Gene for regulating and controlling nitrogen utilization efficiency of rice and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and discloses a gene for regulating and controlling the nitrogen utilization rate of rice and application of the gene. The gene is OsDLN13, the nucleotide sequence of the gene is shown as SEQ ID NO.1, and the gene is located on a first chromosome of rice. The gene OsDLN13 can regulate and control the plant height, tillering, single plant yield and nitrogen absorption capacity of rice. By cloning the gene OsDLN13, constructing a transgenic vector, obtaining an overexpressed plant and measuring the phenotypic character of the transgenic plant, it is found that the plant height, the tiller number, the yield of a single plant and the nitrogen absorption capacity of the overexpressed plant are remarkably changed, and compared with wild type rice, the plant height, the tiller number, the yield of a single plant and the nitrogen absorption capacity of the overexpressed plant are remarkably improved. The overexpressed plant shows the characters of plant height, tillering number, single plant yield, nitrogen absorption capacity increase and the like, which indicates that the OsDLN13 gene plays an important regulation and control role in rice plant height, tillering number, single plant yield and nitrogen absorption capacity.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and discloses a gene for regulating nitrogen utilization efficiency of rice and application thereof. Background Art

[0002] The growth of rice depends on the input of large amounts of chemical fertilizers, mainly nitrogen fertilizers. However, the utilization efficiency of nitrogen fertilizers in my country is only about 30%. The nitrogen fertilizer utilization rate of rice is not high. In order to increase production, measures such as inputting large amounts of chemical fertilizers are generally taken. Long-term reliance on large amounts of chemical fertilizer input to maintain grain production not only brings serious resource waste and environmental problems, but also causes chain problems such as hardening of cultivated land, soil acidification, and decline in grain quality.

[0003] The main components of rice yield are the number of panicles per mu, the number of grains per panicle, and 1000-grain weight. Among the components of rice yield, tillering capacity is highly correlated with nitrogen fertilizer use efficiency. When nitrogen fertilizer supply increases, the number of grains per panicle and 1000-grain weight do not change significantly. However, tillering nitrogen response is the primary factor in improving nitrogen use efficiency (Liu et al., 2021). While regulating tiller number can improve rice yield and nitrogen use efficiency under conditions of reduced nitrogen fertilizer application, further research is needed to identify relevant genes. Increasing rice yield while reducing nitrogen fertilizer application is an urgent issue that needs to be addressed. Summary of the Invention

[0004] The present invention aims to address, at least to some extent, one of the problems in the related art. To this end, the present invention provides a gene for regulating nitrogen use efficiency in rice and its application, thereby improving nitrogen use efficiency in rice by utilizing the gene OsDLN13 (located on rice chromosome 1).

[0005] In order to achieve the above objectives, this application adopts the following technical solutions:

[0006] The purpose of the present invention is to provide a genetic engineering application of a gene OsDLN13, which can increase rice plant height, tillering, single plant yield and nitrogen absorption capacity.

[0007] The purpose of the present invention can be achieved through the following technical solutions.

[0008] The gene for regulating nitrogen utilization efficiency of rice provided by the present invention is OsDLN13, and its nucleotide sequence is shown in SEQ ID NO.1.

[0009] The present invention provides an amino acid for regulating the expression of a gene for nitrogen utilization efficiency in rice, and its amino acid sequence is shown in SEQ ID NO.2.

[0010] The present invention provides a recombinant expression vector into which a gene for regulating the nitrogen utilization efficiency of rice is inserted.

[0011] Furthermore, the recombinant expression vector provided by the present invention includes a pCAMBIA2300-35S-eGFP vector and a gene for regulating nitrogen utilization efficiency of rice.

[0012] The present invention provides a transgenic cell line comprising the recombinant expression vector.

[0013] Furthermore, the host cells of the transgenic cell line are rice callus cells.

