CN110684796B - Method for specifically knocking out soybean lipoxygenase gene by CRISPR-Cas9 and application thereof - Google Patents
Method for specifically knocking out soybean lipoxygenase gene by CRISPR-Cas9 and application thereof Download PDFInfo
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Abstract
The invention discloses a method for specifically knocking out a soybean lipoxygenase gene by CRISPR-Cas9 and application thereof, belonging to the field of soybean molecular breeding and biotechnology. The method comprises the following steps: targeting of highly homologous soybean lipoxygenase gene coding region designGmLox1AndGmLox2sgRNA of (a)GmLox1/2，And targetingGmLox3sgRNA of (a)GmLox3(ii) a Constructing a CRISPR-Cas9 gene knockout vector for each sgRNA independently, and then infecting soybean cotyledonary nodes in a mixed manner; stably genetically transforming soybean to obtain plantGmlox‑28AndGmlox‑60knock out 3 simultaneouslyGmLoxA gene; after selfing for two generations, at T25T-DNA strains separated from editing sites in generation plantsGmLoxAll deletion mutants. The method provides a new strategy for directly and purposefully and rapidly creating a new soybean germplasm without beany flavor.
Description
Technical Field
The invention belongs to the technical field of soybean molecular breeding and biology, and particularly relates to a method for specifically knocking out a soybean lipoxygenase gene by using CRISPR-Cas9 and application thereof.
Background
(iii) semen glycinesGlycine max(L.) Merr.) originated in China, is one of the main cultivated crops in China and even in the world, is an important source for human beings, poultry and livestock to take plant protein and fat, and plays a very important role in agricultural production in China (Korean et al, 2003; Biyindong et al, 2014; Panwenhua and Schwangsi, 2014; Zhang Yao nan, 2017). However, Lipoxygenase (LOX) in the cotyledons of soybeans can specifically catalyze the oxygenation reaction of polyunsaturated fatty acids having a cis-1.4-pentadiene structure during storage and processing of soybeans and products thereof to form hydrogen peroxide derivatives (Axelrod et al, 1981), and finally decompose into volatile substances such as aldehydes, alcohols and ketones of small molecules to generate beany flavor and bitter taste (Brash, 1999; Liavonchanka and Fesnser, 2006), which seriously affect the eating quality, storage and nutritional value (Rackis et al, 1979). The production and processing of soybeans often adopt a heating or microwave treatment methodIt is fishy and requires a lot of money and labor in the process. The economic and permanent method for removing beany flavor is to breed the soybean variety without beany flavor with the deletion of lipoxidase. In conventional breeding, it is often necessary toloxThe mutant is hybridized with a fine variety, and then the soybean variety without beany flavor is bred through multi-generation backcross or selfing, which is a time-consuming and labor-consuming process.
CRISPR-Cas9 (Clustered regular intercurrent short linked templates organized nucleic acid loop 9) is a new generation of gene site-directed editing technology which appears after zinc finger endonuclease (ZFN) and transcription activator like nuclease (TALEN), and due to the advantages of simple operation, short period, high efficiency and the like, once the technology is developed (Cong et al, 2013; Mali et al, 2013) rapidly, the technology is attractive in the fields of gene function analysis, crop genetic improvement, new variety breeding and the like (Upday et al, 2013; Liang et al, 2014; Sun et al, 2016; Cai et al, 2018). The working principle is as follows: in the CRISPR-Cas9 system, an exogenous Single-stranded guide RNA (sgRNA) directs cleavage of the endonuclease protein Cas9 at the target site and generates a Double-stranded DNA break (DSBs) (Jinek et al, 2012); subsequently, DSBs induce a DNA self-Repair mechanism, introducing insertions/deletions of small fragments or precise site-Directed substitutions/insertions at the break sites by non-homologous End Joining (NHEJ) or homologous recombination (HDR), resulting in gene function deletions (Shan et al, 2013; Svitashev et al, 2015). At present, the CRISPR-Cas9 system has been successfully applied to soybeansGmFT2a、FAD2-2AndGmSPL9the creation of isogenic mutants (Cai et al, 2018; Amin et al, 2019; Bao et al, 2019) indicates that the genetic improvement of soybean agronomic traits by using the CRISPR-Cas9 technology is feasible.
