CN111424050B - Mitsubishi tree mutant, method for obtaining same and application thereof - Google Patents
Mitsubishi tree mutant, method for obtaining same and application thereof Download PDFInfo
- Publication number
- CN111424050B CN111424050B CN202010144373.1A CN202010144373A CN111424050B CN 111424050 B CN111424050 B CN 111424050B CN 202010144373 A CN202010144373 A CN 202010144373A CN 111424050 B CN111424050 B CN 111424050B
- Authority
- CN
- China
- Prior art keywords
- vector
- sycamore
- crispr
- cas9
- obtaining
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Botany (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
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
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
Suzuki (Platanus spp.) is a tree species of the deciduous tree of the genus Coulter of the family Convaliaceae. The family is only one genus, about 10-12, all produced abroad. Three types of cultivation are introduced in China: sycamore (p. occidentalis lin., usa), sycamore (p. aceifolia Willd.), and sycamore (p. orientalis lin., france). Among them, the british phoenix tree is a hybrid of the united states phoenix tree and the french phoenix tree (p. occidentalis x p. orientalis), has good heterosis, and has a history of hundreds of years in the introduction of China. Because the tree grows rapidly, the crown is big and shade, the trunk is straight, the dry bark is smooth and clean, the tree is resistant to pruning and easy to reproduce, the tree can adapt to urban environment and various soil conditions, has stronger air pollution resistance, photochemical smog resistance, benzene, ether, hydrogen sulfide and other harmful gases and good dust retention, noise reduction and air purification capabilities, is widely used as greening tree species of roads, streets and factories in various cities of the global temperate zone and subtropical zone, and enjoys the reputation of the king of street trees. In China, sycamore is widely applied to yellow river and Yangtze river basin areas as street trees and shade trees, is deeply loved by vast citizens and garden workers, becomes a garden plant which cannot be replaced by other tree species, and plays an important role in urban garden construction.
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.
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.
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 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.
Preferably, in the step of obtaining the sycamore pollen allergen plaa1 gene by performing a PCR amplification reaction using a fifth primer and a sixth primer, the PCR amplification reaction system is as follows: DNA of sycamore leaf: 5uL, fifth primer: 5uL, sixth primer: 5uL, 2 × Taq mix: 25uL, Mg2+: 1uL of glycerol: 1uL, 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, and the reaction system of the first annealing treatment is as follows: the concentration of the first primer and the second primer is 100mM, and each primer comprises 1uL and 8uL of purified water; the reaction sequence of the first annealing treatment is as follows: denaturation at 95 ℃ for 5min in a PCR instrument and slow cooling to room temperature for 1 h.
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 gRNA target sequence annealing product is obtained by a second annealing treatment, and a reaction system of the second annealing treatment is as follows: the concentration of the third primer and the fourth primer is 100mM, and each primer comprises 1uL and 8uL of purified water; the reaction sequence of the second annealing treatment is as follows: denaturation at 95 ℃ for 5min in a PCR instrument and slow cooling to room temperature for 1 h.
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 performing enzyme digestion treatment on the CRISPR/Cas9 vector plasmid by using the restriction enzyme to obtain the enzyme digested CRISPR/Cas9 vector plasmid, a reaction system of the enzyme digestion treatment is as follows: CRISPR/Cas9 vector plasmid 1ug, 10U BsaI enzyme, add BsaI enzyme buffer to 20 uL.
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 performing ligation treatment on the enzyme-cleaved CRISPR/Cas9 vector plasmid and the gRNA target sequence annealing product by using the ligase to obtain the CRISPR/Cas9 genome editing vector, a reaction system of the ligation treatment is as follows: mu.L of 2X reaction buffer, 1. mu. L T, was added to 20. mu.L of the 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 amplified PCR fragment is cloned, wherein the PCR amplification reaction system is as follows: DNA of sycamore leaf: 5uL, seventh primer: 5uL, eighth primer: 5uL, 2 × Taq mix: 25uL, Mg2+: 1uL of glycerol: 1uL, ddH2O: 12 uL; and, 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 ℃.
