Gene for regulating tillering, dry weight and yield of rice and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and discloses a gene for regulating tillering, dry weight and yield of rice and application of the gene. The gene is an NIP1 gene and is located on the sixth chromosome of rice, the nucleotide sequence of the NIP1 gene is as shown in SEQ ID NO.1, and the amino acid sequence coded by the NIP1 gene is as shown in SEQ ID NO.2. The NIP1 gene is cloned, a transgenic vector is constructed, an NIP1 overexpression plant is obtained, and the NIP1 overexpression plant can regulate and control tillering, dry weight and yield of rice. Through determination of overexpression plant characters, compared with control group rice, overexpression shows tillering, dry weight and yield increase characters under low-nitrogen and high-nitrogen conditions, which indicates that the NIP1 gene plays an important regulation function in the aspects of tillering, dry weight, yield and nitrogen utilization of rice.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and in particular to a gene for regulating tillering, dry weight and yield of rice and application thereof. BACKGROUND

[0002] Nitrogen is an essential mineral element for plant growth and development, and is a component of proteins, nucleic acids and cell structures, and is also involved in energy metabolism and signal transduction. Nitrogen-deficient crops exhibit dwarfism, pale leaves, and reduced photosynthetic efficiency, ultimately affecting yield. Increasing nitrogen fertilizer is considered an important measure to ensure grain yield. However, long-term overuse of nitrogen fertilizer in farmland in China has resulted in generally low nitrogen use efficiency in crops, and has caused ecological and environmental problems such as soil acidification, water eutrophication and greenhouse gas emissions, which have become a major constraint on the green transformation of agriculture. Improving nitrogen use efficiency, breeding nitrogen-efficient varieties and reducing nitrogen fertilizer use have become key approaches to achieving sustainable agricultural development.

[0003] Rice (Oryza sativa L.) is one of the most important food crops in China. Although some nitrogen-efficient genes have been discovered, they still cannot meet the needs of breeding, and the molecular network of nitrogen-efficient utilization in rice still needs to be further elucidated. Therefore, in-depth analysis of the genetic basis of nitrogen use efficiency can systematically mine regulatory genes and provide theoretical support and gene resources for nitrogen-efficient breeding. In particular, transcription factor genes are closely related to plant nitrogen utilization and are important factors affecting nitrogen use efficiency. However, the mining of excellent alleles related to nitrogen fertilizer utilization is still insufficient. Therefore, the discovery of new nitrogen-efficient genes and the elucidation of their functions are of great significance for the cultivation of nitrogen-efficient rice varieties. SUMMARY

[0004] The present application overcomes the deficiencies of the prior art and provides a gene for regulating tillering, dry weight and yield of rice and application thereof. By obtaining overexpression of the NIP1 gene, effective regulation of tillering, dry weight and yield of rice is achieved. The gene can significantly increase the tillering number, dry weight and yield of rice under low-nitrogen and / or high-nitrogen conditions, and has significant potential for yield increase and fertilizer reduction.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions. In a first aspect, a rice NIP1 gene is provided. The nucleotide sequence of the NIP1 gene is shown in SEQ ID NO. 1, and the amino acid sequence encoded by the NIP1 gene is shown in SEQ ID NO. 2.

[0006] In a second aspect, the application of the NIP1 gene in regulating tillering, dry weight and yield of rice is provided. The application includes obtaining overexpression of the NIP1 gene in plants under low-nitrogen or high-nitrogen conditions to increase the tillering, dry weight and yield of rice.

[0007] This gene can effectively reduce the amount of nitrogen fertilizer applied in the field and alleviate ecological problems such as soil compaction, acidification, and eutrophication of water bodies.

[0008] Furthermore, the NIP1 gene overexpressing plants were obtained through the following steps: The NIP1 gene as shown in SEQ ID NO.1 was cloned, and the NIP1 gene was introduced into an expression vector to construct a recombinant expression vector. The recombinant expression vector was introduced into rice recipient cells, and transgenic rice plants overexpressing the NIP1 gene were obtained after screening and cultivation.

