CN111944827B - Preparation method of peanut mutant, peanut mutant gene, protein coded by peanut mutant gene and application of peanut mutant gene - Google Patents

Preparation method of peanut mutant, peanut mutant gene, protein coded by peanut mutant gene and application of peanut mutant gene Download PDF

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CN111944827B
CN111944827B CN202010672354.6A CN202010672354A CN111944827B CN 111944827 B CN111944827 B CN 111944827B CN 202010672354 A CN202010672354 A CN 202010672354A CN 111944827 B CN111944827 B CN 111944827B
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于为常
张旺
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Shenzhen Shenhua Biotechnology Co.,Ltd.
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Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a preparation method of a peanut mutant, a peanut mutant gene, a protein coded by the peanut mutant gene and application of the peanut mutant gene. According to the invention, a CRISPR/Cas9 genome editing system is constructed by designing sgRNA T1-T4, a Cas9 enzyme guided by the sgRNA cuts a peanut desaturation lipase gene AhFAD2B at a gene target point, and insertion of a gene sequence or mutation of a base is generated after DNA repair, so that the targeted mutation of the peanut desaturation lipase gene AhFAD2B is completed, the gene cannot synthesize functional desaturation lipase, and cannot catalyze oleic acid dehydrogenation to generate linoleic acid, thus being beneficial to obtaining peanuts with high oleic acid content and improving the quality of peanut varieties.

Description

Preparation method of peanut mutant, peanut mutant gene, protein coded by peanut mutant gene and application of peanut mutant gene
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a preparation method of a peanut mutant, a peanut mutant gene, protein coded by the peanut mutant gene, application of the peanut mutant gene, and a primer for detecting the peanut mutant gene.
Background
Peanuts are used as raw materials of high-quality edible oil, are important oil crops and economic crops in China, have the total output and consumption accounting for more than 40% of the world, have the export accounting for more than 55% of the world (United nations data center, 2012), and have strong competitiveness internationally. The peanut seeds have rich oil content, the average oil content can reach about 51 percent, and the peanut seeds are widely popular in the market. The peanut oil mainly comprises monounsaturated fatty acid oleic acid (C18: 1, delta 9), polyunsaturated fatty acid linoleic acid (C18: 2, delta 9, delta 12) and saturated fatty acid palmitic acid (C16: 0) and stearic acid (C18:0), wherein the unsaturated fatty acid accounts for about 80% (oleic acid 36-67%, linoleic acid 15-43%), and the saturated fatty acid accounts for 20% (palmitic acid 6-11%, stearic acid 2-6%, arachidic acid 5-7%, and behenic acid 2-3%). The fatty acid composition and the content thereof in the peanut seeds are important indexes for measuring the quality of the peanut seeds. In the aspect of oil stability, linoleic acid belongs to polyunsaturated fatty acid, and oleoyl residues of the linoleic acid are easy to oxidize to cause oil corruption deterioration, thereby seriously influencing the storage period of the oil; the oleic acid molecule only contains a divalent unsaturated bond, the chemical structure is relatively stable, the oxidation resistance is strong, the autoxidation of the oleic acid is 10 times more stable than that of the linoleic acid, the peanut with high oleic acid/linoleic acid ratio is more storage-resistant, the peanut is not easy to deteriorate during refining, storage and frying, and compared with the common peanut, the peanut with high oleic acid content has longer shelf life. In terms of nutritional value, the quality of peanut oil has a significant impact on the health of the stationary consumer population of peanut oil. Oleic acid has a high nutritional value, and can reduce the content of low-density lipoprotein in blood, maintain the level of beneficial high-density lipoprotein, protect cardiovascular and cerebrovascular diseases more effectively, and reverse the inhibition of inflammatory cytokine TNF-alpha on insulin production. Today, food health is more and more valued, edible oil with high oleic acid ratio is a good choice for balancing the use of saturated fatty acid and unsaturated fatty acid in many food processing enterprises. Therefore, increasing the oleic acid content has become an important goal in peanut quality breeding.
Desaturated lipases (. DELTA.12FAD or FAD2) are key enzymes that catalyze the dehydrogenation of oleic acid at the C12 position to yield diunsaturated linoleic acid, which controls the oleic acid, linoleic acid content and their ratio (O/L). There is increasing evidence from molecular biology research that indicates that,AhFAD2is a key gene for generating linoleic acid from oleic acid, and determines the relative content of oleic acid and linoleic acid in peanut seeds. Cultivating peanut (A)Arachis hypogaea) Is an heterotetraploid species with genome a and genome B (2 n-4 x-40),AhFAD2by being located on different genomes2 pairs of non-allelic homologous genesAhFAD2AAndAhFAd2Bthe 2 genes are respectively derived from genomes of wild peanut species A and B, the 2 genes do not belong to seed specific expression genes, the 2 genes or one of the 2 genes can be normally expressed in common peanut varieties with oleic acid content, and in high oleic acid varieties,AhFAD2AandAhFAd2Bthe mutations in all the genes together result in the high oleic acid trait.
The American scientist first identified and screened a high oleic peanut natural mutant F435 with 79% oleic acid content (37O/L) from peanut resources. The sequence alignment of common peanut olein and F435 shows thatAhFAD2AThe coding region of (2) has a single base substitution (G > A) found at 448bp from the initiation codon, so that the 150 th amino acid of the coded protein is converted from aspartic acid (D) into asparagine (N), while the D150 residue is absolutely conserved in all FAD2 and is a key site of high oleic acid character, and the mutation causes that the activity of AhFAD2A enzyme is reduced; in that AhFAD2BA single base insertion (442insA) exists at the 441-442 bp position from the gene initiation codon, so that codon mutation is caused, a translated protein sequence is terminated early, and inactive protein is generated. Later, scientists in the United states utilized chemical mutagenesis to directly breed high oleic acid peanut varieties C458 and M2-225, which were inAhFAD2ABased on natural mutation (G448A), mutagenesis was carried outAhFAD2BMiniature inverted repeat transposable elements MITEs (205bp) are inserted at 665bp and 997bp behind the gene initiation codon respectively, so that the peptide chain is terminated early, and the function of the protein is lost. AboveAhFAD2BThe mutant genes all belong to Nonsense mutation (Nonsense mutation), so that the produced protein is incomplete and loses the functions of the original genes, and the mutant genes become gene sources for peanut high-oleic acid breeding.
However, peanut germplasm resources meeting the requirements of high oleic acid and low palmitic acid are mastered in foreign research institutions and agricultural enterprises for a long time, and the application of the high-quality resources in China is limited by the factors such as international political game, patent protection and the like. Pedigree analysis traces back the source of high oleic acid genes of bred high oleic acid varieties in China, and over 70 percent of high oleic acid gene donors of the varieties are derived from F435. The simplification of the high oleic acid gene source has resulted in relatively narrow genetic base, reduced genetic diversity, and increasingly outstanding contradiction between yield and quality and between high quality and disease resistance of the bred high oleic acid variety. At present, the oleic acid content of peanut varieties applied in large area in domestic production is generally lower (less than 50%), the palmitic acid content is higher (more than 11%), the oleic acid content of domestic peanut germplasm resources does not exceed 70%, and the maximum content is only 67.2%.
In addition to the above-mentioned F435 high oleic gene source internationally, recently, American scientists identified 2 new natural high oleic peanut mutants PI342664 and PI342666, which are naturally occurring mutants of PI342666AhFAD2AThe G > A mutation at position 448 of the coding region of the gene is the same as previously reported, andAhFAD2Bthe gene mutation C301G causes the change of the amino acid H101D to belong to a new mutation site, and finally the oleic acid content of the mutant reaches over 79 percent. Indian peanut breeders adopt gamma-ray mutagenesis of disease-resistant varieties to obtain 2 new high-oleic acid mutants (GM6-1 and GM4-3), the oleic acid content is more than 75%, and researches show thatAhFAD2BThe base substitution G1111A in the gene resulted in a change in amino acid G372S, resulting in a high oleic acid phenotype. In addition, domestic peanut researchers have utilized EMS chemical mutagenesis to obtainAhFAD2BThe new mutation site C313T of the gene causes the mutation of the amino acid sequence H105Y, so that the oleic acid content is increased from 44% to over 60%. However, the traditional breeding method needs to consume a large amount of manpower, material resources and financial resources, and can not complete targeted breeding tasks in a short period of time with great failure possibility by searching for few mutant materials in a large amount of natural populations or mutagenic populations.
