ATP synthase beta subunit gene and expression vector and application thereof

Abstract

The invention discloses an ATP synthase beta subunit gene as well as an expression vector and application thereof, and belongs to the technical field of gene engineering. The nucleotide sequence of the ATP synthase beta subunit gene is as shown in SEQ ID NO. 1. The ATP synthase beta subunit gene AhatpB and the protein AhatpB coded by the ATP synthase beta subunit gene AhatpB are cloned from peanuts for the first time, the gene CDS is inserted into an overexpression vector pCAMBIA1300S, and the obtained recombinant vector pCAMBIA1300S: AhatpB of the gene is transferred into hairy roots of a peanut variety Huayu 22, so that the number of root nodules on the hairy roots of the peanuts can be remarkably increased. The AhatpB is used for genetic engineering breeding and is introduced into a peanut variety with low-efficiency root nodules, so that peanut germplasm with efficient nodulation and nitrogen fixation can be obtained, and the AhatpB has important significance for improving the peanut variety with efficient root nodule nitrogen fixation.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to an ATP synthase β subunit gene, its expression vector, and its applications. Background Technology

[0002] Nitrogen is a major nutrient element required for plant growth and development. However, excessive application of nitrogen fertilizer can cause environmental problems such as soil compaction and degradation, and water pollution, seriously affecting the sustainable development of agricultural production in my country. Legumes fix nitrogen through symbiotic processes with rhizobia at an average annual rate of 55 million tons, accounting for approximately 55% of the total biological nitrogen fixation in Earth's terrestrial ecosystems each year. However, root nodule nitrogen fixation is a highly energy-intensive biological process; fixing one molecule of nitrogen in a legume root nodule requires the consumption of 16 molecules of ATP. Therefore, the appropriate number of root nodules is crucial for the balanced distribution of matter and energy during the symbiotic nitrogen fixation process in legumes.

[0003] Peanut (Arachis hypogaea L.) is a leguminous oilseed crop, and its nitrogen-fixing capacity through root nodules directly affects the aboveground growth of the plant and the final yield. Related studies have reported a close correlation between peanut root nodule number and pod yield. However, the molecular mechanisms regulating peanut root nodule number are currently unclear. Therefore, identifying key genes controlling peanut root nodule number and elucidating their molecular regulatory mechanisms is of great significance for improving peanut varieties with efficient root nodule nitrogen fixation.

[0004] Nodule number is a complex quantitative trait controlled by multiple genes. To date, several QTLs controlling soybean nodule number have been mapped using QTL mapping methods (Nicolás et al., 2006; Zhang Yongfang, 2008; Santos et al., 2013; Hwang et al., 2014; Shi et al., 2018; Huo et al., 2019). In peanut, studies using parental genetic populations to map nodule number QTLs have been reported: Peng et al. (2021) obtained a RIL population by crossing two nodulating peanut materials and found that non-nodulating families appeared in the offspring population. Genetic analysis showed that peanut nodule number is regulated by two independent genes, and when the two recessive alleles recombine together, the root nodule is absent. Huang et al. (2023) identified two major-effect QTLs, qPNA08 and qPNB07, for nodulation in peanuts at A08 and B07, respectively, using high-generation RIL populations and SSR marker linkage maps. However, peanut nodule number is a complex quantitative trait controlled by multiple genes, and trait investigation is extremely difficult. To date, no gene directly controlling peanut nodule number has been cloned, and the molecular mechanism regulating nodule number remains largely unclear. Therefore, elucidating the genetic basis of peanut nodule number requires innovative research strategies. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide an ATP synthase β subunit gene, its expression vector, and its applications, thereby resolving the existing lack of genes capable of effectively increasing the number of peanut root nodules.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] The first objective of this invention is to provide an ATP synthase β subunit gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0008] The beneficial effects of this invention are as follows: This invention cloned an ATP synthase β subunit gene AhatpB from peanut for the first time. The CDS of this gene was inserted into the overexpression vector pCAMBIA1300S. The resulting recombinant vector pCAMBIA1300S:AhatpB was transferred into the hairy roots of peanut variety Huayu 22, which can significantly increase the number of root nodules on peanut hairy roots.

