CN111909938B - Peanut mutant gene, protein coded by same and preparation method of peanut mutant - Google Patents
Peanut mutant gene, protein coded by same and preparation method of peanut mutant Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, and particularly relates to a peanut mutant gene, a protein coded by the peanut mutant gene and a preparation method of a peanut mutant. The nucleotide sequence of the peanut mutant gene is shown as SEQ ID NO. 1 and SEQ ID NO. 3. The peanut allergen gene Arah1 is edited by a genome editing system to generate deletion, insertion or base mutation of a gene sequence, so that targeted mutation of the peanut allergen gene Arah1 is completed, amino acid of a coded protein of the peanut allergen gene Arah is changed, the change of phenotype is further influenced, the peanut mutant with low allergenicity is obtained, targeted mutation of a gene locus can be realized, randomness, contingency and agnosticity in the traditional mutation method are avoided, the peanut mutant has the advantages of high mutation speed and high efficiency, and has great significance for reducing or avoiding allergy caused by peanuts.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a peanut mutant gene Arah1a-3-1-1, a peanut mutant gene Arah1b-7-1-8, a protein coded by the same, a preparation method of a peanut mutant, and a primer for detecting the peanut mutant gene.
Background
Peanuts are important oil and economic crops in China, the total output and the consumption account for 40 percent of the world, the export accounts for 30 percent of the world, and the peanut oil and economic crops have strong competitiveness internationally. The peanut seeds contain a large amount of fatty acid, protein, abundant vitamins and other nutrient substances, can be used for extracting oil, can be directly eaten, and can be processed into various foods such as candies, cakes and the like, thereby being well popular among the masses. The average oil content of the peanut seeds is 51 percent, the peanut seeds are mainly unsaturated fatty acid which is beneficial to human health, wherein oleic acid and linoleic acid account for 80 percent of the total oil content, the peanut seeds have good quality, rich nutrition and fragrant smell, and are one of the main edible oils in the market.
The protein content of peanut seeds is about 25% by weight, of which 90% is basic protein consisting of arachin and congou. The peanut protein has similar nutritive value to animal protein, but does not contain cholesterol, is a high-nutrition plant protein resource, and is the third place in the plant protein resource in the world. However, peanuts are considered to be one of the most serious food allergens in the world, and the number of people allergic to peanuts is about 0.5-1.1% of the total population. Among the eight major food allergens (milk, eggs, fish, shellfish, peanuts, soybeans, nuts and wheat), peanut allergy accounts for 10-47%. Peanut allergy has the characteristics of lethality, low-dose allergenicity, durability and the like. Peanut anaphylaxis is manifested by mild urticaria, facial swelling, abdominal cramps, asthma, allergic dermatitis, anaphylactic shock, and other symptoms, and can cause life risks if diagnosis and treatment are not timely performed. Human allergies to other foods occur most often in the juvenile stage and generally disappear with increasing age, but peanut allergies are different from other food allergies, and most people (-80%) have lifelong allergies. Because of the popularization of peanut food and peanut oil, and because an extremely small amount of peanut allergen (0.1-10 mg) can cause allergy, the allergy caused by peanuts is difficult to avoid, and the allergy caused by peanuts tends to rise. Therefore, the peanut allergy problem is very important to the economy, the society and the health of the public.
At present, 11 peanut allergy-causing proteins are found to be Arah 1-11. According to the WHO/IUIS classification, 7 of these proteins are seed storage proteins, including Arah1, Arah3 and Arah4 of the CUPIN family, Arah2, Arah6 and Arah7 of the Conglutins family, Arah5 of the profilins protein, Arah8 in the PR protein, non-specific lipid transfer proteins (nsLTPs) Arah9 and Oleosins proteins Arah10 and Arah 11. Among them, Arah1, Arah2, Arah3, Arah4, and Arah6 are major allergy proteins because they can cause more than 50% of allergic reactions. Arah3 and Arah4 have very high similarity in protein sequence, and have similar physicochemical properties such as molecular weight (60 Kda), isoelectric point (5.5), etc., and are generally considered as a protein isoform Arah 3/4.
Arah1 accounts for 12-16% of the total peanut protein, is a glycoprotein with molecular weight of 63.5kDa and isoelectric point of 4.55, and belongs to cupin family. Burk et al showed that Arah1 appears as a trimeric complex; clear beta sheets can be found at the level of secondary structure, wherein alpha helix is 31%, beta sheet is 36%, and no coil structure is 33%; when the purified Ara h1 is heated to 80-90 ℃, the secondary structure folding is intensified, and the solubility is reduced. The quaternary structure level is a complex containing 3 monomers. Research results show that the antigenic determinants of Arah1 are amino acid sequences at positions 25-34, 89-98, 393-402, 498-507 and 597-606, wherein the dominant antigenic determinant is peptide at positions 498-507. The key amino acids for the sensitization of Arah1 are arginine at positions 499, 503 and alanine at position 502. Arah1 has the characteristics of strong heat stability, enzymolysis resistance, indigestibility and the like, the main antigen structure of Arah1 cannot be damaged at high temperature, and Arah1 protein after high-temperature denaturation still has similar IgE binding activity to natural Arah1 protein. It has been shown that although the heat-treated allergen Arah1 has changed its protein conformation, its allergenicity is unchanged. Arah1 can still activate dendritic antigen presenting cells (antigen presenting cells) after proteolysis, and can also cause anaphylaxis to the body. Arah1 was also extracted from peanuts and tested for ELISA inhibition, indicating that milling did not alter the binding capacity of Ara h1 to IgE.
Therefore, current treatments for peanut allergy sufferers are mainly prophylactic, i.e. avoid contact with peanut allergens. However, peanuts are added into a lot of food at home and abroad at present, and the standardization of food labels is not yet realized in China for a moment, so that peanut allergy patients are likely to contact peanut allergens. Peanut allergy has the characteristics of lethality, low-dose sensitization, durability and the like, so that great challenge is brought to prevention of peanut allergy. At present, the peanut allergy problem is generally solved by the following three methods: processing, immunotherapy and genetic engineering.
Although the allergenic properties in peanuts are relatively stable, some processing methods can have a significant impact on them. The allergenicity of peanut allergens can be changed by different physical methods such as heat treatment, radiation treatment and the like and by different enzymatic chemical methods, but the methods have great limitations. Although these methods can destroy the structure of allergen protein, it has been found that glycosylation products produced by thermal processing are also important food allergens because allergy is caused by multiple epitopes on allergen protein and single epitope polypeptide can still cause allergic reactions. Therefore, the problem of peanut allergy is difficult to solve fundamentally.
Desensitization therapy, also known as specific immunotherapy, or allergen immunotherapy (allergen immunotherapy), is a treatment that utilizes immunological principles to treat immune allergies. In clinic, after determining the allergen of an allergic disease patient, the allergen is prepared into an allergen extracting solution and prepared into preparations with different concentrations, and the preparations are repeatedly contacted with the patient through repeated injection or other administration routes, the dosage is increased from small to large, and the concentration is increased from low to high, so that the tolerance of the patient to the allergen is improved, and when the patient is contacted with the allergen again, the phenomenon of allergy is not generated or is relieved. Is the only treatment that can affect the natural course of allergic disease except for avoiding exposure to allergens. However, the traditional specific immunotherapy has high risk of desensitization by crude antigen extract, and may cause strong allergic reaction and even death.
