CN111424050A

CN111424050A - Mitsubishi tree mutant, method for obtaining same and application thereof

Info

Publication number: CN111424050A
Application number: CN202010144373.1A
Authority: CN
Inventors: 夏立新; 于为常; 骆超; 周秋艳
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2020-03-04
Filing date: 2020-03-04
Publication date: 2020-07-17
Anticipated expiration: 2040-03-04
Also published as: CN111424050B

Abstract

The invention provides a plane mutant, a method for obtaining the plane mutant and application thereof, wherein a genome editing carrier is constructed by designing a gRNA target sequence of a plane target gene, a transformation product is obtained by transforming the plane target gene into a seed embryo by using a particle gun method, and the plane mutant is obtained by cultivating and identifying the transformation product.

Description

Mitsubishi tree mutant, method for obtaining same and application thereof

Technical Field

The invention relates to the technical field of transgenosis, in particular to a plane mutant, a method for obtaining the plane mutant and application of the plane mutant.

Background

The Platanus orientalis (Platanus spp.) is a tree species of deciduous trees of the genus sycamore of the family sycamore, only one genus of the family is about 10-12 species, and the family is produced abroad, three species of plants are introduced and cultivated in China, namely, one-ball sycamore (p. occidentalis L inn.), two-ball sycamore (p. acerifolia Willd.) and three-ball sycamore (p. france, p. orientalis L inn.), wherein, the sycamore in england is a hybrid of the sycamore and the sycamore (p. occidentalis × p. orientalis), has good hybrid advantages, the species has a history of hundreds of years, and the species can not be widely used as a yellow tree plant replacement for yellow trees, white trees, dry barks, pruning resistance, easy propagation, urban environments and various soil conditions, has strong air pollution resistance, light resistance, smoke resistance, benzene smoke resistance, air pollution resistance, wide popularization as urban yellow tree planting plants in urban gardens, urban yellow street greenbelt construction, and as a vast urban street greening plant replacement for yellow street trees in China.

However, in around one month every year, when old fruits fall off, new flowers and new fruits grow, fruit hairs generated by the falling of the old fruits and a large amount of pollen scattered by the new male inflorescences fall off trees, so that the environment is polluted, respiratory tracts of people are stimulated, respiratory diseases are caused, skin cancer and itching of people are easily caused, allergic diseases such as rhinitis and pollinosis are induced, keratitis is caused by careless falling into eyes, and the life and the body health of people are seriously influenced.

Over the years, researchers have conducted different explorations and attempts on controlling seasonal fruit hair and pollen flying after sycamore blossoms and fruits, wherein the explorations and attempts include breeding of fruitless strains, mutation breeding, polyploid breeding, crown grafting, trimming and fruit control, chemical agent treatment and other methods.

Disclosure of Invention

The invention aims to provide a plane mutant, a method for obtaining the plane mutant and application thereof, and aims to solve the problems of long time and poor directionality of mutation breeding of plane in the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method of obtaining a sycamore mutant comprising the steps of:

obtaining a gene of the order of the sycames and determining a gRNA target sequence;

synthesizing a gRNA target sequence oligonucleotide fragment containing a viscous tail end of a restriction endonuclease and a complementary chain thereof, and performing mixed annealing to generate a DNA annealing product containing the viscous tail ends of the restriction endonuclease at two ends;

providing a CRISPR/Cas9 vector plasmid and a restriction enzyme, and carrying out enzyme digestion treatment on the CRISPR/Cas9 vector plasmid by adopting the restriction enzyme to obtain an enzyme digested CRISPR/Cas9 vector plasmid;

providing a ligase, and adopting the ligase to sequentially connect the enzyme-cut CRISPR/Cas9 vector plasmid and the DNA annealing product to obtain a CRISPR/Cas9 genome editing vector;

and transforming the CRISPR/Cas9 genome editing vector into a seed embryo by a particle gun method to obtain a transformation product, cultivating the transformation product, and screening the sycamore mutant.

And, a mutant of sycamore, which is a mutant producing a mutation of a target gene, including insertion, deletion or alteration of a nucleotide sequence of a gene fragment encoding a gene.

And the method for obtaining the sycamore mutant or the application of the sycamore mutant in sycamore breeding.

According to the invention, a genome editing vector is constructed by designing a gRNA target sequence of a target gene of the plane bollwood, a transformation product is obtained by transforming the target gene into a seed embryo by using a particle gun method, and the transformation product is cultivated and identified to obtain the plane bollwood mutant.

The sycamore mutant is a mutant with a changed target gene sequence, and the mutant can obtain the mutation of a specific target gene in a targeted manner and quickly, thereby being beneficial to subsequent application.

The invention adopts Cas9 enzyme guided by gRNA to cut the genomic DNA of the sycamore at a gene target point, and the deletion, insertion or base mutation of a gene sequence is generated after DNA repair. These mutations can cause amino acid changes, amino acid substitutions, amino acid additions or deletions in the gene-encoded protein, thereby affecting changes in the sycamore phenotype. The method for obtaining the plane tabebuia mutants can carry out targeted mutation on all genes in the plane tabebuia genome, is simple and convenient, has high efficiency, can quickly obtain the mutants of specific genes, and has wide application of the prepared plane tabebuia mutants in plane tabebuia breeding.

Drawings

FIG. 1 is the transformation vector of Plaa1 gene of sycamore provided by the embodiment of the invention.

FIG. 2 is a flow chart for the production of sycamore mutants provided by the examples of the present invention.

Detailed Description

In order to make the objects, technical solutions and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments obtained by a person of ordinary skill in the art without any inventive step in connection with the embodiments of the present invention shall fall within the scope of protection of the present invention.

In the description of the present invention, it is to be understood that the terms "first", "second" and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.

The embodiment of the invention provides a method for obtaining sycamore mutant, as shown in figure 2, comprising the steps of vector construction, gene transformation, transgenic seedling screening and mutant gene sequence analysis, and specifically comprising the following steps:

s01, obtaining a gene of the order Suzuki and determining a gRNA target sequence;

s02, synthesizing a gRNA target sequence oligonucleotide fragment containing a viscous tail end of a restriction endonuclease and a complementary chain thereof, and mixing and annealing to generate a DNA annealing product containing the viscous tail ends of the restriction endonuclease at two ends;

s03, providing a CRISPR/Cas9 vector plasmid and a restriction enzyme, and carrying out enzyme digestion treatment on the CRISPR/Cas9 vector plasmid by adopting the restriction enzyme to obtain an enzyme digested CRISPR/Cas9 vector plasmid;

s04, providing a ligase, and adopting the ligase to sequentially connect the enzyme-cut CRISPR/Cas9 vector plasmid and the DNA annealing product to obtain a CRISPR/Cas9 genome editing vector;

s05, transforming the CRISPR/Cas9 genome editing vector into a seed embryo by adopting a particle gun method to obtain a transformation product, cultivating the transformation product, and screening the sycamore mutant.

Specifically, in step S01, a gene of the order sycames is obtained and the gRNA target sequence is determined.

Preferably, the species of sycamore selected is Platanus x acerifolia. In some embodiments of the present application, plane 1 is the plane pollen allergen of plane. In the preferred embodiment of the invention, leaves of Platanus x acerifolia are taken as samples, a genome extraction kit is adopted to extract genomic DNA of Platanus, and PCR amplification reaction is carried out to obtain the Platanus pollen allergen plaa1 gene.

Preferably, in the step of obtaining the sycamore pollen allergen plaa1 gene, a fifth primer and a sixth primer are used for performing PCR amplification reaction to obtain the sycamore pollen allergen plaa1 gene, wherein the sequence of the fifth primer is shown as seq id No. 3: 5'-tacgcggggaacaaaacaatccaat-3', respectively; the sequence of the sixth primer is shown as SEQ ID No.4, and the sixth primer is: 5'-ccgaagagggccaaatatcataca-3' are provided.

Preferably, in the step of obtaining the sycamore pollen allergen plaa1 gene by performing PCR amplification reaction by using a fifth primer and a sixth primer, the PCR amplification reaction system comprises DNA of sycamore leaves 5u L, the fifth primer 5u L, the sixth primer 5u L, 2 × Taq mix 25u L, and Mg²⁺1u L Glycerol 1u L, ddH₂O：12uL。

Further preferably, the PCR amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 1min, and 40 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.

