Application of gramineous conserved gene REP4 in promoting root growth, improving nitrogen utilization efficiency and / or increasing yield

Abstract

The invention relates to the technical field of gene engineering, and discloses application of a gramineous conserved gene REP4 in promoting root growth, improving nitrogen utilization efficiency and / or increasing yield. According to the invention, the root specific expression small peptide hormone coding gene OsREP4 in rice is identified, and the overexpression and near-isogenic line materials of the gene can promote the growth of the rice root, improve the nitrogen utilization efficiency and increase the yield; the overexpression and near-isogenic line of the corn homologous gene ZmREP4 can also significantly promote the growth of corn roots, improve the nitrogen utilization efficiency and increase the yield. Mature small peptides synthesized according to REP4 protein sequences in gramineous crops such as rice, wheat and corn are crossly applied under extremely low concentration, and the root growth and nitrogen absorption capability of the rice, the corn and the wheat can be remarkably improved. When the OsREP4 mature small peptide secreted and expressed by the bacillus subtilis is directly applied to soil, the growth of a rice root system can be obviously promoted, and the yield can be increased.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and discloses the application of the conserved gene REP4 in the Gramineae family in promoting root growth, improving nitrogen use efficiency, and / or increasing yield. Background Technology

[0002] Rice, corn, and wheat are the world's three major food crops, with nearly 80% of the world's population relying on them as their staple food. Long-term reliance on excessive chemical fertilizers has led to crop nutrient imbalances, delayed maturity, reduced resilience, and severe resource waste and environmental pollution. Furthermore, a large portion of residual nitrogen fertilizer contributes to severe soil acidification and eutrophication of water bodies. The areas affected by soil acidification and eutrophication in our country are showing a year-on-year increasing trend. In addition, nitrogen fertilizer production consumes a large amount of energy, resulting in significant carbon emissions and severe air pollution. Moreover, our country faces a huge food production shortfall and needs to increase grain output year by year. Therefore, the rational use of nitrogen fertilizer poses a strategic challenge to sustainable agricultural development. Improving the nitrogen fertilizer use efficiency of crops and reducing nitrogen fertilizer use in agriculture will be key to solving these problems.

[0003] In recent years, numerous studies have focused on the role of small peptide signaling in plant growth and development, including post-translational modified small peptides, such as most cystatins, and cysteine-rich small peptides, such as RALF-type small peptides. Although some small peptides have been shown to respond to nitrogen uptake and nitrogen starvation in Arabidopsis thaliana and have related physiological effects, the relationship between small peptide hormones and plant nitrogen use efficiency remains unclear.

[0004] This invention identifies the function of a conserved small peptide gene, REP4, in grasses, and discovers that it has conserved functions in crops, such as promoting root growth, improving nitrogen use efficiency, and increasing yield. This is of great significance for breeding new crop varieties with high nutrient utilization efficiency. Summary of the Invention

[0005] The present invention relates to the application of the conserved gene REP4 in grasses in promoting root growth, improving nitrogen use efficiency, and increasing yield. To achieve the above objectives, the present invention employs the following technical measures:

[0006] This invention identified a root-specific expressed small peptide hormone encoding gene, OsREP4, in rice. Overexpression of OsREP4 and its near-isogenic line (NIL) materials both promoted root growth, improved nitrogen use efficiency, and increased yield in rice. Homologous sequence alignment revealed highly homologous genes in maize and wheat, named ZmREP4 and TaREP4-2A / 2B / 2D, respectively. Based on homology, homologous genes SbREP4 in sorghum and HvREP4 in barley were also cloned. Overexpression of ZmREP4 in maize and residual substitution line (RHL) materials both promoted root growth, yield per plant, nitrogen use efficiency, and grain protein content in maize. We synthesized the mature active forms of the small peptide encoded by the REP4 gene in three crops: OsREP4, ZmREP4, and TaREP4. All three peptides, even at extremely low concentrations, enhanced root growth and nitrate uptake in rice, maize, and wheat. In addition, the Bacillus subtilis secretory expression system can achieve high-abundance in vitro expression of mature OsREP4 peptide. In vitro application of Bacillus subtilis fermentation broth containing small OsREP4 peptide can significantly improve the root length, single-plant yield and nitrogen use efficiency of two rice varieties, Huang Huazhan and Hua Moxiang.