The soybean lipoxygenase comprises 3 isozymes Lox1, Lox2 and Lox3, respectivelyGlyma.13g347600（GmLox1）、Glyma.13g347500 （GmLox2) AndGlyma.15g026300（GmLox3) And (4) coding and synthesizing. Simultaneous knockout of 3 through CRISPR-Cas9 systemGmLoxThe gene can rapidly create beany flavor-free big foodNovel germplasm of bean. The CRISPR-Cas9 is an artificially designed sgRNA to mediate the cleavage of a specific site of a genome, so whether an accurately targeted sgRNA can be successfully designed is a key technical problem of the path. The invention aims to solve the technical problem so as to provide a new way for creating a new soybean germplasm without beany flavor.
Disclosure of Invention
The invention aims to provide a method for specifically knocking out soybean lipoxygenase genes by CRISPR-Cas9 and application thereof.
In order to achieve the purpose, the invention adopts the following technical measures:
a method for specifically knocking out soybean lipoxygenase genes by CRISPR-Cas9, comprising the following steps:
(1) designing and synthesizing two sgRNAs at soybean lipoxygenase gene editing sites, sgRNA-GmLox1/2And sgRNA-GmLox3(ii) a Wherein sgRNA-GmLox1/2Targeting simultaneously highly homologousGmLox1AndGmLox2，sgRNA-GmLox3then target toGmLox3。
(2) The soybean-dedicated CRISPR/Cas9 gene editing vector pGES201 is used as a framework to independently construct a CRISPR-Cas9 gene knockout vector for each sgRNA and transform agrobacterium GV 3101;
(3) carrying out mixed infection on soybean cotyledon nodes by agrobacterium GV3101 carrying a CRISPR-Cas9 gene knockout vector in a mixed pool mode to perform stable transformation on soybeans;
(4) at T0Screening positive plants, identifying the sgRNA distribution condition and detecting the editing condition of the targeted gene in the generation stable transformation seedlings;
(5) at T1generation-T2Generation screening of T-DNA separated from editing sitesGmLoxAll deletion mutants.
The accurate positioning of the soybean lipoxygenase gene editing sites in the step (1) is carried out on the No. 13 chromosome of a soybean genome (Wm82. a 2)GmLox1Coding region exon 2 (chromosomal coordinates 43769605-43769627) andGmLox2exon 2 of the coding region (with chromosome coordinates 43762424-4376244), and chromosome 15GmLox3Coding region exon 3: (Chromosome coordinates 2125132 and 2125154); the sgRNA-GmLox1/2The nucleotide sequence of (a) is: 5'-GGAAAGGATACGTTCTTGGAAGG-3', respectively; sgRNA-GmLox3The nucleotide sequence of (a); 5'-CCTTTCCTTATCCTCGTAGGGGG-3', respectively; sgRNA-GmLox1/2And sgRNA-GmLox3Contains AGG or GGG as a PAM sequence specifically recognized by the Cas9 endonuclease, and can guide the endonuclease protein Cas9 to cut at a target site and generate double-stranded DNA break, so that small-fragment insertion/deletion or precise site-specific substitution/insertion is introduced at the break position to cause gene function loss.
The CRISPR-Cas9 gene knockout vector in the step (2) is constructed as follows: annealing the sgRNA sense and antisense oligonucleotide chains into double chains, connecting the sgRNA annealing double chains to a vector pGES201 by using a CRISPR-Cas9 gene editing vector pGES201 as a framework, converting the connection product to an escherichia coli competent cell, detecting to obtain positive clones, extracting recombinant plasmids, and obtaining a CRISPR-Cas9 gene knockout vector; the expression of sgRNA is started by pGmU6 promoter, and the expression of Cas9 is started by soybean endogenous promoter pM 4.
Further, the sgRNA sense and antisense oligonucleotide strands described above, wherein the sgRNA-GmLox1/2The sense oligonucleotide chain of (a) is: 5'-GGATTGGAAAGGATACGTTCTTGGA-3', antisense oligonucleotide strand 5'-AAACTCCAAGAACGTATCCTTTCCA-3'; sgRNA-GmLox3The sense oligonucleotide chain of (a) is: 5'-GGATTGCCTTTCCTTATCCTCGTAGG-3', antisense oligonucleotide strand 5'-AAACCCTACGAGGATAAGGAAAGGCA-3'.
And (3) infecting soybean cotyledon nodes in the step (3), and preferably selecting the soybean variety as No. 6 Huachun.
The method for specifically knocking out the soybean lipoxygenase gene by the CRISPR-Cas9 is applied to breeding soybean germplasm without beany flavor.
The invention has the advantages that:
the sgRNA provided by the invention guides the endonuclease protein Cas9 to perform high-efficiency cutting at a target position and generate double-stranded DNA (deoxyribonucleic acid) breakage, and realizes quick, accurate, high-efficiency and specific knockout of 3 soybeans by combining a mixed pool conversion mode of a sgRNA libraryGmLoxA gene. The invention providesFor a convenient and efficient strategy to construct soybeansGmLoxThe full deletion mutant effectively avoids the problems of long period, high cost and the like in the conventional breeding of the fishy-free soybeans.
Drawings
FIG. 1 is a simplified vector structure diagram of a CRISPR-Cas9 gene editing vector pGES 201.
FIG. 2 the editing sitesGmLoxThe position on the gene.
FIG. 3T0And (5) DNA sequencing result of the generation plant target area.GmLox-28 andGmLox-60 are marked with circles.
FIG. 4T1And (5) determining the enzyme activity of the generation plant LOX 1. A shows the result of color reaction of each sample; b shows the absorbance of each sample; c shows the gene editing condition of each T1 generation mutant; CK (water) represents ultrapure water, HC6 represents "Huachun No. 6" having LOX1 enzyme activity, and WX4 represents a non-fishy soybean variety (five stars No. 4) having no LOX1 enzyme activity; HC6 is colorless, while CK (water), WX4 and strainsGmLox-28 andGmLoxall-60 showed blue color, indicatingGmLox-28 andGmLoxnone of-60 contained LOX1 enzyme activity.
FIG. 5T1And (5) determining the enzyme activity of the generation plant LOX 2.A shows the result of color reaction of each sample; b shows the absorbance of each sample; c shows each T1Generation mutant gene editing conditions; CK (water) represents ultrapure water, HC6 represents "Huachun No. 6" having LOX2 enzyme activity, and WX4 represents a non-fishy soybean variety (five stars No. 4) having no LOX2 enzyme activity; HC6 is colorless, while CK (water), WX4 and strainsGmLox-28 andGmLoxall-60 showed blue color, indicatingGmLox-28 andGmLoxnone of-60 contains LOX2 enzyme activity
FIG. 6T1And (5) determining the enzyme activity of the generation plant LOX 3. A shows the result of color reaction of each sample; b shows the absorbance of each sample; c shows each T1Generation mutant gene editing conditions; CK (water) represents ultrapure water, HC6 represents "Huachun No. 6" having LOX3 enzyme activity, and WX4 represents a non-fishy soybean variety (five stars No. 4) having no LOX3 enzyme activity; HC6 is colorless, while CK (water), WX4 and strainsGmLox-28 andGmLox-60 homographicShows yellow color, indicateGmLox-28 andGmLoxnone of-60 contained LOX3 enzyme activity.
FIG. 7 the results of the transform-free mutant screening. In the figure, Arabic numerals indicate the numbers of different individuals of each strain, "M" indicates a DNA molecular weight standard Marker, "water" indicates ultrapure water, and the gene-free mutant is marked with a circle.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the following examples are only examples of the present invention and do not represent the scope of the present invention defined by the claims.
The technical scheme of the invention is the conventional technology in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available.
Example 1: construction of CRISPR-Cas9 gene knockout vector and soybean stable transformation
Main reagents and material sources:
coli DH5 alpha competent cell, Agrobacterium rhizogenes K599 competent cell and Agrobacterium GV3101 competent cell used in the study are all preserved in the laboratory; the CRISPR-Cas9 gene editing vector pGES201 used was modified from pCAMBIA1300 (Cambia, australia) in this laboratory, pGmU6 promoter started expression of sgRNA, soybean endogenous promoter pM4 started expression of Cas9, and the empty vector carried its own gene "escherichia coli toxin-antitoxin system toxin protein ccdb (control of cell division or death system)", both ends of which had cleavage sites BsaI, respectively (fig. 1).
PCR Mix enzyme PCR Master Mix was purchased from Otsugaku Biotechnology Ltd; BsaI restriction enzymes were purchased from NEB (UK); t4 ligase was purchased from Beijing Quanjin Biotechnology, Inc.; the agarose gel DNA recovery kit and the plasmid small-extraction kit are purchased from Tiangen company; antibiotics such as kanamycin (kanamycin), streptomycin (streptomycin), gentamicin (gentamicin), rifampin (rifampicin), Carbenicillin (Carbenicillin), and glufosinate (Basta) were purchased from Sigma.
Bacterial culture medium reagent: tryptone and yeast extract were from OXOID (USA); other nutrient solution reagents are all domestic or imported analytical pure products.
The method comprises the following operation steps:
1.1 sgRNA design and synthesis: the sgRNA should be designed to follow the following 4 principles, with at least 1 base mismatch to the off-target site guaranteed: 1) designing more than 2 sgRNAs for each target gene so as to screen the sgRNAs with high gene editing efficiency; 2) ensuring that the sgRNA is positioned at the middle upstream of the CDS sequence of the target gene; 3) different positions of the target genes are distributed among multiple sgRNAs of the same gene, and the distance is ensured to be not more than 1000bp as far as possible; 4) according to the experimental requirements, the 'single-knock sgRNA' or 'multi-knock sgRNA' is selected. The term "single-knock sgRNA" means that one sgRNA has only one targeted gene, i.e., the sgRNA can knock out only one gene, and "multiple-knock sgRNA" means that one sgRNA has multiple targeted genes, i.e., multiple genes can be knocked out simultaneously (most of the multiple targeted genes are homologous genes in the species). In the research, the sgRNA is designed by using online software CRISPR-GE (http:// skl.scau.edu.cn/home /), and two sgRNAs are designed in total, wherein the sgRNA is prepared by adding one or more auxiliary materialsGmLox1/2And sgRNA-GmLox3。sgRNA-GmLox1/2Targeting simultaneously highly homologousGmLox1AndGmLox2sgRNA-GmLox3 is targetedGmLox3.4 sgrnas are designed for each sgRNA, a hairy root transformation experiment verifies the editing efficiency of the sgrnas, and then the sgrnas with high gene editing efficiency are selected for the transformation of agrobacterium GV3101 and the stable transformation of soybeans. sgRNA information is as in table 1:
table 1 sgRNA information
1.2 annealing of oligonucleotides to duplexes:
an annealing reaction system was prepared as follows
Sense oligo (10. mu.M, sequence as in Table 2) 5. mu.L
Antisense oligonucleotide (10. mu.M, sequence shown in Table 2) 5. mu.L
NaCl 100 mM (final concentration)
Tris-HCl pH7.450 mM (final concentration)
Adding water to make up to 50 μ L
Mixing the prepared annealing reaction buffer solution repeatedly, placing the mixture on a PCR instrument after short-time centrifugation, and operating the following procedures:
95℃ 4min，
RAMP 0.1℃/S ， 95℃ to 16℃
16℃ ∞
the annealed double-stranded Oligo was used immediately or stored at-20 ℃ for a long period of time.
TABLE 2 Positive antisense oligonucleotides
1.3 pGES201 vector linearization:
enzyme digestion system:
BsaI 2μL
10x cutsmart buffer 5μL
Adding water to make up to 50uL, placing in a metal bath, and setting the temperature at 37 ℃ for 2 hours.
1.4 linking the linearized vector to the annealed oligonucleotide strands:
after annealing, the oligonucleotide chain was 1. mu.L
10x T4 buffer 1μL
T4 ligase 0.5. mu.L
Water was added to 10ul and the ligation was carried out in a 25 ℃ metal bath for 2 hours or overnight at 16 ℃.
1.5 transformation of E.coli DH 5. alpha. and identification of positive clones:
the ligation product was transformed into E.coli competent cells (DH 5. alpha. competent), and the pGES201 vector was resistant to kanamycin in the prokaryotes, and the transformation was accomplished according to the following experimental protocol:
(1) DH5 alpha competent cell is taken out from-80 ℃, insert in ice rapidly, wait for the fungus piece to melt after 5 minutes, add the junctional product and stir EP tube bottom gently and mix evenly with hand, stand in ice for 30 minutes;
(2) heat shock is carried out in 42 ℃ water bath for 60 seconds, the ice is quickly put back on the ice and stands for 2 minutes, and the conversion efficiency is reduced by shaking;
(3) adding 400 mu L of antibiotic-free sterile medium (LB medium) into the centrifuge tube, uniformly mixing, and then recovering for 45-60 minutes at 37 ℃ by a shaking table at 200 rpm;
(4) taking 100-500 mu L supernatant, gently blowing and beating the resuspended strain block and spreading the resuspended strain block on LB culture medium containing corresponding antibiotics (the concentration is 50 mg/L);
(5) the plates were placed upside down in a 37 ℃ incubator overnight.
The positive clone is detected by adopting a PCR method, an artificially synthesized sense oligonucleotide chain is used as a PCR front primer (shown in table 2), and a common rear primer CRISPR-R (with the sequence of 5'-GGCTCACGTTCAAACGTGGC-3') is designed at a position which is about 500bp away from the sgRNA on a carrier. And (4) storing the positive bacteria liquid and extracting plasmids to obtain a CRISPR-Cas9 gene knockout vector.
1.6 Agrobacterium K599 transformation and hairy root transformation experiments:
the CRISPR-Cas9 gene knockout vector is used for transforming agrobacterium K599 competent cells, and the resistance is kanamycin resistance and streptomycin resistance. The experimental procedure was as follows:
(1) taking out K599 competent cells from-80 ℃, quickly inserting into ice, after 5 minutes, adding plasmids and manually dialing the bottom of an EP tube, gently mixing uniformly, and standing in ice for 30 minutes;
(2) putting the mixture into liquid nitrogen for 2 minutes, quickly putting the mixture into a water bath kettle at 37 ℃ and standing for 5 minutes, wherein the conversion efficiency is reduced by shaking;
(3) adding 400 mu L of antibiotic-free sterile medium (LB medium) into the centrifuge tube, uniformly mixing, and recovering for 4 hours at 28 ℃ by a shaking table at 180 rpm;
(4) 100 and 500 mu L of supernatant are taken, and the resuspended strain blocks are lightly blown and spread on LB culture medium containing corresponding antibiotics (the concentration is 50 mg/L).
(5) The plate was placed in an incubator at 28 ℃ overnight with inversion.
Selecting positive clones, and adopting a cotyledonary node infection method to carry out hairy root transformation on the soybeans. Extracting DNA from hairy roots growing for 7-10d, detecting whether the hairy roots are positive by taking an artificially synthesized sense oligonucleotide chain as a PCR front primer (shown in table 2) and a CRISPR-R (sequence is 5'-GGCTCACGTTCAAACGTGGC-3') as a rear primer, selecting 9-12 positive roots with bright bands, carrying out PCR detection and sequencing by using an editing primer, and counting the editing efficiency according to a sequencing result. As shown in the following table, the sgRNA with high editing efficiency was finally selectedGmLox1/2-sg-3 and sgRNA-GmLox3-sg-2 (FIG. 2) for subsequent stable soybean transformation experiments.
Table 3 sgRNA editing efficiency
1.7 Agrobacterium GV3101 transformation and Stable Soybean transformation:
two selected vectors with high editing efficiency (sgRNA-GmLox1/2-sg-3 and sgRNA-GmLox3-sg-2) to respectively transform agrobacterium GV3101 competent cells (the method is the same as the above 1.6), and two agrobacterium liquid solutions are mixed in equal amount to form a 'bank', and the stable transformation of soybean is carried out by adopting an agrobacterium infection cotyledonary node method. The acceptor variety used for stable genetic transformation of the soybean is southern main cultivated variety Huachun No. 6 (national examined soybean variety bred by southern China agricultural university), and the screening resistance is Basta. The specific process is as follows: placing the Huachun No. 6 seed sterilized by chlorine in a culture dish, taking cotyledonary node as explant after germination for agrobacterium infection, and obtaining T from the explant through processes of induction culture, elongation culture, rooting culture and the like0Stably transformed plants.
Example 2: the T-DNA being separated from the editing siteGmLoxSelection of all-deletion mutants
The stable transformation plant is screened by methods of blade smearing Basta, Bar Primer PCR (BPP), SgRNA Specific PCR (SSP), PCR product sequencing and the like, and the identification of the sgRNA distribution condition and the detection of the target gene editing condition are carried out. The results show that: 76T strains060 plants in the generation-stable transformed seedlings are positive plants, wherein 27 plants only comprise sgRNA-GmLox1/222 strain contains only sgRNA-GmLox311 strains simultaneously contain sgRNA-GmLox1/2And sgRNA-GmLox3(ii) a 22 positive plants have occurredGmLoxGene editing (the gene editing efficiency is 36.7%), wherein the positive plantsGmlox-28 andGmlox60 knockdown 3 at the same timeGmLoxGenes (FIG. 3).
Get T0Generation positive plantGmlox-28 andGmlox-60 harvested seeds, cultivated in pots in a growth chamber, obtaining T1And (5) plant generation. Get T1DNA extracted from leaf of plant for detectionGmLoxGene editing, seeds harvested at maturity were used to determine LOX enzyme activity. LOX enzyme activity is measured by colorimetric assay, and the specific process is as follows: 1) the dried seed samples were ground separately into powders. 2) Weighing seed sample powder 15, 30 and 15 mg respectively in 1.5 mL of 0.2 mol L−1Sodium borate buffer (pH 9.0), 1.5 mL of 0.2 mol L−1Sodium phosphate buffer solution (pH 6.0) and 1.5 mL of 0.2 mol L−1Lipoxygenase solutions 1, 2 and 3 (designated LS1, LS2 and LS3, for LOX1, LOX2, LOX3 detection, respectively) were extracted in sodium phosphate buffer solution (pH 6.6), extracted at rest at 4 ℃ for 1 hour, and then centrifuged (12000 rpm, 5 minutes, 4 ℃) to obtain supernatant. 3) Detection of LOX1 enzyme activity: 0.5 mL of LS1 was added to 1.0 mL of substrate solution 1 (containing 0.125 mol L of sodium borate buffer pH 9.0)−1Methylene blue 12.5. mu. mol L−11.375 mmol L of sodium linoleate substrate−1). Detection of LOX2 enzyme activity: 0.5 mL of LS2 was added to 1.0 mL of substrate solution 2 (containing 0.125 mol L of pH 6.0 sodium phosphate buffer solution)−1Methylene blue 12.5. mu. mol L−11.375 mmol L of sodium linoleate substrate−1Dithiothreitol 25 mmol L−1Acetone 12.5%). Detection of LOX3 enzyme activity: 0.5 mL of LS3 was added to 1.0 mL of substrate solution 3 (containing 0.125 mol L of pH 6.6 sodium phosphate buffer solution)−11.375 mmol L of sodium linoleate substrate−1Beta-carotene substrate 12.5%). After mixing, each reaction was incubated in a clear centrifuge tube for 15 minutes and the solution color was recorded (LOX 1 and LOX2 were colorless or blue, LOX3 was colorless or yellow). 4) The absorbance of each sample was measured with a spectrophotometer (UV-1600; Shimadzu, Kyoto, Japan) at 660 nm (for LOX1 and LOX2) and 452 nm (for LOX 3). After the detection, the detection result shows that,Gmlox-28 andGmlox-60 T1all the generation plants areGmLoxAll deletion mutants, none containing LOX enzyme activity (fig. 4-6).
Then, we get fromGmlox-28 andGmlox-60T1selecting two single plants from the generation line, harvesting seeds, planting in a pot in a growth chamber to obtain T2And (5) plant generation. Get T2The DNA of the leaf of the generation plant is extracted, and the separation of the transgenic element is detected by using CRISPR-Cas9 and sgRNA specific primers (the primer sequences are shown in the table below). Finally fromGmlox-28 andGmlox-T of 6025 transgenic-free plants are selected from the generation plantsGmLoxFull deletion mutants (fig. 7).
TABLE 4 transgenic element detection primers
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> CRISPR-Cas9 specific soybean lipoxygenase gene knockout method and application thereof
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<170> PatentIn version 3.3
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<213> sgRNA-GmLox3 Sg-3
<400> 13
aatagcgtaa ccagcgttgg ggg 23
<210> 14
<211> 23
<212> DNA
<213> sgRNA-GmLox3 Sg-4
<400> 14
cggttatcac tggtcaacga cgg 23
<210> 15
<211> 26
<212> DNA
<213> sgRNA-GmLox1/2 Sg-1 sense strand
<400> 15
ggattgagaa ggttgcttcc tgagaa 26
<210> 16
<211> 26
<212> DNA
<213> sgRNA-GmLox1/2 Sg-1 antisense strand
<400> 16
aaacttctca ggaagcaacc ttctca 26
<210> 17
<211> 26
<212> DNA
<213> sgRNA-GmLox1/2 Sg-2 sense strand
<400> 17
ggattgtacc tgcagatctt atcaag 26
<210> 18
<211> 26
<212> DNA
<213> sgRNA-GmLox1/2 Sg-2 antisense strand
<400> 18
aaaccttgat aagatctgca ggtaca 26
<210> 19
<211> 25
<212> DNA
<213> sgRNA-GmLox1/2 Sg-3 sense strand
<400> 19
ggattggaaa ggatacgttc ttgga 25
<210> 20
<211> 25
<212> DNA
<213> sgRNA-GmLox1/2 Sg-3 antisense strand
<400> 20
aaactccaag aacgtatcct ttcca 25
<210> 21
<211> 26
<212> DNA
<213> sgRNA-GmLox1/2 Sg-4 sense strand
<400> 21
ggattgtcgt taccaacttt gggagc 26
<210> 22
<211> 26
<212> DNA
<213> sgRNA-GmLox1/2 Sg-4 antisense strand
<400> 22
aaacgctccc aaagttggta acgaca 26
<210> 23
<211> 26
<212> DNA
<213> sgRNA-GmLox3 Sg-1 sense strand
<400> 23
ggattgaagg aaggattaac tttcag 26
<210> 24
<211> 26
<212> DNA
<213> sgRNA-GmLox3 Sg-1 antisense strand
<400> 24
aaacctgaaa gttaatcctt ccttca 26
<210> 25
<211> 26
<212> DNA
<213> sgRNA-GmLox3 Sg-2 sense strand
<400> 25
ggattgcctt tccttatcct cgtagg 26
<210> 26
<211> 26
<212> DNA
<213> sgRNA-GmLox3 Sg-2 antisense strand
<400> 26
aaaccctacg aggataagga aaggca 26
<210> 27
<211> 26
<212> DNA
<213> sgRNA-GmLox3 Sg-3 sense strand
<400> 27
ggattgaata gcgtaaccag cgttgg 26
<210> 28
<211> 26
<212> DNA
<213> sgRNA-GmLox3 Sg-3 antisense strand
<400> 28
aaacccaacg ctggttacgc tattca 26
<210> 29
<211> 26
<212> DNA
<213> sgRNA-GmLox3 Sg-4 sense strand
<400> 29
ggattgcggt tatcactggt caacga 26
<210> 30
<211> 26
<212> DNA
<213> sgRNA-GmLox3 Sg-4 antisense strand
<400> 30
aaactcgttg accagtgata accgca 26
<210> 31
<211> 25
<212> DNA
<213> pGmU6-F
<400> 31
taaactgaag gcgggaaacg acaat 25
<210> 32
<211> 20
<212> DNA
<213> CRISPR-R
<400> 32
<210> 33
<211> 25
<212> DNA
<213> sgRNA-GmLox1/2-R
<400> 33
aaactccaag aacgtatcct ttcca 25
<210> 34
<211> 26
<212> DNA
<213> sgRNA-GmLox3-R
<400> 34
aaaccctacg aggataagga aaggca 26
Claims (5)
1. A method for specifically knocking out soybean lipoxygenase genes by CRISPR-Cas9, which is characterized by comprising the following steps:
(1) designing and synthesizing two sgRNAs at soybean lipoxygenase gene editing sites, sgRNA-GmLox1/2And sgRNA-GmLox3(ii) a Wherein sgRNA-GmLox1/2Targeting simultaneously highly homologousGmLox1AndGmLox2，sgRNA-GmLox3targetingGmLox3；
(2) The soybean-dedicated CRISPR/Cas9 gene editing vector pGES201 is used as a framework to independently construct a CRISPR-Cas9 gene knockout vector for each sgRNA and transform agrobacterium GV 3101;
(3) carrying out mixed infection on soybean cotyledon nodes by agrobacterium GV3101 carrying a CRISPR-Cas9 gene knockout vector in a mixed pool mode to perform stable transformation on soybeans;
(4) at T0Screening positive plants, identifying the sgRNA distribution condition and detecting the editing condition of the targeted gene in the generation stable transformation seedlings;
(5) at T1generation-T2Generation screening of T-DNA separated from editing sitesGmLoxA full deletion mutant;
the accurate positioning of the soybean lipoxygenase gene editing sites in the step (1) is carried out on the No. 13 chromosome of the soybean genomeGmLox1Coding region exon No. 2, andGmLox2exon No. 2 of coding region; and chromosome 15GmLox3Coding region exon 3; the sgRNA-GmLox1/2The nucleotide sequence of (a) is: 5'-GGAAAGGATACGTTCTTGGAAGG-3', respectively; sgRNA-GmLox3The nucleotide sequence of (a); 5'-CCTTTCCTTATCCTCGTAGGGGG-3', respectively; sgRNA-GmLox1/2And sgRNA-GmLox3Contains AGG or GGG as a PAM sequence specifically recognized by the Cas9 endonuclease, and can guide the endonuclease protein Cas9 to cut at a target site and generate double-stranded DNA break, so that small-fragment insertion/deletion or precise site-specific substitution/insertion is introduced at the break position to cause gene function loss.
2. The method for specifically knocking out soybean lipoxygenase gene by CRISPR-Cas9 according to claim 1, wherein the method comprises the following steps: the CRISPR-Cas9 gene knockout vector in the step (2) is constructed as follows: annealing the sgRNA sense and antisense oligonucleotide chains into double chains, connecting the sgRNA annealing double chains to a vector pGES201 by using a CRISPR-Cas9 gene editing vector pGES201 as a framework, converting the connection product to an escherichia coli competent cell, detecting to obtain positive clones, extracting recombinant plasmids, and obtaining a CRISPR-Cas9 gene knockout vector; the expression of sgRNA is started by pGmU6 promoter, and the expression of Cas9 is started by soybean endogenous promoter pM 4.
3. The method for specifically knocking out soybean lipoxygenase gene by CRISPR-Cas9 according to claim 2, wherein the method comprises the following steps: the sgRNA sense and antisense oligonucleotide strands, wherein the sgRNA-GmLox1/2The sense oligonucleotide chain of (a) is: 5'-GGATTGGAAAGGATACGTTCTTGGA-3', antisense oligonucleotide strand 5'-AAACTCCAAGAACGTATCCTTTCCA-3'; sgRNA-GmLox3The sense oligonucleotide chain of (a) is: 5'-GGATTGCCTTTCCTTATCCTCGTAGG-3', antisense oligonucleotide strand 5'-AAACCCTACGAGGATAAGGAAAGGCA-3'.
4. The method for specifically knocking out soybean lipoxygenase gene by CRISPR-Cas9 according to claim 1, wherein the method comprises the following steps: and (3) infecting soybean cotyledon nodes, wherein the soybean variety is 'Huachun No. 6'.
5. The use of the method of claim 1 for specifically knocking out a soybean lipoxygenase gene with CRISPR-Cas9 in breeding soybean germplasm without beany flavor.
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