Rice C2H2 type zinc finger protein OsZFP7 gene and application of rice C2H2 type zinc finger protein OsZFP7 gene in increasing rice seed size

Abstract

The invention relates to application of a CRISPR / Cas9 technology to targeted editing of a rice C2H2 type zinc finger protein OsZFP7 gene to increase the size of rice seeds.

Description

Technical Field

[0001] This invention relates to the fields of transgenic technology and plant disease control, specifically to the application of CRISPR / Cas9 technology in targeted editing of the rice C2H2 type zinc finger protein OsZFP7 gene and its use in increasing rice grain size. Background Technology

[0002] To achieve the most efficient allocation of resources, the plant hormone signaling cascade must be strategically regulated. Transcriptional repressors play a key regulatory role in the plant hormone signaling cascade and are essential for maintaining the balance between transcriptional activation and repression.

[0003] Particle size is one of the key factors determining grain yield. Transcription factors are an important class of regulatory proteins that play a crucial role in regulating developmental responses. Zinc finger proteins are one of the largest families of transcription factors in plants, and their subfamilies differentiate through the arrangement of Cys and His residues, such as Cys2 / His2 type (C2H2), C3H, C3HC4, C2HC5, C4HC3, C2HC, C4, C6, and C815. Notably, C2H2 type zinc finger proteins have been identified as having significant transcriptional repression and playing a key role in the regulation of various plant life processes. The inventors of this invention used the CRISPR / Cas9 gene editing system to target and edit the OsZFP7 gene, a member of the C2H2 type zinc finger protein family. By constructing the YL-Hu-OsZFP7 knockout vector, callus tissue induced by mature NIP embryos of rice was transformed using rice callus transfection technology. After obtaining T0 generation transgenic rice seeds, they were further propagated to obtain T1 generation seeds. DNA was extracted from the young leaves of T1 generation individual plants, and the sgRNA target site was amplified using target-specific primers and sequenced to obtain stable homozygous mutant transgenic plants.

[0004] The inventors of this application have demonstrated through numerous experiments that OsZFP7 mutant transgenic rice can significantly enhance rice grain size; OsZFP7 mutant plants have great application value in the field of transgenic technology and have achieved unexpected technical results. Summary of the Invention

[0005] This invention relates to the use of CRISPR / Cas9 technology to target and edit rice C2H2 type zinc finger protein OsZFP7.

[0006] On the one hand, the OsZFP7 gene has the nucleotide sequence shown in SEQ ID NO.1.

[0007] .

[0008] On the other hand, the amino acid sequence (SEQ ID NO:2) of rice C2H2 type zinc finger protein OsZFP7 is as follows:

[0009] MERSPEVVQHKAANRAAEDDHTTSIPWLKLGVVDALTAEAGKLPESNPKP

[0010] AVAAPHRTFSCNYCMRKFFS SQALGGHQNAHKRERCAPRKSHGFQQQHLM

[0011] VGLSPTAPSSFLHHMRVNPHATILKVNRGYSSADGVVVAKFHGGQMSSSWVPFAVEHGRGSVWPGSFKASSQEQKKRTEEDLDLSLRL*.

[0012] In some implementations, the preferred gramineous food crops are rice, maize, wheat, oats, and barley; more preferably rice, and most preferably Nipponbare.

[0013] In some implementations, the negative regulation of OsZFP7 gene expression is achieved via a CRISPR / Cas9 system; the target of the CRISPR / Cas9 system is shown in SEQ ID NO:3.

[0014] SEQ ID NO: 3: CCTGAAGTGGTGCAACACAAGGC.

