Application of LjPUB7 protein to regulation and control of plant root nodule nitrogen fixation efficiency and / or regulation and control of plant yield

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of LjPUB7 protein to regulation and control of plant root nodule nitrogen fixation efficiency and / or regulation and control of plant yield. According to the present invention, the LjPUB7 gene is knocked out through the CRISPR-CAS9 technology, and the LjPUB7 mutant plant is obtained through the lotus corniculatus stable transformation technology; after inoculation of rhizobium, compared with a wild type, an infection line of an LjPUB7 gene mutant plant is remarkably improved, which indicates that the LjPUB7 gene regulates and controls development of root nodules; results of nodulation experiments show that after the LjPUB7 is knocked out, the number of root nodules and the density of the root nodules can be remarkably increased, and it shows that the LjPUB7 gene and the protein expressed by the LjPUB7 gene regulate and control the formation of early-stage root nodules and the nitrogen fixation efficiency of leguminous plants. By inhibiting the expression of the gene, the nitrogen fixation efficiency of the leguminous plants can be improved, so that the biomass and the yield of the plants are increased, and the gene has an application prospect in the research of a leguminous crop nitrogen fixation mechanism and agricultural production.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene editing, and in particular relates to the application of LjPUB7 ​​protein in regulating the nitrogen fixation efficiency of plant nodules and / or regulating plant yield. Background Art

[0002] Nitrogen resources in nature are very abundant, mainly in the form of gas (N2) in the atmosphere, but cannot be directly absorbed and utilized by plants and animals. Nitrogen is one of the main limiting factors for crop yields, so sufficient nitrogen sources play an important role in ensuring and improving human living standards. Humans can use chemical synthesis methods to synthesize industrial nitrogen fertilizers, breaking through the bottleneck of low crop yields. However, long-term reliance on chemical synthesis has exposed the defects of industrial nitrogen fertilizers. The energy utilization rate is low and the cost is high. The synthesis of nitrogen fertilizers is accompanied by a large amount of greenhouse gases, and the utilization rate of industrial nitrogen fertilizers by crops is relatively low. The residual nitrogen fertilizers are lost to the water body and other environments, leading to a series of problems such as red tides in large areas, changes in soil structure, and changes in bacterial species. Therefore, the use of genetic crop improvement technology and genetic engineering technology can not only effectively reduce the use of chemical fertilizers, but also help to increase the yield of leguminous crops.

[0003] In the long evolutionary process of nature, legumes can establish a symbiotic relationship with rhizobia. Symbiotic nitrogen fixation in root nodules is the main way of biological nitrogen fixation, which is environmentally friendly, economical, and efficient. Rhizobia use nitrogenase in their own cells to synthesize ammonia, which is absorbed and utilized by plants through nitrogen metabolism and other methods, while the host plant provides a carbon source for nitrogen-fixing bacteria, and the two coexist. Legumes are important sources of food crops and feed, and are also the main raw materials for edible protein for animals and humans. They have important nutritional value and economic benefits. Lotus japonicus is a model legume. It is of great scientific research significance to identify the functional genes in the symbiotic nodulation and nitrogen fixation of Lotus japonicus and explore the specific mechanism in the symbiotic nitrogen fixation signaling pathway. It has very important practical application value for increasing the number of nodules and enhancing the nitrogen fixation efficiency of legumes in agriculture, and also provides a possibility for reducing the use of nitrogen fertilizers, increasing the yield of legumes, and improving the quality of legumes in agricultural production.

[0004] Currently, regarding the application research of PUB7 gene in Lotus japonicus, there is no report on the role of LjPUB7 ​​protein in regulating nitrogen fixation in plant nodules. Summary of the invention

[0005] In order to solve the above problems, the present invention provides the use of LjPUB7 ​​protein in regulating the nitrogen fixation efficiency of plant nodules and / or regulating plant yield. The LjPUB7 ​​protein of the present invention can regulate the nitrogen fixation efficiency of plant nodules, especially the number of nodules, and has broad application prospects in leguminous crops.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a LjPUB7 ​​protein for regulating the nitrogen fixation efficiency of plant nodules and / or regulating plant yield. The amino acid sequence of the LjPUB7 ​​protein is shown in SEQ ID NO.1.

[0008] The present invention also provides a LjPUB7 ​​gene encoding the LjPUB7 ​​protein described in the above technical solution, and the nucleotide sequence of the LjPUB7 ​​gene is shown in SEQ ID NO.2.

[0009] The present invention also relates to biological materials related thereto, wherein the biological materials include one or more of the following: a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the LjPUB7 ​​gene.

[0010] The present invention also provides the use of the LjPUB7 ​​protein, LjPUB7 ​​gene or biological materials related thereto described in the above technical solution in regulating the nitrogen fixation efficiency of plant nodules and / or regulating plant yield.

[0011] The regulation of plant nodule nitrogen fixation includes one or more of the following aspects (1) to 4):

[0012] (1) Regulate plant nodule development;

[0013] (2) Regulate the expression level of early nodulation genes in plants;

[0014] (3) Regulate the number of plant nodules;

[0015] (4) Regulate nitrogen fixation efficiency in plant nodules;

[0016] Preferably, the regulating plant nodule development comprises: negatively regulating the expression of the LjPUB7 ​​gene to increase the number of infection points and infection lines of plant nodules.

[0017] Preferably, the regulating the expression level of the plant early nodulation gene comprises: negatively regulating the expression of the LjPUB7 ​​gene to increase the expression level of the early nodulation gene.

