Genes regulating tillering, yield per plant or nitrogen uptake capacity of rice and applications thereof

Abstract

The present application relates to the field of plant growth and development molecular biology, and particularly relates to genetic engineering application of gene OsDLN194, the gene OsDLN194 has the nucleotide sequence shown in SEQ ID NO.1, the protein sequence of the gene is SEQ ID NO.2, and the gene is located on the 7th chromosome of rice.The application of gene OsDLN194 in regulating at least one trait of rice tillering, yield per plant and nitrogen absorption.The present application obtains overexpression plants by cloning gene OsDLN194, constructing a transgenic vector, determining the phenotype of the transgenic plants, finds that the tiller number of the overexpression plants is significantly changed, and compared with wild type rice, the overexpression plants show the traits of increased tiller number, increased yield per plant, improved nitrogen absorption capacity and the like, which indicates that OsDLN194 gene plays an important regulatory function in the process of rice tillering, yield per plant and nitrogen absorption.

Description

Technical Field

[0001] This invention discloses a genetic engineering application for regulating rice tillering, yield per plant, or nitrogen uptake capacity, belonging to the field of genetic engineering technology, specifically involving applications in increasing rice tillering, yield per plant, or nitrogen uptake. Background Technology

[0002] Ensuring stable grain production is a crucial strategic issue affecting social stability, national security, and the healthy and sustainable development of the economy and society. Rice is one of my country's most important staple crops, with about two-thirds of the population relying on it as their primary food source. With my country's growing population, the rigid demand for rice relies heavily on the input of chemical fertilizers. According to statistics from the Food and Agriculture Organization of the United Nations (FAO), my country consumes more than 30% of the world's total chemical fertilizers for grain production, primarily nitrogen fertilizers. However, my country's nitrogen fertilizer utilization efficiency is only around 30%, far below the average level of developed countries (FAO, 2019). This long-term reliance on large amounts of chemical fertilizers for grain production not only leads to serious resource waste and environmental problems (Yu Fei et al., 2015), but also causes a chain reaction of problems such as soil compaction, soil acidification, and declining grain quality.

[0003] The main components of rice yield are the number of panicles per mu (667 square meters), the number of grains per panicle, and the thousand-grain weight (Sun et al., 2014). Among these components, tillering ability is highly correlated with nitrogen fertilizer use efficiency (Liu et al., 2021). When nitrogen fertilizer supply increases, the number of grains per panicle and the thousand-grain weight do not change significantly, while tillering nitrogen response ability is the main factor in improving nitrogen use efficiency (Liu et al., 2021). Under conditions of reduced nitrogen fertilizer application, it is feasible to increase yield and nitrogen use efficiency by increasing the number of tillers in rice (Wu et al., 2020; Liu et al., 2021). Therefore, discovering new genes that promote tillering and improve nitrogen fertilizer use efficiency is also of great significance for breeding new green rice varieties that are fertilizer-saving, high-yielding, and green. Summary of the Invention

[0004] The purpose of this invention is to provide a genetic engineering application of the gene OsDLN194, which can promote rice tillering, increase yield per plant, and improve nitrogen fertilizer utilization efficiency.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] A gene that regulates rice tillering, yield per plant, or nitrogen uptake, wherein the gene is OsDLN194, and the sequence of the gene OsDLN194 is shown in SEQ ID NO.1.

[0007] The amino acid sequence of the gene OsDLN194 is shown in SEQ ID NO.2.

[0008] An overexpression vector containing the gene OsDLN194.

[0009] Application of the gene OsDLN194 in regulating rice tillering, yield per plant, or nitrogen uptake.

[0010] Application of an overexpression vector for the gene OsDLN194 in regulating rice tillering, yield per plant, or nitrogen uptake.

[0011] Beneficial effects:

[0012] This invention, through the construction of OsDLN194 overexpression material, found that compared with wild-type rice (Zhonghua 11), the overexpression plants had a significantly increased number of tillers under both low-nitrogen and high-nitrogen conditions.

[0013] This invention, through the construction of OsDLN194 overexpression material, found that compared with wild-type rice (Zhonghua 11), the overexpression plants showed a significant increase in yield per plant under both low-nitrogen and high-nitrogen conditions.

[0014] This invention, through the construction of OsDLN194 overexpression material, found that compared with wild-type rice (Zhonghua 11), the overexpression plants have significantly increased nitrogen uptake capacity under both low-nitrogen and high-nitrogen conditions. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0016] Figure 1 This is a graph showing the molecular detection results of the OsDLN194 gene overexpression material in Example 1 of this invention;

[0017] Figure 2 The images show the field phenotypic figures of the OsDLN194 overexpression material and the wild-type material under high nitrogen (HN) and low nitrogen (LN) conditions in the embodiments of the present invention.