[0009] Furthermore, the ATP synthase β subunit gene is derived from peanuts.

[0010] Furthermore, the peanut variety is Huayu 22.

[0011] A second objective of this invention is to provide a protein encoded by the ATP synthase β subunit gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0012] A third objective of this invention is to provide an expression vector for an ATP synthase β subunit gene, comprising the aforementioned ATP synthase β subunit gene.

[0013] Furthermore, the starting vector used to construct the expression vector of the ATP synthase β subunit gene was pCAMBIA1300S.

[0014] Furthermore, the expression vector of the ATP synthase β subunit gene was prepared by inserting the ATP synthase β subunit gene between the BamHI and KpnI restriction sites of pCAMBIA1300S.

[0015] A fourth objective of this invention is to provide an RNAi expression vector for the ATP synthase β subunit gene, comprising a portion of the ATP synthase β subunit gene as described in claim 1.

[0016] Furthermore, the nucleotide sequence of a portion of the fragment is shown in SEQ ID NO.3.

[0017] Furthermore, the RNAi expression vector for the ATP synthase β subunit gene was prepared by inserting a partial fragment into the RNAi vector pBWA(V)HS.

[0018] The fifth objective of this invention is to provide the application of the above-mentioned ATP synthase β subunit gene or expression vector of ATP synthase β subunit gene or RNAi expression vector of ATP synthase β subunit gene in the construction of efficient nodulation and nitrogen fixation peanut varieties.

[0019] The beneficial effects of this invention are as follows: By constructing the ATP synthase β subunit gene into an expression vector and transforming it into peanuts, this invention can effectively increase the number of root nodules on peanut hairy roots. When applied to genetic engineering breeding, it can obtain peanut germplasm with efficient nodulation and nitrogen fixation.

[0020] The present invention has the following beneficial effects:

[0021] This invention marks the first cloning of an ATP synthase β subunit gene, AhatpB, and its encoded protein, AhatpB, from peanut. The CDS of this gene was inserted into the overexpression vector pCAMBIA1300S, and the resulting recombinant vector pCAMBIA1300S:AhatpB was transferred into the hairy roots of the peanut variety Huayu 22, significantly increasing the number of root nodules. Using AhatpB in genetic engineering breeding, and introducing it into peanut varieties with low-efficiency root nodule formation, can yield peanut germplasm with high-efficiency nodule nitrogen fixation, which is of great significance for improving peanut varieties with high-efficiency root nodule nitrogen fixation. Attached Figure Description

[0022] Figure 1 The image shows the pCAMBIA1300S:AhatpB overexpression vector in Example 2;

[0023] Figure 2 This is a map of the AhatpB-RNAi recombinant vector from Example 2;

[0024] Figure 3 The image shows the results of detecting the number of root nodules in peanut hairy roots when peanuts were transformed and infected with different vectors in Example 3. Among them, (A) is the empty pCAMBIA1300S vector group, (B) is the pCAMBIA1300S:AhatpB vector group, (C) is the empty pBWA(V)HS vector group, (D) is the AhatpB-RNAi expression vector group, (E) is a comparison of the number of root nodules in hairy roots between the empty pCAMBIA1300S vector group and the pCAMBIA1300S:AhatpB vector group, and (F) is a comparison of the number of root nodules in hairy roots between the empty pBWA(V)HS vector group and the AhatpB-RNAi expression vector group. Detailed Implementation

[0025] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0026] Example 1: Cloning of candidate genes regulating peanut root nodule number

[0027] In previous studies, our research group used GWAS to identify a QTL locus qNN12 on chromosome 12 that is associated with peanut nodule number. Within this locus region, we screened for the candidate gene Arahy.5XLW1T, which regulates peanut nodule number. This gene encodes the β subunit of ATP synthase and has two typical conserved domains, atpB and atpE, and is therefore named AhatpB.

