WO2023066413A1 - Dmp蛋白及其编码基因与应用 - Google Patents

Dmp蛋白及其编码基因与应用 Download PDF

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WO2023066413A1
WO2023066413A1 PCT/CN2022/140182 CN2022140182W WO2023066413A1 WO 2023066413 A1 WO2023066413 A1 WO 2023066413A1 CN 2022140182 W CN2022140182 W CN 2022140182W WO 2023066413 A1 WO2023066413 A1 WO 2023066413A1
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protein
nucleic acid
molecule
plant
acid molecule
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林浩
王娜
牛丽芳
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中国农业科学院生物技术研究所
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites

Definitions

  • the invention relates to the technical field of genetic engineering, in particular to DMP protein and its coding gene and application.
  • Haploid breeding has become one of the important methods for cultivating new plant varieties. At the same time, improving the haploid induction rate and simplifying the haploid induction procedure are the key steps of haploid breeding technology. With the development and improvement of haploid induction technology, haploid breeding technology has been widely used in the breeding research of many important crops, showing the advantages of rapid gene homozygosity, shortened breeding period, and high breeding efficiency. Leguminous plants are important economic crops, and the in vivo haploid induction system has not yet been developed. If haploid breeding can be realized, it will have a wide application prospect in agricultural production.
  • Medicago truncatula (Medicago truncatula), as a model plant of legumes, has the general characteristics of legumes. Therefore, it is of great application value to study haploid induction of Medicago truncatula and develop an in vivo haploid induction system suitable for legumes.
  • the purpose of the present invention is to provide DMP protein and its coding gene and application.
  • the invention claims a kit of proteins.
  • the set of proteins claimed in the present invention consists of protein A and protein B. Both the protein A and the protein B are from Medicago truncatula, named DMP8 and DMP9 respectively.
  • the protein A (ie DMP8) can be any of the following:
  • (A1) the amino acid sequence is the protein of SEQ ID No.1;
  • A2 The amino acid sequence shown in SEQ ID No.1 undergoes the substitution and/or deletion and/or addition of one or several amino acid residues and has the same function protein;
  • (A3) A protein having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of the amino acid sequence defined in (A1)-(A2) and having the same function;
  • a fusion protein obtained after the N-terminus and/or C-terminus of any one of the proteins defined in (A1)-(A3) is linked to a protein tag.
  • the protein B (ie DMP9) can be any of the following:
  • (B1) amino acid sequence is the protein of SEQ ID No.2;
  • (B3) A protein having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of the amino acid sequence defined in (B1)-(B2) and having the same function;
  • (B4) A fusion protein obtained after the N-terminus and/or C-terminus of any one of the proteins defined in (B1)-(B3) is linked to a protein tag.
  • the above-mentioned proteins can be synthesized artificially, or their coding genes can be synthesized first, and then biologically expressed.
  • the tag refers to a polypeptide or protein that is fused and expressed with the protein of interest using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and/or purification of the protein of interest.
  • the tag can be Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag, etc.
  • the identity refers to the identity of amino acid sequences.
  • Amino acid sequence identities can be determined using homology search sites on the Internet, such as the BLAST webpage of the NCBI homepage. For example, in advanced BLAST2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as Matrix, and set Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default value), retrieve the identity of a pair of amino acid sequences, perform calculations, and then obtain the value (%) of the identity.
  • the above 99% identity may be at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identity.
  • the above 95% identity may be at least 96%, 97%, 98% identity.
  • the above 90% identity may be at least 91%, 92%, 93%, 94% identity.
  • the above 85% identity may be at least 86%, 87%, 88%, 89% identity.
  • the above 80% identity may be at least 81%, 82%, 83%, 84% identity.
  • the invention claims a set of nucleic acid molecules.
  • the set of nucleic acid molecules claimed in the present invention consists of nucleic acid molecule A and nucleic acid molecule B.
  • the nucleic acid molecule A is a nucleic acid molecule capable of expressing the aforementioned protein A;
  • the nucleic acid molecule B is a nucleic acid molecule capable of expressing the aforementioned protein B.
  • the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.
  • nucleic acid molecule A (named MtDMP8) can be any one of the following DNA molecules:
  • (a2) a DNA molecule that hybridizes to the DNA molecule defined in (a1) and encodes said protein A under stringent conditions;
  • (a3) A DNA molecule encoding the protein A having 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity with the DNA sequence defined in (a1) or (a2).
  • the nucleic acid molecule B (named MtDMP9) can be any one of the following DNA molecules:
  • (b2) a DNA molecule that hybridizes to the DNA molecule defined in (b1) and encodes the protein B under stringent conditions;
  • (b3) A DNA molecule encoding the protein B having 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity with the DNA sequence defined in (b1) or (b2).
  • the stringent conditions can be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na PO 4 and 1mM EDTA, at 50°C, 2 ⁇ Rinse in SSC, 0.1% SDS; also: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M Na 3 PO 4 and 1mM EDTA, rinse in 50°C, 1 ⁇ SSC, 0.1% SDS; also Can be: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M Na 3 PO 4 and 1mM EDTA, rinse at 50°C, 0.5 ⁇ SSC, 0.1% SDS; can also be: 50°C, in 7% Hybridize in a mixed solution of SDS, 0.5M Na 3 PO 4 and 1mM EDTA, rinse at 50°C in 0.1 ⁇ SSC, 0.1% SDS; also: 50°C, in 7% SDS
  • homology refers to the identity of nucleotide sequences.
  • Nucleotide sequence identity can be determined using homology search sites on the Internet, such as the BLAST webpage of the NCBI homepage. For example, in advanced BLAST2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as Matrix, and set Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (the default value) and search for the identity of a pair of nucleotide sequences to calculate, and then the value (%) of the identity can be obtained.
  • the above 95% homology may be at least 96%, 97%, or 98% identity.
  • the above 90% homology may be at least 91%, 92%, 93%, 94% identity.
  • the above 85% homology may be at least 86%, 87%, 88%, 89% identity.
  • the above 80% homology may be at least 81%, 82%, 83%, 84% identity.
  • the present invention claims any of the following biological materials:
  • the expression cassette A is an expression cassette containing the aforementioned nucleic acid molecule A
  • the expression cassette B is an expression cassette containing the aforementioned nucleic acid molecule B
  • a complete set of recombinant vectors consisting of a recombinant vector A and a recombinant vector B;
  • the recombinant vector A is a recombinant vector containing the nucleic acid molecule A described above;
  • the recombinant vector B is a recombinant vector containing the nucleic acid molecule B described above;
  • a complete set of recombinant bacteria consisting of recombinant bacteria A and recombinant bacteria B; the recombinant bacteria A is a recombinant bacteria containing the aforementioned nucleic acid molecule A; the recombinant bacteria B is a recombinant bacteria containing the aforementioned nucleic acid molecule B;
  • transgenic cell lines consisting of a transgenic cell line A and a transgenic cell line B;
  • the transgenic cell line A is a transgenic cell line containing the aforementioned nucleic acid molecule A;
  • the transgenic cell line B is a transgenic cell line containing the aforementioned nucleic acid A transgenic cell line for molecule B;
  • a complete set of sgRNA consisting of sgRNA molecule A and sgRNA molecule B;
  • the sgRNA molecule A is the sgRNA molecule used for targeted knockout of the aforementioned nucleic acid molecule A;
  • the sgRNA molecule B is used for the targeted knockout of the aforementioned The sgRNA molecule of the nucleic acid molecule B;
  • CRISPR-Cas9 system a complete set of CRISPR-Cas9 system (product), consisting of CRISPR-Cas9 system A and CRISPR-Cas9 system B;
  • the CRISPR-Cas9 system A is composed of sgRNA molecule A and Cas9 protein described in P5;
  • the CRISPR-Cas9 System B is composed of sgRNA molecule B and Cas9 protein described in P5;
  • P7 CRISPR-Cas9 knockout vector, containing the sgRNA molecule A described in P5, the sgRNA molecule B and the coding gene of the Cas9 protein.
