WO2021139024A1 - 一种植物育性相关蛋白及其应用 - Google Patents

一种植物育性相关蛋白及其应用 Download PDF

Info

Publication number
WO2021139024A1
WO2021139024A1 PCT/CN2020/085004 CN2020085004W WO2021139024A1 WO 2021139024 A1 WO2021139024 A1 WO 2021139024A1 CN 2020085004 W CN2020085004 W CN 2020085004W WO 2021139024 A1 WO2021139024 A1 WO 2021139024A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
gmms1
gene
plant
protein
Prior art date
Application number
PCT/CN2020/085004
Other languages
English (en)
French (fr)
Inventor
蒋炳军
孙�石
陈莉
韩天富
岳岩磊
侯文胜
刘路平
袁珊
武婷婷
Original Assignee
中国农业科学院作物科学研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国农业科学院作物科学研究所 filed Critical 中国农业科学院作物科学研究所
Priority to US17/788,923 priority Critical patent/US20230175007A1/en
Publication of WO2021139024A1 publication Critical patent/WO2021139024A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N15/8241—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
    • C12N15/8289—Male sterility
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N15/8241—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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]

Definitions

  • the invention relates to the field of soybean molecular genetic breeding, in particular to a plant fertility-related protein and its application.
  • Soybean is the most important oil crop and high-protein food crop in the world.
  • soybeans are one of the four major food crops, which play a very important role in ensuring national food security, improving the lives of urban and rural people, and increasing farmers’ income.
  • Soybean is a typical short-day crop, and the suitable planting range of a single variety is mostly between 1-1.5 latitudes. The introduction of different latitudes will cause the flowering time and maturity to advance or delay due to the change in the length of sunlight, resulting in a decline in yield or even particles. No receipt. At the same time, my country has a large geographic area, many ecological types, and uneven breeding levels. It is urgent to strengthen the exchange and application of excellent germplasm resources, improve the overall level of soybean breeding, and increase the level of soybean yield in my country.
  • Soybean is a typical self-pollinated crop with small flowers and difficult emasculation, which is not conducive to the development of traditional artificial hybridization. This severely limits the mining and utilization of soybean germplasm resources and severely restricts the aggregation and utilization of excellent gene loci.
  • the soybean recurrent population selection technology based on soybean male sterile mutants can effectively broaden the genetic basis of soybean germplasm resources and has a wide range of application values.
  • the invention provides a plant fertility-related protein and its application.
  • the present invention protects a method for cultivating male sterile plants, including the following steps: reducing or inhibiting the activity and/or content of GmMS1 protein in the target plant to obtain male sterile plants;
  • the GmMS1 protein is as follows (A1) or (A2) or (A3):
  • a protein consisting of the amino acid sequence shown in sequence 1 in the sequence listing;
  • (A2) is derived from soybeans, the sequence is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to (A1) and has an influence on soybean males Sterile functional protein;
  • A3 A protein in which the amino acid sequence shown in sequence 1 in the sequence listing has undergone one or several amino acid residue substitutions and/or deletions and/or additions and is related to plant male fertility.
  • the above-mentioned protein can be synthesized artificially, or the coding gene can be synthesized first, and then obtained by biological expression.
  • the present invention protects a method for cultivating male sterile plants, including the following steps: silencing or suppressing the expression of GmMS1 gene in the target plant or knocking out the GmMS1 gene to obtain male sterile plants:
  • the GmMS1 gene is any one of the following DNA molecules:
  • (B4) Derived from soybean the sequence and the DNA sequence defined by (B1) or (B2) or (B3) have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % Or more than 99% identity, and a DNA molecule that has the function of affecting soybean male sterility.
  • the stringent conditions may be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4 and 1mM EDTA, and rinsing at 50°C, 2 ⁇ SSC, 0.1% SDS; It can also be: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, and rinsing in 50°C, 1 ⁇ SSC, 0.1% SDS; it can also be: 50°C, in 7% SDS, Hybridize in a mixed solution of 0.5M NaPO4 and 1mM EDTA, and rinse at 50°C, 0.5 ⁇ SSC, 0.1% SDS; also: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, Rinse at 50°C, 0.1 ⁇ SSC, 0.1% SDS; also: 50°C, hybridize in a mixed solution of
  • Said silencing or suppressing the expression of the GmMS1 gene in the target plant or knocking out the GmMS1 gene is to mutate the GmMS1 gene in the target plant to reduce the expression of the GmMS1 gene in the target plant or to cause the GmMS1 gene in the target plant to lose its function.
  • the mutations are deletion mutations and/or insertion mutations and/or substitution mutations that occur in exons.
  • the mutation is the mutation described in (A) or (B) as follows:
  • Sequence 2 of the sequence listing inserts base A between base 51 and base 52 of the 5'end;
  • the mutation is achieved by CRISPR/Cas9 gene editing technology; the target sequence of the CRISPR/Cas9 is shown in sequence 6 of the sequence list.
  • Using CRISPR/Cas9 to make the GmMS1 gene in the target plant undergo the mutation (A) or (B) includes the following steps: introducing the CRISPR/Cas9 knockout vector targeting the target sequence into the target plant to obtain a transgenic plant.
  • the CRISPR/Cas9 vector may specifically be a recombinant vector obtained by inserting a primer dimer into a Cas9/gRNA vector; the primer dimer is a combination of F2 (5'-TTGCGCCGAGGTCTAAGATACAG-3') and primer R2 (5'-AACCTGTATCTTAGACCTCGGCG) -3') formed by annealing.
  • the introduction of the CRISPR/Cas9 knockout vector targeting the target sequence into the target plant may specifically be: transforming plant cells or tissues through conventional biological methods such as Agrobacterium-mediated, and cultivating the transformed plant tissues into plants.
  • CRISPR/Cas9 can also be used to make the GmMS1 gene in the target plant occur as follows (C) or ( D) The mutation:
  • Sequence 7 of the sequence listing has a deletion mutation from base 716 to base 721 at the 5'end.
  • the target sequence of the CRISPR/Cas9 is shown in sequence 4 of the sequence list.
  • CRISPR/Cas9 to cause (C) or (D) the mutation of the GmMS1 gene in the target plant includes the following steps: introducing the CRISPR/Cas9 knockout vector targeting the target sequence into the target plant to obtain a transgenic plant.
