WO2021174441A1 - 悬铃木突变体、获取悬铃木突变体的方法和应用 - Google Patents

悬铃木突变体、获取悬铃木突变体的方法和应用 Download PDF

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WO2021174441A1
WO2021174441A1 PCT/CN2020/077764 CN2020077764W WO2021174441A1 WO 2021174441 A1 WO2021174441 A1 WO 2021174441A1 CN 2020077764 W CN2020077764 W CN 2020077764W WO 2021174441 A1 WO2021174441 A1 WO 2021174441A1
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platanus
gene
vulgaris
vector
obtaining
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PCT/CN2020/077764
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French (fr)
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于为常
夏立新
骆超
周秋艳
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深圳大学
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    • 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
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • 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
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • 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)

Definitions

  • This application relates to the field of transgenic technology, in particular to Platanus vulgaris mutants, methods and applications for obtaining platanus vulgaris mutants.
  • Platanus spp. is a deciduous tree species of Platanaceae. There is only one genus in this family, about 10-12 species, all of which are produced abroad. Three types of platanus (P. occidentalis Linn.), two-ball (P. acerifolia Willd) and three-ball (P. orientalis Linn.) are introduced in China. Among them, the British sycamore is a hybrid of American sycamore and French sycamore (P. occidental is ⁇ P. orientalis), has a good heterosis, and has a history of more than one hundred years of introduction in my country.
  • the purpose of the embodiments of the present application is to provide Platanus vulgaris mutants, and methods and applications for obtaining platanus vulgaris mutants, aiming to solve the problems of short timeliness and poor orientation of mutant breeding of Platanus vulgaris in the prior art.
  • a method for obtaining Platanus vulgaris mutants which includes the following steps:
  • CRISPR/Cas9 vector plasmid Provide a CRISPR/Cas9 vector plasmid and restriction endonucleases, and use the restriction enzymes to digest the CRISPR/Cas9 vector plasmid to obtain a digested CRISPR/Cas9 vector plasmid;
  • a ligase Provide a ligase, and use the ligase to sequentially ligate the digested CRISPR/Cas9 vector plasmid and the DNA annealing product to obtain a CRISPR/Cas9 genome editing vector;
  • the gene gun method is used to transform the CRISPR/Cas9 genome editing vector into a seed embryo to obtain a transformation product, and the transformation product is cultivated to screen for Platanus vulgaris mutants.
  • a Platanus vulgaris mutant is provided.
  • the platanus vulgaris mutant is a mutant that produces a mutation in a target gene.
  • the mutation in the target gene includes the insertion, deletion, or nucleotide sequence change of a gene fragment in the coding region of the gene.
  • a method for obtaining a Platanus vulgaris mutant or an application of the Platanus vulgaris mutant in breeding of Platanus vulgaris is provided.
  • the gRNA target sequence of the target gene of Platanus orientalis is designed to construct a genome editing vector, which is transformed into seed embryos by the gene gun method to obtain transformation products, and the transformation products are cultivated and identified to obtain the platanus mutants.
  • This method It can carry out targeted mutations to specific genes of Platanus vulgaris, with strong orientation and high efficiency.
  • the Platanus vulgaris mutant described in the present application is a mutant with an altered target gene sequence, and the mutant rapidly obtains a specific target gene sequence mutation in a targeted and rapid manner, which is beneficial for subsequent applications.
  • gRNA-guided Cas9 enzyme is used to cut Platanus vulgaris genomic DNA at the gene target point, and after DNA repair, deletion, insertion or base mutation of the gene sequence occurs. These mutations can cause amino acid changes, amino acid substitutions, and amino acid increases or decreases in gene-encoded proteins, thereby affecting the changes in the phenotype of Platanus orientalis.
  • the method for obtaining platanus mutants described in this application can target all genes in the platanus platanus genome. The method is simple and efficient, and can quickly obtain mutants of specific genes. The prepared platanus mutants are useful in platanus breeding Wide range of applications.
  • Fig. 1 is a Plaa1 gene transformation vector provided by an embodiment of the present application.
  • Fig. 2 is a flow chart of generating Platanus vulgaris mutants provided by an embodiment of the present application.
  • first and second are only used for description purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Thus, the features defined with “first” and “second” may explicitly or implicitly include one or more of these features. In the description of this application, “plurality” means two or more than two, unless explicitly defined otherwise.
  • the example of this application provides a method for obtaining Platanus vulgaris mutants.
  • the method is shown in Figure 2 "vector construction-gene transformation-transgenic seedling screening-mutant gene sequence analysis", including the following steps:
  • the gRNA target sequence of the target gene of Platanus orientalis is designed to construct a genome editing vector, which is transformed into seed embryos by the gene gun method to obtain transformation products, and the transformation products are cultivated and identified to obtain the platanus mutants.
  • This method It can carry out targeted mutations to specific genes of Platanus vulgaris, with strong orientation and high efficiency.
  • step S01 the target gene of Platanus vulgaris is obtained and the gRNA target sequence is determined.
  • the selected platanus species is Platanus x acerifolia.
  • the platanus pollen allergen plaa1 is used as the platanus target gene.
  • a leaf of Platanus x acerifolia is taken as a sample, a genome extraction kit is used to extract Platanus vulgaris genomic DNA, and the platanus pollen allergen plaa1 gene is obtained through PCR amplification.
  • the fifth primer and the sixth primer are used to perform PCR amplification reaction to obtain the platanus pollen allergen plaa1 gene, wherein the sequence of the fifth primer is as As shown in SEQ ID No. 3, it is: 5'-tacgcggggaacaaacaatccaat-3'; the sequence of the sixth primer is as shown in SEQ ID No. 4, which is: 5'- ccgaagagggccaaatatcataca-3’.
  • the PCR amplification reaction system is as follows: the DNA of the platanus leaf: 5 uL, the fifth primer: 5 uL, The sixth primer: 5 uL, 2 ⁇ Taq mix: 25 uL, Mg2+: 1 uL glycerol: 1 uL, ddH2O: 12 uL.
  • the PCR amplification reaction conditions are as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, 40 cycles; extension at 72°C for 10 min, and storage at 4°C.
  • the PCR product of the platanus pollen allergen plaa1 gene was obtained by PCR amplification reaction and sequenced and analyzed, and the coding region sequence of the platanus pollen allergen plaa1 gene was obtained as shown in SEQ ID No. 5, which is:
  • the first gRNA target sequence and the second gRNA target sequence are designed according to the sequence of the coding region of the platanus pollen allergen plaa1 gene obtained by sequencing.
  • the screening requirements for the target sequence are: (1)
  • the target sequence mainly includes 15-25 bases, (2) these 15-25 bases are followed by NGG (N is any Base) 3-base PAM (Protospacer adjacent motif, PAM).
  • the gRNA target sequence includes a first gRNA target sequence and a second gRNA target sequence.
  • the first gRNA target sequence is shown in SEQ ID No. 1, which is: 5'-CAGCGCCGATATTGTTCAGG-3';
  • the second gRNA target sequence is shown in SEQ ID No. 2, which is: 5' -TTCTGCGCGAAGTCTCTTGG-3'.
  • the target sequence can be selected from any sequence in the coding region of the gene or upstream or downstream regulatory sequences of gene expression, such as promoter sequences, enhancer sequences and other sequences.
  • the number of the target sequence is selected from one, two or more, and the target sequence is selected from single use or simultaneous use.
  • RNA target sequence oligonucleotide fragment containing the sticky end of the restriction endonuclease and its complementary strand are synthesized, mixed and annealed to produce a DNA annealing product containing the sticky end of the endonuclease at both ends.
  • the method for obtaining the annealing product of the first gRNA target sequence includes the following steps: determining the first primer and the second primer of the first gRNA target sequence, and obtaining the annealing of the first gRNA target sequence through the first annealing treatment product.
  • the first primer of the first gRNA target sequence is shown in SEQ ID No. 6, which is: 5'- ATTG CAGCGCCGATATTGTTCAGG-3';
  • the second primer of the first gRNA target sequence is:
  • the underlined part in the first primer and the second primer of the first gRNA target sequence is the BsaI sticky end sequence of the restriction endonuclease.
  • the annealing product of the first gRNA target sequence is obtained through the first annealing treatment, and the reaction system of the first annealing treatment is as follows: each of the first primer and the second primer with a concentration of 100 mM, 1 uL, 8 uL purified water;
  • the reaction procedure of the first annealing treatment is as follows: denature at 95°C for 5 minutes in a PCR machine, and slowly cool down to room temperature for 1 hour.
  • the method for obtaining the annealing product of the second gRNA target sequence includes the following steps: determining the third primer and the fourth primer of the second gRNA target sequence, and obtaining the annealing of the second gRNA target sequence through a second annealing treatment product.
  • the third primer of the second gRNA target sequence is shown in SEQ ID No. 8, and is: 5'- ATTG CAGCGCCGATATTGTTCAGG-3';
  • the fourth primer of the second gRNA target sequence is:
  • the underlined part in the first primer and the second primer of the first gRNA target sequence is the BsaI sticky end sequence of the restriction endonuclease.
  • the annealing product of the second gRNA target sequence is obtained through the second annealing treatment, and the reaction system of the second annealing treatment is as follows: the first primer and the second primer with a concentration of 100 mM are each 1 uL, 8 uL purified water;
  • the reaction procedure of the second annealing treatment is as follows: denature at 95°C for 5 minutes in a PCR machine, and slowly cool down to room temperature for 1 hour.
  • a CRISPR/Cas9 vector plasmid and a restriction endonuclease are provided, and the CRISPR/Cas9 vector plasmid is digested with the restriction enzyme to obtain a digested CRISPR/Cas9 vector plasmid.
  • the CRISPR/Cas9 vector plasmid includes but is not limited to pKSE401, which can be selected from other genome editing vector systems.
  • the CRISPR/Cas9 vector plasmid is selected from pKSE401, and the pKSE401 vector is fused with green fluorescent protein (GFP), which is conducive to subsequent screening.