  • the target sequence of the sgRNA molecule A is SEQ ID No.5 and SEQ ID No.6.
  • the target sequence of the sgRNA molecule B is SEQ ID No.7 and SEQ ID No.8.
  • the CRISPR-Cas9 knockout vector is MtCRISPR/Cas9::MtDMP8, MtCRISPR/Cas9::MtDMP9 and/or MtCRISPR/Cas9::MtDMP8MtDMP9 in the examples.
  • the present invention claims the use of the set of proteins described in the first aspect above or the set of nucleic acid molecules described in the second aspect above or the biological material described in the third aspect above in any of the following:
  • the expression and/or activity of the set of proteins i.e. the aforementioned protein A and the protein B
  • the expression and/or activity of the set of proteins i.e. the aforementioned protein A and the protein B
  • the expression and/or activity of the set of proteins is reduced (for example, the translation of the corresponding protein is terminated in advance), and the obtained positive Plants can obtain haploids from offspring by selfing or crossing.
  • the present invention claims a method for constructing a plant haploid inducing line.
  • the method for constructing a plant haploid inducible line claimed in the present invention may include the following steps: the expression levels and/or activities of the aforementioned protein A and the protein B in the recipient plant are all reduced (such as making the corresponding protein Translation is terminated prematurely), and then haploids can be obtained from selfed offspring or hybrid offspring.
  • the method may include the following steps: simultaneously inhibiting the expression of the aforementioned nucleic acid molecule A and the nucleic acid molecule B in the recipient plant to obtain a transgenic plant; Haploids are acquired in the offspring.
  • suppressing the expression of the aforementioned nucleic acid molecule A and the nucleic acid molecule B in the recipient plant can be achieved by any technical means, including but not limited to the use of CRISPR-Cas9 technology for all the nucleic acid molecules in the recipient plant.
  • the nucleic acid molecule A and the nucleic acid molecule B are knocked out simultaneously.
  • the complete CRISPR-Cas9 system described in P6 of the third aspect above or the CRISPR-Cas9 knockout vector described in P7 into the recipient plant (due to the insertion or deletion of nucleotides, the reading frame frameshift, the translated protein terminates prematurely).
  • the hybrid progeny is obtained by crossing the transgenic plant with other varieties of the plant.
  • the method may include the following steps:
  • MtCRISPR/Cas9::MtDMP8 Construct the binary expression vector MtCRISPR/Cas9::MtDMP8, MtCRISPR/Cas9::MtDMP9 and MtCRISPR for MtDMP8 and MtDMP9 gene targeting by Agrobacterium tumefaciens-mediated transformation according to the nucleotide sequence of the target sequence /Cas9::MtDMP8MtDMP9
  • the MtCRISPR/Cas9 vector contains an sgRNA expression cassette and a Cas9 nuclease expression cassette, and the sgRNA expression cassette contains the target sequence described above.
  • the binary expression vector MtCRISPR/Cas9::MtDMP8MtDMP9 is introduced into the target plant cell, the sgRNA expression cassette and the Cas9 nuclease expression cassette are co-expressed in the target plant cell, and MtDMP8 and MtDMP9 are cut
  • the target fragment of the double strand of the gene induces the DNA repair function of the target plant cell itself, randomly inserts or deletes bases at the target site to cause a frameshift mutation, and realizes the loss-of-function mutation of the MtDMP8 and MtDMP9 genes in the cell.
  • step (3) Using the loss-of-function mutant cells of the MtDMP8 and MtDMP9 genes obtained in step (3) to regenerate plants.
  • step (4) Perform PCR amplification on the DNA segments of the MtDMP8 and MtDMP9 genes containing the above-mentioned target sequences in the regenerated plants obtained in step (4), and then perform sequencing.
  • the loss-of-function mutation refers to terminator or reading frame shift at the target site in the normal MtDMP8 and MtDMP9 coding sequences.
  • the Cas9 nuclease expression cassette is located in the same vector contained in the sgRNA expression cassette.
  • step (3) the binary expression vectors MtCRISPR/Cas9::MtDMP8, MtCRISPR/Cas9::MtDMP9 and MtCRISPR/Cas9::MtDMP8MtDMP9 are introduced into the target plant cells, so that the cells contain the target fragments described in the step at the same time sgRNA, Cas9 nuclease.
  • the double-stranded target fragments of MtDMP8 and MtDMP9 genes are sheared, and then through the DNA repair function of the target plant cell itself, the random insertion of the target fragments of MtDMP8 and MtDMP9 genes in the cells is finally realized and/or missing at random.
  • the method for introducing the recombinant vector into the target plant cell is Agrobacterium-mediated stable transformation of callus. Because in the process of introducing the obtained recombinant vector into the target plant cell, the method mediated by Agrobacterium is adopted, and the recombinant vector is introduced into the genetic DNA of the target plant, so when cutting, the fragments of the genetic DNA of the target plant subject to shear.
  • the method for regenerating plants is to obtain plants through tissue culture of cells or tissues.
  • DNA fragments containing target fragments of MtDMP8 and MtDMP9 genes in regenerated plants can be cloned by genomic PCR method, and target deep sequencing is performed on the amplified products.
  • the genomic PCR method comprises: designing site-specific primers for the genomic region containing the target fragment, and using the genomic DNA of the regenerated plant as a template to amplify the genomic region containing the target fragment.
  • the present invention claims the application of the method described in the fifth aspect above in plant haploid breeding.
  • the present invention claims to protect the plant haploid induction line constructed by the method described in the fifth aspect above.
  • the plant may be a leguminous plant.
  • the plant may be a Medicago plant.
  • the plant is specifically Medicago truncatula. More specifically, Medicago truncatula R108.
  • the above-mentioned hybrid progeny is the hybrid progeny of the positive plant obtained after knocking out the DMP8 and DMP9 genes in Medicago truncatula R108 and Medicago truncatula A17.
  • Figure 1 shows the pollen staining of Medicago truncatula R108, single mutant dmp8, single mutant dmp9 and double mutant dmp8 dmp9.
  • Figure 2 is the statistics of the number of seeds in each pod of Medicago truncatula R108, single mutant dmp8, single mutant dmp9 and double mutant dmp8 dmp9. ** indicates that there is a very significant difference, P ⁇ 0.01.
  • the sample size is 30 pods.
  • Figure 3 is the haploid plant phenotype induced by the selfed offspring of the double mutant dmp8 dmp9.
  • Figure 4 is the haploid plant phenotype of the hybrid offspring of the double mutant dmp8 dmp9 and Medicago truncatula A17.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.
  • Medicago truncatula R108 was provided by The Nobel Foundation (https://www.nobelprize.org/the-nobel-prize-organisation/the-nobel-foundation/).
  • All Agrobacterium tumefaciens AGL1 were provided by the Institute of Biotechnology, Chinese Academy of Agricultural Sciences (that is, the applicant), and the public can obtain them from the applicant.
  • YEP liquid medium Dissolve 10g of peptone, 10g of yeast extract and 5g of sodium chloride in an appropriate amount of distilled water, then dilute to 1L with distilled water, and autoclave at 121°C for 15 minutes.
  • Callus induction liquid medium dissolve 100mL of macroelement mother solution, 1mL of trace element mother solution, 1mL of organic element mother solution, 20mL of iron salt mother solution, 100mg of inositol, 30g of sucrose, 4mg of auxin and 0.5mg of cytokinin in appropriate amount of distilled water, then Dilute to 1 L with distilled water, adjust the pH to 5.8, and autoclave at 121°C for 15 minutes.