  • the CRISPR/Cas9 vector may specifically be a recombinant vector obtained by inserting a primer dimer into a Cas9/gRNA vector; the primer dimer is a combination of F1 (5'-TTGGACGGGAACACCTGTGGCGG-3') and primer R1 (5'-AACCCGCCACAGGTGTTCCCGTC) -3') formed by annealing.
  • the introduction of the CRISPR/Cas9 knockout vector targeting the target sequence into the target plant may specifically include: transforming plant cells or tissues through conventional biological methods such as Agrobacterium-mediated, and cultivating the transformed plant tissues into plants.
  • the present invention protects the application of GmMS1 protein or its related biological materials in regulating plant fertility
  • the relevant biological material is any one of the following (1)-(3):
  • the GmMS1 protein is as described above.
  • GmMS1 gene The gene encoding the GmMS1 protein (GmMS1 gene) is as described above.
  • the "substance used to silence or inhibit the expression of (1) or knock out (1) in the target plant” or “substance used to reduce or inhibit the activity and/or content of the GmMS1 protein in the target plant” may specifically be CRISPR /Cas9 knockout vector or recombinant bacteria containing the vector; the target sequence of the CRISPR/Cas9 knockout vector is shown in sequence 4 or sequence 6 in the sequence table.
  • the CRISPR/Cas9 vector may specifically be a recombinant vector obtained by inserting a primer dimer into a Cas9/gRNA vector; the primer dimer is a combination of F2 (5'-TTGCGCCGAGGTCTAAGATACAG-3') and primer R2 (5'-AACCTGTATCTTAGACCTCGGCG) -3') formed by annealing.
  • the CRISPR/Cas9 vector may specifically be a recombinant vector obtained by inserting a primer dimer into a Cas9/gRNA vector; the primer dimer is a combination of F1 (5'-TTGGACGGGAACACCTGTGGCGG-3') and primer R1 (5'-AACCCGCCACAGGTGTTCCCGTC) -3') formed by annealing.
  • the present invention protects a specific sgRNA for CRISPER-Cas9 gene editing; the target sequence of the sgRNA is shown in sequence 6 of the sequence list.
  • the invention also provides a vector for CRISPER-Cas9 gene editing, which expresses a specific sgRNA; the target sequence of the sgRNA is shown in sequence 6 of the sequence list.
  • the CRISPR/Cas9 vector may specifically be a recombinant vector obtained by inserting a primer dimer into a Cas9/gRNA vector; the primer dimer is F2 (5'-TTGCGCCGAGGTCTAAGATACAG-3') and primer R2 (5'-AACCTGTATCTTAGACCTCGGCG-3'). ') formed by annealing.
  • the present invention protects male sterile plants.
  • the protection of male sterile plants of the present invention is obtained by reducing or inhibiting the activity and/or content of GmMS1 protein in the target plant;
  • the GmMS1 protein is as follows (A1) or (A2) or (A3):
  • a protein consisting of the amino acid sequence shown in sequence 1 in the sequence listing;
  • (A2) is derived from soybeans, the sequence is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to (A1) and has an influence on soybean males Sterile functional protein;
  • A3 A protein in which the amino acid sequence shown in sequence 1 in the sequence listing has undergone one or several amino acid residue substitutions and/or deletions and/or additions and is related to plant male fertility.
  • the present invention also protects male sterile plants, which is obtained by silencing or inhibiting the expression of GmMS1 gene in the target plant or knocking out the GmMS1 gene;
  • the GmMS1 gene is any one of the following DNA molecules:
  • (B4) Derived from soybean the sequence and the DNA sequence defined by (B1) or (B2) or (B3) have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % Or more than 99% identity, and a DNA molecule that has the function of affecting soybean male sterility.
  • the present invention also protects male sterile plants.
  • the difference from wild-type plants is that the GmMS1 gene has a mutation; the mutation is the mutation described in (A) or (B) as follows:
  • Sequence 2 of the sequence listing inserts base A between base 51 and base 52 of the 5'end;
  • the method for preparing the male sterile plants can be referred to the first and second aspects described above.
  • the present invention protects any of the following applications in plant breeding
  • the soybean may specifically be a soybean variety Jack.
  • Figure 1 shows the sequencing results of GmMS1 gene editing.
  • Figure 2 shows the sterile phenotype of GmMS1 gene-edited plants.
  • 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, unless otherwise specified, are all conventional methods.
  • the test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.
  • the quantitative experiments in the following examples are all set to repeat the experiment three times, and the results are averaged.
  • Germination medium 3.1g/L B5 medium basal salt (Gamborgs Basal Salt Mixture, Phytotech G768), 20g/L sucrose, 1ml/L B5 medium vitamin solution (Gamborgs Vitamin Solution, Phytotech G219), 7g/L agar, pH 5.8.
  • Co-cultivation liquid medium 2.0g/L MS basic salt mixture (Murashige&Skoog Basal Salt Mixture), 3.9g/L morpholine ethanesulfonic acid (MES), 30g/L sucrose, 1ml/L B5 medium vitamin solution (Gamborgs Vitamin Solution, Phytotech G219), 150mg/L dithiothreitol, 40mg/L acetosyringone, 2mg/L zeatin, pH5.4.
  • MS basic salt mixture Murashige&Skoog Basal Salt Mixture
  • MES morpholine ethanesulfonic acid
  • sucrose sucrose
  • 1ml/L B5 medium vitamin solution Gibborgs Vitamin Solution, Phytotech G219)
  • 150mg/L dithiothreitol 40mg/L acetosyringone
  • 2mg/L zeatin pH5.4.
  • Co-cultivation medium 2.0g/L MS basal salt mixture (Murashige&Skoog Basal Salt Mixture), 3.9g/L morpholine ethanesulfonic acid, 30g/L sucrose, 1ml/L B5 medium vitamin solution (Gamborgs Vitamin Solution, Phytotech G219 ), 150mg/L dithiothreitol, 40mg/L acetosyringone, 2mg/L zeatin, 7g/L agar, pH 5.4.
  • Recovery medium 3.1g/L B5 medium basal salt (Gamborgs Basal Salt Mixture, Phytotech G768), 0.98g/L morpholine ethanesulfonic acid, 30g/L sucrose, 1ml/L B5 medium vitamin solution (Gamborgs Vitamin Solution , Phytotech G219), 150mg/L cefotaxime, 450mg/L timentin, 1mg/L 6-Benzylaminopurine, 7g/L agar, pH 5.7.
  • Screening medium 3.1g/L B5 medium basal salt (Gamborgs Basal Salt Mixture, Phytotech G768), 0.98g/L morpholine ethanesulfonic acid, 30g/L sucrose, 1ml/L B5 medium vitamin solution (Gamborgs Vitamin Solution) , Phytotech G219), 150mg/L cefotaxime, 450mg/L timentin, 1mg/L 6-benzylaminopurine, 7g/L agar, 6mg/L glufosinate, pH 5.7.
  • Elongation medium 4.0g/L MS basal salt mixture (Murashige&Skoog Basal Salt Mixture), 0.6g/L morpholine ethanesulfonic acid, 30g/L sucrose, 1ml/L B5 medium vitamin solution (Gamborgs Vitamin Solution, Phytotech G219 ), 150mg/L Cefotaxime, 450mg/L Timentin, 0.1mg/L 3-Indoleacetic acid, 0.5mg/L Gibberellic acid, 1mg/L Zeatin, 7g/L L agar, 6mg/L glufosinate, pH 5.6.
