Application of indica rice OsRNU5 gene

Abstract

The invention discloses an application of an indica rice OsRNU5 gene. The indica rice OsRNU5 gene is applied to improving the nitrogen fertilizer utilization efficiency and yield of rice by regulating and controlling the nitrate nitrogen absorption rate. The difference on the indica rice OsRNU5 gene promoter is one of reasons for causing utilization efficiency and yield of indica rice and japonica rice nitrogen fertilizer, and the indica rice OsRNU5 gene promoter sequence is shown as SEQ ID NO.3. The QTL analysis is carried out on hundreds of replacement line materials carrying IRAT261 single fragments under the background of Huajing indica 74, and the site qRNU5 influencing the nitrate nitrogen absorption rate is positioned. The expression level of the OsRNU5 haplotype gene in indica rice is high, and the nitrate nitrogen absorption rate is high. The excellent allelic variation of the OsRNU5 in the indica rice is introduced into the japonica rice, so that the nitrogen fertilizer utilization efficiency and yield of the japonica rice can be improved.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology and relates to the application of the OsRNU5 gene in indica rice to regulate nitrogen fertilizer use efficiency and yield in rice. Background Technology

[0002] Rice (Oryza sativa L.) is a vital food crop, accounting for more than a quarter of the world's total food production and providing staple food for over half of the global population. Since the 20th century, the world's population has grown rapidly, and it is estimated that by 2050, the world population will approach 9 billion. This means that food production needs to increase by at least 50% to meet the needs of the growing population (Ashikari et al., 2005; Khush, 2001). However, with the increasing industrialization of human society, the natural environment upon which humanity depends for survival has been significantly damaged. Coupled with frequent natural disasters in recent years, the area under rice cultivation on Earth has been decreasing year by year, and water resources are becoming increasingly scarce. Therefore, ensuring food security has become an urgent problem for countries around the world.

[0003] Rice yields have experienced two significant increases to date. In the early 1960s, the first "Green Revolution" was launched, characterized by semi-dwarf breeding. The semi-dwarf gene (sd1) was widely used in indica rice breeding, resulting in short-stalked, fertilizer-tolerant, and lodging-resistant rice varieties, leading to a substantial increase in rice yield (Sasaki et al., 2002). In 1973, the successful development of three-line hybrid rice breeding, utilizing heterosis, led to the widespread adoption of hybrid rice, further increasing rice yields. Both of these significant yield increases relied heavily on the input of chemical fertilizers. While fertilization can increase crop yields, it is important to note that although the yields of semi-dwarf rice varieties and hybrid rice were significantly increased, the rice's growth and development response to nitrogen fertilizer was significantly weakened, greatly reducing the rice's nitrogen use efficiency (NUE) (Pingali, 2012).

[0004] Fertilization is one of the main measures to increase crop yield, and nitrogen fertilizer is the most important fertilizer source, accounting for about 60% of the total fertilizer application (Wang et al., 2018). Nitrogen is one of the mineral nutrients most required by plants and a key factor limiting crop growth, development, and yield. Many important organic compounds in plants, such as proteins, nucleic acids, chlorophyll, enzymes, vitamins, and alkaloids, cannot function without nitrogen, playing a vital role in plant life activities. In agricultural production, nitrogen deficiency leads to poor plant development and ultimately reduced yield. Therefore, in recent decades, increasing nitrogen fertilizer application has become an important means for my country to pursue high crop yields. Since 1961, nitrogen fertilizer application has increased more than eightfold, reaching 2.311 × 10⁷ t in 2017 (Lu & Tian, ​​2017). However, the continuous increase in nitrogen fertilizer application has not led to a significant increase in crop yield. On the contrary, the indiscriminate application of large amounts of nitrogen fertilizer is not only toxic to crops but also leads to a decrease in nitrogen fertilizer utilization efficiency. China's annual nitrogen fertilizer consumption is currently three times the world average, but its nitrogen fertilizer utilization rate is only around 40.2% (Ministry of Agriculture and Rural Affairs). Excessive nitrogen fertilizer application not only increases energy consumption and agricultural production costs, posing a significant obstacle to sustainable agricultural production, but also causes a series of environmental problems, such as declining air, soil, and water quality, and greenhouse gas emissions (Wang, Hsu & Tsay, 2012). Therefore, given the continued need to increase crop yields, reducing nitrogen fertilizer use and improving nitrogen fertilizer utilization efficiency are of great significance for achieving sustainable agricultural development that conserves resources, increases yields and efficiency, and protects the environment.