[0018] Preferably, the regulating the number of plant nodules comprises: negatively regulating the expression of the LjPUB7 ​​gene to increase the number of nodules of the regulated plant.

[0019] Preferably, the regulating the nitrogen fixation efficiency of plant nodules comprises: improving the nitrogen fixation efficiency of plant nodules by negatively regulating the expression of the LjPUB7 ​​gene.

[0020] Preferably, the regulating plant yield comprises: increasing plant yield by negatively regulating the expression of LjPUB7 ​​gene.

[0021] The present invention also provides the use of the LjPUB7 ​​protein described in the above technical solution or the LjPUB7 ​​gene described in the above technical solution in cultivating transgenic plants with improved nitrogen fixation efficiency and / or improved plant yield.

[0022] Preferably, the plant comprises a plant from the family Leguminosae.

[0023] Preferably, the leguminous plants include one or more of soybean, Lotus japonica and alfalfa.

[0024] Beneficial effects:

[0025] The present invention provides a LjPUB7 ​​protein for regulating the nitrogen fixation efficiency of plant nodules and / or regulating plant yield, and the amino acid sequence of the LjPUB7 ​​protein is shown in SEQ ID NO.1. The present invention utilizes CRISPR-CAS9 technology to knock out the LjPUB7 ​​gene, and quickly obtains transgenic plants through transgenic engineering technology. Through rhizobium inoculation experiments, the results show that inhibiting the expression of the LjPUB7 ​​gene can significantly increase the number of plant nodules, and a substantial technical effect has been achieved. It shows that the LjPUB7 ​​gene and the LjPUB7 ​​protein expressed by it have important biological functions in regulating the nitrogen fixation efficiency of plant nodules, and can improve the nitrogen fixation efficiency of leguminous plants, thereby increasing the biomass and yield of plants, which has broad application prospects in the study of nitrogen fixation mechanisms of leguminous crops and agricultural production.

[0026] The present invention proves through experiments that the expression of the LjPUB7 ​​gene is almost undetectable in the LjPUB7 ​​gene mutant plants of Lotus japonicus; at the same time, compared with the wild type, the number of nodules in the LjPUB7 ​​gene mutant plants is significantly increased, indicating that the LjPUB7 ​​protein encoded by the LjPUB7 ​​gene regulates the nitrogen fixation efficiency of the nodules, indicating that the LjPUB7 ​​protein has an important function in regulating the nitrogen fixation efficiency of plant nodules, thereby increasing the biomass and yield of the plants, which has application prospects in the study of the nitrogen fixation mechanism of leguminous crops and agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a diagram of the vector construction strategy for knocking out the LjPUB7 ​​gene by CRSPR-CAS9, where Figure A shows the design of three sgRNAs for LjPUB7, Figure B shows the main components of the pBluescript-SK(+)-LjU6-LjPUB7-sgRNA vector, and Figure C shows the main components of the p1300-sGFP-2x35s-Cas9-U6-LjPUB7 ​​vector.

[0028] Figure 2Figure 2 shows the number of infection lines of Ljpub7 mutant plants and wild-type plants. Five days after the wild-type MG20 and two Ljpub7 mutant lines were inoculated with M. loti MAFF303099 rhizobia carrying GFP fluorescence, the infection points (Figure A) and infection lines (Figure B) were counted respectively.

[0029] Figure 3 To detect the expression level of early nodulation genes by fluorescence quantitative PCR, we used the roots of MG20 and LjPUB7 ​​mutants 2 days after inoculation with rhizobia (M. lotiMAFF303099) as materials, designed different primers according to the genes, and used qRT-PCR to detect the gene expression of NIN (A), Enod40-1 (B), and Enod40-2 (C).

[0030] Figure 4 The nodule number (A) and nodule density (B) of LjPUB7 ​​mutant plants and wild-type plants. Ljpub7 is a gene knockout mutant plant, and MG20 is a wild-type plant.

[0031] In the figure: * indicates that the difference is significant when the significance level is 0.01<p≤0.05; ** indicates that the difference is between significant and extremely significant when the significance level is 0.001<p≤0.01; *** indicates that the difference is extremely significant when p≤0.001. DETAILED DESCRIPTION

[0032] The present invention provides an application of the LjPUB7 ​​gene in regulating the nitrogen fixation efficiency of plant nodules and / or regulating plant yield. The amino acid sequence of the protein encoded by the LjPUB7 ​​gene is shown in SEQ ID NO.1:

[0033] MNDAVSAMMISSGLLPTGTLLDSLIHISNEVGSMEKLPLVHTRNVSSMIRRIKLLSSLFEEI

[0034] QETDSPLPPSSILCFTELYSVITRVKDLIQECKDGSTLWGLIQLELISNQFYVLAKELGRALDI

[0035] LPLKLLNITADTKEQVQLLQKQAKRVELLIDPRELQRREQLLQVMANNSLRNKKNKGFIDF

[0036] GKVEDILSSIGLRTPSDYDGEISNLEVEAQNQAGTGGLIVVSNINNLISLVSYSKSMIFRNGEN

[0037] DRDEEECKPLSLILYNKN HDSSSSSMSISVIPNIPDEFRCPISLDLMRDPVIVSSGHTYDRNSI

[0038] AQAINTG H HTCPKSGQRLIHTALIPNYALKSLVQQWCYENNVPVNEPMNSLLNNSRSKKN

[0039] LKEEAIDHISANKAASDAVKMTAEFLVGKLATGSVDIQRQAAYELRLLAKSGTNNRSIIPEA

[0040] GAIPFLVTLLASQDSRTQEHAVTALFNLSILDNNKILIMVAGAVDNIVEVLESGKTMEAREN

[0041] AAAAIYSLSMVDDCKVQIGACTRAIPALVALLKEGTPIGKRDAASAIFNLAVYNPNKSSIVK

[0042] AGAVPLLVELLMDDKAGITDDVLAVLAVLQGCSEGLEEIRDNRALVPLLIDLLRFGSVKGKE

[0043] NSITLLLGLCKEEGELVARRLLANPRSVPSLQSLAADGSLRARRKADALLRLLNRCCSQPHHSV.