  • Rooting medium 2.0g/L MS basal salt mixture (Murashige&Skoog Basal Salt Mixture), 0.6g/L morpholine ethanesulfonic acid, 20g/L sucrose, 1ml/L B5 medium vitamin solution (Gamborgs Vitamin Solution, Phytotech G219) , 7g/L agar, 3mg/L glufosinate, pH 5.7.
  • GmMS1 gene Sequencing and functional analysis of the soybean genome revealed a soybean male sterility gene, which was named GmMS1 gene. Its CDS is shown in sequence 2 of the sequence list, and the genome sequence is shown in sequence 7 of the sequence list.
  • the protein encoded by the GmMS1 gene is named GmMS1 protein, as shown in sequence 1 of the sequence table.
  • Target 1 GACGGGAACACCTGTGGCGG TGG (sequence 2 from the 3-25th position of the 5'end);
  • Target 2 CGCCGAGGTCTAAGATACAG AGG (sequence 2 from position 35-57 at the 5'end).
  • the underline is the PAM sequence.
  • R1 5'-AACCCGCCACAGGTGTTCCCGTC-3' (sequence 9).
  • F2 5'-TTGCGCCGAGGTCTAAGATACAG-3' (sequence 10);
  • R2 5'-AACCTGTATCTTAGACCTCGGCG-3' (sequence 11).
  • Dilute primer F1 and primer R1 to 10 ⁇ M respectively configure the reaction system: F1 5 ⁇ l, R1 5 ⁇ l, H 2 O 15 ⁇ l. After mixing, react at 95°C for 3 minutes, then cool to 25°C naturally, and then at 16°C for 5 minutes to obtain primer dimer 1.
  • Dilute primer F2 and primer R2 to 10 ⁇ M respectively configure the reaction system: F2 5 ⁇ l, R2 5 ⁇ l, H 2 O 15 ⁇ l. After mixing, react at 95°C for 3 minutes, then cool to 25°C naturally, and then at 16°C for 5 minutes to obtain primer dimer 2.
  • step 2 Take the primer dimer 1 obtained in step 2 to configure the reaction system: Cas9/gRNA carrier 1 ⁇ l, primer dimer 1 1 ⁇ l, Solution1 1 ⁇ l, Solution2 1 ⁇ l, H 2 O 6 ⁇ l. React at 16°C for 2 hours.
  • the above-mentioned vectors and reagents are from Beijing Visunlide Biotechnology Co., Ltd., catalog number VK005-15.
  • the recombinant vector CRISPR/Cas9-GmMS1-1 contains the DNA molecule shown in sequence 3 of the sequence table (verified by sequencing), and expresses sgRNA.
  • the target sequence of sgRNA is sequence 4.
  • step 2 Take the primer dimer 2 obtained in step 2 to configure the reaction system: Cas9/gRNA carrier 1 ⁇ l, primer dimer 2 1 ⁇ l, Solution1 1 ⁇ l, Solution2 1 ⁇ l, H 2 O 6 ⁇ l. React at 16°C for 2 hours.
  • the above-mentioned vectors and reagents are from Beijing Visunlide Biotechnology Co., Ltd., catalog number VK005-15.
  • the recombinant vector CRISPR/Cas9-GmMS1-2 is obtained, and the recombinant expression vector CRISPR/Cas9-GmMS1-2 contains the DNA molecule shown in sequence 5 of the sequence list and expresses sgRNA (verified by sequencing).
  • the target sequence of sgRNA is sequence 6.
  • the recombinant vector CRISPR/Cas9-GmMS1-1 and the recombinant vector CRISPR/Cas9-GmMS1-2 constructed in step 3 and step 4 were respectively introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium GmMS1-1 and recombinant Agrobacterium GmMS1-2 .
  • the seeds of the plump soybean variety Jack with uniform size, no disease spots, no cracks, smooth surface and no wrinkles are selected and put into a glass petri dish. Then put it in the desiccator and open the petri dish. Put a glass beaker in the desiccator, first add 100 mL of sodium hypochlorite, and then drop 4 mL of concentrated hydrochloric acid into the beaker. After applying petroleum jelly on all sides of the dryer cover, cover the dryer to form a sealed state. Then put the desiccator in a fume hood, and sterilize the seeds for 16-20h.
  • step (1) insert the sterilized seed hypocotyl vertically upwards into the germination medium, the petri dish is unsealed, and place it in a tissue culture room at 25°C, 16h light/18h dark for 1d.
  • step (3) After completing step (2), take the cotyledon nodes of the germinated soybean seeds as explants. First peel off the soybean seed coat, separate two cotyledons longitudinally, cut into strip wounds at the junction of cotyledons and hypocotyls (cotyledon nodes), and put the scratched explants into co-cultivation with recombinant Agrobacterium resuspension In the liquid medium (the OD value of the bacterial solution is 0.6-0.8), inoculate at 28°C for 30min. After the infection, inoculate the inner plane of the explants (cotyledons) down in the co-culture medium covered with filter paper at 25°C , Cultivate for 5 days under 16h light/18h dark conditions.
  • the liquid medium the OD value of the bacterial solution is 0.6-0.8
  • step (3) transfer the explants to the recovery medium and culture for 7 days at 25° C., 16 hours of light/18 hours of darkness.
  • step (4) After completing step (4), cut off the main buds produced by the explants, transfer them to a selection medium, and culture for 21 days under the conditions of 25° C., 16 hours of light/18 hours of darkness.
  • step (5) peel off the browned leaves, and transfer the adventitious buds produced to the elongation medium for elongation. It is subcultured once at 15d, and subcultured 2-3 times. During this period, the elongated shoots produced are transferred to the rooting medium for rooting.
  • step (6) After completing step (6), when the roots grow out, the seedlings are taken out of the culture medium and transplanted into a small pot equipped with a substrate to refine the seedlings. After the seedlings were tempered for 1 week, they were transferred to large pots to grow, and T0 generation plants were obtained.
  • T0 generation plants are tested, and the testing methods are as follows:
  • the genomic DNA of transgenic plants and wild-type plants were extracted respectively, and the detection primers were used for PCR amplification using this as a template.
  • F-614 CGCCATAGTGAAGTAGCGGA (sequence 12);
  • R-614 CAGTTGAAAACAAACTTACCGAAGG (sequence 13).
  • PCR reaction conditions pre-denaturation at 95°C for 5min; then 95°C for 30sec, 56°C for 30sec, 72°C for 1min, 35 cycles; then 72°C for 10min extension.
  • the PCR products are sent for sequencing, and the gene-edited plants are screened.
  • Sequencing results showed that after the recombinant Agrobacterium GmMS1-1 was transformed into soybeans, a total of 60 T0 generation plants were obtained, and one of the T1 progeny was a plant with a mutation in the GmMS1 gene and a homozygous mutation. Compared with the wild type The difference between homozygous mutant plants is that there is a 6bp deletion, which is located between the 18th and 23rd positions of sequence 2 (GGCGGT). After the recombinant Agrobacterium GmMS1-2 was transformed into soybeans, a total of 56 T0 generation plants were obtained. Among the T1 progeny, 5 plants were mutated in the GmMS1 gene and were homozygous mutations.
  • Plants to be tested wild-type plants, 5 homozygous mutant plants (insertion mutant plants obtained by transformation into recombinant Agrobacterium GmMS1-2).
  • the invention is of great significance to the research of plant fertility and the breeding of sterile plants.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Peptides Or Proteins (AREA)