[0007] The scope of protection of this invention includes:

[0008] Applications of the conserved gene REP4 in grasses in promoting root growth, improving nitrogen use efficiency, and / or increasing yield.

[0009] Applications of expression cassettes, recombinant vectors, or recombinant microorganisms to enhance the expression of the conserved gramineous gene REP4 in plants that produce plants with high root growth, high nitrogen use efficiency, and / or increased yield.

[0010] Application of molecular markers for detecting sequence variations or expression differences of the conserved gene REP4 in grasses in screening and breeding for root growth, nitrogen use efficiency, and / or yield.

[0011] The application of an active mature peptide encoding a protein of the conserved gene REP4 in the Poaceae family, a recombinant protein obtained by fusing the active mature peptide with a protein tag, or a mixture containing the active mature peptide or recombinant protein in promoting plant root growth, improving nitrogen use efficiency, and / or increasing yield.

[0012] In the above-described applications, preferably, the conserved gene REP4 in the Poaceae family is the conserved gene OsREP4 in rice or its homologous gene REP4 in other Poaceae families, and the protein encoded by the conserved gene REP4 in rice is shown in SEQ ID NO.2.

[0013] In the above-described applications, preferably, the other grasses mentioned are wheat, corn, sorghum, barley, foxtail grass, and other grasses.

[0014] In the above-described applications, preferably, the protein encoded by the maize REP4 gene is shown in SEQ ID NO.5, the protein encoded by the wheat REP4 gene is shown in SEQ ID NO.8, SEQ ID NO.11 or SEQ ID NO.14, the protein encoded by the sorghum REP4 gene is shown in SEQ ID NO.1, the protein encoded by the barley REP4 gene is shown in SEQ ID NO.19, and the protein encoded by the foxtail grass REP4 gene is shown in SEQ ID NO.22.

[0015] In the above-described applications, preferably, the plant is a grass (Poaceae).

[0016] The preferred application described above is achieved by increasing the expression level of the REP4 gene in grass plants.

[0017] In the above-described applications, preferably, the active mature peptide encoded by the conserved Gramineae gene REP4 is shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID NO.17, SEQ ID NO.20 or / and SEQ ID NO.23.

[0018] The preferred application described above involves applying the active mature peptide encoded by the conserved Gramineae gene REP4, a recombinant protein obtained by fusing the active mature peptide with a protein tag, or a mixture containing the active mature peptide or recombinant protein to plants in vitro.

[0019] The preferred application methods described above include: root irrigation, soaking, spraying, or seed coating.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] This invention is the first to discover that the conserved gene REP4 in the Poaceae family can promote root growth, improve nitrogen use efficiency, and increase yield.

[0022] Rice can be transformed using the OsREP4 gene overexpression vector of the present invention to cultivate high-yielding rice varieties with high nitrogen fertilizer utilization efficiency.

[0023] The ZmREP4 gene overexpression vector of the present invention can be used to transform maize to cultivate high-yielding maize varieties with high nitrogen fertilizer utilization efficiency.

[0024] Cross-treatment of seedlings of rice, corn, and wheat using mature small peptides encoded by their respective REP4 genes significantly increased root length and nitrogen uptake capacity, ultimately leading to increased yield.

[0025] The mature small peptide form of REP4 can also be expressed by engineered bacteria such as Bacillus subtilis and applied exogenously to improve the yield of gramineous crops and nitrogen fertilizer utilization efficiency.

[0026] All of the above results demonstrate the conserved functions of the REP4 gene in promoting root growth, improving nitrogen use efficiency, and increasing yield in gramineous crops. Attached Figure Description

[0027] Figure 1 Functional conservation of rice, maize and wheat treated with REP4 small peptides;

[0028] A represents the statistical analysis results of rice seedlings and root lengths treated with REP4 homologous small peptides from rice, corn, and wheat.

[0029] B represents the statistical analysis results of nitrate transport capacity in the roots of rice seedlings treated with REP4 homologous peptides from rice, corn, and wheat.

[0030] C represents the statistical analysis results of maize seedlings and root lengths treated with REP4 homologous small peptides from rice, maize, and wheat.

[0031] D represents the statistical analysis results of nitrate transport capacity in the roots of maize seedlings treated with REP4 homologous peptides from rice, maize, and wheat.