[0028] Using cDNA from Huayu 22 as a template, PCR amplification was performed using primer pairs AhatpB-F: ATGAGAATAAATCCTACCACTTCTGGTCC (SEQ ID NO.4) and AhatpB-R: TTATTGCGAA AGCATATTGATAGCCTCTAC (SEQ ID NO.5) (Table 1). The PCR amplification program was as follows: (1) 98℃ pre-denaturation for 30 seconds; (2) 98℃ denaturation for 10 seconds, 60℃ annealing for 5 seconds, and 72℃ extension for 1 min, this step was repeated 35 times; (3) 72℃ extension for 1 min. After the PCR product was recovered and digested with enzymes (Table 2), it was ligated into the T vector and sequenced.

[0029] Table 1 PCR reaction system

[0030]

[0031] Table 2 Enzyme digestion system

[0032]

[0033]

[0034] The specific nucleotide sequence of the AhatpB gene is shown in SEQ ID NO.1.

[0035]

[0036] The amino acid sequence of the protein AhatpB encoded by the AhatpB gene is shown in SEQ ID NO.2.

[0037] SEQ ID NO. 2: MRINPTTSGPEISVLEKKNLGRIDQIIGPVLDVAFPPG.

[0038] Example 2: Construction of AhatpB gene overexpression vector

[0039] (1) Construction of AhatpB gene overexpression vector

[0040] Primers were designed based on the Arahy.5XLW1T gene sequence from the Peanut Base database.

[0041] The primer sequences are shown below:

[0042] 35S::AhatpB-OE-F: AGCTTTCGCGAGCTCGGTACCATGAGAATAAAT CCTACCACTTCTGGTCC (SEQ ID NO. 6);

[0043] 35S::AhatpB-OE-R: AGGTCGACTCTAGAGGATCCCTTATTGCGAAAGC ATATTGATAGCCTCTAC (SEQ ID NO. 7).

[0044] Using the cDNA of Huayu 22 as a template, PCR amplification was performed using primer pairs 35S::AhatpB-OE-F and 35S::AhatpB-OE-R (PCR conditions and reaction system were the same as in Example 1), and the amplified fragment was recovered.

[0045] The amplification product was double-digested with BamHI and KpnI (digestion system same as in Example 1). The digested product was then inserted into the double-digested vector pCAMBIA1300S with BamHI and KpnI, placing Ahatp B at the multiple cloning site after the 35S promoter. Thus, the target gene AhatpB was cloned downstream of the strong 35S promoter, obtaining the recombinant expression vector pCAMBIA1300S:AhATPb. Figure 1 As shown in the figure. Sequencing verification confirmed that the vector was successfully constructed.

[0046] (2) Construction of AhatpB-RNAi expression vector

[0047] To further verify gene function, we inserted a 250bp fragment of the AhatpB gene (sequence shown in SEQ ID NO.3) into the RNAi vector pBWA(V)HS to construct the recombinant vector AhatpB-RNAi. Using cDNA from Huayu 22 as a template, the target fragment of the AhatpB gene was amplified and inserted at tgtF using primer pairs E6871_0C1(+): cagtGGT CTCacaacgcccctgctacgacatttgcac (SEQ ID NO.8) and E6871_0C1(-): cgatGG TCTCacaggcagttaagcgatcctcttcggataattcg (SEQ ID NO.9). The target fragment tgtF of the AhatpB gene was amplified using primer pairs E6871_2C1(+): cagtGGTCTCagggccagttaagcgatcctcttcggataattcg (SEQ ID NO.10) and E6871_2C1(-): cagtGGTCTCatacagcccctgctacgacatttgcac (SEQ ID NO.11). R; then, the target fragment of the candidate gene is digested and ligated with the vector (Table 3).