Abstract

提供了一种植物育性相关蛋白及其应用，还提供了通过降低或抑制目的植物中GmMS1蛋白的活性和/或含量得到雄性不育植物的方法，以及通过沉默或抑制目的植物中GmMS1基因的表达或敲除GmMS1基因得到雄性不育植物的方法。

Description

一种植物育性相关蛋白及其应用 技术领域
本发明涉及大豆分子遗传育种领域，具体涉及一种植物育性相关蛋白及其应用。
背景技术
大豆是世界上最为重要的油料作物和高蛋白粮食作物。在我国，大豆是四大粮食作物之一，对保证国家粮食安全、改善城乡人民生活和增加农民收入有十分重要的作用。
大豆是典型的短日照作物，单个品种的适种范围大都在1-1.5个纬度之间，不同纬度地区间引种会因日照长度的变化导致开花时间、成熟期提前或延迟，造成产量下降甚至颗粒无收。同时，我国地域范围大，生态类型多，育种水平不均衡，迫切需要加强优异种质资源的交流应用，提升大豆育种的整体水平，提高我国大豆单产水平。
大豆是典型自花授粉作物，花朵小、去雄难，不利于传统的人工杂交工作的开展。这严重限制了大豆种质资源的挖掘利用，严重限制了优异基因位点的聚合利用。基于大豆雄性不育突变体的大豆轮回群体选择技术，可以有效拓宽大豆种质资源的遗传基础，具有广泛的应用价值。
发明公开
本发明提供了一种植物育性相关蛋白及其应用。
第一方面，本发明保护一种培育雄性不育植物的方法，包括如下步骤：降低或抑制目的植物中GmMS1蛋白的活性和/或含量，得到雄性不育植物；
所述GmMS1蛋白是如下(A1)或(A2)或(A3)：
(A1)由序列表中序列1所示的氨基酸序列组成的蛋白质；
(A2)来源于大豆，序列与(A1)具有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％以上同一性且具有影响大豆雄性不育的功能的蛋白质；
(A3)将序列表中序列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/或添加且与植物雄性育性相关的蛋白质。
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。
第二方面，本发明保护一种培育雄性不育植物的方法，包括如下步骤：沉默或抑制目的植物中GmMS1基因的表达或敲除GmMS1基因，得到雄性不育植物：
所述GmMS1基因是如下任一所述的DNA分子：
(B1)编码序列如序列表的序列2所示的DNA分子；
(B2)基因组序列如序列表的序列7所示的DNA分子；
(B3)在严格条件下与(B1)或(B2)限定的DNA分子杂交且编码具有相同功能的蛋白的DNA分子；
(B4)来源于大豆，序列与(B1)或(B2)或(B3)限定的DNA序列具有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％以上同一性，且具有影响大豆雄性不育的功能的DNA分子。
所述严格条件可为如下：50℃，在7％十二烷基硫酸钠(SDS)、0.5M NaPO4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 NaPO4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 NaPO4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 NaPO4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 NaPO4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。
所述沉默或抑制目的植物中GmMS1基因的表达或敲除GmMS1基因，为突变目的植物中GmMS1基因使目的植物中GmMS1基因表达量降低或使目的植物中GmMS1基因发生功能缺失。
所述突变为发生于外显子的缺失突变和/或插入突变和/或替换突变。
所述突变为如下(A)或(B)所述突变：
(A)序列表的序列2自5’端第51位碱基和52位碱基之间插入碱基A；
(B)序列表的序列7自5’端第749位碱基和750位碱基之间插入碱基A。
所述突变是通过CRISPR/Cas9基因编辑技术实现的；所述CRISPR/Cas9的靶序列如序列表的序列6所示。
利用CRISPR/Cas9使目的植物中GmMS1基因发生(A)或(B)所述突变包括如下步骤：将靶向所述靶序列的CRISPR/Cas9敲除载体导入目的植物中，得到转基因植物。所述CRISPR/Cas9载体具体可为将引物二聚体插入Cas9/gRNA载体得到的重组载体；所述引物二聚体是将F2(5’-TTGCGCCGAGGTCTAAGATACAG-3’)和引物R2(5’-AACCTGTATCTTAGACCTCGGCG-3’)退火形成的。
将靶向所述靶序列的CRISPR/Cas9敲除载体导入目的植物，具体可为：通过农杆菌介导等常规生物学方法转化植物细胞或组织，并将转化的植物组织培育成植株。
在本发明的实施例中，所述使目的植物基因组中GmMS1基因表达量降低或使目的植物基因组中GmMS1基因发生功能缺失还可利用CRISPR/Cas9使目的植物中GmMS1基因发生如下(C)或(D)所述突变：
(C)序列表的序列2发生自5’端第18位碱基至23位碱基的缺失突变；
(D)序列表的序列7发生自5’端第716位碱基至721位碱基的缺失突变。
所述CRISPR/Cas9的靶序列如序列表的序列4所示。
所述利用CRISPR/Cas9使目的植物中GmMS1基因发生(C)或(D)所述突变包括如下步骤：将靶向所述靶序列的CRISPR/Cas9敲除载体导入目的植物中，得到转基因植物。所述CRISPR/Cas9载体具体可为将引物二聚体插入Cas9/gRNA载体得到的重组载体；所述引物二聚体是将F1(5’-TTGGACGGGAACACCTGTGGCGG-3’)和引物R1(5’-AACCCGCCACAGGTGTTCCCGTC-3’)退火形成的。
将靶向所述靶序列的CRISPR/Cas9敲除载体导入目的植物，具体可为：通过农杆菌介导等常规生物学方法转化植物细胞或组织，并将转化的植物组织培育成植株。
第三方面，本发明保护GmMS1蛋白或其相关生物材料在调控植物育性中的应用；
所述相关生物材料为如下(1)-(3)中的任一种：
(1)GmMS1蛋白的编码基因；
(2)用于沉默或抑制目的植物中(1)的表达或敲除(1)的物质；
(3)用于降低或抑制目的植物中GmMS1蛋白的活性和/或含量的物质；
所述GmMS1蛋白如前文所述。
所述GmMS1蛋白的编码基因(GmMS1基因)如前文所述。
