Application of Rice OsLPR1, OsLPR3, OsLPR4 and OsLPR5 Genes

Abstract

The present invention belongs to the field of plant genetic engineering technology and specifically relates to the use of rice genes OsLPR1, OsLPR3, OsLPR4, and OsLPR5. The present invention provides the use of rice genes for one or more of the following: (1) regulating rice nitrogen uptake; (2) regulating rice nitrogen utilization efficiency; (3) regulating the colonization of rice nitrogen-fixing microorganisms; and (4) cultivating new rice varieties. The rice genes include the rice OsLPR1 gene, the rice OsLPR3 gene, the rice OsLPR4 gene, and the rice OsLPR5 gene. The present invention finds that the rice genes can regulate rice nitrogen uptake during growth and development, regulate nitrogen content and nitrogen utilization efficiency in the aboveground parts of rice, and regulate the colonization of rice nitrogen-fixing microorganisms.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to the application of rice OsLPR1, OsLPR3, OsLPR4 and OsLPR5 genes. Background Art

[0002] Nitrogen (N) is one of the most important nutrients for rice growth and development. To achieve high rice yields, farmers blindly apply nitrogen fertilizer to the soil. This leads to the loss of large amounts of nitrogen that are not utilized by the rice, directly contributing to eutrophication of water bodies and the emission of the greenhouse gas nitric oxide (NO), causing serious environmental problems.

[0003] Directly determining the amount of nitrogen required for high rice yields through soil testing and formulating fertilizers can improve fertilizer utilization efficiency. However, my country's current rice cultivation model is still dominated by smallholder farmers, with small and scattered plots and relatively extensive management methods. This method is time-consuming and labor-intensive, with high investment costs, making it difficult to implement on a large scale and has not been effectively applied. Although the application of nitrogen-fixing bacteria (NFB) as biofertilizers has achieved positive results, effectively regulating rhizosphere microbial communities and reducing dependence on chemical nitrogen fertilizers, the unstable fertilizer effect and easy loss of effectiveness of bio-organic fertilizers, as well as the potential presence of antibiotics and heavy metals in the raw materials used in their production, have greatly restricted the application of nitrogen-fixing microbial fertilizers. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of rice OsLPR1, OsLPR3, OsLPR4 and OsLPR5 genes to regulate the nitrogen absorption of rice growth and development, regulate the nitrogen content and nitrogen utilization efficiency of rice aboveground parts, and regulate the colonization of rice nitrogen-fixing microorganisms to cultivate new rice varieties.

[0005] The present invention provides the use of rice genes in one or more of the following:

[0006] (1) Regulate nitrogen absorption in rice;

[0007] (2) Regulate nitrogen utilization efficiency of rice;

[0008] (3) regulating the colonization of nitrogen-fixing microorganisms in rice;

[0009] (4) Cultivate new rice varieties;

[0010] The rice genes include rice OsLPR1 gene, rice OsLPR3 gene, rice OsLPR4 gene and rice OsLPR5 gene;

[0011] The nucleotide sequence of the rice OsLPR1 gene is shown in SEQ ID NO.1;

[0012] The nucleotide sequence of the rice OsLPR3 gene is shown in SEQ ID NO.2;

[0013] The nucleotide sequence of the rice OsLPR4 gene is shown in SEQ ID NO.3;

[0014] The nucleotide sequence of the rice OsLPR5 gene is shown in SEQ ID NO.4.

[0015] Preferably, regulating nitrogen absorption of rice includes regulating nitrogen absorption of aboveground parts of rice;

[0016] The regulating the colonization of nitrogen-fixing microorganisms in rice includes regulating the relative abundance and / or quantity of endogenous nitrogen-fixing microorganisms in rice.

[0017] The present invention also provides an sgRNA for knocking out rice OsLPR1 gene, rice OsLPR3 gene, rice OsLPR4 gene and rice OsLPR5 gene, wherein the sgRNA comprises the nucleotide sequence shown in SEQ ID NO.5;

[0018] The nucleotide sequence of the rice OsLPR1 gene is shown in SEQ ID NO.1;

[0019] The nucleotide sequence of the rice OsLPR3 gene is shown in SEQ ID NO.2;

[0020] The nucleotide sequence of the rice OsLPR4 gene is shown in SEQ ID NO.3;

[0021] The nucleotide sequence of the rice OsLPR5 gene is shown in SEQ ID NO.4.