[0015] On the other hand, the present invention relates to a method for preparing a transgenic plant with the dominant trait of larger rice grains, the steps of which include:

[0016] 1) Construction of rice OsZFP7 knockout vector;

[0017] 2) Rice genetic transformation, Agrobacterium-mediated transformation and callus induction culture

[0018] 3) Identification of positive transgenic plants;

[0019] In some embodiments, the preparation method further includes: photographing and quantifying the size of positive transgenic plants: OsZFP7-ko mutant transgenic rice and transgenic rice NIP seeds;

[0020] In some embodiments, the rice OsZFP7 knockout vector construction step further includes:

[0021] Primers were designed based on the OsZFP7 sequence shown in SEQ ID No:1 using the CRISPR-GE website (http: / / skl.scau.edu.cn / ), and the sgRNA fragment was cloned from rice. The PCR product was recovered from the gel. Using the pYLsgRNA-OsU3 vector as a template, the target sequence was ligated to the U3 promoter and gRNA scaffold, respectively. The two PCR products were mixed and used as a template. Overlapping PCR was used to ligate the U3 promoter, target sequence, and gRNA scaffold together to form an sgRNA expression cassette. The gRNA expression cassette product and the uncut pYLCRISPR / Cas9Pubi-H plasmid were mixed and digested with BsaI at 37℃ for 15 min. After digestion, 1.5 μl of 10×DNA Ligase Buffer and 35 U T4 ligase were added, and the digestion and ligation were performed using variable-temperature cycling for 15 cycles: 37℃ for 5 min; 10℃ for 5 min; 20℃ for 5 min. Positive clones were selected and sequenced to confirm the successful construction of the expression vector YL-Hu-OsZFP7. The recombinant plasmid was obtained, and its quality was assessed by 1% agarose gel electrophoresis.

[0022] In some embodiments, the steps of rice genetic transformation, Agrobacterium transformation, and callus induction culture further include:

[0023] Callus induction: First, select mature rice seeds, peel off the husks, pour them into centrifuge tubes, add 75% ethanol for 1 minute for disinfection, then discard the ethanol, rinse three times with sterile water, then add 15 mL of 30% sodium hypochlorite for 20 minutes for disinfection, then discard the sodium hypochlorite and rinse 5-6 times with sterile water; use a pipette to remove excess water, transfer the seeds to the induction medium, and incubate in a 28℃ light incubator for 3 weeks; use pre-sterilized forceps to transfer the callus tissue to the subculture medium, and subculture in a 28℃ light incubator for 1 week;

[0024] Transformation and culture of Agrobacterium: The plasmid containing the target vector was transformed into Agrobacterium rhizogenes GV3101 using the following steps: 5 μL of plasmid was added to 100 μL of competent Agrobacterium GV3101 cells, and the mixture was stirred by pipetting. The mixture was then added to a sterile electrode cup that had been pre-cooled to 4°C. Electroporation was performed at 220V. LB liquid medium without antibiotics was added, and the mixture was incubated at 28°C with shaking for about 3 hours. The mixture was then evenly spread on LB solid medium containing 50 μg / ml Kan and 50 μg / ml Rif, and incubated in the dark at 28°C for 2 days until single colonies appeared.

[0025] Agrobacterium transfection: Use a pipette to aspirate the infection solution and wash off the Agrobacterium on the plate to obtain the Agrobacterium suspension used for co-culture transformation of rice. Select a sufficient number of callus tissues and place them in a 100ml sterile Erlenmeyer flask. Add an appropriate amount of Agrobacterium suspension and incubate at room temperature for 20 minutes, shaking occasionally. Discard the bacterial solution, place the callus tissues on sterile filter paper to absorb excess bacterial solution, and then transfer them to a solid co-culture medium lined with a layer of sterile filter paper. Incubate at 26°C in the dark for 3 days.

[0026] Screening Culture: After 3 days of co-culture, the callus tissue needs to be cleaned. Using a 1ml blue pipette tip, the callus on the co-culture medium is transferred to a sterilized Erlenmeyer flask. Sterile water is added to rinse both sides. For the third rinse, sterile water containing 500ul / L carbenicillin is used. After removing excess water with a pipette, the callus is transferred to sterile filter paper and dried using the air blower in a laminar flow hood for about 30 minutes. After the callus is dried, it is transferred to a medium containing hygromycin B for screening culture. The culture conditions are 28-30℃, dark culture; the screening time is 3-4 weeks.

