Application of sip7 gene and its encoded protein in regulating plant ear type and kernel type

Abstract

This invention discloses SIP7 The application of genes and their encoded proteins in regulating spike and grain type in plants is disclosed, specifically a method for cultivating plants with altered spike and grain types, including the following steps: inhibiting the development of genes and their encoded proteins in the starting plant. SIP7 Gene expression alters the spike and grain shape of plants; SIP7 The gene is a nucleic acid encoding the SIP7 protein; the SIP7 protein is the protein shown in SEQ ID NO:1. This invention has discovered and confirmed that knocking out SIP7 using CRISPR / Cas9 technology... SIP7 After the gene was fully or partially modified, the rice plant height and tillering did not change significantly, but the panicle length, number of primary branches, number of secondary branches and number of grains per panicle decreased significantly, while the grain length and grain length-to-width ratio increased significantly. Therefore, this invention can be used to change the panicle type and grain type of rice, and by finding / creating superior alleles, it can improve the panicle type and grain type of rice, which helps to increase rice yield and variety, and has important economic value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant genetic engineering, in particular, to the application of SIP7 gene and the encoded protein in regulating plant ear type and grain type. BACKGROUND

[0002] Rice is one of the most important food crops in the world, and more than half of the world's population relies on rice as their main food, so there is an urgent need to increase its yield. The yield of rice is determined by three main factors: the number of panicles or effective tillers per plant, the number of grains per panicle, and the grain weight. Ear type and grain type have important influences on the number of grains per panicle and the grain weight, respectively. In addition, due to the different preferences of customers in different regions of the world for grain type, grain type is also an important quality trait of rice seeds. How to further increase the number of grains per panicle to achieve sustainable yield increase, while improving the quality of rice seeds, has always been the focus of breeders and molecular biologists. In recent years, revealing the genetic basis of rice panicle development and seed has become an important direction of theoretical research related to genetic improvement and high-yield breeding of rice, and great progress has been made. At the same time, further exploration and utilization of more gene resources capable of regulating plant ear type and grain type will have important theoretical and practical significance for the genetic improvement of rice. SUMMARY

[0003] The purpose of the present application is to provide the application of SIP7 gene and the encoded protein in regulating plant ear type and grain type.

[0004] In order to achieve the purpose of the present application, in the first aspect, the present application provides the application of SIP7 gene and the encoded protein in regulating plant ear type and grain type.

[0005] Further, inhibiting the expression of SIP7 gene in plants at the transcriptional or translational level can significantly reduce the panicle length, the number of primary branches, the number of secondary branches, and the number of grains per panicle, and significantly increase the grain length and the length-width ratio of the seeds.

[0006] In the present application, the SIP7 gene from rice is a gene encoding the following protein (a) or (b):

[0007] (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; or

[0008] (b) a protein derived from (a) by substituting, deleting, or adding one or more amino acids in the sequence shown in SEQ ID NO: 1 and having equivalent functions.

[0009] Further, the inhibition is achieved by knocking out the full-length or partial segment of SIP7 gene. For example, gene editing techniques such as CRISPR, TALEN, or ZFN can be used to inhibit SIP7 gene.

[0010] CRISPR / Cas9 technology is preferred for inhibiting the SIP7 gene.

[0011] More preferably, the nucleotide sequence of the sgRNA action site is shown in SEQ ID NO:4.

[0012] In this invention, the plants include monocotyledonous plants and dicotyledonous plants.

[0013] Preferably, the plants include, but are not limited to, rice, wheat, corn, soybeans, cotton, and tomatoes.

[0014] Secondly, the present invention provides a method for shortening the panicle length of rice, reducing the number of primary branches, secondary branches and the number of grains per panicle, and increasing the grain length and the length-to-width ratio of the grain, comprising: using genetic engineering methods to weaken or knock out the SIP7 gene in rice.

[0015] Specifically, using the SIP7 gene as a target, a CRISPR / Cas9-based sgRNA sequence is designed, and a DNA fragment containing the sgRNA sequence is ligated into a vector carrying CRISPR / Cas9, which is then transformed into rice to obtain transgenic rice with the gene function lost.

[0016] Furthermore, the vector expresses CAS9 mRNA and OsU6a-sgRNA.

[0017] Preferably, the nucleotide sequence of the OsU6a-sgRNA is shown in SEQ ID NO:6.

[0018] Thirdly, the present invention provides the application of transgenic rice obtained according to the method in plant breeding.

