MiRNA and application thereof in regulating tillering of plants

Abstract

This invention discloses a miRNA and its application in regulating plant tillering, belonging to the field of plant genetic engineering technology. The invention provides a miRNA named miRH1, whose nucleotide sequence is shown in SEQ ID NO.1. Experimental verification shows that miRH1 can regulate the number of tillers in plants. Specifically, overexpression of miRH1 increases the number of tillers in transgenic plants, while inhibiting miRH1 expression decreases the number of tillers. Therefore, miRH1 provides a theoretical basis for elucidating the tillering mechanism and lays the methodological and material foundation for subsequent breeding of new high-yielding plants using genetic engineering technology. In summary, this invention provides a theoretical basis and potential application value for molecular breeding and the improvement of ideal plant architecture.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to a miRNA and its application in regulating plant tillering. Background Technology

[0002] Tillering refers to the branching that occurs in grasses below or near the ground. It generally occurs on the relatively swollen tillering nodes that store abundant nutrients. It is a unique branching pattern that occurs in grasses such as barley, wheat, and rice during their growth and development. It is an important agronomic trait that controls crop morphology, physiological function, and grain yield.

[0003] miRNAs (miRNAs) are a class of endogenous small non-coding RNAs, typically composed of 20-24 nucleotides. They are important regulators of gene expression in most eukaryotes, synthesized in multiple processes including transcription, precursor processing, methylation, and assembly of miRNA-induced silencing complexes. Based on sequence complementarity, miRNAs can directly cleave mRNA, repress translation, and methylate DNA. Recent studies have shown that miRNAs directly function in physiological processes such as root development, morphogenesis, vegetative reproduction, and flowering initiation in plants. They also play important regulatory roles in related gene regulatory networks within plants, participating in many biological processes such as signal transduction, biotic and abiotic stress responses, protein degradation, siRNA pathway feedback regulation, and maintaining genome integrity. Tillering is a key factor affecting yield; exploring the post-transcriptional regulatory network of tillering is not only crucial for breeding new high-yield crop varieties but can also significantly promote the sustainable development of agricultural production. Summary of the Invention

[0004] The purpose of this invention is to provide a miRNA and its application in regulating plant tillering, in order to solve the problems existing in the prior art. Experimental verification shows that miRH1 can regulate the number of tillers in plants. Specifically, overexpression of miRH1 can increase the number of tillers in transgenic plants, while inhibiting the expression of miRH1 will reduce the number of tillers in transgenic plants.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a miRNA, named miRH1, with a nucleotide sequence as shown in SEQ ID NO.1.

[0006] The present invention also provides a precursor of miRH1, pre-miRH1, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0007] This invention also provides the use of the miRH1, the pre-miRH1, or biomaterials that overexpress / inhibit the miRH1 in any of the following: (1) Regulating the number of tillers in plants; (2) Prepare products that regulate the number of tillers in plants; (3) Cultivate plants with a large number of tillers; (4) Cultivate plants with fewer tillers; (5) Improvement of plant tillering traits.

[0008] Optionally, the biological material includes an expression cassette, a recombinant vector, or a recombinant bacterium.

[0009] Optionally, the regulation is positive regulation.

[0010] Optionally, the plant includes barley or rice.

[0011] The present invention also provides a method for regulating the number of tillers in a plant, comprising any one of the following methods: (1) Overexpressing miRH1 in plants increases the number of tillers in the plants; (2) Inhibiting miRH1 expression in plants reduces the number of tillers in the plants; The nucleotide sequence of miRH1 is shown in SEQ ID NO.1.

[0012] The present invention also provides a method for cultivating plants with a large number of tillers, including the step of overexpressing miRH1 in the plant; The nucleotide sequence of miRH1 is shown in SEQ ID NO.1.

[0013] The present invention also provides a method for cultivating plants with few tillers, including the step of inhibiting the expression of miRH1 in the plant; The nucleotide sequence of miRH1 is shown in SEQ ID NO.1.