Preferably, the PCR product of the sycamore pollen allergen plaa1 gene obtained by PCR amplification reaction is subjected to sequencing analysis, and the obtained sequence of the coding region of the sycamore pollen allergen plaa1 gene is shown as SEQ ID No.5 and is:

ATGAAGCTTTCCTTCTCTCTCTGTATCTTCTTCTTCAATCTCCTCCTCCTCCTTCAAGCTGTAATCAGCGCCGATATTGTTCAGGGCACATGCAAGAAAGTTGCTCAGAGAAGCCCAAACGTGAACTACGATTTCTGCGCGAAGTCTCTTGGAGCAGATCCTAAGAGCCACACTGCGGATCTTCAAGGACTTGGGGTCATCTCAGCGAATTTAGCCATACAGCAAGGATCTAAAATCCAAACATTTATTGGTCGCATCTTGAAAAGTAAAGTGGACCCAGCTCTTAAGAAATACTTGAATGATTGCGTGGGACTTTACGCTGATGCGAAGTCTTCAGTTCAAGAGGCCATAGCTGACTTCAATTCCAAGGACTACGCATCAGCTAATGTGAAAATGAGTGCGGCTTTGGACGACTCAGTGACTTGTGAAGATGGGTTTAAGGAGAAGAAAGGTTTAGTATCACCGGTGACGAAGGAGAACAAGGATTATGTACAACTGACTGCAATATCTCTTGCAATTACCAAACTGCTTGGTGCTTGA。

specifically, the gRNA target sequence is designed according to the coding region sequence of the sycamore pollen allergen plaa1 gene obtained by sequencing. According to the requirements of the CRISPR/Cas9 system, the screening requirements of the target sequences are as follows: (1) the target sequence mainly comprises 15-25 bases, and (2) the 15-25 bases are followed by a PAM (Protospace adjacent motif, PAM) region with 3 bases of NGG (N is any base).

Preferably, the gRNA target sequence includes a first gRNA target sequence and a second gRNA target sequence, wherein the first gRNA target sequence is shown in SEQ ID No.1 and is: 5'-CAGCGCCGATATTGTTCAGG-3', respectively; the second gRNA target sequence is shown in SEQ ID No.2 and is: 5'-TTCTGCGCGAAGTCTCTTGG-3' are provided. BsaI restriction enzyme cohesive end sequences are added at the ends of the target sequence and the complementary strand of the target sequence respectively as follows: 5 '-ATTG-3' and 3 '-CAAA-5'.

Subsequent experiments can be performed after the first gRNA target sequence and the second gRNA target sequence are determined. In practice, the target sequence may be selected from any sequence in the coding region of the gene or from upstream or downstream regulatory sequences for gene expression, such as promoter sequences, enhancer sequences and the like. Preferably, the number of the target sequences is selected from 1, 2 or more, and further preferably, the target sequences are selected from single use or simultaneous use.

Specifically, in step S02, an oligonucleotide fragment containing the RNA target sequence with the sticky ends of the restriction enzymes and the complementary strand thereof are synthesized, and after annealing in a mixed manner, DNA annealing products with sticky ends of the restriction enzymes at both ends are generated.

Preferably, the method for obtaining the annealing product of the first gRNA target sequence includes the following steps: and determining a first primer and a second primer of the first gRNA target sequence, and obtaining an annealing product of the first gRNA target sequence through first annealing treatment.

Further preferably, the first primer of the first gRNA target sequence is shown as SEQ ID No.6 and is: 5'-ATTGCAGCGCCGATATTGTTCAGG-3', respectively;

the second primer of the first gRNA target sequence is shown as SEQ ID No.7 and is:

3’-GTCGCGGCTATAACAAGTCCCAAA-5’。

further preferably, the annealing product of the first gRNA target sequence is obtained by a first annealing treatment, the reaction system of the first annealing treatment is that the first primer and the second primer with the concentration of 100mM are respectively purified water with the concentration of 1u L and 8u L, and the reaction program of the first annealing treatment is that the first annealing treatment is denatured for 5 minutes at 95 ℃ in a PCR instrument and slowly cooled to room temperature for 1 hour.

Preferably, the method for obtaining the annealing product of the second gRNA target sequence comprises the following steps: and determining a third primer and a fourth primer of the second gRNA target sequence, and obtaining an annealing product of the second gRNA target sequence through second annealing treatment.

Further preferably, the third primer of the second gRNA target sequence is shown as SEQ ID No.8, and is: 5'-ATTGCAGCGCCGATATTGTTCAGG-3', respectively;

the fourth primer of the second gRNA target sequence is shown as SEQ ID No.9 and is:

3’-GTCGCGGCTATAACAAGTCCCAAA-5’。

further preferably, the second annealing treatment is carried out to obtain the annealing product of the second gRNA target sequence, the reaction system of the second annealing treatment comprises purified water with the concentration of 100mM, 1u L and 8u L respectively, and the reaction program of the second annealing treatment comprises the steps of denaturation at 95 ℃ in a PCR instrument for 5 minutes and slow cooling to room temperature for 1 hour.

Specifically, in step S03, a CRISPR/Cas9 vector plasmid and a restriction enzyme are provided, and the CRISPR/Cas9 vector plasmid is subjected to enzyme digestion by the restriction enzyme to obtain an enzyme-digested CRISPR/Cas9 vector plasmid.

Preferably, the CRISPR/Cas9 vector plasmid includes, but is not limited to, pKSE401, and may be selected from other genome editing vector systems. In the specific embodiment of the application, the CRISPR/Cas9 vector plasmid is selected from pKSE401, and the pKSE401 vector is fused with Green Fluorescent Protein (GFP), which is beneficial for subsequent screening.

Preferably, the restriction enzymes include, but are not limited to, BsaI enzyme, which may be selected from restriction enzymes of gRNA cloning sites carried by other vector systems. In a specific embodiment of the present application, the restriction enzyme is selected from BsaI enzyme, and the digestion treatment is performed by BsaI enzyme, so that the gRNA and the vector can be connected.

Preferably, in the step of carrying out enzyme digestion treatment on the CRISPR/Cas9 vector plasmid by using the restriction endonuclease to obtain the enzyme digested CRISPR/Cas9 vector plasmid, the reaction system of the enzyme digestion treatment is that 1ug of the CRISPR/Cas9 vector plasmid, 10U of BsaI enzyme and 20U of BsaI enzyme buffer solution are added to L.

More preferably, the reaction conditions of the enzyme digestion treatment are as follows: performing enzyme digestion treatment at 50-52 ℃ for 1-1.5 hours, and then heating at 65-68 ℃ for 10 minutes. After the enzyme digestion treatment, heating is performed in order to inactivate the enzyme, thereby completing the enzyme digestion treatment. In the specific embodiment of the present invention, the reaction conditions of the enzyme digestion treatment are as follows: the enzyme was cleaved at 50 ℃ for 1 hour and then heated at 65 ℃ for 10 minutes.

Specifically, in the step S04, providing a ligase, and using the ligase to sequentially perform ligation on the enzyme-cleaved CRISPR/Cas9 vector plasmid and the DNA annealing product to obtain a CRISPR/Cas9 genome editing vector.

Preferably, the ligase is selected from T₄Ligase with T₄And (3) performing ligation by using ligase, and better performing ligation on the digested CRISPR/Cas9 vector plasmid and the DNA annealing product to obtain a CRISPR/Cas9 genome editing vector for ligation so as to construct a CRISPR/Cas9 genome editing vector.

Preferably, in the step of sequentially carrying out ligation treatment on the enzyme-digested CRISPR/Cas9 vector plasmid and the gRNA target point sequence annealing product by adopting the ligase to obtain the CRISPR/Cas9 genome editing vector, a reaction system of the ligation treatment is as follows, 10 mu L2X reaction buffer solution and 1 mu L T reaction buffer solution are added into a 20 mu L reaction system₄DNA ligase, 50ng of the digested vector and 3 times molar amount of annealing product of gRNA target. Further preferably, the reaction procedure of the ligation process is as follows: reacting at 37-38 ℃ for 10-15 min. In a specific embodiment of the present invention, the reaction procedure of the ligation process is as follows: the reaction was carried out at 37 ℃ for 10 min.

Preferably, the prepared CRISPR/Cas9 genome editing vector is transformed into an Escherichia coli competent cell by a heat shock method, and screening and sequencing of positive clones are performed, so that an accurate CRISPR/Cas9 genome editing vector is ensured.

Specifically, in step S05, the CRISPR/Cas9 genome editing vector is transformed into a seed embryo by a particle gun method to obtain a transformation product, and the transformation product is cultivated to screen the sycamore mutant.

Preferably, the method for transforming the CRISPR/Cas9 genome editing vector into a seed embryo to obtain a transformation product by using a particle gun method comprises the following steps:

s051, obtaining fruit seeds in the plant mature period, and culturing germs;

s052, providing a vector to be transformed, wherein the vector to be transformed comprises a plant genome editing vector and metal particles carrying the plant genome editing vector; wherein the diameter of the metal fine particles is 0.6 to 1.8 μm;

s053, adopting a gene gun to convert the vector to be converted into the embryo under the pressure of 450-2200 PSI, and obtaining a conversion product.

In the step S051, the method for obtaining fruit seeds of plant mature period and cultivating embryo includes the following steps:

s0511, obtaining fruit seeds in the mature period of plants,

s0512, after the seeds are subjected to sterilization treatment, dark culture is carried out, and germs of the seeds are obtained.