[0018] Figure 3 The tiller number of OsDLN194 overexpression material and wild-type material in the embodiments of the present invention under high nitrogen (HN) and low nitrogen (LN) conditions;

[0019] Figure 4 The yield per plant of OsDLN194 overexpression material and wild-type material under high nitrogen (HN) and low nitrogen (LN) conditions in the embodiments of the present invention;

[0020] Figure 5This refers to the nitrogen uptake capacity of the OsDLN194 overexpression material and the wild-type material under high nitrogen (HN) and low nitrogen (LN) conditions in the embodiments of the present invention. Detailed Implementation

[0021] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0022] Example 1

[0023] Obtaining genetically modified rice involves the following steps:

[0024] 1) Extraction of total RNA

[0025] Rice plants of the Zhonghua 11 variety were sterilized with 30% NaClO, germinated, and cultured until they reached the two-leaf-one-heart stage. Uniformly sized plants were selected, endosperm removed, and transplanted into a 1 / 2 pH 5.5 IRRI nutrient solution from the International Rice Research Institute. When the plants reached the four-leaf-one-heart stage, the solution was replaced with the IRRI complete nutrient solution from the International Rice Research Institute (Mao D R. The methods of plant nutrition research. Beijing: Beijing Agricultural University Press, 1994). After one week of culture, roots and leaves were quickly frozen in liquid nitrogen. Approximately 0.1g of the sample was weighed, ground with liquid nitrogen, and added to a 1.5mL centrifuge tube. 1mL of Trizol reagent and 0.2mL of chloroform were added. After centrifugation, the supernatant was collected, 0.5mL of isopropanol was added, and after centrifugation, the supernatant was discarded. The precipitate was washed with 70% ethanol. RNA was dissolved in DEPC water (1‰ v / v). RNA quality was detected by 1.0% agarose gel electrophoresis, and the concentration and purity of total RNA were determined using a spectrophotometer. If you pass the test, proceed to the next step.

[0026] 2) Total cDNA synthesis

[0027] 2 μg of each RNA sample was added to 50 μmol·L⁻¹ -1Oligo dT18 was added to a final volume of 10 μL with 1‰ DEPC water. The mixture was incubated at 70°C for 5 min, then placed on ice for 5 min. Next, 0.5 μL of RNase inhibitor, 5 μL of 5xRT buffer, 2.5 μL of 10 mM dNTPs, and 1 μL of M-MLV reverse transcriptase were added sequentially. The final volume was brought to 25 μL with 1‰ DEPC water. The mixture was incubated at 42°C for 60 min, then incubated at 70°C for 10 min to terminate the reaction. (Oligo dT18 was synthesized by GenScript Pharmaceuticals in Nanjing; the reverse transcription kit was purchased from Fermentas, Canada.)

[0028] 3) Obtaining the full-length cDNA of the OsDLN194 gene

[0029] Using the total cDNA obtained from rice Zhonghua 11 as a template, PCR primers were designed. The PCR product contained the complete OsDLN194 reading frame (from the start codon ATG to TGA). The primer sequences are as follows:

[0030] OsDLN194-F:5'-ATGGATGCGGGGGGAGTGGA-3';

[0031] OsDLN194-R:5'-TCAGTTCACGCCAACCTCCGATG-3';

[0032] The PCR program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 56℃ annealing for 45 s, 72℃ annealing and extension for 2 min, 35 cycles, followed by 72℃ for 7 min. The amplified PCR product was detected by 1% agarose gel electrophoresis, and its size was a 1272 bp fragment. After separation by agarose gel electrophoresis, the target PCR product was excised and recovered. The recovered fragment was ligated with the P-easy blunt vector. The total volume of the enzyme ligation system was 5 μL (containing 1 μL of vector and 4 μL of purified PCR product). After adding the sample, the mixture was thoroughly mixed, centrifuged to the bottom of the tube, and incubated at 28℃ for 15 min.

[0033] The enzyme-linked immunosorbent assay (ELISA) system was heat-transferred into *E. coli* DH5α competent cells at 42°C. 500-700 μL of antibiotic-free LB broth was added and the cells were shaken for 1 hour. The cells were then centrifuged at low speed to enrich them, and the mixture was plated onto a plate containing 100 μg / mL kanamycin. -1 After growing on LB solid medium for 12-14 hours, positive colonies were picked for DNA sequencing. The full-length open reading frame (ORF) of OsDLN194 was 1272 bp. The correctly sequenced bacterial solution was added to an equal volume of 50% glycerol and stored at -70℃ for later use. The P vector containing the OsDLN194 ORF was named pOsDLN194inP.

[0034] 4) Construction of pUbi-OsDLN194 overexpression vector

[0035] Based on the cDNA sequence of the rice gene OsDLN194, PCR primers were designed. The PCR product contains the complete reading frame of the OsDLN194 gene (from the start codon ATG to the stop codon TGA). Restriction endonuclease sites KpnI and SpeI were introduced into the upstream and downstream primers, respectively. The primer sequences are as follows:

[0036] overOsDLN194-F:5'-cggGGTACCATGGATGCGGGGGGAGTGGA-3'KpnI;

[0037] overOsDLN194-R:5'-ggACTAGTTCAGTTCACGCCAACCTCCGAT G-3'SpeI;

[0038] Using the pOsDLN194inP plasmid obtained above as a template, the PCR program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 56℃ annealing for 45 s, 72℃ annealing extension for 2 min, 35 cycles, followed by 72℃ for 7 min. The amplified PCR product was detected by 1% agarose gel electrophoresis, and the PCR product size was approximately 1272 bp. The target PCR product was separated by agarose gel electrophoresis and recovered by gel excision. The recovered product was digested with restriction endonucleases KpnI and SpeI. Simultaneously, the plant overexpression vector pTCK303 plasmid was double-digested with KpnI and SpeI. The digested PCR fragment and vector were then recovered separately. The vector was dephosphorylated and recovered again. After recovery, the linearized vector and digested PCR fragment were ligated using T4 ligase at 16℃ overnight. The ligation was then transformed into *E. coli* DH5α competent cells and plated onto a plate containing 50 μg / mL kanamycin. -1 After growing on LB solid medium for 12 hours, positive colonies were picked, plasmids were extracted, and the fragment size was verified by KpnI and SpeI enzyme digestion. The bacterial culture was then sequenced for DNA. The bacterial culture containing the correctly sequenced clone was added to an equal volume of 50% glycerol and stored at -70℃. The positive clone plasmid was extracted and named OsDLN194-OE.

[0039] Finally, the OsDLN194-OE plasmid was transformed into competent Agrobacterium tumefaciens EHA105 cells by electroporation, and the cells were plated on a substrate containing 50 μg / mL kanamycin and streptomycin. -1 After growing on YEP solid medium for 48 hours, positive colonies were picked, plasmids were extracted, and after double digestion with KpnI and SpeI to confirm that they were correct, the bacterial culture was added with an equal volume of 50% glycerol and stored at -70℃ for transgenic use.

[0040] 5) Obtaining transgenic plants

[0041] To avoid cytoplasmic gene mutations in rice during the transgenic process, we conducted transgenic experiments in different batches. From July to October 2021, Agrobacterium tumefaciens carrying the OsDLN194-OE plasmid obtained above was used to infect rice callus tissue and cultured for 3 days. After selection, differentiation, rooting, and hardening of resistant callus tissue, T0 generation transgenic plants from different years and batches were obtained. To avoid changes in plant traits caused by cytoplasmic chimerism due to non-genomic insertion, we propagated all transgenic materials twice to obtain stably inherited T2 generation plants, and then performed physiological measurements on the stably inherited T2 generation plants.

[0042] The specific preparation of transgenic plants is as follows:

[0043] 5.1) Agrobacterium-mediated rice transformation

[0044] Callus induction: Peeled rice seeds (14 seeds per dish) are placed in an Erlenmeyer flask and soaked in 70% ethanol for 1 minute (enough to submerge the seeds). The 70% ethanol is then discarded, and the seeds are rinsed 5-6 times with sterile water. Next, the seeds are soaked in 30% sodium hypochlorite solution for 30 minutes, followed by rinsing with sterile water 5-6 times until the solution is clear. The seeds are then transferred to sterile filter paper with tweezers to absorb excess moisture. Finally, the seeds are placed on the induction medium and cultured in a 32℃ light incubator for 5 days.

[0045] Preparation of Agrobacterium: Agrobacterium strain EHA105 with the appropriate vector was streaked onto AB medium (50 mg / L Kan) and incubated in the dark at 28°C for 3 days. The Agrobacterium colonies were scraped off with a sterile spoon and resuspended in AAM medium (containing As), with an OD600 of approximately 0.1.

[0046] Infection and co-culture of callus: Pick rice callus from the subculture medium and place it into a centrifuge tube, ensuring the callus tissue covers the conical portion of the 50ml centrifuge tube (select pale yellow, rounded, and resilient callus tissue). Transfer 1mL of the cultured bacterial solution to a 1.5mL centrifuge tube, centrifuge at 5000rpm for 1 min at 4℃, and discard the supernatant. Use a solution containing 200μmol·L⁻¹... -1 The collected bacterial cells were prepared into a suspension using 30 mL of acetylsylgenone (As) inoculum. This suspension was then poured into the selected callus tissue and incubated for 5 minutes. The liquid was discarded, and the callus tissue was removed and placed on a sterile culture dish lined with absorbent paper to drain for 30-40 minutes. The callus tissue was then placed on a co-culture medium (with a 9 cm layer of sterile filter paper on top) and incubated in the dark at 25°C for 3 days.

