encoded by the AhPGIP1 gene is shown in SEQ ID NO.2.

[0008] Preferably, the nucleotide sequence of the AhPGIP1 gene is shown in SEQ ID NO.1.

[0009] Preferably, the method for interfering with AhPGIP1 gene expression in peanuts is as follows: constructing a plant expression vector containing an AhPGIP1 gene interference fragment and transforming it into peanut plants using Agrobacterium-mediated transformation.

[0010] Preferably, the nucleotide sequence of the AhPGIP1 gene interference fragment is shown in SEQ ID NO.5.

[0011] The present invention provides a peanut with increased nodule number, wherein the expression of the AhPGIP1 gene in the peanut is suppressed, and the amino acid sequence of the protein encoded by the AhPGIP1 gene is shown in SEQ ID NO.2.

[0012] Compared with the prior art, the present invention has the following beneficial effects: This invention identified an AhPGIP1 gene that negatively regulates nodule number in peanuts. Studies showed that overexpression of the AhPGIP1 gene significantly reduced the number of root nodules on the hairy roots of peanut lines, while interference with the AhPGIP1 gene significantly increased the number of root nodules on the hairy roots, demonstrating that AhPGIP1 negatively regulates nodule number in peanuts. This invention provides a sound theoretical basis and genetic resources for accelerating the cultivation of efficient nitrogen-fixing peanut varieties using molecular techniques.

[0013]

[0014] SEQ ID NO.2: MASTSTISMFLLPIVLLIWVSPAVLSEKCSPHEKKILLQVKQEFGNPSMLSSWNASTDCCTSAWEGIECTTEKPYRVSQIELHGLDLPGPVDLPPSLFNLVDLDELLIFEMPNLKGQIPPQISNLKKLTVLYLFSTNVSGPIPESLAQIKTLTNINLAGNNLSGPLPH LLPSLPSIRVIFADENRISGPIPESYGSFASKSLVTLTLSHNMLSGKIPAALKGLDAELLDLSWNKLEGDGSVLFGAEKKTEEITLAGNMLSFDIGKVEFGRNITRLNLKHNRIYGKLPEQLTQLKQLTRFNVSYNQLCGPIPQGGRLQGYFAIDASSFAHNKCLCGSPLPPCK Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0016] Figure 1 The structural map of the expression vector 35S::AhPGIP1 for overexpressing AhPGIP1; Figure 2 The structural map of the expression vector AhPGIP1-RNAi that interferes with AhPGIP1 expression; Figure 3 The results show the statistical results of the number of root nodules in peanut hairy roots after transformation with different vectors. Detailed Implementation

[0017] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional experimental methods. Unless otherwise specified, the reagents used in the following embodiments were purchased from conventional biochemical reagent companies.

[0018] Example 1: Cloning of the AhPGIP1 gene and construction of an overexpression vector

[0019] 1. Cloning of the AhPGIP1 gene

[0020] Using peanut variety HY22 as gene cloning material, total RNA was extracted from root nodules and reverse transcribed into cDNA first strand as a PCR amplification template. Cloning primers were designed based on the AhPGIP1 gene (nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence encoding the protein as shown in SEQ ID NO.2). The target fragment was amplified using Novizan high-fidelity enzyme 2 × Phanta FlashMaster Mix (P520). The reaction system is shown in Table 1, and the reaction procedure is shown in Table 2. After the reaction, the DNA was purified using the Novizan FastPure Gel DNA Extraction Mini Kit (DC301). The designed cloning primers are as follows: Forward primer: 5'-ATGGCAAGCACCAGTA-3' (SEQ ID NO.3) Reverse primer: 5'-TCACTTGCATGGAGGGA-3' (SEQ ID NO.4) Table 1 PCR reaction system

[0021] Table 2 PCR reaction procedure

[0022] 2. Construction of AhPGIP1 gene overexpression vector

[0023] Using restriction enzymes Kpn I and Bam The pCAMBIA1300S plasmid (pCAMBIA1300-35S-EGFP plasmid) was double-digested with HI, and then ligated with the homologous recombination method to the amplified and purified PCR product with added homologous arms. The ligation product was transformed into E. coli DH5α competent cells and cultured overnight at 37 °C. Single colonies were picked and identified by colony PCR. Positive colonies were selected to obtain the expression vector 35S::AhPGIP1 overexpressing AhPGIP1 (structural diagram shown). Figure 1 (As shown).