Preferably, in step S0511, the fruit seeds of plant mature period are obtained, the seeds of mature period are selected as transformation object, the seeds of mature period contain rich organic matter, have a certain germination ability and strong environmental resistance, and as transformation object, it can be beneficial to effectively transform the recombinant plasmid into seeds, and it is beneficial to improve the gene transformation efficiency.

Preferably, in step S0512, the method for obtaining embryo of said seed by dark culture after sterilizing said seed comprises the following steps:

s05121, after unhairing treatment is conducted on the seeds, conducting first sterilization treatment on the seeds through alcohol with the volume concentration of 75%, and conducting second sterilization treatment through mercuric chloride solution with the mass concentration of 0.1% to obtain sterilized seeds;

s05122, placing the sterilized seeds on wet filter paper, and carrying out dark culture for 2-3 days at the temperature of 25 ℃ to obtain germs of the seeds.

Preferably, in step S05121, the seeds are unhairing treated, and the unhairing treatment mainly removes the seeds on the surface of the seeds to ensure the clean seed surface. Further preferably, the seeds are unhaired by washing powder water, so that the seeds are cleaned.

Preferably, after the first sterilization treatment is carried out by using alcohol with the volume concentration of 75%, the second sterilization treatment is carried out by using mercuric chloride solution with the mass concentration of 0.1% to obtain sterilized seeds; and the seeds are sterilized, so that the seeds for healthy germination and gene transformation tests are sterile and clean, and the gene transformation test can be well completed. Preferably, in the step of performing the first sterilization treatment with alcohol having a volume concentration of 75%, the time for the first sterilization treatment is 40 to 60 seconds. Preferably, in the step of performing the second sterilization treatment by using a mercuric chloride solution with a mass concentration of 0.1%, the time of the second sterilization treatment is 40-50 minutes.

Further preferably, the second sterilization treatment of the seeds further comprises rinsing with sterile water, rinsing with sterile water to obtain sterilized seeds, and removing mercuric chloride solution on the surfaces of the seeds to obtain clean sterilized seeds.

In the step S052, providing a vector to be transformed, wherein the vector to be transformed comprises a plant genome editing vector and metal particles carrying the plant genome editing vector; wherein the diameter of the metal fine particles is 0.6 to 1.8 μm.

Specifically, a transformation vector is provided for transformation, and the vector to be transformed comprises a plant genome editing vector and metal particles carrying the plant genome editing vector.

In a preferred embodiment of the present invention, the method for carrying the metal microparticles of the plant genome editing vector includes a conventional coating method such as dipping. The metal particles carrying the plant genome editing vector are medium for plant gene transformation, and the plant genome editing vector is transformed into a seed embryo by a gene gun method through the medium.

Preferably, the diameter of the metal fine particles is 0.6 to 1.8 μm. The metal particles with the diameters are selected as a medium to carry out a gene transformation test, so that the transformation efficiency can be ensured. If the diameter of the metal particles is too large, the damage to plant seeds is large, the transformation efficiency is influenced, and the survival rate of the seeds is influenced; if the diameter of the metal microparticle is too small, the recombinant plasmid carried by the metal microparticle is too small, and the transformation efficiency is too low. In a preferred embodiment of the invention, the diameter of the metal particles is 0.6 μm.

In a preferred embodiment of the invention, the metal particles are selected from gold powder or tungsten powder. And the metal particles with stable properties are selected for testing, so that the damage to plant seeds is reduced, and the survival rate of the plant seeds is improved.

Preferably, in the step of providing the vector to be transformed, the vector to be transformed is prepared by coating the metal particles and the plant genome editing vector at a mass ratio of 15mg:2 μ g. If the addition amount of the metal particles is too large, plant seeds can be damaged, the conversion efficiency is influenced, and the survival rate of the seeds is influenced; if the amount of the metal fine particles added is too small, the amount of the recombinant plasmid carried by the metal fine particles is too small, and the conversion efficiency is too low.

In the step S053, a gene gun is adopted to convert the carrier to be converted into the embryo under the pressure of 450-2200 PSI, so as to obtain a conversion product.

Preferably, in the step of transforming the vector to be transformed into the germs by using a gene gun under the condition that the pressure is 450-2200 PSI, the vector to be transformed is used for transforming 100-500 germs in proportion. Under the pressure condition, the metal particle inclusion can be converted into the seeds with higher efficiency. In a preferred embodiment of the invention, the conversion pressure is 900 PSI.

Preferably, the gene gun is selected from PDS-1000/He gene guns of BIO-RAD company.

Specifically, in step S05, the transformation product is cultivated, preferably, the transformation product is cultivated in the dark at 25 ℃ for one day and then cultivated in the light for three days, after green buds grow, the transformation product is transplanted into a greenhouse to be cultivated into seedlings, and the seedlings are transplanted and cultivated and screened to obtain the sycamore mutant.

Preferably, the step of screening to obtain the sycamore mutant comprises the following steps of carrying out PCR amplification reaction by using plant genomic DNA of the sycamore mutant as a template and adopting a seventh primer and an eighth primer to verify the sycamore mutant. Further preferably, the sequence of the seventh primer is shown as SEQ ID No.10, and is:

5’-TGTAAAACGACGGCCAGTATGAAGCTTTCCTTCTCTCT-3’；

the sequence of the eighth primer is shown as SEQ ID No.11 and comprises:

5’-CAGGAAACAGCTATGACCTCAAGCACCAAGCAGTTTGG-3’。

preferably, the PCR amplification reaction system comprises 5u L of the DNA of sycamore leaf, 5u L of the seventh primer, 5u L of the eighth primer, 2 × Taq mix, 25u L of the eighth primer, and Mg²⁺1u L Glycerol 1u L, ddH₂12u L, and the PCR amplification reaction conditions are pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 1min, 40 cycles, extension at 72 ℃ for 10min and preservation at 4 ℃.

And selecting monoclonals for sequencing, selecting 10 monoclonals for sequencing from each gene transformation plant, and comparing the sequencing result with a wild-type plant gene sequence (SEQ ID No.5) through an NCBI website B L AST program to identify a mutant gene.

Accordingly, the present embodiments also provide a sycamore mutant, which is a mutant with an altered sequence of a target gene, including a mutant with an inserted, deleted or altered base of a gene fragment. The mutant targetedly and quickly obtains the mutation of a specific target gene sequence, thereby being beneficial to subsequent application.

Correspondingly, the embodiment of the application also provides a method for obtaining the sycamore mutant or application of the sycamore mutant in sycamore breeding.

The Cas9 enzyme guided by gRNA is adopted to cut the genomic DNA of the sycamore at a gene target point, and the mutation of a gene sequence is generated after the DNA is repaired. These mutations can cause amino acid changes, amino acid substitutions, amino acid additions or deletions in the gene-encoded protein, thereby affecting changes in the sycamore phenotype. The method for obtaining the plane tabebuia mutants can carry out targeted mutation on all genes in the plane tabebuia genome, is simple and convenient, has high efficiency, can quickly obtain the mutants of specific genes, and has wide application of the prepared plane tabebuia mutants in plane tabebuia breeding.

Further analysis was performed as detailed in the examples.

Example 1

Cloning and sequencing of sycamore pollen allergen gene Plaa1

(1) Selecting leaves of Platanus x acerifolia as a sample, extracting genomic DNA of Platanus acerifolia by adopting a genome extraction kit, wherein the sequence of a fifth primer is shown as SEQ ID No.3 and is as follows: 5'-tacgcggggaacaaaacaatccaat-3', respectively; the sequence of the sixth primer is shown as SEQ ID No.4 and is as follows: 5'-ccgaagagggccaaatatcataca-3', obtaining the sycamore pollen allergen pla 1 gene by PCR amplification reaction.

The PCR amplification reaction system comprises 5u L of the DNA of sycamore leaf, 5u L of a fifth primer, 5u L of a sixth primer, 25u L of 2 × of Taq mix, 1u L of glycerol, 1u L of Mg2 +: 1u L and 12u L of ddH 2O.

The PCR amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 1min, and 40 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.

(2) And obtaining a PCR product of the sycamore pollen allergen plaa1 gene through PCR amplification reaction for sequencing analysis.

Example 2

Construction of genome editing vector

(1) Designing two sgRNA sequences according to the sycamore gene sequence (SEQ ID No.5), wherein the two sgRNA sequences are respectively a first gRNA target sequence (target T1): CAGCGCCGATATTGTTCAGG and a second gRNA target sequence (target T2): TTCTGCGCGAAGTCTCTTGG are provided.

Respectively synthesizing sense strands and antisense strands of the two sgRNA sequences with joints, wherein the underlined parts are the joint sequences, and after mixed annealing, forming the following two double-stranded DNAs:

designing a first primer of a first gRNA target sequence SEQ ID No. 6: (5' -ATTGCAGCGCCGATATTGTTCAGG-3 ') and a second primer SEQ ID No.7(3 ' -GTCGCGGCTATAACAAGTCCCAAA-5 '), to form a first gRNA target sequence;

designing a third primer of a second gRNA target sequence SEQ ID No. 8: (5' -ATTGTTCTGCGCGAAGTCTCTTGG-3 ') and a fourth primer SEQ ID No.9(3 ' -AAGACGCGCTTCAGAGAACCCAAA-5 '), and performing mixed annealing treatment to form a second gRNA target sequence.