[0022] The present invention also provides a knockout vector for knocking out rice OsLPR1 gene, rice OsLPR3 gene, rice OsLPR4 gene and rice OsLPR5 gene, wherein the knockout vector comprises a basic vector and the sgRNA described in the above technical solution inserted into the basic vector.

[0023] Preferably, the basic vector comprises pYLCRISPR.

[0024] The present invention also provides a recombinant bacterium for knocking out rice OsLPR1, rice OsLPR3, rice OsLPR4 and rice OsLPR5 genes. The recombinant bacterium comprises a base bacterium and the sgRNA or knockout vector described in the above technical solution introduced into the base bacterium.

[0025] Preferably, the basic bacteria include Agrobacterium.

[0026] The present invention also provides the use of the sgRNA or knockout vector or recombinant bacteria described in the above technical solution in one or more of the following:

[0027] (1) Promote nitrogen absorption by rice;

[0028] (2) Improve nitrogen utilization efficiency of rice;

[0029] (3) Promote the colonization of nitrogen-fixing microorganisms in rice;

[0030] (4) Cultivate new rice varieties.

[0031] The present invention also provides a method for promoting one or more of nitrogen absorption by rice, improving nitrogen utilization efficiency of rice, and promoting colonization of nitrogen-fixing microorganisms in rice, comprising the following steps:

[0032] The sgRNA or knockout vector or recombinant bacteria described in the above technical solution is introduced into the recipient rice tissue.

[0033] Preferably, the introduction method includes Agrobacterium-mediated method; and the recipient rice tissue includes rice callus tissue.

[0034] Beneficial effects:

[0035] The present invention discovered that the four rice genes, OsLPR1, OsLPR3, OsLPR4, and OsLPR5, can regulate nitrogen absorption for rice growth and development, regulate nitrogen content and nitrogen use efficiency in rice shoots, and regulate the colonization of nitrogen-fixing microorganisms in rice. The results of the examples showed that compared with wild-type rice, mutant rice plants with co-knockout of the four genes, OsLPR1, OsLPR3, OsLPR4, and OsLPR5, showed significantly improved aboveground nitrogen content and nitrogen use efficiency under normal field management conditions. The aboveground nitrogen content increased by 16.53% to 34.30%, the relative abundance of endophytic nitrogen-fixing microorganisms increased by 776.64% to 1201.58%, and the number of endophytic nitrogen-fixing OTUs increased by 44.57% to 66.76%. The nitrogen use efficiency of the co-knockout rice mutants was approximately 128.06% to 148.51% of that of the wild-type. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0037] Figure 1 is a map of the pYLCRISPR vector;

[0038] Figure 2 This is a map of the single-target gene editing plasmid pYLCRISPR-OsLPR;

[0039] Figure 3 The mutation patterns of OsLPR1, OsLPR3, OsLPR4, and OsLPR5 genes in osLPR1 / 3 / 4 / 5 mutants;

[0040] Figure 4 The nitrogen content of the aboveground parts of wild-type rice and mutant rice in Example 4;

[0041] Figure 5 The nitrogen utilization efficiency of wild-type rice and mutant rice in Example 4;

[0042] Figure 6 This is a schematic diagram of the xylem sap collection device;

[0043] Figure 7 This is a comparison of the relative abundance of endophytic nitrogen-fixing microorganisms in wild-type rice and mutant rice in Example 4;

[0044] Figure 8 The number of endophytic OTUs in rice capable of nitrogen fixation in wild-type rice and mutant rice in Example 4;

[0045] Figure 9 The nitrogen content of the aboveground parts of wild-type rice and mutant rice in Example 5;

[0046] Figure 10 The nitrogen utilization efficiency of wild-type rice and mutant rice in Example 5;

[0047] Figure 11 This is a comparison of the relative abundance of endophytic nitrogen-fixing microorganisms in wild-type rice and mutant rice in Example 5;