[0005] Various forms of nitrogen sources exist in soil, including nitrate nitrogen (NO3). － ), ammonium nitrogen (NH4) + Plants absorb and utilize inorganic nitrogen, including urea and amino acids, and organic nitrogen, including urea and amino acids. However, in the soil environment, available organic nitrogen is scarce, so plants primarily absorb and utilize NO3. － and NH4 + Two major inorganic nitrogen sources. Among them, NO3... － It is the most easily absorbed nitrogen nutrient by plants, and under normal natural conditions, NH4 in the soil... + NO3 will be formed through nitrification by microorganisms. － Approximately 40% of the nitrogen in rice is absorbed and utilized by plants as NO3. － The form is absorbed, therefore for NO3 － Therefore, research on metabolic mechanisms is essential (Li, Fan & Shen, 2008). NO3 －Not only can NO3- be supplied to plants as a nutrient for absorption and utilization, but it can also participate in plant growth, development, and morphogenesis as a signaling molecule (Krouk et al., 2010). Therefore, a deeper understanding of plant NO3- is crucial. － The physiological and molecular mechanisms of absorption, translocation, and assimilation are of great significance for improving the efficiency of nitrogen fertilizer absorption and utilization in crops and achieving high quality and high yield. Summary of the Invention

[0006] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing an application of the OsRNU5 gene in indica rice.

[0007] Another object of the present invention is to provide a marker related to nitrogen fertilizer utilization efficiency in rice.

[0008] Another objective of this invention is to provide a method for improving the nitrogen fertilizer utilization efficiency of rice.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] Application of the OsRNU5 gene in indica rice in improving nitrogen fertilizer use efficiency and yield in rice. The nucleotide sequence of the OsRNU5 gene in indica rice is shown in SEQ ID NO.1.

[0011] Application of the OsRNU5 promoter in rice to improve nitrogen fertilizer use efficiency and yield in rice. The promoter sequence is shown in SEQ ID NO.2.

[0012] A marker associated with nitrogen fertilizer use efficiency in rice is an 8 bp sequence (TGTGTGTA) located at 431-438 bp in the promoter of the OsRNU5 gene shown in SEQ ID NO.2; this marker is present in indica rice and is associated with the efficiency of nitrate nitrogen (NO3) in the roots. - It has strong absorption capacity and high nitrogen fertilizer utilization efficiency; the marker mentioned above is missing in japonica rice, resulting in low expression of the OsRNU5 gene and NO3- in the roots. - It has weak absorption capacity and low nitrogen fertilizer utilization efficiency.

[0013] A method for determining nitrogen fertilizer use efficiency in rice involves detecting the promoter of the rice OsRNU5 gene. If the marker is present, the rice variety has high nitrogen fertilizer use efficiency; if the marker is missing, the rice variety has low nitrogen fertilizer use efficiency.

[0014] As a preferred embodiment of the present invention, the PCR primers used to detect the 8bp marker of the rice OsRNU5 gene promoter are shown in SEQ ID NO.6 and SEQ ID NO.7.

[0015] A method for improving nitrogen fertilizer use efficiency in rice involves enhancing the markers described in this invention within the rice. Using this method, both nitrogen fertilizer use efficiency and yield in rice can be improved.

[0016] OsRNU5, as a transcription factor, regulates NO3-. - This improves the absorption of nitrogen fertilizer and increases its utilization efficiency.

