Rice nitrogen utilization efficiency gene osttg1 and application thereof

Abstract

The application relates to the technical field of plant breeding, and specifically discloses a rice nitrogen fertilizer utilization efficiency gene OsTTG1 and application thereof, a first exon mutation of the rice nitrogen fertilizer utilization efficiency gene OsTTG1, and a nucleotide sequence of the rice nitrogen fertilizer utilization efficiency gene OsTTG1, as shown in SEQ ID NO:1. The application expresses the gene OsTTG1 in a rice variety GYMM plant, finds a new use of the gene OsTTG1 in enhancing the nitrogen fertilizer utilization efficiency of the rice, and provides important gene resources for cultivating a rice variety with high nitrogen fertilizer utilization efficiency.

Description

Technical Field

[0001] This application relates to the field of plant breeding technology, and in particular to a rice nitrogen fertilizer use efficiency gene OsTTG1 and its application. Background Technology

[0002] Nitrogen is the mineral element required in the largest quantity by plants and a key limiting factor for plant growth and development. In rice production, the application of large amounts of nitrogen fertilizer has always been one of the important measures to achieve high yields. However, excessive nitrogen application not only increases production costs but also causes serious damage to the agricultural ecological environment. Improving the nitrogen fertilizer utilization efficiency in rice has become an urgent scientific problem to be solved.

[0003] Developing new rice varieties with high nitrogen fertilizer efficiency is an effective way to reduce agricultural production costs, decrease environmental pollution, and significantly increase ecological benefits. Studies have shown that the gene regulatory network for nitrogen absorption and utilization is highly complex. Although scholars both domestically and internationally have identified several genes related to nitrogen absorption, translocation, and assimilation in rice, the molecular mechanisms regulating nitrogen use efficiency remain unclear, limiting researchers' genetic improvements in rice nitrogen fertilizer efficiency. Therefore, it is essential to discover new key gene loci affecting rice nitrogen use efficiency and elucidate their molecular mechanisms regulating nitrogen use efficiency in rice, providing theoretical and technical support for molecular breeding to improve nitrogen fertilizer use efficiency in rice. Summary of the Invention

[0004] The purpose of this application is to overcome the shortcomings of the prior art and provide a rice nitrogen fertilizer utilization efficiency gene OsTTG1 and its application.

[0005] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0006] This application provides a rice nitrogen fertilizer utilization efficiency gene OsTTG1, wherein the first exon of the rice nitrogen fertilizer utilization efficiency gene OsTTG1 is mutated, and the nucleotide sequence of the rice nitrogen fertilizer utilization efficiency gene OsTTG1 is shown in SEQ ID NO: 1.

[0007] SEQ ID NO: 1 is:

[0008] ATGGAGCAGCCCAAGCCGCCGTCGGTAGCCGCCTCGGCGGCGGAGGCGCAGAACCCGAACGCCTTCACCTGCGAGCTGCCGCACTCCATCTACGCGCTGGCCTTCTCCCCCTCCGCGCCCGTCCTCGCCGCCGGCAGCTTCCTCGAGGACCTCCACAACCGCGTCTCCCTCCTCTCCTTCGACCCCGTCCACCCCACCGCCGCCTCCTTCCGCGCCCTCCCCGCGCTCTCCTTCGACCACCCCTACCCGCCCACCAAGCTCCAGTTCCACCCGCGCGCCGCCTCCGCGCCCCACCTCCTCGCCTCCTCCTCCGACGCGCTGCGGCTCTGGCTTGCCCCGCTCGACGATCTCGCCGCCACCGCCACCGCCGCCGCGCCCGAGCTCCGCTCCGTCCAACGAGGCCGAGCCCCGCCGCATCGGGACCGCCTCCATCGACACCACCTGCACCATCTGGGACATCGAGCGCGGCGTCGTCGAGACGCAGCTCATCGCGCACGACAAGGCCGTGCACGACATCGCCTGGGGGGAGAACGGCATCTTCGCCTCCGTCTCAGCCGACGGCTCCGTCCGCGTCTTCGACCTCCGGGACAAGGAGCATTCCACCATCTTCTACGAGAGCCCCCGCCCGGACACGCCGCTCCTCAGGCTGGCATGGAACCGCTATGACTTCCACTACATGGCCACCCTGCTCATGGACAGCAGCGCCGTCGTCGTGCTCGACATGCGCGCGCCCGGGGTGCCGGTGGCCGAACTACACAGACACCGGGCGTGCGCCAACGCGGTCGCTTGGGCGCCACAGGCCACGAGGCACCTTTGCTCGGCTGGGGACGACGGCCAAGCGCTGATTTGGGAGCTGCCAGCGACGCCCGGCGCAGTGCCGGCCGAGGGGATTGATCCTGTGATGGTGTATGATGCCGGTGCAGAGATAAACCAACTGCAATGGGCGGCAGCTTACCCGGAGTGGATATCGATTGCCTTTGAGAACAAGGTCCAGCTTCTCAGGGTCTGA。