And selecting monoclonals for sequencing, selecting 10 monoclonals for sequencing from each gene transformation plant, comparing the sequencing result with a wild-type plant gene sequence (SEQ ID No.5) through a BLAST program of an NCBI website, and identifying the mutant genes.
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 the following steps: DNA of sycamore leaf: 5uL, fifth primer: 5uL, sixth primer: 5uL, 2 × Taq mix: 25uL, Mg2 +: 1uL of glycerol: 1uL, ddH 2O: 12 uL.
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 adopting the BsaI enzyme to obtain a first CRISPR/Cas9 vector plasmid; wherein the reaction system of the enzyme digestion treatment is as follows: CRISPR/Cas9 vector plasmid 1ug, 10U BsaI enzyme, add BsaI enzyme buffer to 20 uL; 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.
(3) Providing a T4 ligase, and adopting the T4 ligase to perform ligation treatment on the first CRISPR/Cas9 vector plasmid, the first gRNA target sequence annealing product or the second gRNA target sequence annealing product to obtain a CRISPR/Cas9 genome editing vector; wherein the reaction system of the connection treatment is as follows: 50ng of vector plasmid after enzyme digestion; gRNA target sequence annealing product 0.1 ug; 1 μ L of T4 ligase (5U); 2 μ L of T4 DNA ligase buffer; and, the reaction procedure of the ligation process is as follows: the reaction was 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 the following steps: DNA of sycamore leaf: 5uL, seventh primer: 5uL, eighth primer: 5uL, 2 × Taq mix: 25uL, Mg2+: 1uL of glycerol: 1uL, ddH2O: 12 uL; and, 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; stretching at 72 deg.C for 10min, and maintaining at 4 deg.CAnd (4) storing.
And selecting monoclonals for sequencing, selecting 10 monoclonals for sequencing from each gene transformation plant, comparing the sequencing result with a wild-type plant gene sequence (SEQ ID No.5) through a BLAST program of an NCBI website, and identifying the mutant genes.
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:
ATGAAGCTTTCCTTCTCTCTCTGTATCTTCTTCTTCAATCTCCTCCTCCTCCTTCAAGCTGTAATCAGCGCCGATATTGTTCAGGGCACATGCAAGAAAGTTGCTCAGAGAAGCCCAAACGTGAACTACGATTTCTGCGCGAAGTCTCTTGGAGCAGATCCTAAGAGCCACACTGCGGATCTTCAAGGACTTGGGGTCATCTCAGCGAATTTAGCCATACAGCAAGGATCTAAAATCCAAACATTTATTGGTCGCATCTTGAAAAGTAAAGTGGACCCAGCTCTTAAGAAATACTTGAATGATTGCGTGGGACTTTACGCTGATGCGAAGTCTTCAGTTCAAGAGGCCATAGCTGACTTCAATTCCAAGGACTACGCATCAGCTAATGTGAAAATGAGTGCGGCTTTGGACGACTCAGTGACTTGTGAAGATGGGTTTAAGGAGAAGAAAGGTTTAGTATCACCGGTGACGAAGGAGAACAAGGATTATGTACAACTGACTGCAATATCTCTTGCAATTACCAAACTGCTTGGTGCTTGA。
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 LB and RB are the left and right borders of T-DNA on a Ti plasmid, 35Sp-Cas9-NosT and 35Sp-NptII-PolyA are the expression frames of Cas9 gene and NptII gene, U6-26-T1 and U6-29-T2 express two sgrnas, 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 to A (causing the amino acid to change from cysteine C to tyrosine Y), base 140 to T (causing the amino acid to change from alanine A to valine V), base 144 to A (causing no amino acid change), base 225 to T (causing the amino acid to change from glutamine Q to histidine H), base 259 to G (causing the amino acid to change from leucine L to valine V), base 306 to T (causing the amino acid to change from C to T (causing no amino acid change), base 363 to G (causing the amino acid to change from asparagine N to lysine K), base 454 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 as 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> mutant of plane tree, method for obtaining mutant of plane tree 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 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> 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 Ala Ile 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
Lys Val 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
Ser Leu 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 Val Ile 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 (8)
1. A method for obtaining sycamore mutants, comprising the steps of:
obtaining a gene of the order of the plane of the sycames and determining a gRNA target sequence; the gRNA target sequence simultaneously contains a first gRNA target sequence and a second gRNA target sequence, wherein the first gRNA target sequence is shown as 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', respectively;
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; the CRISPR/Cas9 genome editing vector contains a first gRNA target sequence annealing product and a second gRNA target sequence annealing product;
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.