(2) Providing a CRISPR/Cas9 vector plasmid pKSE401 and a restriction endonuclease BsaI enzyme, and carrying out enzyme digestion treatment on the CRISPR/Cas9 vector plasmid pKSE401 by using the BsaI enzyme to obtain a first CRISPR/Cas9 vector plasmid, wherein the reaction system of the enzyme digestion treatment comprises 1ug of the CRISPR/Cas9 vector plasmid, 10U of the BsaI enzyme and 20U L of BsaI enzyme buffer solution, and the reaction condition of the enzyme digestion treatment is that the enzyme digestion treatment is carried out at 50 ℃ for 1 hour, and then the heating is carried out at 65 ℃ for 10 minutes.

(3) Providing T4 ligase, and carrying out ligation treatment on the first CRISPR/Cas9 vector plasmid, the annealing product of the first gRNA target sequence or the annealing product of the second gRNA target sequence by adopting the T4 ligase to obtain a CRISPR/Cas9 genome editing vector, wherein the reaction system of the ligation treatment comprises 50ng of the vector after enzyme digestion, 0.1ug of the annealing product of the gRNA target sequence, 1 mu L of T4 ligase (5U) and 2 mu L of T4 DNA ligase buffer solution, and the reaction program of the ligation treatment is that the reaction is carried out at 37 ℃ for 10 min.

(4) And (3) transforming the prepared CRISPR/Cas9 genome vector into an escherichia coli competent cell by adopting a heat shock method, and screening and sequencing positive clones to ensure that an accurate CRISPR/Cas9 genome editing vector is obtained.

Example 3

Gene transformation

(1) Obtaining seeds of mature cones of the growing strong adult sycamore; cleaning the seeds to remove villi on the surfaces of the seeds, and then sequentially disinfecting the seeds for 40s by using 75% alcohol, disinfecting the seeds for 40min by using 0.1% mercury bichloride and rinsing the seeds for 3 times by using sterile water to obtain sterilized seeds; placing the sterilized seeds on wet filter paper, and carrying out dark culture for 2-3 days at 25 ℃ to obtain seeds with germinated germs;

(2) obtaining embryo buds of seeds, wherein a to-be-converted carrier is gold powder particles with the diameter of 0.6 mu m and coated with genome editing carrier plasmids, the conversion parameters are gold powder with the diameter of 0.6 mu m and the pressure of 900PSI, the distance between the conversion carrier and a sample is 6 cm, and the genome editing carrier is converted into the embryo buds by adopting a PDS-1000/He gene gun of BIO-RAD company to obtain a conversion product.

(3) Culturing the transformation product, culturing the transformation product in the dark at 25 ℃ for one day, then culturing for three days by illumination, transplanting the transformation product into a greenhouse after green buds grow out, culturing into seedlings, transplanting and culturing the seedlings, and culturing in the greenhouse for two months and then identifying.

Example 4

Identification of mutants

And carrying out PCR amplification reaction by using the plant genome DNA of the plane suspending mutant as a template and adopting a seventh primer and an eighth primer so as to verify the plane suspending mutant. Wherein, the sequence of the seventh primer is shown as SEQ ID No.10, and is:

5’-TGTAAAACGACGGCCAGTATGAAGCTTTCCTTCTCTCT-3’；

the sequence of the eighth primer is shown as SEQ ID No.11 and comprises:

5’-CAGGAAACAGCTATGACCTCAAGCACCAAGCAGTTTGG-3’。

cloning the amplified PCR fragment, wherein the PCR amplification reaction system comprises DNA of sycamore leaf 5u L, a seventh primer 5u L, an eighth primer 5u L, 2 × Taq mix 25u L and Mg²⁺1u L Glycerol 1u L, ddH₂12u L, and the PCR amplification reaction conditions are that the PCR amplification reaction is carried out under the conditions of pre-denaturation at 95 ℃ for 5min and denaturation at 95 DEG C30s, annealing at 56 ℃ for 30s, extending at 72 ℃ for 1min, and performing 40 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.

And (4) analyzing results:

and analyzing the result:

sequencing analysis is carried out on a PCR product of the sycamore pollen allergen plaa1 gene obtained by the PCR amplification reaction in the embodiment 1, and the obtained sequence of the coding region of the sycamore pollen allergen plaa1 gene is shown as SEQ ID No.5 and is as follows:

and analyzing the result:

aiming at the construction of the genome editing vector in example 2, the constructed genome editing vector is shown in fig. 1, wherein L B and RB are the left and right boundaries of T-DNA on a Ti plasmid respectively, 35Sp-Cas9-NosT and 35Sp-NptII-PolyA are the expression frames of Cas9 gene and NptII gene respectively, U6-26-T1 and U6-29-T2 express two sgRNAs respectively, and F and R are the upstream and downstream primers of the transgenic plant detection PCR respectively.

And analyzing results:

the gene transformation test of example 3 was analyzed, and the results of transplanting and cultivating seedlings were analyzed, and after transplanting, 52 seedlings survived with a survival rate of 86.7%.

After two months of greenhouse culture, 28 plants were positive by PCR, and the positive rate was 53.8%. The gene transformation efficiency was 46.7%.

And analyzing the result:

analysis was performed for identification of the mutant in example 4, and a total of 28 transgenic seedlings of sycamore were obtained in this experiment, and analysis of the target gene revealed that 2 (18 th and 48 th) plants contained mutations.

10 samples of plant number 18 were sequenced and 5 samples were found to contain 1 or more mutations. Wherein the 225 th base of 18-1 is changed from A to G (no change in the encoded amino acid sequence is caused); thus, the nucleotide sequence and protein sequence of 18-1 are not shown.

18-3, 14 th-15 th base TC is deleted to cause frame shift mutation, the protein is terminated in advance (nucleic acid sequence 5, protein sequence 2), the base sequence of the mutant 18-3 is shown as SEQ ID No.12 and is:

ATGAAGCTTTCCTTCTCTCTGTATCTTCTTCTTCAATCTCCTCCTCCTCCTTCAAGCTGTAATCAGCGCCGATATTGTTCAGGGCACATTCAAGAAAGTTGCTCAGAGAAGCCCAAACGTGAACTACGATTTCTGCGCGAAGTCTCTTGGAGCAGATCCTAAGAGCCACACTGCGGATCTTCAAGGACTTGGGGTCATCTCAGCGAATTTAGCCATACAGCAAGGATCTAAAATCCAAACATTTATTGGTCGCATCTTGAAAAGTAAAGTGGACCCAACTCTTAAGAAATACTTGAATGATTGCGTGGGACTTTACGCTGATGCGAAGTCTTCAGTTCAAGAGGCCATAGCTGACTTCAATTCCAAGGACTACGCATCAGCTAATGTGAAAATGAGTGCGGCTTTGGACGACTCAGTGACTTGTGAAGATGGGTTTAAGGAGAAGAAAGGTTTAGTATCACCGGTGACGAAGGAGAACAAGGATTATGTACAACTGACTGCAATATCTCTTGCAATTACCAAACTGCTTGGTGCTTGA。

the protein sequence of the mutant 18-3 is shown as SEQ ID No.13 and is:

MKLSFSLYLLLQSPPPPSSCNQRRYCSGHIQESCSEKPKRELRFLREVSWSRS。

18-6 at base 401 from C to T (resulting in the encoded amino acid changing from alanine A to valine V);

the base sequence of the mutant 18-6 is shown as SEQ ID No.14 and is:

ATGAAGCTTTCCTTCTCTCTCTGTATCTTCTTCTTCAATCTCCTCCTCCTCCTTCAAGCTGTAATCAGCGCCGATATTGTTCAGGGCACATGCAAGAAAGTTGCTCAGAGAAGCCCAAACGTGAACTACGATTTCTGCGCGAAGTCTCTTGGAGCAGATCCTAAGAGCCACACTGCGGATCTTCAAGGACTTGGGGTCATCTCAGCGAATTTAGCCATACAGCAAGGATCTAAAATCCAAACATTTATTGGTCGCATCTTGAAAAGTAAAGTGGACCCAGCTCTTAAGAAATACTTGAATGATTGCGTGGGACTTTACGCTGATGCGAAGTCTTCAGTTCAAGAGGCCATAGCTGACTTCAATTCCAAGGACTACGCATCAGCTAATGTGAAAATGAGTGTGGCTTTGGACGACTCAGTGACTTGTGAAGATGGGTTTAAGGAGAAGAAAGGTTTAGTATCACCGGTGACGAAGGAGAACAAGGATTATGTACAACTGACTGCAATATCTCTTGCAATTACCAAACTGCTTGGTGCTTGA。

the protein sequence of the mutant 18-6 is shown as SEQ ID No.15 and is:

MKLSFSLCIFFFNLLLLLQAVISADIVQGTCKKVAQRSPNVNYDFCAKSLGADPKSHTADLQGLGVISANLAIQQGSKIQTFIGRILKSKVDPALKKYLNDCVGLYADAKSSVQEAIADFNSKDYASANVKMSVALDDSVTCEDGFKEKKGLVSPVTKENKDYVQLTAISLAITKLLGA。

the 402 th base of 18-7 is changed from G to T (no amino acid change is caused); thus, the nucleotide sequence and protein sequence of 18 to 7 are not shown.