[0009] The amino acid sequence encoded by the rice nitrogen use efficiency gene OsTTG1 is:

[0010] MEQPKPPSVAASAAEAQNPNAFTCELPHSIYALAFSPSAPVLAAGSFLEDLHNRVSSLLSFDPVHPTAASFRALPALSFDHPYPPTKLQFHPRAASAPHLLASSSDALRLWLAPLDDLAATATAAAPELRSVQRGRAPPHRDRLHRHHLHHLGHRARRRRDAAHRARQG RARHRLGGERHLRLRLSRRLRPRLRPPGQGAFHHLLREPPPGHAAPQAGMEPL-LPLHGHPAHGQQRRRRARHARARGAGGRTTQTPGVRQRGRLGATGHEAPLLGWGRRPSADLGAASDARRSAGRGD-SCDGV-CRCRDKPTAMGGSLPGVDIDCL-EQGPASQGL.

[0011] In this application, the OsTTG1 gene was introduced into the rice variety GYMM as the target gene to obtain T0 generation overexpressing plants of the OsTTG1 gene. After continuous self-pollination, the homozygous high-expressing T2 generation line was obtained and named the homozygous line T2-10.

[0012] The chlorate resistance (ClO3) of the homozygous line T2-10 obtained in this application and its control wild GYMM rice plants was detected at the bud stage using KClO3 solution (0.02%). - Resistance (CR), the results show that...

[0013] The wild-type GYMM plant exhibited 99.8% chlorate resistance, while the homozygous T2-10 line showed 63.9% chlorate resistance, suggesting that OsTTG1 may be involved in NO3-related processes. - Metabolic regulation.

[0014] Furthermore, when comparing the nitrate reductase activity of the homozygous line T2-10 with its control wild-type GYMM rice plant, the nitrate reductase activity of the homozygous line T2-10 was significantly higher than that of the wild-type GYMM plant.

[0015] The homozygous line T2-10 and the wild GYMM rice plant of ordinary rice showed significant differences in traits such as tiller number, growth period, plant height, and nitrogen fertilizer absorption and utilization rate.

[0016] This application provides a reagent for detecting mutations in the first exon of the rice nitrogen fertilizer use efficiency gene OsTTG1, the reagent comprising primers with nucleotide sequences as shown in SEQ ID NO: 2-3.

[0017] This application provides the application of the rice nitrogen fertilizer use efficiency gene OsTTG1 or biological materials containing the rice nitrogen fertilizer use efficiency gene OsTTG1 in plant breeding.

[0018] In a preferred embodiment of the application described in this application, the plant includes rice.

[0019] This application provides the application of the rice nitrogen fertilizer use efficiency gene OsTTG1 or biological materials containing the rice nitrogen fertilizer use efficiency gene OsTTG1 in the preparation of rice plants with enhanced nitrogen fertilizer use efficiency.

[0020] This application provides the application of the rice nitrogen fertilizer use efficiency gene OsTTG1 or biomaterials containing the rice nitrogen fertilizer use efficiency gene OsTTG1 in the preparation of rice plants with reduced chlorate sensitivity.

[0021] This application provides the application of the rice nitrogen fertilizer utilization efficiency gene OsTTG1 or biological materials containing the rice nitrogen fertilizer utilization efficiency gene OsTTG1 in the preparation of rice plants with enhanced nitrate reductase activity.

[0022] This application provides the application of the rice nitrogen fertilizer utilization efficiency gene OsTTG1 or biological materials containing the rice nitrogen fertilizer utilization efficiency gene OsTTG1 in the preparation of rice plants with improved tiller number, growth period and plant height.