[0014] The invention provides an application of the gene for regulating nitrogen utilization efficiency of rice, the recombinant expression vector, and the transgenic cell line in cultivating transgenic plants with enhanced nitrogen absorption capacity.

[0015] Furthermore, the use of the gene for regulating nitrogen utilization efficiency of rice, the recombinant expression vector, and the transgenic cell line in cultivating transgenic plants with enhanced nitrogen absorption capacity comprises the following steps:

[0016] A recombinant expression vector is constructed, the recombinant expression vector is transformed into Agrobacterium, and then the transformed Agrobacterium is used to infect host cells to obtain a transgenic cell line. Through selective culture, differentiation, rooting, and seedling hardening, the transgenic plant with enhanced nitrogen absorption capacity is obtained.

[0017] The invention provides an application of the gene for regulating rice nitrogen utilization efficiency, the recombinant expression vector, and the transgenic cell line in cultivating rice with increased plant height and / or increased tillering and / or increased yield.

[0018] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0019] The present invention cloned the OsDLN13 gene, constructed a transgenic vector, obtained overexpression plants, and measured the phenotypic traits of the transgenic plants. It was found that the plant height, tiller number, single plant yield and nitrogen absorption capacity of the overexpression plants changed significantly; under low-nitrogen and high-nitrogen conditions, the overexpression plants showed significantly increased plant height, tiller number, single plant yield and nitrogen absorption capacity compared with wild-type rice (Zhonghua 11). BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0022] In the attached figure:

[0023] Figure 1 is a map of the pCAMBIA2300-35S-eGFP plasmid in Example 1 of the present invention;

[0024] Figure 2 This is a diagram showing the electrophoresis detection results of the PCR products in Example 1 of the present invention;

[0025] Figure 3 This is a diagram showing the molecular detection results of the OsDLN13 gene overexpression material in an embodiment of the present invention;

[0026] Figure 4 Figure 2 is a field phenotype diagram of the OsDLN13 overexpressing material and the wild-type material under high nitrogen (HN) and low nitrogen (LN) conditions in the examples of the present invention;

[0027] Figure 5 Statistical graphs of plant height of OsDLN13 overexpressing materials and wild-type materials under high nitrogen (HN) and low nitrogen (LN) conditions in the examples of the present invention;

[0028] Figure 6 Statistical analysis of tiller numbers of OsDLN13-overexpressing materials and wild-type materials under high nitrogen (HN) and low nitrogen (LN) conditions in the examples of the present invention;

[0029] Figure 7 Statistical analysis of single plant yield of OsDLN13 overexpressing materials and wild-type materials under high nitrogen (HN) and low nitrogen (LN) conditions in the examples of the present invention;

[0030] Figure 8 Statistical analysis of nitrogen absorption capacity of OsDLN13 overexpressing materials and wild-type materials under high nitrogen (HN) and low nitrogen (LN) conditions in the examples of the present invention. DETAILED DESCRIPTION

[0031] The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will appreciate that the following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0032] Example 1

[0033] The acquisition of transgenic rice includes the following steps:

[0034] 1) Total RNA extraction

[0035] The rice inflorescence 11 was disinfected with a 2.5% mass percent NaClO solution and germinated. When the rice plants had two leaves and one heart, they were selected and the endosperm was removed. The rice plants were transplanted into a nutrient solution (pH = 5.5, 1 / 2 IRRI nutrient solution provided by the International Rice Research Institute (i.e., the IRRI nutrient solution provided by the International Rice Research Institute was diluted to 1 / 2 before use). When the rice plants had four leaves and one heart, the solution was replaced with the International Rice Research Institute IRRI complete nutrient solution. After one week of culture, the roots and leaves were collected and frozen in liquid nitrogen for storage as samples. 0.1 g of the sample was weighed, ground with liquid nitrogen, and thoroughly added to a 1.5 mL centrifuge tube. 1 mL of the sample was added. To the Trizol reagent, add 0.2 mL of chloroform, centrifuge, and aspirate the supernatant. Add 0.5 mL of isopropanol and centrifuge, discard the supernatant, and wash the precipitate with 70% ethanol. Centrifuge and discard the supernatant. Air-dry the remaining liquid in the centrifuge tube containing the precipitate to obtain the extracted RNA. Dissolve the RNA in 1‰ DEPC water by volume. Check the RNA quality by electrophoresis on a 1.0% by mass agarose gel, and use a spectrophotometer to determine the concentration and purity of the total RNA. If qualified, proceed to the next step.