[0009] Furthermore, the expression vector is pCAMBIA2300-35S-eGFP.

[0010] Furthermore, the rice recipient variety is Zhonghua 11.

[0011] Furthermore, the NIP1 gene is shown in SEQ ID NO.1.

[0012] Compared with the prior art, the beneficial effects of the present invention are: This invention cloned the NIP1 gene from the Zhonghua 11 rice variety and constructed an overexpression vector, obtaining corresponding transgenic plants. Phenotypic analysis showed that, regardless of whether under low or high nitrogen conditions, the overexpressing plants exhibited significantly higher tillering, dry weight, and yield than the control group (Zhonghua 11). These results clearly demonstrate that using NIP1 overexpressing plants can significantly increase yield while reducing nitrogen fertilizer application, possessing both significant economic and ecological benefits.

[0013] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0014] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[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. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a map of the pCAMBIA2300-35S-eGFP plasmid in an embodiment of the present invention; Figure 2This is an agarose gel electrophoresis image of the NIP1 gene PCR amplification product in an embodiment of the present invention; Figure 3 The results of qRT-PCR detection of gene expression levels in NIP1 gene overexpression lines in this embodiment of the invention; the t-test was used for significant difference analysis, where Relative expression is the relative expression level; Figure 4 This is a comparison of the field phenotypes of NIP1 gene overexpression lines and wild types under low nitrogen (LN) and high nitrogen (HN) conditions, as well as statistical charts of tillering, dry weight, and yield. The t-test was used to analyze the significant differences, where Tillernumber is the number of tillers, Dry weight is the dry weight, and Grain yield per plant is the yield per plant. Detailed Implementation

[0017] 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.

[0018] Example This embodiment discloses the method for obtaining transgenic rice, including the following steps: 1) Extraction of total RNA Rice plants of variety Zhonghua 11 were disinfected with a 2.5% NaClO solution, germinated, and cultured until they reached the two-leaf-one-heart stage. Uniformly sized plants were selected, endosperm removed, and transplanted into a nutrient solution (pH 5.5, 1 / 2 IRRI nutrient solution provided by the International Rice Research Institute (IRRI) – i.e., IRRI nutrient solution provided by the International Rice Research Institute diluted to 1 / 2 concentration before use). When the plants reached the four-leaf-one-heart stage, the solution was replaced with IRRI complete nutrient solution from the International Rice Research Institute. After one week of culture, roots and leaves were collected and frozen in liquid nitrogen as samples. 0.1 g of the sample was weighed, ground thoroughly with liquid nitrogen, and added to a 1.5 mL centrifuge tube. 1 mL of Trizol reagent and 0.2 mL of chloroform were added. After centrifugation, the supernatant was collected and 0.5 mL of Trizol reagent was added. Centrifuge with 1 mL of isopropanol, discard the supernatant, wash the precipitate with 70% ethanol solution, centrifuge again, discard the supernatant, and air-dry the remaining liquid in the centrifuge tube containing the precipitate. The precipitate is the extracted RNA. Dissolve the RNA in 1‰ DEPC water (v / v), and assess the RNA quality using 1 wt% agarose gel electrophoresis. Analyze the total RNA concentration and purity using a spectrophotometer. Proceed to the next step after passing the initial test.

[0019] 2) Total cDNA synthesis 2 µg of RNA sample was added to 50 µmol•L -1 Oligo 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. 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 of 25 µL was made up 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 provided by Nanjing Genscript Biotech Co., Ltd.; the reverse transcription kit was purchased from Fermentas (MBIFermentas) in Canada. The kit included DEPC water, RNase inhibitor, 5xRT buffer, dNTPs, and M-MLV reverse transcriptase.)

