Application of cytochrome P450 gene OsCYP71K4 in rice breeding regulation

Abstract

The application provides application of a cytochrome P450 gene OsCYP71K4 in rice breeding regulation and belongs to the field of botany and biotechnology.The nucleotide sequence of the gene OsCYP71K4 is shown as SEQ ID NO:1, and the amino acid sequence of the encoded protein of the gene OsCYP71K4 is shown as SEQ ID NO:2.OsCYP71K4 has great significance for improving the yield and quality of rice and can be used as a new target for creating new rice grain quality improvement.OsCYP71K4 overexpression plants have significantly increased plant height, and the grain width and the thousand-grain weight are obviously decreased;oscyp71k4 mutant plants have significantly decreased plant height, and the grain length and the thousand-grain weight are obviously increased; and OsCYP71K4 negatively regulates the size change of each rice tissue such as rice grains.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of botany and biotechnology, and particularly relates to application of a cytochrome P450 gene OsCYP71K4 in rice breeding regulation. BACKGROUND

[0002] Rice is one of the most widely planted and most important food crops in the world, and is also one of the model organisms for plant genomics, providing food and energy for more than half of the world's population. With the growing population, global climate change and environmental stress, it is particularly important to breed new varieties of high-yield and high-quality rice. Grain size is one of the most important factors affecting the yield and quality of rice. Exploring new genes and molecular mechanisms that regulate grain size can provide an important theoretical basis for future genetic improvement of rice yield and quality.

[0003] Grain size is one of the important factors affecting the yield of rice. Regulating the growth of endosperm or glume can affect the size of rice grains. Related grain size genes are being widely studied, revealing the mechanisms and pathways of rice grain type. These genes can individually regulate the development of rice grains and play multiple roles in the grain type regulation network. Studies have shown that most grain type-related genes mainly affect the size and number of glume epidermal cells by affecting cell division and expansion, which ultimately determines the size of rice grains. Therefore, exploring key grain type genes can help to further explore the regulation mechanism of rice grain type. Although the regulatory pathways of related genes have been studied, the specific mechanisms of most genes are still unclear and need to be further explored.

[0004] Cytochrome P450 is a large superfamily of enzymes that participates in various primary and secondary metabolic reactions in plants and plays a key role in plant growth and development. So far, most cytochrome P450 genes have not been studied, and the most extensive research on their role in plant hormones is currently focused on their role in gibberellin synthesis. Studies have shown that the semi-dwarf high yield of the "green revolution" in rice is actually caused by the deletion of the OsGA 20 ox2 gene encoding GA 20 oxidase (GA 20 ox). Exploring related genes that control gibberellin synthesis and metabolism may provide a reference for breeding new high-yield and high-quality varieties. Cytochrome P450 monooxygenase CYP714D1 / EUI in rice inactivates gibberellin through epoxidation, and cyp714d1 / eui mutants in rice result in GA inactivation defects, exhibiting higher plant height than wild type. Studies have shown that CYP78A13 can promote cell proliferation and has the potential to increase plant height and grain yield.

[0005] Overall, although some reports have indicated that the cytochrome P450 family is involved in rice growth and development, the role of the OsCYP71K4 gene in rice grain regulation remains unclear. Summary of the Invention

[0006] In order to overcome the problems existing in the related art, the purpose of the present invention is to provide an application of the cytochrome P450 gene OsCYP71K4 in rice breeding regulation.

[0007] The nucleotide sequence of the OsCYP71K4 gene is shown in SEQ ID NO: 1. The amino acid sequence of the protein encoded by the OsCYP71K4 gene is shown in SEQ ID NO: 2.

[0008] The rice breeding regulation includes the regulation of rice grain shape and plant shape improvement.

[0009] In one technical solution of the present invention, the regulation of rice grain and plant shape improvement involves mutating the OsCYP71K4 gene to produce a rice mutant (oscyp71k4) with reduced plant height and increased grain length and 1000-grain weight. The cytochrome P450 gene OsCYP71K4 negatively regulates changes in the size of various rice tissues, including grains. Therefore, the cytochrome P450 gene OsCYP71K4 mutant oscyp71k4 is of great significance for increasing grain size, providing an important genetic resource for increasing rice yield.

[0010] In a preferred technical solution of the present invention, the mutant rice oscyp71k4 is obtained by knocking out the OsCYP71K4 gene of rice through gene editing technology, or by generating one or more deoxynucleotide mutations in the coding region of the OsCYP71K4 gene using gene editing technology.