Recently, the development of plant genetic engineering reduces plant allergens through RNAi, antisense RNA or a technology of directionally inducing local mutation of genome (TILLING), and provides a possibility for fundamentally solving the problem of peanut allergy. The most important allergen Arah2 of peanut is silenced through RNAi technology, so that Arah2 allergen is obviously reduced in transgenic peanut seeds. Western immunoblotting shows that Arah2 cannot be detected in crude protein of some transgenic T0 seeds, the IgE binding capacity of the transgenic seeds is obviously reduced, and feasibility of relieving peanut anaphylactic reaction by utilizing RNAi technology is shown. Also studies have used particle gun methods to silence both peanut protein allergens Arah2 and Arah6 to obtain 3 independent transformation lines, 2 of which are single copy transgene integration and 1 of which contains multiple copies of the transgene. Expression of Arah2 was inhibited in all 3 lines, and expression of Arah6 was reduced in only 2 lines. The seed grain weight and the germination rate of transgenic and non-transgenic segregation progeny have no obvious difference, and the aspergillus flavus infection and the fungus growth condition of the transgenic line and the control have no obvious difference. RNA technology has been successful in reducing some important plant allergens, such as rice 14-16kD protein allergen reduction by antisense RNA technology, and soybean major allergen P34, apple allergen Mald1, tomato allergen Lyce1 and e3 silencing by RNAi technology. However, transgenic crops produced by genetic engineering methods are difficult to be applied to large-scale production in the near future due to the current concerns about transgenic foods. Therefore, a method for producing hypoallergenic peanuts without transgenic components is urgently needed. The TILLING technology can knock out allergen genes or change antigenic determinants through mutation, and then analyze gene mutation of a target area through a PCR technology and CelI enzymolysis. There are studies to obtain an Arah2.02 initiation codon mutant by TILLING technology, which does not detect the protein in the offspring, and also to obtain a mutation of Arah1.02 gene, so that the translation of the gene protein is stopped early, and 63.5KD protein is obtained instead of normal 70.3 KD. Although this technique is theoretically possible, it requires a great deal of work to mutate numerous peanut allergen genes.
Disclosure of Invention
The invention aims to provide a peanut mutant gene Arah1a-3-1-1, a peanut mutant gene Arah1b-7-1-8, a protein coded by the same, a preparation method of a peanut mutant and a primer for detecting the peanut mutant gene, and aims to solve the technical problem of high workload in the conventional method for mutating a peanut allergen gene.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
the invention provides a peanut mutant gene Arah1a-3-1-1 and a protein coded by the same, wherein the nucleotide sequence of the peanut mutant gene Arah1a-3-1-1 is shown as SEQ ID NO. 1, and the amino acid sequence of the coded protein is shown as SEQ ID NO. 2.
On the other hand, the invention provides a peanut mutant gene Arah1b-7-1-8 and a protein coded by the same, wherein the nucleotide sequence of the peanut mutant gene Arah1b-7-1-8 is shown as SEQ ID NO. 3, and the amino acid sequence of the coded protein is shown as SEQ ID NO. 4.
The invention further provides a preparation method of the peanut mutant, which comprises the following steps:
providing a peanut allergen gene Arah1, an amplification primer, a gene editing carrier and peanut embryogenic callus;
Performing PCR amplification by using the peanut allergen gene Arah1 as a template through the amplification primer to obtain a coding region sequence Arah1a of the peanut allergen gene Arah1 and a coding region sequence Arah1b of the peanut allergen gene Arah 1;
taking homologous sequence regions of the Arah1a of the peanut allergen gene Arah1 coding region sequence and Arah1b of the peanut allergen gene Arah1 coding region sequence as CRISPR targets to obtain sgRNA T1 and sgRNA T2;
respectively synthesizing a sense strand and an antisense strand of the sgRNA T1 and the sgRNA T2 to obtain a double-stranded DNA T1 and a double-stranded DNA T2;
inserting the double-stranded DNA T1 into an expression frame of the sgRNA T1, and then cloning the double-stranded DNA T1 into the gene editing vector to obtain a gene editing vector T1;
inserting the double-stranded DNA T2 into an expression frame of the sgRNA T2, and then cloning the double-stranded DNA T2 into the gene editing vector to obtain a gene editing vector T2;
performing gene transformation on the peanut embryonic callus by at least one of the gene editing vector T1 and the gene editing vector T2, and culturing to obtain a peanut mutant;
the nucleotide sequence of the peanut mutant is shown as SEQ ID NO 1 and/or SEQ ID NO 3, the nucleotide sequence of the sgRNA T1 is shown as SEQ ID NO 5, and the nucleotide sequence of the sgRNA T2 is shown as SEQ ID NO 6.
The invention also provides application of the peanut mutant obtained by the peanut mutant gene Arah1a-3-1-1, the peanut mutant gene Arah1b-7-1-8 or the preparation method of the peanut mutant in low-sensitization peanut variety breeding.
The last aspect of the invention provides a primer for detecting peanut mutant genes, and the nucleotide sequence of the primer is shown as SEQ ID NO. 9-10.
The peanut mutant gene Arah1a-3-1-1 provided by the invention has 2 mutant sites, namely, a 52 th base is mutated from T to G, and the encoded amino acid is mutated from serine (S) to alanine (A); deletion of the 85 th base T causes frame shift mutation, so that the translation of the protein is terminated early, and a polypeptide containing 50 amino acids is formed. Compared with a peanut allergen gene Arah1, the protein coded by the peanut mutant gene Arah1a-3-1-1 provided by the invention does not contain peanut allergy protein Arah1, and a peanut mutant with low allergenicity can be obtained through breeding, so that the problem of peanut allergy is fundamentally solved.
The peanut mutant gene Arah1b-7-1-8 provided by the invention has 2 mutant sites, namely, the deletion of the 10 th base A, and the insertion of GAG at the same time, thereby causing frame shift mutation, leading the translation of protein to be terminated in advance and forming a polypeptide containing 8 amino acids. Compared with the peanut allergen gene Arah1, the protein coded by the peanut mutant gene Arah1b-7-1-8 does not contain peanut allergy protein Arah1, and a peanut mutant with low allergenicity can be obtained through breeding, so that the problem of peanut allergy is fundamentally solved.
According to the preparation method of the peanut mutant, through a CRISPR/Cas9 genome editing system, Cas9 enzyme guided by sgRNA cuts a peanut allergen gene Arah1 at a gene target point, and deletion, insertion or base mutation of a gene sequence is generated after DNA repair, so that the targeted mutation of the peanut allergen gene Arah1 is completed, the amino acid of the encoded protein of the peanut allergen gene Arah1 is changed, the change of the phenotype is influenced, and the peanut mutant with low allergenicity is obtained. The preparation method provided by the invention can realize targeted mutation of the gene locus, avoids randomness, contingency and agnostic property in the traditional mutation method, has the advantages of high mutation speed and high efficiency, and has great significance for reducing or avoiding allergy caused by peanuts.