[0036] 2) Total cDNA synthesis

[0037] 2 μg of RNA sample, add 50 μmol·L -1 Oligo dT18 was made up to 10 μL with 1‰ DEPC water, incubated at 70°C for 5 min, and placed on ice for 5 min. Then, 0.5 μL of RNase inhibitor and 5 μL of 5xRT buffer, 2.5 μL of 10 mM dNTPs, and 1 μL of M-MLV reverse transcriptase were added in sequence. 1‰ DEPC water was made up to 25 μL, and the reaction was incubated at 42°C for 60 min. Finally, the reaction was terminated by incubating at 70°C for 10 min (Oligo dT18 was provided by Nanjing GenScript Biotech Co., Ltd.; the reverse transcription kit was purchased from MBIFermentas, Canada, and the kit includes DEPC water, RNase inhibitor, 5xRT buffer, dNTPs, and M-MLV reverse transcriptase).

[0038] 3) Construction of p35S-OsDLN13 overexpression vector

[0039] Based on the cDNA sequence of the rice gene OsDLN13, PCR primers were designed. The PCR product contained the complete OsDLN13 gene reading frame (from the start codon ATG to ACA) after removing the stop codon. Homology arms on the vector containing the restriction endonuclease sites KpnI and XbaI were introduced into the upstream and downstream primers, respectively. The primer sequences are:

[0040] overOsDLN13-F:5'-gagaggacagggtaccATGGCGAGGAAGTGCTCCTA-3'

[0041] KpnI;

[0042] overOsDLN13-R:5'-tagtgtcgactctagaTGTGCCGATGGACGGCGTCGA-3'

[0043] SpeI;

[0044] The total cDNA of Zhonghua 11 obtained above was used as a template. The PCR program was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 56°C for 45 s, annealing and extension at 72°C for 2 min, 35 cycles, and complete extension at 72°C for 7 min. The amplified PCR product was detected by 1 wt% agarose gel electrophoresis. The size of the PCR product was 935 bp (as shown in FIG. Figure 2 The target PCR product was separated by agarose gel electrophoresis and then recovered by gel cutting. At the same time, the plant overexpression vector pCAMBIA2300-35S-eGFP plasmid was double-digested with KpnI and XbaI (the plasmid map is shown in Figure 1 As shown), the enzyme-cut vector was obtained, and then the enzyme-cut vector was recovered, and the enzyme-cut vector was dephosphorylated and recovered again; after recovery, the linearized vector (enzyme-cut vector) and the recovered PCR fragment were homologously recombined at 50°C for 15 minutes by homologous recombinase, and then quickly cooled on ice to obtain the pCAMBIA2300-35S-eGFP plasmid containing OsDLN13, which was transformed into Escherichia coli DH5α competent cells (heat shock transformation method) and coated on a plate containing 100 μg·mL of kanamycin. -1 After growing on LB solid medium for 12 h, positive colonies were picked and shaken for DNA sequencing (DNA sequencing was performed by Guangzhou Youkang Biotechnology Co., Ltd. using the Sanger sequencing method, the same below). The culture solution containing the correctly sequenced clones was added with an equal volume of 50% glycerol and stored at -80°C. The plasmid of the positive clone was extracted and named OsDLN13-OE.