[0044] In the present invention, the gene encoding the LjPUB7 ​​protein is preferably the LjPUB7 ​​gene, and the nucleotide sequence of the LjPUB7 ​​gene is preferably as shown in SEQ ID NO.2, specifically as follows:

[0045]

[0046] The present invention also provides the use of the LjPUB7 ​​protein, LjPUB7 ​​gene and related biological materials described in the above technical scheme in regulating the nitrogen fixation efficiency of plant nodules and / or regulating plant yield; wherein the biological materials include one or more of the following: a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the LjPUB7 ​​gene.

[0047] The regulation of plant nodule nitrogen fixation includes one or more of the following aspects 1) to 4): 1) regulating plant nodule development; 2) regulating the expression level of plant early nodulation genes; 3) regulating the number of plant nodules; 4) regulating the nitrogen fixation efficiency in plant nodules;

[0048] In the present invention, the regulation of plant nodule development includes: negatively regulating the expression of LjPUB7 ​​gene to increase the number of infection points and infection lines of plant nodules.

[0049] In the present invention, the regulating the expression level of the plant early nodulation gene includes: negatively regulating the expression of the LjPUB7 ​​gene to increase the expression level of the early nodulation gene.

[0050] In the present invention, the regulating the number of plant nodules includes: negatively regulating the expression of the LjPUB7 ​​gene to increase the number of nodules of the regulated plant.

[0051] In the present invention, the regulating the nitrogen fixation efficiency of plant nodules includes: improving the nitrogen fixation efficiency of plant nodules by negatively regulating the expression of the LjPUB7 ​​gene.

[0052] In the present invention, regulating plant yield includes: increasing plant yield by negatively regulating the expression of LjPUB7 ​​gene.

[0053] In the present invention, the negative regulation method preferably includes the mutant LjPUB7 ​​gene knocked out by CRISPR-CAS9 technology.

[0054] The present invention also provides the use of the LjPUB7 ​​gene or LjPUB7 ​​protein described in the above technical solution in cultivating transgenic plants with improved nitrogen fixation efficiency and / or improved plant yield.

[0055] In the present invention, the improvement of nitrogen fixation efficiency preferably includes: promoting plant nodule development and / or increasing the number of plant nodules. The present invention can promote plant nodule development and increase the number of plant nodules by negatively regulating the expression of the LjPUB7 ​​gene, thereby improving the nitrogen fixation efficiency of the nodules, allowing the nodules to fix more nitrogen and provide it to the plants, thereby achieving the effect of increasing yield. In the present invention, the improvement of yield preferably includes increasing the biomass and yield of the plants by improving the nitrogen fixation efficiency of the plants.

[0056] In the present invention, the plant preferably includes leguminous plants; the leguminous plants preferably include one or more of soybean, alfalfa and Lotus japonicus.

[0057] To further illustrate the present invention, the application of the LjPUB7 ​​protein provided by the present invention in regulating the nitrogen fixation efficiency of plant nodules and / or regulating plant yield is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0058] Embodiment 1:

[0059] Effects of knockout of LjPUB7 ​​gene in Lotus japonicus on nodule development

[0060] 1. Selection of sgRNA

[0061] sgRNA was designed based on the Lj2g3v2794440.1 sequence of the LjPUB7 ​​genome. The criteria for sgRNA selection were: target efficiency greater than 50% and as close as possible to the downstream of the ATG of the gene coding region, located on the first or second exon. Three sgRNAs were selected, among which the genome sequence of LjPUB7 ​​is shown in SEQ ID NO.3:

[0062]

[0063] The three sgRNA sequences selected are as follows:

[0064] sgRAN1: 5'-GCTTGCAAAGGAGCTAGGAA-3', SEQ ID NO.4;

[0065] sgRNA3: 5'-GAGTAACCGAAGCTCATACG-3', SEQ ID NO.5;

[0066] sgRNA5: 5'-GCTAGAATCAGGAAAAACCA-3', SEQ ID NO. 6.

[0067] 2. Vector Construction

[0068] (1) Fragment amplification: The gRNA scaffold and tRNA fragments were amplified using the pGTR plasmid vector as a template, and then the three fragments were connected by the Gibson one-step method to form a gRNA-tRNA fusion fragment ( Figure 1 );

[0069] Amplification was performed using three different pairs of primers (LjU6-Fa and CRISPR-LjPUB7-sgRNA1-Ra; CRISPR-LjPUB7-sgRNA1-Fb and CRISPR-LjPUB7-sgRNA3-Rb; CRISPR-LjPUB7-sgRNA3-Fc and CRISPR-LjPUB7-sgRNA5-Rc); the reaction system was: Powerpol 2x PCRMix with Dye 25 μL, forward primer 1.5 μL, reverse primer 1.5 μL, template 1 μL and sterile water 21 μL; the concentrations of the forward primer and reverse primer were both 100 μM; the amplification fragment reaction procedures were: 95°C pre-denaturation for 3 min; 95°C denaturation for 10 s, 55°C annealing for 30 s, 72°C extension for 15 s, for a total of 30 cycles; 72°C extension for 5 min;

[0070] The pBluescript-SK+-LjU6 vector was digested with restriction endonuclease BbsⅠ to obtain the pBluescript-SK+-LjU6 linearized vector; the amplified fragments were connected by Gibson reaction to obtain the recombinant intermediate vector pBluescript-SK+-LjU6-LjPUB7.

[0071] For the construction method of the pGTR plasmid vector and the pBluescript-SK+-LjU6 vector, refer to the literature [Feng Y, Wu P, Liu C, Peng L, Wang T, Wang C, Tan Q, Li B, Ou Y, Zhu h, Yuan S, Huang R, Stacey G, Zhang Z, Cao Y. Suppression of LjBAK1 mediated immunity by SymRK promotes rhizobial infection in Lotus japonicus. Molecular Plant, 2021, 14: 1935-1950].

[0072] The nucleotide sequence of the gRNA-tRNA fusion fragment is shown in SEQ ID NO.7, specifically: 5'-AACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGCTTGCAAAGGAGCTAGGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGAGT AACCGAAGCTCATACGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCA CGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGCTAGAATCAGGAAAAACCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC-3';

[0073] The 3 pairs of different primer sequences are as follows:

[0074] LjU6-Fa:5'-CAAATAGTCAGCAACAAGGTTCGAACAAAGCACCAGTGG-3', SEQ IDNO.8;

[0075] CRISPR-LjPUB7-sgRNA1-Ra:5'-TTCCTAGCTCCTTTGCAAGCTGCACCAGCCGGGAATC-3', SEQ ID NO.9;

[0076] CRISPR-LjPUB7-sgRNA1-Fb:5'-GCTTGCAAAGGAGCTAGGAAGTTTTAGAGCTAGAAATAGCAAG-3', SEQ ID NO.10;

[0077] CRISPR-LjPUB7-sgRNA3-Rb:5'-CGTATGAGCTTCGGTTACTCTGCACCAGCCGGGAATC-3', SEQ ID NO.11;

[0078] CRISPR-LjPUB7-sgRNA3-Fc:5'-GAGTAACCGAAGCTCATACGGTTTTAGAGCTAGAAATAGCAAG-3', SEQ ID NO.12;

[0079] CRISPR-LjPUB7-sgRNA5-Rc:5'-AACTTGCTATTTCTAGCTCTAAAACTGGTTTTTCCTGATTCTAGCTGCACCAGCCGGGAATC-3', SEQ ID NO.13;

[0080] The Gibson (Yeasen, 10911ES20) reaction system is 5 μL: 2×Hieff Enzyme Premix 2.5 μL, about 100 ng of gene fragment, about 300 ng of vector and the balance of ddH2O; the Gibson reaction conditions are 50°C, 1 h. The single enzyme digestion system is: 10×FastDigest Green buffer 5 μL, intermediate vector pBluescript SK+-LjU62 μg, BbsⅠ endonuclease 2 μL and the balance of ddH2O to 50 μL; BbsⅠ (Thermo Scientific, ER1011) and single enzyme digestion reagents are purchased from Thermo Scientific; the single enzyme digestion conditions are 37°C, 3 h.

[0081] (2) Vector digestion and ligation: The recombinant intermediate vector pBluescript SK(+)-LjU6-LjPUB7 ​​obtained by the Gibson reaction was double digested with Kpn1 / Xba1 restriction endonucleases after sequencing verification, and the small fragment recovered was the LjU6-sgRNA fragment. The p1300-sGFP-2x35s-Cas9-U6 vector was also double-digested with Kpn1 / Xba1 restriction endonucleases to recover the p1300-sGFP-2x35s-Cas9-U6 linearized vector, which was connected to the LjU6-sgRNA fragment to obtain the p1300-sGFP-2x35s-Cas9-U6-LjPUB7 ​​recombinant vector; the CRISPR-Cas9 and LjU6-LjPUB7-sgRNA elements on the p1300-sGFP-2x35s-Cas9-U6-LjPUB7 ​​vector were then digested together with EcoR1 / Xba1 restriction endonucleases, and the recovered DNA fragment was 6905 bp in size. The p1300-Hyg vector was also double-digested with EcoR1 / Xba1 restriction endonucleases to recover the linearized p1300-Hyg vector, which was then connected to the obtained DNA fragment to obtain the final p1300-Hyg-2x35s-Cas9-U6-LjPUB7 ​​vector.

[0082] The construction methods of p1300-sGFP-2x35s-Cas9-U6 vector and p1300-Hyg vector can be found in the literature [Feng Y, Wu P, Liu C, Peng L, Wang T, Wang C, Tan Q, Li B, Ou Y, Zhu h, Yuan S, Huang R, Stacey G, Zhang Z, Cao Y. Suppression of LjBAK1 mediated immunity by SymRK promotes rhizobialinfection in Lotus japonicus. Molecular Plant, 2021, 14: 1935-1950].

[0083] The double enzyme digestion system was: 5 μL of 10×FastDigest Green buffer, 4 μg of vector, 4 μL of each of the two restriction endonucleases, and the remaining amount of ddH2O to a total volume of 50 μL; the enzyme digestion reagents were purchased from Thermo Scientific; and the enzyme digestion conditions were 37°C for 3 h.