  • GFP green fluorescent protein
  • the restriction endonuclease includes but is not limited to BsaI enzyme, which can be selected from restriction endonucleases of gRNA cloning sites carried by other vector systems.
  • the restriction endonuclease is selected from the BsaI enzyme, and the BsaI enzyme is used for digestion treatment to ensure that the gRNA and the carrier can be connected.
  • the reaction system of the digestion treatment is as follows: CRISPR/Cas9 vector plasmid 1 ug, 10 U BsaI enzyme, add BsaI enzyme buffer to 20 uL.
  • the reaction conditions of the enzyme digestion treatment are as follows: the digestion treatment at 50-52°C for 1 to 1.5 hours, and then heating at 65-68°C for 10 minutes. Heating is performed after the digestion treatment, in order to inactivate the enzyme and end the digestion treatment.
  • the reaction conditions for the digestion treatment are as follows: digestion treatment at 50°C for 1 hour, and then heating at 65°C for 10 minutes.
  • a ligase is provided, and the CRISPR/Cas9 vector plasmid after digestion and the DNA annealing product are sequentially ligated using the ligase to obtain a CRISPR/Cas9 genome editing vector.
  • the ligase is selected from T 4 ligase, and T 4 ligase is used for ligation treatment, which can better ligate the CRISPR/Cas9 vector plasmid and the DNA annealing product after digestion to obtain the CRISPR/Cas9 genome
  • the editing vector is connected to construct a CRISPR/Cas9 genome editing vector.
  • the ligase is used to sequentially ligate the digested CRISPR/Cas9 vector plasmid and the annealing product of the gRNA target sequence to obtain the CRISPR/Cas9 genome editing vector.
  • the processed reaction system is as follows: add 10 ⁇ L 2X reaction buffer, 1 ⁇ L T 4 DNA ligase, 50 ng digested vector and 3 times molar amount of gRNA target annealing product to the 20 ⁇ L reaction system.
  • connection treatment is as follows: react at 37 ⁇ 38°C for 10 ⁇ 15 min.
  • reaction procedure of the ligation treatment is as follows: react at 37°C for 10 min.
  • the prepared CRISPR/Cas9 genome editing vector is transformed into E. coli competent cells by the heat shock method, and positive clones are screened and sequenced to ensure that an accurate CRISPR/Cas9 genome editing vector is obtained.
  • step S05 the CRISPR/Cas9 genome editing vector is transformed into the seed embryo to obtain the transformation product by the particle bombardment method, the transformation product is cultivated, and the Platanus vulgaris mutant is screened.
  • the method for transforming the CRISPR/Cas9 genome editing vector into the seed embryo to obtain the transformation product by the particle bombardment method includes the following steps:
  • the vector to be transformed includes a plant genome editing vector and metal particles carrying the plant genome editing vector; wherein the diameter of the metal particles is 0.6 ⁇ m to 1.8 ⁇ m;
  • step S051 the method of obtaining the fruit seeds of the plant at the mature stage and cultivating the embryos includes the following steps:
  • the mature fruit seeds of the plant are obtained, and the mature seeds are selected as the transformation objects.
  • the mature seeds are rich in organic matter, have certain germination ability and strong environmental resistance.
  • As the transformation objects they can have It is beneficial to effectively transform the recombinant plasmid into seeds, and is beneficial to improve the efficiency of gene transformation.
  • step S0512 after the seeds are sterilized, the method of dark culture to obtain the embryos of the seeds includes the following steps:
  • the first sterilization treatment is performed with 75% alcohol by volume, and then the second sterilization treatment is performed with a mercury liter solution with a mass concentration of 0.1%. Seed after fungus
  • the seeds are dehaired, and the dehairing treatment is mainly to remove the seed hair on the surface of the seed to ensure that the surface of the seed is clean, which is conducive to seed germination and gene transformation. Dehairing the seeds with washing powder water to ensure that the seeds are cleaned.
  • the second sterilization treatment with 0.1% mercury solution by mass concentration is used to obtain the sterilized seeds; the seeds are sterilized to ensure Seed germination and the embryos for gene transformation experiments are sterile and clean, and the gene transformation experiments can be completed well.
  • the time for the first sterilization treatment is 40-60 seconds.
  • the time for the second sterilization treatment is 40-50 minutes.
  • the seeds after the second sterilization treatment also include sterile water rinsing, using sterile water for rinsing to obtain sterilized seeds, and removing the mercury solution on the surface of the seeds to obtain clean sterilized seeds.
  • a vector to be transformed includes a plant genome editing vector and metal particles carrying the plant genome editing vector; wherein the diameter of the metal particles is 0.6 ⁇ m to 1.8 ⁇ m.
  • a transformation vector is provided for transformation, and the vector to be transformed includes a plant genome editing vector and metal particles carrying the plant genome editing vector.
  • the carrying method includes conventional coating methods such as soaking.
  • the metal particles carrying the plant genome editing vector are a medium for plant gene transformation, through which the plant genome editing vector is transformed into seed embryos by a gene bombardment method.
  • the diameter of the metal particles is 0.6 ⁇ m to 1.8 ⁇ m. Selecting metal particles with the above diameter as the medium for gene transformation experiments can ensure the transformation efficiency. If the diameter of the metal particles is too large, it will cause greater damage to the plant seeds, affect the transformation efficiency, and affect the survival rate of the seeds; if the diameter of the metal particles is too small, it will cause the metal particles to carry too few recombinant plasmids, resulting in transformation The efficiency is too low. In a preferred embodiment of the present application, the diameter of the metal particles is 0.6 ⁇ m.
  • the metal particles are selected from gold powder or tungsten powder. Select metal particles with more stable properties for testing, reduce the damage to plant seeds, and improve the survival rate of plant seeds.
  • the mass ratio of the metal particles to the plant genome editing vector is 15 mg: 2 ⁇ g for coating, and the plasmid to be transformed is prepared. If the addition amount of metal particles is too much, it will cause damage to plant seeds, affect the transformation efficiency, and affect the survival rate of seeds; if the addition amount of metal particles is too small, it will cause too few recombinant plasmids carried by the metal particles, resulting in The conversion efficiency is too low.
  • a gene gun is used to transform the vector to be transformed into the embryo under the condition of a pressure of 450-2200 PSI to obtain a transformed product.
  • the plasmid to be transformed is used to transform 100 to 500 embryos for transformation. Under the above-mentioned pressure conditions, it can be ensured that the metal particle inclusions are transformed into the seeds with higher efficiency.
  • the conversion pressure is 900 PSI.
  • the gene gun is selected from the PDS-1000/He gene gun of BIO-RAD.
  • the transformation product is cultivated.
  • the transformation product is cultured in the dark at 25°C for one day and then cultivated under light for three days. After the green shoots have grown, they are transplanted into the greenhouse and cultivated into seedlings, and the seedlings are transplanted.
  • the platanus mutants were cultivated and screened.
  • the step of screening and obtaining the platanus mutant includes the following method, using the plant genomic DNA of the platanus mutant as a template, and using the seventh primer and the eighth primer to perform a PCR amplification reaction to verify the platanus mutant.
  • the sequence of the seventh primer is shown in SEQ ID No. 10, which is:
  • the sequence of the eighth primer is shown in SEQ ID No. 11, which is:
  • the seventh primer and the eighth primer were used to amplify PCR cloning, wherein the PCR amplification reaction system was as follows: Platanus vulgaris leaf DNA: 5 uL, seventh primer: 5 uL, eighth primer: 5 uL, 2 ⁇ Taq mix: 25 uL, Mg2+: 1 uL glycerol: 1 uL, ddH2O: 12 uL; and the PCR amplification reaction conditions are as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 56°C annealing for 30 s Extend at 72°C for 1 min, 40 cycles; extend at 72°C for 10 min, store at 4°C.
  • Each gene-transformed plant selects 10 single clones for sequencing.
  • the sequencing results are compared with the wild-type plant gene sequence (SEQ ID No. 5) through the BLAST program of the NCBI website to identify the mutant gene.
  • the embodiments of the present application also provide a Platanus vulgaris mutant, which is a mutant with an altered target gene sequence, and the mutant with an altered target gene sequence includes gene fragment insertions, deletions, or base changes. mutant.
  • the mutants can obtain specific target gene mutations in a targeted and rapid manner, which is beneficial for subsequent applications.
  • the embodiments of the present application also provide a method for obtaining a Platanus vulgaris mutant or an application of the Platanus vulgaris mutant in breeding of Platanus vulgaris.
  • gRNA-guided Cas9 enzyme is used to cut Platanus vulgaris genomic DNA at the gene target point, and mutation of the gene sequence is generated after DNA repair. These mutations can cause amino acid changes, amino acid substitutions, and amino acid increases or decreases in gene-encoded proteins, thereby affecting the changes in the phenotype of Platanus orientalis.
  • the method for obtaining platanus mutants described in this application can target all genes in the platanus platanus genome. The method is simple and efficient, and can quickly obtain mutants of specific genes. The prepared platanus mutants are useful in platanus breeding Wide range of applications.
  • the PCR amplification reaction system is as follows: DNA from Platanus vulgaris leaves: 5 uL, fifth primer: 5 uL, sixth primer: 5 uL, 2 ⁇ Taq mix: 25 uL, Mg2+:1 uL glycerol: 1 uL, ddH2O: 12 uL.
  • the PCR amplification reaction conditions are as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, 40 cycles; extension at 72°C for 10 min, and storage at 4°C.
  • the PCR product of the platanus pollen allergen plaa1 gene is obtained through the PCR amplification reaction, and the PCR product is sequenced and analyzed.
  • Target T1 5'-CAGCGCCGATATTGTTCAGG-3'
  • second gRNA target sequence 5'-TTCTGCGCGAAGTCTCTTGG-3'.