[0048] Figure 12 The number of endophytic OTUs in rice that can fix nitrogen in wild-type rice and mutant rice in Example 5. DETAILED DESCRIPTION

[0049] The present invention provides the use of rice genes in one or more of the following:

[0050] (1) Regulate nitrogen absorption in rice;

[0051] (2) Regulate nitrogen utilization efficiency of rice;

[0052] (3) regulating the colonization of nitrogen-fixing microorganisms in rice;

[0053] (4) Cultivate new rice varieties;

[0054] The rice genes include rice OsLPR1 gene, rice OsLPR3 gene, rice OsLPR4 gene and rice OsLPR5 gene;

[0055] The nucleotide sequence of the rice OsLPR1 gene is shown in SEQ ID NO.1;

[0056] The nucleotide sequence of the rice OsLPR3 gene is shown in SEQ ID NO.2;

[0057] The nucleotide sequence of the rice OsLPR4 gene is shown in SEQ ID NO.3;

[0058] The nucleotide sequence of the rice OsLPR5 gene is shown in SEQ ID NO.4.

[0059] In the present invention, the (1) regulation of rice nitrogen absorption preferably includes regulating nitrogen absorption in the aboveground part of rice; the regulation is preferably negatively regulating the rice gene to promote nitrogen absorption of rice, and more preferably negatively regulating the rice gene to promote nitrogen absorption in the aboveground part of rice. The (2) regulation of rice nitrogen utilization rate preferably includes regulating nitrogen utilization rate in the aboveground part of rice; the regulation is preferably negatively regulating the rice gene to improve the nitrogen utilization rate of rice, and more preferably negatively regulating the rice gene to improve the nitrogen utilization rate of the aboveground part of rice. The (3) regulation of rice nitrogen-fixing microbial colonization preferably includes regulating the relative abundance and / or quantity of endophytic nitrogen-fixing microorganisms in rice; the regulation is preferably negatively regulating the rice gene to promote the colonization of rice nitrogen-fixing microorganisms, and more preferably negatively regulating the rice gene to improve the relative abundance and / or quantity of endophytic nitrogen-fixing microorganisms in rice. The aboveground part of rice in the present invention is preferably rice straw and leaves. While the present invention is described in the examples using the japonica rice variety Tongjing 981 as an example, this should not be construed as the sole embodiment of the present invention. The present invention utilizes the rice gene to regulate nitrogen absorption in all rice varieties. The nucleotide sequences of SEQ ID NOs. 1 to 4 are as follows:

[0060]

[0061]

[0062]

[0063]

[0064] The present invention also provides an sgRNA for knocking out rice OsLPR1, rice OsLPR3, rice OsLPR4, and rice OsLPR5 genes, the sgRNA comprising the nucleotide sequence shown in SEQ ID NO. 5. The nucleotide sequence shown in SEQ ID NO. 5 of the present invention is specifically 5'-GGTACCACGACCACGCCCT-3'.

[0065] The present invention also provides a knockout vector for co-knockout of the rice OsLPR1, OsLPR3, OsLPR4, and OsLPR5 genes. The knockout vector comprises a base vector and the sgRNA described in the above technical solution inserted into the base vector. In the present invention, the base vector preferably comprises pYLCRISPR. The present invention preferably inserts the sgRNA into the Bsa I restriction enzyme site of pYLCRISPR. The present invention has no strict requirements for the method of insertion; conventional methods in the art, such as enzyme digestion and ligation, can be employed. The present invention has no particular restrictions on the source of the sgRNA; sgRNA can be obtained by artificially synthesizing nucleotide sequences using methods well known in the art.

[0066] The present invention also provides a recombinant bacterium for co-knockout of the rice OsLPR1, OsLPR3, OsLPR4, and OsLPR5 genes, comprising a base bacterium and the sgRNA or knockout vector described in the above technical solution introduced into the base bacterium. In the present invention, the base bacterium preferably comprises Agrobacterium, more preferably Agrobacterium EHA105.