18-8, base 310 changed from G to A (resulting in amino acid change from glycine G to arginine R), mutant 18-8 has the base sequence shown in SEQ ID No.16 as:

ATGAAGCTTTCCTTCTCTCTCTGTATCTTCTTCTTCAATCTCCTCCTCCTCCTTCAAGCTGTAATCAGCGCCGATATTGTTCAGGGCACATGCAAGAAAGTTGCTCAGAGAAGCCCAAACGTGAACTACGATTTCTGCGCGAAGTCTCTTGGAGCAGATCCTAAGAGCCACACTGCGGATCTTCAAGGACTTGGGGTCATCTCAGCGAATTTAGCCATACAGCAAGGATCTAAAATCCAAACATTTATTGGTCGCATCTTGAAAAGTAAAGTGGACCCAGCTCTTAAGAAATACTTGAATGATTGCGTGAGACTTTACGCTGATGCGAAGTCTTCAGTTCAAGAGGCCATAGCTGACTTCAATTCCAAGGACTACGCATCAGCTAATGTGAAAATGAGTGCGGCTTTGGACGACTCAGTGACTTGTGAAGATGGGTTTAAGGAGAAGAAAGGTTTAGTATCACCGGTGACGAAGGAGAACAAGGATTATGTACAACTGACTGCAATATCTCTTGCAATTACCAAACTGCTTGGTGCTTGA。

the protein sequence of the mutant 18-8 is shown as SEQ ID No.17 and is:

MKLSFSLCIFFFNLLLLLQAVISADIVQGTCKKVAQRSPNVNYDFCAKSLGADPKSHTADLQGLGVISANLAIQQGSKIQTFIGRILKSKVDPALKKYLNDCVRLYADAKSSVQEAIADFNSKDYASANVKMSAALDDSVTCEDGFKEKKGLVSPVTKENKDYVQLTAISLAITKLLGA。

10 samples of plant No. 48 were sequenced and 4 were found to contain the gene mutation. Base 160 from C to T at 48-2 (resulting in the amino acid change from proline P to serine S);

the base sequence of the mutant gene 48-2 is shown as SEQ ID No.18 and is:

ATGAAGCTTTCCTTCTCTCTCTGTATCTTCTTCTTCAATCTCCTCCTCCTCCTTCAAGCTGTAATCAGCGCCGATATTGTTCAGGGCACATGCAAGAAAGTTGCTCAGAGAAGCCCAAACGTGAACTACGATTTCTGCGCGAAGTCTCTTGGAGCAGATTCTAAGAGCCACACTGCGGATCTTCAAGGACTTGGGGTCATCTCAGCGAATTTAGCCATACAGCAAGGATCTAAAATCCAAACATTTATTGGTCGCATCTTGAAAAGTAAAGTGGACCCAGCTCTTAAGAAATACTTGAATGATTGCGTGGGACTTTACGCTGATGCGAAGTCTTCAGTTCAAGAGGCCATAGCTGACTTCAATTCCAAGGACTACGCATCAGCTAATGTGAAAATGAGTGCGGCTTTGGACGACTCAGTGACTTGTGAAGATGGGTTTAAGGAGAAGAAAGGTTTAGTATCACCGGTGACGAAGGAGAACAAGGATTATGTACAACTGACTGCAATATCTCTTGCAATTACCAAACTGCTTGGTGCTTGA。

the protein sequence of the mutant gene 48-2 is shown as SEQ ID No.19 and is:

MKLSFSLCIFFFNLLLLLQAVISADIVQGTCKKVAQRSPNVNYDFCAKSLGADSKSHT ADLQGLGVISANLAIQQGSKIQTFIGRILKSKVDPALKKYLNDCVGLYADAKSSVQEAIAD FNSKDYASANVKMSAALDDSVTCEDGFKEKKGLVSPVTKENKDYVQLTAISLAITKLLGA。

base 140 from C to T at 48-3 (resulting in the amino acid changing from alanine A to valine V),

the base sequence of the mutant gene 48-3 is shown as SEQ ID No.20 and is:

ATGAAGCTTTCCTTCTCTCTCTGTATCTTCTTCTTCAATCTCCTCCTCCTCCTTCAAGCTGTAATCAGCGCCGATATTGTTCAGGGCACATGCAAGAAAGTTGCTCAGAGAAGCCCAAACGTGAACTACGATTTCTGCGTGAAATCTCTTGGAGCAGATCCTAAGAGCCACACTGCGGATCTTCAAGGACTTGGGGTCATCTCAGCGAATTTAGCCATACAGCAAGGATCTAAAATCCAAACATTTATTGGTCGCATCTTGAAAAGTAAAGTGGACCCAGCTCTTAAGAAATACTTGAATGATTGCGTGGGACTTTACGCTGATGCAAAGTCTTCAGTTCAAGAGGCCATAGCTGACTTCAATTCCAAGGACTACGCATCAGCTAATGTGAAAATGAGTGCGGCTTTGGACGACTCAGTGACTTGTGAAGATGGGTTTAAGGAGAAGAAAGGTTTAGTATCACCGGTGACGAAGGAGAACAAGGATTATGTACAACTGACTGCAATATCTCTTGCAATTACCAAACTGCTTGGTGCTTGA。

the protein sequence of the mutant gene 48-3 is shown as SEQ ID No.21 and is:

MKLSFSLCIFFFNLLLLLQAVISADIVQGTCKKVAQRSPNVNYDFCVKSLGADPKSHTADLQGLGVISANLAIQQGSKIQTFIGRILKSKVDPALKKYLNDCVGLYADAKSSVQEAIADFNSKDYASANVKMSAALDDSVTCEDGFKEKKGLVSPVTKENKDYVQLTAISLAITKLLGA*。

base 144 of 48-6 changed from G to A (no amino acid change induced), base 327 from G to A (no amino acid change induced); therefore, the nucleotide sequence and protein sequence are not shown.

Base 154 of 48-7 changed from G to A (resulting in the amino acid changing from alanine A to threonine T); the base sequence of the mutant gene 48-7 is shown as SEQ ID No.22 and is:

ATGAAGCTTTCCTTCTCTCTCTGTATCTTCTTCTTCAATCTCCTCCTCCTCCTTCAAGCTGTAATCAGCGCCGATATTGTTCAGGGCACATGCAAGAAAGTTGCTCAGAGAAGCCCAAACGTGAACTACGATTTCTGCGCGAAGTCTCTTGGAACAGATCCTAAGAGCCACACTGCGGATCTTCAAGGACTTGGGGTCATCTCAGCGAATTTAGCCATACAGCAAGGATCTAAAATCCAAACATTTATTGGTCGCATCTTGAAAAGTAAAGTGGACCCAGCTCTTAAGAAATACTTGAATGATTGCGTGGGACTTTACGCTGATGCGAAGTCTTCAGTTCAAGAGGCCATAGCTGACTTCAATTCCAAGGACTACGCATCAGCTAATGTGAAAATGAGTGCGGCTTTGGACGACTCAGTGACTTGTGAAGATGGGTTTAAGGAGAAGAAAGGTTTAGTATCACCGGTGACGAAGGAGAACAAGGATTATGTACAACTGACTGCAATATCTCTTGCAATTACCAAACTGCTTGGTGCTTGA.

the protein sequence of the mutant gene 48-7 is shown as SEQ ID No.23 and is:

MKLSFSLCIFFFNLLLLLQAVISADIVQGTCKKVAQRSPNVNYDFCAKSLGTDPKSHTADLQGLGVISANLAIQQGSKIQTFIGRILKSKVDPALKKYLNDCVGLYADAKSSVQEAIADFNSKDYASANVKMSAALDDSVTCEDGFKEKKGLVSPVTKENKDYVQLTAISLAITKLLGA*。

48-8 base 137 changed from G to A (causing the amino acid to change from cysteine C to tyrosine Y), base 140 changed from C to T (causing the amino acid to change from alanine A to valine V), base 144 changed from G to A (causing no amino acid change), base 225 changed from A to T (causing the amino acid to change from glutamine Q to histidine H), base 259 changed from T to G (causing the amino acid to change from leucine L to valine V), base 306 changed from C to T (causing no amino acid change), base 363 changed from T to G (causing the amino acid to change from asparagine N to lysine K), base 454 changed 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 as SEQ ID No.24 and is:

ATGAAGCTTTCCTTCTCTCTCTGTATCTTCTTCTTCAATCTCCTCCTCCTCCTTCAAGCTGTAATCAGCGCCGATATTGTTCAGGGCACATGCAAGAAAGTTGCTCAGAGAAGCCCAAACGTGAACTACGATTTCTACGTGAAATCTCTTGGAGCAGATCCTAAGAGCCACACTGCGGATCTTCAAGGACTTGGGGTCATCTCAGCGAATTTAGCCATACAGCATGGATCTAAAATCCAAACATTTATTGGTCGCATCGTGAAAAGTAAAGTGGACCCAGCTCTTAAGAAATACTTGAATGATTGTGTGGGACTTTACGCTGATGCGAAGTCTTCAGTTCAAGAGGCCATAGCTGACTTCAAGTCCAAGGACTACGCATCAGCTAATGTGAAAATGAGTGCGGCTTTGGACGACTCAGTGACTTGTGAAGATGGGTTTAAGGAGAAGAAAGGTATAGTATCACCGGTGACGAAGGAGAACAAGGATTATGTACAACTGACTGCAATATCTCTTGCAATTACCAAACTGCTTGGTGCTTGA。

the protein sequence of the mutant gene 48-8 is shown in SEQ ID No.25 and is:

MKLSFSLCIFFFNLLLLLQAVISADIVQGTCKKVAQRSPNVNYDFYVKSLGADPKSHTADLQGLGVISANLAIQHGSKIQTFIGRIVKSKVDPALKKYLNDCVGLYADAKSSVQEAIADFKSKDYASANVKMSAALDDSVTCEDGFKEKKGIVSPVTKENKDYVQLTAISLAITKLLGA*。

the invention relates to a sycamore gene mutation technology edited by CRISPR/CAS9 genome, which can be applied to the mutation of all genes of sycamore, such as allergic protein genes affecting health; flowering genes affecting flowering and balling, floral development genes, fruit and seed development genes, seed coat hair development genes; genes affecting rapid growth of trees; drought resistance, waterlogging resistance, salt and alkali resistance, high and low temperature resistance, disease resistance, insect resistance, virus resistance and the like which influence the resistance of trees.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