[0011] In another technical solution of the present invention, the regulation of rice grain shape and plant shape improvement is: the OsCYP71K4 gene is overexpressed, and the resulting overexpression strain OE-OsCYP71K4 has increased plant height and decreased grain width and 1000-grain weight.

[0012] In a preferred technical solution of the present invention, the rice is japonica rice.

[0013] In a preferred technical solution of the present invention, the japonica rice is Zhonghua 11.

[0014] The beneficial effects of the present invention are:

[0015] (1) The present invention cloned the OsCYP71K4 gene from rice using PCR (the corresponding locus number corresponds to LOC_Os02g09400 published in the Rice Genome Annotation Project). The OsCYP71K4 gene is of great significance for improving rice grain size and can serve as a new target for rice yield trait research.

[0016] (2) The present invention mutated OsCYP71K4 through CRISPR / Cas9 to obtain the mutant plant oscyp71k4. The phenotype of the mutant plant oscyp71k4 was analyzed in detail. The results showed that compared with the wild type (WT), the plant height of the mutant plant oscyp71k4 was significantly reduced, the grains became larger, and the 1000-grain weight increased. Through in-depth research, OsCYP71K4 overexpression plants were constructed, and it was found that compared with WT, the plant height of the overexpression plant OE-OsCYP71K4 was significantly increased, the grain width was reduced, and the 1000-grain weight was significantly reduced. OsCYP71K4 negatively regulated the size changes of various rice tissues such as grains.

[0017] (3) The present invention can explore the expression regulation of the OsCYP71K4 gene through in-depth research. In combination with gene editing technology, it can further cultivate ultra-long grains, high-yield, and high-quality rice by molecularly manipulating grain length genes and OsCYP71K4 (such as gene aggregation). Based on previous research, it may be possible to improve the stress resistance of plants and improve the adaptability of rice to the environment, especially to adverse environments. In summary, we believe that the OsCYP71K4 gene is a potential candidate new target for synergistically improving rice yield and stress resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Gene editing of OsCYP71K4: sequence analysis diagram of WT and mutant oscyp71k4; CCCGCGATATCATGAGGACC represents the target site, the dotted line indicates the deleted base, and the font T pointed by the red triangle represents the inserted base T.

[0019] Figure 2 Identification of gene overexpression of OsCYP71K4: The gene expression levels of WT and overexpressed OE-OsCYP71K4 are shown in Figure a, and the protein expression levels of WT and overexpressed plants OE-OsCYP71K4 are shown in Figure b. Two strains with higher expression, OE-OsCYP71K4-5 and OE-OsCYP71K4-6, were selected for subsequent experiments.

[0020] Figure 3The results of OsCYP71K4 phenotypic analysis are shown in Figure a. From top to bottom, Figure a shows the grain length and width results of wild-type WT, overexpression plant OE-OsCYP71K4 and mutant plant oscyp71k4, with a scale of 1 cm. From left to right, Figure b shows the ear shape results of wild-type WT, overexpression plant OE-OsCYP71K4 and mutant plant oscyp71k4, with a scale of 5 cm. From left to right, Figure c shows the grain length and width results of wild-type WT, overexpression plant OE-OsCYP71K4 and mutant plant oscyp71k4, with a scale of 5 cm. Figures 2 and 3 show plant height results for oscyp71k4 (scale equals 5 cm). Figures d and l show phenotypic data for the wild-type (WT), OE-OsCYP71K4 overexpressing plants, and the oscyp71k4 mutant, respectively. Figure d shows spike length; Figure e shows grain number per spike; Figure f shows primary stalk number; Figure g shows plant height; Figure h shows tiller number; Figure i shows 1000-grain weight; Figure j shows secondary stalk number; Figure k shows spike length; and Figure l shows spike length. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences compared with the WT. n = 3.

[0021] Figure 4 Figures show the results of electron microscopy (SEM) analysis of OsCYP71K4. Figure a shows, from left to right, SEM images of glumes from the wild-type WT, an OE-OsCYP71K4 overexpressing plant, and an oscyp71k4 mutant. Scale bar equals 20 μm. Figure b shows the statistical results of glumes from the wild-type WT, an OE-OsCYP71K4 overexpressing plant, and an oscyp71k4 mutant. From left to right, the cell width, total number of cells in the horizontal direction, cell length, and total number of cells in the vertical direction are shown. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences compared with the WT. n = 3. DETAILED DESCRIPTION

[0022] The preferred embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although preferred embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. Rather, these embodiments are provided to make the present invention more thorough and complete and to fully convey the scope of the present invention to those skilled in the art.