[0014] Optionally, the plant includes barley or rice.

[0015] The present invention discloses the following technical effects: This invention discovered a tillering-related miRNA—miRH1. Experimental verification showed that miRH1 can regulate the number of tillers in plants. Specifically, overexpression of miRH1 increases the number of tillers in transgenic plants, while inhibiting miRH1 expression decreases the number of tillers. Therefore, miRH1 provides a theoretical basis for elucidating the tillering mechanism and lays the methodological and material foundation for subsequent breeding of new high-yielding plants using genetic engineering techniques. In conclusion, this invention provides a theoretical basis and potential application value for molecular breeding and the improvement of ideal plant architecture. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 A schematic diagram of the secondary structure folding of the miRH1 precursor sequence; Figure 2 The expression levels of miRH1 in barley varieties Bowman and GSHO1990 at different time points are shown; where A represents the RNA-seq expression level of miRH1 and B represents the relative expression level of miRH1. Figure 3 The expression level of miRH1 in different barley tissues; Figure 4 The figure shows the functional verification results of miRH1 in rice; where A represents the phenotype of miRH1 overexpressing plants and wild-type plants; B represents the relative expression level of miRH1 in transgenic rice lines; and C represents the statistical analysis of the number of tillers in overexpressing plants and wild-type plants. Figure 5 Figure 1 shows the results of miRH1 functional validation in barley. In this figure, A represents the phenotype of miRH1 overexpressing plants and wild-type plants; B represents the phenotype of miRH1 suppressed expression plants and wild-type plants; C represents the relative expression level of miRH1 in transgenic barley lines; and D represents the statistical analysis of tiller number in overexpressing plants, suppressed expression plants, and wild-type plants. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Example 1: Discovery of miRH1, a tillering-related miRNA Bioinformatics analysis identified 1608 miRNAs in a small RNA library from tillering nodes of two barley varieties, Bowman and GSHO1990, from the two-leaf stage to the five-leaf stage, including 207 novel miRNAs (refer to Zhou et al., A novel microRNA miRP81 regulates plant height and contributes to yield improvement in both rice and barley, Plant Physiology and Biochemistry 229(2025) 110459). Based on this, novel barley miRNAs with differential expression among the materials were screened using the following method.

[0024] First, differentially expressed miRNAs at the same developmental stage were screened among the Bowman / GSHO1990 materials, retaining results with a fold change (|Log2FC|≥1) and a significance factor (FDR) <0.01. After obtaining the differentially expressed miRNAs at each stage, miRNAs that were stably differentially expressed in three or more consecutive developmental stages were screened, and miRNAs with an original expression level (readscount <100) were removed. A new differentially expressed miRNA, miRH1, was screened that was stably upregulated in the Bowman material and stably downregulated in the GSHO1990 material, and its nucleotide sequence is shown in SEQ ID NO.1.

[0025] SEQ ID NO. 1: CAGCTCAGCTCGAAAAAAGCTA.

[0026] miRNA precursors exhibit a typical stem-loop structure, allowing for the prediction of new miRNA sequence information and secondary structure using secondary structure folding models. The RNA Folding Form website (http: / / unafold.rna.albany.edu / ?q=mfold / rna-folding-form) was used to predict the secondary structure folding of the miRNA precursor sequence (nucleotide sequence shown in SEQ ID NO.2). The precursor sequence (pre-miRH1) has a minimum free energy of -107.40 kcal / mol, a pairing number of 19 between miRH1 and its antisense strand, and a convex loop of 2, all meeting the conditions for forming a relatively stable hairpin loop structure, thus satisfying the criteria for the formation of mature miRNA. Figure 1 ).

[0027] SEQ ID NO.2: TCCTAGGAAATAGTTTGCTAGCTTTTCTGAGCTGAGCTAATAGCCGGCGAGTCACGGGCGATCGGGCGGCTGTCGATGTCGTTGAGGCGGTGACGGGCGTCGACGGCTGCCCGGATGGCCAGAGCCTCGTCAGCTAGCAGCTCAGCTCGAAAAAGCTACCAAATTATTTTCTAACACA.