[0067] The above-mentioned sgRNA or knockout vector or recombinant bacteria provided by the present invention can achieve the joint knockout of the four genes OsLPR1, OsLPR3, OsLPR4 and OsLPR5 in rice, thereby increasing the nitrogen content in the aboveground part of rice under normal field management conditions, improving nitrogen utilization efficiency, promoting the colonization of rice nitrogen-fixing microorganisms, and increasing the relative abundance and number of endophytic nitrogen-fixing microorganisms in rice; the aboveground part of rice is preferably rice straw and leaves.

[0068] In view of the advantages of the sgRNA or knockout vector or recombinant bacteria provided by the present invention, the use of the sgRNA or knockout vector or recombinant bacteria in one or more of promoting nitrogen absorption by rice, improving nitrogen utilization efficiency of rice, promoting colonization of nitrogen-fixing microorganisms in rice and cultivating new rice varieties also falls within the scope of protection of the present invention. The promotion of nitrogen absorption by rice described in the present invention is preferably to promote nitrogen absorption by the aboveground part of rice; the aboveground part of rice is preferably rice straw and leaves; the promotion of colonization of nitrogen-fixing microorganisms in rice preferably includes increasing the relative abundance and / or number of endophytic nitrogen-fixing microorganisms in rice. The characteristics of the new rice variety described in the present invention preferably include being similar to wild-type rice, and the new rice variety has a high nitrogen absorption rate, a high nitrogen utilization rate, a large relative abundance of nitrogen-fixing microorganism colonization and a large number of nitrogen-fixing microorganism colonization.

[0069] The present invention also provides a method for promoting one or more of nitrogen absorption by rice, improving nitrogen utilization efficiency of rice, and promoting colonization of nitrogen-fixing microorganisms in rice, comprising the following steps:

[0070] The sgRNA or knockout vector or recombinant bacteria described in the above technical solution is introduced into the recipient rice tissue.

[0071] In the present invention, the introduction method preferably includes Agrobacterium-mediated introduction; the recipient rice tissue preferably includes rice callus. After the sgRNA, knockout vector, or recombinant bacteria are introduced into the recipient rice, the recipient rice is preferably cultured, and the transgenic seeds are screened and harvested. The present invention does not specifically limit the methods of culture, screening, and harvesting, and conventional operating methods familiar to those skilled in the art can be used. The introduction of the sgRNA, knockout vector, or recombinant bacteria into the recipient rice of the present invention can co-knockout the four rice genes OsLPR1, OsLPR3, OsLPR4, and OsLPR5, which can promote nitrogen absorption in rice, especially promoting nitrogen absorption in the aboveground part of rice, improving nitrogen utilization efficiency, and promoting the colonization of rice nitrogen-fixing microorganisms. The results of the examples show that compared with wild-type rice, the mutant rice with the four genes OsLPR1, OsLPR3, OsLPR4, and OsLPR5 co-knockout significantly improved the aboveground nitrogen content and nitrogen utilization efficiency under normal field management conditions.

[0072] To further illustrate the present invention, the application of co-knockout of rice OsLPR1, OsLPR3, OsLPR4 and OsLPR5 genes provided by the present invention is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0073] Example 1

[0074] Construction of gene editing vector for co-knockout of OsLPR1 (LOC_Os01g03530), OsLPR3 (LOC_Os01g03630), OsLPR4 (LOC_Os01g03620), and OsLPR5 (LOC_Os01g03640) genes

[0075] (1) Synthesize the forward strand of sgRNA and its complementary single-stranded DNA containing the sticky ends of the Bsa I restriction enzyme digestion product, phosphorylate the single-stranded DNA and anneal to form a double-stranded sgRNA containing sticky ends (the nucleotide sequence of the sgRNA forward strand without Bsa I restriction enzyme digestion is shown in SEQ ID NO.5); linearize the pYLCRISPR vector with Bsa I restriction enzyme ( Figure 1 ) and dephosphorylated using alkaline phosphatase.

[0076] (2) T4 ligase was used to connect the double-stranded sgRNA and linearized pYLCRISPR to construct a recombinant vector to obtain a single-target gene editing plasmid pYLCRISPR-OsLPR that can knock out four genes, OsLPR1, OsLPR3, OsLPR4, and OsLPR5. The map is shown in Figure 2. Figure 2 shown.