Because the proteins coded by the peanut mutant gene Arah1a-3-1-1 and the peanut mutant gene Arah1b-7-1-8 do not contain peanut allergic protein Arah1, and the peanut mutant prepared by the preparation method of the peanut mutant has low allergenicity, when the mutant gene or the peanut mutant is applied to peanut variety breeding, a peanut variety with low allergenicity can be obtained, and the peanut mutant has an important effect on improving peanut quality.
The primer for detecting the peanut mutant gene provided by the invention can be used for accurately and quickly identifying the mutant gene and the mutant site in the peanut mutant plant by amplifying the genomic DNA of the peanut mutant plant as a template and comparing the amplified product with the gene sequence of a wild plant, so that the accuracy of peanut breeding character identification can be obviously improved, and the breeding process can be accelerated.
Drawings
FIG. 1 shows the gene homology sequence region and CRISPR targets T1 and T2 of Arah1a and Arah1b gene sequences of the invention;
FIG. 2 is a flow chart of the construction of the genome editing vector of the present invention;
fig. 3 is a flow diagram of the present invention for making peanut mutants by CRISPR/CAS9 genome editing;
FIG. 4 is a base comparison graph of the peanut allergen gene Arah1a and the mutant gene Arah1a-3-1-1 according to the present invention;
FIG. 5 is a base comparison graph of the peanut allergen gene Arah1b and the mutant gene Arah1 b-7-1-8.
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 are clearly and completely described, and the embodiments described below are a part of the embodiments of the present invention, but not all of the 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. Those whose specific conditions are not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In the description of the present invention, the term "and/or" describes an association relationship of associated objects, which means that there may be three relationships, for example, a and/or B, which may mean: a alone, A and B together, and B alone. Wherein A and B can be singular or plural. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
It should be understood that the weight of the related components mentioned in the embodiments of the present invention may not only refer to the specific content of each component, but also represent the proportional relationship of the weight among the components, and therefore, it is within the scope of the disclosure that the content of the related components is scaled up or down according to the embodiments of the present invention. Specifically, the weight described in the embodiments of the present invention may be a unit of mass known in the chemical field such as μ g, mg, g, kg, etc.
In addition, unless the context clearly uses otherwise, an expression of a word in the singular is to be understood as including the plural of the word. The terms "comprises" or "comprising" are intended to specify the presence of stated features, quantities, steps, operations, elements, portions, or combinations thereof, but are not intended to preclude the presence or addition of one or more other features, quantities, steps, operations, elements, portions, or combinations thereof.
It should be noted that the molecular biology experimental methods not specifically described in the examples of the present invention are all performed according to the specific methods listed in "molecular cloning experimental manual" (third edition) j. sambrook, or according to the kit and product instructions; the relevant reagents and biological materials, unless otherwise specified, are commercially available.
The embodiment of the invention provides a peanut mutant gene Arah1a-3-1-1, the nucleotide sequence of which is shown in SEQ ID NO. 1. Correspondingly, the amino acid sequence of the protein (peanut mutant 3-1-1) coded by the peanut mutant gene Arah1a-3-1-1 is shown as SEQ ID NO: 2.
The nucleotide sequence of the peanut mutant gene Arah1a-3-1-1 (SEQ ID NO: 1):
in SEQ ID NO. 1, the new gene coding region is underlined, and the start codon ATG and the stop codon TGA are bold.
Amino acid sequence of peanut mutant 3-1-1 (SEQ ID NO: 2):
MRGRVSPLMLLLGILVLASVSATQAKSPTGKQRTPAPRGASR VVNRNRTT*
the peanut mutant gene Arah1a-3-1-1 provided by the embodiment of the invention has 2 mutant sites, wherein the 52 th nucleotide is mutated from T to G, so that the coded amino acid is mutated from serine (S) to alanine (A); deletion of nucleotide 85, T, results in a frame shift mutation that prematurely terminates protein translation to form a 50 amino acid polypeptide. Compared with the peanut allergen gene Arah1, the protein coded by the peanut mutant gene Arah1a-3-1-1 provided by the embodiment of the invention does not contain peanut allergy protein Arah1, and a peanut mutant with low allergenicity can be obtained through breeding, so that the problem of peanut allergy is fundamentally solved.
The embodiment of the invention also provides a peanut mutant gene Arah1b-7-1-8, the nucleotide sequence of which is shown in SEQ ID NO. 3. Correspondingly, the amino acid sequence of the protein (peanut mutant 7-1-8) coded by the peanut mutant gene Arah1b-7-1-8 is shown as SEQ ID NO. 4.
The nucleotide sequence of the peanut mutant gene Arah1b-7-1-8 (SEQ ID NO: 3):
in SEQ ID NO 3, the new gene coding region is underlined, and the start codon ATG and the stop codon TGA are bold.
Amino acid sequence of peanut mutant 7-1-8 (SEQ ID NO: 4):
MRGEGFLH*
the peanut mutant gene Arah1b-7-1-8 provided by the embodiment of the invention has 2 mutation sites, namely, the deletion of the 10 th nucleotide A, and the insertion of GAG simultaneously to cause frame shift mutation, so that the translation of protein is terminated early, and a polypeptide containing 8 amino acids is formed. Compared with the peanut allergen gene Arah1, the protein coded by the peanut mutant gene Arah1b-7-1-8 provided by the embodiment of the invention does not contain peanut allergy protein Arah1, and a peanut mutant with low allergenicity can be obtained through breeding, so that the problem of peanut allergy is fundamentally solved.
The peanut mutants 3-1-1 and 7-1-8 provided by the embodiment of the invention can be prepared by the following preparation method.
Correspondingly, the embodiment of the invention also provides a preparation method of the peanut mutant, which comprises the following steps:
s1, providing a peanut allergen gene Arah1, an amplification primer, a gene editing carrier and peanut embryogenic callus;
s2, performing PCR amplification by using a peanut allergen gene Arah1 as a template through an amplification primer to obtain a peanut allergen gene Arah1 coding region sequence Arah1a and a peanut allergen gene Arah1 coding region sequence Arah1 b;
s3, taking a homologous sequence region of a coding region sequence Arah1a of a peanut allergen gene Arah1 and a coding region sequence Arah1b of a peanut allergen gene Arah1 as CRISPR targets to obtain sgRNA T1 and sgRNA T2;
s4, respectively synthesizing sense chains and antisense chains of sgRNA T1 and sgRNA T2 to obtain double-stranded DNA T1 and double-stranded DNA T2;
s5, inserting the double-stranded DNA T1 into an expression frame of sgRNA T1, and then cloning the double-stranded DNA T1 into a gene editing vector to obtain a gene editing vector T1;
s6, inserting the double-stranded DNA T2 into an expression frame of sgRNA T2, and then cloning the double-stranded DNA T2 into a gene editing vector to obtain a gene editing vector T2;
s7, performing gene transformation on the peanut embryogenic callus by at least one of a gene editing vector T1 and a gene editing vector T2, and culturing to obtain a peanut mutant;
The nucleotide sequence of the peanut mutant is shown in SEQ ID NO. 1 and/or SEQ ID NO. 3, the nucleotide sequence of sgRNA T1 is shown in SEQ ID NO. 5, and the nucleotide sequence of sgRNA T2 is shown in SEQ ID NO. 6.