所述“用于沉默或抑制目的植物中(1)的表达或敲除(1)的物质”或“用于降低或抑制目的植物中GmMS1蛋白的活性和/或含量的物质”具体可为CRISPR/Cas9敲除载体或含有所述载体的重组菌；所述CRISPR/Cas9敲除载体的靶序列如序列表的序列4或序列6所示。所述CRISPR/Cas9载体具体可为将引物二聚体插入Cas9/gRNA载体得到的重组载体；所述引物二聚体是将F2(5’-TTGCGCCGAGGTCTAAGATACAG-3’)和引物R2(5’-AACCTGTATCTTAGACCTCGGCG-3’)退火形成的。所述CRISPR/Cas9载体具体可为将引物二聚体插入Cas9/gRNA载体得到的重组载体；所述引物二聚体是将F1(5’-TTGGACGGGAACACCTGTGGCGG-3’)和引物R1(5’-AACCCGCCACAGGTGTTCCCGTC-3’)退火形成的。
第四方面，本发明保护一种用于CRISPER-Cas9基因编辑的特异sgRNA；所述sgRNA的靶序列如序列表的序列6所示。
本发明还一种用于CRISPER-Cas9基因编辑的载体，表达特异sgRNA；所述sgRNA的靶序列如序列表的序列6所示。
所述CRISPR/Cas9载体具体可为将引物二聚体插入Cas9/gRNA载体得到的重组载体；引物二聚体是将F2(5’-TTGCGCCGAGGTCTAAGATACAG-3’)和引物R2(5’-AACCTGTATCTTAGACCTCGGCG-3’)退火形成的。
第五方面，本发明保护雄性不育植物。
本发明保护雄性不育植物，是降低或抑制目的植物中GmMS1蛋白的活性和/或含量得到的；
所述GmMS1蛋白是如下(A1)或(A2)或(A3)：
(A1)由序列表中序列1所示的氨基酸序列组成的蛋白质；
(A2)来源于大豆，序列与(A1)具有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％以上同一性且具有影响大豆雄性不育的功 能的蛋白质；
(A3)将序列表中序列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/或添加且与植物雄性育性相关的蛋白质。
本发明还保护雄性不育植物，是沉默或抑制目的植物中GmMS1基因的表达或敲除GmMS1基因得到的；
所述GmMS1基因是如下任一所述的DNA分子：
(B1)编码序列如序列表的序列2所示的DNA分子；
(B2)基因组序列如序列表的序列7所示的DNA分子；
(B3)在严格条件下与(B1)或(B2)限定的DNA分子杂交且编码具有相同功能的蛋白的DNA分子；
(B4)来源于大豆，序列与(B1)或(B2)或(B3)限定的DNA序列具有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％以上同一性，且具有影响大豆雄性不育的功能的DNA分子。
本发明还保护雄性不育植物，与野生型植物的区别在于：GmMS1基因发生突变；所述突变为如下(A)或(B)所述突变：
(A)序列表的序列2自5’端第51位碱基和52位碱基之间插入碱基A；
(B)序列表的序列7自5’端第749位碱基和750位碱基之间插入碱基A。
上述雄性不育植物的制备方法可参照前文第一方面和第二方面所述。
第六方面，本发明保护下述任一在植物育种中的应用；
(C1)前文任一所述方法；
(C2)前文任一所述的GmMS1蛋白或其相关生物材料；
(C3)前文任一所述的特异sgRNA或载体；
(C4)前文任一所述的雄性不育植物。
以上任一所述植物为(D1)或(D2)或(D3)：
(D1)双子叶植物或单子叶植物；
(D2)豆科植物；
(D3)大豆。
所述大豆具体可为大豆品种Jack。
附图说明
图1为GmMS1基因编辑的测序检测结果。
图2为GmMS1基因编辑植株的不育表型。
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。
发芽培养基：3.1g/L B5培养基基础盐(Gamborgs Basal Salt Mixture，Phytotech G768)，20g/L蔗糖，1ml/L B5培养基维生素溶液(Gamborgs Vitamin Solution，Phytotech G219)，7g/L琼脂，pH 5.8。
共培养液体培养基：2.0g/L MS基础盐混合物(Murashige&Skoog Basal Salt Mixture)，3.9g/L吗啉乙磺酸(MES)，30g/L蔗糖，1ml/L B5培养基维生素溶液(Gamborgs Vitamin Solution，Phytotech G219)，150mg/L二硫苏糖醇，40mg/L乙酰丁香酮，2mg/L玉米素，pH5.4。
共培养培养基：2.0g/L MS基础盐混合物(Murashige&Skoog Basal Salt Mixture)，3.9g/L吗啉乙磺酸，30g/L蔗糖，1ml/L B5培养基维生素溶液(Gamborgs Vitamin Solution，Phytotech G219)，150mg/L二硫苏糖醇，40mg/L乙酰丁香酮，2mg/L玉米素，7g/L琼脂，pH 5.4。
恢复培养基：3.1g/L B5培养基基础盐(Gamborgs Basal Salt Mixture，Phytotech G768)，0.98g/L吗啉乙磺酸，30g/L蔗糖，1ml/L B5培养基维生素溶液(Gamborgs Vitamin Solution，Phytotech G219)，150mg/L头孢噻肟(cefotaxime)，450mg/L特美汀(timentin)，1mg/L 6-苄氨基嘌呤(6-Benzylaminopurine)，7g/L琼脂，pH 5.7。
筛选培养基：3.1g/L B5培养基基础盐(Gamborgs Basal Salt Mixture，Phytotech G768)，0.98g/L吗啉乙磺酸，30g/L蔗糖，1ml/L B5培养基维生素溶液(Gamborgs Vitamin Solution，Phytotech G219)，150mg/L头孢噻肟，450mg/L特美汀，1mg/L 6-苄氨基嘌呤，7g/L琼脂，6mg/L草丁膦(glufosinate)，pH 5.7。
伸长培养基：4.0g/L MS基础盐混合物(Murashige&Skoog Basal Salt Mixture)，0.6g/L吗啉乙磺酸，30g/L蔗糖，1ml/L B5培养基维生素溶液(Gamborgs Vitamin Solution，Phytotech G219)，150mg/L头孢噻肟，450mg/L特美汀，0.1mg/L吲哚乙酸(3-Indoleacetic acid)，0.5mg/L赤霉素(Gibberellic acid)，1mg/L玉米素，7g/L琼脂，6mg/L草丁膦，pH 5.6。
生根培养基：2.0g/L MS基础盐混合物(Murashige&Skoog Basal Salt Mixture)，0.6g/L吗啉乙磺酸，20g/L蔗糖，1ml/L B5培养基维生素溶液(Gamborgs Vitamin Solution，Phytotech G219)，7g/L琼脂，3mg/L草丁膦，pH 5.7。