Disclosure of Invention
The invention aims to provide a preparation method of a peanut mutant, a peanut mutant gene, a protein coded by the peanut mutant gene and application of the peanut mutant gene, and a primer for detecting the peanut mutant gene, and aims to solve the technical problem of large workload in the conventional breeding of high-oleic-acid peanuts.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
the invention provides a preparation method of a peanut mutant, which comprises the following steps:
providing a peanut desaturation lipase geneAhFAD2BThe coding region sequence, CRISPR/Cas9 gene editing vector and peanut embryogenic callus;
according to the peanut desaturation lipase geneAhFAD2BObtaining sgRNA target sequences T1-T4;
respectively synthesizing a sgRNA target sequence nucleotide fragment containing a sticky end and a complementary chain thereof according to the sgRNA target sequence T1-T4, and performing mixed annealing to obtain double-stranded DNA T1-T4 of which two ends contain the sticky ends;
connecting the double-stranded DNA T1-T4 with the CRISPR/Cas9 gene editing vector respectively to obtain a gene editing vector T1-T4;
performing gene transformation on the peanut embryonic callus by at least one gene editing vector T1-T4, and culturing to obtain the peanut mutant;
wherein the sgRNA target sequence T1-T4 has nucleotide sequences shown in SEQ ID NO 1-4, and the peanut desaturation lipase geneAhFAD2BThe sequence of the coding region is shown as SEQ ID NO. 5.
In another aspect, the invention provides a peanut mutant gene, which is a peanut desaturation lipase gene AhFAD2BThe coding region sequence of (a) is mutated such that the gene is unable to synthesize a functional desaturated lipase. The nucleotide sequence of the peanut mutant gene is shown in SEQ ID NO. 8-16.
The invention also provides the protein coded by the peanut mutant gene, and the amino acid sequence of the protein is shown as SEQ ID NO. 17-25.
The invention also provides the peanut mutant prepared by the preparation method of the peanut mutant, the peanut mutant gene and application of the protein coded by the peanut mutant gene in high-oleic-acid peanut variety breeding.
In the last aspect of the invention, a primer for detecting peanut mutant genes is provided, and the nucleotide sequence of the primer is shown as SEQ ID NO. 6-7.
In the preparation method of the peanut mutant provided by the invention, a CRISPR/Cas9 genome editing system is constructed by designing a CRISPR target sequence sgRNA T1-T4,cas9 enzyme guided by sgRNA at gene target point for peanut desaturation lipase geneAhFAD2BCutting, and generating insertion of gene sequence or base mutation after DNA repair to complete the desaturation of the peanut lipase geneAhFAD2BThe gene can not synthesize functional desaturated fatty acid enzyme by targeted mutation, so that the reaction of catalyzing oleic acid to linoleic acid is influenced, and the peanut mutant with high oleic acid content is obtained. The preparation method provided by the invention can realize targeted mutation of the gene locus, reduces randomness, contingency and indefiniteness in the traditional mutation method, has the advantages of convenience in operation, high mutation speed and high efficiency, and the obtained peanut mutant can be stably inherited and has important significance for improving the quality of peanuts and enriching the genetic diversity of the peanuts.
The peanut mutant gene provided by the invention is a peanut desaturation lipase geneAhFAD2BOn the basis of the coding region sequence, a plurality of peanut mutant genes are obtained according to at least one mutation mode of mutation modes such as mutation of different basic groups, insertion of basic groups and the like, so that the genes can not synthesize functional desaturated lipase, and the desaturated lipase can not catalyze oleic acid dehydrogenation to generate linoleic acid, thereby being beneficial to obtaining peanuts with high oleic acid content and improving the quality of peanut varieties.
The protein coded by the peanut mutant gene provided by the invention is a peanut desaturation lipase geneAhFAD2BThe protein encoded by the coding region sequence of (1) is obtained by amino acid mutation, premature termination of protein translation, frame shift mutation or the like. Compared with wild peanuts, the protein coded by the peanut mutant gene provided by the invention can be used for obtaining a peanut mutant through breeding, so that the activity of the desaturated lipase of the peanuts is influenced, the desaturated lipase can not catalyze oleic acid to dehydrogenate to generate linoleic acid, the high-oleic-acid-content peanuts can be obtained, and the quality of peanut varieties can be improved.
The peanut mutant gene and the protein coded by the same provided by the invention are all used for desaturating lipase genes of peanuts AhFAD2BThe coding sequence and the coding protein thereof are mutated, and the peanut mutant prepared by the preparation method of the peanut mutant has high oleic acid contentThe quantity is characterized, so that when the mutant gene or the peanut mutant is applied to peanut variety breeding, a peanut variety with high oleic acid content can be obtained.
The primer for detecting the peanut mutant gene provided by the invention can accurately and quickly identify 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 identifying the peanut breeding character can be obviously improved, and the breeding process is accelerated.
Drawings
FIG. 1 is a flow chart of construction of a genome editing vector according to an embodiment of the present invention, wherein FIG. 1A is a process of cloning a gene editing vector T1 into a vector; FIG. 1B shows the cloning of the gene editing vector T1-T4 into the same vector;
fig. 2 is a flow chart of an embodiment of the invention for preparing peanut mutants by CRISPR/CAS9 genome editing;
FIG. 3 shows the peanut mutant gene and the peanut desaturation lipase gene obtained in the example of the invention AhFAD2BA nucleotide sequence alignment map of (a) (first part);
FIG. 4 shows the peanut mutant gene and the peanut desaturation lipase gene obtained in the example of the inventionAhFAD2BA nucleotide sequence alignment map of (a) (second part);
FIG. 5 shows the peanut mutant gene and the peanut desaturation lipase gene obtained in the example of the inventionAhFAD2BNucleotide sequence alignment map of (a third part);
FIG. 6 shows the peanut mutant gene and the peanut desaturation lipase gene obtained in the example of the inventionAhFAD2BNucleotide sequence alignment map of (d) (fourth section).
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" describing an association relationship of associated objects means that there may be three relationships, for example, a and/or B, may mean: a is present alone, A and B are present simultaneously, and B is present 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 performed by referring to the specific methods listed in the molecular cloning experimental manual (third edition) j. sambrook, or according to the kit and the product specification; the relevant reagents and biological materials, unless otherwise specified, are commercially available.
The embodiment of the invention provides a preparation method of a peanut mutant, which comprises the following steps:
s1 provides the peanut desaturation fatty acid enzyme geneAhFAD2BThe coding region sequence, CRISPR/Cas9 gene editing vector and peanut embryogenic callus;
s2, based on the peanut desaturation lipase geneAhFAD2BObtaining sgRNA target sequences T1-T4;
s3, synthesizing a sgRNA target sequence nucleotide fragment containing a sticky end and a complementary chain thereof according to the sgRNA target sequence T1-T4 respectively, and performing mixed annealing to obtain double-stranded DNA T1-T4 containing sticky ends at two ends;
s4, connecting the double-stranded DNA T1-T4 with a CRISPR/Cas9 gene editing vector respectively to obtain a gene editing vector T1-T4;
s5, performing gene transformation on the peanut embryogenic callus by at least one gene editing vector T1-T4, and culturing to obtain a peanut mutant;
Wherein, the sgRNA target sequence T1-T4 nucleotide sequences are respectively shown in SEQ ID NO 1-4, and the peanut desaturation lipase geneAhFAD2BThe sequence of the coding region of (1) is shown in SEQ ID NO. 5.
In the preparation method of the peanut mutant provided by the embodiment of the invention, a CRISPR/Cas9 genome editing system is constructed by designing a CRISPR target sequence sgRNA T1-T4, and a Cas9 enzyme guided by the sgRNA is used for constructing a peanut desaturation lipase gene at a gene target pointAhFAD2BCutting, and generating insertion of gene sequence or base mutation after DNA repair to complete the desaturated lipase gene of peanutAhFAD2BThe gene can not synthesize functional desaturation lipase and catalyze oleic acid dehydrogenation to generate linoleic acid, so that the peanut with high oleic acid content can be obtained, and the quality of peanut varieties can be improved. The preparation method provided by the embodiment of the invention can realize targeted mutation of gene loci, reduces randomness, contingency and indefiniteness in the traditional mutation method, has the advantages of convenient operation, high mutation speed and high efficiency, and the obtained peanut mutant can be stably inherited and has important significance for improving the quality of peanuts and enriching the genetic diversity of the 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 2. Specifically, in S1, the coding region sequence of the peanut desaturation lipase gene AhFAD2B can be obtained by PCR amplification. In some embodiments, primers for amplification include a forward amplification primer FAD2B-f and a reverse amplification primer FAD2B-r, and the nucleotide sequence of the forward amplification primer is shown as SEQ ID NO:6 and the nucleotide sequence of the reverse amplification primer is shown as SEQ ID NO:7, with FAD (Arachis Hypogaea cubvar Huayu81 delta-12 Fatty Acid Desaturase (FAD) mRNA, complete cds, GenBank: EF 186911.1) as a template.
Furthermore, by optimizing a PCR reaction system and reaction conditions, the PCR reaction efficiency can be improved, and the occurrence of non-specific amplification can be avoided. Specifically, the reaction system for PCR amplification contained double distilled water (ddH) per 50. mu.l of PCR amplification2O) 13. mu.l, amplification primer 5. mu.l: (1FAD2B-f andFAD2B2.5. mu.l each of-f), a peanut desaturation lipase geneAhFAD2B 5μl、2×Taq mix 25μl、Mg2+1 μ l and glycerol 1 μ 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.