[0084] The reaction system for the connection was: 0.5 μL of 10×T4 buffer, 0.5 μL of T4 DNA ligase, about 300 ng of linearized vector, about 100 ng of small fragment and 2 μL of sterile water; the reagents for the connection were purchased from Thermo Scientific; the reaction conditions for the connection were 22° C., 1 h.

[0085] 3. Stable transformation of Lotus japonicus

[0086] (1) Seed germination: Add seeds to a 50mL sterile conical flask and then pour in concentrated H2SO4. Shake evenly until the seeds are covered. The treatment time is 8-12 minutes. Then carefully pipette the concentrated H2SO4 into a glass beaker filled with water, and add sterile water to the remaining seeds to quickly rinse the seeds. Repeat the rinsing 3 times until the color of the sterile water does not change. Then add 2% sodium hypochlorite solution (effective chlorine concentration), cover the seeds, and shake gently. The treatment time is 8-12 minutes. After treatment, discard the waste liquid, add sterile water again to rinse the seeds 3 times until the color of the sterile water does not change. Finally, leave a little water to immerse the seeds, put them in a 4℃ refrigerator, and place them for 1-2 days for vernalization. Move the seeds to the MS culture medium plate and culture them in the dark (upright) at 23℃ for 24-36 hours, and then culture them in the light (upright) for 2-3 days. When the cotyledons grow up and the true leaves have not grown, and the hypocotyl is about 1-2 cm long, they can be infected and transformed.

[0087] Wild-type seeds of Lotus japonicus were published in [Feng Y, Wu P, Liu C, Peng L, Wang T, Wang C, Tan Q, Li B, Ou Y, Zhuh, Yuan S, Huang R, Stacey G, Zhang Z, Cao Y. Suppression of LjBAK1mediated immunity by SymRK promotes rhizobial infection in Lotus japonicus. Molecular Plant, 2021, 14: 1935-1950].

[0088] MS medium was prepared as follows: 4.43 g MS salts (Sigma, M5519-1L), pH adjusted to 6.0, volume made up to 1 L with dd H2O, 1.5 wt.% agar powder added as solid, and sterilized at 115°C for 20 min.

[0089] (2) Infection and co-cultivation: First, transform the p1300-Hyg-2x35s-Cas9-U6-LjPUB7 ​​into the EHA105 recombinant strain, then activate the EHA105 recombinant strain transformed with the p1300-Hyg-2x35s-Cas9-U6-LjPUB7 ​​plasmid by streaking, and grow a single colony at 28°C for 1-2 days. Then inoculate the single colony into 5mL LB liquid culture medium, and culture it at 28°C, 200r / min for 18h to obtain the culture solution. Inoculate the culture solution into 50mL LB liquid culture medium containing a final concentration of 50μg / ml Kana antibiotics at an inoculum volume of 2% (1mL of bacterial solution is added to 50mL of culture medium), and continue to culture at 28°C for 6h until the OD 600 The cell was collected by centrifugation at 4000r / min for 5min, the supernatant was discarded, and 1 / 10 B5 co-culture medium was added to suspend the cell, and cultured at 28°C for 30min to obtain a bacterial solution.

[0090] The hypocotyls of the stem segments of the seedlings germinated in step (1) are cut into a length of about 0.3 cm and immersed in the bacterial solution for 30 minutes; then the bacterial solution is aspirated, 10 filter papers are placed in a culture dish, and liquid 1 / 10 B5 co-culture medium is added until the filter paper is completely wetted, the hypocotyls are placed on the 1 / 10 B5 co-culture medium, and placed in a dark incubator at 23° C. for culturing for 5-7 days to obtain explants after co-culture.

[0091] LB liquid medium was prepared as follows: 10 g / L NaCl + 10 g / L Tryptone + 5 g / L Yeast Extract, (1.2% Agar), adjusted to pH 7.0. Reagent sources: Tryptone (Sigma, T2559), Yeast Extract (Sigma, Y0875), Agar (Yeasan, 70101ES76).

[0092] Kana solution was prepared as follows: 1.00 g of Kanamycin sulfate (Yeasan, 60206ES10) was weighed and dissolved in 10 mL of ultrapure water. After filtration and sterilization, the solution was dispensed into sterile 1.5 mL centrifuge tubes and stored at -20°C.

[0093] The EHA105 recombinant strain is disclosed in [Feng Y, Wu P, Liu C, et al. Suppression of LjBAK1-mediated immunity by SymRK promotes rhizobial infection in Lotus japonicus, 2021, 14: 1935-1950].

[0094] 1 / 10 B5 co-culture medium was prepared as follows: 0.387 g / L B5 basal salts, 2 g / L Sucrose, pH 5.5, 0.03% Phytagel sterilized at 115°C for 20 min. Before use, add MES (1 M pH 5.2) 5 mL, AS (100 mM) 1 mL, 6-BA (1 mg / mL) 500 μL, NAA (1 mg / mL) 50 μL, 1000× B5 vita min stock 100 μL.

[0095] 1M MES was prepared as follows: 1.952 g MES was dissolved in 10 mL dd H2O, solid KOH was added to adjust the pH to 5.6, filtered to sterilize, and dispensed into 1.5 mL centrifuge tubes and stored at -20°C.

[0096] 100 mM acetosyringone (AS) was prepared as follows: 196.2 mg of acetosyringone was diluted to 10 mL with dimethyl sulfoxide (DMSO), sterilized by filtration, and stored at -20°C.