大豆品种Jack的种子：参考文献：Wei Liu,Bingjun Jiang,Liming Ma,Shouwei Zhang,Hong Zhai,Xin Xu,Wensheng Hou,Zhengjun Xia,Cunxiang Wu,Shi Sun,Tingting Wu,Li Chen,Tianfu Han,Functional diversification of Flowering Locus T homologs in soybean:GmFT1a and GmFT2a/5a have opposite roles in controlling flowering and maturation,New Phytologist,2018,217(3):1335-1345.；国家种质库保存号：WDD01579，公众可以从中国农业科学院作物科学研究所获得。
实施例1、大豆雄性核不育基因GmMS1的获得
对大豆基因组进行测序和功能分析，发现一个大豆雄性核不育基因，将其命名为GmMS1基因，其CDS如序列表的序列2所示，基因组序列如序列表的序列7所示。所述GmMS1基因编码的蛋白质命名为GmMS1蛋白，如序列表的序列1所示。
实施例2、GmMS1基因在调控大豆育性中的应用
一、Crisper/CAS9基因编辑载体的构建
1、gRNA靶点设计与合成
设计两个靶点序列，分别为
靶点1：GACGGGAACACCTGTGGCGG TGG(序列2自5’端第3-25位)；
靶点2：CGCCGAGGTCTAAGATACAG AGG(序列2自5’端第35-57位)。
靶点1和靶点2中，下划线为PAM序列。
根据靶点序列分别设计两对引物：
靶点1引物：
F1:5’-TTGGACGGGAACACCTGTGGCGG-3’(序列8)；
R1:5’-AACCCGCCACAGGTGTTCCCGTC-3’(序列9)。
靶点2引物：
F2:5’-TTGCGCCGAGGTCTAAGATACAG-3’(序列10)；
R2:5’-AACCTGTATCTTAGACCTCGGCG-3’(序列11)。
2、引物二聚体的形成
将引物F1和引物R1分别稀释成10μM，配置反应体系：F1 5μl、R1 5μl、H 2O 15μl。混匀后95℃反应3min，然后自然冷却至25℃，然后16℃，5min，得到引物二聚体1。
将引物F2和引物R2分别稀释成10μM，配置反应体系：F2 5μl、R2 5μl、H 2O 15μl。混匀后95℃反应3min，然后自然冷却至25℃，然后16℃，5min，得到引物二聚体2。
3、取步骤2得到的引物二聚体1配置反应体系：Cas9/gRNA载体1μl、引物二聚体1 1μl、Solution1 1μl、Solution2 1μl、H 2O 6μl。16℃反应2小时。
上述载体和试剂来自北京唯尚立德生物科技有限公司，货号VK005-15。
反应结束，得到重组载体CRISPR/Cas9-GmMS1-1，重组载体CRISPR/Cas9-GmMS1-1中含有序列表的序列3所示的的DNA分子(已经测序验证)，表达sgRNA。sgRNA的靶序列为序列4。
4、取步骤2得到的引物二聚体2配置反应体系：Cas9/gRNA载体1μl、引物二聚体2 1μl、Solution1 1μl、Solution2 1μl、H 2O 6μl。16℃反应2小时。
上述载体和试剂来自北京唯尚立德生物科技有限公司，货号VK005-15。
反应结束，得到重组载体CRISPR/Cas9-GmMS1-2，重组表达重组载体CRISPR/Cas9-GmMS1-2中含有序列表的序列5所示的DNA分子，表达sgRNA (已经测序验证)。sgRNA的靶序列为序列6。
二、重组农杆菌的制备
将步骤3和步骤4构建的重组载体CRISPR/Cas9-GmMS1-1和重组载体CRISPR/Cas9-GmMS1-2分别导入导入根癌农杆菌EHA105，得到重组农杆菌GmMS1-1和重组农杆菌GmMS1-2。
三、基因编辑大豆的获得
取步骤二得到的重组农杆菌，采用根癌农杆菌介导的大豆子叶节转化方法转化大豆，具体转化方法如下：
(1)选取大小均匀一致、无病斑、无裂纹，表面光滑、无褶皱的饱满大豆品种Jack的种子，将其装入玻璃培养皿中。然后放入干燥器内，敞开培养皿。在干燥器中放入一个玻璃烧杯，先加入100mL次氯酸钠，再向烧杯中滴入4mL的浓盐酸。凡士林涂于干燥器盖的四周后盖上干燥器，形成密封状态。然后将干燥器置于通风橱中，种子消毒16-20h。
(2)完成步骤(1)后，将灭菌的种子下胚轴垂直向上接入发芽培养基中，培养皿不封口，放在25℃，16h光照/18小时黑暗的组培间培养1d。
(3)完成步骤(2)后，以萌发的大豆种子的子叶节为外植体。首先剥去大豆种皮，纵切分离两片子叶，在子叶与胚轴交接部位(子叶节)划成条状伤口，将划伤的外植体放入含重组农杆菌重悬液的共培养液体培养基(菌液OD值为0.6—0.8)中，28℃浸染30min，侵染后，将外植体(子叶)内平面向下接种于表面铺有滤纸的共培养培养基中，25℃、16h光照/18小时黑暗条件下培养5天。
(4)完成步骤(3)后，将外植体转入恢复培养基中，25℃、16h光照/18小时黑暗条件下培养7d。
(5)完成步骤(4)后，切除外植体产生的主芽，将其转入筛选培养基中，25℃、16h光照/18小时黑暗条件下培养21d。
(6)完成步骤(5)后，剥去褐化的叶片，将产生的不定芽转入伸长培养基中，进行伸长。15d继代一次，继代2-3次。期间将产生的伸长苗转入生根培养基中，进行生根。
(7)完成步骤(6)，待根长出，将苗子从培养基中取出，移栽到装有基质的小盆中进行炼苗。炼苗1周后，转入大盆生长，获得T0代植株。
(8)对T0代植株进行检测，检测方法如下：
分别提取转基因植株和野生型植株基因组DNA，以此为模板，用检测引物进行PCR扩增。
F-614:CGCCATAGTGAAGTAGCGGA(序列12)；
R-614:CAGTTGAAAACAAACTTACCGAAGG(序列13)。
PCR反应条件：先95℃预变性5min；然后95℃ 30sec，56℃ 30sec，72℃ 1min，35个循环；再72℃延伸10min。将PCR产物送去测序，筛选基因编辑植株。
测序结果显示，重组农杆菌GmMS1-1转化大豆后，共得到60株T0代植株，在T1后代中得到1株为在GmMS1基因中发生突变且为纯合型突变的植株，与野生型相比，纯合突变植株的差异在于：存在6bp的缺失，该缺失位于序列2的第18位和第23位之间(GGCGGT)。重组农杆菌GmMS1-2转化大豆后，共得到56株T0代植株，在T1后代中得到5株为在GmMS1基因中发生突变且为纯合型突变的植株，与野生型相比，纯合突变植株的差异在于：存在一个核苷酸的差异(即发生了一个插入突变且为纯合型，该插入位于序列2的第51位和52位之间，插入的单碱基为A，测序结果见图1)，该核苷酸的差异引起移码，从而不能有效表达GmMS1蛋白(图1)。
四、大豆育性检测
待测植株：野生型植株、5株纯合型突变植株(转入重组农杆菌GmMS1-2得到的插入突变植株)。
室外自然条件下，盆栽种植待测植株。
结果如图2所示。结果显示，野生型对照植株结荚正常，籽粒饱满，而GmMS1基因编辑移码突变的纯合植株为雄性不育植株，不能正常结荚。这些结果说明对GmMS1基因进行编辑可以导致雄性不育。
工业应用
本发明对于植物育性研究及不育植物的育种有重要意义。