[0020] 3) Construction of the overexpression vector (p35S-NIP1) Based on the cDNA sequence of the NIP1 gene, PCR primers were designed. The PCR product contains the complete NIP1 gene reading frame (from start codon ATG to TGA) after the stop codon is removed. Homologous arms from the vector containing restriction endonuclease sites KpnI and XbaI were introduced into the upstream and downstream primers, respectively. The primer sequences are as follows: overNIP1-F:5'-atttggagaggacaGGGTAccATGAAGTTTGTCCCTCGTTTCCA-3'KpnI; overNIP1-R:5'-CCCTTGCTCACCATGGATCCAGATTGTGTAGGACTGGATGTCAC-3'XbaI; Using the total cDNA of Zhonghua 11 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℃ final extension for 7 min. The amplified PCR product was detected by 1wt% agarose gel electrophoresis. The PCR product size was 342 bp (e.g., ...). Figure 2 (As shown). The target PCR product was separated by agarose gel electrophoresis and then recovered by gel extraction. Simultaneously, the plant overexpression vector pCAMBIA2300-35S-eGFP plasmid was digested with KpnI and XbaI (plasmid map shown). Figure 1As shown in the figure, the enzyme-digested vector was obtained, and then the enzyme-digested vector was recovered. The enzyme-digested vector was dephosphorylated and recovered again. After recovery, the linearized vector (enzyme-digested vector) and the recovered PCR fragment were homologously recombinated with homologous recombination enzyme at 50℃ for 15 min, and then rapidly cooled on ice to obtain the pCAMBIA2300-35S-eGFP plasmid containing the NIP1 gene. This plasmid was transformed into E. coli DH5α competent cells (heat shock transformation method) and plated on a substrate containing 100 µg•mL kanamycin. -1 After growing on LB solid medium for 12 h, positive colonies were picked, and the bacterial culture was sequenced for DNA after shaking (DNA sequencing was performed by Guangzhou Youkang Biotechnology Co., Ltd., using the Sanger sequencing method, the same below). The bacterial culture containing the correctly sequenced clone was added to an equal volume of 50% glycerol and stored at -80℃. The plasmid of the positive clone was extracted and named NIP1-OE. Finally, the NIP1-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 h, positive colonies were picked, and after shaking, the bacterial culture was DNA sequenced (DNA sequencing was performed by Guangzhou Youkang Biotechnology Co., Ltd., and the sequencing method was Sanger dideoxy chain termination). The bacterial culture containing the correct sequence was added to an equal volume of 50% glycerol and stored at -80℃ for transgenic preparation. 4) Obtaining transgenic plants To avoid cytoplasmic gene mutations in rice during the transgenic process, this invention conducted transgenic experiments in different batches. From May to June 2023, Agrobacterium tumefaciens carrying the NIP1-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, different batches of T0 generation transgenic plants were obtained. To avoid changes in plant traits caused by cytoplasmic chimerism due to non-genomic insertion, this invention performed two propagations on all transgenic materials to obtain stably inherited T2 generation plants, and physiological measurements were performed on the stably inherited T2 generation materials.

[0021] The specific preparation of transgenic plants is as follows: 4.1) Agrobacterium-mediated rice transformation Callus induction: Peeled rice seeds (14 seeds per dish) were placed in an Erlenmeyer flask and soaked in 70% ethanol for 1 min (enough to submerge the seeds). The ethanol was then discarded, and the seeds were rinsed 5-6 times with sterile water. The seeds were then soaked in 2.5% NaClO solution for 30 min, followed by rinsing with sterile water 5-6 times until clear. The seeds were transferred to sterile filter paper with tweezers to absorb excess moisture. Finally, the seeds were placed on induction medium (NB medium containing 2 mg / L 2,4-D) and cultured in a 32℃ light incubator for 5 days to obtain callus tissue.

[0022] Preparation of Agrobacterium: Agrobacterium EHA105 strain carrying the appropriate vector (NIP1-OE) was streaked onto AB medium (50 mg / L kanamycin (Kan)) and incubated in the dark at 28°C for 3 days. Agrobacterium colonies were scraped off with a sterile spatula and then resuspended in AAM medium (containing 200 μmol•L). -1 As), OD600 is 0.1.