  • the sense and antisense strands of the two sgRNA sequences with linkers are synthesized separately, and the following two double-stranded DNAs are formed after mixing and annealing:
  • first primer SEQ ID No. 6 of the first gRNA target sequence (5'- ATTG CAGCGCCGATATTGTTCAGG-3') and the second primer SEQ ID No. 7 (3'-GTCGCGGCTATAACAAGTCC CAAA -5'), and perform mixed annealing Processing to form the first gRNA target sequence;
  • the underlined sequence is the linker sequence. After annealing, a double-stranded DNA with a BsaI restriction endonuclease cohesive end is formed, which can be cloned into the following vector after being cut by the BsaI enzyme.
  • the reaction system is as follows: CRISPR/Cas9 vector plasmid 1 ug, 10 U BsaI enzyme, add BsaI enzyme buffer to 20 uL; and the reaction conditions for the digestion treatment are as follows: digestion at 50°C for 1 hour, and then at 65 Heat at °C for 10 minutes.
  • reaction system of the ligation treatment is as follows: vector plasmid 50 ng digested vector; gRNA target sequence annealing product 0.1 ug; T4 ligase (5 U) 1 ⁇ L; T4 DNA ligase buffer Liquid 2 ⁇ L; and, the reaction procedure of the ligation treatment is as follows: react at 37° C. for 10 min.
  • the prepared CRISPR/Cas9 genome vector is transformed into E. coli competent cells by heat shock, and positive clones are screened and sequenced to ensure that an accurate CRISPR/Cas9 genome editing vector is obtained.
  • the plasmid to be transformed is a gold powder particle with a diameter of 0.6 ⁇ m coated with a genome editing vector.
  • the transformation parameter is set to 900 PSI pressure, the distance between the transformation vector and the sample is 6 cm, and the BIO-RAD company is used.
  • the PDS-1000/He gene gun transforms the genome editing vector into the embryo to obtain the transformed product.
  • the seventh primer and the eighth primer are used to perform a PCR amplification reaction to verify the platanus mutant.
  • the sequence of the seventh primer is shown in SEQ ID No. 10, which is:
  • the sequence of the eighth primer is as SEQ ID No.11 is shown as:
  • the amplified PCR fragments were cloned.
  • the PCR amplification reaction system was as follows: Platanus leaf DNA: 5 uL, seventh primer: 5 uL, eighth primer: 5 uL, 2 ⁇ Taq mix: 25 uL, Mg2+:1 uL glycerol: 1 uL, ddH2O: 12 uL; and the PCR amplification reaction conditions are as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 1 min, 40 cycles ; Extend at 72°C for 10 min, store at 4°C.
  • Each gene-transformed plant selects 10 single clones for sequencing.
  • the sequencing results are compared with the wild-type plant gene sequence (SEQ ID No. 5) through the BLAST program of the NCBI website to identify the mutant gene.
  • the PCR amplification reaction obtained the PCR product of the platanus pollen allergen plaa1 gene for sequencing analysis, and the coding region sequence of the platanus pollen allergen plaa1 gene was obtained as shown in SEQ ID No. 5, which is:
  • the constructed genome editing vector is shown in Figure 1, where LB and RB are the left and right boundaries of the T-DNA on the Ti plasmid, 35Sp-Cas9-NosT and 35Sp-NptII-PolyA These are the cassettes of Cas9 gene and NptII gene.
  • U6-26-T1 and U6-29-T2 express two sgRNAs respectively.
  • F and R are the upstream and downstream primers of transgenic plant detection PCR.
  • Example 4 According to the identification of the mutants in Example 4, a total of 28 platanus transgenic seedlings were obtained in this experiment. Through the analysis of target genes, it was found that 2 strains (No. 18 and No. 48) contained mutations.
  • the 14-15th base TC of 18-3 is missing, causing a frameshift mutation, and the protein is terminated prematurely (nucleic acid sequence 5, protein sequence 2).
  • the base sequence of mutant 18-3 is shown in SEQ ID No. 12, which is:
  • mutant 18-3 is shown in SEQ ID No. 13, which is:
  • mutant 18-6 is shown in SEQ ID No. 14, which is:
  • mutant 18-6 is shown in SEQ ID No. 15, which is:
  • the 402th base of 18-7 was changed from G to T (no amino acid change was caused); therefore, the base sequence and protein sequence of 18-7 were not listed.
  • the 310th base of 18-8 changed from G to A (causing the amino acid to change from glycine G to arginine R).
  • the base sequence of mutant 18-8 is shown in SEQ ID No. 16, which is:
  • mutant 18-8 is shown in SEQ ID No. 17, which is:
  • mutant gene 48-2 is shown in SEQ ID No. 18, which is:
  • mutant gene 48-2 is shown in SEQ ID No. 19, which is:
  • mutant gene 48-3 is shown in SEQ ID No. 20, which is:
  • mutant gene 48-3 is shown in SEQ ID No. 21, which is:
  • the 144th base 48-6 changed from G to A (no amino acid change was caused), and the 327th base changed from G to A (no amino acid change was caused); therefore, the base sequence and protein sequence were not listed.