Claims (18)

  1. 一种培育雄性不育植物的方法，包括如下步骤：降低或抑制目的植物中GmMS1蛋白的活性和/或含量，得到雄性不育植物；
    所述GmMS1蛋白是如下(A1)或(A2)或(A3)：
    (A1)由序列表中序列1所示的氨基酸序列组成的蛋白质；
    (A2)来源于大豆，序列与(A1)具有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％以上同一性且具有影响大豆雄性不育的功能的蛋白质；
    (A3)将序列表中序列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/或添加且与植物雄性育性相关的蛋白质。
  2. 一种培育雄性不育植物的方法，包括如下步骤：沉默或抑制目的植物中GmMS1基因的表达或敲除GmMS1基因，得到雄性不育植物：
    所述GmMS1基因是如下任一所述的DNA分子：
    (B1)编码序列如序列表的序列2所示的DNA分子；
    (B2)基因组序列如序列表的序列7所示的DNA分子；
    (B3)在严格条件下与(B1)或(B2)限定的DNA分子杂交且编码具有相同功能的蛋白的DNA分子；
    (B4)来源于大豆，序列与(B1)或(B2)或(B3)限定的DNA序列具有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％以上同一性，且具有影响大豆雄性不育的功能的DNA分子。
  3. 如权利要求2所述的方法，其特征在于：所述沉默或抑制目的植物中GmMS1基因的表达或敲除GmMS1基因，为突变目的植物中GmMS1基因使目的植物中GmMS1基因表达量降低或使目的植物中GmMS1基因发生功能缺失。
  4. 如权利要求3所述的方法，其特征在于：所述突变为发生于GmMS1基因外显子的缺失突变和/或插入突变和/或替换突变。
  5. 如权利要求4所述的方法，其特征在于：所述突变为如下(A)或(B)所述突变：
    (A)序列表的序列2自5’端第51位碱基和52位碱基之间插入碱基A；
    (B)序列表的序列7自5’端第749位碱基和750位碱基之间插入碱基A。
  6. 如权利要求5所述的方法，其特征在于：所述突变是通过CRISPR/Cas9基因编辑技术实现的；所述CRISPR/Cas9的靶序列如序列表的序列6所示。
  7. 如权利要求1-6任一所述的方法，其特征在于：所述植物为(D1)或(D2)或(D3)：
    (D1)双子叶植物或单子叶植物；
    (D2)豆科植物；
    (D3)大豆。
  8. GmMS1蛋白或其相关生物材料在调控植物育性中的应用；
    所述相关生物材料为如下(1)-(3)中的任一种：
    (1)GmMS1蛋白的编码基因；
    (2)用于沉默或抑制目的植物中(1)的表达或敲除(1)的物质；
    (3)用于降低或抑制目的植物中GmMS1蛋白的活性和/或含量的物质；
    所述GmMS1蛋白是如下(A1)或(A2)或(A3)：
    (A1)由序列表中序列1所示的氨基酸序列组成的蛋白质；
    (A2)来源于大豆，序列与(A1)具有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％以上同一性且具有影响大豆雄性不育的功能的蛋白质；
    (A3)将序列表中序列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/或添加且与植物雄性育性相关的蛋白质。
  9. 如权利要求8所述的应用，其特征在于：所述植物为(D1)或(D2)或(D3)：
    (D1)双子叶植物或单子叶植物；
    (D2)豆科植物；
    (D3)大豆。
  10. 一种用于CRISPER-Cas9基因编辑的特异sgRNA；所述sgRNA的靶序列如序列表的序列6所示。
  11. 一种用于CRISPER-Cas9基因编辑的载体，表达特异sgRNA；所述 sgRNA的靶序列如序列表的序列6所示。
  12. 雄性不育植物，是降低或抑制目的植物中GmMS1蛋白的活性和/或含量得到的；
    所述GmMS1蛋白是如下(A1)或(A2)或(A3)：
    (A1)由序列表中序列1所示的氨基酸序列组成的蛋白质；
    (A2)来源于大豆，序列与(A1)具有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％以上同一性且具有影响大豆雄性不育的功能的蛋白质；
    (A3)将序列表中序列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/或添加且与植物雄性育性相关的蛋白质。
  13. 雄性不育植物，是沉默或抑制目的植物中GmMS1基因的表达或敲除GmMS1基因得到的；
    所述GmMS1基因是如下任一所述的DNA分子：
    (B1)编码序列如序列表的序列2所示的DNA分子；
    (B2)基因组序列如序列表的序列7所示的DNA分子；
    (B3)在严格条件下与(B1)或(B2)限定的DNA分子杂交且编码具有相同功能的蛋白的DNA分子；
    (B4)来源于大豆，序列与(B1)或(B2)或(B3)限定的DNA序列具有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％以上同一性，且具有影响大豆雄性不育的功能的DNA分子。
  14. 雄性不育植物，与野生型植物的区别在于：GmMS1基因发生突变；所述突变为如下(A)或(B)所述突变：
    (A)序列表的序列2自5’端第51位碱基和52位碱基之间插入碱基A；
    (B)序列表的序列7自5’端第749位碱基和750位碱基之间插入碱基A。
  15. 如权利要求12-14任一所述的雄性不育植物，其特征在于：所述植物为(D1)或(D2)或(D3)：
    (D1)双子叶植物或单子叶植物；
    (D2)豆科植物；
    (D3)大豆。
  16. 下述任一在植物育种中的应用；
    (C1)权利要求1-7任一所述方法；
    (C2)权利要求8或9中所述的GmMS1蛋白或其相关生物材料；
    (C3)权利要求10或11所述的特异sgRNA或载体；
    (C4)权利要求12-15任一所述的雄性不育植物。
  17. 如权利要求16所述的应用，其特征在于：所述育种的目的是培育雄性不育的植物。
  18. 如权利要求16或17所述的应用，其特征在于：所述植物为(D1)或(D2)或(D3)：
    (D1)双子叶植物或单子叶植物；
    (D2)豆科植物；
    (D3)大豆。
PCT/CN2020/085004 2020-01-09 2020-04-16 一种植物育性相关蛋白及其应用 WO2021139024A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/788,923 US20230175007A1 (en) 2020-01-09 2020-04-16 A plant fertility-associated protein and its application thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010021592.0 2020-01-09
CN202010021592.0A CN112592932B (zh) 2020-01-09 2020-01-09 一种植物育性相关蛋白及其应用

Publications (1)

Publication Number Publication Date
WO2021139024A1 true WO2021139024A1 (zh) 2021-07-15