[0024] The 35S::AhPGIP1 was further transformed into Agrobacterium rhizogenes K599 competent cells. Single clones were picked and identified by Agrobacterium culture PCR. The reaction system and procedure are shown in Tables 1 and 2, and the primers used are shown in SEQ ID NO. 3 and SEQ ID NO. 4. The correctly PCR-converted bacterial cultures were retained for subsequent peanut hairy root transformation.

[0025] Example 2: Design of AhPGIP1 gene interference fragment and construction of interference vector

[0026] 1. Design of AhPGIP1 gene interference fragment

[0027] An interference fragment (SEQ ID NO.5) and its amplification primers (SEQ ID NO.6 and SEQ ID NO.7) were designed based on the AhPGIP1 gene sequence.

[0028] The interference fragment is: TCCATGCTCTCTTCCTGGAACGCTTCCACCGACTGCTGCACCAGCGCCTGGGAAGGCATCGAATGCACCACCGAGAAGCCTTACCGTGTTAGCCAAATAGAGCTACATGGGCTGGACCTCCCCGGCCCAGTTGACCTCCCTCCCTCCCTATTTAACCTCGTTGATCTTGATGAGCTCTTAATTTTTGAAATGCCCAA CCTAAAAGGCCAAATTCCTCCCCAGATCTCAAATCTCAAAAAACTTACAGTCCTTTACTTATTCAGCACCAATGTCCAGGCCCAATACCAGAATCTCTAGCCCAAATCAAGACCCTCACGAACATTAACCTCGCCGGCAACAATCTCTCCGGCCCACTCCCTCATCTCCTCCCCTCACTACCCAGCATCAGGGTGATCT (SEQ ID NO.5)

[0029] Forward amplification primers for the interfering fragment (SEQ ID NO.6): 5'-ggggacaagtttgtacaaaaaagcaggctgcTCCATGCTCTCTTCCTG- 3' Reverse amplification primers for the interfering fragment (SEQ ID NO.7): 5'-ggggaccactttgtacaagaaagctgggtgAGATCACCCTGATGCTG- 3' 2. Construction of an expression vector interfering with the AhPGIP1 gene Using primers as shown in SEQ ID NO. 6 and SEQ ID NO. 7, PCR amplification was performed according to the PCR reaction systems and procedures shown in Tables 1 and 2. After the amplified fragments were recovered and confirmed to be correct by electrophoresis, the PCR products were recombined in vitro with the Phellsgate4 plasmid according to the reaction system shown in Table 3 to obtain the expression vector AhPGIP1-RNAi that interferes with AhPGIP1 expression (structural map shown in Table 3). Figure 2 (As shown).

[0030] Table 3 Reaction system of the interference carrier

[0031] AhPGIP1-RNAi was further transformed into Agrobacterium rhizogenes K599 competent cells. Single colonies were picked and identified by Agrobacterium culture PCR. The reaction system and procedure are shown in Tables 1 and 2, and the primers used are shown in SEQ ID NO. 6 and SEQ ID NO. 7. Colonies initially identified as positive by PCR were extracted and their plasmids were digested with XhoI and XbaI enzymes, respectively. If the digested band was 200-300 bp larger than the target band, the ligation was successful. The correctly PCR-positive bacterial culture was retained for subsequent peanut hairy root transformation.

[0032] Example 3: Regulation of peanut nodule number by the AhPGIP1 gene

[0033] To clarify the function of the AhPGIP1 gene, transgenic hairy roots were obtained and molecularly identified using Agrobacterium rhizogenes-mediated transformation of peanut hairy roots. The tested peanut variety was HY22, and two-leaf-one-heart peanut seedlings were obtained 7 days after sowing as recipients for Agrobacterium rhizogenes infection.

[0034] Obtaining the infecting bacterial culture: The *Agrobacterium rhizogenes* K599 strains containing 35S::AhPGIP1, AhPGIP1-RNAi, and *Agrobacterium rhizogenes* K599 strains transformed with EV-O overexpression vector and EV-R interference vector were activated in YEB solid medium containing kanamycin resistance (50 µg / mL). Single colonies were obtained by incubation at 28 ℃ for 48 h. Each colony was then transferred to 1 mL of YEB liquid medium containing kanamycin resistance for propagation. After overnight incubation, the cells were centrifuged, the supernatant was discarded, and the cells were evenly spread onto YEB solid medium containing kanamycin resistance. Once a bacterial film formed on the medium, the cells were collected, and an appropriate amount of acetylsyleugenone (0.5 mol / L) was added. The mixture was then allowed to stand for 2 h for later use.