[0019] Breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0020] Fourthly, the present invention provides the application of a substance for inhibiting the expression of the SIP7 gene in plants in the cultivation of plants with altered spike and grain shapes.

[0021] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0022] This invention reveals for the first time the biological function of the SIP7 gene. By knocking out / inserting a portion of the SIP7 gene using CRISPR / Cas9 technology, the panicle length, number of primary branches, number of secondary branches, and number of grains per panicle in rice are significantly reduced, while the grain length and grain length-to-width ratio are significantly increased. This invention can be used to regulate rice panicle type and grain type, thereby controlling rice yield, and has significant economic value. Attached Figure Description

[0023] Figure 1 The target sites and editing sites of the rice SIP7 gene in the preferred embodiment of the present invention are as follows: (A) the structure of the SIP7 gene and the editing target sites and editing sites; (B) the sequencing results of the editing sites in the sip7-1 and sip7-2 mutants.

[0024] Figure 2 The comparison (A) and statistical analysis (B) of the strain types of Nipponbare (Nip) and sip7-1 and sip7-2 mutants in the preferred embodiment of the present invention are shown. The scale bar is 10 cm.

[0025] Figure 3 The spikelet type comparison (A) and statistical analysis (B) of the Nipponbare (Nip) and sip7-1 and sip7-2 mutants in the preferred embodiment of the present invention are shown in the figure. The scale bar is 1 cm.

[0026] Figure 4 The particle type comparison (AB) and statistical analysis (C) of Nipponbare (Nip) and sip7-1 and sip7-2 mutants in the preferred embodiment of the present invention are shown in the figure. The scale bar is 1 cm.

[0027] In the figure, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. Detailed Implementation

[0028] This invention aims to provide the application of the SIP7 gene and its encoded SIP7 protein in regulating plant spike and grain type.

[0029] The present invention adopts the following technical solution:

[0030] This invention provides a method for cultivating plants with altered spike and grain shapes, comprising the following steps: inhibiting the expression of the SIP7 gene in the starting plant, thereby altering the spike and grain shapes of the plant;

[0031] The SIP7 gene is a nucleic acid that encodes the SIP7 protein;

[0032] The SIP7 protein is either (a1) or (a2) or (a3) ​​as follows:

[0033] (a1) The protein shown in SEQ ID NO:1;

[0034] (a2) A protein that has the same function as (a1) by substitution and / or deletion and / or addition of one or more amino acid residues;

[0035] (a3) is a protein derived from rice that has 99%, 95%, 90%, 85%, or 80% homology with (a1) and has the same function.

[0036] The SIP7 gene is one of the following (b1) or (b2) or (b3) or (b4):

[0037] (b1) A DNA molecule with a coding region as shown in SEQ ID NO:2;

[0038] (b2) The DNA molecule shown in SEQ ID NO:3;

[0039] (b3) A DNA molecule that hybridizes under stringent conditions with a DNA molecule defined in (b1) or (b2) and encodes the SIP7 protein;

[0040] (b4) A DNA molecule derived from rice and having 99%, 95%, 90%, 85% or more or 80% homology with (b1) or (b2) and encoding the SIP7 protein.

[0041] The above stringent conditions can be achieved by hybridization at 65°C using a solution of 6×SSC and 0.5% SDS, followed by washing the membrane once each with 2×SSC and 0.1% SDS and 1×SSC and 0.1% SDS.

[0042] "Suppressing the expression of the SIP7 gene in the starting plant" is achieved by knocking out the entire length or a portion of the SIP7 gene.

[0043] "Inhibition of SIP7 gene expression in the starting plant" is achieved through CRISPR / Cas9 technology. The CRISPR / Cas9 technology uses, but is not limited to, OsU6a-sgRNA, the sequence of which is shown in SEQ ID NO:6, and the target sequence is shown in SEQ ID NO:4.

[0044] "Inhibition of SIP7 gene expression in the starting plant" is achieved by introducing an interference vector; the interference vector expresses CAS9 mRNA and OsU6a-sgRNA, as shown in SEQ ID NO:6, and the target sequence is shown in SEQ ID NO:4. Specifically, the interference vector can be a recombinant plasmid obtained by inserting the double-stranded DNA molecule shown in SEQ ID NO:5 into the multiple cloning site (e.g., the MluⅠ restriction site) of the pYLCRISPR / Cas9Pubi-H plasmid.

[0045] The present invention also provides the application of the SIP7 gene as a target for silencing in the cultivation of plants with altered spike and grain shapes.