[0045] Finally, the OsDLN13-OE plasmid was transformed into competent cells of Agrobacterium tumefaciens EHA105 by electroporation and plated on a plate containing 50 μg mL-1 of kanamycin and 50 μg mL-1 of streptomycin. -1 After growing on YEP solid medium for 48 h, positive colonies were picked and shaken to perform DNA sequencing (DNA sequencing was completed by Guangzhou Youkang Biotechnology Co., Ltd., using the Sanger dideoxy chain termination method). The culture containing the correct sequence was added with an equal volume of 50% glycerol and stored at -80°C for genetic modification.

[0046] 4) Obtaining transgenic plants

[0047] To avoid cytoplasmic gene mutations in rice during the transgenic process, the present invention conducted transgenic experiments in different batches. From July to October 2021, the Agrobacterium obtained above, carrying the OsDLN13-OE plasmid, was used to infect rice callus tissue and co-cultured for 3 days. After selective culture, differentiation, rooting, and seedling hardening of resistant callus, different batches of T0-generation transgenic plants were obtained. To avoid changes in plant traits due to cytoplasmic mosaics caused by non-genomic insertions, the present invention carried out two propagation propagations on all transgenic materials to obtain stable T2 generations, and physiological assays were performed on these stable T2 generation materials.

[0048] The specific preparation of transgenic plants is as follows:

[0049] 4.1) Agrobacterium-mediated rice transformation

[0050] Callus induction: Peeled rice seeds (14 seeds per tray) were placed in a conical flask and soaked in 70% ethanol for 1 minute (covering the seeds). The 70% ethanol was discarded, and the seeds were rinsed 5-6 times with sterile water. Then, the seeds were soaked in 2.5% NaClO solution for 30 minutes, and then rinsed 5-6 times with sterile water until clear. Using tweezers, the seeds were transferred to sterile filter paper, blotted dry, and placed on induction medium (NB medium containing 2 mg / L 2,4-D) and cultured in a 32°C light incubator for 5 days to obtain callus.

[0051] Preparation of Agrobacterium: Agrobacterium EHA105 strain carrying the corresponding vector (OsDLN13-OE) was streaked onto AB medium (50 mg / L kanamycin (Kan)) and cultured in the dark at 28°C for 3 days. Agrobacterium colonies were scraped off with a sterile spoon and then suspended in AAM culture medium (containing 200 μmol·L -1 As), OD600 was 0.1.

[0052] Infection of callus and co-cultivation: Pick out rice callus from the induction medium and place it in a centrifuge tube. The amount of callus should not exceed the conical part of the 50ml centrifuge tube (select light yellow, round and tough callus). Take 1mL of the cultured Agrobacterium solution and place it in a 1.5mL centrifuge tube. Centrifuge at 4℃ and 5000rpm for 1 minute. Remove the supernatant. -1 30mL of acetosyringone (As) solution (containing 200μmol·L -1 Prepare a suspension of collected cells using AAM medium (containing acetosyringone). Pour this suspension into the selected callus and infect for 5 minutes. Discard the liquid, remove the callus, and place it on a sterile Petri dish lined with absorbent paper to drain for 30-40 minutes. Place the callus on co-cultivation medium (provided by Wuhan Boyuan Biotechnology Co., Ltd.) and line it with a 9 cm layer of sterile filter paper. Incubate in the dark at 25°C for 3 days.

[0053] Bacteria washing and antibiotic screening culture: Remove the callus from the co-culture medium and wash it with sterile water 5 times, shaking it for 5 minutes each time. -1 Soak in sterile water containing carbenicillin (CAR) for 40-60 minutes. Finally, place on sterile filter paper to drain for 2 hours. First round of screening: Transfer the dried callus tissue to a sterile water containing 400mg·L -1 Carbenicillin (car) and 50 mg·L -1 The first selection was performed on hygromycin (Hyg) selective medium and cultured at 32°C in the light for two weeks;

[0054] Second round of screening: The actively growing callus tissue was transferred to the medium containing 50 mg·L -1 Hygromycin B and 250 mg·L -1 Differentiation was induced on carboxybenzyl differentiation medium at 28°C with continuous light for about two weeks.