  • Callus induction solid medium 100mL of macroelement mother solution, 1mL of trace element mother solution, 1mL of organic element mother solution, 20mL of iron salt mother solution, 100mg of inositol, 30g of sucrose, 4mg of auxin, 0.5mg of cytokinin, 200mg of cephalosporin, Temei Dissolve Phytagel 250mg, glufosinate 2mg and Phytagel 3.2g with appropriate amount of distilled water, then dilute to 1L with distilled water, adjust the pH value to 5.8, and autoclave at 121°C for 15min.
  • Differentiation medium mix 100mL macroelement mother liquor, 1mL trace element mother liquor, 1mL organic element mother liquor, 20mL iron salt mother liquor, inositol 100mg, sucrose 20g, cephalosporin 200mg, Timentin 250mg, glufosinate 2mg and Phytagel 3.2g with appropriate amount Dissolve in distilled water, then dilute to 1 L with distilled water, adjust the pH to 5.8, and autoclave at 121°C for 15 minutes.
  • Rooting medium Dissolve 2.215 g of Murashige & Skoog Basal Medium with Vitamins (product of PhytoTechnology Laboratories, product number 16B0519138A) with an appropriate amount of distilled water, then adjust the volume to 1 L with distilled water, adjust the pH value to 5.8, and autoclave at 121 °C for 15 min.
  • Iron salt mother liquor Dissolve 37.3 mg of disodium edetate and 27.8 mg of ferrous sulfate heptahydrate with an appropriate amount of distilled water, and then dilute to 1 L with distilled water.
  • Macroelement mother liquor Dissolve 1.85g of magnesium sulfate heptahydrate, 28.3g of potassium nitrate, 4.63g of ammonium sulfate, 1.66g of calcium chloride dihydrate and 4g of potassium dihydrogen phosphate with appropriate amount of distilled water, then dilute to 1L with distilled water.
  • Trace element mother liquor Dissolve 1g of manganese sulfate monohydrate, 500mg of boric acid, 100mg of zinc sulfate heptahydrate, 100mg of potassium iodide, 10mg of sodium molybdate dihydrate, 20mg of copper sulfate pentahydrate and 10mg of cobalt chloride hexahydrate with appropriate amount of distilled water. Dilute to 1L.
  • Organic element mother liquor Dissolve 500mg of niacin, 500mg of thiamine hydrochloride and 500mg of pyridoxine hydrochloride in appropriate amount of distilled water, and then dilute to 1L with distilled water.
  • Alexander stain formula 95% ethanol 5mL, 1% malachite green 500 ⁇ L, 1% acid fuchsin 2.5mL, 1% orange G 250 ⁇ L, glycerin 12.5mL, glacial acetic acid 2mL, then distilled water to 50mL.
  • LB01 buffer 1.5 mL of 1M tris(hydroxymethyl)aminomethane (pH 7.5), 0.5M ethylenediaminetetraacetic acid (pH 8.0), 8 mL of 1M potassium chloride, 400 ⁇ L of 5M sodium chloride, 5 mM spermine tetrahydrochloride, 200 ⁇ L of ⁇ -mercaptoethanol and 100 ⁇ L of polyethylene glycol octylphenyl ether.
  • step 1 use the cDNA of Medicago truncatula R108 as a template to amplify DMP8 and DMP9 genes respectively, wherein DMP8 uses primers DMP8-attB1-F and DMP8-attB2-R to amplify the first round, and then use the first The round product was used as a template, and the attBa adapter-F and attBa adapter-R primers were used to amplify the second round. DMP9 can be amplified for one round with primers DMP9-attB1-F and DMP9-attB2-R. Both genes obtained a PCR amplification product of about 657bp, and the PCR product was recovered and subjected to BP reaction with the vector pDONR207 to obtain two intermediate vectors.
  • DMP8-attB1-F 5'-caaaaaagcaggcttcATGGAACAAACACAACAAG-3';
  • DMP8-attB2-R 5'-caagaaagctgggtcGGCAGACATGCATCCAAT-3'.
  • DMP9-attB1-F 5'-ggggacaagtttgtacaaaaagcaggcttcATGGAACAAACTCAACAAG-3';
  • DMP9-attB2-R 5'-ggggaccactttgtacaagaaagctgggtcGGAAGACATGCATCCAAT-3'.
  • AttB adapter-F 5'GTGGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3';
  • AttB adapter-R 5'GTGGGGACCACTTTGTACAAGAAAGCTGGGTC-3'.
  • Sequencing results show that the intermediate vector targeting the DMP8 gene contains the DNA molecule shown in SEQ ID No.3.
  • the DNA molecule shown in SEQ ID No.3 is the DMP8 gene, which encodes the DMP8 protein shown in SEQ ID No.1.
  • the intermediate vector targeting the DMP9 gene contains the DNA molecule shown in SEQ ID No.4.
  • the DNA molecule shown in SEQ ID No.3 is the DMP9 gene, encoding the DMP9 protein shown in SEQ ID No.2.
  • Example 2 the acquisition of T 0 generation MtDMP8MtDMP9 gene knockout Medicago truncatula
  • the two targets for the MtDMP8 gene are:
  • the two targets for the MtDMP9 gene are:
  • Fragment 1 The primer combination is CmYLCV+MtDMP8-B_gRNA1;
  • Fragment 2 The combination of primers is MtDMP8-C_gRNA1+MtDMP8-D_gRNA2;
  • Fragment 3 The primer combination is MtDMP8-C_gRNA2+oCsy-E;
  • Fragment 4 The primer combination is CmYLCV+MtDMP9-D2_gRNA3;
  • Fragment 5 The combination of primers is MtDMP9-C_gRNA3+MtDMP9-D_gRNA4;
  • Fragment 6 The primer combination is MtDMP9-C_gRNA4+oCsy-E;
  • Fragment 7 The primer combination is MtDMP8-C_gRNA2+MtDMP9-D_gRNA3.
  • CmYLCV 5'-TGCTCTTCGCGCTGGCAGACATACTGTCCCAC-3';
  • MtDMP8-B_gRNA1 5'-TCGTCTCCTCTTGTGGTGGCCTGCCTATACGGCAGTGAACCTG-3';
  • MtDMP8-C_gRNA1 5'-TCGTCTCAAAAGAAGCCATGGGTTTTTAGAGCTAGAAATAGC-3';
  • MtDMP8-D_gRNA2 5'-TCGTCTCATAGGAACGGCCACTGCCTATACGGCAGTGAAC-3';
  • MtDMP8-C_gRNA2 5'-TCGTCTCACCTATAGATCGAGTTTTTAGAGCTAGAAATAGC-3';
  • MtDMP9-D_gRNA3 5'-TCGTCTCACTCTTGTGGTGGCTGCCTATACGGCAGTGAAC-3';
  • MtDMP9-C_gRNA3 5'-TCGTCTCAAGAGGCCATAGGGTTTTGAGCTAGAAAT AGC-3';
  • MtDMP9-D_gRNA4 5'-TCGTCTCAAAACTATCGGTACTGCCTATACGGCAGTGAAC-3'
  • MtDMP9-C_gRNA4 5'-TCGTCTCAGTTTTCACGGCGGTTTTAGAGCTAGAAATAGC-3';
  • MtDMP9-D2_gRNA3 5'-TCGTCTCACTCTTGTGGTGGCTGCCTATACGGCAGTGAACCTG-3';
  • oCsy-E 5'-TGCTCTTCTGACCTGCCTATACGGCAGTGAAC-3'.
  • reaction program is: 20 ⁇ (37°C/5min+25°C/10min)+4°C hold If necessary, the number of cycles of connection can be increased.
  • MtCRISPR/Cas9::MtDMP8 The correct vectors verified by sequencing were named MtCRISPR/Cas9::MtDMP8, MtCRISPR/Cas9::MtDMP9 and MtCRISPR/Cas9::MtDMP8MtDMP9 according to the different insert fragments.