[0017] Beneficial effects:

[0018] This invention utilizes over one hundred single-fragment substitution system materials from IRAT261 in the background of Huajingxian 74 (HJX74). C hromosome Segment Substitution L ines, CSSLs) to conduct NO3 - Absorption rate detection identified a regulator of NO3. - Material 034, whose absorption rate decreased significantly, was backcrossed with HJX74 to construct a genetic population. Map-based cloning was used to locate the regulator of NO3-. - The key gene for uptake is RNU5 (Regulator of Nitrate Uptake 5). This invention also discovered OsRNU5 in the HJX74 variety. HJX74 Compared to the 1.5kb promoter (SEQ ID NO.2) of the gene, the allele OsRNU5 IRAT261 A single 8bp deletion fragment is predominantly present in its 1.5kb promoter (SEQ ID NO.4). The inventors sequenced and analyzed the promoter region within 2kb of the OsRNU5 gene in 12 indica and 12 japonica rice samples from their laboratory. The results showed that the 8bp deletion in the promoter was absent in japonica rice but present in indica rice. This 8bp deletion leads to low expression of the OsRNU5 gene, thus reducing NO3- expression in indica rice roots. - Increased absorption capacity. Therefore, this sequence can serve as a marker for judging nitrogen fertilizer use efficiency in rice. Attached Figure Description

[0019] Figure 1 The study showed that NO3 was present in single-segment substitution lines (SSSLs) constructed from indica rice HJX74 and japonica rice IRAT261. - Materials with significantly reduced absorption rates. (a) Field phenotype of a single-fragment material; (b) NO3 in a single-fragment material. - Absorption rate; HJX74 is an abbreviation for Huajingxian 74.

[0020] Figure 2 Map-based cloning of candidate gene OsRNU5.

[0021] Figure 3The OsRNU5 gene promoter exhibits differences between indica and japonica rice varieties. (a) INDEL analysis of the OsRNU5 promoter region in 12 indica and 12 japonica rice varieties; (b) The effect of INDEL on the transcriptional level of OsRNU5; (c) and (d) Based on the INDEL of the OsRNU5 promoter region, 3010 rice accessions were divided into three haplotypes, with indica rice being Hap. I and III, and japonica rice mainly being Hap. II.

[0022] Figure 4 Validation of OsRNU5 transgenic materials. (a) Transcription level; (b) Mutation location of knockout materials; (c) Nitrate uptake rate of OsRNU5 transgenic materials.

[0023] Figure 5 Field phenotypic analysis of OsRNU5 transgenic materials. (a) Field phenotype of OsRNU5 transgenic materials; (b) Plant height; (c) Tillers; (d) Number of primary branches; (e) Number of secondary branches; (f) Number of grains per spike; (g) Yield per plant; Detailed Implementation

[0024] In the following examples, HJX74 is the abbreviation for the indica rice variety Huajingxian 74, and ZH11 is the abbreviation for Zhonghua 11.

[0025] Example 1: Screening for NO3 in rice SSSLs materials - Materials with significant differences in absorption rate