[0046] This invention also provides a substance for inhibiting the expression of the SIP7 gene, and its application in cultivating plants with altered spike and grain shapes. The substance for inhibiting the expression of the SIP7 gene can be a substance that inhibits the expression of the SIP7 gene using CRISPR / Cas9 technology. The CRISPR / Cas9 technology uses, but is not limited to, OsU6a-sgRNA, the sequence of which is shown in SEQ ID NO:6, and the target sequence as shown in SEQ ID NO:4.

[0047] The OsU6a-sgRNA is shown in SEQ ID NO:6. The substance that inhibits the expression of the SIP7 gene can be an interference vector. The interference vector expresses CAS9 mRNA and OsU6a-sgRNA; the target sequence is shown in SEQ ID NO:4; the OsU6a-sgRNA is shown in SEQ ID NO:6. Specifically, the interference vector can be a recombinant plasmid obtained by inserting the double-stranded DNA molecule shown in SEQ ID NO:5 into the multiple cloning site (e.g., the MluⅠ restriction site) of the pYLCRISPR / Cas9Pubi-H plasmid.

[0048] The present invention also provides a method for cultivating plants with altered spike and grain shapes, comprising the following steps: reducing the content of the SIP7 protein in the plant and / or inhibiting the activity of the SIP7 protein in the plant, thereby altering the spike and grain shapes of the plant.

[0049] The present invention also provides applications of the SIP7 protein, selected from the following (c1), (c2), or (c3):

[0050] (c1) Regulates plant spike and grain shape;

[0051] (c2) Regulates plant yield;

[0052] (c3) Used as a target for editing to cultivate plants with altered spike and grain shapes.

[0053] The present invention also protects substances for reducing the content of the SIP7 protein in plants and / or inhibiting the activity of the SIP7 protein in plants, and their use in cultivating plants with altered spike and grain shapes.

[0054] Ear shape and grain shape have a significant impact on yield, therefore this invention can be used to regulate plant yield.

[0055] Any of the plants described above can be monocotyledonous or dicotyledonous. Further, the monocotyledonous plant can be a grass (Poaceae). More specifically, the grass is rice. More specifically, the rice is japonica rice, such as Nipponbare.

[0056] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0057] The pYLCRISPR / Cas9Pubi-H and pYLgRNA-OsU6a plasmids used in the following examples were kindly provided by Professor Yaoguang Liu's research group at South China Agricultural University. See "Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YA robust CRISPR / Cas9 system for convenient, high-efficiency multiplexgenome editing in monocot and dicot plants. Molecular Plant, 2015, 8: 1274–1284". These plasmids are also commercially available to the public.

[0058] Escherichia coli Trans5a was purchased from Beijing TransGen Biotech Co., Ltd.

[0059] Agrobacterium tumefaciens EHA105 was purchased from Beijing Bomed Gene Technology Co., Ltd.

[0060] Nipponbare, short for Japanese rice, is a japonica rice variety originating from the China National Rice Research Institute.

[0061] The example demonstrates how gene editing of the SIP7 gene alters the panicle and grain type of rice.

[0062] A protein that interacts with the known rice panicle-type protein SMAP1 was discovered in rice (Oryza sativa L.) and named SIP7 (SMAP1-Interacting Protein 7), as shown in SEQ ID NO:1. The gene encoding the SIP7 protein was named the SIP7 gene, the open reading frame in its cDNA is shown in SEQ ID NO:2, and the gene sequence in the genome is shown in SEQ ID NO:3.

[0063] I. Construction of CRISPR-Cas9 gene knockout vector

[0064] 1. Design SIP7-specific targets

[0065] Using the CRISPR-P website (http: / / crispr.hzau.edu.cn / CRISPR2 / ), target sequences were screened in the SIP7 gene. After specific comparison, the target sequences (SEQ ID NO:4 and...) were determined. Figure 1 A) is:

[0066] 5'-GGATTCGGAAGTCGGTACCGGGG-3'.