The CRISPR/Cas9 gene editing vector is used for preparing peanut mutants by using a CRISPR/Cas9 gene editing system, and correspondingly, the used vector is the CRISPR/Cas9 gene editing vector. It can be understood that the gene editing vector contains a Cas 9-responsive 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, the lipase gene is derived from peanut desaturationAhFAD2BThe target sequence is designed to obtain the sgRNA target sequence T1-T4. Specifically, the design requirements for the target sequence are: (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). The obtained sgRNA target sequence is an sgRNA target sequence T1, an sgRNA target sequence T2, an sgRNA target sequence T3 and an sgRNA target sequence T4.
It should be noted that, the CRISPR target of the embodiment of the present invention is designed by taking the homologous sequence region of the gene as an example to illustrate the steps, and is not a limitation on the selection of the target sequence of the present invention. In fact, the target site can be any sequence in the coding region of the 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 the target points are used, the target points can be used separately or together.
In S3, sgRNA target sequence nucleotide fragments containing sticky ends and complementary strands thereof are synthesized respectively according to the sgRNA target sequences T1-T4. In some embodiments, the sticky end isBsaIThe cohesive end cut by the restriction endonuclease, correspondingly, the nucleotide fragment of the sgRNA target sequence containing the cohesive end and the end sequences of the complementary strand thereof are 5 '-ATTG-3' and 3 '-CAAA-5', respectively.
And (3) annealing the sgRNA target sequence nucleotide fragment containing the sticky end and the complementary strand thereof in a mixed manner to obtain double-stranded DNA T1-T4 with the sticky ends at two ends.
The sequence of the double-stranded DNA T1 is as follows:
5’-ATTGTTGTCTATGATCTCTTAATGG
AACAGATACTAGAGAATTACCCAAA-5’
the sequence of the double-stranded DNA T2 is as follows:
5’-ATTGAGCAATCACCCAAACACCGG
TCGTTAGTGGGTTTGTGGCCCAAA-5’
the sequence of the double-stranded DNA T3 is as follows:
5’-ATTGACCTTTGATTTTGGTTTCGG
TGGAAACTAAAACCAAAGCCCAAA-5’
the sequence of the double-stranded DNA T4 is as follows:
5’-ATTGCACAATGCTTTGTAAACTGG
GTGTTACGAAACATTTGACCCAAA-5’
In S4, double-stranded DNA T1-T4 are respectively connected with CRISPR/Cas9 gene editing vectors to respectively obtain gene editing vectors T1-T4. A flowchart of the construction of the gene editing vector T1-T4 is shown in FIG. 1. In some embodiments, the CRISPR/Cas9 gene editing vector is subjected to enzymatic cleavage with a restriction enzyme. In particular, restriction enzymes are preferredBsaIThe enzyme is characterized in that the sgRNA target sequence T1-T4 nucleotide fragment and the end of the complementary strand thereof pass throughBsaIRestriction enzyme cleavage, therefore selectionBsaIEnzyme digestion treatment of the CRISPR/Cas9 gene editing vector by enzyme is beneficial to connection of the double-stranded DNA T1-T4 and the CRISPR/Cas9 gene editing vector.
In S5, at least one gene editing vector T1-T4 is used for gene transformation of peanut embryogenic callus, and the peanut embryogenic callus is cultured to obtain peanut mutants. The gene editing vectors T1-T4 can be transformed separately or in a mixed way, and the two vectors 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 of (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. 2014 Nov 29;14(1): 327), which is not repeated herein.
The embodiment of the invention also provides a peanut mutant gene which is a peanut desaturation lipase geneAhFAD2BThe coding region sequence of (a) is mutated such that the gene is unable to synthesize a functional desaturated lipase. The peanut mutant gene provided by the embodiment of the invention is a peanut desaturation lipase geneAhFAD2BOn the basis of the coding region sequence, a plurality of peanut mutant genes are obtained according to at least one mutation mode of mutation modes such as mutation of different basic groups, insertion of basic groups and the like, so that the genes cannot synthesize functional desaturated lipase, and cannot catalyze oleic acid dehydrogenation to generate linoleic acid, the high-oleic-acid-content peanuts can be obtained, and the quality of peanut varieties can be improved. The following are the proteins (peanut mutants) coded by the peanut mutant genes and the amino acid sequences thereof provided by the embodiment of the invention:
correspondingly, the embodiment of the invention also provides the peanut mutant prepared by the preparation method of the peanut mutant, and the application of the peanut mutant gene and the protein coded by the peanut mutant gene in the breeding of high-oleic acid peanut varieties.
The peanut mutant gene and the protein coded by the same provided by the embodiment of the invention are all genes for the peanut desaturation lipase AhFAD2BThe coding sequence and the coding protein thereof are mutated, so that the gene can not synthesize functional desaturated fatty acid enzyme and can not catalyze oleic acid dehydrogenation to generate linoleic acid, and the peanut mutant prepared by the preparation method of the peanut mutant provided by the embodiment of the invention has the characteristic of high oleic acid content, so that when the mutant gene or the peanut mutant is applied to peanut variety breeding, a peanut variety with high oleic acid content can be obtained.
The embodiment of the invention also provides a primer for detecting the peanut mutant geneFAD2B-f andFAD2B-r, the nucleotide sequence of which is shown in SEQ ID NO 6-7.
The primer for detecting the peanut mutant gene provided by the embodiment of the invention can accurately and quickly identify 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 is accelerated.
In order to make the details and operation of the above-mentioned embodiments of the present invention clearly understood by those skilled in the art, and to make the progress of the preparation method of the peanut mutant, the peanut mutant gene and its encoded protein and application obvious, the above-mentioned technical solutions are illustrated by the following examples.
Examples
1. Peanut desaturation lipase geneAhFAD2BCloning and sequencing of
According to the cultivation of peanut (Arachis hypogaea) design of PCR primers for delta-12 Fat Acid Desaturase (FAD) mRNA sequence (GenBank: EF 186911.1)FAD2B-f andFAD2B-r, the nucleotide sequences of which are shown as SEQ ID NO 6 and SEQ ID NO 7, respectively.
Taking peanut leaves, extracting genome DNA by the kit, and amplifying by PCRAhFAD2BA gene. 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 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.
DNA sequencing is carried out on the PCR product to obtainAhFAD2BThe nucleotide sequences of the gene coding regions are respectively shown as SEQ ID NO. 5.
2. Construction of genome editing vector
Cas-Designer (http:// www.rgenome.net /) using CRISPR Designer program according to the aboveAhFAD2BThe gene coding region sequence is wild peanutArachis ipaensisA genome sequence (PeatBase v 1.0) is a reference genome, and 4 sgRNA target sequences T1-T4 are designed, and the nucleotide sequences are respectively shown as SEQ ID NO: 1-4. Respectively synthesizing a sense strand and an antisense strand of the 4 sgRNA sequences with joints, and after mixed annealing, forming 4 double-stranded DNAs as follows:
The sequence of the double-stranded DNA T1 is as follows:
5’-ATTGTTGTCTATGATCTCTTAATGG
AACAGATACTAGAGAATTACCCAAA-5’
the sequence of the double-stranded DNA T2 is as follows:
5’-ATTGAGCAATCACCCAAACACCGG
TCGTTAGTGGGTTTGTGGCCCAAA-5’
the sequence of the double-stranded DNA T3 is as follows:
5’-ATTGACCTTTGATTTTGGTTTCGG
TGGAAACTAAAACCAAAGCCCAAA-5’
the sequence of the double-stranded DNA T4 is as follows:
5’-ATTGCACAATGCTTTGTAAACTGG
GTGTTACGAAACATTTGACCCAAA-5’
then inserted into the sgRNA expression cassettesBsaIThe sites (shown in FIG. 1) were cloned into pKSE401 binary vector to obtain gene editing vector T1-T4, and the gene was transformed (FIG. 1A). 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 (FIG. 1B), or more targets are designed for transformation, and a plurality of genes or gene loci are knocked out simultaneously. 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 plants, BMC Plant biol. 2014 Nov 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 20 min 2 times, and 3 times with sterile water. Cutting peanut embryo (plumule), removing cotyledon and radicle, placing on MS5PG induction culture medium, culturing at 26-28 deg.C in dark, and inducing 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: 4.4 g/L MS basal salts (Sigma-Aldrich # M5519), sucrose 30.0 g/L, glutamine 1.0 g/L, Picloram 5.0 mg/L, agar powder 8 g/L, pH 5.8, autoclaving and packaging.
MS3 PG: 4.4 g/L MS basal salts (Sigma-Aldrich # M5519), sucrose 30.0 g/L, glutamine 1.0 g/L, Picloram 3.0 mg/L, agar powder 8 g/L, pH 5.8, autoclaving and packaging.