[0097] 1 mg / mL 6-BA was prepared as follows: 10 mg 6-BA was dissolved in 1 mL 1 mol / L KOH, the volume was made up to 10 mL with ddH2O, and after filtration and sterilization, the solution was dispensed into sterile 1.5 mL centrifuge tubes and stored at -20°C.

[0098] 1 mg / mL NAA was prepared as follows: 10 mg NAA was dissolved in 1 mL 1 mol / L KO H, the volume was made up to 10 mL with ddH2O, and after filtration and sterilization, the solution was dispensed into sterile 1.5 mL centrifuge tubes and stored at -20°C.

[0099] (3) Screening and subculture:

[0100] The explants after co-cultivation are moved into callus induction medium to screen positive callus. Subculture once a week until green callus grows (i.e., the plate is moved once a week, and the total time is 5-8 weeks). The green callus is transferred to the bud induction medium and subcultured once a week until buds emerge to obtain callus containing small buds for 3-5 weeks. The callus containing small buds is successively moved to the stem elongation medium to make the small buds develop into the aboveground part, which can be subcultured once every 7-10 days together with the bud induction. Only 2-3 small buds grown from the same callus are taken, and the small buds are cut off and placed in the root induction medium to continue to cultivate, subcultured once a week, until more roots grow, and then the seedlings are transplanted to a 32-hole hole plate of a mixed matrix of vermiculite and perlite (ratio is 2:1) for identification.

[0101] B5 basal medium was prepared as follows: 3.87 g / L B5 basal salts, 20 g / L Sucrose, 1 mL 1000×B5 vitamin stock, 0.03% Phytagel, pH 5.5, and sterilized at 115°C for 20 min.

[0102] The callus induction medium was prepared as follows: B5 basal medium, before use, add 6-BA (1 mg / mL) 500 μL, NAA (1 mg / mL) 50 μL, 1000×B5 vitamin stock 1 mL, Timentin (300 mg / mL) 1 mL, and Hygromycin (300 mg / mL) 300 μL.

[0103] The bud induction medium was prepared as follows: B5 basal medium, before use, add 6-BA (1 mg / mL) 500 μL, NAA (1 mg / mL) 50 μL, 1000×B5 vitamin stock 1 mL, (NH4)2SO4 (2 mol / L) 2.5 mL, Timentin (300 mg / mL) 1 mL, and Hygromycin (300 mg / mL) 150 μL.

[0104] The stem elongation medium was prepared as follows: B5 basal medium, with 500 μL of 6-BA (1 mg / mL), 1 mL of 1000×B5 vitamin stock, 1 mL of Timentin (300 mg / mL), and 150 μL of Hygromycin (300 mg / mL) added before use.

[0105] The root induction medium was prepared as follows: B5 basal medium, with 1 mL of NAA (1 mg / mL), 1 mL of 1000×B5 vitamin stock, and 1 mL of Timentin (300 mg / mL) added before use.

[0106] Reagent sources: Phytagel (Thermo, VG0100B), B5 basal salts (Phytotech, G768-50L), B5 Vitaminstock (Mkbio, MS0620-500G), Timentin (Yeasan, 60230ES60), Hygromycin (Yeasan, 60225ES10), 6-BA (Sigma, B3408-25G), NAA (Sigma, N0640-25G).

[0107] (4) Genome extraction and identification:

[0108] Take one cotyledon of the Lotus japonicus leaf grown from the subcultured plant in step (3), add 300 μL of CTAB extract preheated at 65°C, grind thoroughly or grind with a sample grinder, mix thoroughly, place in a 65°C warm bath for 10 min-20 min, and gently invert once every 10 min to mix thoroughly. Add 300-400 μL of chloroform, mix by inverting, centrifuge at 12000 r / min for 10 min, take 200 μL of the upper liquid (to prevent aspiration of the middle layer) into a new 1.5 mL centrifuge tube, add 160 μL of isopropanol (or 400 μL of anhydrous ethanol) and 20 μL of NaAc (3 mol / L, pH 5.2), mix by inverting, let stand at -20°C or room temperature for 20-30 min, centrifuge at 12000 r / min for 10-15 min, discard the supernatant, add 500 μL of 70% (v / v) ethanol precooled at 4°C to thoroughly wash the precipitate. Centrifuge again at 12000r / min for 5min, discard the supernatant, then invert onto absorbent paper, place the centrifuge tube on the operating plate and leave at room temperature to allow the DNA precipitate to dry naturally, or place the centrifuge tube in a 65℃ oven to quickly blow dry or use a dryer to dry it (both times are 15-20min); dissolve it with 50μL ddH2O, determine the concentration, and store it briefly at 4℃ for PCR identification, or store it long-term at -20℃ for future use.

[0109] The extracted DNA was used as a template and PCR was performed with specific primers. A 15 μL reaction system was used: 2×PCRmix buffer, 0.3 μL of each 10 μmol / L primer, 1-3 μL of template DNA, and ultrapure water was added to 15 μL. PCR reaction program: 94°C 5min; 95°C 30s, 50°C-55°C 30s, 72°C 30s, 32 cycles, 72°C 10min. For CRISPR gene knockout detection and sequencing, 3 μL DNA / 30 μL PCR system was used, Tm 58°C. Plant genomic DNA was extracted, and after PCR identification and DNA sequencing, the positive seedlings were transplanted into a 14 cm diameter plastic pot filled with a mixed matrix of vermiculite and perlite in a ratio of 2:1, and placed in a greenhouse, 23°C, 16h / 8h photoperiod culture.

[0110] (5) Transplanting seedlings and inoculating rhizobia:

[0111] After subculture and sequencing identification, Ljpub7 stably transformed plants were finally obtained. The nodulation experiment was carried out using the Ljpub7 stably transformed homozygous mutant plants obtained by the CRISPR-CAS9 system as materials and the wild-type MG20 as a control.

[0112] Vermiculite was moistened and sterilized at 121°C for 30 min, and then cooled to room temperature. The seeds were treated with concentrated sulfuric acid and sodium hypochlorite and then kept in the dark at 4°C overnight. After germination, they were planted in sterilized perlite: vermiculite (ratio 1:2) culture soil. After the two cotyledons of the seedlings opened, rhizobia (Mesorhizobium lotiMAFF303099 / pHC60) marked with GFP were inoculated. The rhizobia were cultured in TY liquid medium at 28°C with shaking until the OD 600 The value was about 1.0, and the cells were collected by centrifugation at 5000 rpm / min for 5 min, and the culture medium was nitrogen-free nutrient solution B&D until the OD 600 =0.04, inoculated into the roots of Lotus japonicus seedlings.

[0113] Mesorhizobium lotiMAFF303099 / pHC60 is disclosed in [FengY, WuP, Liu C, Peng L, WangT, WangC, Tan Q, Li B, Ou Y, Zhu h, Yuan S, Huang R, Stacey G, Zhang Z, CaoY. Suppression of LjBAK1-mediated immunity by SymRK promotes rhizobialinfection in Lotus japonicus.MolecularPlant,2021,14:1935-1950].

[0114] TY medium was prepared as follows: 3 g yeast extract, 5 g tryptone, 0.69 g CaCl2, pH adjusted to 7.0, fixed to 1 L with ddH2O, and sterilized at 121°C for 20 min.

[0115] The nitrogen-free nutrient solution B&D culture solution is prepared as follows: 1.0mM CaCl2·2H2O, 0.5mM KH2PO4, 10μM Fe-Citrate, 0.25mM MgSO4·7H2O, 0.25mM K2SO4, 1.0μM MnSO4·H2O, 2.0μM H3BO3, 0.5μM ZnSO4·7H2O, 0.2μM CuSO4·5H2O, 0.1μM CoSO4·7H2O, 0.1μM Na2MoO4·2H2O.

[0116] (6) Count the number of infection lines:

[0117] Five days after inoculation with rhizobia, the plants were dug out from the vermiculite, the roots of the plants were washed, and the number of infection points (IPs) and infection lines (ITs) was counted under a Nikon SMZ18 fluorescent stereo microscope. The results showed that the average number of IPs and ITs in the Ljpub7 mutant was significantly increased compared with the wild type ( Figure 2 ), which indicates that reducing LjPUB7 ​​gene expression can promote the formation of infection lines, which is beneficial to nodule development, and LjPUB7 ​​can negatively regulate the early nodule symbiosis process.

[0118] Embodiment 2:

[0119] qRT-PCR analysis of the expression of early nodulation genes in roots of Ljpub7 mutants

[0120] 1. Preparation of Total RNA

[0121] In order to detect the effect of LjPUB7 ​​mutation on early symbiotic nodulation-related genes, the roots of MG20 and Ljpub7 mutants 2 days after inoculation with rhizobia were used as materials, and the gene expression levels of NIN, Enod40-1, and Enod40-2 were analyzed by qRT-PCR. The plant RNA extraction kit (CW0588S) of Kangwei Century Company was used for total RNA extraction. The specific operation steps are detailed in the instructions. The RNA concentration was determined by NanoDrop micro-spectrophotometer. The OD260 / OD280 value was between 1.8 and 2.2, which was the best. A value less than this indicated the presence of impurities, and a value greater than this indicated RNA degradation. Add 2 μL 5×LoadingBuffer, 1% Agrose gel (Yeasan, 10208ES76), and electrophoresis at 110V for 20-25min to detect RNA integrity.

[0122] 2. qRT-PCR detection of early nodulation gene expression

[0123] The total RNA extracted from plant tissues was reverse transcribed using Primescript RT Reagent kit DRR047A (Takara, Japan), and qRT-PCR was performed according to the one-step SYBR PrimeScript RT-PCR kit II (Takara). The specific methods of the experiment refer to the corresponding kit operation manual. The Roche LightCycler apparatus fluorescent quantitative PCR instrument was used, and the PCR reaction program was: 50℃2min; 95℃10min; 95℃15sec, 60℃1min, 40 cycles. The relative quantitative ΔΔCt method was used to standardize the gene expression level with Lotus japonicus Ubiquitin (Genbank ID: AW720576) and ATPase (Genbank ID: AW719841) as internal references to detect the expression levels of early nodulation genes LjNIN, Enod40-1, and Enod40-2. The results showed that the mRNA levels of NIN, Enod40-1, and Enod40-2 in the roots of the LjPUB7 ​​mutant were significantly higher than those in MG20 ( Figure 3 ). These results indicate that the loss of LjPUB7 ​​enhances the early infection signal of rhizobia.

[0124] The amplification primers in the fluorescent quantitative PCR are:

[0125] Ubiquitin-F: 5'-TTCACCTTGTGCTCCGTCTTC-3', SEQ ID NO. 14;

[0126] Ubiquitin-R: 5'-AACAACCAGCACACACAGACAATC-3', SEQ ID NO.15;

[0127] ATPase-F: 5'-CAATGTCGCCAAGGCCATGGTG-3', SEQ ID NO.16;

[0128] ATPase-R: 5'-AACACCACTCTCGATCATTCTCTG-3', SEQ ID NO. 17.

[0129] NIN-F: 5'-AGGAGCCCAAGTGAGTGCTA-3', SEQ ID NO.18;

[0130] NIN-R: 5'-GCCATCAAGGTATATGACGAG-3', SEQ ID NO.19;

[0131] Enod40-1-F: 5'-CCTCTGAACCAATCCATCAAATCCA-3', SEQ ID NO. 20;

[0132] Enod40-1-R: 5'-AGGAGTGTGAGAGGTGACAGCA-3', SEQ ID NO. 21.

[0133] Enod40-2-F: 5'-GTCGCACTTGCAGTTGTGGATC-3', SEQ ID NO. 22;

[0134] Enod40-2-R: 5'-GACTTGCCGGTTCGCCAGGCTG-3', SEQ ID NO. 23.

[0135] Embodiment 3:

[0136] Effects of knockout of LjPUB7 ​​gene in Lotus japonicus on the number of nodules

[0137] 1. Seed germination is the same as in Example 1.

[0138] 2. Transplanting seedlings and inoculating rhizobia are the same as in Example 1.

[0139] The inoculated rhizobium (M. loti MAFF303099) was disclosed in [Feng Y, Wu P, Liu C, Peng L, Wang T, Wang C, Tan Q, Li B, Ou Y, Zhu h, Yuan S, Huang R, Stacey G, Zhang Z, Cao Y. Suppression of LjBAK1 mediated immunity by SymRK promotes rhizobialinfection in Lotus japonicus. Molecular Plant, 2021, 14: 19351950].

[0140] 3. Count the number and density of nodules.

[0141] The wild-type MG20 and LjPUB7 ​​homozygous mutant were used as materials, and phenotypic statistics were performed 7 days after inoculation with rhizobia (M. loti MAFF303099). The experimental results showed that compared with the wild-type MG20, the number of nodules in the LjPUB7 ​​mutant increased significantly. Figure 4 A), further statistical analysis of nodule density revealed that similar to the results of nodule number, the nodule density of the LjPUB7 ​​mutant was also significantly higher than that of MG20 ( Figure 4 B) This result indicates that LjPUB7 ​​is involved in the early nodule development process.

[0142] In summary, the LjPUB7 ​​gene of Lotus japonicus has an important function in regulating nitrogen fixation efficiency. Knocking out the LjPUB7 ​​gene enhanced the expression of early symbiotic nodulation genes, and the number of infection lines increased significantly, indicating that the LjPUB7 ​​gene regulated the early development process of nodules. Through nodulation experiments, compared with wild-type plants, the number and density of nodules in mutant plants increased significantly, indicating that the LjPUB7 ​​gene has achieved significant technical effects in improving the nitrogen fixation efficiency and yield of Lotus japonicus, which is of great significance.

[0143] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of the Lotus japonicus LjPUB7 ​​gene or biological materials related thereto in regulating the nitrogen fixation efficiency of plant nodules and / or regulating plant yield, characterized in that: The nucleotide sequence of the LjPUB7 ​​gene is shown in SEQ ID NO.2, and the amino acid sequence of the protein encoded by the LjPUB7 ​​gene is shown in SEQ ID NO.1; the biological material includes one or more of the following: a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the LjPUB7 ​​gene.

2. The use according to claim 1, characterized in that: The regulation of plant nodule nitrogen fixation includes one or more of the following aspects (1) to (4): (1) Regulate plant nodule development; (2) Regulate the expression level of early nodulation genes in plants; (3) Regulate the number of plant nodules; (4) Regulate nitrogen fixation efficiency in plant nodules.

3. The use according to claim 2, characterized in that: The method for regulating plant nodule development comprises: increasing the number of infection lines of plant nodules by negatively regulating the expression of LjPUB7 ​​gene.

4. The use according to claim 2, characterized in that: The regulating the expression level of the plant early nodulation gene comprises: increasing the expression level of the early nodulation gene by negatively regulating the expression of the LjPUB7 ​​gene.

5. The use according to claim 2, characterized in that: The method for regulating the number of plant nodules comprises: increasing the number of plant nodules by negatively regulating the expression of the LjPUB7 ​​gene.

6. The use according to claim 2, characterized in that: The regulating the nitrogen fixation efficiency of plant nodules comprises: improving the nitrogen fixation efficiency of plant nodules by negatively regulating the expression of LjPUB7 ​​gene.

7. The use according to claim 1, characterized in that: The regulating plant yield comprises: increasing plant yield by negatively regulating the expression of LjPUB7 ​​gene.

8. Use of the LjPUB7 ​​gene according to claim 1 in cultivating transgenic plants with improved nitrogen fixation efficiency and / or improved plant yield, characterized in that: The amino acid sequence of the protein encoded by the LjPUB7 ​​gene is shown in SEQ ID NO.1, and the nucleotide sequence of the LjPUB7 ​​gene is shown in SEQ ID NO.

2.

9. The use according to claim 8, characterized in that: The improvement of nitrogen fixation efficiency and / or plant yield includes: increasing the number of plant nodules.

10. The use according to any one of claims 1 to 9, characterized in that: The plants include leguminous plants; the leguminous plants include one or more of soybean, Lotus japonicus and alfalfa.

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