[0023] Plant materials: The test material for this study was japonica rice (Oryza sativa L.). The wild type (WT) was Zhonghua 11 (ZH11) (a publicly available rice variety). The plant overexpression vector pRHV was provided by the research group of Wang Guoliang of the Chinese Academy of Agricultural Sciences (the plant overexpression vector pRHVcGFP was disclosed in the supplementary data of He F, Zhang F, Sun W, et al. A Versatile Vector Toolkit for Functional Analysis of Rice Genes [J]. Rice, 2018, 11(1): 27.); mutants were constructed using the CRISPR / Cas9 technology described in Zeng D, Ma X, Xie X, et al. A protocol for CRISPR / Cas9-based multi-gene editing and sequence decoding of mutant sites in plants [J]. Scientia Sinica Vitae, 2018, 48(7): 783-794. The genetic transformation of the above genetic materials was constructed by BioGene Technology Co., Ltd.

[0024] Chemical reagents: Restriction endonucleases were purchased from New England Biolabs; high-fidelity enzyme KOD FX was purchased from TOYOBO; AG reverse transcription kit was purchased from Aikerui Bioengineering Co., Ltd.; gel recovery kit and plasmid extraction kit were purchased from Jifan Biotechnology (Beijing) Co., Ltd.; 2×Taq PCR StarMix was purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd.

[0025] Nucleotide sequence of rice OsCYP71K4:

[0026]

[0027] Amino acid sequence of the protein encoded by the rice OsCYP71K4 gene:

[0028] MAGGAMPLVVLLLATIPLLFFTIKRSAQRRGGGGGGEGRLPPGPWALPVIGHLHHLAGDLPHRALSALARRHGALMLLRLGEVQAVVASSPDAARDIMRTHDAAFASRPLSPMQQLAYGRDAEGVIFAPY GDGRHLRKICTAELLSARRVQSFRPVREAELGRLLRSVAEATSSSSASLVNLTELISAFVADSTVRAIIGSRFEHRDAYLRMLQDGLKIVPGMTLPDLFPSSRLALFLSRVPGRIEHHRQGMQRFIDA IIVEHQEKRAAAAANDDDDEDEDFLDVLLKLQKEEMGSQHPLTTANIKTVMLDMFGAGSESSATVLQWTMAELMRNPRVMQKAQDEVRRALAGHDKVTEPNLTNLPYLRLVIKETLRLHPPAPLLLPRKCG STCKILGFDVPEGVMVIVNAWAIGRDLTYWDKPEEFVPERFEHNGRDFKGMDFEFIPFGAGRRICPGITFGMAHVELVLSALLYHFDWELPQGMAAKDLDMTEDFGVTTQRRSNLLVRPIHRVSVPVE*.

[0029] Example 1: Construction of overexpression strains

[0030] The plant overexpression vector pRHVcGFP was provided by the research group of Wang Guoliang of the Chinese Academy of Agricultural Sciences (the plant overexpression vector pRHVcGFP has been disclosed in the supplementary data of He F, Zhang F, Sun W, et al. A Versatile Vector Toolkit for Functional Analysis of Rice Genes [J]. Rice, 2018, 11(1): 27.).

[0031] 1. Construction of pRHVcGFP-OsACR8 overexpression vector

[0032] (1) Target gene amplification

[0033] The cDNA of WT leaves was used as a template (according to the instructions of the AG reverse transcription kit), primers were designed according to the target gene (Table 1), and the target gene was obtained by PCR amplification. The PCR amplification system is shown in Table 2:

[0034] Table 1: Primers for PCR amplification of target fragments

[0035]

[0036] Table 2: Target gene PCR reaction system

[0037] Reaction solution Volume (μL) Template 2 μL 2x KOD FX buffer 25 μL Primer OsCYP71K4-OE-F / R (10 μM) 1.5 μL 2 mM dNTPs 10 μL High-fidelity enzyme KOD FX 1 μL <![CDATA[ddH2O]]> 9 μL Total volume 50 μL

[0038] The amplification procedure was as follows: 94°C initial denaturation for 5 minutes; 32 cycles of denaturation at 98°C for 10 seconds, annealing at 55°C for 30 seconds, and extension at 68°C for 60 seconds; a total extension at 68°C for 5 minutes, followed by a 1-minute hold at 16°C. After the reaction, 50 μL of the amplified product was electrophoresed on a 1% agarose gel containing a nucleic acid dye. After electrophoresis, the gel was imaged on a UV imager to detect amplified bands. If the target band was amplified, the amplified fragment likely contained the target gene. The target band was excised and the product was purified by gel extraction (according to the gel extraction kit instructions) and concentration was determined.