[0032] E represents the statistical analysis results of wheat seedlings and root lengths treated with REP4 homologous peptides from rice, corn, and wheat.

[0033] F represents the statistical analysis results of nitrate transport capacity in wheat seedling roots treated with REP4 homologous peptides from rice, corn, and wheat.

[0034] Figure 2 Phenotype of rice plants overexpressing the OsREP4 gene;

[0035] Image A shows rice plants overexpressing the OsREP4 gene under normal and low nitrogen conditions.

[0036] Image B shows rice panicles overexpressing the OsREP4 gene under normal and low nitrogen conditions.

[0037] C represents the statistical analysis results of single-plant yield of rice with overexpression of the OsREP4 gene under normal and low nitrogen conditions;

[0038] D represents the statistical analysis results of nitrogen use efficiency in rice with overexpression of the OsREP4 gene under normal and low nitrogen conditions.

[0039] Figure 3 Phenotype of near-isogenic rice lines with the OsREP4 gene;

[0040] A represents the near-isogenic line material HHZ of the OsREP4 gene. OsREP4G and its control HHZ OsREP4A Photographs of rice plant architecture in the field under normal and low nitrogen conditions;

[0041] B represents the statistical analysis results of root system images, root length, and total root area;

[0042] C represents the statistical analysis results of grain images of representative single plants, plot yield, and nitrogen use efficiency.

[0043] Figure 4 Phenotype of maize plants overexpressing the ZmREP4 gene;

[0044] A shows maize root images and root length statistical analysis results under normal and low nitrogen conditions with overexpression of the ZmREP4 gene. B shows representative ears of the above maize plants. C shows the statistical analysis results of kernel weight, nitrogen use efficiency, and kernel protein content of individual ears of the above maize plants.

[0045] Figure 5 Phenotype of the remaining heterozygous line of the maize ZmREP4 gene;

[0046] A represents the remaining heterozygous line material RHL for the ZmREP4 gene. ZmREP4G and its control group RHL ZmREP4T Photos of maize plant architecture in the field under normal and low nitrogen conditions;

[0047] B is a representative photo of the ears of corn from the aforementioned corn plant;

[0048] C represents the statistical analysis results of the individual ear kernel weight, nitrogen use efficiency, and kernel protein content of the above-mentioned maize plants.

[0049] Figure 6 The effect of OsREP4 small peptide in microbial fertilizer application;

[0050] A is a schematic diagram of the vector for expressing the mature OsREP4 peptide in the Bacillus subtilis secretory expression system;

[0051] B shows the dot blot hybridization results of OsREP4 mature peptide expressed by the Bacillus subtilis secretory expression system and the corresponding control.

[0052] C represents the statistical analysis results of root system, representative single plant grains, root length, and single plant yield of rice Huanghuazhan treated with positive strain (B.sub 113#), empty vector control (B.sub EV), and blank control (Mock).

[0053] D represents the statistical analysis results of root system, representative single-plant grains, root length, and single-plant yield of *B. sub 113#*, *B. sub EV*, and blank control (Mock) in *B. sub 113#* rice. Detailed Implementation

[0054] The following examples define the present invention and describe the methods for cloning DNA fragments containing the complete coding region of the OsREP4 / ZmREP4 gene and verifying the function of the OsREP4 / ZmREP4 gene. Based on the following description and these examples, those skilled in the art can determine the essential characteristics of the invention, and various changes and modifications can be made to adapt it to different uses and conditions without departing from the spirit and scope of the invention. Unless otherwise specified, all methods are conventional, and materials or reagents are commercially available.

[0055] Example 1:

[0056] Discovery of the rice OsREP4 gene and its homologous protein REP4 in grasses:

[0057] The applicant located the OsREP4 gene in rice through genome-wide association analysis of nitrogen use efficiency under low nitrogen conditions and its relationship with root length. The coding region of the OsREP4 gene has a nucleotide length of 270 bp (as shown in SEQ ID NO.1), and the encoded protein is shown in SEQ ID NO.2. We identified the active mature peptide of OsREP4 small peptide by submerging OsREP4 overexpressing seedlings in liquid medium, extracting peptides from the medium, and performing mass spectrometry analysis. The corresponding sequence is shown in SEQ ID NO.3.