[0077] Example 2

[0078] Rice transformation

[0079] 1. 0.5 μg of the single-target gene editing plasmid pYLCRISPR-OsLPRs prepared in Example 1 was transformed into the competent cells of Agrobacterium tumefaciens strain EHA105, followed by ice bath for 30 min, heat shock at 42 ° C for 90 min, and ice bath for 2 min. Antibiotic-free LB culture medium was added and activated in a shaker at 28 ° C for 1 h to obtain an Agrobacterium strain containing the pYLCRISPR-OsLPRs gene editing plasmid.

[0080] 2. Transform callus tissue of japonica rice Tongjing 981 with Agrobacterium tumefaciens strain containing the pYLCRISPR-OsLPRs gene editing plasmid. The specific steps are as follows:

[0081] (1) Agrobacterium containing the pYLCRISPR-OsLPRs gene editing plasmid was cultured in AAM liquid medium containing 50 mg / L kanamycin (Kan) and 25 mg / L rifampicin (Rif) at 28°C in the dark with shaking at 200 rpm until the OD 600The nm was 0.4-0.6, and the cells were collected by centrifugation and treated with liquid NB-As (NB basic medium + 100 μmol / L acetosyringone, pH 5.2-5.5, wherein the NB basic medium includes: 2830 mg / L KNO3, 463 mg / L (NH4)2SO4, 400 mg / L KH2PO4, 185 mg / LMgSO4.7H2O, 166 mg / LCaCl2·2H2O, 27.8 mg / LFeSO4·7H2O, 37.5 mg / LNa2-EDTA, 10 mg / LMnSO4·4H2O, 3 mg / LH3BO3, 2 mg / LZnSO4·7H2O, 0.25 mg / LNa2MoO4·2H2O, 0.025 mg / L CuSO4·5H2O, 0.025 mg / L CoCl2·6H2O, 0.75 mg / L KI, 10 mg / L thiamine hydrochloride, 1 mg / L pyridoxine hydrochloride, 1 mg / L niacin, 100 mg / L inositol, 300 mg / L hydrolyzed casein, 500 mg / L glutamine, 500 mg / L proline, 30000 mg / L sucrose) and resuspended to OD 600 nm=0.1;

[0082] (2) Select callus tissue of japonica rice strain Tongjing 981 of appropriate size and good condition, immerse it in a 50mL centrifuge tube containing Agrobacterium resuspension for 30 minutes, absorb the excess bacterial solution on sterile filter paper, cover the surface of the callus with sterile filter paper and air dry it in a clean bench. After air drying, transfer the callus tissue to solid NBD-As medium (NB basic medium without glutamine and proline + 2.0mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) + 100μmol / L acetosyringone + 10g / L glucose + 2.6mg / LPhytagel plant gel + 2mg / L inositol, pH 5.2-5.5) covered with sterile filter paper and culture in the dark at 25℃. After three days, the calli were soaked in sterile water containing 150 mg / L Timentin for 10 minutes. Excess water was then blotted off, the calli were covered with sterile filter paper, and air-dried in a clean bench. The calli were then transferred to NBD-As medium for recovery culture at 32°C under light for 4 days. The calli were then transferred to NBD-T medium (NB minimal medium + 2.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) + 500 mg / L cefotaxine + 50 mg / L hygromycin B + 2.6 mg / L Phytagel plant gel + 2 mg / L inositol, pH 5.8) containing the selection antibiotic hygromycin for continued selection and culture, with subculture every two weeks. When the catalyzed adventitious buds grew to 3–5 cm, the seedlings were cut and transferred to rooting medium RE2 for rooting induction to obtain seedlings.

[0083] Example 3

[0084] Molecular detection of target gene knockout

[0085] (1) Leaves of the seedlings obtained in Example 2 were selected and plant genomic DNA was extracted. PCR identification of the hygromycin resistance gene was performed using GreenMix of Novezan Biotechnology Co., Ltd., using conventional procedures well known to those skilled in the art, to obtain positive seedlings that were successfully transformed. Then, PCR amplification of the OsLPR1, OsLPR3, OsLPR4, and OsLPR5 genes was performed using GreenMix of Novezan Biotechnology Co., Ltd., using conventional procedures well known to those skilled in the art. The primer information for the hygromycin resistance gene, OsLPR1, OsLPR3, OsLPR4, and OsLPR5 genes is shown in Table 1.