[0039] (2) Preparation of linearized vector

[0040] 3 μg of each of the overexpression vector pRHVcGFP and the target gene were taken (according to the instructions of the plasmid extraction kit). The corresponding restriction endonucleases were added to both reaction systems and double-digested at 37°C for 20 minutes. The reaction systems for the overexpression vector pRHVcGFP and the target gene are shown in Table 3:

[0041] Table 3: Enzyme digestion reaction system

[0042] Reaction solution Amount 10x CutSmart buffer 5 μL Sac I endonuclease 1 μL kpn I endonuclease 1 μL Overexpression vector pRHVcGFP / target gene 3 μg Total volume make up with dd H2O to 50 μL

[0043] Take 50 μL of the reaction product and add it to 1% agarose gel containing nucleic acid dye for electrophoresis. After the electrophoresis is completed, the gel is imaged on a UV imager. After the target band is separated, the target band is cut and the gel is continued to recover the purified product and determine the concentration.

[0044] (3) Recombination reaction

[0045] Use 2×Hieff MultiS Enzyme Premix (derived from the Multi-Fragment One-Step Rapid Cloning Kit, purchased from Shanghai Yisheng Biotechnology Co., Ltd.) homologous recombinase recombined the insert and vector in appropriate proportions to generate the pRHVcGFP-OsCYP71K4 overexpression vector. The optimal vector-to-insert molar ratio is 1:(2-3). The DNA mass corresponding to these molar ratios can be roughly calculated using the following formula: Optimal vector dosage X = [0.02 × number of vector base pairs] ng (0.03 pmol). Optimal insert dosage Y = [0.04 × number of insert base pairs] ng (0.06 pmol) or [0.06 × number of insert base pairs] ng (0.09 pmol). The recombination reaction system is shown in Table 4.

[0046]

[0047] Table 4: Recombination reaction system

[0048] After the system is prepared, gently pipette to mix the components. Briefly centrifuge to collect the reaction mixture at the bottom of the tube. Incubate at 50°C for 20 minutes. The reaction product can be used for transformation directly or stored at -20°C and thawed for transformation when needed.

[0049] (4) Transformation and plating of recombinant products

[0050] Thaw cloning competent DH5α cells on ice. Add 10 μL of the cooled recombinant product (containing the pRHVcGFP-OsCYP71K4 overexpression vector) to 100 μL of competent cells. Gently tap the tube several times to mix thoroughly. Place on ice for 30 minutes. Heat shock at 42°C for 60 seconds, incubate on ice for 2 minutes, add 900 μL of LB medium, shake at 37°C at 200 rpm for 30 minutes, centrifuge at 5000 rpm for 3 minutes, and discard the supernatant. Resuspend the cells in the remaining medium and gently spread evenly on a plate containing kan resistance using a sterile spreader. Once the suspension is absorbed, invert the plate and incubate overnight at 37°C.

[0051] (5) Clone identification

[0052] The most convenient and quickest method is colony PCR. Use a sterile pipette tip or toothpick to pick a single colony into 500 μL of LB medium and mix thoroughly. Take 1 μL directly as the PCR template, and use the remaining culture for sequencing and verification. The PCR reaction system is shown in Table 5.

[0053] Table 5: Colony PCR reaction system

[0054]

[0055]

[0056] The amplification procedure was as follows: 94°C pre-denaturation for 5 minutes; 25 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds; a total extension at 72°C for 5 minutes, followed by a 1-minute hold at 16°C. After the reaction, 20 μL of the amplified product was electrophoresed on a 1% agarose gel containing a nucleic acid dye. After electrophoresis, the gel was imaged on a UV imager to detect amplified bands. If amplified bands were detected, the clone was sent to a sequencing company for sequencing (sequencing was performed by Guangzhou Qingke Biotechnology Co., Ltd.). The sequencing primers were:

[0057] UbiP-seq:TTTTAGCCCTGCCTTCATACGC;

[0058] GFP-seqR:AACTTGTGGCCGTTTACGTCG.