SEQUENCE LISTING

<110> Shenzhen university

<120> plane mutant, method for obtaining plane mutant and application

<130>2020-03-02

<160>25

<170>PatentIn version 3.3

<210>1

<211>20

<212>DNA

<213> Artificial Synthesis

<400>1

cagcgccgat attgttcagg 20

<210>2

<211>20

<212>DNA

<213> Artificial Synthesis

<400>2

ttctgcgcga agtctcttgg 20

<210>3

<211>25

<212>DNA

<213> Artificial Synthesis

<400>3

tacgcgggga acaaaacaat ccaat 25

<210>4

<211>24

<212>DNA

<213> Artificial Synthesis

<400>4

ccgaagaggg ccaaatatca taca 24

<210>5

<211>540

<212>DNA

<213> Artificial Synthesis

<400>5

atgaagcttt ccttctctct ctgtatcttc ttcttcaatc tcctcctcct ccttcaagct 60

gtaatcagcg ccgatattgt tcagggcaca tgcaagaaag ttgctcagag aagcccaaac 120

gtgaactacg atttctgcgc gaagtctctt ggagcagatc ctaagagcca cactgcggat 180

cttcaaggac ttggggtcat ctcagcgaat ttagccatac agcaaggatc taaaatccaa 240

acatttattg gtcgcatctt gaaaagtaaa gtggacccag ctcttaagaa atacttgaat 300

gattgcgtgg gactttacgc tgatgcgaag tcttcagttc aagaggccat agctgacttc 360

aattccaagg actacgcatc agctaatgtg aaaatgagtg cggctttgga cgactcagtg 420

acttgtgaag atgggtttaa ggagaagaaa ggtttagtat caccggtgac gaaggagaac 480

aaggattatg tacaactgac tgcaatatct cttgcaatta ccaaactgct tggtgcttga 540

<210>6

<211>24

<212>DNA

<213> Artificial Synthesis

<400>6

attgcagcgc cgatattgtt cagg 24

<210>7

<211>24

<212>DNA

<213> Artificial Synthesis

<400>7

gtcgcggcta taacaagtcc caaa 24

<210>8

<211>24

<212>DNA

<213> Artificial Synthesis

<400>8

attgcagcgc cgatattgtt cagg 24

<210>9

<211>24

<212>DNA

<213> Artificial Synthesis

<400>9

gtcgcggcta taacaagtcc caaa 24

<210>10

<211>38

<212>DNA

<213> Artificial Synthesis

<400>10

tgtaaaacga cggccagtat gaagctttcc ttctctct 38

<210>11

<211>38

<212>DNA

<213> Artificial Synthesis

<400>11

caggaaacag ctatgacctc aagcaccaag cagtttgg 38

<210>12

<211>538

<212>DNA

<213> Artificial Synthesis

<400>12

atgaagcttt ccttctctct gtatcttctt cttcaatctc ctcctcctcc ttcaagctgt 60

aatcagcgcc gatattgttc agggcacatt caagaaagtt gctcagagaa gcccaaacgt 120

gaactacgat ttctgcgcga agtctcttgg agcagatcct aagagccaca ctgcggatct 180

tcaaggactt ggggtcatct cagcgaattt agccatacag caaggatcta aaatccaaac 240

atttattggt cgcatcttga aaagtaaagt ggacccaact cttaagaaat acttgaatga 300

ttgcgtggga ctttacgctg atgcgaagtc ttcagttcaa gaggccatag ctgacttcaa 360

ttccaaggac tacgcatcag ctaatgtgaa aatgagtgcg gctttggacg actcagtgac 420

ttgtgaagat gggtttaagg agaagaaagg tttagtatca ccggtgacga aggagaacaa 480

ggattatgta caactgactg caatatctct tgcaattacc aaactgcttg gtgcttga 538

<210>13

<211>53

<212>PRT

<213> Artificial Synthesis

<400>13

Met Lys Leu Ser Phe Ser Leu Tyr Leu Leu Leu Gln Ser Pro Pro Pro

1 5 10 15

Pro Ser Ser Cys Asn Gln Arg Arg Tyr Cys Ser Gly His Ile Gln Glu

20 25 30

Ser Cys Ser Glu Lys Pro Lys Arg Glu Leu Arg Phe Leu Arg Glu Val

35 40 45

Ser Trp Ser Arg Ser

50

<210>14

<211>540

<212>DNA

<213> Artificial Synthesis

<400>14

atgaagcttt ccttctctct ctgtatcttc ttcttcaatc tcctcctcct ccttcaagct 60

gtaatcagcg ccgatattgt tcagggcaca tgcaagaaag ttgctcagag aagcccaaac 120

gtgaactacg atttctgcgc gaagtctctt ggagcagatc ctaagagcca cactgcggat 180

cttcaaggac ttggggtcat ctcagcgaat ttagccatac agcaaggatc taaaatccaa 240

acatttattg gtcgcatctt gaaaagtaaa gtggacccag ctcttaagaa atacttgaat 300

gattgcgtgg gactttacgc tgatgcgaag tcttcagttc aagaggccat agctgacttc 360

aattccaagg actacgcatc agctaatgtg aaaatgagtg tggctttgga cgactcagtg 420

acttgtgaag atgggtttaa ggagaagaaa ggtttagtat caccggtgac gaaggagaac 480

aaggattatg tacaactgac tgcaatatct cttgcaatta ccaaactgct tggtgcttga 540

<210>15

<211>179

<212>PRT

<213> Artificial Synthesis

<400>15

Met Lys Leu Ser Phe Ser Leu Cys Ile Phe Phe Phe Asn Leu Leu Leu

1 5 10 15

Leu Leu Gln Ala Val Ile Ser Ala Asp Ile Val Gln Gly Thr Cys Lys

20 25 30

Lys Val Ala Gln Arg Ser Pro Asn Val Asn Tyr Asp Phe Cys Ala Lys

35 40 45

Ser Leu Gly Ala Asp Pro Lys Ser His Thr Ala Asp Leu Gln Gly Leu

50 55 60

Gly Val Ile Ser Ala Asn Leu Ala Ile Gln Gln Gly Ser Lys Ile Gln

65 70 75 80

Thr Phe Ile Gly Arg Ile Leu Lys Ser Lys Val Asp Pro Ala Leu Lys

85 90 95

Lys Tyr Leu Asn Asp Cys Val Gly Leu Tyr Ala Asp Ala Lys Ser Ser

100 105 110

Val Gln Glu Ala Ile Ala Asp Phe Asn Ser Lys Asp Tyr Ala Ser Ala

115 120 125

Asn Val Lys Met Ser Val Ala Leu Asp Asp Ser Val Thr Cys Glu Asp

130 135 140

Gly Phe Lys Glu Lys Lys Gly Leu Val Ser ProVal Thr Lys Glu Asn

145 150 155 160

Lys Asp Tyr Val Gln Leu Thr Ala Ile Ser Leu Ala Ile Thr Lys Leu

165 170 175

Leu Gly Ala

<210>16

<211>540

<212>DNA

<213> Artificial Synthesis

<400>16

atgaagcttt ccttctctct ctgtatcttc ttcttcaatc tcctcctcct ccttcaagct 60

gtaatcagcg ccgatattgt tcagggcaca tgcaagaaag ttgctcagag aagcccaaac 120

gtgaactacg atttctgcgc gaagtctctt ggagcagatc ctaagagcca cactgcggat 180

cttcaaggac ttggggtcat ctcagcgaat ttagccatac agcaaggatc taaaatccaa 240

acatttattg gtcgcatctt gaaaagtaaa gtggacccag ctcttaagaa atacttgaat 300

gattgcgtga gactttacgc tgatgcgaag tcttcagttc aagaggccat agctgacttc 360

aattccaagg actacgcatc agctaatgtg aaaatgagtg cggctttgga cgactcagtg 420

acttgtgaag atgggtttaa ggagaagaaa ggtttagtat caccggtgac gaaggagaac 480

aaggattatg tacaactgac tgcaatatct cttgcaatta ccaaactgct tggtgcttga 540

<210>17

<211>179

<212>PRT

<213> Artificial Synthesis

<400>17

Met Lys Leu Ser Phe Ser Leu Cys Ile Phe Phe Phe Asn Leu Leu Leu

1 5 10 15

Leu Leu Gln Ala Val Ile Ser Ala Asp Ile Val Gln Gly Thr Cys Lys

20 25 30

Lys Val Ala Gln Arg Ser Pro Asn Val Asn Tyr Asp Phe Cys Ala Lys

35 40 45

Ser Leu Gly Ala Asp Pro Lys Ser His Thr Ala Asp Leu Gln Gly Leu

50 55 60

Gly Val Ile Ser Ala Asn Leu Ala Ile Gln Gln Gly Ser Lys Ile Gln

65 70 75 80

Thr Phe Ile Gly Arg Ile Leu Lys Ser Lys Val Asp Pro Ala Leu Lys

85 90 95

Lys Tyr Leu Asn Asp Cys Val Arg Leu Tyr Ala Asp Ala Lys Ser Ser

100 105 110

Val Gln Glu Ala Ile Ala Asp Phe Asn Ser Lys Asp Tyr Ala Ser Ala

115 120 125

Asn Val Lys Met Ser Ala Ala Leu Asp Asp Ser Val Thr Cys Glu Asp

130 135 140

Gly Phe Lys Glu Lys Lys Gly Leu Val Ser Pro Val Thr Lys Glu Asn

145 150 155 160

Lys Asp Tyr Val Gln Leu Thr AlaIle Ser Leu Ala Ile Thr Lys Leu

165 170 175

Leu Gly Ala

<210>18

<211>540

<212>DNA

<213> Artificial Synthesis

<400>18

atgaagcttt ccttctctct ctgtatcttc ttcttcaatc tcctcctcct ccttcaagct 60

gtaatcagcg ccgatattgt tcagggcaca tgcaagaaag ttgctcagag aagcccaaac 120

gtgaactacg atttctgcgc gaagtctctt ggagcagatt ctaagagcca cactgcggat 180

cttcaaggac ttggggtcat ctcagcgaat ttagccatac agcaaggatc taaaatccaa 240

acatttattg gtcgcatctt gaaaagtaaa gtggacccag ctcttaagaa atacttgaat 300

gattgcgtgg gactttacgc tgatgcgaag tcttcagttc aagaggccat agctgacttc 360

aattccaagg actacgcatc agctaatgtg aaaatgagtg cggctttgga cgactcagtg 420

acttgtgaag atgggtttaa ggagaagaaa ggtttagtat caccggtgac gaaggagaac 480

aaggattatg tacaactgac tgcaatatct cttgcaatta ccaaactgct tggtgcttga 540

<210>19

<211>179

<212>PRT

<213> Artificial Synthesis

<400>19

Met Lys Leu Ser Phe Ser Leu Cys Ile Phe Phe Phe Asn Leu Leu Leu

1 5 10 15

Leu Leu Gln Ala Val Ile Ser Ala Asp Ile Val Gln Gly Thr Cys Lys

20 25 30

Lys Val Ala Gln Arg Ser Pro Asn Val Asn Tyr Asp Phe Cys Ala Lys

35 40 45

Ser Leu Gly Ala Asp Ser Lys Ser His Thr Ala Asp Leu Gln Gly Leu

50 55 60

Gly Val Ile Ser Ala Asn Leu Ala Ile Gln Gln Gly Ser Lys Ile Gln

65 70 75 80

Thr Phe Ile Gly Arg Ile Leu Lys Ser Lys Val Asp Pro Ala Leu Lys

85 90 95

Lys Tyr Leu Asn Asp Cys Val Gly Leu Tyr Ala Asp Ala Lys Ser Ser

100 105 110

Val Gln Glu Ala Ile Ala Asp Phe Asn Ser Lys Asp Tyr Ala Ser Ala

115 120 125

Asn Val Lys Met Ser Ala Ala Leu Asp Asp Ser Val Thr Cys Glu Asp

130 135 140

Gly Phe Lys Glu Lys Lys Gly Leu Val Ser Pro Val Thr Lys Glu Asn

145 150 155 160

Lys Asp Tyr Val Gln Leu Thr Ala Ile Ser Leu Ala Ile Thr Lys Leu

165 170 175

Leu Gly Ala

<210>20

<211>540

<212>DNA

<213> Artificial Synthesis

<400>20

atgaagcttt ccttctctct ctgtatcttc ttcttcaatc tcctcctcct ccttcaagct 60

gtaatcagcg ccgatattgt tcagggcaca tgcaagaaag ttgctcagag aagcccaaac 120

gtgaactacg atttctgcgt gaaatctctt ggagcagatc ctaagagcca cactgcggat 180

cttcaaggac ttggggtcat ctcagcgaat ttagccatac agcaaggatc taaaatccaa 240

acatttattg gtcgcatctt gaaaagtaaa gtggacccag ctcttaagaa atacttgaat 300

gattgcgtgg gactttacgc tgatgcaaag tcttcagttc aagaggccat agctgacttc 360

aattccaagg actacgcatc agctaatgtg aaaatgagtg cggctttgga cgactcagtg 420

acttgtgaag atgggtttaa ggagaagaaa ggtttagtat caccggtgac gaaggagaac 480

aaggattatg tacaactgac tgcaatatct cttgcaatta ccaaactgct tggtgcttga 540

<210>21

<211>179

<212>PRT

<213> Artificial Synthesis