[0086] Table 1 Primer information

[0087]

[0088] (2) The PCR product obtained in step (1) was sent to Sangon Biotech Co., Ltd. for sequencing. The sequencing results were compared and analyzed using Snapgene, and the mutant strains were analyzed for possible off-target sites using conventional operation methods well known to those skilled in the art. A homozygous osLPR1 / 3 / 4 / 5 mutant strain with common mutations in the four genes OsLPR1, OsLPR3, OsLPR4 and OsLPR5 and no off-target sites was obtained. The editing efficiency was 27.45%. The mutation patterns of the four genes OsLPR1, OsLPR3, OsLPR4 and OsLPR5 in this strain are as follows: Figure 3 As shown, the osLPR1 / 3 / 4 / 5 mutant was selected for subsequent aboveground nitrogen content detection.

[0089] Example 4

[0090] Seeds of wild-type Tongjing 981 (WT) and the osLPR1 / 3 / 4 / 5 mutant Tongjing 981 (osLPR1 / 3 / 4 / 5) obtained in Example 3 were cultivated in farmland soil 1 (collected from the Zijingang Agricultural Experimental Station of Zhejiang University (30.30°N, 120.08°E)) according to conventional farmer cultivation methods. Agronomic management was carried out according to farmers' daily management methods during the growth period.

[0091] 1. Detection of nitrogen content in the aboveground part of rice

[0092] After rice is harvested at the tillering stage, the straw is washed and dried, then crushed and sieved using a grinder. The straw powder is weighed and digested using a concentrated sulfuric acid-hydrogen peroxide method. The completely lysed samples are collected in clean tubes, and the nitrogen content is determined using the indigo blue colorimetric method. The nitrogen utilization efficiency (NUEbala) is then calculated using the balance method. This is the ratio of crop nitrogen uptake to total nitrogen input. The calculation formula is: NUEbala = crop nitrogen uptake / total nitrogen input × 100%. The results showed that the aboveground nitrogen content of wild-type Tongjing 981 was approximately 7.07 g / kg, while the aboveground nitrogen content of the OsLPR1 / 3 / 4 / 5 mutant Tongjing 981 was 9.51 g / kg, an increase of 34.30% ( ) compared to wild-type Tongjing 981. Figure 4 The nitrogen utilization efficiency of the OsLPR1 / 3 / 4 / 5 mutant Tongjing 981 was approximately 148.51% of that of the wild type 981 ( Figure 5 ).

[0093] 2. Extraction and Detection of Rice Endophytic Microorganisms

[0094] (1) During the rice tillering period, the xylem sap is extracted from the rice using a xylem sap collecting device; wherein the xylem sap collecting device is as follows: Figure 6 As shown, the main body consists of the following three parts: a 5mL centrifuge tube, a 200μL pipette tip, and absorbent cotton. Use scissors to cut off the tip of the pipette tip and place it in the centrifuge tube as a base. Take an appropriate amount of cotton and insert it into the centrifuge tube until the base is completely fixed. After high temperature and high pressure sterilization, it can be used for subsequent experiments.

[0095] The extraction procedure was as follows: Under high humidity conditions between 9:00 AM and 3:00 PM, five rice stems were selected from each plant. The stems were scrubbed with a sterile cotton ball dipped in 75% ethanol for 60 seconds, then rinsed three times with sterile water. Excess moisture was blotted off with sterile filter paper. Scissors were sterilized with 75% ethanol and cut approximately 10 cm from the stem base. Juice exuded during the first 10 minutes was discarded. The cut was rinsed with sterile water, blotted dry with sterile filter paper, and mounted for extraction for 6 hours. The extraction apparatus was centrifuged at 15,000 × g for 5 minutes to collect the sap from the cotton. Juice collected from five stems was pooled into one sample, and four biological replicates were collected for each treatment. Endophytic microorganisms were then enriched by filtration using a sterile 0.22 μm filter.