[0048] Table 3 Enzyme digestion and ligation system

[0049]

[0050] Table 4 Enzyme digestion and ligation reaction conditions

[0051]

[0052] The ligation product was transformed into competent DH5α cells. Single clones were picked and identified by PCR using primer F: tTCATTTGGAGAGAACACGGGggac (SEQ ID NO.12) and primer R: cagtGGTCTCatacagcccctgctacgacatttgcac (SEQ ID NO.13). Positive single clones were confirmed by sequencing, indicating successful vector construction.

[0053] SEQ ID NO.3:GCCCCTGCTACGACATTTGCACATTTGGATGCTACG ACCGTACTATCAAGAGGATTGGCTGCCAAAGGTATCTATCCAGCTGTAGATCCTTTAGATTCAACGTCAACTATGCTCCAACCTCGGATTGTTGGTGAAGAACATTATGAAACTGCGCAAAGAGTTAAACAAACTTTACAGCATTACAAAGAACTTCAGGACATTATAGCTATCCTTGGGTTGGACGAATTATCCGAAGAGGATCGCTTAACTG.

[0054] Example 3: Functional analysis of the AhatpB gene regulating the number of peanut root nodules

[0055] The hairy root transient transformation method is a reliable and rapid method for identifying gene function in roots. This invention utilizes this method to transform pCAMBIA1300S:AhatpB plasmid DNA into Agrobacterium rhizogenes. Using Agrobacterium-mediated genetic transformation, candidate genes are transformed into peanut roots. Rhizobia are then inoculated, and changes in root nodule number are observed and statistically analyzed to clarify the biological function of candidate genes in regulating peanut root nodules.

[0056] I. pCAMBIA1300S:AhatpB vector DNA was transformed into Agrobacterium rhizogenes and used to infect peanuts.

[0057] The peanut variety used in the experiment was Huayu 22, donated by the Shandong Peanut Research Institute. Peanut seeds were first surface-sterilized with 10% sodium hypochlorite for 10 minutes, then soaked overnight. After the seeds showed signs of sprouting, they were removed, rinsed with deionized water, and sown in peat moss in the dark for germination. Seedlings were cultured at 26℃ for 7 days to obtain peanut seedlings with two leaves and one bud, which served as recipients for Agrobacterium rooting bacterium infection. During the formation of hairy roots, the peanuts inoculated with Agrobacterium were cultured in a rooting nutrient solution (composition shown in Table 5). After the hairy roots formed, they were cultured in a low-nitrogen nutrient solution (composition shown in Table 6).

[0058] Table 5 Composition of Rooting Nutrient Solution

[0059]

[0060] Table 6 Composition of Low-Nitrogen Nutrient Solution

[0061]

[0062] The nutrient solution was changed every three days, and the pH was maintained at around 5.8. Once the fine roots had emerged, inoculation with rhizobia was initiated. Peanut cultivation was conducted in an artificial climate chamber with a photoperiod / dark period of 16h / 8h (light / dark), corresponding to temperatures of 26℃ / 22℃ (day / night), and a relative humidity of 60%. The peanut rhizobia strain (Bradyrhizobium) was isolated and preserved in the laboratory.

[0063] Take 5 μL of vector (pCAMBIA1300S:AhatpB vector or AhatpB-RNAi expression vector obtained in Example 2) and gently mix it with 100 μL of Agrobacterium rhizogenes (CC96315 K599, Tolobio) competent cells. Incubate on ice for 10 min, then freeze in liquid nitrogen for 5 min, followed by a 37°C water bath for 5 min, and an ice bath for 5 min. Add 900 μL of YEB medium to Agrobacterium, and culture at 28°C with shaking at 220 r / min for 3 h. Centrifuge at 6000 rpm for 1 min, collect the bacterial solution, and spread it evenly on YEB solid medium containing kanamycin (50 mg / L). Culture at 28°C for 2-3 days to obtain successfully transformed strains.