[0055] Induction of differentiation and rooting of resistant callus: Pick the bright yellow resistant callus and move it into a differentiation tank filled with differentiation medium, place it in a constant temperature culture room, and wait for differentiation into seedlings (about 30 days, the culture conditions in the tissue culture room are 24-30℃, 14h light / 10h dark). When the seedlings grow to about 5cm, place them in rooting medium to strengthen the seedlings.

[0056] Hardening and transplanting of transgenic seedlings: Pick out the test tubes with relatively complete differentiation of the roots, stems and leaves of the seedlings (open the lid in time when the seedlings grow to the top of the test tube), open the sealing film, add sterile water (to prevent bacteria from growing in the culture medium), harden the seedlings for about 3 to 7 days, then wash off the agar and transplant them to the greenhouse for hydroponic or soil culture.

[0057] 4.2) Rapid detection of transgenic seedlings using hygromycin to obtain T0 generation plants

[0058] Cut and collect fresh green leaves of about 1 cm long from the seedlings to be tested (with cuts left at both ends), and place them flat on a plate containing hygromycin (80 mg·L -1 Positive plants were identified if their leaves remained bright green after 48 hours of culture at 30°C under a 16h / 8h (light / dark) cycle, while negative seedlings showed massive necrosis on their leaves. Twenty positive T0 plant lines were obtained by hygromycin screening. From November 2021 to April 2022, the overexpressing material (positive T0 plant lines) was cultivated in Ledong Li Autonomous County, Hainan Province, and T0 seeds were obtained.

[0059] 4.3) Molecular identification of OsDLN13 overexpressing strains

[0060] After T0 generation seeds germinated, two T1 generation transgenic seedlings (OsDLN13-OE-1 and OsDLN13-OE-2) were selected. At the seedling stage of 11 flowers in the transgenic seedlings (OsDLN13-OE-1 and OsDLN13-OE-2) and the wild-type material, roots were collected for RNA extraction (RNA extraction method was the same as above). After reverse transcription (reverse transcription method was the same as above), qRT-PCR was performed (using a qRT-PCR kit for the operation method, refer to the kit manual. The manufacturer of the qRT-PCR kit is Beijing Quanshijin Biotechnology Co., Ltd., and the primers are shown below) for quantitative PCR identification (the results are shown in Figure 2). Figure 3 Stable genetic transgenic lines OE-1 and OE-2 were obtained.

[0061] qRTOsDLN13-F: 5'-GCAGCATCGGCAAGAAGAA-3';

[0062] qRTOsDLN13-R: 5'-GGTCTCGTGCGTGTTCATC-3';

[0063] Test example

[0064] The above OE-1 and OE-2 transgenic lines were cultivated to the T2 generation, and then subjected to low nitrogen and high nitrogen treatments in the field respectively with the wild type (Zhonghua 11). Grouping: 48 OE-1 transgenic lines were divided into the low nitrogen OE-1 group, 48 OE-2 transgenic lines were divided into the low nitrogen OE-2 group, 48 Zhonghua 11 lines were divided into the low nitrogen wild type group, 48 OE-1 transgenic lines were divided into the high nitrogen OE-1 group, 48 OE-2 transgenic lines were divided into the high nitrogen OE-2 group, and 48 Zhonghua 11 lines were divided into the high nitrogen wild type group. Among them, the low nitrogen condition was 50 kg ha -1 Urea; 150 kg ha for high nitrogen conditions-1 Urea (fertilizers are applied once, urea is used 15 The other conditions of the above 6 groups were the same. The 6 groups were cultivated until the rice matured, and the field phenotypes, plant height, tiller number, single plant yield, and nitrogen absorption capacity of the 6 groups were observed (using 15 The N isotope labeling method was used, and then the gas chromatography-mass spectrometry (GC / MS) was used to determine the 15 N) statistics. The results are as follows Figure 4 、 Figure 5 、 Figure 6 、 Figure 7 、 Figure 8 As shown. Among them, Figure 5 、 Figure 6 、 Figure 7 、 Figure 8 The results of each group are the average values ​​of the relevant data of each group. Figure 3 、 Figure 5 、 Figure 6 、 Figure 7 、 Figure 8 a, b, and c in the table indicate significant differences. If the differences are significant (p<0.05), different letters are used; if the differences are not significant, the same letters are used.