[0023] Preferably, the biomaterial includes a gene editing vector.

[0024] This application expresses the gene OsTTG1 in rice cultivar GYMM and discovers a new use for enhancing nitrogen fertilizer use efficiency in rice, providing an important gene resource for breeding rice varieties with high nitrogen fertilizer use efficiency.

[0025] This application provides a method for constructing rice with improved nitrogen fertilizer use efficiency, comprising the following steps:

[0026] S1. Construct an expression vector containing the rice nitrogen fertilizer use efficiency gene OsTTG1, wherein the first exon of the rice nitrogen fertilizer use efficiency gene OsTTG1 is mutated, and the nucleotide sequence of the rice nitrogen fertilizer use efficiency gene OsTTG1 is shown in SEQ ID NO: 1.

[0027] S2. The overexpression vector is transferred into Agrobacterium competent cells to construct a genetically engineered bacterium containing the OsTTG1 gene overexpression vector;

[0028] S3. The Agrobacterium engineered bacteria were transformed into rice callus tissue to cultivate homozygous lines that overexpressed OsTTG1 protein and were stably inherited.

[0029] In a preferred embodiment of the method for constructing rice with improved nitrogen fertilizer utilization efficiency as described in this application, in step S1, primers with the nucleotide sequences shown in SEQ ID NO: 2-3 are used for amplification during the construction of the expression vector containing the rice nitrogen fertilizer utilization efficiency gene OsTTG1.

[0030] This application also utilizes transgenic technology to provide a method for constructing rice with improved nitrogen fertilizer use efficiency, and obtains homozygous plants with high nitrogen fertilizer use efficiency by overexpressing the OsTTG1 gene.

[0031] Compared with the prior art, this application has the following beneficial effects:

[0032] This application provides a rice nitrogen fertilizer use efficiency gene, OsTTG1, and its application. Mutation of exon 1 of the OsTTG1 gene in the rice variety GYMM revealed a significant difference in chlorate resistance between wild-type GYMM plants and the homozygous line T2-10 (mutant). The chlorate resistance of wild-type GYMM plants was 99.8%, while that of the homozygous line T2-10 (mutant) was 63.9%. The nitrate reductase activity of wild-type GYMM plants was lower than that of the homozygous line T2-10 (mutant). Significant differences were also found between wild-type GYMM plants and the homozygous line T2-10 (mutant) in traits such as tiller number, growth period, and plant height. Furthermore, the dynamic changes in biomass (fresh weight and dry weight) throughout the entire growth period of wild-type GYMM plants and the homozygous line T2-10 (mutant) were investigated, showing significant differences. The total nitrogen content of the aboveground parts of each rice line was determined by the semi-micro Kjeldahl method, and the nitrogen use efficiency was calculated. The nitrogen fertilizer absorption and utilization rates of wild GYMM rice plants and homozygous line T2-10 (mutant) were 4.3% and 39.6%, respectively, with significant differences. Attached Figure Description

[0033] Figure 1 The results show the sensitivity of wild GYMM plants and homozygous line T2-10 to KClO3.

[0034] Figure 2 Figure showing the comparison of nitrate reductase activity between wild GYMM plants and homozygous line T2-10 at different growth and development stages;

[0035] Figure 3 Figure showing the differences in agronomic traits, aboveground biomass, and yield per plant between wild GYMM plants and the homozygous line T2-10. Detailed Implementation

[0036] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0037] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0038]

[0039] To identify mutations in the first exon of the OsTTG1 gene in gene-edited lines, PCR amplification and sequencing were performed on the adjacent sequences of the OsTTG1 target site in T0 generation plants using primers F: CTGCCGCACTCCATCTACG (SEQ ID NO: 2) and R: CGTTCCAGTCGAAGGAGGTGA (SEQ ID NO: 3). (Primer usage conditions: 20 μL reaction system, including 10 μL 2×Taq PCR Master Mix (Beijing, Zhongkerui), 0.5 μL Forward primer (Shenzhen, BGI), 0.5 μL Reverse primer (Shenzhen, BGI), 1 μL DNA, 8 μL...) ddH2O was used. The reaction conditions were: 94℃ pre-denaturation for 5 min; 35 cycles of (94℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s). The PCR products were then detected by 1.5% agarose gel electrophoresis (Guangzhou, Shuopu Biotechnology). The products were purified using an agarose gel recovery kit (Beijing, Tiangen Biotech) and sent to a company for sequencing (Shenzhen, BGI Genomics).