[0064] During the germination and seedling development of peanut seeds, Agrobacterium containing the expression vector was activated using YEB solid medium. Before inoculation, bacterial cells were collected using a disposable spreader, and a small amount of 0.5 mol / L acetylsyl syringone was added to the bacterial cells and mixed well to obtain a bacterial suspension. When peanut seedlings reach the stage of two leaves and one bud (generally 7 days after sowing), remove the seedlings and make 3-4 vertical ring cuts on the hypocotyl using a scalpel dipped in disinfectant. Then, inject the bacterial solution into the ring cuts using a disposable syringe. Next, inoculate the seedlings into vermiculite irrigated with rooting nutrient solution and cultivate for 7-10 days until peanut hairy roots form. Afterward, irrigate with rooting nutrient solution mixed with 50 mg / L hygromycin every 5 days for 3-4 treatments to inhibit the growth of non-GMO hairy roots. After 15-20 days, when the peanut hairy roots reach 5-8 cm in length, remove the taproot, leaving only one root at the ring cut. Plant the infected peanuts in soil irrigated with low-nitrogen nutrient solution (substrate: vermiculite: quartz sand: soil = 1:1:1:1, mass ratio). After 2-3 days of seedling establishment, activate the peanut rhizobium on solid YMA medium in advance. Select single colonies and propagate them on liquid YMA medium to obtain a peanut rhizobium culture solution. Then, use the peanut rhizobium culture solution (approximately 1×10⁻⁶) 7 Irrigate with a low-nitrogen nutrient solution (1 cell / mL) for 24-28 days until mature root nodules are formed.

[0065] II. Results Analysis

[0066] The pCAMBIA1300S:AhatpB vector and the AhatpB-RNAi expression vector obtained in Example 2 were used to transform Agrobacterium rhizogenes and infect peanuts. The number of root nodules formed on the peanut rhizogenes was detected by using their respective blank vectors as blank controls.

[0067] The results are as follows Figure 3 As shown, the results revealed that, compared with the empty vector, the number of hairy root nodules transformed with the pCAMBIA1300S:AhatpB vector significantly increased by 60.0% (e.g., ...). Figure 3 As shown in Figures (A), (B), and (E), the number of hairy root nodules transformed with the AhatpB-RNAi expression vector was significantly reduced (e.g., ...). Figure 3 Figures (C), (D), and (F) show that the AhatpB gene can effectively regulate peanut nodule formation and increase the number of nodules on peanut hairy roots, which is of great significance for improving peanut varieties with efficient nodule nitrogen fixation.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An ATP synthase β subunit gene, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

1.

2. A protein encoded by the ATP synthase β subunit gene according to claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. An expression vector for an ATP synthase β subunit gene, characterized in that: The invention comprises the ATP synthase β subunit gene according to claim 1.

4. The expression vector of the ATP synthase β subunit gene according to claim 3, characterized in that The starting vector used to construct the expression vector of ATP synthase β subunit gene is pCAMBIA1300S.

5. The expression vector of the ATP synthase β subunit gene according to claim 4, characterized in that The expression vector of the ATP synthase beta subunit gene is prepared by inserting the ATP synthase beta subunit gene into the BamHI and KpnI restriction sites of pCAMBIA1300S.

6. An RNAi expression vector for ATP synthase β subunit gene, characterized in that: The invention comprises a partial fragment of the ATP synthase β subunit gene according to claim 1.

7. The RNAi expression vector of the ATP synthase β subunit gene according to claim 6, characterized in that: The nucleotide sequence of the partial fragment is shown in SEQ ID NO.

3.

8. The RNAi expression vector of the ATP synthase β subunit gene according to claim 7, characterized in that: The RNAi expression vector of the ATP synthase beta subunit gene is prepared by inserting the partial fragment into the RNAi vector pBWA(V)HS.

9. Use of the ATP synthase β subunit gene according to claim 1, the expression vector of the ATP synthase β subunit gene according to any one of claims 4 to 6, or the RNAi expression vector of the ATP synthase β subunit gene according to any one of claims 6 to 8 in constructing a high-efficiency nodulation and nitrogen-fixing peanut variety.

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