[0065] from Figure 3 It can be seen that the gene expression of OsDLN13 in the T2 generation OsDLN13 gene overexpression materials (OE-1 and OE-2) was significantly upregulated compared with the wild type (Zhonghua 11).

[0066] from Figure 4 、 Figure 5 and Figure 6 It can be seen that the T2 generation OsDLN13 gene overexpression materials (OE-1 and OE-2) showed a significant difference compared with the wild type (Zhonghua 11) under low nitrogen (LN, 50 kg ha -1 Urea) and high nitrogen (HN, 150 kg ha -1 In the figure, ZH11 represents the wild type (Zhonghua 11), OE-1 represents the OE-1 transgenic line, and OE-2 represents the OE-2 transgenic line.

[0067] from Figure 7 It can be seen that the T2 generation OsDLN13 gene overexpression materials (OE-1 and OE-2) had significantly increased yield per plant compared with the wild type (Zhonghua 11) under low nitrogen (LN) and high nitrogen (HN) conditions.

[0068] from Figure 8It can be seen that the T2 generation OsDLN13 gene overexpression materials (OE-1 and OE-2) showed a significantly higher yield than the wild type (Zhonghua 11) under low nitrogen (LN) and high nitrogen (HN) conditions. 15 The nitrogen absorption capacity was significantly improved.

[0069] In summary, the OsDLN13 gene has a significant effect on plant height, tiller number, yield per plant and nitrogen use efficiency.

[0070] It is understandable that the above embodiments only express the preferred implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, the above technical features can be freely combined without departing from the concept of the present invention, and several deformations and improvements can be made, which all fall within the scope of protection of the present invention. Therefore, all equivalent changes and modifications made to the scope of the claims of the present invention should fall within the scope of coverage of the claims of the present invention.

Claims

1. A gene for regulating nitrogen utilization efficiency in rice, characterized in that: The gene is OsDLN13, and the nucleotide sequence of the gene OsDLN13 is shown in SEQ ID NO.

1.

2. An amino acid for regulating the expression of a gene for nitrogen utilization efficiency in rice according to claim 1, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that: The gene for regulating nitrogen utilization efficiency of rice as claimed in claim 1 is inserted.

4. The recombinant expression vector according to claim 3, characterized in that The invention comprises a pCAMBIA2300-35S-eGFP vector and the gene for regulating the nitrogen utilization efficiency of rice according to claim 1.

5. A transgenic cell line, characterized in that Comprising the recombinant expression vector according to any one of claims 3 to 4.

6. The transgenic cell line according to claim 5, characterized in that The host cells of the transgenic cell line are rice callus cells.

7. Use of the gene for regulating nitrogen utilization efficiency of rice according to claim 1, the recombinant expression vector according to any one of claims 3 to 4, and the transgenic cell line according to any one of claims 5 to 6 in cultivating transgenic plants with enhanced nitrogen absorption capacity.

8. The use according to claim 7, characterized in that The steps include: A recombinant expression vector is constructed, the recombinant expression vector is transformed into Agrobacterium, and then the transformed Agrobacterium is used to infect host cells to obtain a transgenic cell line. Through selective culture, differentiation, rooting, and seedling hardening, the transgenic plant with enhanced nitrogen absorption capacity is obtained.

9. Use of the gene for regulating nitrogen utilization efficiency of rice according to claim 1, the recombinant expression vector according to any one of claims 3 to 4, or the transgenic cell line according to any one of claims 5 to 6 in cultivating rice with increased plant height and / or increased tillering and / or increased yield.

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