  • the MtCRISPR/Cas9::MtDMP8MtDMP9 binary vector was introduced into Agrobacterium tumefaciens AGL1 to obtain a recombinant Agrobacterium, named AGL1/MtCRISPR/Cas9::MtDMP8MtDMP9.
  • the MtCRISPR/Cas9::MtDMP8 binary vector was introduced into Agrobacterium tumefaciens AGL1 to obtain a recombinant Agrobacterium, which was named AGL1/MtCRISPR/Cas9::MtDMP8.
  • the MtCRISPR/Cas9::MtDMP9 binary vector was introduced into Agrobacterium tumefaciens AGL1 to obtain a recombinant Agrobacterium, named AGL1/MtCRISPR/Cas9::MtDMP9.
  • step (1) inoculate 500 ⁇ L of cultured bacteria solution 1 into 5 mL of YEP liquid medium, then add 5 ⁇ L of acetosyringone aqueous solution with a concentration of 100 mg/mL, and shake culture at 28 ° C and 200 rpm to obtain an OD600nm value of 0.8 of the culture solution 2.
  • step (2) After completing the step (2), take the culture bacteria liquid 2, centrifuge at 3800rpm for 15min, and collect the bacteria.
  • step (3) the bacteria were taken and resuspended in a callus induction liquid medium containing 100 mg/L acetosyringone to obtain an infection solution with an OD600nm value of 0.2.
  • step (1) After completing step (1), place the small leaf pieces in the infection solution obtained in step 1, and shake in the dark for 30 minutes.
  • step (3) After completing step (2), the leaf pieces were transferred to solid medium for callus induction, and cultured in dark at 24° C. for 4 weeks (the medium was replaced every 2 weeks) to obtain white embryogenic callus.
  • step (3) the white embryogenic callus was transferred to a differentiation medium, and cultured in alternating light and dark at 24° C. for 4 weeks (the medium was replaced every 2 weeks), and green embryoids were differentiated.
  • step (4) After completing step (4), transfer the green embryoid body to the rooting medium, culture it alternately in light and dark at 24°C (replace the medium every 2 weeks), and move it to vermiculite after rooting and growing leaves until it becomes mature. Seedling.
  • step 3 single mutant dmp8, single mutant dmp9 and double mutant dmp8dmp9
  • the wild type (Melfalfa truncatula R108) were subjected to pollen Alexander staining and in vitro germination tests, respectively.
  • the dmp8 dmp9 double mutant was crossed with Medicago truncatula A17, and the hybrid offspring were analyzed by flow cytometry.
  • the results showed that crossing dmp8 dmp9 double mutants with different ecotypes of Medicago truncatula parents could induce haploid materials from the maternal parent, and the growth of haploid plants was consistent with the leaf phenotype of A17 plants.
  • the result is shown in Figure 4.
  • the present invention creates a haploid induction system of Medicago truncatula by designing sgRNA specifically targeting the DMP8 and DMP9 genes of Medicago truncatula, and then using the CRISPR-Cas9 system to knock out the DMP8 and DMP9 genes of Medicago truncatula.
  • the invention has great significance for the breeding of leguminous plants, and can effectively shorten the years of plant breeding.

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Abstract

本发明公开了DMP蛋白及其编码基因与应用；公开了成套蛋白，由蛋白质A（即DMP8）和蛋白质B（即DMP9）组成，所述蛋白质A的氨基酸序列为SEQ ID No.1，所述蛋白质B的氨基酸序列为SEQ ID No.2；本发明还公开一种构建植物单倍体诱导系的方法及其应用，以及由该方法构建的植物单倍体诱导系。

Description

DMP蛋白及其编码基因与应用 技术领域
本发明涉及基因工程技术领域，具体涉及DMP蛋白及其编码基因与应用。
背景技术
单倍体育种已经成为培育植物新品种的重要方法之一，与此同时提高单倍体诱导率和简化单倍体诱导程序是单倍体育种技术的关键步骤。随着单倍体诱导技术的发展与改进，单倍体育种技术已经被广泛应用于许多重要农作物的育种研究中，展现出基因快速纯合、育种年限缩短、育种效率高等优势。豆科植物是重要的经济作物，目前尚未开发体内单倍体诱导体系，若能实现单倍体育种，在农业生产中将会有广泛的应用前景。
蒺藜苜蓿(Medicago truncatula)作为豆科的模式植物，具有豆科植物的普遍特征，因此，研究蒺藜苜蓿单倍体诱导，进而开发适用于豆科的体内单倍体诱导体系具有重要的应用价值。
发明公开
本发明的目的是提供DMP蛋白及其编码基因与应用。
第一方面，本发明要求保护一种成套蛋白质。