[0040] The results showed that 11 positive plants had mutations in the exon regions, all resulting in amino acid frameshift mutations. In the transgenic T1 generation, the OsTTG1 target site of these plants was sequenced. Primers F: CTGCCGCACTCCATCTACG (SEQ ID NO: 2) and R: CGTTCCAGTCGAAGGAGGTGA (SEQ ID NO: 3) were used to further amplify the adjacent sequences of the OsTTG1 target site in the T1 generation plants for PCR amplification and sequencing. The primer usage conditions were as follows: a 20 μL reaction system, including 10 μL 2×Taq PCR Master Mix (Beijing, Zhongke Rui), 0.5 μL Forward primer (Shenzhen, BGI Genomics), 0.5 μL Reverse primer (Shenzhen, BGI Genomics), 1 μL DNA, and 8 μL ddH2O. The reaction conditions were: 94℃ pre-denaturation for 5 min; 35 cycles (94℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s). Then, the PCR products were detected by 1.5% agarose gel electrophoresis (Guangzhou, Shuopu Biotechnology). The PCR products were purified using an agarose gel recovery kit (Beijing, Tiangen Biotech) and sent to a company for sequencing (Shenzhen, BGI Genomics). A homozygous mutant strain (T2-10) was obtained through screening, and subsequent experiments were conducted using the homozygous line T2-10. The nucleotide sequence of the OsTTG1 gene in the homozygous line T2-10 is shown in SEQ ID NO: 1.

[0041] The amino acid sequence encoding the rice nitrogen fertilizer use efficiency gene OsTTG1 is as follows: MEQPKPPSVAASAAEAQNPNAFTCELPHSIYALAFSPSAPVLAAGSFLEDLHNRVSLLSFDPVHPTAASFRALPALSFDHPYPPTKLQFHPRAASAPHLLASSSDALRLWLAPLDDLAATATAAAPELRSVQRGRAPPHRDRLHRHHLHHLGHRARRRRDAAHRARQGRARHRLGGERHLRLRLSRRLRPRLRPPGQGAFHHLLREPPPGHAAPQAGMEPL-LPLHGHPAHGQQRRRRARHARA RGAGGRTTQTPGVRQRGRLGATGHEAPLLGWGRRPSADLGAASDARRSAGRGD-SCDGV-CRCRDKPTAMGGSLPGVDIDCL-EQGPASQGL.

[0042] Example 2: Chlorate sensitivity analysis of wild GYMM plants and homozygous line T2-10:

[0043] Due to ClO3 - With NO3 - Similar in molecular structure, it is a competitive inhibitor of nitrate reductase (NR), inhibiting the reduction of nitrate and blocking NO3. - Utilization. Meanwhile, ClO3 - Excessive accumulation of chlorate can be toxic to plants and detrimental to their normal growth. In previous studies, the applicant of this application referred to the experimental method published by Gao et al. (Gao et al., Nat Commun, 2019, 10:5027) and used KClO3 solution (0.02%) to detect the chlorate resistance (ClO3) of wild GYMM rice plants and its gene-edited line (homozygous line T2-10) at the bud stage. - resistance, CR).

[0044] The calculation formula is: CR%=(ClO3) - (Average seedling length in solution / Average seedling length in clear water) × 100.

[0045] like Figure 1 As shown, the chlorate resistance of wild-type GYMM is 99.8% ( Figure 1 -a), the chlorate resistance of the gene-edited line T2-10 was 63.9% ( Figure 1 -b). This suggests that OsTTG1 may be involved in NO3. - Metabolic regulation.

[0046] Example 3: Comparison of nitrate reductase activity between wild GYMM plants and homozygous line T2-10

[0047] Different forms of nitrogen absorbed by plant roots need to undergo a series of assimilation processes before they can be utilized by the plant. Nitrate reductase (NR) plays an important role in this process.