[0028] Example 2: Analysis of miRNA Expression Characteristics 1. Extraction of miRH1 Take an appropriate amount of plant tissue into a 2 mL centrifuge tube containing steel balls and grind the sample using a cryogenic homogenizer. After thorough grinding, add 1 mL of lysis buffer MZ for homogenization. Incubate the lysis buffer mixture at room temperature for 5 min to separate the nucleic acid-protein complex. If the plant tissue has many nodules, centrifuge at 4°C and 12000 r / min for 5 min, then transfer the supernatant to a new enzyme-free centrifuge tube. Add 200 μL of chloroform, cap the tube, and vortex vigorously for 15 sec, then incubate at room temperature for 5 min. Then centrifuge at 4°C and 12000 r / min for 15 min. At this point, the sample will separate into three layers, with RNA mainly in the upper aqueous phase. Measure the volume of the aqueous phase and transfer it to a new enzyme-free centrifuge tube. Slowly add 1.5 times the volume of the transfer buffer in anhydrous ethanol and mix well. Precipitation may occur at this point. Transfer the resulting solution and precipitate together into an adsorption column miRspin, centrifuge at 12000 r / min for 30 sec at room temperature, discard the eluent, and retain the adsorption column. Add 500 μL of protein-removing buffer (MRD) to the miRspin adsorption column, incubate at room temperature for 2 min, centrifuge at 12000 rpm for 30 sec, discard the waste liquid, and retain the adsorption column. Add 500 μL of wash buffer (RW) to the miRspin adsorption column, incubate at room temperature for 2 min, centrifuge at 12000 rpm for 30 sec, discard the waste liquid, and repeat the wash once more. Transfer the miRspin adsorption column (after two washes) back to the matching collection tube, centrifuge at 12000 rpm for 1 min at room temperature, and discard the waste liquid. To ensure accurate subsequent reverse transcription, place the adsorption column on a clean bench for a short period to allow it to air dry completely. Then transfer the miRspin adsorption column to a new RNase-free 1.5 mL centrifuge tube, add 30–100 μL of nuclease-free water, incubate at room temperature for 2 min to fully dissolve the RNA, centrifuge at 12000 rpm for 2 min at room temperature, and retain the RNA in the 1.5 mL centrifuge tube. The extracted RNA was tested for concentration and purity using a Nanodrop spectrophotometer and stored at -80°C to prevent degradation.

[0029] 2. Reverse transcription The miRH1 stem-loop primer RT-miRH1 (nucleotide sequence shown in SEQ ID NO.3) was designed, and reverse transcription was performed using the Abclonal ABScript II cDNA First-Strand Synthesis Kit. The reaction system is shown in Table 1. The reaction program was: 25℃ for 5 min, 42℃ for 60 min, and 80℃ for 5 min.

[0030] SEQ ID NO. 3: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTAGCTTT.

[0031] Table 1 Reverse transcription system Note: Random primers were obtained from the ABScript II cDNA First-Strand Synthesis Kit.

[0032] 3. RT-qPCR Specific primers miRH1-QF and miRH1-QR (nucleotide sequences shown in SEQ ID NO. 4 and 5) were designed. RT-qPCR was performed using cDNA as a template with Abclonal 2× Universal SYBR Green Fast qPCR Mix reagent. The reaction system is shown in Table 2. The reaction program was: 95℃ for 30 s; 95℃ for 10 s, 60℃ for 1 min, for 40 cycles.

[0033] SEQ ID NO.4: TCACAGTCCAGCTCGAAAAAG; SEQ ID NO. 5: CTCAACTGGTGTCGTGGAGTCGG.

[0034] Table 2 RT-qPCR system Use 2 -△△Ct The relative expression level of miRH1 was calculated to determine the differences in expression levels among treatments.

[0035] In this embodiment, total RNA was extracted from barley from the two-leaf stage to the five-leaf-one-heart stage to analyze the expression characteristics of miRH1 in barley. The RNA-seq analysis results are as follows ( Figure 2 (A) showed that miRH1 expression levels were higher in Bowman than in the GSHO1990 mutant material across five consecutive time periods, and the difference between the two sequencing materials was significant. P <0.01). To validate the expression characteristics predicted by sequencing, miRH1 expression levels were detected and analyzed in tillers from the same five stages of both materials. Figure 2 As shown in Figure B, miRH1 expression levels in Bowman were higher than those in GSHO1990 across all five developmental stages examined. Except for the four-leaf stage, significant differences in miRH1 expression were observed between the two materials. P <0.01), which is consistent with the bioinformatics expectation of this invention.

[0036] Secondly, the relative expression levels of miRH1 in different tissues of barley Golden Promise plants were examined, and the results are as follows: Figure 3 As shown, miRH1 exhibits significant tissue specificity in expression: its transcript abundance is highest in tillering nodes, significantly higher than in other tissues; it maintains moderate expression levels in leaves, axillary buds, stems, and spikelets; while its expression is extremely low or almost nonexistent in roots, florets, stems at the jointing stage, spike neck nodes, and rachis. Overall, miRH1 expression is highly enriched in the core tillering region, suggesting that miRH1 may act as a key regulatory factor involved in the initiation and development of tillers.

[0037] Example 3 Construction and transformation of miRH1 transgenic vector 1. Construction of miRH1 overexpression vector The flanking sequences of the miRH1 precursor sequence (SEQ ID NO. 2) were extended by more than 200 bp each using the Ensembl Plant database to obtain the target gene DNA sequence required for constructing the recombinant vector containing the miRH1 precursor sequence in this embodiment. The nucleotide sequence is shown in SEQ ID NO. 6. Then, specific primers premiRH1-F and premiRH1-R (nucleotide sequences shown in SEQ ID NO. 7 and 8) were designed. The miRH1 precursor sequence was amplified using barley genomic DNA as a template. The amplification system is shown in Table 3. PCR program: 95℃ 5 min; 95℃ 30 s, 60℃ 30 s, 72℃ 30 s, 35 cycles; 72℃ 8 min; 12℃ hold.

[0038] SEQ ID NO.6: .

[0039] SEQ ID NO.7: ACGGGGGACTCTTGACCATGGCCAGCCTCCTCTTGTATCCAC; SEQ ID NO. 8: TAGAAATTTACCCTCAGATCTAATCTTCTAACCAGTCCACCCG.

[0040] Table 3 miRH1 precursor sequence amplification system Agarose gel electrophoresis was then performed to confirm the correct band size, and the gel was cut and recovered using Omega EZNA gel recovery. ® Gel Extraction Kit (D2500-01) reagent kit.

[0041] choose Bgl II and NcoI. The pCAMBIA1301 vector was double-digested with the following enzymes: pCAMBIA1301 2 μg, BglⅡ 1 μL, NcoⅠ 1 μL, NEBuffer 3.1 5 μL, and ddH2O to a final volume of 50 μL. The digested products were then gel-cleaved and the recovered target band was ligated to the digested vector using the Vazyme ClonExpress II One Step Cloning Kit. The ligation system is shown in Table 4.

[0042] Table 4 Connection System After gently mixing all components of the system by pipetting, the mixture was placed in a 37°C water bath for 30 min. Immediately after the reaction, the reaction tube was placed on ice for 5 min to cool. The tube was then transformed into *E. coli* DH5α. The specific procedure was as follows: When the DH5α had just thawed on ice, the ligation product was added. After incubating on ice for 30 min, the tube was heat-shocked in a 42°C water bath for 40 s, placed on ice for 3 min, and then 600 μL of antibiotic-free LB liquid medium was added. After shaking the culture on a shaker at 37°C for 90 min, 600 μL of the bacterial culture was plated onto LB agar plates containing kanamycin. After culturing for 12 h, positive single clones were picked and sent to Qingke Biotechnology Co., Ltd. for sequencing to obtain the miRH1 overexpression vector.

[0043] 2. Construction of miRH1 short tandem target overexpression vector The short tandem target mimic (STTM) technology was used to effectively inhibit the activity of endogenous miRNAs in barley. Based on the mature miRH1 sequence as a template, a three-base protrusion was inserted into the miRH1 sequence and connected by a stem-loop structure. The short tandem target mimic was designed, and the sequence is shown in SEQ ID NO.9.

[0044] SEQ ID NO.9: CAGCTCAGCTCTAGGAAAAAGCTAATTCTTCTTCTTTAGACCATATTTAAATTAGACCATAACAACAACAACCAGCTCAGCTCTAGGAAAAAGCTA.

[0045] The construction method of the miRH1 short tandem target (STTM) vector is the same as the construction process in "1. Construction of miRH1 overexpression vector". Specifically, homologous recombination is used to simulate the short tandem target sequence and... Bgl II and NcoI. Homologous recombination was performed on the pCAMBIA1301 vector after double enzyme digestion. Subsequent recombination reaction system and conditions, E. coli transformation, and antibiotic screening and sequencing identification of positive clones were all performed according to "1. Construction of miRH1 overexpression vector". The final result was a miRH1 short tandem target overexpression vector with the correct sequence.

[0046] 3. Genetic transformation The miRH1 overexpression vector was sent to the company for genetic transformation in rice (receptor: kittake) and barley (receptor: Golden Promise), and the company returned T0 generation seedlings.

[0047] The miRH1 short tandem target overexpression vector was sent to the company for barley genetic transformation. The receptor was GoldenPromise, and the company returned T0 generation seedlings.

[0048] Example 4: Phenotypic Statistics of miRH1 Transgenic Plants 1. Extraction of plant genomic DNA Place an appropriate amount of fresh plant tissue in a 2 mL centrifuge tube containing steel balls, freeze in liquid nitrogen, and homogenize using a cryo-mixer. Then add 800 μL of CTAB separation buffer, mix by inverting, and incubate in a 65°C water bath for 30 min, gently shaking every 5 min. Add 800 μL of chloroform:isoamyl alcohol (24:1), mix by inverting, centrifuge at 12000 r / min for 10 min at 4°C, and transfer the supernatant to a new centrifuge tube. Add 0.5 volume of isopropanol, mix gently, and incubate at -20°C for 30 min to precipitate. Centrifuge at 12000 r / min for 10 min at 4°C, discard the supernatant, add 500 μL of 70% ethanol to wash the precipitate, centrifuge at 12000 r / min for 5 min at room temperature, and discard the supernatant. Add 500 μL of anhydrous ethanol to wash the precipitate, centrifuge at 12000 r / min for 5 min at room temperature, and discard the supernatant. Place in a well-ventilated area for 2-3 hours or at room temperature overnight to allow residual alcohol to evaporate. After the alcohol has evaporated, add an appropriate amount of ddH2O to dissolve the precipitate, and then test the concentration and mass.

[0049] 2. Positive identification of transgenic plants For transgenic rice and barley plants, each transgenic material was tagged and numbered, and DNA was extracted from leaves. Wild-type plants were used as negative controls, and positive transgenic lines were screened using PCR with specific primers. The primers used for detecting overexpression plants were 1301-F and miRH1-R, with nucleotide sequences shown in SEQ ID NO. 10 and 11; the primers used for detecting suppressed expression plants were STTM-F and STTM-R, with nucleotide sequences shown in SEQ ID NO. 12 and 13. The PCR amplification reaction system is shown in Table 5. The PCR program was: 95℃ for 5 minutes; 95℃ for 30 seconds, 60℃ for 30 seconds, 72℃ for 30 seconds, repeated 34 times; 72℃ for 7 minutes. The PCR products were analyzed by 1.2% agarose gel electrophoresis to detect DNA bands.