本发明要求保护的成套蛋白质由蛋白质A和蛋白质B组成。所述蛋白质A和所述蛋白质B均来自于蒺藜苜蓿，分别命名为DMP8和DMP9。
所述蛋白质A(即DMP8)可为如下任一：
(A1)氨基酸序列为SEQ ID No.1的蛋白质；
(A2)将SEQ ID No.1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质；
(A3)与(A1)-(A2)中任一所限定的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且具有相同功能的蛋白质；
(A4)在(A1)-(A3)中任一所限定的蛋白质的N端和/或C端连接蛋白标签后得到的融合蛋白。
所述蛋白质B(即DMP9)可为如下任一：
(B1)氨基酸序列为SEQ ID No.2的蛋白质；
(B2)将SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质；
(B3)与(B1)-(B2)中任一所限定的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且具有相同功能的蛋白质；
(B4)在(B1)-(B3)中任一所限定的蛋白质的N端和/或C端连接蛋白标签后得到的融合蛋白。
上述蛋白质可人工合成，也可先合成其编码基因，再进行生物表达得到。
上述蛋白质中，所述标签(tag)是指利用DNA体外重组技术，与目的蛋白 一起融合表达的一种多肽或者蛋白，以便于目的蛋白的表达、检测、示踪和/或纯化。所述标签可为Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。
上述蛋白质中，同一性是指氨基酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性，如NCBI主页网站的BLAST网页。例如，可在高级BLAST2.1中，通过使用blastp作为程序，将Expect值设置为10，将所有Filter设置为OFF，使用BLOSUM62作为Matrix，将Gap existence cost，Per residue gap cost和Lambda ratio分别设置为11，1和0.85(缺省值)，检索一对氨基酸序列的同一性，进行计算，然后即可获得同一性的值(％)。
上述蛋白质中，所述99％以上的同一性可为至少99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％、99.9％或100％的同一性。所述95％以上的同一性可为至少96％、97％、98％的同一性。所述90％以上的同一性可为至少91％、92％、93％、94％的同一性。所述85％以上的同一性可为至少86％、87％、88％、89％的同一性。所述80％以上的同一性可为至少81％、82％、83％、84％的同一性。
第二方面，本发明要求保护一种成套核酸分子。
本发明要求保护的成套核酸分子由核酸分子A和核酸分子B组成。
所述核酸分子A为能够表达前文所述蛋白质A的核酸分子；
所述核酸分子B为能够表达前文所述蛋白质B的核酸分子。
所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
进一步地，所述核酸分子A(命名为MtDMP8)可为如下任一所示DNA分子：
(a1)SEQ ID No.3所示的DNA分子；
(a2)在严格条件下与(a1)限定的DNA分子杂交且编码所述蛋白质A的DNA分子；
(a3)与(a1)或(a2)限定的DNA序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码所述蛋白质A的DNA分子。
所述核酸分子B(命名为MtDMP9)可为如下任一所示DNA分子：
(b1)SEQ ID No.4所示的DNA分子；
(b2)在严格条件下与(b1)限定的DNA分子杂交且编码所述蛋白质B的DNA分子；
(b3)与(b1)或(b2)限定的DNA序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码所述蛋白质B的DNA分子。
上述核酸分子中，所述严格条件可为如下：50℃，在7％十二烷基硫酸钠(SDS)、0.5M Na 3PO 4和1mM EDTA的混合溶液中杂交，在50℃，2×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M Na 3PO 4和1mM EDTA的混合溶液中杂交，在50℃，1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M Na 3PO 4和1mM EDTA的混合溶液中杂交，在50℃，0.5×SSC，0.1％SDS中漂洗；还可为： 50℃，在7％SDS、0.5M Na 3PO 4和1mM EDTA的混合溶液中杂交，在50℃，0.1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M Na 3PO 4和1mM EDTA的混合溶液中杂交，在65℃，0.1×SSC，0.1％SDS中漂洗；也可为：在6×SSC，0.5％SDS的溶液中，在65℃下杂交，然后用2×SSC，0.1％SDS和1×SSC，0.1％SDS各洗膜一次。
上述核酸分子中，同源性是指核苷酸序列的同一性。可使用国际互联网上的同源性检索站点测定核苷酸序列的同一性，如NCBI主页网站的BLAST网页。例如，可在高级BLAST2.1中，通过使用blastp作为程序，将Expect值设置为10，将所有Filter设置为OFF，使用BLOSUM62作为Matrix，将Gap existence cost，Per residue gap cost和Lambda ratio分别设置为11，1和0.85(缺省值)并进行检索一对核苷酸序列的同一性进行计算，然后即可获得同一性的值(％)。
上述核酸分子中，所述95％以上的同源性可为至少96％、97％、98％的同一性。所述90％以上的同源性可为至少91％、92％、93％、94％的同一性。所述85％以上的同源性可为至少86％、87％、88％、89％的同一性。所述80％以上的同源性可为至少81％、82％、83％、84％的同一性。
第三方面，本发明要求保护如下任一生物材料：
P1、成套表达盒，由表达盒A和表达盒B组成；所述表达盒A为含有前文所述核酸分子A的表达盒；所述表达盒B为含有前文所述核酸分子B的表达盒；
P2、成套重组载体，由重组载体A和重组载体B组成；所述重组载体A为含有前文所述核酸分子A的重组载体；所述重组载体B为含有前文所述核酸分子B的重组载体；
P3、成套重组菌，由重组菌A和重组菌B组成；所述重组菌A为含有前文所述核酸分子A的重组菌；所述重组菌B为含有前文所述核酸分子B的重组菌；
P4、成套转基因细胞系，由转基因细胞系A和转基因细胞系B组成；所述转基因细胞系A为含有前文所述核酸分子A的转基因细胞系；所述转基因细胞系B为含有前文所述核酸分子B的转基因细胞系；
P5、成套sgRNA，由sgRNA分子A和sgRNA分子B组成；所述sgRNA分子A为用于靶向敲除前文所述核酸分子A的sgRNA分子；所述sgRNA分子B为用于靶向敲除前文所述核酸分子B的sgRNA分子；
P6、成套CRISPR-Cas9系统(产品)，由CRISPR-Cas9系统A和CRISPR-Cas9系统B组成；所述CRISPR-Cas9系统A由P5中所述sgRNA分子A和Cas9蛋白组成；所述CRISPR-Cas9系统B由P5中所述sgRNA分子B和Cas9蛋白组成；
P7、CRISPR-Cas9敲除载体，含有P5中所述sgRNA分子A、所述sgRNA分子B和Cas9蛋白的编码基因。
在本发明的具体实施方式中，P5-P7中，所述sgRNA分子A的靶序列为SEQ ID No.5和SEQ ID No.6。所述sgRNA分子B的靶序列为SEQ ID No.7和SEQ ID No.8。所述CRISPR-Cas9敲除载体即为实施例中的MtCRISPR/Cas9：：MtDMP8、 MtCRISPR/Cas9：：MtDMP9和/或MtCRISPR/Cas9：：MtDMP8MtDMP9。
第四方面，本发明要求保护前文第一方面所述成套蛋白质或前文第二方面所述成套核酸分子或前文第三方面所述生物材料在如下任一中的应用：
Q1、构建植物单倍体诱导系；
Q2、植物单倍体育种；
在所述应用中，降低所述植物体内的所述成套蛋白质(即前文所述蛋白质A和所述蛋白质B)的表达量和/或活性(如使相应蛋白的翻译提前终止)，得到的阳性植株通过自交或者杂交从后代中都可获得单倍体。
第五方面，本发明要求保护一种构建植物单倍体诱导系的方法。