  • the 154th base 48-7 changed from G to A (causing the amino acid to change from alanine A to threonine T); the base sequence of the mutant gene 48-7 is shown in SEQ ID No. 22, which is:
  • mutant gene 48-7 is shown in SEQ ID No. 23, which is:
  • the 137th base of 48-8 changed from G to A (causing the amino acid to change from cysteine C to tyrosine Y), and the 140th base from C to T (causing the amino acid to change from alanine A to valine) Acid V), base 144 changed from G to A (no amino acid change), base 225 changed from A to T (causing amino acid change from glutamine Q to histidine H), base 259 changed from T changed to G (causing amino acid change from leucine L to valine V), base 306 changed from C to T (no change in amino acid), base 363 changed from T to G (causing amino acid to change from day) Paraffin N changes to Lysine K), the 454th base changes from T to A (causing the amino acid to change from Leucine L to Isoleucine I),
  • the base sequence of the mutant gene 48-8 is shown in SEQ ID No. 24, which is:
  • mutant gene 48-8 is shown in SEQ ID No. 25, which is:
  • This application is a gene mutation technology of Platanus vulgaris through CRISPR/CAS9 genome editing, which can be applied to the mutation of all genes of Platanus vulgaris, such as allergic protein genes that affect health; flowering genes that affect flowering and heading, flower development genes, fruit and Seed development genes, seed coat development genes; genes that affect the rapid growth of trees; genes that affect resistance to drought, waterlogging, salinity, high and low temperature, disease, insects, and viruses.

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Abstract

一种悬铃木突变体、获取悬铃木突变体的方法和应用，通过设计悬铃木的目的基因的第一gRNA靶点序列及所述第二gRNA靶点序列，构建得到基因组编辑载体，利用基因枪法转化处理至种子胚中得到转化产物，对所述转化产物进行培育、鉴定得到所述悬铃木突变体，该方法可对悬铃木的特定基因进行定向突变，定向性强，效率高。

Description

悬铃木突变体、获取悬铃木突变体的方法和应用 技术领域
本申请涉及转基因技术领域，具体涉及悬铃木突变体、获取悬铃木突变体的方法和应用。
 背景技术
悬铃木（Platanus spp.）系悬铃木科悬铃木属落叶乔木树种。该科仅有一属，约10-12种，均产于国外。我国引入栽培三种：一球悬铃木（美国梧桐， P. occidentalis Linn. ）、二球悬铃木（英国梧桐，P. acerifolia Willd ）和三球悬铃木（法国梧桐，P. orientalis Linn.）。其中，英国梧桐为美国梧桐和法国梧桐的杂交种（P. occidental is ×P. orientalis），具有良好的杂种优势，引种我国己有百余年的历史。因其生长迅速、冠大荫浓、树干通直、干皮光洁、耐修剪、易繁殖,能适应城市环境和各种土壤条件,具有较强的抗空气污染，抗光化学烟雾、苯、乙醚、硫化氢等有害气体及良好的滞尘减噪、净化空气的能力,全球温带及亚热带各大城市普遍用作公路、街道和工厂绿化树种，并享有“行道树之王”的美誉。在我国，悬铃木作为行道树和庭荫树广泛应用于黄河及长江流域地区,深受广大市民及园林工作者喜爱，成为一种别的树种所无法替代的园林植物，在城市园林建设中占有十分重要的地位。
但是，但每年一月份左右，当老果脱落，新花、新果生长之际，老果脱落产生的果毛、新生的雄花序散落的大量花粉都从树上飘落下来，不仅污染环境，而且会刺激人们的呼吸道，引发呼吸系统疾病，易导致人们皮肤癌痒、诱发产生鼻炎、花粉症等过敏性病症，不慎落入眼中还会引发角膜炎，严重影响人们的生活及身体健康。
多年来，科研工作者们对控制悬铃木开花结果后季节性果毛、花粉飞扬做了不同的探索和尝试，其中包括进行无果品系的选育、诱变育种、多倍体育种、树冠嫁接、修剪控果、化学药剂处理等方法，虽然都有一定的效果，但都具有处理周期长、时效短、定向性差等弊端和不足，难以彻底解决问题。
 技术问题
本申请实施例的目的在于：提供悬铃木突变体、获取悬铃木突变体的方法和应用，旨在解决现有技术中对悬铃木进行突变育种时效短、定向性差的问题。
 技术解决方案
本申请实施例是这样实现的，
第一方面，提供了一种获得悬铃木突变体的方法，包括以下步骤：
获取悬铃木目的基因并确定所述gRNA靶点序列；
合成含限制性内切酶粘性末端的gRNA靶点序列寡核苷酸片段和及其互补链，混合退火后产生两端含内切酶粘性末端的DNA退火产物；
提供CRISPR/Cas9载体质粒和限制性内切酶，采用所述限制性内切酶对所述CRISPR/Cas9载体质粒进行酶切处理得到酶切后的CRISPR/Cas9载体质粒；
提供连接酶，采用所述连接酶依次将所述酶切后的CRISPR/Cas9载体质粒、所述DNA退火产物进行连接处理得到CRISPR/Cas9基因组编辑载体；
采用基因枪法将所述CRISPR/Cas9基因组编辑载体转化至种子胚中得到转化产物，对所述转化产物进行培育，筛选悬铃木突变体。
第二方面，提供了一种悬铃木突变体，所述悬铃木突变体为目的基因产生突变的突变体，所述目的基因产生突变包括基因编码区基因片段的插入，缺失或核苷酸序列的改变。
第三方面，提供了一种所述的获得悬铃木突变体的方法或所述的悬铃木突变体在悬铃木育种中的应用。
 有益效果
本申请实施例提供的一种获得悬铃木突变体的方法的有益效果在于：
本申请通过设计悬铃木目的基因的gRNA靶点序列，构建得到基因组编辑载体，利用基因枪法转化处理至种子胚中得到转化产物，对所述转化产物进行培育、鉴定得到所述悬铃木突变体，该方法可对悬铃木的特定基因进行定向突变，定向性强，效率高。
本申请实施例提供的一种悬铃木突变体的有益效果在于：
本申请所述悬铃木突变体为目的基因序列改变的突变体，所述突变体靶向性地、快速地获得了特定的目的基因序列的突变，有利于进行后续的应用。
本申请实施例提供的一种获得悬铃木突变体的方法的应用的有益效果在于：
本申请采用gRNA引导的Cas9酶在基因靶点对悬铃木基因组DNA进行切割，经DNA修复后而产生基因序列的缺失、插入或碱基的突变。这些突变可引起基因编码蛋白的氨基酸变化，氨基酸替代，氨基酸增加或减少，从而影响悬铃木表现型的变化。本申请所述的获得悬铃木突变体的方法可对悬铃木基因组中所有的基因进行靶向突变，方法简捷，效率高，可快速获得特定基因的突变体，制备得到的悬铃木突变体在悬铃木育种中的应用广泛。
 附图说明
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例或示范性技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其它的附图。
图1是本申请实施例提供的悬铃木plaa1基因转化载体。
图2是本申请实施例提供的产生悬铃木突变体的流程图。
 本发明的实施方式
为了使本申请的目的、技术方案及优点更加清楚明白，以下结合实施例，对本申请进行进一步详细说明。应当理解，此处所描述的实施例仅仅用以解释本申请，并不用于限定本申请。
在本申请的描述中，需要理解的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。在本申请的描述中，“多个”的含义是两个或两个以上，除非另有明确的限定。
为了说明本申请所述的技术方案，以下结合附图及实施例进行详细说明。
本申请实例提供一种获得悬铃木突变体的方法，所述方法如图2所示“载体构建-基因转化-转基因苗筛选-突变体基因序列分析”，包括以下步骤：
S01. 获取悬铃木目的基因并确定所述gRNA靶点序列；
S02. 合成含限制性内切酶粘性末端的gRNA靶点序列寡核苷酸片段和及其互补链，混合退火后产生两端含内切酶粘性末端的DNA退火产物；
S03. 提供CRISPR/Cas9载体质粒和限制性内切酶，采用所述限制性内切酶对所述CRISPR/Cas9载体质粒进行酶切处理得到酶切后的CRISPR/Cas9载体质粒；
S04. 提供连接酶，采用所述连接酶依次将所述酶切后的CRISPR/Cas9载体质粒、所述DNA退火产物进行连接处理得到CRISPR/Cas9基因组编辑载体；
S05. 采用基因枪法将所述CRISPR/Cas9基因组编辑载体转化至种子胚中得到转化产物，对所述转化产物进行培育，筛选悬铃木突变体。
本申请通过设计悬铃木目的基因的gRNA靶点序列，构建得到基因组编辑载体，利用基因枪法转化处理至种子胚中得到转化产物，对所述转化产物进行培育、鉴定得到所述悬铃木突变体，该方法可对悬铃木的特定基因进行定向突变，定向性强，效率高。
在上述步骤S01中，获取悬铃木目的基因并确定所述gRNA靶点序列。
所选悬铃木的品种为Platanus x acerifolia。在本申请一些实施例中，以悬铃木花粉过敏原plaa1作为所述悬铃木目的基因。在一些实施例，取悬铃木Platanus x acerifolia的叶片为样本，采用基因组提取试剂盒提取悬铃木基因组DNA，通过PCR扩增反应得到所述悬铃木花粉过敏原plaa1基因。