Genetic transformation of peanut embryogenic callus: performing osmotic pressure pretreatment on the peanut embryogenic callus on an MS3PGO culture medium for 4 hours, and transforming by using a gene gun PDS-1000/HE (Berkeley corporation), wherein gold powder particles are 0.6 micron gold powder, the use amount of each transformation plasmid DNA is 2 micrograms, the transformation pressure is 650 PSI, 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 (MS 3PG medium supplemented with 50 mg/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 calli were transferred to MSC differentiation medium (4.4 g/L MS basal salts (Sigma-Aldrich # M5519), sucrose 20.0 g/L, activated carbon 1.0 g/L, kanamycin 50 mg/L, agar powder 8 g/L, pH 5.8, subpackaged after autoclaving), light-cultured at 26-28 ℃ for 4-6 weeks (16 hours per day), embryoid was transferred to MS10 medium (4.4 g/L MS basal salts (Sigma-Aldrich # M5519), sucrose 20.0 g/L, benzylaminopurine 10 mg/L, kanamycin 50 mg/L, agar powder 8 g/L, pH 5.8, subpackaged after autoclaving), light-cultured at 26-28 ℃ for 16 hours per day, passage once every 3-4 weeks, until the seedlings grow into plantlets, the plantlets are transferred into MSOR culture medium (4.4 g/L MS basal salts (Sigma-Aldrich # M5519), sucrose 20.0 g/L, naphthylacetic acid 1.0 mg/L, kanamycin 50 mg/L, agar powder 8 g/L, pH 5.8, subpackaged after autoclaving), are subjected to illumination culture at 26-28 ℃ (16 hours per day), induced to root, rooted seedlings with 3-4 leaves are transplanted into flowerpot soil, and are subjected to illumination culture in a culture room until flowering and fruiting are achieved.
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: 26) and Cas9r (nucleotide sequence shown as SEQ ID NO: 27) are designed according to the Cas9 gene sequence on the pKSE401 vector.
The transgene was verified by PCR using genomic DNA of peanut seedlings as template. Among 11 transgenic seedlings, 11 were positive, and the positive rate was 100%.
5. Identification of mutants
Using transgenic plant genome DNA as template and amplification primer asFAD2B-f (nucleotide sequence shown in SEQ ID NO: 6) andFAD2B-r (nucleotide sequence shown in SEQ ID NO: 7). Cloning the amplified PCR fragment, and then selecting a monoclonal clone for sequencing. Selecting 10 monoclonals from each gene transformation plant for sequencing, comparing the sequencing result with a wild plant gene sequence (SEQ ID NO: 5) through a BLAST program of an NCBI website, and identifying a mutant gene; at the same time, the gene sequence of the wild plant gene (such as SEQ ID NO: 28) is compared. The results of the identification and the site of the mutated gene are shown in FIGS. 3 to 6.
Statistics were performed on the mutation sites as shown in table 1.
TABLE 1AhFAD2BSite of gene mutation
Figure 498165DEST_PATH_IMAGE001
As can be seen from the comparison results of FIGS. 3-6 and Table 1, 11 transgenic peanuts were obtained in total in the examples of the present invention, and 30 mutations were found by analyzing the target gene. Of these, 13 mutations did not cause changes in the encoded protein sequence and 17 mutations caused changes in the protein sequence. Wherein, the base of the mutant 12-2, 13-5, 148-2, 148-6, 148-10, 149-4, 150-12 at 451 is changed from G to T to cause the corresponding codon to be changed from GAA to TAA, so that the translation of the protein is terminated early, and 150 amino acids of protein are generated; the mutant 12-2, 12-5, 13-5, 148-2, 148-6, 148-9, 148-10 has 7 bases of CTCAGGA inserted in 668 th position, and can cause frame shift mutation of protein. The coding nucleotide sequences of the mutants are shown as SEQ ID NO 8-16, and the amino acid sequences of the mutants are shown as SEQ ID NO 17-25. These mutationsThe fatty acid desaturase structural domain positioned at 79-340 amino acids is destroyed, so that the generated polypeptide loses the function of catalyzing the desaturation of oleic acid to generate linoleic acid, which is beneficial to obtaining peanuts with high oleic acid content and improving the quality of peanut varieties. Other mutations are all single amino acid mutations. Thus, influencing by the above means AhFAD2BThe change of the gene coding protein sequence influences the activity of the peanut desaturation lipase, so that the linoleic acid generated by oleic acid catalytic dehydrogenation is reduced or avoided, the oleic acid content of the peanut is increased, and the peanut desaturation lipase can be applied to high-oleic acid peanut breeding.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood 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 should be subject to the appended claims.
Sequence listing
<110> Shenzhen university
<120> preparation method of peanut mutant, peanut mutant gene, protein coded by peanut mutant gene and application of peanut mutant gene
<160> 28
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ttgtctatga tctcttaatg g 21
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agcaatcacc caaacaccgg 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
acctttgatt ttggtttcgg 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cacaatgctt tgtaaactgg 20
<210> 5
<211> 1140
<212> DNA
<213> peanut (Arachis hypogaea)
<400> 5
atgggagctg gagggcgtgt cactaagatt gaagctcaaa agaagcctct ttcaagggtt 60
ccacattcaa accctccatt cagtgttggc caactcaaga aagcaattcc accacattgc 120
tttgaacgtt ctcttttcat atcattctca tatgttgtct atgatctctt aatggcctac 180
ttactcttct acattgccac cacttatttc cacaagcttc catacccatt ttccttcctt 240
gcttggccaa tctattgggc catccaaggc tgcattctca ccggtgtttg ggtgattgct 300
catgagtgtg gccaccatgc cttcagcaag taccaacttg ttgatgacat ggttggtttg 360
acccttcact cttgtctatt agttccttat ttctcatgga aaatcagcca ccgccgccac 420
cactccaaca caggttccct cgaccgcgac gaagtgtttg tcccgaaacc aaaatcaaag 480
gtatcatggt ataacaagta catgaacaat ccaccaggga gggctatttc ccttttcatc 540
acactcacac taggatggcc cttgtacttg gccttcaatg tttctggcag accctatgat 600
agatttgcaa gccactatga cccttatgct cccatatact ctaacaggga aaggcttcta 660
atttatgtct cagattcatc tgtctttgct gtaacatatc tgctatatca catagcaact 720
ttgaaaggtt tgggttgggt ggtatgtgtt tatggggtgc cattgctcat tgtgaatggg 780
tttctagtta ccataaccta tttgcagcac acacatgcat cattgcctca ctatgattca 840
tccgaatggg actggttaag aggagcattg gcaacagtgg acagagatta tgggatactg 900
aataaggcat ttcatcatat aactgatacg catgtggctc atcatttgtt ctcaacaatg 960
cctcattacc atgcaatgga agcaaccaat gcaataaagc caatattggg tgattactac 1020
caatttgatg gcaccccagt ttacaaagca ttgtggagag aagccaaaga gtgcctctat 1080
gtggagccag atgatggagc ttctcagaag ggtgtttatt ggtacaagaa caagttctga 1140
<210> 6