[0059] 2. Genetic transformation and identification of transgenic plants

[0060] The constructed pRHVcGFP-OsCYP71K4 overexpression vector was sent to BioGene Technology Co., Ltd. for genetic transformation to obtain T0 generation plants. The obtained transgenic plants were identified. Since the pRHVcGFP-OsCYP71K4 overexpression vector carries the HYG hygromycin tag, it is only necessary to identify whether it is a transgenic plant by identifying whether the hygromycin tag is present. The total genomic DNA of the leaves of the test plant was extracted and PCR amplified using the primer HYG (Table 6). The band size of the PCR product was determined by agarose gel electrophoresis to determine whether it met the requirements. If it met the requirements, it was a transgenic plant containing the target gene (i.e., an OsCYP71K4 gene overexpressing plant). The PCR amplification method referred to the PCR reaction system and PCR amplification procedure of step (1) cloning and identification in the method for constructing the plant pRHVcGFP-OsCYP71K4 overexpression vector. After the reaction, 5 μL of the amplified product was added to a 1% agarose gel containing a nucleic acid dye for electrophoresis. After the electrophoresis, the gel was imaged on a UV imager to observe whether there was a target band. The T0 generation plants were self-pollinated to obtain the T1 generation plants, and the T1 generation plants were self-pollinated to obtain the T2 generation homozygous OsCYP71K4 gene overexpressing plants (OE-OsCYP71K4).

[0061] Table 6: HYG identification primers

[0062]

[0063] The OsCYP71K4 gene is cloned from rice by the method of PCR in this embodiment, and the OsCYP71K4 gene has important significance for regulating rice grain development and can be used as a new target for improving rice grain traits. The OsCYP71K4 gene is introduced into rice by genetic transformation, and a series of overexpression lines are screened. The phenotype of the overexpression lines is analyzed in detail, and the results are shown in Figure 3 .

[0064] Example 2: Construction of mutant lines

[0065] The oscyp71k4 mutant is constructed according to the CRISPR / Cas9 technology described in Zeng D, Ma X, Xie X, et al. A protocol for CRISPR / Cas9-based multi-gene editing and sequence decoding of mutant sites in plants [J]. Scientia Sinica Vitae, 2018, 48(7): 783-794.

[0066] 1. Target sequence selection and primer design

[0067] The OsCYP71K4 gene of the wild type plant is taken as the target site with 20bp size CCCGCGATATCATGAGGACC, and the primer is designed according to the target site. gRT2: GGTCCTCATGATATCGCGGGgttttagagctagaaat; OsU3T2: CCCGCGATATCATGAGGACCTgccacggatcatctgc; gRT1: CGTGCTCTTGA AGCTCCAAAgttttagagctagaaat; OsU3T1: TTTGGAGCTTCAAGAGCACGTgccacggatcatctgc.

[0068] 2. Overlapping PCR

[0069] (1) First round of PCR. The purpose of this step is to introduce the target sequence into the downstream of the U3 / U6 promoter and the upstream of the sgRNA sequence, respectively.

[0070] 7.5 μl of 2x Phanta Max Buffer; 0.25 μl of 10 mM dNTPs Mix; 0.2 μl of Phanta Max Polymerase; 2-5 ng of YLgRNA-U6 / U3; 0.3 μl each of 10 μM UF and UT (reaction 1); 0.3 μl each of 10 μM gR-T and gR-R (reaction 2); make up to 15 μl with ddH2O; perform 25-26 PCR cycles: 95°C for 10 seconds, 58°C for 15 seconds, and 72°C for 15 seconds. Check 3-5 μl of the PCR product by 1.5% agarose gel electrophoresis (the product in reaction 2 is approximately 140 bp long). If the amplified product is weak, continue with a second round of PCR.

[0071] (2) Second round of PCR. The purpose of this step is to construct the promoter, target and sgRNA into a complete expression cassette. When using, synthesize two pairs of primers, Pps-R / Pgs-2 and Pps-2 / Pgs-L, according to the two targets, and pre-mix the universal primer pairs into a working solution (10 μM each). Pps-R: TTCAGAGGTCTCTaccgACTAGTCACGCGTATG GAATCGGCAGCAAA; Pgs-2: AGCGTGggtctcGtcagggTCCATCCACTCCAAGCTC; Pps-2: TTCAGAggtctcTctgacacTGGAATCGGCAGCAAAGG; Pgs-L: AGCGTGggtctc GctcgACGCGTATCCATCCACTCCAAGC.

[0072] (3) For each expression cassette in the second round of PCR, select a 30 μl system. Take 1 μl of each product from reaction 1 and reaction 2 in the first round of PCR, add them to 8 μl of ddH2O, and dilute them 10-fold. Take 1 μl as the template for the second round of PCR. Take 3 μl of the PCR product and perform electrophoresis to check the approximate concentration of the sample. Based on the concentration of the PCR fragments of each expression cassette in the second round, mix all the expression cassette fragments in roughly equal amounts and purify them using a PCR product purification kit.