Family

ID=75180101

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/085004 WO2021139024A1 (zh) 2020-01-09 2020-04-16 一种植物育性相关蛋白及其应用

Country Status (3)

Country Link
US (1) US20230175007A1 (zh)
CN (1) CN112592932B (zh)
WO (1) WO2021139024A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112391395A (zh) * 2020-12-03 2021-02-23 吉林省农业科学院 一种大豆不育基因突变体、应用及大豆不育系的构建方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008022782A (ja) * 2006-07-21 2008-02-07 Tokyo Univ Of Agriculture & Technology 雄性不稔植物体およびその作出方法
CN102634522A (zh) * 2012-03-07 2012-08-15 四川农业大学 控制水稻育性的基因及其编码蛋白和应用
CN104080914A (zh) * 2011-06-21 2014-10-01 先锋国际良种公司 产生雄性不育植物的组合物和方法
CN104131012A (zh) * 2014-07-10 2014-11-05 安徽农业大学 一种鉴定大豆细胞核雄性不育系的分子标记及其鉴定方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107072165B (zh) * 2014-09-26 2021-05-28 先锋国际良种公司 小麦Ms1多核苷酸、多肽以及使用方法
DE102016106656A1 (de) * 2016-04-12 2017-10-12 Kws Saat Se Kernkodierte männliche Sterilität durch Mutation in Cytochrom P450 Oxidase
CN110386967B (zh) * 2018-03-26 2021-04-06 中国农业科学院作物科学研究所 与植物雄性育性相关的蛋白SiMS1及其编码基因与应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008022782A (ja) * 2006-07-21 2008-02-07 Tokyo Univ Of Agriculture & Technology 雄性不稔植物体およびその作出方法
CN104080914A (zh) * 2011-06-21 2014-10-01 先锋国际良种公司 产生雄性不育植物的组合物和方法
CN102634522A (zh) * 2012-03-07 2012-08-15 四川农业大学 控制水稻育性的基因及其编码蛋白和应用
CN104131012A (zh) * 2014-07-10 2014-11-05 安徽农业大学 一种鉴定大豆细胞核雄性不育系的分子标记及其鉴定方法

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
"PREDICTED: Glycine soja kinesin-like protein KIN-7B (LOC114381111), mRNA", GENEBANK ACCESSION NUMBER: XM_028340292.1, 12 March 2019 (2019-03-12) *
BIONE N. C. P., PAGLIARINI M. S., DE ALMEIDA L. A., SEIFERT A. L.: "An ms2 male-sterile, female-fertile soybean sharing phenotypic expression", PLANT BREEDING, vol. 121, no. 4, 1 August 2002 (2002-08-01), pages 307 - 313, XP055826627, ISSN: 0179-9541, DOI: 10.1046/j.1439-0523.2002.726806.x *
DATABASE Nucleotide 12 March 2009 (2009-03-12), ANONYMOUS: "Glycine max strain Williams 82 clone GM_WBc0099F23, complete sequence", XP055826624, retrieved from NCBI Database accession no. AC235472 *
DATABASE Nucleotide 12 March 2019 (2019-03-12), ANONYMOUS: "PREDICTED: Glycine soja kinesin-like protein KIN-7B (LOC114381111), mRNA", XP055826618, retrieved from NCBI Database accession no. XM_028340292 *
DATABASE Protein 19 April 2021 (2021-04-19), ANONYMOUS: "Kinesin-like protein KIN-7B [Glycine max]", XP055826615, retrieved from NCBI Database accession no. XP_003541319 *
YANG JING-TING;LIU ZHEN-HUA;LI SHUO;XIANG FENG-NING: "Research Progress on Identification of Sterility-Related QTLs and Functional Genes in Soybean", PLANT PHYSIOLOGY JOURNAL, vol. 55, no. 10, 20 October 2019 (2019-10-20), pages 1436 - 1448, XP055826700, ISSN: 2009-1108, DOI: 10.13592/j.cnki.ppj.2019.0100 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112391395A (zh) * 2020-12-03 2021-02-23 吉林省农业科学院 一种大豆不育基因突变体、应用及大豆不育系的构建方法

Also Published As

Publication number Publication date
CN112592932B (zh) 2022-03-22
CN112592932A (zh) 2021-04-02
US20230175007A1 (en) 2023-06-08

Similar Documents

Publication Publication Date Title
Santosh Kumar et al. CRISPR-Cas9 mediated genome editing of drought and salt tolerance (OsDST) gene in indica mega rice cultivar MTU1010
ES2883229T3 (es) Gen para la inducción de partenogénesis, un componente de la reproducción apomíctica
CN107245480B (zh) 具有除草剂抗性的乙酰乳酸合酶突变蛋白及其应用
CN107475210B (zh) 一种水稻白叶枯病抗性相关基因OsABA2及其应用
CN113652416A (zh) 烟草植物中腋芽生长的遗传控制
CN111333707B (zh) 一种植物粒型相关蛋白及其编码基因与应用
CN117904170A (zh) 与大豆中锈病抗性相关联的新颖的遗传基因座
US20220106607A1 (en) Gene for parthenogenesis
CN110257421B (zh) 一种甘蓝型油菜基因突变体ptg8的构建方法及其应用
WO2020052415A1 (zh) 隐性抗烟草脉带花叶病毒的烟草eIFiso4E-S基因及其应用
KR101497774B1 (ko) 형질전환 동안 ｂｂｍ의 유도를 통해 가임 식물을 제공하는 방법
CN108003227B (zh) 一种水稻早花时相关蛋白及其编码基因
WO2021139024A1 (zh) 一种植物育性相关蛋白及其应用
CN111286504A (zh) 调控油菜种子含油量的基因orf188
EP4069853A1 (en) Cca gene for virus resistance
BR112015020368B1 (pt) Uso de primeiro e segundo ácidos nucleicos que codificam polipeptídeos si como marcadores genéticos moleculares, kit para controlar hibridização ou autoincompatibilidade em plantas, construto genético e método para manipular autoincompatibilidade em uma planta
CN106589085A (zh) 一种植物淀粉合成相关蛋白OsFLO8及其编码基因与应用
WO2013082865A1 (zh) 一种制备育性减低植物的方法
CN107937363B (zh) 一种水稻穗顶退化相关蛋白激酶及其编码基因
CN105950598B (zh) 一个水稻休眠性相关蛋白及其编码基因与应用
Karmacharya Molecular Mapping of QTL for Genetic Transformation-Related Trait and CRISPR/Cas9-Mediated Gene Editing in Wheat
CN113512551B (zh) 调控大豆籽粒大小基因的克隆及应用
US11795469B2 (en) Scaevola plants with radially symmetrical flowers
Phillip et al. Application of biotechnology to Lotus breeding
WO2024043238A1 (ja) 超開花性ムギとその生産方法および評価方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20912385

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20912385

Country of ref document: EP

Kind code of ref document: A1