[0050] SEQ ID NO.10: AGACGTTCCAACCACGTCTT; SEQ ID NO.11: CACCCGGCGCATGAATTTAG; SEQ ID NO.12: ACGGGGGACTCTTGACCATGG; SEQ ID NO. 13: TAGAAATTTACCCTCAGATCT.

[0051] Table 5 PCR amplification reaction system 3. Results After multiple generations of greenhouse propagation and positive identification, a stable homozygous positive line of generation T3 was finally obtained. The expression level of miRH1 in the miRH1 transgenic line was first detected, such as... Figure 4 As shown, in rice, the expression level of miRH1 in the three overexpression lines was significantly increased compared to the wild type. Figure 4 (B) Subsequently, phenotypic observation, image collection, and trait statistics were performed on wild-type kittake and three miRH1 overexpression homozygous positive rice lines. The results showed that the number of tillers in miRH1 overexpressing rice plants was significantly higher than that in wild-type (B). Figure 4 (A and C). The expression level of miRH1 in the miRH1 transgenic lines of barley was also detected, such as... Figure 5 As shown, the relative expression levels of miRH1 in the three overexpression lines were significantly higher than those in the wild type, while the relative expression levels of miRH1 in the three suppressed expression lines were significantly lower than those in the wild type. Figure 5 Phenotypic observation, image collection, and trait statistics were performed on wild-type Golden Promise and three homozygous positive miRH1 overexpression and suppression lines. The results showed that, compared with wild-type, miRH1 overexpressing barley plants had a significantly increased number of tillers (C). Figure 5 (A and D), while miRH1-inhibited barley plants showed a significantly reduced number of tillers ( Figure 5 (B and D in the middle).

[0052] The above results indicate that miRH1 can regulate the number of tillers in plants, and overexpression of miRH1 can enhance the number of tillers in transgenic plants; inhibiting the expression of miRH1 will reduce the number of tillers in transgenic plants. This invention provides a theoretical basis and potential application value for molecular breeding and the improvement of ideal plant type.

[0053] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A miRNA, characterized in that, The miRNA is named miRH1, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. The pre-miRH1 precursor of miRH1 as described in claim 1, characterized in that, The nucleotide sequence of the pre-miRH1 is shown in SEQ ID NO.

2.

3. The use of the miRH1 of claim 1, the pre-miRH1 of claim 2, or the biomaterial that overexpresses / inhibits the miRH1 of claim 1 in any of the following: (1) Regulating the number of tillers in plants; (2) Prepare products that regulate the number of tillers in plants; (3) Cultivate plants with a large number of tillers; (4) Cultivate plants with fewer tillers; (5) Improvement of plant tillering traits.

4. The application as described in claim 3, characterized in that, The biomaterials include expression cassettes, recombinant vectors, or recombinant bacteria.

5. The application as described in claim 3, characterized in that, The regulation mentioned is a positive regulation.

6. The application as described in claim 3, characterized in that, The plants mentioned include barley or rice.

7. A method for regulating the number of tillers in a plant, characterized in that, Including any of the following methods: (1) Overexpressing miRH1 in plants increases the number of tillers in the plants; (2) Inhibiting miRH1 expression in plants reduces the number of tillers in the plants; The nucleotide sequence of miRH1 is shown in SEQ ID NO.

1.

8. A method for cultivating plants with a large number of tillers, characterized in that, This includes steps for overexpressing miRH1 in plants; The nucleotide sequence of miRH1 is shown in SEQ ID NO.

1.

9. A method for cultivating plants with few tillers, characterized in that, This includes steps to inhibit miRH1 expression in plants; The nucleotide sequence of miRH1 is shown in SEQ ID NO.

1.

10. The method according to any one of claims 7-9, characterized in that, The plants mentioned include barley or rice.

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