本发明要求保护的构建植物单倍体诱导系的方法，可包括如下步骤：使受体植物体内前文所述蛋白质A和所述蛋白质B的表达量和/或活性均降低(如使相应蛋白的翻译提前终止)，然后从自交后代或者杂交后代中可以获得单倍体。
进一步地，所述方法可包括如下步骤：对所述受体植物体内前文所述核酸分子A和所述核酸分子B同时进行抑制表达，得到转基因植物；从所述转基因植物的自交后代或者杂交后代中获得单倍体。
其中，对所述受体植物体内前文所述核酸分子A和所述核酸分子B同时进行抑制表达可以通过任何技术手段实现，包括但不限于采用CRISPR-Cas9技术对所述受体植物体内的所述核酸分子A和所述核酸分子B同时进行敲除。
如，可通过向所述受体植物中导入前文第三方面的P6所述成套CRISPR-Cas9系统或P7中所述CRISPR-Cas9敲除载体实现(因核苷酸的插入或者缺失，导致阅读框移码，翻译的蛋白提前终止)。
所述杂交后代为将所述转基因植物与所述植物的其他品种进行杂交后所得。
具体而言，所述方法可包括如下步骤：
(1)根据MtDMP8和MtDMP9基因的外显子区域选取靶标片段；其中，所述靶标片段的双链结构中的一条链具有NGG结构，其中N代表碱基A、T、C、G中的任意一种。
(2)按照靶标序列的核苷酸排列顺序，构建用于MtDMP8和MtDMP9基因打靶的由根癌农杆菌介导转化的双元表达载体MtCRISPR/Cas9：：MtDMP8、MtCRISPR/Cas9：：MtDMP9和MtCRISPR/Cas9：：MtDMP8MtDMP9，所述MtCRISPR/Cas9载体包含sgRNA表达框和Cas9核酸酶表达框，所述sgRNA表达框包含前文所述靶序列。
(3)将所述双元表达载体MtCRISPR/Cas9：：MtDMP8MtDMP9导入目的植物细胞，使所述sgRNA表达框和所述Cas9核酸酶表达框在所述目的植物细胞中共同表达，剪切MtDMP8和MtDMP9基因的双链的所述靶标片段，诱发所述目的植物细胞自身的DNA修复功能，在靶标位点随机插入或缺失碱基造成移码突变，实现细胞内MtDMP8和MtDMP9基因的功能缺失突变。
(4)用步骤(3)中所得MtDMP8和MtDMP9基因的功能缺失突变的细胞再生植株。
(5)将步骤(4)中所得的再生植株中MtDMP8和MtDMP9基因包含前文所述靶序列的DNA区段进行PCR扩增后，进行测序。
(6)选择两个等位基因都出现功能缺失突变的再生植株，进行表型鉴定。
其中，所述功能缺失突变指的是正常MtDMP8和MtDMP9编码序列在靶标位点出现终止子或阅读框移位。
所述Cas9核酸酶表达框位于包含在所述sgRNA表达框的同一载体中。
在步骤(3)中将所述双元表达载体MtCRISPR/Cas9：：MtDMP8、MtCRISPR/Cas9：：MtDMP9和MtCRISPR/Cas9：：MtDMP8MtDMP9导入所述目的植物细胞，从而使细胞同时含有步骤所述靶标片段的sgRNA，Cas9核酸酶。在sgRNA和Cas9核酸酶的共同作用下，MtDMP8和MtDMP9基因的双链靶标片段被剪切，再通过所述目的植物细胞自身的DNA修复功能，最终实现细胞内MtDMP8和MtDMP9基因靶标片段的随机插入和/或随机缺失。
所述方法中，将重组载体导入目的植物细胞的方法为农杆菌介导的愈伤组织稳定转化。由于在将所获重组载体导入目的植物细胞的过程中，是采用农杆菌介导的方法，重组载体被导入到目的植物的遗传DNA中，所以在进行剪切时使得目的植物的遗传DNA的片段受到剪切。
在本发明中，所述再生植物的方法为细胞或组织经过组织培养，获得植株。
在步骤(5)中，可以通过基因组PCR方法克隆再生植株中MtDMP8和MtDMP9基因包含靶标片段的DNA片段，并对扩增产物进行靶点深度测序。所述基因组PCR方法为，针对包含靶标片段的基因组区域，设计位点特异性引物，以再生植株的基因组DNA为模板，扩增所述包含靶标片段的基因组区域。
第六方面，本发明要求保护前文第五方面所述方法在植物单倍体育种中的应用。
第七方面，本发明要求保护利用前文第五方面所述方法构建得到的植物单倍体诱导系。
在上述各方面中，所述植物可为豆科植物。
进一步地，所述植物可为苜蓿属植物。
在本发明的具体实施方式中，所述植物具体为蒺藜苜蓿。更加具体地，为蒺藜苜蓿R108。相应的，前文所述杂交后代为敲除蒺藜苜蓿R108中DMP8和DMP9基因后得到的阳性植株与蒺藜苜蓿A17的杂交后代。
附图说明
图1为蒺藜苜蓿R108、单突变体dmp8、单变体dmp9和双突变体dmp8 dmp9花粉染色。
图2为蒺藜苜蓿R108、单突变体dmp8、单变体dmp9和双突变体dmp8 dmp9每个果荚内种子数目统计。**表示表示有极显著性差异，P<0.01。样本数为30 个果荚。
图3为双突变体dmp8 dmp9自交后代诱导出的单倍体植株表型。
图4为双突变体dmp8 dmp9与蒺藜苜蓿A17杂交后代的单倍体植株表型。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
蒺藜苜蓿R108由The NobelFoundation(网址为：https://www.nobelprize.org/the-nobel-prize-organisation/the-nobel-fo undation/)提供。
根癌农杆菌AGL1均由中国农业科学院生物技术所(即申请人处)提供，公众可以从申请人处获得。
YEP液体培养基：将蛋白胨10g、酵母提取物10g和氯化钠5g用适量蒸馏水溶解，然后用蒸馏水定容至1L，121℃高压灭菌15min。
愈伤诱导液体培养基：将大量元素母液100mL、微量元素母液1mL、有机元素母液1mL、铁盐母液20mL、肌醇100mg、蔗糖30g、生长素4mg和细胞分裂素0.5mg用适量蒸馏水溶解，然后用蒸馏水定容至1L，调节pH值至5.8，121℃高压灭菌15min。
愈伤诱导固体培养基：将大量元素母液100mL、微量元素母液1mL、有机元素母液1mL、铁盐母液20mL、肌醇100mg、蔗糖30g、生长素4mg、细胞分裂素0.5mg、头孢200mg、特美汀250mg、草胺磷2mg和Phytagel 3.2g用适量蒸馏水溶解，然后用蒸馏水定容至1L，调节pH值至5.8，121℃高压灭菌15min。
分化培养基：将大量元素母液100mL、微量元素母液1mL、有机元素母液1mL、铁盐母液20mL、肌醇100mg、蔗糖20g、头孢200mg、特美汀250mg、草胺磷2mg和Phytagel 3.2g用适量蒸馏水溶解，然后用蒸馏水定容至1L，调节pH值至5.8，121℃高压灭菌15min。
生根培养基：将2.215g Murashige&Skoog Basal Medium with Vitamins(PhytoTechnology Laboratories公司的产品，货号为16B0519138A)用适量蒸馏水溶解，然后用蒸馏水定容至1L，调节pH值至5.8，121℃高压灭菌15min。
铁盐母液：将乙二胺四乙酸二钠37.3mg和七水合硫酸亚铁27.8mg用适量蒸馏水溶解，然后用蒸馏水定容至1L。
大量元素母液：将七水合硫酸镁1.85g、硝酸钾28.3g、硫酸铵4.63g、二水合氯化钙1.66g和磷酸二氢钾4g用适量蒸馏水溶解，然后用蒸馏水定容至1L。
微量元素母液：将一水合硫酸锰1g、硼酸500mg、七水合硫酸锌100mg、碘化钾100mg、二水合钼酸纳10mg、五水合硫酸铜20mg和六水合氯化钴10mg用适量蒸馏水溶解，然后用蒸馏水定容至1L。
有机元素母液：将烟酸500mg、盐酸硫胺素500mg和盐酸吡哆醇500mg用适量蒸馏水溶解，然后用蒸馏水定容至1L。
亚历山大染液配方：95％乙醇5mL、1％孔雀石绿500μL、1％酸性品红2.5mL、1％橙黄G 250μL、甘油12.5mL、冰醋酸2mL，然后用蒸馏水定容至50mL。
LB01缓冲液：1M三(羟甲基)氨基甲烷(pH 7.5)1.5mL、0.5M乙二胺四乙酸(pH 8.0)、1M氯化钾8mL、5M氯化钠400μL、5mM四盐酸精胺、β-巯基乙醇200μL、聚乙二醇辛基苯基醚100μL。
实施例1、MtDMP8和MtDMP9基因的克隆
一、MtDMP8和MtDMP9基因的克隆
1、提取蒺藜苜蓿R108植株上盛开花的总RNA，然后反转录，得到蒺藜苜蓿R108的cDNA。
2、完成步骤1后，以蒺藜苜蓿R108的cDNA为模板，分别扩增DMP8和DMP9基因，其中DMP8采用引物为DMP8-attB1-F和DMP8-attB2-R扩增第一轮，然后以第一轮产物为模板，采用attB adaptor-F和attB adaptor-R引物扩增第二轮。DMP9采用引物为DMP9-attB1-F和DMP9-attB2-R扩增一轮即可。两个基因都得到约657bp的PCR扩增产物，将PCR产物回收后和载体pDONR207进行BP反应，得到两个中间载体。