<400>21

Met Lys Leu Ser Phe Ser Leu Cys Ile Phe Phe Phe Asn Leu Leu Leu

1 5 10 15

Leu Leu Gln Ala Val Ile Ser Ala Asp Ile Val Gln Gly Thr Cys Lys

20 25 30

LysVal Ala Gln Arg Ser Pro Asn Val Asn Tyr Asp Phe Cys Val Lys

35 40 45

Ser Leu Gly Ala Asp Pro Lys Ser His Thr Ala Asp Leu Gln Gly Leu

50 55 60

Gly Val Ile Ser Ala Asn Leu Ala Ile Gln Gln Gly Ser Lys Ile Gln

65 70 75 80

Thr Phe Ile Gly Arg Ile Leu Lys Ser Lys Val Asp Pro Ala Leu Lys

85 90 95

Lys Tyr Leu Asn Asp Cys Val Gly Leu Tyr Ala Asp Ala Lys Ser Ser

100 105 110

Val Gln Glu Ala Ile Ala Asp Phe Asn Ser Lys Asp Tyr Ala Ser Ala

115 120 125

Asn Val Lys Met Ser Ala Ala Leu Asp Asp Ser Val Thr Cys Glu Asp

130 135 140

Gly Phe Lys Glu Lys Lys Gly Leu Val Ser Pro Val Thr Lys Glu Asn

145 150 155 160

Lys Asp Tyr Val Gln Leu Thr Ala Ile Ser Leu Ala Ile Thr Lys Leu

165 170 175

Leu Gly Ala

<210>22

<211>540

<212>DNA

<213> Artificial Synthesis

<400>22

atgaagcttt ccttctctct ctgtatcttc ttcttcaatc tcctcctcct ccttcaagct 60

gtaatcagcg ccgatattgt tcagggcaca tgcaagaaag ttgctcagag aagcccaaac 120

gtgaactacg atttctgcgc gaagtctctt ggaacagatc ctaagagcca cactgcggat 180

cttcaaggac ttggggtcat ctcagcgaat ttagccatac agcaaggatc taaaatccaa 240

acatttattg gtcgcatctt gaaaagtaaa gtggacccag ctcttaagaa atacttgaat 300

gattgcgtgg gactttacgc tgatgcgaag tcttcagttc aagaggccat agctgacttc 360

aattccaagg actacgcatc agctaatgtg aaaatgagtg cggctttgga cgactcagtg 420

acttgtgaag atgggtttaa ggagaagaaa ggtttagtat caccggtgac gaaggagaac 480

aaggattatg tacaactgac tgcaatatct cttgcaatta ccaaactgct tggtgcttga 540

<210>23

<211>179

<212>PRT

<213> Artificial Synthesis

<400>23

Met Lys Leu Ser Phe Ser Leu Cys Ile Phe Phe Phe Asn Leu Leu Leu

1 5 10 15

Leu Leu Gln Ala Val Ile Ser Ala Asp Ile Val Gln Gly Thr Cys Lys

20 25 30

Lys Val Ala Gln Arg Ser Pro Asn Val Asn Tyr Asp Phe Cys Ala Lys

35 40 45

SerLeu Gly Thr Asp Pro Lys Ser His Thr Ala Asp Leu Gln Gly Leu

50 55 60

Gly Val Ile Ser Ala Asn Leu Ala Ile Gln Gln Gly Ser Lys Ile Gln

65 70 75 80

Thr Phe Ile Gly Arg Ile Leu Lys Ser Lys Val Asp Pro Ala Leu Lys

85 90 95

Lys Tyr Leu Asn Asp Cys Val Gly Leu Tyr Ala Asp Ala Lys Ser Ser

100 105 110

Val Gln Glu Ala Ile Ala Asp Phe Asn Ser Lys Asp Tyr Ala Ser Ala

115 120 125

Asn Val Lys Met Ser Ala Ala Leu Asp Asp Ser Val Thr Cys Glu Asp

130 135 140

Gly Phe Lys Glu Lys Lys Gly Leu Val Ser Pro Val Thr Lys Glu Asn

145 150 155 160

Lys Asp Tyr Val Gln Leu Thr Ala Ile Ser Leu Ala Ile Thr Lys Leu

165 170 175

Leu Gly Ala

<210>24

<211>540

<212>DNA

<213> Artificial Synthesis

<400>24

atgaagcttt ccttctctct ctgtatcttc ttcttcaatc tcctcctcct ccttcaagct 60

gtaatcagcg ccgatattgt tcagggcaca tgcaagaaag ttgctcagag aagcccaaac 120

gtgaactacg atttctacgt gaaatctctt ggagcagatc ctaagagcca cactgcggat 180

cttcaaggac ttggggtcat ctcagcgaat ttagccatac agcatggatc taaaatccaa 240

acatttattg gtcgcatcgt gaaaagtaaa gtggacccag ctcttaagaa atacttgaat 300

gattgtgtgg gactttacgc tgatgcgaag tcttcagttc aagaggccat agctgacttc 360

aagtccaagg actacgcatc agctaatgtg aaaatgagtg cggctttgga cgactcagtg 420

acttgtgaag atgggtttaa ggagaagaaa ggtatagtat caccggtgac gaaggagaac 480

aaggattatg tacaactgac tgcaatatct cttgcaatta ccaaactgct tggtgcttga 540

<210>25

<211>179

<212>PRT

<213> Artificial Synthesis

<400>25

Met Lys Leu Ser Phe Ser Leu Cys Ile Phe Phe Phe Asn Leu Leu Leu

1 5 10 15

Leu Leu Gln Ala Val Ile Ser Ala Asp Ile Val Gln Gly Thr Cys Lys

20 25 30

Lys Val Ala Gln Arg Ser Pro Asn Val Asn Tyr Asp Phe Tyr Val Lys

35 40 45

Ser Leu Gly Ala Asp Pro Lys Ser His Thr Ala Asp Leu Gln Gly Leu

50 55 60

Gly ValIle Ser Ala Asn Leu Ala Ile Gln His Gly Ser Lys Ile Gln

65 70 75 80

Thr Phe Ile Gly Arg Ile Val Lys Ser Lys Val Asp Pro Ala Leu Lys

85 90 95

Lys Tyr Leu Asn Asp Cys Val Gly Leu Tyr Ala Asp Ala Lys Ser Ser

100 105 110

Val Gln Glu Ala Ile Ala Asp Phe Lys Ser Lys Asp Tyr Ala Ser Ala

115 120 125

Asn Val Lys Met Ser Ala Ala Leu Asp Asp Ser Val Thr Cys Glu Asp

130 135 140

Gly Phe Lys Glu Lys Lys Gly Ile Val Ser Pro Val Thr Lys Glu Asn

145 150 155 160

Lys Asp Tyr Val Gln Leu Thr Ala Ile Ser Leu Ala Ile Thr Lys Leu

165 170 175

Leu Gly Ala

Claims

1. A method for obtaining sycamore mutants, comprising the steps of:

2. The method of obtaining sycamore mutants of claim 1, wherein the gRNA target sequences include a first gRNA target sequence and a second gRNA target sequence, wherein,

the sequence of the first gRNA target point is shown in SEQ ID No.1 and is as follows:

5’-CAGCGCCGATATTGTTCAGG-3’；

the second gRNA target sequence is shown in SEQ ID No.2 and is:

5’-TTCTGCGCGAAGTCTCTTGG-3’。

3. the method for obtaining sycamore mutants as claimed in claim 1, wherein the method for transforming the CRISPR/Cas9 genome editing vector into seed embryos to obtain transformation products by using a particle gun method comprises the following steps:

obtaining fruit seeds in the mature period of the plant, and culturing germs;

providing a vector to be transformed, wherein the plasmid vector to be transformed comprises the plant genome editing vector and metal particles carrying the plant genome editing vector; wherein the diameter of the metal fine particles is 0.6 to 1.8 μm;

and (3) converting the carrier to be converted into the embryo by adopting a gene gun under the condition that the pressure is 450-2200 PSI to obtain a conversion product.

4. The method for obtaining sycamore mutants as claimed in claim 3, wherein in the step of providing the vectors to be transformed, the vectors to be transformed are prepared by coating the metal particles and the plant genome editing vectors at a mass ratio of 15mg:2 μ g.

5. The method for obtaining the sycamore mutant as claimed in claim 3, wherein the step of transforming the vector to be transformed into the embryo is carried out by using a gene gun under the condition of the pressure of 450-2200 PSI, and the vector to be transformed is used for transforming the embryo in the proportion of 100-500 embryos.

6. The method for obtaining sycamore mutants according to any one of claims 1 to 3, wherein the CRISPR/Cas9 vector plasmid comprises pKSE 401; and/or the presence of a gas in the gas,

the restriction enzymes include BsaI enzyme.

7. The method for obtaining the plane mutant according to any one of claims 1 to 3, wherein in the step of obtaining the plane target gene of plane, the plane target gene is a plane pollen allergen pla 1 gene of plane, and a PCR amplification reaction is performed by using a fifth primer and a sixth primer to obtain the plane pollen allergen pla 1 gene, wherein the sequence of the fifth primer is shown as SEQ ID No.3 and is: 5'-tacgcggggaacaaaacaatccaat-3', respectively; the sequence of the sixth primer is shown as SEQ ID No.4, and the sixth primer is: 5'-ccgaagagggccaaatatcataca-3' are provided.

8. The method for obtaining the sycamore mutant according to any one of claims 1 to 3, wherein in the step of performing enzyme digestion treatment on the CRISPR/Cas9 vector plasmid by using the restriction endonuclease to obtain the enzyme digested CRISPR/Cas9 vector plasmid, the reaction system of the enzyme digestion treatment comprises 1ug of the CRISPR/Cas9 vector plasmid, 10U of BsaI enzyme, and 20U L of BsaI enzyme buffer solution, and the reaction condition of the enzyme digestion treatment is that the enzyme digestion treatment is performed at 50-52 ℃ for 1-1.5 hours, and then the heating is performed at 65-68 ℃ for 10 minutes, and/or,

in the step of sequentially carrying out ligation treatment on the enzyme-digested CRISPR/Cas9 vector plasmid and the gRNA target point sequence annealing product by adopting the ligase to obtain the CRISPR/Cas9 genome editing vector, the reaction system of the ligation treatment is as follows, 10 mu L2X reaction buffer solution and 1 mu L T are added into a 20 mu L reaction system₄DNA ligase, 50ng of the vector after enzyme digestion and 3 times molar amount of annealing products of gRNA targets; and the reaction conditions of the ligation process are as follows: reacting at 37-38 ℃ for 10-15 min.

9. A sycamore mutant, which is a mutant generating mutation of a target gene, wherein the mutant generating mutation of the target gene comprises an insertion, deletion or change of nucleotide sequence of a gene segment of a gene coding region.

10. The method for obtaining the sycamore mutant as claimed in any one of claims 1 to 8 or the use of the sycamore mutant as claimed in claim 9 in sycamore breeding.