[0058] We then analyzed the OsREP4 protein sequence in the Ensembl Plants database and found that the homologous protein of OsREP4 exists only in grasses, indicating that OsREP4 is a conserved protein in grasses.

[0059] The homologous gene in maize is named ZmREP4, the coding region nucleotides are shown in SEQ ID NO.4, the sequence of the encoded protein is shown in SEQ ID NO.5, and the corresponding active mature peptide is shown in SEQ ID NO.6.

[0060] There are three corresponding homologous genes in wheat:

[0061] TaREP4-2A, the coding nucleotide region is shown in SEQ ID NO.7, the encoded protein is shown in SEQ ID NO.8, and the corresponding active mature peptide is shown in SEQ ID NO.9.

[0062] TaREP4-2B, the coding nucleotide region is shown in SEQ ID NO.10, the encoded protein is shown in SEQ ID NO.11, and the corresponding active mature peptide is shown in SEQ ID NO.12.

[0063] TaREP4-2D, the coding nucleotide region is shown in SEQ ID NO.13, the encoded protein is shown in SEQ ID NO.14, and the corresponding active mature peptide is shown in SEQ ID NO.9.

[0064] The corresponding homologous gene in sorghum is SbREP4, the coding region nucleotides are shown in SEQ ID NO.15, the sequence of the encoded protein is shown in SEQ ID NO.16, and the corresponding active mature peptide is shown in SEQ ID NO.17.

[0065] The corresponding homologous gene in barley is HvREP4, the coding region nucleotides are shown in SEQ ID NO.18, the sequence of the encoded protein is shown in SEQ ID NO.19, and the corresponding active mature peptide is shown in SEQ ID NO.20.

[0066] The corresponding homologous gene in the representative forage grass of the Poaceae family, foxtail grass, is SvREP4, the coding region nucleotide is shown in SEQ ID NO.21, the sequence of the encoded protein is shown in SEQ ID NO.22, and the corresponding active mature peptide is shown in SEQ ID NO.23.

[0067] Example 2:

[0068] Application of REP4 small peptide in promoting root growth and nitrogen absorption in gramineous plants:

[0069] Mature bioactive peptides of artificially synthesized OsREP4, ZmREP4, and TaREP4 have sequences of SEQ ID NO.3, SEQ ID NO.6, and SEQ ID NO.12, respectively.

[0070] Each small peptide was used to treat rice variety Zhonghua 11, maize variety B73, and wheat variety Zhongguochun under hydroponic conditions. The concentration of each small peptide was 20 nM. A mock control without any small peptides was also set up.

[0071] The hydroponic solution is a modified version of Kimura nutrient solution, containing the following components: 2.5 mM KNO3, 0.36 mM CaCl2·4H2O, 0.54 mM MgSO4·7H2O, 0.18 mM KH2PO4, 40 μM Fe(II)-EDTA, 18.8 μM H3BO3, 13.4 μM MnCl2·4H2O, 0.32 μM CuSO4·5H2O, 0.3 μM ZnSO4·4H2O, and 0.03 μM Na2MoO4·4H2O.

[0072] The results of small peptide cross-treatment showed that, in the control treatment, the root lengths of rice, corn, and wheat seedlings were 8.3 cm, 12.8 cm, and 11.2 cm, respectively. After treatment with OsREP4 peptide in the experimental group, the root lengths of the three plants became 10.0 cm, 16.8 cm, and 12.7 cm, respectively. After treatment with ZmREP4 peptide in the experimental group, the root lengths of the three plants became 11.3 cm, 18.7 cm, and 12.8 cm, respectively. After treatment with TaREP4 peptide in the experimental group, the root lengths of the three plants became 9.8 cm, 18.2 cm, and 12.9 cm, respectively. Figure 1 (AC). Based on this, it can be concluded that the three small peptides OsREP4 / ZmREP4 / TaREP4 can all promote root growth in the three plants.

[0073] To demonstrate that the REP4 peptide is related to nitrogen uptake efficiency, the nitrate uptake capacity of roots before and after peptide treatment was determined using KNO3 labeled with a stable isotope.

[0074] Rice, corn, and wheat seedlings were cultured in a hydroponic solution with a small peptide concentration of 20 nM for 10 days, then transferred to a modified Kimura nutrient solution containing 2.5 mM 15N-KNO3 for 3 hours of absorption. Afterward, they were treated with 0.1 mM CaSO4 for 1 minute, rinsed three times with deionized water, and the aboveground and underground parts were collected and dried at 70℃. The dried samples were ground into powder, and the 15N content was determined using isotope ratio mass spectrometry. The results showed that, in the control (Mock) treatment, the root nitrate uptake capacity of rice, corn, and wheat seedlings was 0.043, 0.028, and 0.011 N, respectively. 15 After treatment with the OsREP4 peptide in the experimental group (mMg-1root DW h-1), the nitrate uptake capacity of the experimental group became 0.054, 0.032, and 0.014, respectively. After treatment with the ZmREP4 peptide in the experimental group, the nitrate uptake capacity became 0.055, 0.038, and 0.015, respectively (unit: N). 15 After treatment with TaREP4 peptide (mM g-1root DW h-1), the nitrate uptake capacity of the experimental group became 0.055, 0.033, and 0.014 (unit: Nm2, g-1, root DW h-1), respectively.15 mMg-1root DWh-1). Based on this, it can be concluded that the three small peptides OsREP4 / ZmREP4 / TaREP4 can all promote the root nitrate uptake capacity of the three plants. Figure 1 (DF).

[0075] Example 3:

[0076] Yield phenotype of OsREP4 gene overexpression in rice materials

[0077] To gain a more comprehensive understanding of the stress-resistance function of the OsREP4 gene, the applicant previously obtained overexpression materials of the OsREP4 gene in rice in the patent "Application of OsREP4 Gene in Controlling Drought Resistance in Rice" (patent publication number CN109112135B). The construction method of the overexpression vector and the genetic transformation process have been described in detail in the aforementioned patent and will not be repeated in this patent. We planted two overexpression families, OsREP4-OE#1 and OsREP4-OE#2, and a wild-type control under field conditions of normal nitrogen (150 kg net nitrogen / ha) and low nitrogen (50 kg net nitrogen / ha). The rice plants and panicles were as follows: Figure 2 China A and Figure 2 As shown in Figure B. We investigated the yield-related traits of the above materials. The results showed that under normal nitrogen conditions, the yield per plant in the two overexpression families and the wild-type control were 14.59 g, 15.91 g, and 16.24 g, respectively, and the nitrogen use efficiency (NUE) for the three was 28.01, 30.55, and 31.19, respectively (unit: yield g / net nitrogen g). Under low nitrogen conditions, the yield per plant in the three was 11.08 g, 13.81 g, and 13.35 g, respectively, and the nitrogen use efficiency was 85.08, 106.1, and 102.5, respectively (unit: yield g / net nitrogen g). Figure 2 (C-2, D). Based on this, it can be concluded that the OsREP4 overexpression material showed significantly improved single-plant yield and nitrogen use efficiency under normal and low nitrogen conditions compared to the wild-type control.

[0078] Example 4:

[0079] Construction of near-isogenic lines (NILs) of OsREP4 in rice and their yield phenotypes

[0080] Near-isogenic lines were constructed using wild rice (Oryza rufipogon) as the donor parent and the indica rice variety Huanghuazhan (HHZ) as the recipient parent. The hybrid was backcrossed with Huanghuazhan four times to obtain BC4F4. PCR polymorphic markers distributed across 12 chromosomes were used to identify the backcross progeny to obtain the near-isogenic line HHZ containing a strongly functional fragment. OsREP4GIts receptor material is HHZ containing a weakly functional fragment. OsREP4A We have nearly isomorphic line HHZ OsREP4G and receptor HHZ OsREP4A Planting was carried out under field conditions with normal nitrogen (150 kg net nitrogen / ha) and low nitrogen (50 kg net nitrogen / ha). Images of plants and root systems are shown below. Figure 3 As shown in Figure A, we investigated the correlation between root system and yield traits. The results showed that the near-isogenic line HHZ... OsREP4G Under normal and low nitrogen conditions, the root length, root area per tiller, plot yield, and nitrogen use efficiency (NUE) were 26.5 and 22.7 cm, and 116.3 and 117.6 cm, respectively. 2 6.84 and 5.00 kg, 35.1 and 76.9 (units are yield g / net nitrogen g), receptor HHZ OsREP4A Under normal and low nitrogen conditions, the root length, root area per tiller, plot yield, and nitrogen use efficiency (NUE) were 21.8 and 17.4 cm, and 86.4 and 89.6 cm, respectively. 2 5.88 and 4.14 kg, 30.2 and 63.7 (units are yield g / net nitrogen g). Based on the results, the near-isogenic line HHZ... OsREP4G Both are more than the receptor HHZ OsREP4A Significant improvement ( Figure 3 (Chinese BC).

[0081] Example 5:

[0082] Yield phenotype of ZmREP4 gene overexpression in maize

[0083] The overexpression vector for the ZmREP4 gene was developed by Weimi Biotechnology (Jiangsu) Co., Ltd., and the genetic transformation of the maize overexpression material was also completed by Weimi Biotechnology (Jiangsu) Co., Ltd. The recipient used for maize transformation in this experiment was maize KN5585. We planted two overexpression families, ZmREP4-OE#2 and ZmREP4-OE#3, and a wild-type control under field conditions of normal nitrogen (100 kg net nitrogen / ha) and low nitrogen (50 kg net nitrogen / ha). The maize root system and ears... Figure 4As shown in Figures A and B, we investigated the correlation between root length and yield. The results showed that under normal nitrogen conditions, the root lengths of the two overexpression families and the wild-type control were 33.3, 38.4, and 40.6 cm, respectively; the yield per plant was 61.7, 75.0, and 81.7 g, respectively; and the nitrogen use efficiency (NUE) was 41.1, 50.0, and 54.5, respectively (unit: yield g / net nitrogen g). Under low nitrogen conditions, the root lengths of the two overexpression families and the wild-type control were 30.3, 38.5, and 37.5 cm, respectively; the yield per plant was 41.8, 69.5, and 73.4 g, respectively; and the nitrogen use efficiency was 55.8, 92.6, and 97.9, respectively (unit: yield g / net nitrogen g). Figure 4 (AC). Based on this, it can be concluded that the ZmREP4 overexpression material showed significantly improved root length, single-plant yield, and nitrogen use efficiency compared to the wild-type control under normal and low nitrogen conditions.

[0084] Example 6:

[0085] Yield phenotype of the ZmREP4 gene in remaining heterozygous maize lines

[0086] We screened a residual heterozygous line RHL from the recombinant inbred line RIL of maize B73 / BY804, which was still heterozygous in the ZmREP4 gene region, and isolated a strongly functional RHL from it. ZmREP4G and weak-function RHL ZmREP4T Materials. Both materials were planted under normal nitrogen (100 kg net nitrogen / ha) and low nitrogen (50 kg net nitrogen / ha) field conditions to examine yield-related traits. Results showed that the high-performance RHL... ZmREP4G Under normal and low nitrogen conditions, the yield per plant was 74.5 g and 73.5 g, respectively, with nitrogen use efficiencies of 49.7% and 98.0% (yield g / net nitrogen g), and protein content of 11.7% and 11.0%, respectively, showing little difference between the two conditions. However, the weakly functional RHL... ZmREP4T Under normal and low nitrogen conditions, the yield per plant was 73.5 g and 62.4 g, respectively, with nitrogen use efficiencies of 49.0% and 83.2% (yield g / net nitrogen g), and protein content of 10.6% and 8.0%, respectively. This indicates that the high-functionality RHL... ZmREP4G The material outperformed the weakly functional RHL in terms of low-nitrogen (50 kg net nitrogen / ha) yield, nitrogen use efficiency (NUE), and grain protein content. ZmREP4T The materials have been significantly improved. Figure 5 (AC).

[0087] Example 7:

[0088] OsREP4 peptide expressed in prokaryotes promotes root growth and increases yield in rice.