DMP8-attB1-F：5’-caaaaaagcaggcttcATGGAACAAACACAACAAG-3’；
DMP8-attB2-R：5’-caagaaagctgggtcGGCAGACATGCATCCAAT-3’。
DMP9-attB1-F：5’-ggggacaagtttgtacaaaaaagcaggcttcATGGAACAAACTCAACAAG-3’；
DMP9-attB2-R：5’-ggggaccactttgtacaagaaagctgggtcGGAAGACATGCATCCAAT-3’。
attB adaptor-F：5’GTGGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3’；
attB adaptor-R：5’GTGGGGACCACTTTGTACAAGAAAGCTGGGTC-3’。
3、将步骤2得到的中间载体进行测序。
测序结果表明，针对DMP8基因的中间载体中含有SEQ ID No.3所示的DNA分子。SEQ ID No.3所示的DNA分子即DMP8基因，编码SEQ ID No.1所示的DMP8蛋白。针对DMP9基因的中间载体中含有SEQ ID No.4所示的DNA分子。SEQ ID No.3所示的DNA分子即DMP9基因，编码SEQ ID No.2所示的DMP9蛋白。
实施例2、T 0代MtDMP8MtDMP9基因敲除蒺藜苜蓿的获得
一、MtCRISPR/Cas9：：MtDMP8MtDMP9双元载体的构建
1、目的基因靶序列的选择，每个基因分别设计两个靶点。
其中，针对MtDMP8基因的两个靶点为：
5’-GCCACCACAAGAAGCCATGGGGG-3’(SEQ ID No.5)；
5’-TGGCCGTTCCTATAGATCGAAGG-3’(SEQ ID No.6)。
针对MtDMP9基因的两个靶点为：
5’-CCACCACAAGAGGCCATAGGCGG-3’(SEQ ID No.7)；
5’-TACCGATAGTTTTCACGGCGCGG-3’(SEQ ID No.8)。
2、以pDIRECT-22C载体(北京中源合聚生物科技有限公司，货号为91135-ADG)为模板，利用KOD高保真酶扩增以下片段：
片段1：采用引物组合为CmYLCV+MtDMP8-B_gRNA1；
片段2：采用引物组合为MtDMP8-C_gRNA1+MtDMP8-D_gRNA2；
片段3：采用引物组合为MtDMP8-C_gRNA2+oCsy-E；
片段4：采用引物组合为CmYLCV+MtDMP9-D2_gRNA3；
片段5：采用引物组合为MtDMP9-C_gRNA3+MtDMP9-D_gRNA4；
片段6：采用引物组合为MtDMP9-C_gRNA4+oCsy-E；
片段7：采用引物组合为MtDMP8-C_gRNA2+MtDMP9-D_gRNA3。
上述各引物信息如下：
CmYLCV：5’-TGCTCTTCGCGCTGGCAGACATACTGTCCCAC-3’；
MtDMP8-B_gRNA1：5’-TCGTCTCCTCTTGTGGTGGCCTGCCTATACGGCAGTGAACCTG-3’；
MtDMP8-C_gRNA1：5’-TCGTCTCAAAGAAGCCATGGGTTTTAGAGCTAGAAATAGC-3’；
MtDMP8-D_gRNA2：5’-TCGTCTCATAGGAACGGCCACTGCCTATACGGCAGTGAAC-3’；
MtDMP8-C_gRNA2：5’-TCGTCTCACCTATAGATCGAGTTTTAGAGCTAGAAATAGC-3’；
MtDMP9-D_gRNA3：5’-TCGTCTCACTCTTGTGGTGGCTGCCTATACGGCAGTGAAC-3’；
MtDMP9-C_gRNA3：5’-TCGTCTCAAGAGGCCATAGGGTTTTAGAGCTAGAAAT AGC-3’；
MtDMP9-D_gRNA4：5’-TCGTCTCAAAACTATCGGTACTGCCTATACGGCAGTGAAC-3’
MtDMP9-C_gRNA4：5’-TCGTCTCAGTTTTCACGGCGGTTTTAGAGCTAGAAATAGC-3’；
MtDMP9-D2_gRNA3：5’-TCGTCTCACTCTTGTGGTGGCTGCCTATACGGCAGTGAACCTG-3’；
oCsy-E：5’-TGCTCTTCTGACCTGCCTATACGGCAGTGAAC-3’。
3、用回收试剂盒回收上述7个片段，电泳检测并测浓度。每个片段加入5-7ng，pDIRECT-22C载体加入50ng，SapI酶加入0.5μL,Esp3I酶加入0.5μL，T7DNA Ligase酶加入1μL，2×T7DNA Ligase buffer加入10μL，最后dd H 2O补充至20μL。其中MtDMP8单敲除突变体构建需要加入片段1,2,3；MtDMP9单敲除突变体构建需要加入片段4,5,6；MtDMP8MtDMP9双敲除突变体构建需要加入片段1,2,5,6,7。
4、反应程序为：20×(37℃/5min+25℃/10min)+4℃ hold必要时可以增加连接的循环数。
将经测序验证正确后的载体依据插入片段的不同，分别命名为MtCRISPR/Cas9：：MtDMP8、MtCRISPR/Cas9：：MtDMP9和MtCRISPR/Cas9：： MtDMP8MtDMP9。
二、重组农杆菌的获得
将MtCRISPR/Cas9：：MtDMP8MtDMP9双元载体导入根癌农杆菌AGL1，得到重组农杆菌，命名为AGL1/MtCRISPR/Cas9：：MtDMP8MtDMP9。
将MtCRISPR/Cas9：：MtDMP8双元载体导入根癌农杆菌AGL1，得到重组农杆菌，命名为AGL1/MtCRISPR/Cas9：：MtDMP8。
将MtCRISPR/Cas9：：MtDMP9双元载体导入根癌农杆菌AGL1，得到重组农杆菌，命名为AGL1/MtCRISPR/Cas9：：MtDMP9。
三、T 0代MtDMP8和MtDMP9基因敲除突变体的获得
1、侵染液的制备
(1)将AGL1/MtCRISPR/Cas9：：MtDMP8、AGL1/MtCRISPR/Cas9：：MtDMP9和AGL1/MtCRISPR/Cas9：：MtDMP8MtDMP9单菌落分别接种于含50mg/mL利福平和50mg/mL卡那霉素的YEP液体培养基，28℃、200rpm振荡培养过夜，得到培养菌液1。
(2)完成步骤(1)后，将500μL培养菌液1接种于5mL YEP液体培养基，再加入5μL浓度为100mg/mL的乙酰丁香酮水溶液，28℃、200rpm振荡培养，得到OD 600nm值为0.8的培养菌液2。
(3)完成步骤(2)后，取培养菌液2，3800rpm离心15min，收集菌体。
(4)完成步骤(3)后，取菌体，用含100mg/L乙酰丁香酮的愈伤诱导液体培养基重悬，得到OD 600nm值为0.2的侵染液。
2、T 0代MtDMP8和MtDMP9基因敲除蒺藜苜蓿的获得
(1)取生长至4周的蒺藜苜蓿R108植株的复叶，并用刀片将叶片切割4-5个切口。
(2)完成步骤(1)后，将所述叶片小块置于步骤1所得的侵染液中，黑暗摇30min。
(3)完成步骤(2)后，将所述叶片小块转移至愈伤诱导固体培养基，24℃暗培养4周(每隔2周更换一次培养基)，得到白色胚性愈伤组织。
(4)完成步骤(3)后，将白色胚性愈伤组织转移至分化培养基，24℃光暗交替培养4周(每隔2周更换一次培养基)，分化出绿色胚状体。
(5)完成步骤(4)后，将绿色胚状体转移至生根培养基，24℃光暗交替培养(每隔2周更换一次培养基)，生根长叶后移至蛭石中，直至成苗。
(6)利用CTAB法提取所获的含有所述蒺藜苜蓿MtDMP8和MtDMP9基因CRISPR/Cas9打靶的重组载体的转基因蒺藜苜蓿植株的基因组DNA。以该DNA为模板，用2×Rapid Taq Master MixPCR扩增包含靶标区域的序列，并送测序。
经测序后证实得到单突变体dmp8、单突变体dmp9和双突变体dmp8 dmp9(因核苷酸的插入或者缺失，导致阅读框移码，翻译的蛋白提前终止)。
四、MtDMP8和MtDMP9敲除突变体的表型分析
1、对步骤三获得的突变体(单突变体dmp8、单突变体dmp9和双突变体dmp8dmp9)和野生型(蒺藜苜蓿R108)分别进行花粉亚历山大染色和体外萌发试验。
花粉亚历山大染色步骤：
(1)取蒺藜苜蓿花粉已经成熟但尚未散粉花的花药置于适量卡诺氏固定液(无水乙醇:冰乙酸＝3:1)中，室温固定3-4h，必要时可以固定过夜。