[0096] (2) The microbial DNA on the filter membrane was extracted using a microbial DNA extraction method well known in the art, and the concentration and quality of the extracted microbial group DNA were determined using a NanoDrop spectrophotometer (NanoDrop2000, Thermo Fisher Scientific, Waltham, MA, USA). The bacterial genome was amplified using 799F / 1193R primers (799F: 5'-AACMGGATTAGATACCCKG-3' (SEQ ID NO.16, where M represents A or C, K represents G or T) and 1193R: 5'-ACGTCATCCCCACCTTCC-3' (SEQ ID NO.17)). A barcode was added to the 5' end of the 1193R primer to distinguish different samples. The PCR product was purified and recovered, quantified using a NanoDrop spectrophotometer, and then sent to Guangzhou Meige Gene Technology Co., Ltd. HiSeq 2500 platform (Illumina Inc) for high-throughput sequencing.

[0097] 16S rRNA gene sequences were processed using QIIME v.1.9.1 and USEARCH v.10.0. Illumina paired-end reads were quality-checked using FastQC v.0.11.5 and then processed using USEARCH using the following steps: concatenating paired-end reads and relabeling sequencing names; removing barcodes and primers; filtering low-quality reads; and finding non-redundant reads. Unique reads with a similarity of 97% were clustered into OTUs. Representative sequences were selected using UPARSE. OTUs were aligned to the SILVA 138 database to remove chimeras and host plastid sequences. OTU tables were generated using USEARCH. FAPROTAX v.1.2.1 was used to assign bacterial OTUs to multiple functional groups. The results showed that the relative abundance of endophytic nitrogen-fixing microorganisms in wild-type Tongjing 981 was 0.5872%, while that in the OsLPR1 / 3 / 4 / 5 mutant Tongjing 981 was 0.045%, which was an increase of 1201.58% compared with that in wild-type Tongjing 981 ( Figure 7 The number of endogenous OTUs in wild-type Tongjing 981 capable of nitrogen fixation was logarithmically transformed, i.e., log(X+1), and the result was 4.19. The number of endogenous OTUs in OsLPR1 / 3 / 4 / 5 mutant Tongjing 981 capable of nitrogen fixation was logarithmically transformed, i.e., log(X+1), and the result was 6.05, which was 44.58% higher than that in wild-type Tongjing 981 ( Figure 8 ).

[0098] Example 5

[0099] Seeds of wild-type Tongjing 981 (WT) and the osLPR1 / 3 / 4 / 5 mutant Tongjing 981 (osLPR1 / 3 / 4 / 5) obtained in Example 3 were cultivated in farmland soil 2 (collected from Hangzhou Academy of Agricultural Sciences, Zhejiang Province (30.13°N, 120.14°E)) according to conventional farmer cultivation methods. During the growth period, agronomic management was carried out according to farmers' daily management methods.

[0100] 1. Detection of nitrogen content in the aboveground part of rice

[0101] The nitrogen content of the aboveground part of rice was detected by referring to the method of Example 4. The results showed that the aboveground nitrogen content of wild-type Tongjing 981 was about 9.55 g / kg, and the aboveground nitrogen content of OsLPR1 / 3 / 4 / 5 mutant Tongjing 981 was 11.13 g / kg, which was 16.53% higher than that of wild-type Tongjing 981 ( Figure 9 The nitrogen utilization efficiency of the OsLPR1 / 3 / 4 / 5 mutant Tongjing 981 was approximately 128.06% of that of the wild type 981 ( Figure 10 ).

[0102] 2. Extraction and Detection of Rice Endophytic Microorganisms

[0103] The extraction and detection of endophytic microorganisms in rice were carried out in the same manner as in Example 4. The results showed that the relative abundance of endophytic nitrogen-fixing microorganisms in wild-type Tongjing 981 was 0.010%, and the relative abundance of endophytic nitrogen-fixing microorganisms in the OsLPR1 / 3 / 4 / 5 mutant Tongjing 981 was 0.092%, which was 776.64% higher than that in wild-type Tongjing 981 ( Figure 11 The logarithmic transformation of the number of endogenous OTUs in wild-type Tongjing 981 capable of nitrogen fixation, i.e., log(X+1), resulted in a value of 3.96. The logarithmic transformation of the number of endogenous OTUs in OsLPR1 / 3 / 4 / 5 mutant Tongjing 981 capable of nitrogen fixation, i.e., log(X+1), resulted in a value of 6.60. This was an increase of 66.76% compared to wild-type Tongjing 981 ( Figure 12 ).