[0089] We constructed a Bacillus subtilis strain to express the mature OsREP4 peptide using a commercially available Bacillus subtilis secretory expression system (Takara Cat. #3380). Specific methods and steps are detailed in the product manual. The general steps are as follows: First, we optimized the coding sequence for the mature OsREP4 peptide using codons specific to Bacillus subtilis. Then, we synthesized the optimized sequence at Beijing Qingke Biotechnology Co., Ltd., and ligated it into the expression vector pBE-S to obtain the pBE-S OsREP4 vector. Figure 6 (A). Then, a mixture of 173 Bacillus subtilis signal peptides was ligated into the pBE-S OsREP4 vector and transformed into Bacillus subtilis competent cells to obtain a Bacillus subtilis expression library. By dot blot hybridization, the strain B. subtilis subtilis 113# was selected as the one with the highest OsREP4 secretion expression in the supernatant. Figure 6 (B). Subsequently, using strain B. sub 113#, which highly expresses OsREP4, and strain B. sub EV, which only expresses the empty vector, along with a blank control (Mock), we transplanted rice varieties Huanghuazhan and Huamoxiang into soil for pot experiments after 4 weeks of seedling cultivation. B. sub 113#, which highly expresses OsREP4 (OD value of approximately 0.8), and B. sub EV, which only expresses the empty vector, were simultaneously poured into the potting soil, with an average of about 50 ml of bacterial solution applied to the roots of each plant. One month later, when the rice plants were in the peak tillering stage, the bacterial solution treatment was repeated until the root length and yield per plant were finally measured. The results showed that the root length of the OsREP4-highly expressing strain B. sub 113# in rice variety Huanghuazhan was 47.5, 42.7, and 38.6 cm, respectively, compared with the empty vector control and blank control, and the yield per plant was 18.2, 15.3, and 13.0 g, respectively. In the treated rice variety Huamoxiang, the root length was 51.6, 44.2, and 41.8 cm, respectively, and the yield per plant was 18.4, 12.5, and 12.1 g, respectively. These results indicate that the OsREP4-highly expressing strain B. sub 113# treatment significantly promoted root growth and yield per plant in rice varieties Huanghuazhan and Huamoxiang compared with the empty vector control and blank control. Figure 6 Medium CD).

Claims

1. A conserved gene of the family Poaceae REP4 for use in promoting root growth, increasing nitrogen use efficiency and / or increasing yield in plants.

2. Expression cassette for increasing expression of grass conserved genes REP4 Use of the recombinant vector or the recombinant microorganism in creating a plant with increased root growth, high nitrogen use efficiency and / or increased yield.

3. Detection of a conserved gene of the family Poaceae REP4 Use of molecular markers of sequence variation or expression differences in the selection breeding of plants for root growth, nitrogen use efficiency and / or yield.

4. Poaceae conserved genes REP4 Use of an active mature peptide encoding a protein, a recombinant protein obtained after fusion of the active mature peptide with a protein tag, or a mixture containing the active mature peptide or the recombinant protein, for promoting root growth, increasing nitrogen use efficiency, and / or increasing yield in plants.

5. The use according to any one of claims 1 to 4, characterized in that: The conserved gene of the Poaceae REP4 The conserved gene of the Poaceae OsREP4 Or the REP4 homologous gene thereof in other Poaceae REP4 The encoded protein is shown as SEQ ID NO.

2.

6. The use according to claim 5, wherein the other grasses are wheat, corn, sorghum, barley, dogtail grass and the like.

7. Use according to claim 6, characterized in that: The corn REP4 The protein encoded by the gene is shown as SEQ ID NO. 5, wheat REP4 The protein encoded by the gene is shown as SEQ ID NO. 8, SEQ ID NO. 11 or SEQ ID NO. 14, sorghum REP4 The protein encoded by the gene is shown as SEQ ID NO. 1, barley REP4 The protein encoded by the gene is shown as SEQ ID NO. 19, grasses Cynodon dactylon REP4 The protein encoded by the gene is shown as SEQ ID NO.

22.

8. The use of any one of claims 1-4, wherein: The plants are grasses.

9. Use according to claim 4, characterized in that: The conserved gene of the family Poaceae REP4 The active mature peptide encoding protein is SEQ ID NO. 3 , SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12, SEQ ID NO. 17, SEQ ID NO. 20 or / and SEQ ID NO.

23.

10. Use according to claim 4, characterized in that: The application process described involves using conserved genes from the Poaceae family. REP4 The active mature peptide encoding the protein, the recombinant protein obtained by fusing the active mature peptide with a protein tag, or a mixture containing the active mature peptide or the recombinant protein are applied to plants in vitro.

11. Use according to claim 10, wherein the method of in vitro administration comprises: Drenching, soaking, spraying or seed coating.

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