[0027] Seedling rooting: When the differentiated seedlings grow to about 2-3cm and have obvious roots, they can be transferred to rooting medium to allow them to grow. The rooting medium should be poured into a relatively tall bottle or tube so that the rooted seedlings have enough space to grow tall. Rooting culture conditions: 28-30℃, sterile light culture.

[0028] In some embodiments, the identification of the positive transgenic plants further includes: taking young leaves from T0 and T1 generation plants, extracting DNA, amplifying the sgRNA target site using target-specific primers, detecting the amplified products by 1% gel electrophoresis, recovering the specific and correctly sized fragments from the gel, and sequencing them. These were named OsZFP7-ko (OsZFP7-2# and OsZFP7-4#), demonstrating the successful construction of the transgenic rice line.

[0029] In some implementation schemes, a photographic measurement step is also included, as follows: after obtaining homozygous positive mutant plants, the length of every ten grains of the OsZFP7-ko line and NIP is measured, and five groups are measured and photographed.

[0030] In some embodiments, the induction medium comprises: N6 medium 24.1 g / L, 2 mg / L 2,4-D, pH 5.8.

[0031] In some embodiments, the subculture medium comprises: N6 medium 24.1 g / L, 2 mg / L 2,4-D, 50 mg / L hygromycin, 300 mg / mL cephalosporin, pH 5.8.

[0032] In some embodiments, the rooting medium comprises: 1 / 2 MS 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, pH 5.8.

[0033] In some embodiments, the co-culture medium comprises: 24.1 g / L N6 medium, 2 mg / L 2,4-D, 200 μmol / L acetylsylgenone, and pH 5.2.

[0034] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0035] This invention constructs the YL-Hu-OsZFP7 vector and introduces it into the rice variety (Nipponbare) using Agrobacterium-mediated transformation, obtaining two stably heritable transgenic rice varieties, named OsZFP7-2# and OsZFP7-4#, respectively. Testing confirmed that the obtained transgenic rice varieties exhibited a significant difference in NIP grain size compared to wild-type rice, with grains significantly larger than the control group, achieving unexpected technical results. Attached Figure Description

[0036] Figure 1 Results of OsZFP7 mutation in OsZFP7-ko mutant transgenic rice.

[0037] Figure 2 Comparison of grain size between OsZFP7-ko mutant transgenic rice and transgenic rice NIP.

[0038] Figure 3 : Quantitative diagram of the size of transgenic rice seeds from the OsZFP7-ko mutant and transgenic rice NIP.

[0039] NIP: Nipponbare rice.

[0040] The following examples are provided to better illustrate the content of the present invention, but are not intended to limit the scope of the invention to the illustrated examples. Non-essential improvements and adjustments made to the implementation schemes by those skilled in the art based on the above description are still within the protection scope of the present invention. Examples: Detailed Implementation

[0041] The rice variety used in this series of experiments is: Nipponbare.

[0042] The relevant culture medium components are as follows:

[0043] Induction medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: HBZ0601) 24.1g / L, 2mg / L 2,4-D, pH=5.8.

[0044] Subculture medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: 15HBZ0601) 24.1 g / L, 2,4-D, 50 mg / L hygromycin, 300 mg / mL cephalosporin, pH=5.8.

[0045] Co-culture medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: HBZ0601) 24.1 g / L, 2 mg / L 2,4-D, 200 μmol / L acetylsylgenone, pH=5.2.

[0046] Rooting medium: 1 / 2 MS (Manufacturer: Haibo Biotechnology Co., Ltd., Product No.: HB8469-6)

[0047] 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, pH = 5.8.

[0048] Example 1: Construction of rice YL-Hu-OsZFP7 vector

[0049] The CRISPR / Cas9 gene editing vector construction protocol employed overlapping PCR to construct the sgRNA expression cassette, with the target sequence being CCTGAAGTGGTGCAACACAAGGC (SEQ ID NO:3). The PCR primer sequences were as follows:

[0050] SEQ ID NO:4

[0051] YL-Hu-OsZFP7-F:cagtGGTCTCatgcaCGATCGATTTGTCCATGCCCGGG;

[0052] SEQ ID NO:5

[0053] YL-Hu-OsZFP7-R:cagtGGTCTCaaaacCCCGGGCATGGACAAATCGATCG

[0054] After product recovery and purification, the sgRNA expression cassette was ligated to the YL-Hu expression vector using a ligation-by-cleavage method. The ligation product was transformed into competent E. coli cells using the heat shock method, and positive clones were selected for sequencing. The recombinant vector (YL-Hu-OsZFP7) with correct sequencing results was transformed into Agrobacterium GV3101.