[0104] Based on the above content, it can be seen that the present invention utilizes the rice OsLPR1, OsLPR3, OsLPR4 and OsLPR5 genes to regulate the nitrogen absorption of rice growth and development, regulate the nitrogen content and nitrogen utilization efficiency of the aboveground parts of rice, and regulate the colonization of nitrogen-fixing microorganisms in rice. Co-knockout of the above genes can improve the nitrogen absorption of rice growth and development, increase the nitrogen content of the aboveground parts of rice, and the relative abundance of endophytic nitrogen-fixing microorganisms in rice, thereby promoting plant nitrogen utilization.

[0105] Although the above embodiment provides a detailed description of the present invention, 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 scope of protection of the present invention.

Claims

1. Application of rice genes in one or more of the following: (1) regulating nitrogen absorption of rice; the regulating nitrogen absorption of rice is to co-knock out the rice gene to promote nitrogen absorption of rice; (2) regulating the nitrogen utilization rate of rice; the regulating the nitrogen utilization rate of rice is to co-knock out the rice gene to improve the nitrogen utilization rate of rice; (3) regulating the colonization of rice nitrogen-fixing microorganisms; the regulating the colonization of rice nitrogen-fixing microorganisms is to co-knock out the rice gene to promote the colonization of rice nitrogen-fixing microorganisms; (4) Cultivate new rice varieties; The rice genes include rice OsLPR1 gene, rice OsLPR3 gene, rice OsLPR4 gene and rice OsLPR5 gene; The nucleotide sequence of the rice OsLPR1 gene is shown in SEQ ID NO. 1; The nucleotide sequence of the rice OsLPR3 gene is shown in SEQ ID NO. 2; The nucleotide sequence of the rice OsLPR4 gene is shown in SEQ ID NO. 3; The nucleotide sequence of the rice OsLPR5 gene is shown in SEQ ID NO.

4.

2. The use according to claim 1, characterized in that The rice nitrogen absorption includes nitrogen absorption by the aboveground part of the rice; The rice nitrogen-fixing microbial colonization includes the relative abundance and / or quantity of endophytic nitrogen-fixing microorganisms in rice.

3. Use of sgRNA, knockout vector, or recombinant bacteria in one or more of the following: (1) Promote nitrogen absorption by rice; (2) Improve nitrogen utilization efficiency of rice; (3) Promote the colonization of nitrogen-fixing microorganisms in rice; (4) Cultivate new rice varieties; The nucleotide sequence of the sgRNA is shown in SEQ ID NO. 5; The knockout vector includes a basic vector and the sgRNA inserted into the basic vector; The recombinant bacteria include a basic bacteria and the sgRNA or the knockout vector introduced into the basic bacteria.

4. The use according to claim 3, characterized in that The basic vector includes pYLCRISPR.

5. The use according to claim 3, characterized in that The basic bacteria include Agrobacterium.

6. A method for promoting one or more of nitrogen absorption by rice, improving nitrogen utilization efficiency of rice, and promoting colonization of nitrogen-fixing microorganisms in rice, characterized in that: The steps include: Introduce sgRNA or knockout vector or recombinant bacteria into recipient rice tissue; The nucleotide sequence of the sgRNA is shown in SEQ ID NO. 5; The knockout vector includes a basic vector and the sgRNA inserted into the basic vector; The recombinant bacteria include a basic bacteria and the sgRNA or the knockout vector introduced into the basic bacteria.

7. The method according to claim 6, characterized in that The basic vector includes pYLCRISPR.

8. The method according to claim 6, characterized in that The basic bacteria include Agrobacterium.

9. The method according to claim 6, characterized in that The introduction method includes Agrobacterium-mediated method; the recipient rice tissue includes rice callus tissue.

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