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
acggagcttt aacaacacaa caatg 25
<210> 7
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
acgtcacacc aagctagtat ttcac 25
<210> 8
<211> 1147
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atgggagctg gagggcgtgt cactaagatt gaagctcaag agaagcctct ttcaagggtt 60
ccacattcaa accctccatt cagtgttggc caactcaaga aagcaattcc accacattgc 120
tttgaacgtt ctcttttcat atcattctcc tatgttgtct atgatctctt aatggcctac 180
ttactcttct acattgccac cacttatttc caccagcttc catacccatt ttccttcctt 240
gcttggccaa tctattgggc catccaaggc tgcattctca ctggtgtttg ggtgattgct 300
catgagtgtg gccaccatgc cttcagcaag tatcaacttg ttgatgacat ggttggtttg 360
atccttcact cttgtctatt agtcccttat ttctcatgga aaatcagcca ccgccacctc 420
cactccaaca ccggttccct cgaccgcgac taagtgtttg tcctgaaacc aaaatcaaag 480
gtatcacggt ataacaagta catgaacaat ccactagaga gggctatttc ccttttcatc 540
acactcacac taggatggcc cttgtacttg gccttcaatg tttctggcag accctatgat 600
agatttgcaa gccactatga cccttatgct cccatatact ctaacaggga aaggcttcta 660
atttatgtct caggactcag attcatctgt ctttgctgta acatatctgc tatatcacat 720
agcaactttg aaaggtttgg gttgggtggt atgtgtttat ggggtgccat tgctcattgt 780
gaatgggttt ctagttacca taacctgttt gcaacacaca catgcatcat tgcctcacta 840
tgattcatcc gaatgggact ggttaagagg agcattggca acagtggaca gagattatgg 900
gatactgaat aagacatttc atcatataac tgatacacat gtggctcatc atttgttctc 960
aacaatgcca cattaccatg caatggaagc aaccaatgca ataaagccaa tattgggtga 1020
ttattaccaa tttgatggca ccccatttta caaggcattg tggagagaag ccaaagagtg 1080
cctctatgtg gagccagatg atggagcttc tcagaagggt gtttattggt acaagaacaa 1140
gttctga 1147
<210> 9
<211> 1147
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atgggagctg gagggcgtgt cactaagatt gaagctcaag agaagcctct ttcaagggtt 60
ccacattcaa accctccatt cagtgttggc caactcaaga aagcaattcc accacattgc 120
tttgaacgtt ctcttttcat atcattctca tatgttgtct atgatctctt aatggcctac 180
ttactcttct acattgccac cacttatttc cacaagcttc catacccatt ttccttcctt 240
gcttggccaa tctattgggc catccaaggc tgcattctca ccggtgtttg ggtgattgct 300
catgagtgtg gccaccatgc cttcagcaag taccaacttg ttgatgacat ggttggtttg 360
acccttcact cttgtctatt agttccttat ttctcatgga aaatcagcca ccgccgccac 420
cactccaaca caggttccct cgaccgcgac gaagtgtttg tcccgaaacc aaaatcaaag 480
gtatcatggt ataacaagta catgaacaat ccaccaggga gggctatttc ccttttcatc 540
acactcacac taggatggcc cttgtacttg gccttcaatg tttctggcag accctatgat 600
agatttgcaa gccactatga cccttatgct cccatatact ctaacaggga aaggcttcta 660
atttatgtct caggactcag attcatctgt ctttgctgta acatatctgc tatatcacat 720
agcaactttg aaaggtttgg gttgggtggt atgtgtttat ggggtgccat tgctcattgt 780
gaatgggttt ctagttacca taacctgttt gcaacacaca catgcatcat tgcctcacta 840
tgattcatcc gaatgggact ggttaagagg agcattggca acagtggaca gagattatgg 900
gatactgaat aaggcatttc atcatataac tgatacgcat gtggctcatc atttgttctc 960
aacaatgcct cattaccatg caatggaagc aaccaatgca ataaagccaa tattgggtga 1020
ttactaccaa tttgatggca ccccagttta caaagcattg tggagagaag ccaaagagtg 1080
cctctatgtg gagccagatg atggagcttc taagaagggt gtttattggt acaagaacaa 1140
gttctga 1147
<210> 10
<211> 1147
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atgggagctg gagggcgtgt cactaagatt gaagctcaaa agaagcctct ttcaagggtt 60
ccacattcaa accctccatt cagtgttggc caactcaaga aagcaattcc accacattgc 120
tttgaacgtt ctcttttcat atcattctca tatgttgtct atgatctctt aatggcctac 180
ttactcttct acattgccac cacttatttc cacaagcttc catacccatt ttccttcctt 240
gcttggccaa tctattgggc catccaaggc tgcattctca ctggtgtttg ggtgattgct 300
catgagtgtg gccaccatgc cttcagcaag tatcaacttg ttgatgacat ggttggtttg 360
atccttcact cttgtctatt agtcccttat ttctcatgga aaatcagcca ccgccacctc 420
cactccaaca ccggttccct cgaccgcgac taagtgtttg tcctgaaacc aaaatcaaag 480
gtatcacggt ataacaagta catgaacaat ccactagaga gggctatttc ccttttcatc 540
acactcacac taggatggcc cttgtacttg gccttcaatg tttctggcag accctatgat 600
agatttgcaa gccactatga cccttatgct cccatatact ctaacaggga aaggcttcta 660
atttatgtct caagactcag attcatctgt ctttgctgta acatatctgc tatatcacat 720
agcaactttg aaaggtttgg gttgggtggt atgtgtttat ggggtgccat tgctcattgt 780
gaatgggttt ctagttacca taacctgttt gcaacacaca catgcatcat tgcctcacta 840
tgattcatcc gaatgggact ggttaagagg agcattggca acagtggaca gagattatgg 900
gatactgaat aaggcatttc atcatataac tgatacacat gtggctcatc atttgttctc 960
aacaatgcca cattaccatg caatggaagc aaccaatgca ataaagccaa tattgggtga 1020
ttattaccaa tttgatggca ccccatttta caaggcattg tggagagaag ccaaagagtg 1080
cctctatgtg gagccagatg atggagcttc tcagaagggt gtttattggt acaagaacaa 1140
gttctga 1147
<210> 11
<211> 1147
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
atgggagctg gagggcgtgt cactaagatt gaagctcaag agaagcctct ttcaagggtt 60
ccacattcaa accctccatt cagtgttggc caactcaaga aagcaattcc accacattgc 120
tttgaacgtt ctcttttcat atcattctcc tatgttgtct atgatctctt aatggcctac 180
ttactcttct acattgccac cacttatttc caccagcttc catacccatt ttccttcctt 240
gcttggccaa tctattgggc catccaaggc tgcattctca ctggtgtttg ggtgattgct 300
catgagtgtg gccaccatgc cttcagcaag tatcaacttg ttgatgacat ggttggtttg 360
atccttcact cttgtctatt agtcccttat ttctcatgga aaatcagcca ccgccacctc 420
cactccaaca ccggttccct cgaccgcgac taagtgtttg tcctgaaacc aaaatcaaag 480
gtatcacggt ataacaagta catgaacaat ccactagaga gggctatttc ccttttcatc 540
acactcacac taggatggcc cttgtacttg gccttcaatg tttctggcag accctatgat 600
agatttgcaa gccactatga cccttatgct cccatatact ctaacaggga aaggcttcta 660
atttatgtct caggactcag attcatctgt ctttgctgta acatatctgc tatatcacat 720
agcaactttg aaaggtttgg gttgggtggt atgtgtttat ggggtgccat tgctcattgt 780
gaatgggttt ctagttacca taacctgttt gcaacacaca catgcatcat tgcctcacta 840
tgattcatcc gaatgggact ggttaagagg agcattggca acagtggaca gagattatgg 900
gatactgaat aaggcatttc atcatataac tgatacacat gtggctcatc atttgttctc 960
aacaatgcca cattaccatg caatggaagc aaccaatgca ataaagccaa tattgggtga 1020
ttattaccaa tttgatggca ccccatttta caaggcattg tggagagaag ccaaagagtg 1080
cctctatgtg gagccagatg atggagcttc tcagaagggt gtttattggt acaagaacaa 1140
gttctga 1147
<210> 12
<211> 1147
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
atgggagctg gagggcgtgt cactaagatt gaagctcaaa agaagcctct ttcaagggtt 60
ccacattcaa accctccatt cagtgttggc caactcaaga aagcaattcc accacattgc 120
tttgaacgtt ctcttttcat atcattctca tatgttgtct atgatctctt aatggcctac 180
ttactcttct acattgccac cacttatttc cacaagcttc catacccatt ttccttcctt 240
gcttggccaa tctattgggc catccaaggc tgcattctca ccggtgtttg ggtgattgct 300
catgagtgtg gccaccatgc cttcagcaag tatcaacttg ttgatgacat ggttggtttg 360
atccttcact cttgtctatt agtcccttat ttctcatgga aaatcagcca ccgccacctc 420
cactccaaca ccggttccct cgaccgcgac taagtgtttg tcctgaaacc aaaatcaaag 480
gtatcacggt ataacaagta catgaacaat ccactagaga gggctatttc ccttttcatc 540
acactcacac taggatggcc cttgtacttg gccttcaatg tttctggcag accctatgat 600
agatttgcaa gccactatga cccttatgct cccatatact ctaacaggga aaggcttcta 660