[0026] Previous laboratory studies have found that NO3 in the roots of indica rice variety HJX74 and japonica rice variety IRAT261 - Absorption capacity varies. Therefore, the inventors constructed Single Segment Substitution Lines (SSSLs) materials under the HJX74 background and conducted hydroponic experiments on over one hundred SSSLs to determine the effects of different SSSLs on absorption capacity. 15 N-labeled NO3 - Absorption rate was determined, and QTL analysis was performed. NO3 -The specific method for determining the absorption rate was as follows: HJX74 and SSSLs seeds were disinfected with a 20% sodium hypochlorite solution for 30 minutes. Afterwards, they were placed in a 37℃ incubator for 24 hours to absorb water and swell. The water was drained, and the seeds were transferred to a 28℃ incubator for germination. Once the seeds showed signs of germination, they were transferred to perforated 96-well plates and cultured for 7 days. Seedlings with uniform growth were selected and transferred to a 40L nutrient solution containing (1.25mM NH4NO3, 0.5mM NaH2PO4·2H2O, 0.75mM K2SO4, 1mM CaCl2, 1.667mM MgSO4·7H2O, 40μM Fe-EDTA(Na), 19μM H3BO3, 9.1μM MnSO4·H2O, 0.15μM ZnSO4·7H2O, 0.16μM CuSO4, and 0.52μM (NH4)3Mo7O). 24 The solution is placed in a blue box containing 4H2O (pH 5.5). When treating with different nitrogen concentrations, the 1N (1.25mM NH4NO3) in the standard nutrient solution needs to be replaced with 0.6N (0.75mM NH4NO3), 0.3N (0.375mM NH4NO3), and 0.15N (0.1875mM NH4NO3). The solution is cultured for 4 weeks, with the pH adjusted every 2 days.

[0027] After 4 weeks of cultivation, the rice roots were immersed in 0.1 mM CaSO4 for 1 minute, and then transferred to a solution containing 2.5 mM K. 15 Immerse the roots in a nutrient solution containing NO3 for 5 minutes, then transfer to 0.1 mM CaSO4 for 1 minute. Blot the roots dry with filter paper or gauze, cut off the roots, dry them, grind them, and then measure the moisture content. 15 Nitrogen content (computed by Li Yuzhong's laboratory at the Chinese Academy of Agricultural Sciences, using an Isoprime 100 instrument).

[0028] The results showed that the material SSSL-034, carrying the candidate fragment, exhibited a field phenotype characterized by reduced plant height and fewer tillers. Figure 1 a) The nitrate nitrogen uptake rate of SSSL-034 roots decreased significantly. Figure 1 b).

[0029] Example 2: Identification of NO3- causative agents - The key site for differences in absorption rate is OsRNU5.

[0030] In order to precisely target and regulate NO3 - For candidate genes with differential uptake rates, we backcrossed SSSL-034 and HJX74 to construct a finely mapped population. The candidate genes were mapped to a length range of 4.7 kb, between molecular markers BJ18 and BJ19. Figure 2According to the Gramene website, only one gene exists in this segment, which we named OsRNU5.

[0031] The molecular markers used in this study were PCR-based, including SSR markers and self-designed InDel markers. All SSR markers were derived from the microsatellite marker linkage map published by McCouch et al. (2001, 2002). STS markers were selected by analyzing clone sequences using an SSR analysis tool (http: / / www.gramene.org / gramene / searches / ssrtool) to identify SSR target sequences with good microsatellite reproducibility, and then primers were designed for these target sequences using Primer5 analysis software. The polymorphic marker primer sequences used for fine mapping and map-based cloning are detailed in Table 1.

[0032] Table 1

[0033]

[0034]

[0035] The PCR procedure was slightly modified from the method of Panaud et al. (1996). Specifically, each 20 μL amplification reaction tube contained: 0.15 μM SSR primers, 200 μM dNTPs, 1× PCR reaction buffer (50 mM KCl, 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 0.01% gelatin), 50–100 ng template DNA, and 1 U Taq enzyme. The reaction program was: DNA denaturation at 94℃ for 5 min, followed by 36 cycles of (94℃ 1 min, 56℃ 1 min, 72℃ 1 min) and a final extension at 72℃ for 5 min. The amplified PCR products were electrophoresed on a 6% polyacrylamide denaturing gel, and the gel was imaged after electrophoresis.