在一些实施例中，获取悬铃木花粉过敏原plaa1基因的步骤中，采用第五引物和第六引物进行PCR扩增反应得到所述悬铃木花粉过敏原plaa1基因，其中，所述第五引物的序列如SEQ ID No.3所示，为：5’-tacgcggggaacaaaacaatccaat-3’；所述第六引物的序列如SEQ ID No.4所示，为：5’- ccgaagagggccaaatatcataca-3’。
采用第五引物和第六引物进行PCR扩增反应得到所述悬铃木花粉过敏原plaa1基因的步骤中，所述PCR扩增反应体系如下：悬铃木叶片的DNA：5 uL，第五引物：5 uL，第六引物：5 uL，2×Taq mix：25 uL，Mg2+：1 uL甘油：1 uL，ddH2O：12 uL。所述PCR扩增反应条件如下：95℃预变性5 min；95℃变性30 s，56℃退火30 s，72℃延伸1 min，40个循环；72℃延伸10 min，4℃保存。
通过PCR扩增反应得到所述悬铃木花粉过敏原plaa1基因的PCR产物进行测序分析，得到所述悬铃木花粉过敏原plaa1基因编码区序列如SEQ ID No.5所示，为：
ATGAAGCTTTCCTTCTCTCTCTGTATCTTCTTCTTCAATCTCCTCCTCCTCCTTCAAGCTGTAATCAGCGCCGATATTGTTCAGGGCACATGCAAGAAAGTTGCTCAGAGAAGCCCAAACGTGAACTACGATTTCTGCGCGAAGTCTCTTGGAGCAGATCCTAAGAGCCACACTGCGGATCTTCAAGGACTTGGGGTCATCTCAGCGAATTTAGCCATACAGCAAGGATCTAAAATCCAAACATTTATTGGTCGCATCTTGAAAAGTAAAGTGGACCCAGCTCTTAAGAAATACTTGAATGATTGCGTGGGACTTTACGCTGATGCGAAGTCTTCAGTTCAAGAGGCCATAGCTGACTTCAATTCCAAGGACTACGCATCAGCTAATGTGAAAATGAGTGCGGCTTTGGACGACTCAGTGACTTGTGAAGATGGGTTTAAGGAGAAGAAAGGTTTAGTATCACCGGTGACGAAGGAGAACAAGGATTATGTACAACTGACTGCAATATCTCTTGCAATTACCAAACTGCTTGGTGCTTGA。
根据测序得到的悬铃木花粉过敏原plaa1基因编码区序列设计所述第一gRNA靶点序列及所述第二gRNA靶点序列。根据CRISPR/Cas9系统的要求，所述靶点序列的筛选要求为：（1）靶点序列主要包括15-25个碱基，（2）这15-25个碱基后面是NGG（N 为任意碱基）3 个碱基的PAM 区（Protospacer adjacent motif，PAM）。
在一些实施例中，所述gRNA靶点序列包括第一gRNA靶点序列和第二gRNA靶点序列。其中，所述第一gRNA靶点序列如SEQ ID No.1所示，为：5’-CAGCGCCGATATTGTTCAGG-3’；所述第二gRNA靶点序列如SEQ ID No.2所示，为：5’-TTCTGCGCGAAGTCTCTTGG-3’。确定所述第一gRNA靶点序列及所述第二gRNA靶点序列之后，可进行后续试验。在实际应用中，靶序列可选自基因编码区任意序列或基因表达的上游或下游调控序列，如启动子序列，增强子序列等序列。其中，所述靶点序列的数量选自1个，2个或多个，所述靶点序列选自单一使用或同时使用。
在上述步骤S02中，合成含限制性内切酶粘性末端的RNA靶点序列寡核苷酸片段和及其互补链，混合退火后产生两端含内切酶粘性末端的DNA退火产物。
获取第一gRNA靶点序列退火产物的方法中，包括如下步骤：确定所述第一gRNA靶点序列的第一引物和第二引物，通过第一退火处理得到所述第一gRNA靶点序列退火产物。
所述第一gRNA靶点序列的第一引物如SEQ ID No.6所示，为：5’- ATTGCAGCGCCGATATTGTTCAGG-3’；
所述第一gRNA靶点序列的第二引物如SEQ ID No.7所示，为：
3’-GTCGCGGCTATAACAAGTCC CAAA-5’。
其中所述第一gRNA靶点序列的第一引物和第二引物中下划线部分为限制性内切酶BsaI粘性末端序列。
通过第一退火处理得到所述第一gRNA靶点序列退火产物，所述第一退火处理的反应体系如下：浓度为100 mM的第一引物和第二引物各1 uL，8 uL 纯净水；所述第一退火处理的反应程序如下：PCR仪中95℃变性5分钟，缓慢降温至室温1小时。
获取第二gRNA靶点序列退火产物的方法中，包括如下步骤：确定所述第二gRNA靶点序列的第三引物和第四引物，通过第二退火处理得到所述第二gRNA靶点序列退火产物。
所述第二gRNA靶点序列的第三引物如SEQ ID No.8所示，为：5’- ATTGCAGCGCCGATATTGTTCAGG-3’；
所述第二gRNA靶点序列的第四引物如SEQ ID No.9所示，为：
3’-GTCGCGGCTATAACAAGTCC CAAA-5’。
其中所述第一gRNA靶点序列的第一引物和第二引物中下划线部分为限制性内切酶BsaI粘性末端序列。
通过第二退火处理得到所述第二gRNA靶点序列退火产物，所述第二退火处理的反应体系如下：浓度为100 mM的第一引物和第二引物各1 uL，8 uL 纯净水；所述第二退火处理的反应程序如下：PCR仪中95℃变性5分钟，缓慢降温至室温1小时。
在上述步骤S03中，提供CRISPR/Cas9载体质粒和限制性内切酶，采用所述限制性内切酶对所述CRISPR/Cas9载体质粒进行酶切处理得到酶切后的CRISPR/Cas9载体质粒。
在一些实施例中，所述CRISPR/Cas9载体质粒包括但不限于pKSE401，可以选自其他基因组编辑载体系统。在本申请具体实施例中，所述CRISPR/Cas9载体质粒选自pKSE401，pKSE401载体融合了绿色荧光蛋白（GFP），有利于后续进行筛选。
在一些实施例中，所述限制性内切酶包括但不限于BsaI酶，可以选自其他载体系统所携带的gRNA克隆位点的限制性内切酶。在本申请具体实施例中，所述限制性内切酶选自BsaI酶，采用BsaI酶进行酶切处理，确保gRNA与载体能够进行连接。
采用所述限制性内切酶对所述CRISPR/Cas9载体质粒进行酶切处理得到酶切后的CRISPR/Cas9载体质粒的步骤中，所述酶切处理的反应体系如下：CRISPR/Cas9载体质粒 1 ug，10 U BsaI酶，加BsaI酶缓冲液至20 uL。
所述酶切处理的反应条件如下：50~52℃酶切处理1~1.5小时，再于65~68℃加热10分钟。在酶切处理后进行加热，目的是为了使酶失活，结束酶切处理。在本申请一些实施例中，所述酶切处理的反应条件如下：50℃酶切处理1小时，再于65℃加热10分钟。
在上述步骤S04中，提供连接酶，采用所述连接酶依次将所述酶切后的CRISPR/Cas9载体质粒、所述DNA退火产物进行连接处理得到CRISPR/Cas9基因组编辑载体。
所述连接酶选自T 4连接酶，采用T 4连接酶进行连接处理，能够较好地将所述酶切后的CRISPR/Cas9载体质粒、所述DNA退火产物进行连接处理得到CRISPR/Cas9基因组编辑载体进行连接，构建得到CRISPR/Cas9基因组编辑载体。
在一些实施例中，采用所述连接酶依次将所述酶切后的CRISPR/Cas9载体质粒、所述gRNA靶点序列退火产物进行连接处理得到CRISPR/Cas9基因组编辑载体的步骤中，所述连接处理的反应体系如下：在 20 μL反应体系中加入10 μL 2X反应缓冲液，1 μL T 4 DNA连接酶，50 ng酶切后的载体和3倍摩尔量的gRNA靶点退火产物。
所述连接处理的反应程序如下：37~38℃反应10~15min。在本申请一些实施例中，所述连接处理的反应程序如下：37℃反应10 min。
将制备得到的CRISPR/Cas9基因组编辑载体采用热激法转化至大肠杆菌感受态细胞中，并进行阳性克隆子的筛选和测序，确保得到准确的CRISPR/Cas9基因组编辑载体。
在上述步骤S05中，采用基因枪法将所述CRISPR/Cas9基因组编辑载体转化至种子胚中得到转化产物，对所述转化产物进行培育，筛选悬铃木突变体。
采用基因枪法将所CRISPR/Cas9基因组编辑载体转化至种子胚中得到转化产物的方法中，包括如下步骤：
S051. 获取植物成熟期果实种子，培育出胚芽；
S052. 提供待转化载体，所述待转化载体包括植物基因组编辑载体以及携带所述植物基因组编辑载体的金属微粒；其中，所述金属微粒的直径为0.6 μm~1.8 μm；
S053. 采用基因枪，在压力为450~2200 PSI的条件下将所述待转化载体转化至所述胚芽中，得到转化产物。
在上述步骤S051中，获取植物成熟期果实种子，培育出胚芽的方法，包括如下步骤：
S0511. 获取植物成熟期果实种子，
S0512. 将所述种子进行灭菌处理后，暗培养得到所述种子的胚芽。
在上述步骤S0511中，获取植物成熟期果实种子，选择成熟期的种子作为转化对象，成熟期的种子含有丰富的有机质，具有一定的发芽能力和较强的环境抵抗能力，作为转化对象，能够有利于将重组质粒有效地转化至种子中，有利提高基因转化效率。
在上述步骤S0512中，将所述种子进行灭菌处理后，暗培养得到所述种子的胚芽的方法，包括如下步骤：
S05121. 将所述种子进行去毛处理后，用体积浓度为75%的酒精进行第一次灭菌处理后，再用质量浓度为0.1%的升汞溶液进行第二次灭菌处理，得到灭菌后的种子；
S05122.将所述灭菌后的种子放置于湿润的滤纸上，于25℃的条件下进行暗培养2~3天，得到种子的胚芽。
在上述步骤S05121中，将所述种子进行去毛处理，去毛处理主要是去除种子表面的种毛，确保种子表面干净，有利于种子萌发和进行基因转化。采用洗衣粉水对所述种子进行去毛处理，确保所述种子清洗干净。
用体积浓度为75%的酒精进行第一次灭菌处理后，再用质量浓度为0.1%的升汞溶液进行第二次灭菌处理得到灭菌后的种子；对种子进行灭菌处理，确保种子萌发和进行基因转化试验的胚芽无菌干净，能够较好完成基因转化试验。用体积浓度为75%的酒精进行第一次灭菌处理的步骤中，所述第一次灭菌处理的时间为40~60秒。再用质量积浓度为0.1%的升汞溶液进行第二次灭菌处理的步骤中，所述第二次灭菌处理的时间为40~50分钟。
对经过第二次灭菌处理后的种子还包括无菌水漂洗，采用无菌水进行漂洗得到灭菌后的种子，除去种子表面的升汞溶液，得到干净的灭菌后的种子。
在上述步骤S052中，提供待转化载体，所述待转化载体包括植物基因组编辑载体以及携带所述植物基因组编辑载体的金属微粒；其中，所述金属微粒的直径为0.6 μm~1.8 μm。
提供转化载体进行转化，所述待转化载体包括植物基因组编辑载体以及携带所述植物基因组编辑载体的金属微粒。
在一些实施例中，携带所述植物基因组编辑载体的金属微粒的方法中，所述携带的方法包括浸泡等常规的包覆方法。所述携带所述植物基因组编辑载体的金属微粒为植物基因转化的介质，通过所述介质，将所述植物基因组编辑载体通过基因枪法转化至种子胚中。
其中，所述金属微粒的直径为0.6 μm~1.8 μm。选择上述直径的金属微粒作为介质进行基因转化试验，能够保证转化效率。若金属微粒的直径过大，会导致植物种子的伤害较大，影响转化效率，影响种子的成活率；若金属微粒的直径过小，则会导致金属微粒所携带的重组质粒过少，造成转化效率过低。在本申请优选实施例中，所述金属微粒的直径为0.6 μm。