atttatgtct caggactcag attcatctgt ctttgctgta acatatctgc tatatcacat 720
agcaactttg aaaggtttgg gttgggtggt atgtgtttat ggggtgccat tgctcattgt 780
gaatgggttt ctagttacca taacctgttt gcaacacaca catgcatcat tgcctcacta 840
tgattcatcc gaatgggact ggttaagagg agcattggca acagtggaca gagattatgg 900
gatactgaat aaggcatttc atcatataac tgatacacat gtggctcatc atttgttctc 960
aacaatgcca cattaccatg caatggaagc aaccaatgca ataaagccaa tattgggtga 1020
ttattaccaa tttgatggca ccccatttta caaggcattg tggagagaag ccaaagagtg 1080
cctctatgtg gagccagatg atggagcttc tcagaagggt gtttattggt acaagaacaa 1140
gttctga 1147
<210> 13
<211> 1147
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
atgggagctg gagggcgtgt cactaagatt gaagctcaag agaagcctct ttcaagggtt 60
ccacattcaa accctccatt cagtgttggc caactcaaga aagcaattcc accacattgc 120
tttgaacgtt ctcttttcat atcattctca tatgttgtct atgatctctt aatggcctac 180
ttactcttct acattgccac cacttatttc cacaagcttc catacccatt ttccttcctt 240
gcttggccaa tctattgggc catccaaggc tgcattctca ccggtgtttg ggtgattgct 300
catgagtgtg gccaccatgc cttcagcaag taccaacttg ttgatgacat ggttggtttg 360
acccttcact cttgtctatt agttccttat ttctcatgga aaatcagcca ccgccgccac 420
cactccaaca caggttccct cgaccgcgac gaagtgtttg tcccgaaacc aaaatcaaag 480
gtatcatggt ataacaagta catgaacaat ccaccaggga gggctatttc ccttttcatc 540
acactcacac taggatggcc cttgtacttg gccttcaatg tttctggcag accctatgat 600
agatttgcaa gccactatga cccttatgct cccatatact ctaacaggga aaggcttcta 660
atttatgtct caggactcag attcatctgt ctttgctgta acatatctgc tatatcacat 720
agcaactttg aaaggtttgg gttgggtggt atgtgtttat ggggtgccat tgctcattgt 780
gaatgggttt ctagttacca taacctgttt gcaacacaca catgcatcat tgcctcacta 840
tgattcatcc gaatgggact ggttaagagg agcattggca acagtggaca gagattatgg 900
gatactgaat aaggcatttc atcatataac tgatacgcat gtggctcatc atttgttctc 960
aacaatgcct cattaccatg caatggaagc aaccaatgca ataaagccaa tattgggtga 1020
ttactaccaa tttgatggca ccccagttta caaagcattg tggagagaag ccaaagagtg 1080
cctctatgtg gagccagatg atggagcttc tcagaagggt gtttattggt acaagaacaa 1140
gttctga 1147
<210> 14
<211> 1147
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
atgggagctg gagggcgtgt cactaagatt gaagctcaaa agaagcctct ttcaagggtt 60
ccacattcaa accctccatt cagtgttggc caactcaaga aagcaattcc accacattgc 120
tttgaacgtt ctcttttcat atcattctcc tatgttgtct atgatctctt agtggcctac 180
ttactcttct acattgccac cacttatttc cacaagcttc catacccatt ttccttcctt 240
gcttggccaa tctattgggc catccaaggc tgcattctca ctggtgtttg ggtgattgct 300
catgagtgtg gccaccatgc cttcagcaag tatcaacttg ttgatgacat ggttggtttg 360
atccttcact cttgtctatt agtcccttat ttctcatgga aaatcagcca ccgccacctc 420
cactccaaca ccggttccct cgaccgcgac taagtgtttg tcctgaaacc aaaatcaaag 480
gtatcacggt ataacaagta catgaacaat ccactagaga gggctatttc ccttttcatc 540
acactcacac taggatggcc cttgtacttg gccttcaatg tttctggcag accctatgat 600
agatttgcaa gccactatga cccttatgct cccatatact ctaacaggga aaggcttcta 660
atttatgtct caggactcag attcatctgt ctttgctgta acatatctgc tatatcacat 720
agcaactttg aaaggtttgg gttgggtggt atgtgtttat ggggtgccat tgctcattgt 780
gaatgggttt ctagttacca taacctgttt gcaacacaca catgcatcat tgcctcacta 840
tgattcatcc gaatgggact ggttaagagg agcattggca acagtggaca gagattatgg 900
gatactgaat aaggcatttc atcatataac tgatacacat gtggctcatc atttgttctc 960
aacaatgcca cattaccatg caatggaagc aaccaatgca ataaagccaa tattgggtga 1020
ttattaccaa tttgatggca ccccatttta caaggcattg tggagagaag ccaaagagtg 1080
cctctatgtg gagccagatg atggagcttc tcagaagggt gtttattggt acaagaacaa 1140
gttctga 1147
<210> 15
<211> 1140
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
atgggagctg gagggcgtgt cactaagatt gaagctcaag agaagcctct ttcaagggtt 60
ccacattcaa accctccatt cagtgttggc caactcaaga aagcaattcc accacattgc 120
tttgaacgtt ctcttttcat atcattctcc tatgttgtct atgatctctt aatggcctac 180
ttactcttct acattgccac cacttatttc caccagcttc catacccatt ttccttcctt 240
gcttggccaa tctattgggc catccaaggc tgcattctca ctggtgtttg ggtgattgct 300
catgagtgtg gccaccatgc cttcagcaag tatcaacttg ttgatgacat ggttggtttg 360
atccttcact cttgtctatt agtcccttat ttctcatgga aaatcagcca ccgccacctc 420
cactccaaca ccggttccct cgaccgcgac taagtgtttg tcctgaaacc aaaatcaaag 480
gtatcacggt ataacaagta catgaacaat ccactagaga gggctatttc ccttttcatc 540
acactcacac taggatggcc cttgtacttg gccttcaatg tttctggcag accctatgat 600
agatttgcaa gccactatga cccttatgct cccatatact ctaacaggga aaggcttcta 660
atttatgtct cagattcatc tgtctttgct gtaacatatc tgctatatca catagcaact 720
ttgaaaggtt tgggttgggt ggtatgtgtt tatggggtgc cattgctcat tgtgaatggg 780
tttctagtta ccataaccta tttgcagcac acacatgcat cattgcctca ctatgattca 840
tccgaatggg actggttaag aggagcattg gcaacagtgg acagagatta tgggatactg 900
aataaggcat ttcatcatat aactgatacg catgtggctc atcatttgtt ctcaacaatg 960
cctcattacc atgcaatgga agcaaccaat gcaataaagc caatattggg tgattactac 1020
caatttgatg gcaccccagt ttacaaagca ttgtggagag aagccaaaga gtgcctctat 1080
gtggagccag atgatggagc ttctcagaag ggtgtttatt ggtacaagaa caagttctga 1140
<210> 16
<211> 1140
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
atgggagctg gagggcgtgt cactaagatt gaagctcaaa agaagcctct ttcaagggtt 60
ccacattcaa accctccatt cagtgttggc caactcaaga aagcaattcc accacattgc 120
tttgaacgtt ctcttttcat atcattctca tatgttgtct atgatctctt aatggcctac 180
ttactcttct acattgccac cacttatttc cacaagcttc catacccatt ttccttcctt 240
gcttggccaa tctattgggc catccaaggc tgcattctca ctggtgtttg ggtgattgct 300
catgagtgtg gccaccatgc cttcagcaag tatcaacttg ttgatgacat ggttggtttg 360
atccttcact cttgtctatt agtcccttat ttctcatgga aaatcagcca ccgccacctc 420
cactccaaca ccggttccct cgaccgcgac taagtgtttg tcctgaaacc aaaatcaaag 480
gtatcacggt ataacaagta catgaacaat ccactagaga gggctatttc ccttttcatc 540
acactcacac taggatggcc cttgtacttg gccttcaatg tttctggcag accctatgat 600
agatttgcaa gccactatga cccttatgct cccatatact ctaacaggga aaggcttcta 660
atttatgtct cagattcatc tgtctttgct gtaacatatc tgctatatca catagcaact 720
ctgaaaggtt tgggttgggt ggtatgtgtt tatggggtgc cattgctcat tgtgaatggg 780
tttctagtta ccataaccta tttgcagcac acacatgcat cattgcctca ctatgattca 840
tccgaatggg actggttaag aggagcattg gcaacagtgg acagagatta tgggatactg 900
aataaggcat ttcatcatat aactgatacg catgtggctc atcatttgtt ctcaacaatg 960
cctcattacc atgcaatgga agcaaccaat gcaataaagc caatattggg tgattactac 1020
caatttgatg gcaccccatt ttacaaagca ttgtggagag aagccaaaga gtgcctctat 1080
gtggagccag atgatggagc ttctaagaag ggtgtttatt ggtacaagaa caagttctga 1140
<210> 17
<211> 150
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Met Gly Ala Gly Gly Arg Val Thr Lys Ile Glu Ala Gln Glu Lys Pro
1 5 10 15
Leu Ser Arg Val Pro His Ser Asn Pro Pro Phe Ser Val Gly Gln Leu
20 25 30
Lys Lys Ala Ile Pro Pro His Cys Phe Glu Arg Ser Leu Phe Ile Ser
35 40 45
Phe Ser Tyr Val Val Tyr Asp Leu Leu Met Ala Tyr Leu Leu Phe Tyr
50 55 60
Ile Ala Thr Thr Tyr Phe His Gln Leu Pro Tyr Pro Phe Ser Phe Leu