[0073] 3. Clone the sgRNA expression cassette into the pYLCRISPR / Cas9 vector

[0074] This step uses the "Golden Gate" cloning method based on Bsa I enzyme digestion and ligation to assemble sgRNA expression cassette to pYLCRISPR / Cas9 vector by "cut and paste" method. Prepare 15 μl reaction system: 10x CutSmart Buffer 1.5 μl; 10 mM ATP 1.5 μl (1.5 μl of 10x T4 DNA ligase buffer can also be added instead of ATP); pYLCRISPR / Cas9 plasmid 60-80 ng; purified mixed sgRNA expression cassette, 10-15 ng per expression cassette, a total of 20-30 ng for 2 target points; BsaI-HF 10U; T4 DNA ligase 35U; ddH2O to 15 μl. Use variable temperature cycling (PCR instrument can be used) to perform cut and paste reaction for 10-15 cycles (37°C 5 min, 10°C 5 min, 20°C 5 min); finally 37°C 5 min.

[0075] 4. Recombination reaction and transformation

[0076] Method same as overexpression vector construction.

[0077] 5. Genetic transformation and identification of transgenic plants

[0078] The constructed CRISPR / Cas9-OsCYP71K4 vector is sent to Baige Gene Technology Co., Ltd. for genetic transformation to obtain T0 generation plants. The obtained transgenic plants are identified, and since the CRISPR / Cas9-OsCYP71K4 vector carries a HYG hygromycin tag, only the presence or absence of the hygromycin marker needs to be identified to determine whether it is a transgenic plant. The detection method is the same as the overexpression material identification in the above, after the PCR amplification program is completed, 5 μL of the amplification product is added to a 1% agarose gel containing a nucleic acid dye for electrophoresis, after electrophoresis, the gel is imaged on an ultraviolet imager, and whether the target band is observed. The remaining PCR stock solution is sent to a sequencing company for sequencing and compared with the wild type nucleotide sequence to determine the mutation type of the mutant. The T0 generation plants are selfed to obtain T1 generation plants, at this time, whether the plants carry the CRISPR / Cas9 vector is detected, and the T1 generation seeds are harvested from the plant lines that do not contain the vector, the T1 generation plants are selfed, and T2 generation homozygous OsCYP71K4 gene mutant plants (oscyp71k4) are obtained. The phenotype of the mutant plants is analyzed in detail, and the results are shown in Figure 3 .

[0079] Example 3: Expression amount detection of overexpression plants

[0080] The present invention sowed wild-type japonica rice (WT) and OsCYP71K4 overexpression strain (OE-OsCYP71K4) in a farm of South China Agricultural University in Guangzhou, Guangdong Province, and extracted RNA and protein from mature rice leaves for detection.

[0081] 1. Detection of OE-OsCYP71K4 gene expression

[0082] The specific steps are as follows:

[0083] Step 1: Extraction of total RNA from rice

[0084] Cut an appropriate amount of fresh rice leaves and place them in a pre-chilled mortar (previously sterilized). Add liquid nitrogen and grind rapidly and thoroughly in liquid nitrogen. Weigh 100 mg of the ground powder into a 1.5 mL RNase-free centrifuge tube. Add 1 mL of Trizol and vortex to mix thoroughly. Alternatively, the sample can be minced and then ground directly in Trizol. Let the lysed sample or homogenate stand at room temperature for 5-10 minutes to completely separate the nucleoprotein from the nucleic acid. Add 0.2 mL of chloroform, vortex for 15 seconds, and let stand at room temperature for 3 minutes. Centrifuge at 12,000 rpm at 4°C for 10 minutes. Transfer the upper aqueous phase to a new RNase-free centrifuge tube, add an equal volume of isopropanol, mix thoroughly, and let stand at room temperature for 20 minutes. Centrifuge at 12,000 rpm at 4°C for 10 minutes and discard the supernatant. Wash the pellet with 1 mL of 75% ethanol. Centrifuge at 12,000 rpm at 4°C for 3 minutes and discard the supernatant. Dry at room temperature for 5-10 minutes (do not heat or overdry, as RNA will be difficult to dissolve if completely dried). Add 30-50 μL of RNase-free ddH2O to fully dissolve the RNA. Store the resulting RNA solution at -80°C or use it for subsequent experiments.

[0085] Step 2: Synthesis of the first strand of cDNA

[0086] In this experiment, the Evo M-MLV Reverse Transcription Kit II (Accurate Biotechnology) was used to reverse transcribe the previously extracted RNA to obtain the desired cDNA. The specific steps are as follows: Remove RNA from the -80°C freezer, thaw on ice, and measure the RNA concentration after complete dissolution. Calculate the amount of RNA used in the system as X = 1 μg RNA / concentration × volume. Remove any residual DNA from the RNA using gDNA Clean Reagent. The reaction system includes: 1 μL gDNA Clean Reagent; 2 μL 5× gDNA Clean Buffer; 2000 ng RNA; and add RNase-free H2O to a volume of 10 μL.