在一些实施例中，所述金属微粒选自金粉或钨粉。选择性质较稳定的金属微粒进行试验，降低对植物种子的伤害，提高植物种子的存活率。
其中，提供待转化载体的步骤中，按照所述金属微粒与所述植物基因组编辑载体的质量比为15 mg: 2 µg进行包被，制备得到所述待转化质粒。若金属微粒的添加量过多，则会对植物种子造成伤害，影响转化效率，影响种子的成活率；若金属微粒的添加量过少，则会导致金属微粒所携带的重组质粒过少，造成转化效率过低。
在上述步骤S053中，采用基因枪，在压力为450~2200 PSI的条件下将所述待转化载体转化至所述胚芽中，得到转化产物。
其中，采用基因枪，在压力为450~2200 PSI的条件下将所述待转化载体转化至所述胚芽中的步骤中，所述待转化质粒用于转化100~500个胚芽的比例进行转化。在上述压力条件下，可保证金属微粒包裹体较高效率地转化至种子中。在本发明优选实施例中，所述转化压力为900 PSI。
其中，所述基因枪选自BIO-RAD 公司的 PDS-1000/He 基因枪。
在上述步骤S05中，对所述转化产物进行培育，对所述转化产物在25℃暗培养一天后光照培养三天，待长出绿芽后移栽到温室中培养成幼苗，对幼苗进行移栽培育并筛选得到所述悬铃木突变体。
筛选得到所述悬铃木突变体的步骤包括如下方法，以所述悬铃木突变体的植物基因组DNA为模板，采用第七引物和第八引物进行PCR扩增反应以验证所述悬铃木突变体。所述第七引物的序列如SEQ ID No.10所示，为：
5’-TGTAAAACGACGGCCAGTATGAAGCTTTCCTTCTCTCT-3’；
所述第八引物的序列如SEQ ID No.11所示，为：
5’-CAGGAAACAGCTATGACCTCAAGCACCAAGCAGTTTGG-3’。
采用所述第七引物和第八引物进行扩增PCR克隆，其中，所述PCR扩增反应体系如下：悬铃木叶片的DNA：5 uL，第七引物：5 uL，第八引物：5 uL，2×Taq mix：25 uL，Mg2+：1 uL甘油：1 uL，ddH2O：12 uL；且，所述PCR扩增反应条件如下：95℃预变性5 min；95℃变性30 s，56℃退火30 s，72℃延伸1 min，40个循环；72℃延伸10 min，4℃保存。
再挑选单克隆进行测序，每个基因转化植株挑选10个单克隆进行测序，测序结果通过NCBI网站BLAST程序和野生型植株基因序列（SEQ ID No.5）比对，鉴定突变基因。
相应的，本申请实施例还提供了一种悬铃木突变体，所述悬铃木突变体为目的基因序列改变的突变体，所述目的基因序列改变的突变体包括基因片段插入、缺失或碱基改变的突变体。所述突变体靶向性地、快速地获得了特定的目的基因的突变，有利于进行后续的应用。
相应的，本申请实施例还提供了一种所述的获得悬铃木突变体的方法或所述的悬铃木突变体在悬铃木育种中的应用。
本申请采用gRNA引导的Cas9酶在基因靶点对悬铃木基因组DNA进行切割，经DNA修复后而产生基因序列的突变。这些突变可引起基因编码蛋白的氨基酸变化，氨基酸替代，氨基酸增加或减少，从而影响悬铃木表现型的变化。本申请所述的获得悬铃木突变体的方法可对悬铃木基因组中所有的基因进行靶向突变，方法简捷，效率高，可快速获得特定基因的突变体，制备得到的悬铃木突变体在悬铃木育种中的应用广泛。
为了使本申请要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合实施例，对本申请进行进一步详细说明。应当理解，此处所描述的实施例仅仅用以解释本申请，并不用于限定本申请。
为了说明本申请所述的技术方案，下面通过实施例来进行说明。
实施例1
悬铃木花粉过敏原基因Plaa1的克隆及测序
（1）选择悬铃木Platanus x acerifolia的叶片为样本，采用基因组提取试剂盒提取悬铃木基因组DNA，第五引物的序列如SEQ ID No.3所示，为：5’-tacgcggggaacaaaacaatccaat-3’；第六引物的序列如SEQ ID No.4所示，为：5’- ccgaagagggccaaatatcataca-3’，通过PCR扩增反应得到所述悬铃木花粉过敏原plaa1基因。
所述PCR扩增反应体系如下：悬铃木叶片的DNA：5 uL，第五引物：5 uL，第六引物：5 uL，2×Taq mix：25 uL，Mg2+：1 uL甘油：1 uL，ddH2O：12 uL。
所述PCR扩增反应条件如下：95℃预变性5 min；95℃变性30 s，56℃退火30 s，72℃延伸1 min，40个循环；72℃延伸10 min，4℃保存。
（2）通过PCR扩增反应得到所述悬铃木花粉过敏原plaa1基因的PCR产物进行测序分析。
实施例2
基因组编辑载体的构建
（1）根据所述悬铃木基因序列（SEQ ID No.5）设计两条sgRNA序列，分别为第一gRNA靶点序列（靶点T1）：5’-CAGCGCCGATATTGTTCAGG-3’和第二gRNA靶点序列（靶点T2）：5’-TTCTGCGCGAAGTCTCTTGG-3’。
分别合成带接头的该两条sgRNA序列的正义链和反义链，混合退火后形成如下两条双链DNA：
设计第一gRNA靶点序列的第一引物SEQ ID No.6：（5’- ATTGCAGCGCCGATATTGTTCAGG-3’）及第二引物SEQ ID No.7（ 3’-GTCGCGGCTATAACAAGTCC CAAA-5’），进行混合退火处理，形成第一gRNA靶点序列；
设计第二gRNA靶点序列的第三引物SEQ ID No.8：（5’- ATTGTTCTGCGCGAAGTCTCTTGG-3’）及第四引物SEQ ID No.9（ 3’- AAGACGCGCTTCAGAGAACC CAAA-5’），进行混合退火处理，形成第二gRNA靶点序列。
其中下划线序列为接头序列，在退火后形成带有BsaI限制性内切酶粘性末端的双链DNA，可克隆至由BsaI酶切割后的下述载体。
（2）提供CRISPR/Cas9载体质粒pKSE401和限制性内切酶BsaI酶，采用所述BsaI酶对所述CRISPR/Cas9载体质粒pKSE401进行酶切处理得到CRISPR/Cas9载体质粒；其中，酶切处理的的反应体系如下：CRISPR/Cas9载体质粒 1 ug，10 U BsaI酶，加BsaI酶缓冲液至20 uL；且，所述酶切处理的反应条件如下：50℃酶切处理1小时，再于65℃加热10分钟。
（3）提供T4连接酶，采用所述T4连接酶将所述CRISPR/Cas9载体质粒、所述第一gRNA靶点序列退火产物或所述第二gRNA靶点序列退火产物进行连接处理得到CRISPR/Cas9基因组编辑载体；其中，所述连接处理的反应体系如下：载体质粒 50 ng酶切后的载体； gRNA靶点序列退火产物 0.1 ug； T4 连接酶（5 U）1 μL；T4 DNA连接酶缓冲液 2 μL；且，所述连接处理的反应程序如下：37℃反应10min。
（4）将制备得到的CRISPR/Cas9基因组载体采用热激法转化至大肠杆菌感受态细胞中，并进行阳性克隆子的筛选和测序，确保得到准确的CRISPR/Cas9基因组编辑载体。
实施例3
基因转化
（1）获取生长健壮的成年悬铃木的成熟球果的种子；将种子进行清洗去除种子表面的绒毛，再用依次用75%酒精消毒 40 s、0.1%升汞消毒 40 min、无菌水漂洗 3 次得到灭菌后的种子；将所述灭菌后的种子放置于湿润的滤纸上，于25℃的条件下进行暗培养2~3天，得到萌发胚芽的种子；
（2）获取种子的胚芽，待转化质粒为包覆基因组编辑载体质粒的直径为0.6 μm金粉微粒，设置转化参数为900 PSI 压力，转化载体距离与样品间距离为6厘米，采用BIO-RAD 公司的 PDS-1000/He 基因枪将所述基因组编辑载体转化至所述胚芽中得到转化产物。
（3）对所述转化产物进行培育，对所述转化产物在25℃暗培养一天后光照培养三天，待长出绿芽后移栽到温室中培养成幼苗，对幼苗进行移栽培育，在进行温室培养两个月再进行鉴定。
实施例4
突变体的鉴定
以所述悬铃木突变体的植物基因组DNA为模板，采用第七引物和第八引物进行PCR扩增反应以验证所述悬铃木突变体。其中，所述第七引物的序列如SEQ ID No.10所示，为：
5’-TGTAAAACGACGGCCAGTATGAAGCTTTCCTTCTCTCT-3’；
所述第八引物的序列如SEQ ID No.11所示，为：
5’-CAGGAAACAGCTATGACCTCAAGCACCAAGCAGTTTGG-3’。
将扩增PCR片段进行克隆，所述PCR扩增反应体系如下：悬铃木叶片的DNA：5 uL，第七引物：5 uL，第八引物：5 uL，2×Taq mix：25 uL，Mg2+：1 uL甘油：1 uL，ddH2O：12 uL；且，所述PCR扩增反应条件如下：95℃预变性5 min；95℃变性30 s，56℃退火30 s，72℃延伸1 min，40个循环；72℃延伸10 min，4℃保存。
再挑选单克隆进行测序，每个基因转化植株挑选10个单克隆进行测序，测序结果通过NCBI网站BLAST程序和野生型植株基因序列（SEQ ID No.5）比对，鉴定突变基因。
结果分析：
结果分析一：
针对实施例1 PCR扩增反应得到所述悬铃木花粉过敏原plaa1基因的PCR产物进行测序分析，得到所述悬铃木花粉过敏原plaa1基因编码区序列如SEQ ID No.5所示，为：
ATGAAGCTTTCCTTCTCTCTCTGTATCTTCTTCTTCAATCTCCTCCTCCTCCTTCAAGCTGTAATCAGCGCCGATATTGTTCAGGGCACATGCAAGAAAGTTGCTCAGAGAAGCCCAAACGTGAACTACGATTTCTGCGCGAAGTCTCTTGGAGCAGATCCTAAGAGCCACACTGCGGATCTTCAAGGACTTGGGGTCATCTCAGCGAATTTAGCCATACAGCAAGGATCTAAAATCCAAACATTTATTGGTCGCATCTTGAAAAGTAAAGTGGACCCAGCTCTTAAGAAATACTTGAATGATTGCGTGGGACTTTACGCTGATGCGAAGTCTTCAGTTCAAGAGGCCATAGCTGACTTCAATTCCAAGGACTACGCATCAGCTAATGTGAAAATGAGTGCGGCTTTGGACGACTCAGTGACTTGTGAAGATGGGTTTAAGGAGAAGAAAGGTTTAGTATCACCGGTGACGAAGGAGAACAAGGATTATGTACAACTGACTGCAATATCTCTTGCAATTACCAAACTGCTTGGTGCTTGA。
结果分析二：
针对实施例2基因组编辑载体的构建，构建得到的基因组编辑载体如图1所示，其中，LB和RB分别为Ti质粒上T-DNA的左右边界，35Sp-Cas9-NosT和35Sp-NptII-PolyA分别为Cas9基因和NptII基因的表达框，U6-26-T1和U6-29-T2分别表达两个sgRNA，F和R分别为转基因植物检测PCR上游和下游引物。
结果分析三：
针对实施例3基因转化试验进行分析，对幼苗进行移栽培育的结果进行分析，分析得到，移栽后，有52株幼苗成活，成活率为86.7%。
温室培养两个月后，通过 PCR进行验证，有28株为阳性，阳性率为53.8%。基因转化效率为46.7%。
结果分析四：