65 70 75 80
Ala Trp Pro Ile Tyr Trp Ala Ile Gln Gly Cys Ile Leu Thr Gly Val
85 90 95
Trp Val Ile Ala His Glu Cys Gly His His Ala Phe Ser Lys Tyr Gln
100 105 110
Leu Val Asp Asp Met Val Gly Leu Ile Leu His Ser Cys Leu Leu Val
115 120 125
Pro Tyr Phe Ser Trp Lys Ile Ser His Arg His Leu His Ser Asn Thr
130 135 140
Gly Ser Leu Asp Arg Asp
145 150
<210> 18
<211> 280
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Met Gly Ala Gly Gly Arg Val Thr Lys Ile Glu Ala Gln Glu Lys Pro
1 5 10 15
Leu Ser Arg Val Pro His Ser Asn Pro Pro Phe Ser Val Gly Gln Leu
20 25 30
Lys Lys Ala Ile Pro Pro His Cys Phe Glu Arg Ser Leu Phe Ile Ser
35 40 45
Phe Ser Tyr Val Val Tyr Asp Leu Leu Met Ala Tyr Leu Leu Phe Tyr
50 55 60
Ile Ala Thr Thr Tyr Phe His Lys Leu Pro Tyr Pro Phe Ser Phe Leu
65 70 75 80
Ala Trp Pro Ile Tyr Trp Ala Ile Gln Gly Cys Ile Leu Thr Gly Val
85 90 95
Trp Val Ile Ala His Glu Cys Gly His His Ala Phe Ser Lys Tyr Gln
100 105 110
Leu Val Asp Asp Met Val Gly Leu Thr Leu His Ser Cys Leu Leu Val
115 120 125
Pro Tyr Phe Ser Trp Lys Ile Ser His Arg Arg His His Ser Asn Thr
130 135 140
Gly Ser Leu Asp Arg Asp Glu Val Phe Val Pro Lys Pro Lys Ser Lys
145 150 155 160
Val Ser Trp Tyr Asn Lys Tyr Met Asn Asn Pro Pro Gly Arg Ala Ile
165 170 175
Ser Leu Phe Ile Thr Leu Thr Leu Gly Trp Pro Leu Tyr Leu Ala Phe
180 185 190
Asn Val Ser Gly Arg Pro Tyr Asp Arg Phe Ala Ser His Tyr Asp Pro
195 200 205
Tyr Ala Pro Ile Tyr Ser Asn Arg Glu Arg Leu Leu Ile Tyr Val Ser
210 215 220
Gly Leu Arg Phe Ile Cys Leu Cys Cys Asn Ile Ser Ala Ile Ser His
225 230 235 240
Ser Asn Phe Glu Arg Phe Gly Leu Gly Gly Met Cys Leu Trp Gly Ala
245 250 255
Ile Ala His Cys Glu Trp Val Ser Ser Tyr His Asn Leu Phe Ala Thr
260 265 270
His Thr Cys Ile Ile Ala Ser Leu
275 280
<210> 19
<211> 150
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Met Gly Ala Gly Gly Arg Val Thr Lys Ile Glu Ala Gln Lys Lys Pro
1 5 10 15
Leu Ser Arg Val Pro His Ser Asn Pro Pro Phe Ser Val Gly Gln Leu
20 25 30
Lys Lys Ala Ile Pro Pro His Cys Phe Glu Arg Ser Leu Phe Ile Ser
35 40 45
Phe Ser Tyr Val Val Tyr Asp Leu Leu Met Ala Tyr Leu Leu Phe Tyr
50 55 60
Ile Ala Thr Thr Tyr Phe His Lys Leu Pro Tyr Pro Phe Ser Phe Leu
65 70 75 80
Ala Trp Pro Ile Tyr Trp Ala Ile Gln Gly Cys Ile Leu Thr Gly Val
85 90 95
Trp Val Ile Ala His Glu Cys Gly His His Ala Phe Ser Lys Tyr Gln
100 105 110
Leu Val Asp Asp Met Val Gly Leu Ile Leu His Ser Cys Leu Leu Val
115 120 125
Pro Tyr Phe Ser Trp Lys Ile Ser His Arg His Leu His Ser Asn Thr
130 135 140
Gly Ser Leu Asp Arg Asp
145 150
<210> 20
<211> 150
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Met Gly Ala Gly Gly Arg Val Thr Lys Ile Glu Ala Gln Glu Lys Pro
1 5 10 15
Leu Ser Arg Val Pro His Ser Asn Pro Pro Phe Ser Val Gly Gln Leu
20 25 30
Lys Lys Ala Ile Pro Pro His Cys Phe Glu Arg Ser Leu Phe Ile Ser
35 40 45
Phe Ser Tyr Val Val Tyr Asp Leu Leu Met Ala Tyr Leu Leu Phe Tyr
50 55 60
Ile Ala Thr Thr Tyr Phe His Gln Leu Pro Tyr Pro Phe Ser Phe Leu
65 70 75 80
Ala Trp Pro Ile Tyr Trp Ala Ile Gln Gly Cys Ile Leu Thr Gly Val
85 90 95
Trp Val Ile Ala His Glu Cys Gly His His Ala Phe Ser Lys Tyr Gln
100 105 110
Leu Val Asp Asp Met Val Gly Leu Ile Leu His Ser Cys Leu Leu Val
115 120 125
Pro Tyr Phe Ser Trp Lys Ile Ser His Arg His Leu His Ser Asn Thr
130 135 140
Gly Ser Leu Asp Arg Asp
145 150
<210> 21
<211> 150
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Met Gly Ala Gly Gly Arg Val Thr Lys Ile Glu Ala Gln Lys Lys Pro
1 5 10 15
Leu Ser Arg Val Pro His Ser Asn Pro Pro Phe Ser Val Gly Gln Leu
20 25 30
Lys Lys Ala Ile Pro Pro His Cys Phe Glu Arg Ser Leu Phe Ile Ser
35 40 45
Phe Ser Tyr Val Val Tyr Asp Leu Leu Met Ala Tyr Leu Leu Phe Tyr
50 55 60
Ile Ala Thr Thr Tyr Phe His Lys Leu Pro Tyr Pro Phe Ser Phe Leu
65 70 75 80
Ala Trp Pro Ile Tyr Trp Ala Ile Gln Gly Cys Ile Leu Thr Gly Val
85 90 95
Trp Val Ile Ala His Glu Cys Gly His His Ala Phe Ser Lys Tyr Gln
100 105 110
Leu Val Asp Asp Met Val Gly Leu Ile Leu His Ser Cys Leu Leu Val
115 120 125
Pro Tyr Phe Ser Trp Lys Ile Ser His Arg His Leu His Ser Asn Thr
130 135 140
Gly Ser Leu Asp Arg Asp
145 150
<210> 22
<211> 280
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Met Gly Ala Gly Gly Arg Val Thr Lys Ile Glu Ala Gln Glu Lys Pro
1 5 10 15
Leu Ser Arg Val Pro His Ser Asn Pro Pro Phe Ser Val Gly Gln Leu
20 25 30
Lys Lys Ala Ile Pro Pro His Cys Phe Glu Arg Ser Leu Phe Ile Ser
35 40 45
Phe Ser Tyr Val Val Tyr Asp Leu Leu Met Ala Tyr Leu Leu Phe Tyr
50 55 60
Ile Ala Thr Thr Tyr Phe His Lys Leu Pro Tyr Pro Phe Ser Phe Leu
65 70 75 80
Ala Trp Pro Ile Tyr Trp Ala Ile Gln Gly Cys Ile Leu Thr Gly Val
85 90 95
Trp Val Ile Ala His Glu Cys Gly His His Ala Phe Ser Lys Tyr Gln
100 105 110
Leu Val Asp Asp Met Val Gly Leu Thr Leu His Ser Cys Leu Leu Val
115 120 125
Pro Tyr Phe Ser Trp Lys Ile Ser His Arg Arg His His Ser Asn Thr
130 135 140
Gly Ser Leu Asp Arg Asp Glu Val Phe Val Pro Lys Pro Lys Ser Lys
145 150 155 160
Val Ser Trp Tyr Asn Lys Tyr Met Asn Asn Pro Pro Gly Arg Ala Ile
165 170 175
Ser Leu Phe Ile Thr Leu Thr Leu Gly Trp Pro Leu Tyr Leu Ala Phe
180 185 190
Asn Val Ser Gly Arg Pro Tyr Asp Arg Phe Ala Ser His Tyr Asp Pro
195 200 205
Tyr Ala Pro Ile Tyr Ser Asn Arg Glu Arg Leu Leu Ile Tyr Val Ser
210 215 220
Gly Leu Arg Phe Ile Cys Leu Cys Cys Asn Ile Ser Ala Ile Ser His
225 230 235 240
Ser Asn Phe Glu Arg Phe Gly Leu Gly Gly Met Cys Leu Trp Gly Ala
245 250 255
Ile Ala His Cys Glu Trp Val Ser Ser Tyr His Asn Leu Phe Ala Thr
260 265 270
His Thr Cys Ile Ile Ala Ser Leu
275 280
<210> 23
<211> 150
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Met Gly Ala Gly Gly Arg Val Thr Lys Ile Glu Ala Gln Lys Lys Pro
1 5 10 15
Leu Ser Arg Val Pro His Ser Asn Pro Pro Phe Ser Val Gly Gln Leu
20 25 30
Lys Lys Ala Ile Pro Pro His Cys Phe Glu Arg Ser Leu Phe Ile Ser
35 40 45
Phe Ser Tyr Val Val Tyr Asp Leu Leu Val Ala Tyr Leu Leu Phe Tyr
50 55 60
Ile Ala Thr Thr Tyr Phe His Lys Leu Pro Tyr Pro Phe Ser Phe Leu
65 70 75 80
Ala Trp Pro Ile Tyr Trp Ala Ile Gln Gly Cys Ile Leu Thr Gly Val
85 90 95
Trp Val Ile Ala His Glu Cys Gly His His Ala Phe Ser Lys Tyr Gln
100 105 110
Leu Val Asp Asp Met Val Gly Leu Ile Leu His Ser Cys Leu Leu Val
115 120 125
Pro Tyr Phe Ser Trp Lys Ile Ser His Arg His Leu His Ser Asn Thr
130 135 140
Gly Ser Leu Asp Arg Asp
145 150
<210> 24
<211> 150
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Met Gly Ala Gly Gly Arg Val Thr Lys Ile Glu Ala Gln Glu Lys Pro
1 5 10 15
Leu Ser Arg Val Pro His Ser Asn Pro Pro Phe Ser Val Gly Gln Leu
20 25 30
Lys Lys Ala Ile Pro Pro His Cys Phe Glu Arg Ser Leu Phe Ile Ser
35 40 45
Phe Ser Tyr Val Val Tyr Asp Leu Leu Met Ala Tyr Leu Leu Phe Tyr
50 55 60