[0087] Pipette the reaction mixture to mix thoroughly, centrifuge briefly, and then place in a PCR instrument. Set the reaction mixture to 42°C for 2 minutes. Remove the reaction mixture and immediately place it on ice for the next step. For further experiments, reverse transcribe the reaction mixture into cDNA. Pipette the mixture to mix thoroughly, centrifuge briefly, and then place it in a PCR instrument. PCR reaction conditions: 37°C for 15 minutes; 85°C for 5 seconds. After the reaction, place in a -20°C refrigerator until ready to use. Reaction system: Evo M-MLV RTase Enzyme Mix 1μL; RT Primer Mix 10μL; 5× RTase Reaction Buffer Mix I 4μL; RNAase-free H2O 4μL.

[0088] Step 3: qRT-PCR analysis

[0089] The cDNA obtained by reverse transcription was used as a template and adjusted to the same concentration. The primers for qRT-PCR were shown in Table 7. qPCR SYBR Green Master Mix Kit. Place a 96-well PCR plate on ice and add samples in the dark. Each sample requires three biological replicates and three technical replicates, and set the corresponding internal control. Seal the plate with film, centrifuge and mix thoroughly, and place in a real-time fluorescence quantitative PCR instrument. Reaction system: qPCR SYBR Green Master Mix 10 μL; Primer F / R 0.4 μL; cDNA template 1 μL; ddH2O 8.2 μL. Amplification program: 95°C 2 min; 95°C 10 s; 60°C 30 s; 95°C 15 s; 60-95°C 0.1°C / s.

[0090] Table 7 qRT-PCR expression analysis primers

[0091]

[0092]

[0093] 2. Detection of OE-OsCYP71K4 protein expression

[0094] Step 1: Protein Extraction

[0095] Place an appropriate amount of rice leaves in a liquid nitrogen-cooled mortar and pestle. Grind the leaves quickly and thoroughly in liquid nitrogen. Weigh 0.1 g of the ground powder into a 1.5 mL centrifuge tube and resuspend in 500 mL of IP buffer (Tris-MES, pH 8.0, 50 mmol / L; Sucrose, 500 mmol / L; MgCl2, 1 mmol / L; EDTA, 10 mmol / L; DTT, 5 mmol / L; PMSF, 1 mmol / L; Cocktail, 100×). Mix thoroughly with a shaker. Let stand on ice for 30 min to fully lyse the leaves. Centrifuge in a pre-cooled benchtop centrifuge at 14,000 × g for 30 min at 4°C. Pipette the supernatant into a new 2 mL centrifuge tube on ice (all subsequent steps are performed at 4°C). Add an appropriate amount of 2× SDS protein loading buffer to the tube. Boil at 95°C for 5-10 minutes. Centrifuge at 5,000 × g for 1 minute and prepare for western blotting.

[0096] Step 2: Western blotting

[0097] (1) Prepare electrophoresis gel: Fix the gel plate, prepare the separation gel of appropriate concentration according to the size of the protein, inject the prepared separation gel into the gel plate with a pipette, and press the upper layer with pure alcohol until the separation gel solidifies.

[0098] (2) After solidification for 40-60 minutes, pour off the alcohol and carefully absorb the remaining alcohol with filter paper. Prepare the concentrated gel and carefully add it to the upper layer of the separation gel. Insert the comb (be careful not to have bubbles).

[0099] (3) After the concentrated gel is completely solidified, remove the comb, fix the gel in the electrophoresis tank, add electrophoresis solution, load the prepared samples in sequence, and perform SDS-PAGE electrophoresis until the marker bands are completely separated and the target band does not run out of the gel.

[0100] (4) After the SDS-PAGE electrophoresis is completed, use a thin plate to gently pry apart the two glass plates of the gel plate, leaving the gel on one of the glass plates. Carefully cut the separation gel with a blade along the junction of the separation gel and the concentrated gel, and cut off a small corner of the separation gel to mark the order of spotting. Then carefully transfer the gel into the transfer buffer. Cut a 0.45μm PVDF membrane of the same size as the separation gel and soak it in methanol for 5s. Take 6 pieces of filter paper of the same size and soak them in transfer buffer for 15min at the same time as the PVDF membrane and gel. Place the sponge gasket, filter paper, gel, membrane, filter paper, and sponge gasket (from bottom to top) in the transfer device from the negative pole (black bottom) to the positive pole. Be sure to remove bubbles when placing them. Constant current 200mA, transfer for 40-60min. After the transfer is completed, take out the PVDF membrane, carefully cut off a corner at the same position as the gel, and mark the electrophoresis direction and the front and back of the membrane.

[0101] (5) Blocking the membrane: Incubate with 5% skim milk powder at 37°C with slow shaking for 1-2 h. After blocking, wash the membrane. Add 20 mL of TBST to a square container and place the PVDF membrane in it, ensuring that the TBST covers the membrane. Wash once by shaking on a shaker at low speed for 10 min.

[0102] (6) Dilute the primary antibody with blocking solution according to the dilution factor recommended in the antibody instructions. After aspirating the blocking solution with a pipette, immediately add the diluted primary antibody and incubate slowly on a shaker at room temperature for 1 hour. After the primary antibody incubation is complete, aspirate the primary antibody with a pipette, add TBST to a square fresh-keeping box, place the PVDF membrane in the fresh-keeping box, make sure the TBST covers the PVDF membrane, shake at low speed on a shaker for 10 minutes, and repeat the wash three times.

[0103] (7) Dilute the secondary antibody to an appropriate concentration using blocking buffer according to the secondary antibody instructions. Immerse the PVDF membrane in a square fresh-keeping box containing the secondary antibody and incubate slowly on a shaker at room temperature for 1 hour. After the secondary antibody incubation is complete, remove the secondary antibody with a pipette and add TBST to the square fresh-keeping box. Place the PVDF membrane in the fresh-keeping box so that the TBST covers the PVDF membrane. Shake at low speed on a shaker for 10 minutes and repeat the wash three times.

[0104] (8) Prepare the luminescent solution (liquid A and liquid B) in a 1:1 ratio (be careful to avoid light). Use a pipette to draw an appropriate amount of luminescent solution to cover the PVDF membrane. Expose and collect images on an ECL luminometer.

[0105] (9) Use Western Blot with primary and secondary antibody removal buffer to wash away the antibodies on the PVDF membrane. Add TBST to a square container and place the PVDF membrane in the container until the TBST covers the membrane. Shake the membrane on a shaker at low speed for 10 minutes and repeat the wash three times. Then, follow steps (6)-(8) to incubate with another labeled antibody for development.

[0106] Example 4: Scanning electron microscopy analysis of rice grain hull cells

[0107] Tested rice varieties: wild type WT, mutant oscyp71k4, and overexpressed OE-OsCYP71K4.

[0108] The specific steps are as follows: Select wild-type WT, mutant oscyp71k4, and overexpressing OE-OsCYP71K4 seeds and cut the husks of appropriate size, then prepare the samples, spray gold, mount them, observe the size of rice husk cells using a scanning electron microscope, take pictures and count them. Figure 4 .

[0109] Unless otherwise specifically stated, the relative arrangement, numerical expression and numerical value of the parts and steps set forth in these embodiments do not limit the scope of the application. In all examples shown and discussed here, any specific value should be interpreted as merely exemplary, rather than as a restriction. Therefore, other examples of exemplary embodiments can have different values. It should be noted that: similar reference numerals and letters represent similar items in the accompanying drawings below, and therefore, once a certain item is defined in an accompanying drawing, it does not need to be further discussed in the accompanying drawings subsequently.

[0110] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Knockout OsCYP71K4 The application of genes in rice breeding regulation is characterized by: described OsCYP71K4 The nucleotide sequence of the gene is shown in SEQ ID NO:

1. The rice breeding regulation is the regulation of rice grain shape and plant type improvement; the regulation of rice grain shape and plant type improvement is: OsCYP71K4 The mutant rice was obtained by mutating the gene oscyp71k4 ; The mutant rice oscyp71k4 The phenotypes are: reduced plant height, increased grain length and 1000-grain weight.

2. according to claim 1 OsCYP71K4 The application of genes in rice breeding regulation is characterized by: described OsCYP71K4 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

3. according to claim 1 OsCYP71K4 Application of gene in rice breeding regulation, the mutant rice oscyp71k4 Knockout of rice using gene editing technology OsCYP71K4 genes, or using gene editing technology in OsCYP71K4 It is caused by more than one deoxynucleotide mutation in the coding region of the gene.

4. The method according to any one of claims 1 to 3 OsCYP71K4 The application of genes in rice breeding regulation is characterized by: The rice is japonica rice.

5. according to claim 4 OsCYP71K4 The application of genes in rice breeding regulation is characterized by: The japonica rice is Zhonghua No. 11.

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