[0023] Infection and co-culture of callus: Pick rice callus from the induction medium and place it into a centrifuge tube, ensuring the callus tissue covers the conical portion of the 50 ml centrifuge tube (select pale yellow, rounded, and resilient callus tissue). Take 1 mL of the cultured Agrobacterium culture into a 1.5 mL centrifuge tube and centrifuge at 4℃ and 5000 rpm for 1 min, discarding the supernatant. Use 30 mL of inoculum containing 200 μmol•L⁻¹ acetylsylgenin (As) as the inoculum. -1 The collected bacterial cells were prepared into a suspension using AAM culture medium containing acetylsyleugenol. This suspension was then poured into the selected callus tissue and inoculated for 5 minutes. The liquid was discarded, and the callus tissue was removed and placed on a sterile culture dish containing absorbent paper to drain for 30-40 minutes. The callus tissue was then placed on co-culture medium (provided by Wuhan Boyuan Biotechnology Co., Ltd.), covered with a 9 cm layer of sterile filter paper, and incubated in the dark at 25°C for 3 days.

[0024] 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 min 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; Second round of screening: Vigorously growing callus tissue was transferred to a solution containing 50 mg•L... -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.

[0025] 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℃, 14 h light / 10 h dark). When the seedlings grow to about 5 cm, place them in a rooting medium to strengthen them.

[0026] 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 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 cultivation.

[0027] 4.2) Rapid detection of hygromycin to obtain T0 generation plants from transgenic seedlings 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 On culture medium, plants whose leaves remained bright green after 16 h / 8 h (light / dark) incubation for 48 h at 30℃ were considered positive, while negative seedlings showed patchy necrosis of leaves. Twenty positive T0 plant lines were obtained through hygromycin screening. From August to November 2023, the overexpressed materials (positive T0 plant lines) were planted in Guangzhou, Guangdong Province, and T1 generation seeds were obtained. From December 2023 to April 2024, the overexpressed materials (positive T0 plant lines) were planted in Ledong Li Autonomous County, Hainan Province, and T2 generation seeds were obtained.

[0028] 4.3) Molecular identification of NIP1 overexpression lines After T2 generation seeds germinated, two overexpression lines (NIP1-OE-1 and NIP1-OE-2) were selected. RNA was extracted from roots of transgenic seedlings (NIP1-OE-1 and NIP1-OE-2) and wild-type material Zhonghua 11 at the seedling stage (RNA extraction method as described above). Reverse transcription was performed (reverse transcription method as described above), followed by qRT-PCR (using a qRT-PCR kit, following the kit's instructions; the kit is manufactured by Beijing TransGen Biotech Co., Ltd., primers are shown below). Quantitative PCR identification was then performed (results are shown below). Figure 3 As shown in the figure, stable genetically inherited OE-1 and OE-2 transgenic lines were obtained.

[0029] qRTNIP1-F: 5'-AAAAGGGTGGGAATGGGCAA-3'; qRTNIP1-R: 5'-GGATGTCACGCAGCAAAACC-3'; Test case The aforementioned 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 wild-type (Zhonghua 11). Grouping: 48 OE-1 transgenic lines were divided into a low-nitrogen OE-1 group, 48 OE-2 transgenic lines into a low-nitrogen OE-2 group, 48 Zhonghua 11 lines into a low-nitrogen wild-type group, 48 OE-1 transgenic lines into a high-nitrogen OE-1 group, 48 OE-2 transgenic lines into a high-nitrogen OE-2 group, and 48 Zhonghua 11 lines into a high-nitrogen wild-type group. The low-nitrogen condition was 50 kg ha. -1 Urea; high nitrogen conditions: 150 kg ha -1 Urea. All other conditions were the same for the above six groups. The six groups were cultivated until rice maturity, and field phenotypes, dry weight, tillering, and yield were observed for each group. Results are as follows: Figure 3 and Figure 4 As shown. Among them, Figure 3 and Figure 4 The results for each group are the average values ​​of the relevant data for that group. Figure 3 and Figure 4 In the text, a, b, and c indicate significant differences. If the difference is significant (p<0.05), different letters are used; if the difference is not significant, the same letter is used.