[0036] Example 3: Identification of the superior allele OsRNU5 HJX74 SNP analysis of promoter regions

[0037] Compared with OsRNU5 in variety HJX74 HJX74 Compared to the 1.5kb promoter (SEQ ID NO.2) of the gene, the allele OsRNU5 IRAT261 A single 8bp deletion is predominantly present in its 1.5kb promoter (SEQ ID NO.4). Sequencing analysis of the 2kb promoter region of the OsRNU5 gene in 12 indica and 12 japonica rice samples collected in the laboratory revealed that the 8bp deletion in the promoter was present in indica rice but absent in japonica rice. In japonica rice, the 8bp deletion resulted in low expression of the OsRNU5 gene. Figure 3 ), combined with previous laboratory findings on NO3 in indica rice roots - The absorption capacity is generally high; therefore, it can be considered that different allelic variations of the OsRNU5 gene in indica and japonica rice lead to the NO3 absorption capacity in these two rice varieties. - Differences in absorption capacity. This 8bp difference can be detected by PCR to determine NO3 levels in rice roots. - Absorption capacity.

[0038] To investigate the universality of this difference, the inventors performed haplotype analysis on SNPs in the promoter region of the OsRNU5 gene within a 2kb range from 3010 materials in a rice database. The results showed the existence of three different haplotypes: Hap.Ⅰ-Hap.Ⅲ. Figure 3 Among them, Hap. I and III are indica rice types, containing the aforementioned 8bp sequence; japonica rice is mainly Hap. II, which lacks the aforementioned 8bp sequence. The above data demonstrate that the OsRNU5 gene exhibits a clear differentiation between indica and japonica varieties.

[0039] Example 4: Construction and identification of OsRNU5-related transgenic materials

[0040] To further verify that OsRNU5 can regulate NO3 in rice roots -To identify key sites for uptake rate, we constructed the OsRNU5 overexpression line pAct::OsRNU5-Flag and the knockout material rnu5 in the ZH11 background. We used the gateway system to construct OsRNU5 overexpression vectors. Gateway technology does not rely on restriction endonucleases; instead, it utilizes specific recombination sites and recombinases present on the vector to efficiently and rapidly clone the target gene into the expression vector. First, the target fragment of OsRNU5 was amplified by PCR using cDNA from rice materials. Then, according to the manufacturer's instructions (Invitrogen, Carlsbad, CA; 11789100 and 11791100, USA), it was inserted into the pAct:Flag-nos vector using Gateway recombination cloning technology (Siyu Zhang; Limei Zhu; Chengbo Shen; Zhe Ji; Haipeng Zhang; Tao Zhang; Yu Li; Jianping Yu; Ning Yang; Yubing He; Yanan Tian; Kun Wu; Juyou Wu; Nicholas P. Harberd; Yunde Zhao; Xiangdong Fu; Shaokui Wang; Shan Li; Natural allelic variation in a modulator of auxinhomeostasis improves grain yield and nitrogen use efficiency in rice, The Plant Cell, 2021, 33(3):566-580). Knockout materials of OsRNU5 were constructed using the CRISPR-Cas9 system. The CRISPR / Cas9 system consists of trans-activating crRNA (TracrRNA), crRNA, and the Cas9 protein. The crRNA-tracrRNA structure guides the Cas9 endonuclease to recognize and bind to the target gene fragment, cutting the foreign DNA containing a 20-nt complementary sequence to the crRNA before the PAM (protospacer adjacent motif, characterized by NGG, where N can be any base). This results in a double-stranded break (DSB). The DSB is then repaired primarily through the error-prone non-homologous end-joining (NHEJ) pathway. NHEJ easily leads to single-base insertions, deletions, or substitutions. Therefore, gene editing can be achieved through double-strand breaks and subsequent DNA repair. The CRISPR-Cas9 system was used to construct rnu5 knockout material.First, the OsRNU5 target sequence was designed using the CRISPR-P website. PCR amplification was performed using a transcription unit with the rice U6+U3 promoter as a template (SEQ ID NO.5). The PCR product was recovered from the gel and ligated to a TKC vector that had been completely digested with Spe I (Yubing He; Min Zhu; Lihao Wang; Qiaoyan Wang; Rongchen Wang; Yunde Zhao; Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR / Cas9-Edited Rice Plants. Rice Science; 2019, 26(2):109-11). Finally, positive clones were obtained by transformation into E. coli and sent for sequencing. The constructed OsRNU5 overexpression and knockout vectors were transformed using Agrobacterium, that is, the target gene was inserted into the genome of Agrobacterium. Through Agrobacterium infection of plants, the foreign gene was transferred and integrated into plant cells, and introduced into japonica rice ZH11. The primer sequences for constructing the OsRNU5 overexpression and knockout vectors are detailed in Table 2.