It should be noted that, although the steps S5 and S6 describe the preparation of the peanut mutants in a specific order, it is not required that the steps be performed in the specific order, and the steps may be performed simultaneously or sequentially.
Nucleotide sequence of sgRNA T1 (SEQ ID NO: 5):
CAGAGAACCCCTGCGCCCAGAGG
nucleotide sequence of sgRNA T2 (SEQ ID NO: 6):
CCTCCAGGGGAGCGGACACGTGG
according to the preparation method of the peanut mutant provided by the embodiment of the invention, through a CRISPR/Cas9 genome editing system, Cas9 enzyme guided by sgRNA cuts a peanut allergen gene Arah1 at a gene target point, and deletion, insertion or base mutation of a gene sequence is generated after DNA repair, so that the targeted mutation of the peanut allergen gene Arah1 is completed, the amino acid of the encoded protein of the peanut allergen gene Arah1 is changed, the change of the phenotype is influenced, and the peanut mutant with low allergenicity is obtained. The preparation method provided by the embodiment of the invention can realize targeted mutation of the gene locus, avoids randomness, contingency and agnostic property in the traditional mutation method, has the advantages of high mutation speed and high efficiency, and has great significance for reducing or avoiding allergy caused by peanuts.
The preparation method of the peanut mutant provided by the embodiment of the invention and the flow for identifying the obtained mutant are shown in figure 3. Specifically, in S1, GenBank accession No. AB440237.1 of peanut allergen gene Arah 1. The protein Arah1 coded by the gene is shown to be one of the main sensitization proteins in peanuts, and the main antigen structure cannot be damaged by high-temperature and physical grinding treatment, so that the allergenicity cannot be changed. Therefore, in the embodiment of the invention, the peanut allergen gene Arah1 is edited by a CRISPR/CAS9 genome editing system, and the obtained peanut mutant can change the allergenicity of the protein Arah1 and reduce or avoid the allergy caused by peanuts.
Amplification primers, used in the present examples for PCR amplification of the peanut allergen gene Arah 1. In some embodiments, the amplification primers are Arah1-F (nucleotide sequence shown in SEQ ID NO:7) and Arah1-R (nucleotide sequence shown in SEQ ID NO:8), which are designed based on the first exon sequence of the peanut allergen gene Arah 1.
The nucleotide sequence of Arah1-F (SEQ ID NO: 7):
ATGAGAGGGAGGGTTTCTCCAC
the nucleotide sequence of Arah1-R (SEQ ID NO: 8):
CTTGCTGGATAACAAGGATGTTATCAG
the gene editing vector is used for preparing the peanut mutant by adopting the CRISPR/Cas9 gene editing system, and correspondingly, the used vector is the gene editing vector. It is understood that the gene editing vector contains a Cas9 system, and after being transferred into a host cell, the artificially constructed guide RNA generated by the gene editing vector can guide the Cas9 protein to cut a specific DNA sequence of the host cell, thereby playing a role in gene editing. In some embodiments, a pKSE401 binary vector is selected as the gene editing vector. The reason is that the peanut belongs to dicotyledonous plants, the pKSE401 binary vector is a gene editing vector suitable for the dicotyledonous plants, genome editing can be efficiently induced, and the vector is fused with green fluorescent protein, so that efficient screening in the follow-up process is facilitated.
Peanut embryogenic callus, in the present examples, was used to induce peanut mutants.
In S2, peanut allergen gene Arah1 is used as a template, PCR amplification is carried out through the amplification primers Arah1-F and Arah1-R, and a coding region sequence Arah1a of the peanut allergen gene Arah1 and a coding region sequence Arah1b of the peanut allergen gene Arah1 are obtained. In some embodiments, the efficiency of the PCR reaction can be improved and non-specific amplification can be avoided by optimizing the system and reaction conditions of the PCR reaction. Specifically, the reaction system for PCR amplification comprises double distilled water (ddH) per 50. mu.l of PCR amplification2O) 13. mu.l, amplification primers 5. mu.l (2.5. mu.l each of Arah1-F and Arah 1-R), peanut allergen gene Arah 15. mu.l, 2 XTaq mix 25. mu.l, Mg 2+1. mu.l and glycerol 1. mu.l; the reaction conditions for PCR amplification were: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 56 deg.C for 30s, and extension at 72 deg.C for 1min, circulating for 40 times, completely extending at 72 deg.C for 10min, and storing at 4 deg.C.
By amplification, two sequences of coding regions of peanut allergen genes Arah1 were obtained, two subgenomic homologous genes from peanut A and B, respectively: the sequence Arah1a (the nucleotide sequence is shown as SEQ ID NO: 11) of the coding region sequence Arah1 of the peanut allergen gene and the sequence Arah1b (the nucleotide sequence is shown as SEQ ID NO: 12) of the coding region sequence Arah1 of the peanut allergen gene. By sequencing, the sequence Arah1a had 99% homology to the mRNA of the peanut allergen Arah1(LOC112711772) in the NCBI database and the sequence Arah1b had 100% homology to the mRNA of the peanut allergen Arah1(LOC112776552) in the NCBI database.