[0030] from Figure 3 It can be seen that the NIP1 gene expression in the T2 generation NIP1 overexpression materials (OE-1 and OE-2) is significantly upregulated compared with the wild type (Zhonghua 11). OE-1 refers to the OE-1 transgenic line, and OE-2 refers to the OE-2 transgenic line.

[0031] from Figure 4 It can be seen that, compared with the wild type (Zhonghua 11), the T2 generation NIP1 gene overexpression materials (OE-1 and OE-2) showed better performance under low nitrogen (LN, 50 kg ha) conditions. -1 Urea) and high nitrogen (HN, 150 kg ha) -1 Under urea conditions, dry weight, tillering, and yield per unit area all increased significantly.

[0032] In summary, overexpression of NIP1 has a significant impact on tillering, dry weight, and yield.

[0033] The nucleotide sequence of the NIP1 gene is shown in SEQ ID NO.1: ATGAAGTTTGTCCCTCGTTTCCAGGATTGTTGGCGGCCTCAATCAAATGCCAGCCCACCCACGGGAAATGGGCCAGGATTGAGGCCTGCTGAACAAGGTGTAGAGGAGGAAGCAGGTCGTCCCTTGCCCAAGTTTGGTGAATGGGATGTCAACGACCCAGCGTCCGCTGAT GGATTCACAGTGATATTCAACAAAGCCAGAGATGAGAAAAAGGGTGGGAATGGGCAAGATACTGATTCACCCTGCAAAGAAACTAGGACTGAGAGGGTGGAATCATATGCCCCCAAGACAAACTCGAAGAAATGGTTTTGCTGCGTGACATCCAGTCCTACACAATCTTGA The amino acid sequence encoded by the NIP1 gene is shown in SEQ ID NO.2: MKFVPRFQDCWRPQSNASPPTGNGPGLRPAEQGVEEEAGRPLPKFGEWDVNDPASADGFTVIFNKARDEKKGGNGQDTDSPCKETRTERVESYAPKTNSKKWFCCVTSSPTQS* It is understood that the above embodiments only illustrate preferred embodiments of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can freely combine the above technical features without departing from the concept of the present invention, and can also make several modifications and improvements, all of which fall within the protection scope of the present invention. Therefore, all equivalent transformations and modifications made with respect to the scope of the claims of the present invention should fall within the scope of the claims of the present invention.

Claims

1. A rice NIP1 gene, characterized in that, The nucleotide sequence of the NIP1 gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by the NIP1 gene is shown in SEQ ID NO.

2.

2. The application of the NIP1 gene in regulating rice tillering, dry weight, and yield, characterized by: The applications include improving rice tillering, dry weight, and yield by obtaining NIP1 gene-overexpressing plants under low- or high-nitrogen conditions.

3. The application of the NIP1 gene according to claim 2 in regulating rice tillering, dry weight, and yield, characterized in that, The NIP1 gene-overexpressing plants were obtained through the following steps: The NIP1 gene as shown in SEQ ID NO.1 was cloned, and the NIP1 gene was introduced into an expression vector to construct a recombinant expression vector. The recombinant expression vector was introduced into rice recipient cells, and transgenic rice plants overexpressing the NIP1 gene were obtained after screening and cultivation.

4. The application of the NIP1 gene according to claim 3 in regulating rice tillering, dry weight, and yield, characterized in that, The expression vector is pCAMBIA2300-35S-eGFP.

5. The application of the NIP1 gene according to claim 3 in regulating rice tillering, dry weight, and yield, characterized in that, The rice recipient variety is Zhonghua 11.

6. The application of the NIP1 gene according to any one of claims 2-5 in regulating rice tillering, dry weight, and yield, characterized in that, The NIP1 gene is shown in SEQ ID NO.1.

Images