(2)在通风厨里吸走固定液，然后加入适量体积的亚历山大染色液，放于37℃培养箱里黑暗染色过夜。
(3)次日将染色的花药转移到含有10％甘油的离心管中室温条件下脱色45min，最后在显微镜下观察花粉的着色情况。
结果如图1所示。由图可见，dmp8单突变体和dmp9单突变体花粉活性并未受到影响，但是dmp8dmp9双突变体花粉活性部分受到影响。
2、对步骤三获得的突变体(单突变体dmp8、单变体dmp9和双突变体dmp8dmp9)和野生型(蒺藜苜蓿R108)每个果荚内的种子数目进行统计，结果如图2所示。由图可见，与野生型相比，突变体的果荚内的种子数目均有所降低，但是dmp8dmp9双突变体果荚内种子数目降低的更多
五、自交后代单倍体植株的表型分析
对dmp8、dmp9和dmp8 dmp9突变体自交后代进行流式细胞仪分析。流式细胞仪检测细胞核DNA含量的具体步骤：
(1)取蒺藜苜蓿未展开的复叶或者刚展开的叶片放入1mL LB01缓冲液中，用新的锋利的刀片切割叶片2-3min。
(2)将第一步得到的匀浆通过70μm滤膜过滤到1.5mL离心管中，135g 5min离心收集裂解出来的细胞核。
(3)丢掉上清,加入450μL LB01缓冲液重悬沉淀，再向其中加入25μL1mg/mL碘化丙啶(PI)，处于黑暗环境冰上染色10min。
(4)染色后的样品通过流式细胞仪分析细胞核中的DNA含量。
结果显示：在单突变体dmp8、单变体dmp9和野生型R108自交后代中未观察到单倍体。而在双突变体dmp8 dmp9自交后代获得了单倍体植株，并对其进行表型分析，结果如图3所示。
六、杂交后代单倍体表型分析
将dmp8 dmp9双突变体与蒺藜苜蓿A17进行杂交，并对杂交后代进行流式细胞仪分析。结果显示dmp8 dmp9双突变体与不同生态型蒺藜苜蓿亲本杂交可以诱导产生母本来源的单倍体材料，其单倍体植株的长势和A17植株叶片表型相一致。结果如图4所示。
序列表
Figure PCTCN2022140182-appb-000001
Figure PCTCN2022140182-appb-000002
Figure PCTCN2022140182-appb-000003
Figure PCTCN2022140182-appb-000004
Figure PCTCN2022140182-appb-000005
工业应用
本发明通过设计特异性靶向蒺藜苜蓿DMP8和DMP9基因的sgRNA，然后利用CRISPR-Cas9系统敲除蒺藜苜蓿DMP8和DMP9基因的方法，创造出蒺藜苜蓿单倍体诱导体系。本发明对于豆科植物的育种具有重要意义，可有效缩短植物育种年限。

Claims (17)

  1. 成套蛋白质，由蛋白质A和蛋白质B组成；
    所述蛋白质A为如下任一：
    (A1)氨基酸序列为SEQ ID No.1的蛋白质；
    (A2)将SEQ ID No.1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质；
    (A3)与(A1)-(A2)中任一所限定的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且具有相同功能的蛋白质；
    (A4)在(A1)-(A3)中任一所限定的蛋白质的N端和/或C端连接蛋白标签后得到的融合蛋白；
    所述蛋白质B为如下任一：
    (B1)氨基酸序列为SEQ ID No.2的蛋白质；
    (B2)将SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质；
    (B3)与(B1)-(B2)中任一所限定的氨基酸序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且具有相同功能的蛋白质；
    (B4)在(B1)-(B3)中任一所限定的蛋白质的N端和/或C端连接蛋白标签后得到的融合蛋白。
  2. 成套核酸分子，由核酸分子A和核酸分子B组成；
    所述核酸分子A为能够表达权利要求1中所述的蛋白质A的核酸分子；
    所述核酸分子B为能够表达权利要求1中所述的蛋白质B的核酸分子。
  3. 根据权利要求2所述的成套核酸分子，其特征在于：所述核酸分子A为如下任一所示DNA分子：
    (a1)SEQ ID No.3所示的DNA分子；
    (a2)在严格条件下与(a1)限定的DNA分子杂交且编码所述蛋白质A的DNA分子；
    (a3)与(a1)或(a2)限定的DNA序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码所述蛋白质A的DNA分子；
    所述核酸分子B为如下任一所示DNA分子：
    (b1)SEQ ID No.4所示的DNA分子；
    (b2)在严格条件下与(b1)限定的DNA分子杂交且编码所述蛋白质B的DNA分子；
    (b3)与(b1)或(b2)限定的DNA序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同一性且编码所述蛋白质B的DNA分子。
  4. 如下任一生物材料：
    P1、成套表达盒，由表达盒A和表达盒B组成；所述表达盒A为含有权利 要求2或3中所述核酸分子A的表达盒；所述表达盒B为含有权利要求2或3中所述核酸分子B的表达盒；
    P2、成套重组载体，由重组载体A和重组载体B组成；所述重组载体A为含有权利要求2或3中所述核酸分子A的重组载体；所述重组载体B为含有权利要求2或3中所述核酸分子B的重组载体；
    P3、成套重组菌，由重组菌A和重组菌B组成；所述重组菌A为含有权利要求2或3中所述核酸分子A的重组菌；所述重组菌B为含有权利要求2或3中所述核酸分子B的重组菌；
    P4、成套转基因细胞系，由转基因细胞系A和转基因细胞系B组成；所述转基因细胞系A为含有权利要求2或3中所述核酸分子A的转基因细胞系；所述转基因细胞系B为含有权利要求2或3中所述核酸分子B的转基因细胞系；
    P5、成套sgRNA，由sgRNA分子A和sgRNA分子B组成；所述sgRNA分子A为用于靶向敲除权利要求2或3中所述核酸分子A的sgRNA分子；所述sgRNA分子B为用于靶向敲除权利要求2或3中所述核酸分子B的sgRNA分子；
    P6、成套CRISPR-Cas9系统，由CRISPR-Cas9系统A和CRISPR-Cas9系统B组成；所述CRISPR-Cas9系统A由P5中所述sgRNA分子A和Cas9蛋白组成；所述CRISPR-Cas9系统B由P5中所述sgRNA分子B和Cas9蛋白组成；
    P7、CRISPR-Cas9敲除载体，含有P5中所述sgRNA分子A、所述sgRNA分子B和Cas9蛋白的编码基因。
  5. 权利要求1所述成套蛋白质或权利要求2或3所述成套核酸分子或权利要求4所述生物材料在如下任一中的应用：
    Q1、构建植物单倍体诱导系；
    Q2、植物单倍体育种。
  6. 根据权利要求5所述的应用，其特征在于：所述植物为豆科植物。
  7. 根据权利要求6所述的应用，其特征在于：所述植物为苜蓿属植物。
  8. 根据权利要求7所述的应用，其特征在于：所述植物为蒺藜苜蓿。
  9. 一种构建植物单倍体诱导系的方法，包括如下步骤：使受体植物体内权利要求1中所述蛋白质A和所述蛋白质B的表达量和/或活性均降低，然后从自交后代或者杂交后代中获得单倍体诱导系。
  10. 根据权利要求9所述的方法，其特征在于：所述方法包括如下步骤：对所述受体植物体内权利要求2或3中所述核酸分子A和所述核酸分子B同时进行抑制表达，得到转基因植物；从所述转基因植物的自交后代或者杂交后代中获得单倍体诱导系。
  11. 根据权利要求10所述的方法，其特征在于：所述方法中，采用CRISPR-Cas9技术对所述受体植物体内的所述核酸分子A和所述核酸分子B同时进行敲除。
  12. 根据权利要求11所述的方法，其特征在于：是通过向所述受体植物中 导入权利要求4中P6所述成套CRISPR-Cas9系统或P7中所述成套CRISPR-Cas9敲除载体实现的。
  13. 根据权利要求9-12中任一所述的方法，其特征在于：所述植物为豆科植物。
  14. 根据权利要求13所述的方法，其特征在于：所述植物为苜蓿属植物。
  15. 根据权利要求14所述的方法，其特征在于：所述植物为蒺藜苜蓿。
  16. 权利要求9-15中任一所述方法在植物单倍体育种中的应用。
  17. 利用权利要求9-15中任一所述方法构建得到的植物单倍体诱导系。
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