[0035] Using a disposable syringe, the bacterial solution was drawn up and the hypocotyl of the peanut seedling was scratched while the syringe was pushed to allow the bacterial solution to soak into the wound on the hypocotyl. The seedlings were then left at room temperature for 1 hour. The infected peanut seedlings were planted in a substrate (vermiculite:quartz sand:soil = 1:1:1, v / v / v). Two weeks later, the taproot was removed, leaving only one root at the infection site. The slow-growing rhizobium of peanut was propagated in advance on liquid YMA medium. The rhizobium solution was dissolved in a low-nitrogen nutrient solution (formulation shown in Table 4) and the plants were irrigated. Mature root nodules formed after 21 days. Positive transgenic hairy roots of peanut were identified, and the phenotype of the transgenic hairy roots was statistically analyzed.

[0036] Table 4 Composition of Low-Nitrogen Nutrient Solution

[0037] The expression of 35S::AhPGIP1 in hairy roots was identified using specific primers as shown in SEQ ID NO. 8 and SEQ ID NO. 9; the expression of AhPGIP1-RNAi in hairy roots was identified using specific primers as shown in SEQ ID NO. 10 and SEQ ID NO. 11. The reaction system and procedure for PCR identification are shown in Tables 1 and 2.

[0038] Forward primer: 5'-ATTTGTGTACGCCCGACAGT-3' (SEQ ID NO.8)

[0039] Reverse primer: 5'-CGGGGATTCCCAATACGAGG-3' (SEQ ID NO.9)

[0040] Forward primer: 5'-AAAAGCGGCCATTTTCCACC-3' (SEQ ID NO.10)

[0041] Reverse primer: 5'- GACCACCAAGCGAAACATCG-3' (SEQ ID NO.11)

[0042] Figure 3 The table shows the statistical results of root nodule numbers in peanut hairy roots transformed with different vectors. (a)–(d) show peanut hairy roots transformed with EV-O overexpression empty vector, 35S::AhPGIP1 vector, EV-R interference empty vector, and AhPGIP1-RNAi vector, respectively, with a scale bar of 1 cm. (e) shows the statistical results of root nodule numbers in hairy roots transformed with EV-O overexpression empty vector and 35S::AhPGIP1 vector. (f) shows the statistical results of root nodule numbers in hairy roots transformed with EV-R interference empty vector and AhPGIP1-RNAi vector. It can be seen that overexpression of the AhPGIP1 gene significantly reduces the number of root nodules in peanut hairy roots, while interference with AhPGIP1 gene expression significantly reduces the number of root nodules in peanut hairy roots, indicating that the AhPGIP1 gene can negatively regulate peanut nodule formation.

[0043] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of the AhPGIP1 gene in regulating peanut nodule number, characterized in that, The AhPGIP1 gene negatively regulates the number of peanut nodules; the amino acid sequence of the protein encoded by the AhPGIP1 gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the AhPGIP1 gene is shown in SEQ ID NO.

1.

3. A method for increasing peanut nodule number by interfering with the expression of the AhPGIP1 gene in peanuts, characterized in that, The amino acid sequence of the protein encoded by the AhPGIP1 gene is shown in SEQ ID NO.

2.

4. The method according to claim 3, characterized in that, The nucleotide sequence of the AhPGIP1 gene is shown in SEQ ID NO.

1.

5. The method according to claim 3, characterized in that, The method for interfering with AhPGIP1 gene expression in peanuts is as follows: construct a plant expression vector containing an AhPGIP1 gene interference fragment, and then transform it into peanut plants using Agrobacterium-mediated transformation.

6. The method according to claim 5, characterized in that, The nucleotide sequence of the AhPGIP1 gene interference fragment is shown in SEQ ID NO.

5.

7. A peanut with increased nodule count, characterized in that, The expression of the AhPGIP1 gene in the peanut was suppressed, and the amino acid sequence of the protein encoded by the AhPGIP1 gene is shown in SEQ ID NO.2.

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