针对实施例4突变体的鉴定进行分析，本实验共计得到28株悬铃木转基因苗，通过对靶基因分析，发现2株（18号和48号）含有突变。
对18号植株10个样本进行测序，发现5个样本含有1个或多个突变。其中，18-1第225个碱基由A变为G（没有引起编码氨基酸序列变化）；故没有列举18-1的碱基序列和蛋白质序列。
18-3第14-15碱基TC缺失，引起移码突变，蛋白质提前终止（核酸序列5，蛋白质序列2），突变体18-3的碱基序列如SEQ ID No.12所示，为：
ATGAAGCTTTCCTTCTCTCTGTATCTTCTTCTTCAATCTCCTCCTCCTCCTTCAAGCTGTAATCAGCGCCGATATTGTTCAGGGCACATTCAAGAAAGTTGCTCAGAGAAGCCCAAACGTGAACTACGATTTCTGCGCGAAGTCTCTTGGAGCAGATCCTAAGAGCCACACTGCGGATCTTCAAGGACTTGGGGTCATCTCAGCGAATTTAGCCATACAGCAAGGATCTAAAATCCAAACATTTATTGGTCGCATCTTGAAAAGTAAAGTGGACCCAACTCTTAAGAAATACTTGAATGATTGCGTGGGACTTTACGCTGATGCGAAGTCTTCAGTTCAAGAGGCCATAGCTGACTTCAATTCCAAGGACTACGCATCAGCTAATGTGAAAATGAGTGCGGCTTTGGACGACTCAGTGACTTGTGAAGATGGGTTTAAGGAGAAGAAAGGTTTAGTATCACCGGTGACGAAGGAGAACAAGGATTATGTACAACTGACTGCAATATCTCTTGCAATTACCAAACTGCTTGGTGCTTGA。
突变体18-3的蛋白质序列如SEQ ID No.13所示，为：
MKLSFSLYLLLQSPPPPSSCNQRRYCSGHIQESCSEKPKRELRFLREVSWSRS。
18-6第401碱基由C变为T（引起编码氨基酸由丙氨酸A变为缬氨酸V）；
突变体18-6的碱基序列如SEQ ID No.14所示，为：
ATGAAGCTTTCCTTCTCTCTCTGTATCTTCTTCTTCAATCTCCTCCTCCTCCTTCAAGCTGTAATCAGCGCCGATATTGTTCAGGGCACATGCAAGAAAGTTGCTCAGAGAAGCCCAAACGTGAACTACGATTTCTGCGCGAAGTCTCTTGGAGCAGATCCTAAGAGCCACACTGCGGATCTTCAAGGACTTGGGGTCATCTCAGCGAATTTAGCCATACAGCAAGGATCTAAAATCCAAACATTTATTGGTCGCATCTTGAAAAGTAAAGTGGACCCAGCTCTTAAGAAATACTTGAATGATTGCGTGGGACTTTACGCTGATGCGAAGTCTTCAGTTCAAGAGGCCATAGCTGACTTCAATTCCAAGGACTACGCATCAGCTAATGTGAAAATGAGTGTGGCTTTGGACGACTCAGTGACTTGTGAAGATGGGTTTAAGGAGAAGAAAGGTTTAGTATCACCGGTGACGAAGGAGAACAAGGATTATGTACAACTGACTGCAATATCTCTTGCAATTACCAAACTGCTTGGTGCTTGA。
突变体18-6的蛋白质序列如SEQ ID No.15所示，为：
MKLSFSLCIFFFNLLLLLQAVISADIVQGTCKKVAQRSPNVNYDFCAKSLGADPKSHTADLQGLGVISANLAIQQGSKIQTFIGRILKSKVDPALKKYLNDCVGLYADAKSSVQEAIADFNSKDYASANVKMSVALDDSVTCEDGFKEKKGLVSPVTKENKDYVQLTAISLAITKLLGA*。
18-7第402碱基由G变为T（没有引起氨基酸的变化）；故没有列举18-7的碱基序列和蛋白质序列。
18-8第310碱基由G变为A（引起氨基酸由甘氨酸G变为精氨酸R），突变体18-8的碱基序列如SEQ ID No.16所示，为：
ATGAAGCTTTCCTTCTCTCTCTGTATCTTCTTCTTCAATCTCCTCCTCCTCCTTCAAGCTGTAATCAGCGCCGATATTGTTCAGGGCACATGCAAGAAAGTTGCTCAGAGAAGCCCAAACGTGAACTACGATTTCTGCGCGAAGTCTCTTGGAGCAGATCCTAAGAGCCACACTGCGGATCTTCAAGGACTTGGGGTCATCTCAGCGAATTTAGCCATACAGCAAGGATCTAAAATCCAAACATTTATTGGTCGCATCTTGAAAAGTAAAGTGGACCCAGCTCTTAAGAAATACTTGAATGATTGCGTGAGACTTTACGCTGATGCGAAGTCTTCAGTTCAAGAGGCCATAGCTGACTTCAATTCCAAGGACTACGCATCAGCTAATGTGAAAATGAGTGCGGCTTTGGACGACTCAGTGACTTGTGAAGATGGGTTTAAGGAGAAGAAAGGTTTAGTATCACCGGTGACGAAGGAGAACAAGGATTATGTACAACTGACTGCAATATCTCTTGCAATTACCAAACTGCTTGGTGCTTGA。
突变体18-8的蛋白质序列如SEQ ID No.17所示，为：
MKLSFSLCIFFFNLLLLLQAVISADIVQGTCKKVAQRSPNVNYDFCAKSLGADPKSHTADLQGLGVISANLAIQQGSKIQTFIGRILKSKVDPALKKYLNDCVRLYADAKSSVQEAIADFNSKDYASANVKMSAALDDSVTCEDGFKEKKGLVSPVTKENKDYVQLTAISLAITKLLGA。
对48号植株10个样本进行测序，发现4个含有基因突变。48-2第160碱基由C变为T（引起氨基酸由脯氨酸P变为丝氨酸S）；
突变基因48-2的碱基序列如SEQ ID No.18所示，为：
ATGAAGCTTTCCTTCTCTCTCTGTATCTTCTTCTTCAATCTCCTCCTCCTCCTTCAAGCTGTAATCAGCGCCGATATTGTTCAGGGCACATGCAAGAAAGTTGCTCAGAGAAGCCCAAACGTGAACTACGATTTCTGCGCGAAGTCTCTTGGAGCAGATTCTAAGAGCCACACTGCGGATCTTCAAGGACTTGGGGTCATCTCAGCGAATTTAGCCATACAGCAAGGATCTAAAATCCAAACATTTATTGGTCGCATCTTGAAAAGTAAAGTGGACCCAGCTCTTAAGAAATACTTGAATGATTGCGTGGGACTTTACGCTGATGCGAAGTCTTCAGTTCAAGAGGCCATAGCTGACTTCAATTCCAAGGACTACGCATCAGCTAATGTGAAAATGAGTGCGGCTTTGGACGACTCAGTGACTTGTGAAGATGGGTTTAAGGAGAAGAAAGGTTTAGTATCACCGGTGACGAAGGAGAACAAGGATTATGTACAACTGACTGCAATATCTCTTGCAATTACCAAACTGCTTGGTGCTTGA。
突变基因48-2的蛋白质序列如SEQ ID No.19所示，为：
MKLSFSLCIFFFNLLLLLQAVISADIVQGTCKKVAQRSPNVNYDFCAKSLGADSKSHTADLQGLGVISANLAIQQGSKIQTFIGRILKSKVDPALKKYLNDCVGLYADAKSSVQEAIADFNSKDYASANVKMSAALDDSVTCEDGFKEKKGLVSPVTKENKDYVQLTAISLAITKLLGA*。
48-3第140碱基由C变为T（引起氨基酸由丙氨酸A变为缬氨酸V），
突变基因48-3的碱基序列如SEQ ID No.20所示，为：
ATGAAGCTTTCCTTCTCTCTCTGTATCTTCTTCTTCAATCTCCTCCTCCTCCTTCAAGCTGTAATCAGCGCCGATATTGTTCAGGGCACATGCAAGAAAGTTGCTCAGAGAAGCCCAAACGTGAACTACGATTTCTGCGTGAAATCTCTTGGAGCAGATCCTAAGAGCCACACTGCGGATCTTCAAGGACTTGGGGTCATCTCAGCGAATTTAGCCATACAGCAAGGATCTAAAATCCAAACATTTATTGGTCGCATCTTGAAAAGTAAAGTGGACCCAGCTCTTAAGAAATACTTGAATGATTGCGTGGGACTTTACGCTGATGCAAAGTCTTCAGTTCAAGAGGCCATAGCTGACTTCAATTCCAAGGACTACGCATCAGCTAATGTGAAAATGAGTGCGGCTTTGGACGACTCAGTGACTTGTGAAGATGGGTTTAAGGAGAAGAAAGGTTTAGTATCACCGGTGACGAAGGAGAACAAGGATTATGTACAACTGACTGCAATATCTCTTGCAATTACCAAACTGCTTGGTGCTTGA
突变基因48-3的蛋白质序列如SEQ ID No.21所示，为：
MKLSFSLCIFFFNLLLLLQAVISADIVQGTCKKVAQRSPNVNYDFCVKSLGADPKSHTADLQGLGVISANLAIQQGSKIQTFIGRILKSKVDPALKKYLNDCVGLYADAKSSVQEAIADFNSKDYASANVKMSAALDDSVTCEDGFKEKKGLVSPVTKENKDYVQLTAISLAITKLLGA*。
48-6第144碱基由G变为A（没有引起氨基酸变化），第327碱基由G变为A（没有引起氨基酸变化）；故没有列举其碱基序列和蛋白质序列。
48-7第154碱基由G变为A（引起氨基酸由丙氨酸A变为苏氨酸T）；突变基因48-7的碱基序列如SEQ ID No.22所示，为：
ATGAAGCTTTCCTTCTCTCTCTGTATCTTCTTCTTCAATCTCCTCCTCCTCCTTCAAGCTGTAATCAGCGCCGATATTGTTCAGGGCACATGCAAGAAAGTTGCTCAGAGAAGCCCAAACGTGAACTACGATTTCTGCGCGAAGTCTCTTGGAACAGATCCTAAGAGCCACACTGCGGATCTTCAAGGACTTGGGGTCATCTCAGCGAATTTAGCCATACAGCAAGGATCTAAAATCCAAACATTTATTGGTCGCATCTTGAAAAGTAAAGTGGACCCAGCTCTTAAGAAATACTTGAATGATTGCGTGGGACTTTACGCTGATGCGAAGTCTTCAGTTCAAGAGGCCATAGCTGACTTCAATTCCAAGGACTACGCATCAGCTAATGTGAAAATGAGTGCGGCTTTGGACGACTCAGTGACTTGTGAAGATGGGTTTAAGGAGAAGAAAGGTTTAGTATCACCGGTGACGAAGGAGAACAAGGATTATGTACAACTGACTGCAATATCTCTTGCAATTACCAAACTGCTTGGTGCTTGA.