[0055] Example 2: Genetic transformation of rice

[0056] 1) Callus induction and subculture: Select newly harvested mature rice seeds, remove the husks, pour into 50ml centrifuge tubes, add 15mL of 75% ethanol for 1 minute for sterilization, discard the ethanol, rinse three times with sterile water, discard the ethanol, then add 15mL of 30% sodium hypochlorite for 20 minutes for sterilization, discard the sodium hypochlorite, and rinse 5-6 times with sterile water. Use a pipette to remove excess water, transfer the seeds to induction medium, and incubate in a 28℃ light incubator for 3 weeks. Transfer the grown callus tissue to subculture medium using pre-sterilized forceps, and subculture in a 28℃ light incubator for 1 week.

[0057] 2) Transformation and culture of Agrobacterium: The plasmid containing the target vector was transformed into Agrobacterium rhizogenes GV3101 (manufacturer: Shanghai Weidi, catalog number: AC1003S) using the following steps: 5 μL of plasmid was added to 100 μL of competent Agrobacterium GV3101 cells, and the mixture was mixed by pipetting. The mixture was then added to a sterile electrode cup that had been pre-cooled to 4°C. Electroporation was performed at 220V. LB liquid medium without antibiotics was added, and the mixture was cultured in a shaker at 28°C for about 3 hours. The culture was then evenly spread on LB solid medium containing 50 μg / ml Kan and 50 μg / ml Rif, and cultured in the dark at 28°C for 2-3 days until single colonies appeared.

[0058] 3) Agrobacterium transfection of callus: Use a pipette to aspirate the infection solution and wash off the Agrobacterium from the plate to prepare the Agrobacterium suspension needed for rice transformation. Select a sufficient number of callus tissues (the callus should be in good condition, bright yellow in color, round and firm in texture, and the particle diameter should be about 3 mm), place them in a 100 ml sterile Erlenmeyer flask, add an appropriate amount of Agrobacterium suspension (ensuring sufficient contact between the bacterial solution and the material), and incubate at room temperature for 20 minutes, shaking occasionally. Discard the bacterial solution, place the callus tissues on sterile filter paper to absorb excess bacterial solution, and then transfer them to a solid co-culture medium lined with a layer of sterile filter paper. Incubate at 26°C in the dark for 3 days.

[0059] 4) Screening Culture: After 3 days of co-culture, the callus tissue needs to be cleaned. Using a 1ml blue pipette tip, transfer the callus from the co-culture medium to a sterile Erlenmeyer flask and rinse twice with sterile water. Rinse a third time with sterile water containing 500ul / L carbenicillin. After aspirating excess water with a pipette, transfer the callus to sterile filter paper and dry it using the airflow from a laminar flow hood for approximately 30 minutes. Once dry, transfer the callus to screening medium for screening culture at 28-30℃ in the dark for 3-4 weeks.

[0060] 5) Seedling Rooting: When the seedlings grow to about 2-3 cm and have a noticeable root system, they can be transferred to a rooting medium to continue growing. The rooting medium should be poured into taller bottles or tubes so that the seedlings have enough space to grow upwards. The optimal rooting conditions are 28-30℃ under sterile light.