2. 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 plasmid 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 particles is 0.6-1.8 μm;
and transforming the plasmid vector to be transformed into the embryo by using a gene gun under the condition that the pressure is 450-2200 PSI to obtain a transformation product.
3. The method for obtaining the sycamore mutant according to claim 2, wherein in the step of providing the plasmid vector to be transformed, the plasmid vector to be transformed is prepared by coating the metal particles and the plant genome editing vector in a mass ratio of 15mg:2 μ g.
4. The method for obtaining the sycamore mutant as claimed in claim 2, wherein the step of transforming the plasmid 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 plasmid vector to be transformed is used for transforming the embryo in the proportion of 100-500 embryos.
5. The method of obtaining sycamore mutants of claim 1 or 2, 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.
6. The method for obtaining the sycamore mutant as claimed in claim 1 or 2, wherein in the step of obtaining the sycamore target gene, the sycamore target gene is sycamore pollen allergen plaa1 gene, and the sycamore pollen allergen plaa1 gene is obtained by performing PCR amplification reaction by using a fifth primer and a sixth primer, 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.
7. The method for obtaining the sycamore mutant as claimed in claim 1 or 2, wherein in the step of obtaining the CRISPR/Cas9 carrier plasmid after enzyme digestion by using the restriction endonuclease to perform enzyme digestion treatment on the CRISPR/Cas9 carrier plasmid, the reaction system of the enzyme digestion treatment is as follows: CRISPR/Cas9 vector plasmid 1ug, 10U BsaI enzyme, add BsaI enzyme buffer to 20 uL; the reaction conditions of the enzyme digestion treatment are as follows: carrying out enzyme digestion treatment at 50-52 ℃ for 1-1.5 hours, and then heating at 65-68 ℃ for 10 minutes; and/or the presence of a gas in the gas,
in the step of sequentially carrying out ligation treatment on the enzyme-cut CRISPR/Cas9 vector plasmid and the annealing product of the gRNA target sequence by adopting the ligase to obtain the CRISPR/Cas9 genome editing vector, a reaction system of the ligation treatment is as follows: mu.L of 2X reaction buffer, 1. mu. L T, was added to 20. mu.L of the 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.