Ile Ala Thr Thr Tyr Phe His Gln Leu Pro Tyr Pro Phe Ser Phe Leu
65 70 75 80
Ala Trp Pro Ile Tyr Trp Ala Ile Gln Gly Cys Ile Leu Thr Gly Val
85 90 95
Trp Val Ile Ala His Glu Cys Gly His His Ala Phe Ser Lys Tyr Gln
100 105 110
Leu Val Asp Asp Met Val Gly Leu Ile Leu His Ser Cys Leu Leu Val
115 120 125
Pro Tyr Phe Ser Trp Lys Ile Ser His Arg His Leu His Ser Asn Thr
130 135 140
Gly Ser Leu Asp Arg Asp
145 150
<210> 25
<211> 150
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Met Gly Ala Gly Gly Arg Val Thr Lys Ile Glu Ala Gln Lys Lys Pro
1 5 10 15
Leu Ser Arg Val Pro His Ser Asn Pro Pro Phe Ser Val Gly Gln Leu
20 25 30
Lys Lys Ala Ile Pro Pro His Cys Phe Glu Arg Ser Leu Phe Ile Ser
35 40 45
Phe Ser Tyr Val Val Tyr Asp Leu Leu Met Ala Tyr Leu Leu Phe Tyr
50 55 60
Ile Ala Thr Thr Tyr Phe His Lys Leu Pro Tyr Pro Phe Ser Phe Leu
65 70 75 80
Ala Trp Pro Ile Tyr Trp Ala Ile Gln Gly Cys Ile Leu Thr Gly Val
85 90 95
Trp Val Ile Ala His Glu Cys Gly His His Ala Phe Ser Lys Tyr Gln
100 105 110
Leu Val Asp Asp Met Val Gly Leu Ile Leu His Ser Cys Leu Leu Val
115 120 125
Pro Tyr Phe Ser Trp Lys Ile Ser His Arg His Leu His Ser Asn Thr
130 135 140
Gly Ser Leu Asp Arg Asp
145 150
<210> 26
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gacaagaagt actcgatcgg cctcg 25
<210> 27
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
tcgtagggta cttctcgtgg taggc 25
<210> 28
<211> 379
<212> PRT
<213> peanut (Arachis hypogaea)
<400> 28
Met Gly Ala Gly Gly Arg Val Thr Lys Ile Glu Ala Gln Lys Lys Pro
1 5 10 15
Leu Ser Arg Val Pro His Ser Asn Pro Pro Phe Ser Val Gly Gln Leu
20 25 30
Lys Lys Ala Ile Pro Pro His Cys Phe Glu Arg Ser Leu Phe Ile Ser
35 40 45
Phe Ser Tyr Val Val Tyr Asp Leu Leu Met Ala Tyr Leu Leu Phe Tyr
50 55 60
Ile Ala Thr Thr Tyr Phe His Lys Leu Pro Tyr Pro Phe Ser Phe Leu
65 70 75 80
Ala Trp Pro Ile Tyr Trp Ala Ile Gln Gly Cys Ile Leu Thr Gly Val
85 90 95
Trp Val Ile Ala His Glu Cys Gly His His Ala Phe Ser Lys Tyr Gln
100 105 110
Leu Val Asp Asp Met Val Gly Leu Thr Leu His Ser Cys Leu Leu Val
115 120 125
Pro Tyr Phe Ser Trp Lys Ile Ser His Arg Arg His His Ser Asn Thr
130 135 140
Gly Ser Leu Asp Arg Asp Glu Val Phe Val Pro Lys Pro Lys Ser Lys
145 150 155 160
Val Ser Trp Tyr Asn Lys Tyr Met Asn Asn Pro Pro Gly Arg Ala Ile
165 170 175
Ser Leu Phe Ile Thr Leu Thr Leu Gly Trp Pro Leu Tyr Leu Ala Phe
180 185 190
Asn Val Ser Gly Arg Pro Tyr Asp Arg Phe Ala Ser His Tyr Asp Pro
195 200 205
Tyr Ala Pro Ile Tyr Ser Asn Arg Glu Arg Leu Leu Ile Tyr Val Ser
210 215 220
Asp Ser Ser Val Phe Ala Val Thr Tyr Leu Leu Tyr His Ile Ala Thr
225 230 235 240
Leu Lys Gly Leu Gly Trp Val Val Cys Val Tyr Gly Val Pro Leu Leu
245 250 255
Ile Val Asn Gly Phe Leu Val Thr Ile Thr Tyr Leu Gln His Thr His
260 265 270
Ala Ser Leu Pro His Tyr Asp Ser Ser Glu Trp Asp Trp Leu Arg Gly
275 280 285
Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile Leu Asn Lys Ala Phe
290 295 300
His His Ile Thr Asp Thr His Val Ala His His Leu Phe Ser Thr Met
305 310 315 320
Pro His Tyr His Ala Met Glu Ala Thr Asn Ala Ile Lys Pro Ile Leu
325 330 335
Gly Asp Tyr Tyr Gln Phe Asp Gly Thr Pro Val Tyr Lys Ala Leu Trp
340 345 350
Arg Glu Ala Lys Glu Cys Leu Tyr Val Glu Pro Asp Asp Gly Ala Ser
355 360 365
Gln Lys Gly Val Tyr Trp Tyr Lys Asn Lys Phe
370 375

Claims (9)

1. The preparation method of the peanut mutant is characterized by comprising the following steps:
providing a coding region sequence of a peanut desaturation lipase gene AhFAD2B, a CRISPR/Cas9 gene editing vector and peanut embryogenic callus;
Obtaining an sgRNA target sequence T1-T4 according to a coding region sequence of the peanut desaturation lipase gene AhFAD 2B;
respectively synthesizing a sgRNA target sequence nucleotide fragment containing a sticky end and a complementary chain thereof according to the sgRNA target sequence T1-T4, and performing mixed annealing to obtain double-stranded DNA T1-T4 of which two ends contain the sticky ends;
respectively connecting the double-stranded DNA T1-T4 with the CRISPR/Cas9 gene editing vector to obtain a gene editing vector T1-T4;
performing gene transformation on the peanut embryogenic callus by using at least one gene editing vector T1-T4, and culturing to obtain the peanut mutant;
the sgRNA target sequences T1-T4 are respectively shown as SEQ ID NO. 1-4, and the coding region sequence of the peanut desaturation lipase gene AhFAD2B is shown as SEQ ID NO. 5.
2. The preparation method of the peanut mutant as claimed in claim 1, wherein the coding region sequence of the peanut desaturation lipase gene AhFAD2B is obtained by performing PCR amplification through an amplification primer by using the peanut desaturation lipase gene FAD as a template, wherein the amplification primer comprises a forward amplification primer and a reverse amplification primer, the nucleotide sequence of the forward amplification primer is shown as SEQ ID NO. 6, and the nucleotide sequence of the reverse amplification primer is shown as SEQ ID NO. 7.
3. The method for preparing the peanut mutant as claimed in claim 2, wherein in the step of performing PCR amplification by the amplification primer, every 50 μ l of reaction system for PCR amplification comprises 13 μ l of double distilled water, 5 μ l of the amplification primer, 5 μ l of the peanut desaturation lipase gene AhFAD2B 5, 2 xTaq mix25 μ l, Mg2+1 μ l and glycerol 1 μ l.
4. The method for preparing peanut mutants according to claim 2, wherein in the step of performing PCR amplification by using the amplification primers, 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.
5. The method for preparing the peanut mutant as claimed in any one of claims 1 to 4, wherein the CRISPR/Cas9 gene editing vector is a pKSE401 binary vector.
6. The method for preparing peanut mutants according to any one of claims 1-4, wherein the cohesive end is a BsaI restriction endonuclease cohesive end.
7. A peanut mutant gene is characterized in that the nucleotide sequence of the peanut mutant gene is shown in SEQ ID NO. 8-16.
8. The peanut mutant gene coded protein of claim 7, characterized in that the amino acid sequence is shown as SEQ ID NO. 17-25.
9. The peanut mutant prepared by the method for preparing the peanut mutant as claimed in any one of claims 1 to 6, the peanut mutant gene as claimed in claim 7 and the application of the protein coded by the peanut mutant gene in the breeding of high-oleic acid peanut varieties.
CN202010672354.6A 2020-07-14 2020-07-14 Preparation method of peanut mutant, peanut mutant gene, protein coded by peanut mutant gene and application of peanut mutant gene Active CN111944827B (en)

Priority Applications (1)

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