Application of OsHUB1 and / or OsHUB2 protein and encoding gene thereof in regulation and control of rice plant height

Abstract

The invention relates to a rice protein OsHUB1 and / or OsHUB2 and application of a coding gene of the rice protein OsHUB1 and / or OsHUB2 to regulation and control of rice plant height. The invention further relates to a method for regulating and controlling the plant height of the rice. The method comprises the step of losing the protein functions of the OsHUB1 and the OsHUB2. The plant height of the rice can be reduced due to OsHUB1 and / or OsHUB2 protein function deficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and relates to OsHUB1 and / or OsHUB2 proteins, their encoding genes, and their use in regulating the plant height of rice. Background Art

[0002] Rice (Oryza sativa L.) is an important food crop, providing staple food for half of the world's population. Rice is the main source of nutrition for more than about 50% of humans [1]. 95% of the world's rice is produced and consumed in Asia, accounting for 40 - 80% of calories in the Asian diet [2]. Since 1960, the rapid growth of the population and the sharp reduction of cultivated land area have made it increasingly difficult to meet people's food needs, leading to a global food crisis and thus initiating the green revolution of crops.

[0003] In modern agriculture, plant height is one of the important traits determining grain yield [3], and it plays an important role in the photosynthesis, lodging resistance, and mechanical harvesting of food crops. The "green revolution" of rice has had a positive impact on improving the yield potential of rice, represented by the breeding of dwarf varieties [4]. The rice semi-dwarf gene SD1 plays a very important role in modern rice breeding. Semi-dwarf plants can effectively prevent lodging and increase nitrogen fertilizer utilization efficiency [5, 6].

[0004] Currently, SD1 is still the most important dwarf gene. Due to the lack of other available dwarf genes, there is a potential risk of narrow genetic background during the breeding process, which also restricts the development of molecular breeding. Therefore, exploring new genes controlling rice plant height and revealing the molecular regulation mechanism of rice dwarfing are beneficial to improving the diversity of rice dwarfing genetic resources and also to the improvement of rice varieties, and have important application value for guiding the cultivation of dwarf rice. Summary of the Invention

[0005] In the research of the present invention, according to the Arabidopsis thaliana HUB1 and HUB2 genes, their orthologous genes were found in rice and identified as the rice protein genes OsHUB1 and OsHUB2. The loss of function of the proteins expressed by these genes can reduce the plant height of rice. By targeted gene editing, the genes were functionally inactivated in rice. After verification through a breeding mutation system, it was proved that OsHUB1 and / or OsHUB2 proteins and their encoding genes are involved in controlling the plant height of rice. To overcome the deficiencies of the prior art, the purpose of the present invention is to provide the application of OsHUB1 and / or OsHUB2 proteins and their encoding genes in regulating the plant height of rice, providing new dwarfing genetic resources for rice plant type breeding, and having good application potential in improving the morphology of rice plants and enhancing the lodging resistance of rice.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides genes related to rice plant height, named OsHUB1 and OsHUB2, whose nucleotide coding sequences are SEQ ID No:1 and SEQ ID No:2 respectively; the OsHUB1 and OsHUB2 genes encode OsHUB1 and OsHUB2 proteins respectively, and their amino acid sequences are SEQ ID No:3 and SEQ ID No:4 respectively; the loss of function of the proteins expressed by the OsHUB1 and / or OsHUB2 genes can reduce the plant height of rice.

[0008] In one aspect, the present invention provides the use of rice proteins in regulating rice plant height, and the proteins are OsHUB1 and OsHUB2 proteins respectively, and their amino acid sequences are as shown in SEQ ID No:3 and SEQ ID No:4.

[0009] In one embodiment, the nucleotide coding sequences of the OsHUB1 and OsHUB2 proteins are as shown in SEQ ID No:1 and SEQ ID No:2.

[0010] In one embodiment, the regulation of rice plant height is to reduce the plant height of rice.

[0011] In another aspect, the present invention provides a method for regulating rice plant height, which includes the step of making the OsHUB1 and OsHUB2 proteins lose their functions, wherein the amino acid sequences of the OsHUB1 and OsHUB2 proteins are SEQ ID No:3 and SEQ ID No:4.

[0012] In one embodiment, the regulation of rice plant height is to reduce the plant height of rice.

[0013] In one embodiment, the step of making the OsHUB1 and / or OsHUB2 proteins lose their functions is achieved by methods such as gene knockout, gene knockdown, frameshift mutation, etc.

[0014] In one embodiment, the gene knockout and / or frameshift mutation uses a CRISPR / Cas9 vector. In one embodiment, the method further includes selecting NGG within the 1st - 19th exons in the genomic nucleotide sequence of the OsHUB1 gene and within the 1st - 18th exons in the genomic nucleotide sequence of the OsHUB2 gene. Because the NGG is selected at a relatively forward position in the genome, the CRISPR / Cas9 gene editing technology can cause frameshift mutation of the target gene at a relatively forward position, resulting in the loss of function of the target protein.