[0041] Table 2

[0042]

[0043] The expression levels of OsRNU5 were detected in ZH11, the overexpression line pAct::OsRNU5-Flag, and the knockout material rnu5.

[0044] The expression level of OsRNU5 was detected in root tip tissues from ZH11, the overexpression line pAct::OsRNU5-Flag, and the knockout material rnu5 after three weeks of hydroponics using qRT-PCR experiments. The results showed that, compared with ZH11, the transcriptional level of OsRNU5 was significantly increased in the overexpression line pAct::OsRNU5-Flag and decreased in the knockout material rnu5. Figure 4 a). The OsRNU5 overexpression line pAct::OsRNU5-Flag had a higher nitrate uptake rate than ZH11, while the knockout line rnu5 had the lowest rate. Figure 4 c).

[0045] Example 5: Comparative analysis of agronomic traits such as yield in OsRNU5-related transgenic materials

[0046] In the field yield test, ZH11, the overexpression line pAct::OsRNU5-Flag, and the knockout material rnu5 were planted in a field with normal nitrogen application (nitrogen application rate of 210 kg / ha), and various important agronomic traits were observed and statistically analyzed.

[0047] Specific statistical methods: Plant height statistics: After rice matures, the height of 15 plants are measured in the field. Number of grains per panicle statistics: After rice matures, panicles from 12 main tillers are collected in the field, and the number of grains per panicle is counted and recorded. Yield per plant statistics: After the rice is fully mature, 12 individual plants in the plot are threshed. The harvested seeds are dried at a constant temperature of 37℃ and then weighed to obtain the yield per plant. Three replicate experiments are required.

[0048] Statistical results show that pAct::OsRNU5-Flag increases plant height, tillers, primary and secondary branches, and the number of grains per ear, ultimately leading to an increase in yield per plant. Figure 5 ).

Claims

1. Application of indica rice OsRNU5 gene in improving nitrogen use efficiency and yield of rice, characterized in that, The nucleotide sequence of the indica rice OsRNU5 gene is shown as SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, Overexpression of the indica rice OsRNU5 gene can improve the nitrogen utilization efficiency and yield of rice.

3. Application of the indica rice OsRNU5 gene promoter in improving the nitrogen utilization efficiency and yield of rice, wherein the sequence of the indica rice OsRNU5 gene promoter is shown as SEQ ID NO.

2.

4. A marker associated with nitrogen use efficiency in rice, characterized in that, The marker is an 8bp sequence of 431-438bp in the OsRNU5 gene promoter shown in SEQ ID NO 2: TGTGTGTA; the marker exists in indica rice, and the NO3 - absorption capacity is strong, and the nitrogen utilization efficiency is high; the marker is deleted in japonica rice, resulting in low expression of the OsRNU5 gene, and the NO3 - absorption capacity of the root system is reduced, and the nitrogen utilization efficiency is low.

5. A method for judging nitrogen fertilizer use efficiency in rice, characterized by, Detecting the promoter of the rice OsRNU5 gene, if the marker of claim 4 exists, the nitrogen utilization efficiency of the rice variety is high, and if the marker of claim 4 is absent, the nitrogen utilization efficiency of the rice variety is low.

6. The method of claim 4, wherein, The PCR primers used for detecting the promoter of the rice OsRNU5 gene are shown as SEQ ID NO. 6 and SEQ ID NO.

7.

7. A method for enhancing root development and nitrogen use efficiency in rice, characterized by, Enhancing the marker of claim 4 in rice.

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