[0061] Example 3: Identification of positive transgenic plants

[0062] Young leaves from T0 and T1 generation plants were collected, and DNA was extracted using the CTAB method. sgRNA targeting sites were amplified using target-specific primers. The amplification products were detected by 1% gel electrophoresis. Fragments with specific bands and correct sizes were recovered from the gel and sequenced. The sequencing primers are as follows:

[0063] SEQ ID NO:6

[0064] OsZFP7-F:AAAGGGTAGAACAGGTAT

[0065] SEQ ID NO:7

[0066] OsZFP7-R:CATGTGGTGAAGAAACGA

[0067] Experimental results are as follows Figure 1As shown, sequencing results indicate that OsZFP7-4# has a two-base TG deletion at the target site, causing OsZFP7 to terminate prematurely at the 20th amino acid site. In contrast, OsZFP7-2# has a one-base insertion at the target site, causing OsZFP7 to terminate prematurely at the 19th amino acid site. This demonstrates the successful construction of the transgenic rice line.

[0068] Example 4: Comparative Study of Rice Grain Size Between Mutants and Control Groups

[0069] After obtaining homozygous positive mutant plants, the length of every ten seeds in the OsZFP7-ko lines (OsZFP7-2# and OsZFP7-4#) and the control NIP was measured, with 5 groups measured and photographed. Specific results are as follows: Figure 2 and Figure 3 As shown in the figure. The experimental results show that the seed length of the OsZFP7 mutant transgenic plant is larger than that of the wild-type control NIP plant. Therefore, the OsZFP7 mutant transgenic rice can significantly enhance the seed size of rice, and this difference is statistically significant; the OsZFP7 mutant plant has great application value in the field of transgenic technology.

[0070] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of the rice C2H2 type zinc finger protein OsZFP7 gene in increasing rice grain size, the gene sequence of which is shown in SEQ ID NO:

1.

2. The application according to claim 1, wherein the protein sequence encoding the OsZFP7 gene is shown in SEQ ID NO:

2.

3. The application according to any one of claims 1-2, wherein the crop is preferably rice, corn, wheat, oats and barley; more preferably rice, and most preferably Nipponbare.

4. The application according to any one of claims 1-3, which increases rice grain size by negatively regulating the expression of the OsZFP7 gene.

5. The application according to any one of claims 4, wherein the negative regulation of OsZFP7 gene expression is achieved by a CRISPR / Cas9 system; the target of the CRISPR / Cas9 system is shown in SEQ ID NO:

3.

6. A method for preparing transgenic rice with increased grain size, comprising the following steps: 1) Construction of rice OsZFP7 gene knockout vector; 2) Rice genetic transformation, Agrobacterium-mediated transformation and callus induction culture; 3) Identification of positive transgenic plants.

7. The method of claim 6, wherein the construction of the rice OsZFP7 knockout vector comprises the following steps: Primers were designed based on the OsZFP7 sequence shown in SEQ ID NO:1, and the sgRNA fragment was cloned from rice. The PCR product was recovered from the gel. Using the pYLsgRNA-OsU3 vector as a template, the target sequence was ligated to the U3 promoter and gRNAscaffold, respectively. The two PCR products were mixed and used as a template. Overlap PCR was used to ligate the U3 promoter, target sequence, and gRNAscaffold together to form an sgRNA expression cassette. The gRNA expression cassette product and the uncut pYLCRISPR / Cas9Pubi-H plasmid were mixed and digested with BsaI at 37℃ for 15 min. After digestion, 1.5 μl of 10×DNA Ligase Buffer and 35 U T4 ligase were added, and the digestion and ligation were performed for 15 cycles using variable temperature cycling. The cycling program was 37℃ for 5 min, 10℃ for 5 min, and 20℃ for 5 min. Positive clones were selected and sequenced to confirm the successful construction of the rice OsZFP7 gene knockout expression vector YL-Hu-OsZFP7.

8. The method of claim 7, wherein the primer sequence is as follows: YL-Hu-OsZFP7-F: CAGTGGTCTCATGCACGATCGATTTGTCCATGCCCGGG; YL-Hu-OsZFP7-R: CAGTGGTCTCAAAACCCCGGGCATGGACAAATCGATCG.

9. A method for increasing rice grain size, comprising using CRISPR / Cas9 technology to target and edit the rice C2H2 type zinc finger protein OsZFP7 gene, wherein the nucleotide sequence of the OsZFP7 gene is shown in SEQ ID NO.

1.

10. An sgRNA having a target sequence binding region as shown in SEQ ID NO:3.

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