[0015] In one embodiment, where the CRISPR / Cas9 vector contains a target sequence for gene knockout, the nucleotide sequence of the target sequence is as shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[0016] In another aspect, the present invention provides a CRISPR / Cas9 vector comprising the target sequence as described in any one of the above.

[0017] In another aspect, the present invention provides mutant genes of OsHUB1 and OsHUB2, and their nucleotide sequences are:

[0018] (a) The nucleotide sequence with an eleven-nucleotide deletion of GCGGCTGGCGG between positions 87 and 97 of SEQ ID No:1 (the resulting sequence is as shown in SEQ ID No:27); or

[0019] (b) The nucleotide sequence with a deletion of G at position 2243 of SEQ ID No:1 (the resulting sequence is as shown in SEQ ID No:28); or

[0020] (c) The nucleotide sequence with an insertion of an A between positions 33 and 34 of SEQ ID No:2 (the resulting sequence is as shown in SEQ ID No:29); or

[0021] (d) The nucleotide sequence with a deletion of seven nucleotides ACTGGAT between positions 2164 and 2170 of SEQ ID No:2 (the resulting sequence is as shown in SEQ ID No:30).

[0022] For SEQ ID No:27, due to the above frameshift mutation in SEQ ID No:1, the nucleotide sequence at positions 117 - 119 of SEQ ID No:1 can be transcribed into a stop codon. For SEQ ID No:28, due to the above frameshift mutation in SEQ ID No:1, the nucleotide sequence at positions 2489 - 2491 of SEQ ID No:1 can be transcribed into a stop codon. For SEQ ID No:29, due to the above frameshift mutation in SEQ ID No:2, the nucleotide sequence at positions 108 - 110 of SEQ ID No:2 can be transcribed into a stop codon. For SEQ ID No:30, due to the above frameshift mutation in SEQ ID No:2, the nucleotide sequence at positions 2192 - 2194 of SEQ ID No:2 can be transcribed into a stop codon.

[0023] After the transcription of incorrect RNA, the self-clearing mode of the cell is initiated, clearing the incorrectly transcribed RNA, thereby not expressing the OsHUB1 and OsHUB2 proteins and / or clearing the incorrectly transcribed OsHUB1 and OsHUB2 proteins.

[0024] In one embodiment, the mutant gene as described above is used to reduce the plant height of rice.

[0025] The present invention has the following advantages and effects compared with the prior art: The present invention uses CRISPR / Cas9 gene editing technology and genotype identification to confirm the role of OsHUB1 and / or OsHUB2 proteins and their coding genes in regulating the plant height of rice. The present invention helps to understand the mechanism of action of the OsHUB1 and OsHUB2 genes, laying a foundation for further understanding the regulation of rice plant height. The OsHUB1 and / or OsHUB2 proteins and their coding genes provided by the present invention have great application value in improving the morphology of rice plants and increasing the yield potential of rice. Brief Description of the Drawings

[0026] Figure 1 It is the map of the pHUNC411-OsHUB1 site1-OsHUB2 site1 vector.

[0027] Figure 2 It is the map of the pHUNC411-OsHUB1 site2-OsHUB2 site2 vector.

[0028] Figure 3 It is the genotypes of OsHUB1 and OsHUB2 in the OsHUB1 and OsHUB2 mutants.

[0029] Figure 4 It is the phenotypic observation of the OsHUB1 and OsHUB2 mutant plants.

[0030] Figure 5 It is the plant height statistics of the OsHUB1 and OsHUB2 mutant plants. The values shown in the figure are mean ± standard error (N = 30), and the method for differential analysis test is T-test. Different letters indicate significant differences between data. Detailed Embodiments

[0031] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the following further describes the present invention in detail with reference to specific embodiments and the accompanying drawings. However, the implementation manners of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0032] The research technical steps of the present invention are as follows:

[0033] (1) Cloning and functional identification of OsHUB1 and OsHUB2 genes:

[0034] 1) According to the principle of CRISPR / Cas9 gene editing technology and the cDNA sequences of OsHUB1 and OsHUB2 genes, through database and tool website analysis (http: / / skl.scau.edu.cn / ), the specific gRNA target sequences of OsHUB1 and OsHUB2 genes were obtained, adapter primers were synthesized, the target DNA sequences were amplified using the intermediate vector pMsg-OsU6 (purchased from Anhui Academy of Agricultural Sciences) as a template, and the target sequences were cloned into the CRISPR / Cas9 gene editing vector pHUNC411 (purchased from Anhui Academy of Agricultural Sciences) to obtain the pHUNC411-OsHUB1 gene editing vector ( Figure 1 ) and the pHUNC411-OsHUB2 gene editing vector ( Figure 2 ).

[0035] 2) Genotype identification of OsHUB1 and OsHUB2:

[0036] The phenotypes of the obtained transgenic offspring T1 generation plants were observed throughout the growth period, and the genomic regions near the gRNA target sequences of all T1 generation plants were amplified and sequenced using specific primers to identify the target sequences and mutations near them. Through genotype comparison and phenotype analysis, the mutant system plants of OsHUB1 and OsHUB2 were obtained.

[0037] The present invention uses the CRISPR / Cas9 gene editing technology to knockout the OsHUB1 and OsHUB2 genes in the rice genome. Through genotype identification, the phenotype of reduced plant height was obtained, laying a foundation for improving the rice plant morphology and increasing the rice yield potential.

[0038] Example

[0039] The present invention will be further described in detail below in combination with examples and accompanying drawings, but the implementation manners of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0040] Example 1: Cloning and sequencing of rice OsHUB1 and OsHUB2 genes in this example, including:

[0041] 1. RNA extraction

[0042] The rice used in this invention was purchased from the Anhui Academy of Agricultural Sciences, and the rice variety is Nipponbare. The total RNA of the rice was extracted using the Trizol method.

[0043] 2. Synthesis of cDNA

[0044] (1) Take about 8 μg of the above RNA into a 1.5 mL EP tube, add 5 μL of 10× reaction buffer, 5 μL of DNaseI, 5 μL of DNaseI, supplement DEPC water to 50 μL, and incubate at 37 °C for 30 min to remove DNA.

[0045] (2) Add 170 μL of EDPC water to the above EP tube, and add an equal volume of phenol-chloroform. Vigorously shake and mix well, and centrifuge at 4 °C for 10 min at a speed of 12,000 rpm;

[0046] (3) Transfer the supernatant (about 200 μL) to a new 1.5 mL EP tube, add 1 / 10 volume of 3M NaAC, 2.5 times volume of pre-cooled absolute ethanol and 1.5 μL of glycogen, invert and mix well, and place at -20 °C for more than 30 min;

[0047] (4) Centrifuge at 4 °C for 15 min at a speed of 12,000 rpm, remove the supernatant, and wash twice with 70% ethanol pre-cooled on ice to remove residual ethanol;

[0048] (5) Invert and air dry, add 25 μL of DEPC water and 2 μL of olig(dT)18, shake and dissolve well, and centrifuge briefly. Incubate at 65 °C for 5 min, and quickly place on ice to cool, to obtain a mixture of RNA and oligdT.

[0049] (6) Use the reverse transcription kit from Novizan Company, and add each component according to Table 1:

[0050] [Table 1]

[0051] System components Volume RNA and oligo dT mixture 27 μL M-MLV 2 μL 5× Buffer 8 μL RNA Inhibitor 1 μL dNTP 2 μL

[0052] (7) After incubating in a 55 °C water bath for 1 h, heat-treat at 80 °C for 5 min. Then add 160 μL of DEPC water to dissolve, to obtain the cDNA of Nipponbare rice, and store at -20 °C.

[0053] 3. Design of PCR primers

[0054] Based on the Arabidopsis thaliana HUB1 gene, a comparison was made in the rice database (http: / / rice.plantbiology.msu.edu / ) to find the gene homologous to Arabidopsis thaliana HUB1, named OsHUB1 gene (LOC_Os04g46450, whose nucleotide sequence is shown in SEQ ID NO:1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:3). Primers for the OsHUB1 gene were designed using software. Forward primer: ATGGGGAGCACGGGGGAGCCCGAC (shown in SEQ ID NO:9), reverse primer: TCATATGTAGATAGGTTTCACGTC (shown in SEQ ID NO:10)

[0055] Based on the Arabidopsis thaliana HUB2 gene, a comparison was made in the rice database (http: / / rice.plantbiology.msu.edu / ) to find the gene homologous to Arabidopsis thaliana HUB2, named OsHUB2 gene (LOC_Os10g41590, whose nucleotide sequence is shown in SEQ ID NO:2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:4). Primers for the OsHUB2 gene were designed using software. Forward primer: ATGGATGCCGCAGCTCTTCAGTAT (shown in SEQ ID NO:11), reverse primer: TCAGATCTTCACCTCCCGAACGTC (shown in SEQ ID NO:12)

[0056] 4. PCR Amplification

[0057] Using the cDNA of Nipponbare rice obtained in the above cDNA synthesis as a template, PCR amplification was carried out using the above corresponding primers. The reaction system of PCR is shown in Table 2:

[0058] [Table 2]

[0059] System components Volume 2X Taq Master Mix 10 μL cDNA template of Nipponbare rice 200 ng Forward primer F (10 μM) 0.2 μL Reverse primer R (10 μM) 0.2 μL Water Make up to 20 μL

[0060] The reaction procedure of PCR is as follows: pre-denaturation at 98°C for 5 min, denaturation at 98°C for 15 s, annealing at 58°C for 30 s, extension at 72°C for 2 min, 35 cycles of reaction, post-extension at 72°C for 8 min, and hold at 4°C. After PCR, it was recovered and purified using the DNA recovery kit from merga company to obtain the cDNA of OsHUB1 and OsHUB2 genes.

[0061] 5. Sequencing of OsHUB1 and OsHUB2 Genes

[0062] The above purified DNA fragments were ligated into the vector pGEM-T (purchased from Promega), and transformed into competent E. coli DH5ɑ cells. Positive clones were selected for plasmid extraction, and then sequenced by BGI. An OsHUB1 cDNA fragment of 2655 bp and an OsHUB2 cDNA fragment of 2535 bp were obtained, which had the DNA sequences shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0063] Example 2: Construction of rice pHUNC411-OsHUB1 and pHUNC411-OsHUB2 gene editing vectors

[0064] According to the principle of CRISPR / Cas9 gene editing technology, target sites were analyzed and designed through databases and tool websites. NGG was selected within the first exon and the eighteenth exon of the genomic nucleotide sequence of the OsHUB1 gene, and the target sequence 1 of the specific gRNA of the OsHUB1 gene was obtained as TTCTCCTGCCAAGCGGCTGG (shown in SEQ ID NO:5, which is within the first exon of the OsHUB1 gene genome), and the target sequence 2 was TCAGTATGAGAACCAGAAGC (shown in SEQ ID NO:6, which is within the eighteenth exon of the OsHUB1 gene genome); NGG was selected within the first exon and the sixteenth exon of the genomic nucleotide sequence of the OsHUB2 gene, and the target sequence 3 of the specific gRNA of the OsHUB2 gene was obtained as TCAGTATGAGAACCAGAAGC (shown in SEQ ID NO:7, which is within the first exon of the OsHUB2 gene genome), and the target sequence 4 was AGTGGCTTCGGTCCGCTAC (shown in SEQ ID NO:8, which is within the sixteenth exon of the OsHUB1 gene genome). According to the gRNA target sequences 1 and 3, the target adapter primers were designed as follows:

[0065] F: 5-AATAATGGTCTCTGGCATTCTCCTGCCAAGCGGCTGG

[0066] GTTTTAGAGCTATGCTGAAAAG-3 (shown in SEQ ID NO:13),

[0067] R: 5-ATTATTGGTCTCTAAACGCTTCTGGTTCTCATACTGA

[0068] CACACAAGCGACAGCGCGCGGG-3 (shown in SEQ ID NO:14). According to the gRNA target sequences 2 and 4, the target adapter primers were designed as follows:

[0069] F: 5-AATAATGGTCTCTGGCAGGCACGTGAATCAGCAGTCCGTTTTAGAGCTATGCTGAAAAG-3 (as shown in SEQ ID NO:15)

[0070] R: 5-ATTATTGGTCTCTAAACGTAGCGGACCGAAGCCACTT

[0071] CACACAAGCGACAGCGCGCGGG-3 (as shown in SEQ ID NO:16).

[0072] Amplify the intermediate vector pMsg-OsU6 (purchased from Anhui Academy of Agricultural Sciences) to obtain the DNA sequence 1 of OsHUB1 site 1 and OsHUB2 site 1 (as shown in SEQ ID NO:17) with a length of 450bp and the DNA sequence 2 of OsHUB1 site 2 and OsHUB2 site 2 (as shown in SEQ ID NO:18).

[0073] Clone the DNA sequence 1 and DNA sequence 2 into the CRISPR / Cas9 gene editing vector pHUNC411 to obtain the gene editing vector pHUNC411-OsHUB1 site1-OsHUB2 site1( Figure 1 ) and the gene editing vector pHUNC411-OsHUB1site2-OsHUB2 site2( Figure 2 ).

[0074] Example 3: Breeding and identification of the OsHUB1 and OsHUB2 mutant systems

[0075] Adopt the method of Agrobacterium-mediated transformation of rice mature embryo callus. Among them, Agrobacterium EHA105 is purchased from Anhui Academy of Agricultural Sciences, and the gene editing vectors pHUNC411-OsHUB1 site1-OsHUB2 site1 and pHUNC411-OsHUB1site2-OsHUB2 site2 are transferred into rice mature embryos.

[0076] 1. Breeding of the mutant system

[0077] (1) Induction of rice mature embryo callus: Dehull the mature Nipponbare seeds (purchased from Anhui Academy of Agricultural Sciences), then surface sterilize them with 70-75% alcohol for 1-2 min, then soak them in 30% NaClO solution for 15 min and repeat 2 times, and then wash them with sterile water 4-5 times. Then place the seeds on the induction medium and culture them in the dark at 26-28°C to induce callus for transformation.

[0078] (2) Co - cultivation of rice callus and Agrobacterium: Activate, enrich, and resuspend the EHA105 strain identified to contain the gene - editing vectors pHUNC411 - OsHUB1site1 - OsHUB2 site1 and pHUNC411 - OsHUB1 site2 - OsHUB2 site2, and adjust OD600 = 0.5 - 0.6. Collect the callus in a 50 - ml sterile centrifuge tube, pour in the resuspended Agrobacterium suspension, and infect the callus. After soaking for 15 - 30 min, pour out the suspension, and place the infected callus on sterile filter paper to absorb the excess Agrobacterium liquid. Then place the callus in a petri dish lined with sterile filter paper and culture it in the dark at 26°C for 2 - 3 days.

[0079] (3) Screening of resistant callus: After co - cultivation, transfer the callus to a selection medium containing 50 - 100 mg / ml of G418 antibiotic and conduct resistance screening at 26 - 28°C.

[0080] (4) Differentiation of resistant callus: Place the callus with good growth status in the selection medium into the differentiation medium, and conduct differentiation culture under the conditions of 16 - hour light / 8 - hour dark and an environmental temperature between 26 - 28°C until small seedlings grow out.

[0081] (5) Rooting of differentiated small seedlings: When the differentiated small seedlings grow to 2 - 5 cm in height, transfer the small seedlings to the rooting medium for rooting culture. Transplant the small seedlings with roots to a greenhouse or transgenic nursery for growth.

[0082] 2. Mutation detection of transgenic plants

[0083] Use the following specific primers to perform specific - primer PCR amplification and sequencing on the genomic regions near the target sequences 1, 2, 3, and 4 of gRNA in Nipponbare transformed plants and wild - type Nipponbare plants to identify mutations in the target sequences and their adjacent regions. The amplification reaction program is as follows: Pre - denaturation at 98°C for 5 min, denaturation at 98°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 30 s, 35 cycles of reaction, and post - extension at 72°C for 8 min. The PCR products are separated by 2% agarose gel electrophoresis and stained with ethidium bromide, and the target bands are cut and sent to BGI for sequencing. The specific primer sequences for PCR are as follows:

[0084] Primer sequence for target sequence 1 of gRNA:

[0085] OsHUB1 - S1F: 5 - ATCAGTCACCATGCCACCATCTAGCTA - 3 (as shown in SEQ ID NO:19)

[0086] OsHUB1 - S1R: 5 - TAGCTAGATGGTGGCATGGTGACTGAT - 3 (as shown in SEQ ID NO:20)

[0087] Primer sequences for target sequence 2 of gRNA:

[0088] OsHUB1 - S2F: 5 - CAAGAGGATGGATGGCAACAATCTG - 3 (as shown in SEQ ID NO:21)

[0089] OsHUB1 - S2R: 5 - TATGTAGATAGGTTTCACGTCATT - 3 (as shown in SEQ ID NO:22)

[0090] Primer sequences for target sequence 3 of gRNA:

[0091] OsHUB2 - S1F: 5 - TACTACTGCCTGACATCATCTGTTG - 3 (as shown in SEQ ID NO:23)

[0092] OsHUB2 - S1R: 5 - AGCAGCACTAAATCATCAATCAGCT - 3 (as shown in SEQ ID NO:24)

[0093] Primer sequences for target sequence 4 of gRNA:

[0094] OsHUB2 - S2F: 5 - GCTACAGAAGCAACTTCAGCATGT - 3 (as shown in SEQ ID NO:25)

[0095] OsHUB2 - S2R: 5 - CAGTTTCTGAGGTTAGCTCACTGAC - 3 (as shown in SEQ ID NO:26)

[0096] Through the analysis of sequencing results, two types of OsHUB1 and two types of homozygous mutants of OsHUB1 were obtained, which can be stably inherited. The genotypes I and II of OsHUB1 mutants and genotypes I and II of OsHUB2 mutants corresponding to the target sequences 1, 2, 3, and 4 of gRNA are as Figure 3 shown.

[0097] Genotype I of OsHUB1 mutant is the deletion of eleven nucleotides GCGGCTGGCGG at positions 87 and 97 of OsHUB1, which leads to the nucleotide sequence at positions 117 - 119 of SEQ ID No:1 being encoded into a stop codon (correspondingly, the nucleotide sequence of genotype I of OsHUB1 mutant is as shown in SEQ ID NO:27);

[0098] The genotype II of the OsHUB1 mutant is a deletion of G at position 2243 of OsHUB1, which results in the nucleotide sequence at positions 2489 - 2491 of SEQ ID No:1 encoding a stop codon (correspondingly, the nucleotide sequence of the OsHUB1 mutant genotype II is shown in SEQ ID NO:28).

[0099] The genotype I of the OsHUB2 mutant is an insertion of an A at positions 33 and 34 of OsHUB2, which results in the nucleotide sequence at positions 108 - 110 of SEQ ID No:2 encoding a stop codon (correspondingly, the nucleotide sequence of the OsHUB2 mutant genotype I is shown in SEQ ID NO:29);

[0100] The genotype II of the OsHUB2 mutant is a deletion of seven nucleotides ACTGGAT between positions 2164 and 2170 of OsHUB2, which results in the nucleotide sequence at positions 2192 - 2194 of SEQ ID No:2 encoding a stop codon (correspondingly, the nucleotide sequence of the OsHUB2 mutant genotype II is shown in SEQ ID NO:30).

[0101] After the transcription of incorrect RNA, the self - clearance mode of the cell is initiated, clearing the transcriptionally incorrect RNA, which leads to the non - expression of the OsHUB1 protein in the OsHUB1 - I and OsHUB1 - II mutants and / or the clearance of the transcriptionally incorrect OsHUB1 protein, and the non - expression of the OsHUB2 protein in the OsHUB2 - I and OsHUB2 - II mutants and / or the clearance of the transcriptionally incorrect OsHUB2 protein. The above - mentioned sequencing results were repeated 3 times, and the same results were obtained each time.

[0102] Example 4: Phenotypic identification of rice plants with OsHUB1 and OsHUB2 mutants

[0103] The plants of the OsHUB1 and OsHUB2 mutants I and II lines of rice and the wild - type rice Nipponbare plants were planted in the field. The phenotypic differences between the plants of the OsHUB1 and OsHUB2 mutant lines of rice and the wild - type rice Nipponbare (abbreviated as WT) plants were observed throughout the growth cycle, and the plant heights of 30 plants each of the wild - type and mutants were statistically analyzed. The observation results are as Figure 4 and 5As shown in the figure, at the heading stage of rice, compared with the average plant height of WT plants, the plants of OsHUB1 and OsHUB2 mutants I and II showed dwarf phenotypes. Among them, the average plant height of OsHUB1 mutant I and mutant II was about 30% shorter than that of WT, the average plant height of OsHUB2 mutant I was about 34% shorter than that of WT, and the plants of OsHUB2 mutant II were about 30% shorter than that of WT. After T-test, it was shown that the mutants were significantly shorter than WT, as Figure 5 shown. This result proved that OsHUB1 and OsHUB2 genes are involved in regulating the plant height of rice.

[0104] References:

[0105] 1. White P T. Rice: The essential harvest[J]. Natl. Geogr., 1994, 185: 48 - 79.

[0106] 2. Paramita Bhattacharjee, Rekha S Singhal, Pushpa R Kulkarni. Basmati rice: a review[J]. International journal of food science & technology, 2002, 37(1): 1 - 12.

[0107] 3. Tomoaki Sakamoto, Makoto Matsuoka. Identifying and exploiting grain yield genes in rice[J]. Current opinion in plant biology, 2008, 11(2): 209 - 214.

[0108] 4. Thomas R Hargrove, Victoria L Cabanilla. The impact of semi - dwarf varieties on Asian rice - breeding programs[J]. BioScience, 1979, 29(12): 731 - 735.

[0109] 5. Peter R Jennings. Plant Type as a Rice Breeding Objective 1[J]. Crop Science, 1964, 4(1): 13 - 15.

[0110] 6. JJ Walcott, DR Laing. Some physiological aspects of growth and yield in wheat crops: a comparison of a semidwarf and a standard height cultivar[J]. Australian Journal of Experimental Agriculture, 1976, 16(81): 578 - 587.

[0111] Sequence Listing

[0112] SEQ ID No:1

[0113]

[0114] SEQ ID No:2

[0115]

[0116] SEQ ID No:3

[0117] MGSTGEPDRKRRLSSSVAPGGGAPVSPAKRLAVAPTSEDKKLDFTVLKYKNQKLSEQLEAHKFEYRALENKFAGLKEKQRTHNETLSLVNSSWEQLVADLKSRSFCKSGSPNSSPGSGHNNVQKDGTCAPIERDTLRSLVESGATESSGCLPGCHLGSDAPPLHLSTANALGDIFFPSSDLLQANEECALAALTKLPENDRSKQLQSTSSNLLSSLNNVVQALSNLQLKHKQLAEDYQNQRDSSARKRAEHRRLKEELASAASELEETNYKLAALKAQRDNTQGARIPYPTLGNKNMPEDKVRDKQREMQDLEATHKELSELISKRLVEIKRLHEERIEILNKIATFQNILMDFKSIRSSKAFQLVNDRLQKSQAELDHYQTLLEKLQVDKDKFVWQERQFNLKVDLAEIPERVSTYCESSIADLKKDIQKLCDEKNMLILKLEEASREPGRNQVITKFKALVSSIPREMGAMQSEMTKHKEASLELNSLRAEVHSLSRILSRKERDNEEASCRSARAGSDITQLQSVISDLKQTNKELKLFADMYKRESTDSREIMESRDREFLEWAHVHALKSSLDESKLEQRVKAANEAEAITQQRLATAEAEIAESGQKLGTSRKDLVSLSHMLKSKQEECEAYRVEVECIGQAYEDIQAQNQQLLQQIIERDDDNTKIFMEGVKAKQTQDALHLETYSLRRNLQQESSLMDLYNQKIVSLEDQLKMWSDRVGKLQEDGWQQSVSLSNYQRKLVDVHRDAQKLMQSLDGIQANVGSSRLEVADLLIELEKERFSKKRIEDDLEVMSRKASSLRAKARESAVLEKLRHEVKEYRGILKCGICHDRQKEVVITKCYHLFCNQCIQKSLGNRQRRCPSCSLSFGANDVKPIYI

[0118] SEQ ID No:4

[0119] MDAAALQYENQKLVQQLEAQKSKMRALEGKFKELRDEQCSYDNTLICLNKMWNQLIDDLVLLGVRAGGDLNGLQALDHEEMSEESLESCPSEEIFLFRLLNSRNFRNNDDSSLSKLVEEALALRYSTTVTLMKSLQEAFAVQQARSESLSLALNGQNSSEDVIVALENHNDYLKEVVDNLRQAVSIINRKHEKYLDEIEAFKNNQSRELHEVKCLSGELEESMAELEESRRKLAVLQLQTGGGSLMNTSAPNGVNGSVSTDKSSDKGMGWRDLKDAVEEAKTLAANRLFELHETQEDNLILSKQLEDIQDQLKDENYIVTSKPYTILSDQLHLNAEIERYRGLVEVLQNEKDQLMQKEEEMLAKAESDVAVQQSITTYKAKIEDLEHEIQKLMAEKDLEIKAEEALQDSGKKDFKDEIHV MAASLSKEMELLDNQMNRSKDAASEALAREADYLRTLLAKKIDEQKEISDRYNTQVTEIKSLKALIETLDQEKQELQFIVDMLGKECSESRAISEIEESENRARKQAEYLRKCLEEHNLELRVKAANEAETACQQRLSIAEALEDLRAKVDASERDVMKLKESIRIKEAEVDGHISEIETIGQAYEDMQTQNQHLLQQVADRDDFNIKLVSDSVKMKQAYGSLAEKNMLQKQLQHVNSSLESSKLKITSGEEQMKTYVAQAMKSSSENRHLAISLERTMLEVSDAEKELKWLRSATGSAEKEYEINQKKIAELKMELERERNERIKLEEEEYEEVKNEVSELTSETEETTIQKLQDEIKECKAILKCGVCFDRPKEVVITKCFHLFCSPCIQRNLEIRHRKCPGCGTPFGQSDVREVKI

[0120] SEQ ID No:5

[0121] 5-TTCTCCTGCCAAGCGGCTGG-3

[0122] SEQ ID No:6

[0123] 5-GGCACGTGAATCAGCAGTCC-3

[0124] SEQ ID No:7

[0125] 5-TCAGTATGAGAACCAGAAGC-3

[0126] SEQ ID No:8

[0127] 5-AGTGGCTTCGGTCCGCTAC-3

[0128] SEQ ID No:9

[0129] 5-ATGGGGAGCACGGGGGAGCCCGAC-3

[0130] SEQ ID No:10

[0131] 5-TCATATGTAGATAGGTTTCACGTC-3

[0132] SEQ ID No:11

[0133] 5-ATGGATGCCGCAGCTCTTCAGTAT-3

[0134] SEQ ID No:12

[0135] 5-TCAGATCTTCACCTCCCGAACGTC-3

[0136] SEQ ID No:13

[0137] 5-AATAATGGTCTCTGGCATTCTCCTGCCAAGCGGCTGGGTTTTAGAGCTATGCTGAAAAG-3

[0138] SEQ ID No:14

[0139] 5-ATTATTGGTCTCTAAACGCTTCTGGTTCTCATACTGACACACAAGCGACAGCGCGCGGG-3

[0140] SEQ ID No:15

[0141] 5-AATAATGGTCTCTGGCAGGCACGTGAATCAGCAGTCC GTTTTAGAGCTATGCTGAAAAG-3

[0142] SEQ ID No:16

[0143] 5-ATTATTGGTCTCTAAACGTAGCGGACCGAAGCCACTTCACACAAGCGACAGCGCGCGGG-3

[0144] SEQ ID No:17

[0145] AATAATGGTCTCTGGCATTCTCCTGCCAAGCGGCTGGGTTTTAGAGCTATGCTGAAAAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTAGTAGTAGCATCTGACGGTGAAGGGGGCGGCCGCGGAGGGATCATGAACCAACGGCCTGGCTGTATTTGGTGGTTGTGTAGGGAGATGGGGAGAAGAAAAGCCCGATTCTCTTCGCTGTGATGGGCTGGATGCATGCGGGGGAGCGGGAGGCCCAAGTACGTGCACGGTGAGCGGCCCACAGGGCGAGTGTGAGCGCGAGAGGCGGGAGGAACAGTTTAGTACCACATTGCCCAGCTAACTCGAACGCGACCAACTTATAAACCCGCGCGCTGTCGCTTGTGTGCCAGCCGCTTGGCAGGAGAATGCCAGAGACCATTATT

[0146] SEQ ID No:18

[0147] AATAATGGTCTCTGGCAGGCACGTGAATCAGCAGTCCGTTTTAGAGCTATGCTGAAAAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG AGTCGGTGCTTTTTTTTAGTAGTAGCATCTGACGGTGAAGGGGGCGGCCGCGGAGGGATCATGAACCAACGGCCTGGCTGTATTTGGTGGTTGTGTAGGGAGATGGGGAGAAG AAAAGCCCGATTCTCTTCGCTGTGATGGGCTGGATGCATGCGGGGAGCGGGAGGCCCAAGTACGTGCACGGTGAGCGGCCCACAGGGCGAGTGAGCGCGAGAGGCGGGA GGAACAGTTTAGTACCACATTGCCCAGCTAACTCGAACGCGACCAACTTATAAACCCGCGCGCTGTCGCTTGTGAAGTGGCTTCGGTCCGCTACGTTTAGACCAATAAT

[0148] SEQ ID No:19

[0149] 5-ATCAGTCACCATGCCACCATCTAGCTA-3

[0150] SEQ ID No:20

[0151] 5-TAGCTAGATGGTGGCATGGTGACTGAT-3

[0152] SEQ ID No:21

[0153] 5-CAAGAGGATGGATGGCAACAATCTG-3

[0154] SEQ ID No:22

[0155] 5-TATGTAGATAGGTTTCACGTCATT-3

[0156] SEQ ID No:23

[0157] 5-TACTACTGCCTGACATCATCTGTTG-3

[0158] SEQ ID No:24

[0159] 5-AGCAGCACTAAATCATCAATCAGCT-3

[0160] SEQ ID No:25

[0161] 5-GCTACAGAAGCAACTTCAGCATGT-3

[0162] SEQ ID No:26

[0163] 5-CAGTTTCTGAGGTTAGCTCACTGAC-3

[0164] SEQ ID No:27

[0165]

[0166] SEQ ID No:28

[0167]

[0168] SEQ ID No:29

[0169]

[0170] SEQ ID No:30

[0171]

[0172] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of OsHUB1 and / or OsHUB2 protein in regulating rice plant growth, wherein the amino acid sequence of the OsHUB1 protein is shown in SEQ ID No: 3, and the amino acid sequence of the OsHUB2 protein is shown in SEQ ID No:

4.

2. Use of the OsHUB1 gene encoding the OsHUB1 protein of claim 1 and / or the OsHUB2 gene encoding the OsHUB2 protein of claim 1 in regulating plant growth in rice.

3. The use according to claim 2, characterized in that The nucleotide coding sequence of the OsHUB1 gene is shown in SEQ ID No: 1, and the nucleotide coding sequence of the OsHUB2 gene is shown in SEQ ID No:

2.

4. The use according to claim 1 or 2, wherein the regulating the plant height of rice is to reduce the plant height of rice.

5. A method for regulating rice plant height, comprising the step of causing loss of OsHUB1 and / or OsHUB2 protein function. The method according to claim 5 , wherein the regulating the plant height of rice is to reduce the plant height of rice.

7. The method according to any one of claims 5 or 6, wherein the step of causing loss of function of OsHUB1 and / or OsHUB2 protein is achieved by gene knockout, gene knockdown or frameshift mutation.

8. The method of claim 7, wherein the gene knockout and / or frameshift mutation uses a CRISPR / Cas9 vector.

9. The method of claim 8, further comprising selecting NGG within exons 1 to 19 within the genomic nucleotide sequence of the OsHUB1 gene or within exons 1 to 18 within the genomic nucleotide sequence of the OsHUB2 gene for functional deletion of the OsHUB1 gene or OsHUB2 gene.

10. The method of claim 8, wherein the CRISPR / Cas9 vector comprises a target sequence for gene knockout, and the nucleotide sequence of the target sequence is shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:

8.

11. A nucleotide sequence selected from the group consisting of: The nucleotide sequence of genotype I of the OsHUB1 mutant gene is shown in SEQ ID NO:27; the nucleotide sequence of genotype II of the OsHUB1 mutant gene is shown in SEQ ID NO:28; the nucleotide sequence of genotype I of the OsHUB2 mutant gene is shown in SEQ ID NO:29; and the nucleotide sequence of genotype II of the OsHUB2 mutant gene is shown in SEQ ID NO:

30.

12. Use of the mutant gene according to claim 11 for reducing the plant height of rice.

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