[0067] Design specific target primers and other primers required for vector construction based on the target sequence:

[0068] SIP7-U6a-F: 5'-GATTCGGAAGTCGGTACCGgttttagagctagaaat-3',

[0069] SIP7-U6a-R: 5'-CGGTACCGACTTCCGAATCCggcagccaagccagca-3';

[0070] U6a-F:5'-CTCCGTTTTACCTGTGGAATCG-3'

[0071] gR-R:5'-CGGAGGAAAATTCCATCCAC-3'

[0072] Pps-GGL:5'-TTCAGAggtctcTctcgACTAGTATGGAATCGGCAGCAAAG G-3'

[0073] Pgs-GGR:5'-AGCGTGggtctcGaccgACGCGTATCCATCCACTCCAAGC TC-3'

[0074] SP-L1:5'-CCCGACATAGATGCAATAACTTC-3'

[0075] SP-R:5'-GCGCGGTGTCATCTATGTTACT-3'

[0076] 2. First round of PCR reaction

[0077] Using 2-5 ng of pYLgRNA-OsU6a plasmid as a template, four primers were used in one reaction: 0.2 μM each of U6a-F and gR-R, and 0.1 μM each of SIP7-U6a-F and SIP7-U6a-R. Cycles were performed for 25-28 times: 94℃ for 10 s, 58℃ for 15 s, and 68℃ for 20 s. During amplification, in the first few cycles, UF / SIP7-U6a-R amplified the U6a promoter-target sequence, and gR-R / SIP7-U6a-F amplified the target-sgRNA sequence. In later cycles, overlap PCR was used to generate a merged sgRNA expression cassette fragment.

[0078] 3. Second round of PCR reaction

[0079] Take 1 μl of the first-round PCR product and dilute it 10-fold with sterile water. Use 1 μl as template in a 50 μl system. The primers are Pps-GGL+PGS-GGR, and the final concentration is 0.15 μM. Use an appropriate amount of KOD-Plus or other high-fidelity PCR enzymes.

[0080] Amplification cycle 17-20 (adjusted according to actual situation): 95℃ 10s, 58℃ 15s, 68℃ 20s.

[0081] Take 2-3 μl for electrophoresis and purify using a PCR product purification kit.

[0082] 4. Enzyme digestion-ligation reaction of binary vector and sgRNA expression cassette

[0083] The double-stranded DNA molecule shown in SEQ ID NO:5 was inserted into the MluⅠ restriction site of the pYLCRISPR / Cas9Pubi-H plasmid to obtain the recombinant plasmid pYLCRISPR / Cas9Pubi-H-SIP7.

[0084] In SEQ ID NO:5, nucleotides 1-447 form the promoter, and nucleotides 448-547 form the coding sequence for OsU6a-sgRNA (OsU6a-sgRNA is shown in SEQ ID NO:6).

[0085] The reaction system is as follows:

[0086]

[0087] Enzyme digestion and ligation were performed using variable temperature cycling for approximately 10-15 cycles: 37°C for 5 min; 10°C for 5 min, 20°C for 5 min; and finally 37°C for 5 min.

[0088] 5. Transformation of ligation products: All of the above ligation products were added to 100 μL of competent cells for saturation, and then transformed into Escherichia coli Trans5α by heat shock transformation.

[0089] 6. Extract plasmids, perform PCR identification using SP-L1 and SP-R, and then perform sequencing detection.

[0090] II. Creating sip7 mutants

[0091] 1. The recombinant plasmid pYLCRISPR / Cas9Pubi-H-SIP7 was introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium.

[0092] 2. Using the recombinant Agrobacterium obtained in step 1, genetic transformation was performed on immature embryo callus tissue of Nipponbare as the transformation recipient to obtain regenerated plants.

[0093] 3. Cross the regenerated plants with Nipponbare to obtain the F1 generation plants. The offspring obtained by self-pollination of the F1 generation plants are the F2 generation plants.

[0094] 3. Screening for rice sip7 mutants with SIP7 gene mutations and without CAS9 editing elements from the F2 generation plants obtained in step 3 by molecular identification.

[0095] (1) Take leaves and extract genomic DNA using the CTAB method.

[0096] (2) Using the genomic DNA obtained in step (1) as a template, PCR amplification was performed using primer pairs consisting of SIP7-CAS9-F and SIP7-CAS9-R. The PCR amplification products were subjected to electrophoresis, and then the specific bands were recovered and sequenced. Genomic DNA from Nipponbare was used as a control.

[0097] SIP7-CAS9-F: 5'-AAGGAAGGTACCTCCAGAAG-3';

[0098] SIP7-CAS9-R: 5'-TCTGAACCTGAACTTGGCTC-3'.

[0099] The PCR amplification product of Nipponbare's genomic DNA is 379 bp, as shown in nucleotides 3698-4076 of SEQ ID NO:3.