[0047] Bacterial washing and antibiotic screening culture: The callus tissue was removed from the co-culture medium and rinsed 5 times with sterile water, shaking continuously for 5 minutes each time. Then it was rinsed with a solution containing 500 mg / L... -1 Soak the callus tissue in sterile water with carbenicillin (CAR) for 40-60 minutes. Finally, drain on sterile filter paper for 2 hours. First round of screening: Transfer the dried callus tissue into a solution containing 400 mg / L... -1 Carbenicillin (CAR) and 50 mg / L -1 The first selection was carried out on hygromycin (Hyg) selective medium, and cultured at 32°C under light for two weeks;

[0048] Second round of screening: Transferring vigorous callus to a solution containing 50 mg·L⁻¹ -1 Hygromycin B and 250 mg·L -1 Differentiation was induced on carboxybenzyl differentiation medium and continuously illuminated at 28°C for about two weeks.

[0049] Induction and rooting of resistant callus: Select bright yellow resistant callus and transfer it into a differentiation tank containing differentiation medium. Place it in a constant temperature culture room and wait for it to differentiate into seedlings (about 30 days, culture conditions in the tissue culture room are 24-30℃, 14h light / 10h dark). When the seedlings grow to about 5cm, place them in a rooting medium to strengthen them.

[0050] Hardening and transplanting of transgenic seedlings: Select test tubes with well-differentiated roots and stems (open the cap in time when the seedlings grow to the top of the test tube), open the sealing film, add an appropriate amount of sterile water (to prevent bacterial growth on the culture medium), harden the seedlings for about 3 to 7 days, then wash off the agar and transplant them to a greenhouse for hydroponic or soil culture for growth and testing.

[0051] 5.2) Rapid detection of hygromycin to obtain T0 generation plants from transgenic seedlings

[0052] Cut and collect fresh green leaves about 1 cm long from the seedlings to be tested (leaving cuts at both ends), and lay them flat on a container containing hygromycin (80 mg / L). -1 Plants whose leaves remained bright green after culturing at 30℃ for 16h / 8h (light / dark) for 48h on culture medium were considered positive, while negative seedlings showed patchy necrosis of leaves (Zheng Ye. Establishment and Application of High-Efficiency Transgenic Rice System. 2008). Twenty positive T0 lines were obtained through hygromycin screening. From November 2021 to April 2022, the overexpressed materials were planted to obtain T0 generation seeds.

[0053] 5.3) Molecular identification of OsDLN194 overexpression lines

[0054] After the T0 generation seeds germinated, T1 generation transgenic seedlings were obtained. RNA was extracted from the sword leaves of the transgenic material OsDLN194-OE and the wild-type material Zhonghua 11 at the tillering stage. After reverse transcription, qRT-PCR was performed and quantitative PCR was conducted to identify the stable genetically inherited OE-1 and OE-2 transgenic lines.

[0055] Test case

[0056] from Figure 2 and 3 It can be seen that the number of tillers in the T2 generation OsDLN194 gene overexpression materials (OE-1, OE-2) was significantly increased compared with the wild type (Zhonghua 11) under both low nitrogen (LN) and high nitrogen (HN) conditions.

[0057] from Figure 4 It can be seen that the T2 generation OsDLN194 gene overexpression materials (OE-1, OE-2) showed a significant increase in single-plant yield compared with the wild type (Zhonghua 11) under both low nitrogen (LN) and high nitrogen (HN) conditions.

[0058] from Figure 5 It can be seen that, compared with the wild type (Zhonghua 11), the T2 generation OsDLN194 gene overexpression materials (OE-1, OE-2) showed better performance under both low nitrogen (LN) and high nitrogen (HN) conditions. 15 The nitrogen absorption capacity is significantly improved.

[0059] In summary, the OsDLN194 gene has a significant impact on tiller number, yield per plant, and nitrogen use efficiency (i.e., nitrogen uptake capacity and yield per plant).

[0060] Although the invention has been illustrated and described with reference to specific embodiments, it should be understood that many other changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, it is intended that all such changes and modifications falling within the scope of the invention be included in the appended claims.

Claims

1. Application of a gene OsDLN194 overexpression vector in promoting the number of tillers, yield per plant or nitrogen absorption capacity of rice, characterized in that, The sequence of the gene OsDLN194 is shown as SEQ ID NO.

1. The sequence of the gene OsDLN194 is shown as SEQ ID NO.

1. The sequence of the

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