Nucleotide sequence of the coding region sequence Arah1a of peanut allergen gene Arah1 (SEQ ID NO: 11):
ATGAGAGGGAGGGTTTCTCCACTGATGCTGTTGCTTGGGATCCTTGTCCTGTCTTCAGTTTCTGCAACGCAGGCCAAGTCACCTTACCGGAAAACAGAGAACCCCTGCGCCCAGAGGTGCCTCCAGAGTTGTCAACAGGAACCGGACGACTTGAAGCAAAAGGCATGCGAGTCTCGCTGCACCAAGCTCGAGTATGATCCTCGTTGTGTCTATGACACTGGCGCCACCAACCAACGTCACCCTCCAGGGGAGCGGACACGTGGCCGCCAACCCGGAGACTACGATGATGACCGCCGTCAACCCCGAAGAGAGGAAGGAGGCCGATGGGGACCAGCTGAACCGAGGGAGCGTGAAAGAGAAGAAGACTGGAGACAACCAAGAGAAGATTGGAGGCGACCAAGTCATCAGCAGCCACGGAAAATAAGGCCCGAAGGAAGAGAAGGAGAACAAGAGTGGGGAACACCAGGCAGCGAGGTGAGGGAAGAAACATCACGGAACAACCCTTTCTACTTCCCGTCAAGGCGGTTTAGCACCCGCTACGGGAACCAAAACGGTAGGATCCGCGTCCTGCAGAGGTTTGACCAAAGGTCAAAGCAGTTTCAGAATCTCCAGAATCACCGTATTGTGCAGATCGAGGCCAGACCTAACACTCTTGTTCTTCCCAAGCACGCTGATGCTGATAACATCCTTGTTATCCAGCAAG
nucleotide sequence of the coding region sequence Arah1b of peanut allergen gene Arah1 (SEQ ID NO: 12):
ATGAGAGGGAGGGTTTCTCCACTGATGCTGTTGCTAGGGATCCTTGTCCTGGCTTCAGTTTCTGCAACGCATGCCAAGTCATCACCTTACCAGAAGAAAACAGAGAACCCCTGCGCCCAGAGGTGCCTCCAGAGTTGTCAACAGGAACCGGATGACTTGAAGCAAAAGGCATGCGAGTCTCGCTGCACCAAGCTCGAGTATGATCCTCGTTGTGTCTATGATCCTCGAGGACACACTGGCACCACCAACCAACGTTCCCCTCCAGGGGAGCGGACACGTGGCCGCCAACCCGGAGACTACGATGATGACCGCCGTCAACCCCGAAGAGAGGAAGGAGGCCGATGGGGACCAGCTGGACCGAGGGAGCGTGAAAGAGAAGAAGACTGGAGACAACCAAGAGAAGATTGGAGGCGACCAAGTCATCAGCAGCCACGGAAAATAAGGCCCGAAGGAAGAGAAGGAGAACAAGAGTGGGGAACACCAGGTAGCCATGTGAGGGAAGAAACATCTCGGAACAACCCTTTCTACTTCCCGTCAAGGCGGTTTAGCACCCGCTACGGGAACCAAAACGGTAGGATCCGGGTCCTGCAGAGGTTTGACCAAAGGTCAAGGCAGTTTCAGAATCTCCAGAATCACCGTATTGTGCAGATCGAGGCCAAACCTAACACTCTTGTTCTTCCCAAGCACGCTGATGCTGATAACATCCTTGTTATCCAGCAAG
in S3, a Blast alignment is performed on the Arah1a coding region sequence of peanut allergen gene Arah1 and the Arah1b coding region sequence of peanut allergen gene Arah1, so as to obtain a gene homologous sequence region, and two small guide RNA sequences (sgRNA T1 and sgRNA T2) are designed in the homologous sequence region and are used as CRISPR target T1 and target T2 (as shown in fig. 1). Wherein the nucleotide sequence of sgRNA T1 is shown in SEQ ID NO. 5, and the nucleotide sequence of sgRNA T2 is shown in SEQ ID NO. 6.
Nucleotide sequence of sgRNA T1 (SEQ ID NO: 5):
CAGAGAACCCCTGCGCCCAGAGG
nucleotide sequence of sgRNA T2 (SEQ ID NO: 6):
CCTCCAGGGGAGCGGACACGTGG
in the CRISPR target of the embodiments of the present invention, the steps are illustrated by designing the gene homologous sequence region, which is not a limitation on the selection of the target sequence of the present invention. In fact, the target may be any sequence in the coding region of a gene, as well as upstream or downstream regulatory sequences, such as promoter sequences, enhancer sequences, etc., which influence the expression of the gene. In addition, the number of the target points can be 1, or 2 or more, and when in use, the target points can be used individually or together.
In S4, a sense strand and an antisense strand of the two sgRNA sequences with a linker are synthesized based on the sgRNA T1 and the sgRNA T2, respectively, and a double-stranded DNA T1 and a double-stranded DNA T2 are obtained after annealing in a mixed manner. Wherein the sequence of the double-stranded DNA T1 is as follows:
5’-ATTGCAGAGAACCCCTGCGCCCAGAGG
GTCTCTTGGGGACGCGGGTCTCCCAAA-5’
the sequence of the double-stranded DNA T2 is as follows:
5’-ATTGCCTCCAGGGGAGCGGACACGTGG
GGAGGTCCCCTCGCCTGTGCACCCAAA-5’
in S5, the double-stranded DNA T1 was inserted into the expression cassette of sgRNA T1 (insertion site is BsaI), and then cloned into a gene editing vector, resulting in a gene editing vector T1.
In S6, the double-stranded DNA T2 was inserted into the expression cassette of sgRNA T2 (insertion site is BsaI), and then cloned into a gene editing vector, resulting in a gene editing vector T2.
FIGS. 2 show the construction schemes of the gene-editing vectors T1 and T2 of S5 and S6.
In S7, peanut embryogenic callus is subjected to gene transformation by using the gene editing vector T1 obtained from S5 and/or the gene editing vector T2 obtained from S6, and the peanut mutants are obtained by culturing. The gene editing vector T1 and the gene editing vector T2 can be transformed separately or in a mixed way, and can be cloned to the same vector for transformation or more targets are designed for transformation, and simultaneously, a plurality of genes or gene loci are knocked out, and a vector containing more than 2 targets is designed. The specific method can be performed by referring to the published method (Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ.A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant biol.2014Nov29; 14(1): 327), which is not described herein again. In addition, besides the CRISPR/Cas9 gene editing system, gene mutation can be carried out by TALEN technology, and the principle is similar to that of the CRISPR/Cas9 gene editing system of the embodiment of the invention.
Correspondingly, the embodiment of the invention also provides application of the peanut mutant prepared by the peanut mutant gene Arah1a-3-1-1, the peanut mutant gene Arah1b-7-1-8 or the preparation method of the peanut mutant in breeding of hypoallergenic peanut varieties.
Because the proteins encoded by the peanut mutant gene Arah1a-3-1-1 and the peanut mutant gene Arah1b-7-1-8 provided by the embodiment of the invention do not contain peanut allergic protein Arah1, and the peanut mutant prepared by the preparation method of the peanut mutant provided by the embodiment of the invention has low allergenicity, when the mutant gene or the peanut mutant is applied to peanut variety breeding, a peanut variety with low allergenicity can be obtained, and the peanut mutant has an important effect on improving peanut quality.
The embodiment of the invention also provides primers for detecting the peanut mutant genes, wherein the primers are Arah1-F '(the nucleotide sequence is shown as SEQ ID NO:9) and Arah 1-R' (the nucleotide sequence is shown as SEQ ID NO: 10).
The nucleotide sequence of Arah 1-F' (SEQ ID NO: 9):
ATGAGAGGGAGGGTTTCTCCACTGA
the nucleotide sequence of Arah 1-R' (SEQ ID NO: 10):
CTTGCTGGATAACAAGGATGTTATC
the primer for detecting the peanut mutant gene provided by the embodiment of the invention can be used for amplifying the genome DNA of a peanut mutant plant as a template, and comparing an amplification product with the gene sequence of a wild plant, so that the mutant gene and the mutant site in the peanut mutant plant can be accurately and quickly identified, the accuracy of peanut breeding character identification can be obviously improved, and the breeding process can be accelerated.