[0100] The PCR amplification product of the genomic DNA of a regenerated plant is 378 bp, as shown in SEQ ID NO:7 and Figure 1 As shown in B, this plant was named the sip7-1 mutant; the PCR amplification product of the genomic DNA of another regenerated plant was 380 bp, as shown in SEQ ID NO:8 and Figure 1As shown in B, this plant was named the sip7-2 mutant. Both the deletion and insertion occurred in the SIP7 gene and caused a frameshift, resulting in premature termination of translation and the inability to form the full-length SIP7 protein.

[0101] The sip7 mutant isolated from the F2 generation was self-crossed to obtain the F3 generation plants, which were used for subsequent phenotypic observation and analysis.

[0102] III. Statistical analysis of plant type, ear type, and grain type phenotypes

[0103] The sip7-1 and sip7-2 mutant plants identified in the above steps were soaked with wild-type Nipponbare rice seeds at 25℃ for 3 days, followed by germination at 28℃ for 1 day. The rice seeds that had sprouted were sown in seedbeds for conventional seedling cultivation. After one month of cultivation, one seedling per hill was transplanted to paddy fields, with a planting spacing of 17cm × 20cm. The experimental sites were the Beiqijia Experimental Farm in Changping District, Beijing, and the Nanfan Base in Yelin Town, Lingshui Li Autonomous County, Hainan Province. Field management, including water, fertilizer, and pest and disease control, was conducted according to local production conditions. At rice maturity, plant height and panicle length were measured with a tape measure. The number of tillers, primary branches, secondary branches, and grains per panicle were counted. The length, width, and length-to-width ratio of the grains were analyzed using the Wanshen SC-E Rice Appearance Quality Detection and Analysis System. Data were collected from 10-12 plants for each experimental material.

[0104] Statistical results showed that, compared with wild-type Nipponbare, the plant height and tiller number of the sip7-1 and sip7-2 mutants did not change significantly. Figure 2 The length of the panicle, the number of primary branches, the number of secondary branches, and the number of grains per panicle were significantly reduced. Figure 3 Grain length and grain length-to-width ratio increased significantly. Figure 4 ).

[0105] This invention uses rice as a model plant to demonstrate the function of the SIP7 gene through experiments. Since rice is a model plant, genes that can function in rice have similar effects in a variety of crops. Therefore, the SIP7 gene of this invention can be used in monocotyledonous crops such as wheat and corn, and dicotyledonous plants such as soybean, cotton, and tomato to regulate the spike and grain types of plants.

[0106] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. SIP7 Application of genes and their encoded proteins in regulating rice grain shape; Inhibiting rice at the transcriptional or translational level SIP7 Gene expression significantly increases rice grain length and grain length-to-width ratio; among them, From rice SIP7 The protein encoded by the gene consists of the amino acid sequence shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The inhibition is achieved by knocking out SIP7 This is achieved through the full length of the gene or a partial segment.

3. The application according to claim 1 or 2, characterized in that, Using CRISPR, TALEN, or ZFN gene editing technologies to inhibit SIP7 Gene.

4. The application according to claim 3, characterized in that, Using CRISPR / Cas9 technology to suppress SIP7 Gene.

5. The application according to claim 4, characterized in that, The nucleotide sequence of the sgRNA action site is shown in SEQ ID NO:

4.

6. A method for increasing the length and length-to-width ratio of rice grains, characterized in that, include: Using genetic engineering techniques to weaken or knock out [certain substances] in rice SIP7 Genes; among which, SIP7 The protein encoded by the gene consists of the amino acid sequence shown in SEQ ID NO:1; Among them, with SIP7 Using the gene as a target, a CRISPR / Cas9-based sgRNA sequence is designed. A DNA fragment containing the sgRNA sequence is ligated into a vector carrying CRISPR / Cas9, and rice is transformed to obtain transgenic rice with the gene function missing.

7. The method according to claim 6, characterized in that, The vector expresses CAS9 mRNA and OsU6a-sgRNA; The nucleotide sequence of the OsU6a-sgRNA is shown in SEQ ID NO:

6.

8. The application of transgenic rice obtained according to the method of claim 6 or 7 in rice grain type breeding; The grain shape changed as grain length and the length-to-width ratio of the grain increased significantly.

9. The application according to claim 8, characterized in that, Breeding methods include transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

10. Used to inhibit [the growth of certain substances] in rice. SIP7 Application of gene expression substances in the cultivation of rice with altered grain shape; the grain shape alteration is characterized by a significant increase in grain length and grain length-to-width ratio; in, SIP7 The protein encoded by the gene consists of the amino acid sequence shown in SEQ ID NO:1.

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