[0048] The applicant used 1mM NH4NO3 nutrient solution (93g NaH2PO4.2H2O, 52.4g K2SO4, 33.3g CaCl2, 122g MgCl2.6H2O, 21.3g FeNaEDTA.3H2O, 3.01g H2BO3, 1.52g MnSO4.H2O, 75mg CuSO4.7H2O, 200.5mg ZnSO4.7H2O, 24mg Na2MO4.2H2O) to cultivate wild GYMM plants and homozygous line T2-10. The applicant began sampling 20 days after seed germination of both wild GYMM plants and homozygous line T2-10, with sampling intervals of 20 days. Nitrate reductase activity was measured at various stages by mixing samples from stems, leaf sheaths, leaves, and young spikelets. Nitrate reductase activity was determined spectrophotometrically. Before 80 days, the nitrate reductase activity of the homozygous T2-10 line was significantly higher than that of the wild-type GYMM line. Figure 2 This suggests that OsTTG1 may affect the nitrate assimilation pathway in rice.

[0049] Example 4: The effect of OsTTG1 on the biomass yield and nitrogen fertilizer uptake and utilization rate of rice.

[0050] Wild GYMM plants and the homozygous line T2-10 were planted under normal nitrogen application levels (80% KNO3 + 20% (NH4)2SO4, 180 kg / ha). Significant differences were found between the wild GYMM plants and the homozygous line T2-10 in traits such as tiller number, growth period, and plant height. Figure 3 -a).

[0051] In addition, the dynamic changes in biomass (fresh weight and dry weight) of near-isogenic lines throughout the entire growth period were investigated, and the differences were significant. Figure 3 -b, Figure 3 -c). Subsequently, the applicant used the semi-micro Kjeldahl method to determine the total nitrogen in the aboveground parts of each line and calculated the nitrogen use efficiency. The nitrogen fertilizer absorption and utilization rates of wild GYMM plants and homozygous line T2-10 were 4.3% and 39.6%, respectively, with significant differences.

[0052] This application provides a rice nitrogen fertilizer use efficiency gene, OsTTG1, and its application. Mutation of exon 1 of the OsTTG1 gene in the rice variety GYMM revealed a significant difference in chlorate resistance between wild-type GYMM plants and the homozygous line T2-10 (mutant). The chlorate resistance of wild-type GYMM plants was 99.8%, while that of the homozygous line T2-10 (mutant) was 63.9%. The nitrate reductase activity of wild-type GYMM plants was lower than that of the homozygous line T2-10 (mutant). Significant differences were also found between wild-type GYMM plants and the homozygous line T2-10 (mutant) in traits such as tiller number, growth period, and plant height. Furthermore, the dynamic changes in biomass (fresh weight and dry weight) throughout the entire growth period of wild-type GYMM plants and the homozygous line T2-10 (mutant) were investigated, showing significant differences. The total nitrogen content of the aboveground parts of each rice line was determined by the semi-micro Kjeldahl method, and the nitrogen use efficiency was calculated. The nitrogen fertilizer absorption and utilization rates of wild GYMM rice plants and homozygous line T2-10 (mutant) were 4.3% and 39.6%, respectively, with significant differences.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A method for constructing rice varieties with improved nitrogen fertilizer utilization efficiency, characterized in that, Includes the following steps: S1. Construct a CRISPR / Cas9 gene knockout vector targeting the first exon of the rice OsTTG1 gene, wherein the CDS of the OsTTG1 gene is shown in SEQ ID NO:1; S2. The gene knockout vector is transferred into Agrobacterium competent cells to obtain Agrobacterium engineered bacteria containing the OsTTG1 gene knockout vector. S3. The Agrobacterium-mediated transformation of rice callus tissue was carried out, and homozygous lines with loss of OsTTG1 gene function were screened and cultivated.

2. The construction method as described in claim 1, characterized in that, The primers shown in SEQ ID NO:2~3 were used to amplify the sequences adjacent to the target site in order to identify the editing mutation sites of the OsTTG1 gene.

3. Application of knockout of rice OsTTG1 gene in improving nitrogen fertilizer use efficiency in rice, wherein the nucleotide sequence of rice OsTTG1 gene is shown in SEQ ID NO:

1.

4. The application as described in claim 3, characterized in that, The rice OsTTG1 gene knockout was achieved by mutating the first exon of the OsTTG1 gene.

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