In order to make the above implementation details and operations of the present invention clearly understood by those skilled in the art and to make the progress of the mutant peanut genes, the encoded proteins thereof, and the preparation methods of the peanut mutants obvious, the above technical solutions are illustrated by the following examples.
Examples
1. Cloning and sequencing of peanut allergen gene Arah1
PCR primers Arah1-F and Arah1-R are designed according to the first exon sequence of peanut allergen gene Arah1(GenBank: AB440237.1), and the nucleotide sequences are respectively shown as SEQ ID NO. 7 and SEQ ID NO. 8.
Taking peanut leaves, extracting genome DNA by using the kit, and amplifying an Arah1 gene segment by PCR. The PCR reaction system is as follows:
a50. mu.l reaction included: ddH2O: 13. mu.l, primer: 5. mu.l, DNA: 5. mu.l, 2 XTaq mix: 25 μ l of Mg2+:1 μ l, glycerol: 1 μ l.
The reaction conditions were as follows: pre-denaturation at 95 ℃ for 5min, then denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 1min are used as a cycle, and after 40 cycles, complete extension at 72 ℃ for 10min is carried out.
And performing DNA sequencing on the PCR product to obtain two Arah1 gene coding region sequences which are respectively from two subgenomic homologous genes Arah1a and Arah1B of peanut A and peanut B, wherein the nucleotide sequences are respectively shown as SEQ ID NO. 11 and SEQ ID NO. 12. By sequencing, the sequence Arah1a had 99% homology to the mRNA of the peanut allergen Arah1(LOC112711772) in the NCBI database and the sequence Arah1b had 100% homology to the mRNA of the peanut allergen Arah1(LOC112776552) in the NCBI database.
2. Construction of genome editing vectors
The Arah1a and Arah1b gene sequences are compared by Blast to obtain the homologous sequence region of the gene (as shown in FIG. 1).
Two sgRNA sequences are designed in the homologous region, namely sgRNA T1 and sgRNA T2, and the nucleotide sequences are respectively shown as SEQ ID NO. 5 and SEQ ID NO. 6. Respectively synthesizing sense strands and antisense strands of the two sgRNA sequences with joints, and forming the following two double-stranded DNAs after mixed annealing:
DNA T1:
5’-ATTGCAGAGAACCCCTGCGCCCAGAGG
GTCTCTTGGGGACGCGGGTCTCCCAAA-5’
DNA T2:
5’-ATTGCCTCCAGGGGAGCGGACACGTGG
GGAGGTCCCCTCGCCTGTGCACCCAAA-5’
then respectively inserted into BsaI sites of sgRNA expression frames (shown in figure 2), and then cloned to a pKSE401 binary vector to obtain a gene editing vector T1 and a gene editing vector T2 for gene transformation. The gene editing vector T1 and the gene editing vector T2 can be transformed independently or in a mixed manner, or the gene editing vector T1 and the gene editing vector T2 can be cloned to the same vector for transformation, or more targets are designed for transformation, and a plurality of genes or gene loci are knocked out at the same time. A gene editing vector containing 2 or more targets was designed by referring to the method disclosed in (Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ.A CRISPR/Cas9 toolkit for multiplex genome editing in planta.BMC Plant biol.2014Nov 29; 14(1): 327.).
3. Induction and culture of peanut embryonic callus
Surface disinfection of mature peanut seeds: 20% Clorox (containing 1% w/v sodium hypochlorite) for 2 times for 20 minutes and 3 times with sterile water. Cutting peanut embryo (plumule), removing cotyledon and radicle, culturing in MS5PG inducing culture medium at 26-28 deg.C in dark to induce embryogenic callus. The embryogenic callus is subcultured for 1 time every 2-3 weeks, and transferred to MS3PG culture medium for embryoid amplification and maintenance after 4-6 weeks.
Induction medium composition:
MS5 PG: 4.4g/L MS basal salts (Sigma-Aldrich # M5519), sucrose 30.0g/L, glutamine 1.0g/L, Picloram 5.0mg/L, agar powder 8g/L, pH 5.8, autoclaving and packaging.
MS3 PG: 4.4g/L MS basal salts (Sigma-Aldrich # M5519), sucrose 30.0g/L, glutamine 1.0g/L, Picloram 3.0mg/L, agar powder 8g/L, pH 5.8, autoclaving and packaging.
Genetic transformation of peanut embryogenic callus: peanut embryonic callus is subjected to osmotic pressure pretreatment on an MS3PGO culture medium for 4 hours, transformation is carried out by utilizing a gene gun PDS-1000/HE of the Berle company, gold powder particles are 0.6 micron gold powder of the Berle company, the DNA dosage of each transformation plasmid is 2 micrograms, the transformation pressure is 650PSI, and the transformation distance is 6 centimeters. After transformation, the cells were maintained overnight on the permeation medium, and then transferred to MS3PG medium again, after dark culture at 26-28 ℃ for 7 days, the cells were transferred to the selection medium MS3PGK50(MS3PG medium supplemented with 50mg/L kanamycin), dark culture at 26-28 ℃ for 8-10 weeks, and the medium was changed every 3-4 weeks.
Differentiation of resistant calli: after 8-10 weeks, the selected resistant callus was transferred to MSC differentiation medium (4.4g/L MS basal salts (Sigma-Aldrich # M5519), sucrose 20.0g/L, charcoal 1.0g/L, kanamycin 50mg/L, agar powder 8g/L, pH 5.8, subpackaged after autoclaving), light culture at 26-28 ℃ for 4-6 weeks (16 hr light daily), embryoid was transferred to MS10 medium (4.4g/L MS basal salts (Sigma-Aldrich # M5519), sucrose 20.0g/L, benzylaminopurine 10mg/L, kanamycin 50mg/L, agar powder 8g/L, pH 5.8, subpackaged after autoclaving), light culture at 26-28 ℃ for 16 hr light daily, passage once every 3-4 weeks until plantlet grows into plantlet, and then transfer plantlet into MSOR medium (MS 4.4 g/L) (Sigma-Aldrich # M5519) 20.0g/L of cane sugar, 1.0mg/L of naphthylacetic acid, 50mg/L of kanamycin, 8g/L of agar powder, pH 5.8, subpackaging after autoclaving, performing illumination culture at 26-28 ℃ (illumination is performed for 16 hours every day), inducing rooting, transplanting 3-4 large rooted seedlings into flower pot soil, and performing illumination culture in a culture room until flowering and fruiting.
4. Identification of transgenic plants
Transplanting the survived seedlings, taking seedling leaves, and extracting genome DNA by using the kit. PCR primers Cas9f (nucleotide sequence shown as SEQ ID NO: 13) and Cas9r (nucleotide sequence shown as SEQ ID NO: 13) are designed according to the Cas9 gene sequence on the pKSE401 vector.
Nucleotide sequence of Cas9f (SEQ ID NO: 13):
GACAAGAAGTACTCGATCGGCCTCG
nucleotide sequence of Cas9r (SEQ ID NO: 14):
TCGTAGGGTACTTCTCGTGGTAGGC
the genomic DNA of the peanut seedlings is used as a template, and the transgenosis is verified by PCR. Among 11 transgenic seedlings, 11 transgenic seedlings were positive, and the positive rate was 100%.
5. Identification of mutants
The transgenic plant genome DNA is taken as a template, and amplification primers are Arah1-F '(the nucleotide sequence is shown as SEQ ID NO: 9) and Arah 1-R' (the nucleotide sequence is shown as SEQ ID NO: 10). Cloning the amplified PCR fragment, and then selecting a monoclonal clone for sequencing. 10 monoclonals are selected from each gene transformation plant for sequencing, and the sequencing result is compared with the gene sequences (SEQ ID NO:11 and SEQ ID NO:12) of the wild type plants through a BLAST program of an NCBI website to identify the mutant genes. The identification results and the mutant gene sites are shown in FIGS. 4 and 5.
In the embodiment of the invention, 8 peanut transgenic seedlings are obtained, and 3 peanut transgenic seedlings (No. 3, No. 5 and No. 7) are found to contain mutation by analyzing the target gene. The sequencing result of No. 3 plant shows that Arah1a gene (Arah1a-3-1-1, SEQ ID NO:1) contains 2 mutation sites (shown in figure 4), which are respectively: nucleotide 52 changed from T to G, resulting in a change of the encoded amino acid from serine (S) to alanine (A), and nucleotide 85, T, deleted, resulting in a frame shift mutation that prematurely terminates protein translation to form a polypeptide of 50 amino acids (SEQ ID NO: 2); arah1b gene (5-1-7) of No. 5 plant generates single base mutation, G is changed into A at position 9, but does not cause the change of the coding protein sequence; plant No. 7 contains 3 mutants, respectively: the 7-1-2Arah1a gene was changed from T to G at position 52, resulting in the change of the coding amino acid from serine (S) to alanine (A), C to T at position 468 (NO change in the coding amino acid), A to G at position 336 (NO change in the coding amino acid) of the 7-1-7Arah1b gene, 7-1-8Arah1b gene (Arah1b-7-1-8, SEQ ID NO:3) was deleted at nucleotide A at position 10, and GAG sequence (as shown in FIG. 5) was inserted, resulting in a frameshift mutation that prematurely terminates protein translation to form a polypeptide of 8 amino acids (SEQ ID NO: 4).
According to the results, the peanut allergen gene Arah1 is cut through a CRISPR/Cas9 gene editing system, the peanut allergen gene Arah1 is subjected to targeted mutation, amino acids of encoded proteins of the peanut allergen gene Arah1 are changed, the change of phenotypes is further influenced, and the low-allergenicity peanut mutant is obtained.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Shenzhen university
<120> peanut mutant gene, protein coded by same and preparation method of peanut mutant
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 705
<212> DNA
<213> peanut (Arachis hypogaea)
<400> 1
atgatgagag ggagggtttc tccactgatg ctgttgcttg ggatccttgt cctggcttca 60
gtttctgcaa cgcaggccaa gtcacctacc ggaaaacaga gaacccctgc gcccagaggt 120
gcctccagag ttgtcaacag gaaccggacg acttgaagca aaaggcatgc gagtctcgct 180
gcaccaagct cgagtatgat cctcgttgtg tctatgacac tggcgccacc aaccaacgtc 240
accctccagg ggagcggaca cgtggccgcc aacccggaga ctacgatgat gaccgccgtc 300
aaccccgaag agaggaagga ggccgatggg gaccagctga accgagggag cgtgaaagag 360
aagaagactg gagacaacca agagaagatt ggaggcgacc aagtcatcag cagccacgga 420
aaataaggcc cgaaggaaga gaaggagaac aagagtgggg aacaccaggt agcgaggtga 480
gggaagaaac atcacggaac aaccctttct acttcccgtc aaggcggttt agcacccgct 540
acgggaacca aaacggtagg atccgcgtcc tgcagaggtt tgaccaaagg tcaaagcagt 600
ttcagaatct ccagaatcac cgtattgtgc agatcgaggc cagacctaac actcttgttc 660
ttcccaagca cgctgatgct gataacatcc ttgttatcca gcaag 705
<210> 2
<211> 50
<212> PRT
<213> peanut (Arachis hypogaea)
<400> 2
Met Arg Gly Arg Val Ser Pro Leu Met Leu Leu Leu Gly Ile Leu Val
1 5 10 15
Leu Ala Ser Val Ser Ala Thr Gln Ala Lys Ser Pro Thr Gly Lys Gln
20 25 30
Arg Thr Pro Ala Pro Arg Gly Ala Ser Arg Val Val Asn Arg Asn Arg
35 40 45
Thr Thr
50
<210> 3
<211> 723
<212> DNA
<213> peanut (Arachis hypogaea)
<400> 3
atgagagggg aggggtttct ccactgatgc tgttgctagg gatccttgtc ctggcttcag 60
tttctgcaac gcatgccaag tcatcacctt accagaagaa aacagagaac ccctgcgccc 120
agaggtgcct ccagagttgt caacaggaac cggatgactt gaagcaaaag gcatgcgagt 180
ctcgctgcac caagctcgag tatgatcctc gttgtgtcta tgatcctcga ggacacactg 240
gcaccaccaa ccaacgttcc cctccagggg agcggacacg tggccgccaa cccggagact 300
acgatgatga ccgccgtcaa ccccgaagag aggaaggagg ccgatgggga ccagctggac 360
cgagggagcg tgaaagagaa gaagactgga gacaaccaag agaagattgg aggcgaccaa 420
gtcatcagca gccacggaaa ataaggcccg aaggaagaga aggagaacaa gagtggggaa 480
caccaggtag ccatgtgagg gaagaaacat ctcggaacaa ccctttctac ttcccgtcaa 540
ggcggtttag cacccgctac gggaaccaaa acggtaggat ccgggtcctg cagaggtttg 600
accaaaggtc aaggcagttt cagaatctcc agaatcaccg tattgtgcag atcgaggcca 660
aacctaacac tcttgttctt cccaagcacg ctgatgctga taacatcctt gttatccagc 720
aag 723
<210> 4
<211> 8
<212> PRT
<213> peanut (Arachis hypogaea)
<400> 4
Met Arg Gly Glu Gly Phe Leu His
1 5
<210> 5
<211> 23
<212> DNA
<213> target sequence T1(Single-guide RNA)
<400> 5
cagagaaccc ctgcgcccag agg 23
<210> 6
<211> 23
<212> DNA
<213> target sequence T2(Single-guide RNA)
<400> 6
cctccagggg agcggacacg tgg 23
<210> 7
<211> 22
<212> DNA
<213> primers (Primer)
<400> 7
atgagaggga gggtttctcc ac 22
<210> 8
<211> 27
<212> DNA
<213> primers (Primer)
<400> 8
cttgctggat aacaaggatg ttatcag 27
<210> 9
<211> 25
<212> DNA
<213> primers (Primer)
<400> 9
atgagaggga gggtttctcc actga 25
<210> 10
<211> 25
<212> DNA
<213> primers (Primer)
<400> 10
cttgctggat aacaaggatg ttatc 25
<210> 11
<211> 703
<212> DNA
<213> peanut (Arachis hypogaea)
<400> 11
atgagaggga gggtttctcc actgatgctg ttgcttggga tccttgtcct gtcttcagtt 60
tctgcaacgc aggccaagtc accttaccgg aaaacagaga acccctgcgc ccagaggtgc 120
ctccagagtt gtcaacagga accggacgac ttgaagcaaa aggcatgcga gtctcgctgc 180
accaagctcg agtatgatcc tcgttgtgtc tatgacactg gcgccaccaa ccaacgtcac 240
cctccagggg agcggacacg tggccgccaa cccggagact acgatgatga ccgccgtcaa 300
ccccgaagag aggaaggagg ccgatgggga ccagctgaac cgagggagcg tgaaagagaa 360
gaagactgga gacaaccaag agaagattgg aggcgaccaa gtcatcagca gccacggaaa 420
ataaggcccg aaggaagaga aggagaacaa gagtggggaa caccaggcag cgaggtgagg 480
gaagaaacat cacggaacaa ccctttctac ttcccgtcaa ggcggtttag cacccgctac 540
gggaaccaaa acggtaggat ccgcgtcctg cagaggtttg accaaaggtc aaagcagttt 600
cagaatctcc agaatcaccg tattgtgcag atcgaggcca gacctaacac tcttgttctt 660
cccaagcacg ctgatgctga taacatcctt gttatccagc aag 703
<210> 12
<211> 721
<212> DNA
<213> peanut (Arachis hypogaea)
<400> 12
atgagaggga gggtttctcc actgatgctg ttgctaggga tccttgtcct ggcttcagtt 60
tctgcaacgc atgccaagtc atcaccttac cagaagaaaa cagagaaccc ctgcgcccag 120
aggtgcctcc agagttgtca acaggaaccg gatgacttga agcaaaaggc atgcgagtct 180
cgctgcacca agctcgagta tgatcctcgt tgtgtctatg atcctcgagg acacactggc 240
accaccaacc aacgttcccc tccaggggag cggacacgtg gccgccaacc cggagactac 300
gatgatgacc gccgtcaacc ccgaagagag gaaggaggcc gatggggacc agctggaccg 360
agggagcgtg aaagagaaga agactggaga caaccaagag aagattggag gcgaccaagt 420
catcagcagc cacggaaaat aaggcccgaa ggaagagaag gagaacaaga gtggggaaca 480
ccaggtagcc atgtgaggga agaaacatct cggaacaacc ctttctactt cccgtcaagg 540
cggtttagca cccgctacgg gaaccaaaac ggtaggatcc gggtcctgca gaggtttgac 600
caaaggtcaa ggcagtttca gaatctccag aatcaccgta ttgtgcagat cgaggccaaa 660
cctaacactc ttgttcttcc caagcacgct gatgctgata acatccttgt tatccagcaa 720
<210> 13
<211> 25
<212> DNA
<213> primers (Primer)
<400> 13
gacaagaagt actcgatcgg cctcg 25
<210> 14
<211> 25
<212> DNA
<213> primers (Primer)
<400> 14
tcgtagggta cttctcgtgg taggc 25
Claims (8)
1. Mutant gene of peanutArah1a-3-1-1The nucleotide sequence is shown as SEQ ID NO. 1.
2. The peanut mutant gene of claim 1Arah1a-3-1-1The coded protein is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 2.
3. Mutant gene of peanutArah1b-7-1-8The nucleotide sequence is shown as SEQ ID NO. 3.
4. The peanut mutant gene of claim 3Arah1b-7-1-8The coded protein is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 4.
5. The preparation method of the peanut mutant is characterized by comprising the following steps:
providing peanut allergen genesArah1Amplification primers, a gene editing vector and peanut embryogenic callus; wherein the GenBank accession number of the peanut allergen gene Arah1 is AB 440237.1;
the peanut allergen gene Arah1As a template, carrying out PCR amplification by using the amplification primer to obtain the peanut allergen geneArah1Coding region sequenceArah1aAnd peanut allergen geneArah1Coding region sequenceArah1b；
The peanut allergen geneArah1Coding region sequenceArah1aAnd the peanut allergen geneArah1Coding region sequenceArah1bThe homologous sequence region of the target is used as a CRISPR target point to obtain sgRNA T1 and sgRNA T2;
respectively synthesizing a sense strand and an antisense strand of the sgRNA T1 and the sgRNA T2 to obtain a double-stranded DNA T1 and a double-stranded DNA T2;
inserting the double-stranded DNA T1 into an expression frame of the sgRNA T1, and then cloning the double-stranded DNA T1 into the gene editing vector to obtain a gene editing vector T1;
inserting the double-stranded DNA T2 into an expression frame of the sgRNA T2, and then cloning the double-stranded DNA T2 into the gene editing vector to obtain a gene editing vector T2;
performing gene transformation on the peanut embryonic callus by at least one of the gene editing vector T1 and the gene editing vector T2, and culturing to obtain a peanut mutant;
wherein the nucleotide sequence of the peanut mutant is shown as SEQ ID NO 1 and/or SEQ ID NO 3, the nucleotide sequence of the sgRNA T1 is shown as SEQ ID NO 5, and the nucleotide sequence of the sgRNA T2 is shown as SEQ ID NO 6; the amplification primers comprise a forward amplification primer and a reverse amplification primer, the nucleotide sequence of the forward amplification primer is shown as SEQ ID NO. 7, and the nucleotide sequence of the reverse amplification primer is shown as SEQ ID NO. 8;
In the step of carrying out PCR amplification by the amplification primer, every 50 mul of reaction system for PCR amplification comprises 13 mul of double distilled water, 5 mul of the amplification primer, 25 mul of peanut allergen gene Arah 15 mul, 2 XTaq mix, Mg2+ 1 mul and 1 mul of glycerol; the reaction conditions of the PCR amplification are as follows: pre-denaturation at 95 ℃ for 5min, then denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 1min are used as a cycle, and after 40 cycles, complete extension at 72 ℃ for 10min is carried out.
6. The method for preparing the peanut mutant as claimed in claim 5, wherein the gene editing vector is pKSE401 binary vector.
7. The peanut mutant gene of claim 1 or 2Arah1a-3-1-1The peanut mutant gene of claim 3 or 4Arah1b-7-1-8The application of the peanut mutant prepared by the method for preparing the peanut mutant as claimed in any one of claims 5 to 6 in the breeding of hypoallergenic peanut varieties.
8. A primer for detecting peanut mutant genes is characterized in that the nucleotide sequence of the primer is shown as SEQ ID NO. 9-10.
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| CN102584966A (en) * | 2011-11-14 | 2012-07-18 | 广州医学院第二附属医院 | Recombinant peanut allergen and mutant and preparation method and application of recombinant peanut allergen and mutant |
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