突变基因48-7的蛋白质序列如SEQ ID No.23所示，为：
MKLSFSLCIFFFNLLLLLQAVISADIVQGTCKKVAQRSPNVNYDFCAKSLGTDPKSHTADLQGLGVISANLAIQQGSKIQTFIGRILKSKVDPALKKYLNDCVGLYADAKSSVQEAIADFNSKDYASANVKMSAALDDSVTCEDGFKEKKGLVSPVTKENKDYVQLTAISLAITKLLGA*。
48-8第137碱基由G变为A（引起氨基酸由半胱氨酸C变为酪氨酸Y），第140碱基由C变为T（引起氨基酸由丙氨酸A变为缬氨酸V），第144碱基由G变为A（没有引起氨基酸变化），第225碱基由A变为T（引起氨基酸由谷氨酰胺Q变为组氨酸H），第259碱基由T变为G（引起氨基酸由亮氨酸L变为缬氨酸V），第306碱基由C变为T（没有引起氨基酸变化），第363碱基由T变为G（引起氨基酸由天冬酰胺N变为赖氨酸K），第454碱基由T变为A（引起氨基酸由亮氨酸L变为异亮氨酸I），
突变基因48-8的碱基序列如SEQ ID No.24所示，为：
ATGAAGCTTTCCTTCTCTCTCTGTATCTTCTTCTTCAATCTCCTCCTCCTCCTTCAAGCTGTAATCAGCGCCGATATTGTTCAGGGCACATGCAAGAAAGTTGCTCAGAGAAGCCCAAACGTGAACTACGATTTCTACGTGAAATCTCTTGGAGCAGATCCTAAGAGCCACACTGCGGATCTTCAAGGACTTGGGGTCATCTCAGCGAATTTAGCCATACAGCATGGATCTAAAATCCAAACATTTATTGGTCGCATCGTGAAAAGTAAAGTGGACCCAGCTCTTAAGAAATACTTGAATGATTGTGTGGGACTTTACGCTGATGCGAAGTCTTCAGTTCAAGAGGCCATAGCTGACTTCAAGTCCAAGGACTACGCATCAGCTAATGTGAAAATGAGTGCGGCTTTGGACGACTCAGTGACTTGTGAAGATGGGTTTAAGGAGAAGAAAGGTATAGTATCACCGGTGACGAAGGAGAACAAGGATTATGTACAACTGACTGCAATATCTCTTGCAATTACCAAACTGCTTGGTGCTTGA。
突变基因48-8的蛋白质序列如SEQ ID No.25所示，为：
MKLSFSLCIFFFNLLLLLQAVISADIVQGTCKKVAQRSPNVNYDFYVKSLGADPKSHTADLQGLGVISANLAIQHGSKIQTFIGRIVKSKVDPALKKYLNDCVGLYADAKSSVQEAIADFKSKDYASANVKMSAALDDSVTCEDGFKEKKGIVSPVTKENKDYVQLTAISLAITKLLGA*。
本申请为一种通过CRISPR/CAS9基因组编辑的悬铃木基因突变技术，可以应用于悬铃木的所有基因的突变，如影响健康的过敏蛋白基因；影响开花及结球的开花基因，花发育基因，果实及种子发育基因，种皮毛发育基因；影响树木快速生长的基因；影响树木抗性的抗干旱、抗涝、抗盐碱、抗高低温、抗病、抗虫、抗病毒基因等。
以上仅为本申请的可选实施例而已，并不用于限制本申请。对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的权利要求范围之内。

Claims (15)

  1. 一种获得悬铃木突变体的方法，其特征在于，包括如下步骤：
    获取悬铃木目的基因并确定所述gRNA靶点序列；
    合成含限制性内切酶粘性末端的gRNA靶点序列寡核苷酸片段和及其互补链，混合退火后产生两端含内切酶粘性末端的DNA退火产物；
    提供CRISPR/Cas9载体质粒和限制性内切酶，采用所述限制性内切酶对所述CRISPR/Cas9载体质粒进行酶切处理得到酶切后的CRISPR/Cas9载体质粒；
    提供连接酶，采用所述连接酶依次将所述酶切后的CRISPR/Cas9载体质粒、所述DNA退火产物进行连接处理得到CRISPR/Cas9基因组编辑载体；
    采用基因枪法将所述CRISPR/Cas9基因组编辑载体转化至种子胚中得到转化产物，对所述转化产物进行培育，筛选悬铃木突变体。
  2. 根据权利要求1所述的获得悬铃木突变体的方法，其特征在于，所述gRNA靶点序列包括第一gRNA靶点序列和第二gRNA靶点序列，其中，
    所述第一gRNA靶点序列如SEQ ID No.1所示，为：
    5’-CAGCGCCGATATTGTTCAGG-3’；
    所述第二gRNA靶点序列如SEQ ID No.2所示，为：
    5’-TTCTGCGCGAAGTCTCTTGG-3’。
  3. 根据权利要求1所述的获得悬铃木突变体的方法，其特征在于，采用基因枪法将所CRISPR/Cas9基因组编辑载体转化至种子胚中得到转化产物的方法中，包括如下步骤：
    获取植物成熟期果实种子，培育出胚芽；
    提供待转化载体，所述待转化载体包括所述植物基因组编辑载体以及携带所述植物基因组编辑载体的金属微粒；其中，所述金属微粒的直径为0.6 μm~1.8 μm；
    采用基因枪，在压力为450~2200 PSI的条件下将所述待转化载体转化至所述胚芽中，得到转化产物。
  4. 根据权利要求3所述的获得悬铃木突变体的方法，其特征在于，，提供待转化载体的步骤中，按照所述金属微粒与所述植物基因组编辑载体的质量比为15 mg: 2 µg进行包被，制备得到所述待转化载体。
  5. 根据权利要求3所述的获得悬铃木突变体的方法，其特征在于，采用基因枪，在压力为450~2200 PSI的条件下将所述待转化质粒转化至所述胚芽中的步骤中，所述待转化载体用于转化100~500个胚芽的比例进行转化。
  6. 根据权利要求3所述的获得悬铃木突变体的方法，其特征在于，获取植物成熟期果实种子，培育出胚芽的方法，包括如下步骤：获取植物成熟期果实种子，将所述种子进行灭菌处理后，暗培养得到所述种子的胚芽。
  7. 根据权利要求6所述的获得悬铃木突变体的方法，其特征在于，对将所述种子进行灭菌处理后，暗培养得到所述种子的胚芽的方法，包括如下步骤：
    将所述种子进行去毛处理，用体积浓度为75%的酒精进行第一次灭菌处理后，再用质量浓度为0.1%的升汞溶液进行第二次灭菌处理，得到灭菌后的种子；
    将所述灭菌后的种子放置于湿润的滤纸上，于23~25℃的条件下进行暗培养2~3天，得到种子的胚芽。
  8. 根据权利要求3所述的获得悬铃木突变体的方法，其特征在于，所述金属微粒选自金粉或钨粉。
  9. 根据权利要求1~3任一所述的获得悬铃木突变体的方法，其特征在于，所述CRISPR/Cas9载体质粒包括pKSE401；和/或，
    所述限制性内切酶包括BsaI酶。
  10. 根据权利要求1~3任一所述的获得悬铃木突变体的方法，其特征在于，获取悬铃木目的基因的步骤中，所述悬铃木目的基因为悬铃木花粉过敏原plaa1基因，采用第五引物和第六引物进行PCR扩增反应得到所述悬铃木花粉过敏原plaa1基因，其中，所述第五引物的序列如SEQ ID No.3所示，为：5’-tacgcggggaacaaaacaatccaat-3’；所述第六引物的序列如SEQ ID No.4所示，为：5’- ccgaagagggccaaatatcataca-3’。
  11. 根据权利要求10所述的获得悬铃木突变体的方法，其特征在于，采用第五引物和第六引物进行PCR扩增反应得到所述悬铃木花粉过敏原plaa1基因的步骤中，所述PCR扩增反应体系如下：悬铃木叶片的DNA：5 uL，上游引物：5 uL，下游引物：5 uL，2×Taq mix：25 uL，Mg2+：1 uL甘油：1 uL，ddH2O：12 uL；且，所述PCR扩增反应条件如下：95℃预变性5 min；95℃变性30 s，56℃退火30 s，72℃延伸1 min，40个循环；72℃延伸10 min，4℃保存。
  12. 根据权利要求1~4任一所述的获得悬铃木突变体的方法，其特征在于，采用所述限制性内切酶对所述CRISPR/Cas9载体质粒进行酶切处理得到酶切后的CRISPR/Cas9载体质粒的步骤中，所述酶切处理的反应体系如下：CRISPR/Cas9载体质粒 1 ug，10 U BsaI酶，加BsaI酶缓冲液至20 uL；且，所述酶切处理的反应条件如下：50~52℃酶切处理1~1.5小时，再于65~68℃加热10分钟。
  13. 根据权利要求1~4任一所述的获得悬铃木突变体的方法，其特征在于，采用所述连接酶依次将所述酶切后的CRISPR/Cas9载体质粒、所述gRNA靶点序列退火产物进行连接处理得到CRISPR/Cas9基因组编辑载体的步骤中，所述连接处理的反应体系如下：在 20 μL反应体系中加入10 μL 2X反应缓冲液，1 μL T 4 DNA 连接酶，50 ng酶切后的载体和3倍摩尔量的gRNA靶点退火产物；且所述连接处理的反应条件如下：37~38℃反应10~15min。
  14. 一种悬铃木突变体，其特征在于，所述悬铃木突变体为目的基因序列改变的突变体，其中，所述目的基因产生突变的突变体包括基因编码区基因片段的插入，缺失或核苷酸序列的改变的突变体。
  15. 权利要求1~13任一所述的获得悬铃木突变体的方法或权利要求14所述的悬铃木突变体在悬铃木育种中的应用。
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