8. Use of the method for obtaining a mutant of sycamore as claimed in any of claims 1 to 7 in sycamore breeding.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010144373.1A CN111424050B (en) | 2020-03-04 | 2020-03-04 | Mitsubishi tree mutant, method for obtaining same and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010144373.1A CN111424050B (en) | 2020-03-04 | 2020-03-04 | Mitsubishi tree mutant, method for obtaining same and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111424050A CN111424050A (en) | 2020-07-17 |
| CN111424050B true CN111424050B (en) | 2022-02-22 |
Family
ID=71547570
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010144373.1A Active CN111424050B (en) | 2020-03-04 | 2020-03-04 | Mitsubishi tree mutant, method for obtaining same and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111424050B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105821075A (en) * | 2016-04-22 | 2016-08-03 | 湖南农业大学 | Establishment method of caffeine synthetase CRISPR/Cas9 genome editing vector |
| CN110592134A (en) * | 2018-05-24 | 2019-12-20 | 中国科学院上海生命科学研究院 | Application of SDG40 gene or coding protein thereof |
-
2020
- 2020-03-04 CN CN202010144373.1A patent/CN111424050B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105821075A (en) * | 2016-04-22 | 2016-08-03 | 湖南农业大学 | Establishment method of caffeine synthetase CRISPR/Cas9 genome editing vector |
| CN110592134A (en) * | 2018-05-24 | 2019-12-20 | 中国科学院上海生命科学研究院 | Application of SDG40 gene or coding protein thereof |
Non-Patent Citations (6)
| Title |
|---|
| CRISPR/Cas9 in allergic and immunologic disease;Goodman M.A.et al;《Expert Review of Clinical Immunology》;20161024;第13卷(第1期);5-9 * |
| CRISPR/Cas9技术发展及其应用进展;欧阳乐军 等;《西北农林科技大学学报（自然科学版）》;20191031;第47卷(第10期);34-40 * |
| Gene editing the major cat allergen, Fel d1, using CRISPR-Cas9;Brackett N.et al;《J Allergy Clin Immunol》;20200229;第145卷(第2期);1 * |
| 应用CRISPR/Cas9技术在杨树中高效敲除多个靶基因;刘婷婷 等;《遗传》;20151031;第37卷(第10期);1044-1052 * |
| 法国梧桐变应原Pla a1和Pla a2基因的克隆和原核表达;程训佳 等;《中华医学会2011年中国***反应学术会议论文集》;20111014;129 * |
| 编码二球悬铃木花粉主要变应原Plala cDNA的基因克隆及性质研究;齐名 等;《第八届全国中西医结合实验医学研讨会论文汇编》;20061001;153-157 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111424050A (en) | 2020-07-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111549055A (en) | Application of maize MS2 gene | |
| JPH05507415A (en) | Rose plants and their production methods and genetic transformation | |
| CN113528537A (en) | NtQPT2 gene mutant for reducing nicotine content in tobacco leaves and application thereof | |
| CN110862445B (en) | NtOEP1 gene influencing tobacco pigment content and application thereof | |
| CN107058335A (en) | A kind of gene mutation bodies of NteIF4E 1 of generation PVY resistances and its application | |
| CN112143738B (en) | Tobacco receptor protein gene and cloning method and application thereof | |
| CN111424050B (en) | Mitsubishi tree mutant, method for obtaining same and application thereof | |
| CN113265403A (en) | Soybean Dt1 gene editing site and application thereof | |
| CN102586250A (en) | Promoter of terpene floral scent gene Hctps1 in hedychium gardneranum and application of promoter | |
| CN107177603A (en) | Tobacco growing element transport protein NtPIN4 and its application | |
| CN116732070A (en) | CGBE single base editor capable of realizing base transversion and application thereof | |
| CN114657188B (en) | Gene PK1 for regulating cadmium accumulation of rice, protein and application thereof | |
| CN114854766B (en) | NtAIDP1 gene mutant for reducing nicotine content in tobacco leaves and application thereof | |
| CN113461794B (en) | Kit and method for regulating seed germination and application thereof | |
| CN112646016B (en) | Gene and method for changing flowering period of corn | |
| CN113151296B (en) | Tobacco heat shock protein related gene and application thereof | |
| WO2021174441A1 (en) | Platanus mutant, method for obtaining platanus mutant, and application | |
| CN107573411A (en) | Application of the wheat TaZIM1 7A albumen in crop heading stage is regulated and controled | |
| CN111560055B (en) | Application of rice gene OsLAT3 in regulation of absorption and accumulation of diquat | |
| CN107459564A (en) | The application of rice ZYGO1 albumen and its encoding gene in pollen fertility is regulated and controled | |
| CN113563439A (en) | Fruit shape development related protein and coding gene and application thereof | |
| CN114181966A (en) | Method for creating corn dwarfing material based on Zm00001d008708 gene | |
| CN112724216B (en) | Gene and method for changing flowering period of corn | |
| CN112661823B (en) | Gene and method for changing flowering period of corn | |
| CN112646014B (en) | Gene and method for changing flowering period of corn |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |