CN112063651A - Method for directionally editing common head cabbage gene by using CRISPR/Cas9 and application - Google Patents

Method for directionally editing common head cabbage gene by using CRISPR/Cas9 and application Download PDF

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CN112063651A
CN112063651A CN202010843518.7A CN202010843518A CN112063651A CN 112063651 A CN112063651 A CN 112063651A CN 202010843518 A CN202010843518 A CN 202010843518A CN 112063651 A CN112063651 A CN 112063651A
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王神云
余方伟
王红
张伟
李建斌
于利
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Nanjing Lihua Agricultural Technology Co ltd
Jiangsu Academy of Agricultural Sciences
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Abstract

The invention relates to a method for directionally editing common head cabbage genes by using CRISPR/Cas9 and application thereof, belonging to the technical field of biology. The method comprises the steps of designing sgRNA by taking a common head cabbage BoGASA gene sequence as a reference, constructing a sgRNA vector of a BoGASA gene, transforming agrobacterium by recombinant plasmids, mediating genetic transformation of common head cabbage stalk cotyledons and molecular detection of transformed plants, establishing a set of complete common head cabbage gene editing technical system on the basis, and having important significance for developing reverse genetics research of the common head cabbage and analyzing gene functions of other cruciferous plants.

Description

Method for directionally editing common head cabbage gene by using CRISPR/Cas9 and application
Technical Field
The invention relates to the technical field of biotechnology, in particular to a method for directionally editing common head cabbage genes by using CRISPR/Cas9 and application thereof.
Background
Common head cabbage (Brassica oleracea L. var. capitata L.) belongs to Brassica crops of Brassicaceae, is a variety of cabbage (Brassica oleracea L.), is commonly planted throughout the country, and is one of important vegetables in China. With the rapid development of high-throughput sequencing technology and the disclosure of genome data of common head cabbage, an effective molecular biological means is urgently needed to analyze a large number of unknown functional genes. Compared with model plants such as rice, arabidopsis thaliana, tomatoes and the like, most of horticultural plants lack an effective regeneration system and a genetic transformation system, and the research on the gene function of the horticultural plants is seriously hindered.
The emergence of the CRISPR/Cas9 gene editing technology provides a convenient method for the research of the gene function of the common head cabbage. The system was first discovered in bacteria and archaea as a weapon for cleaving foreign nucleic acids and defending against infection by foreign viruses. The II-type CRISPR/Cas system can be used for directional gene editing after being modified, and the sgRNA guides the targeted cutting of Cas9 protein to cause DNA double-strand break, trigger a non-homologous recombination end connection repair mechanism and realize gene mutation. Compared with the technologies such as ZFN, TALEN and the like, the CRISPR/Cas9 technical vector is simple to construct and easy to operate, and target gene editing can be realized by matching with a perfect plant regeneration system, so that a material with relatively stable heredity is obtained.
Therefore, how to provide a method for directionally editing a common head cabbage gene by using CRISPR/Cas9 is a problem to be solved urgently by those skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a method for directionally editing a common head cabbage gene by using CRISPR/Cas9 and an application thereof.
On the basis, a set of complete gene editing technical system is established, and the method has important significance for development of horticultural plant reverse genetics research and gene function analysis.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for directionally editing a common head cabbage gene using CRISPR/Cas9, comprising the steps of:
(1) designing and pairing primers: the designed forward and reverse primers and ddH2Preparing reaction liquid by using O, ligase and polynucleotide kinase, and carrying out pairing treatment to obtain a reaction product;
wherein the forward primer sequence is BoGASA-F: 5'-GATTGCTTCCTCACCTCACACAGAA-3', SEQ ID NO. 1;
the reverse primer sequence is BoGASA-R: 5'-AAACTTCTGTGTGAGGTGAGGAAGC-3', SEQ ID NO. 2;
(2) and (3) plasmid digestion: digesting and recovering the psgR-Cas9-At plasmid to obtain a linear plasmid;
(3) connecting: connecting the reaction product obtained in the step (1) and the linear plasmid obtained in the step (2) to obtain a recombinant plasmid;
(4) enzyme digestion, connection and transformation: performing double enzyme digestion on the recombinant plasmid obtained in the step (3) by adopting Hind III and Kpn I to obtain a sgRNA-Cas9 fragment, and connecting the sgRNA-Cas9 fragment with the linearized pCAMBIA1301 to obtain pCAMBIA1301-sgRNA-Cas 9;
(5) genetic transformation: infecting the explant by bacterial liquid containing the plasmid pCAMBIA1301-sgRNA-Cas9 in the step (4) and culturing to obtain a complete plant;
(6) and (3) gene editing plant detection: extracting and editing plant leaf DNA as a template, and carrying out PCR amplification and sequence detection.
Preferably: the step (1) is specifically as follows: mu.L of 100. mu.M forward primer, 1. mu.L of 100. mu.M reverse primer, 1. mu.L of 10 XT 4 DNA ligase buffer, 6.5. mu.L of ddH2O, 0.5 mu L of 10U/mu L T4 polynucleotide kinase, preparing 10 mu L reaction solution, cooling to 25 ℃ at 37 ℃ for 30min and 95 ℃ for 5min, and then cooling to 25 ℃ at 0.2 ℃/s; ddH for reaction products2Diluting by times with O for later use.
Further, the dilution factor was 250-fold.
Preferably: the enzyme in the step (2) is BbsI, and the enzymeThe cutting system is as follows: 20 μ L of psgR-Cas9-At, 10U/. mu.L of BbsI 1 μ L, 10U/. mu.L of bovine small intestine alkaline phosphatase 1 μ L, 4 μ L of 10 XNEBuffer 2.1, 14 μ L of ddH2O。
Further, the enzyme digestion time is 2 h.
Preferably: the step (3) is specifically as follows: mu.L of the linearized plasmid obtained in step (2), 1. mu.L of the reaction product obtained in step (1), 1. mu.L of 10 XT 4 DNA ligase buffer, 1. mu.L of 400U/. mu. L T4 DNA ligase, 6. mu.L of ddH2O, ligation was performed overnight, and the resulting recombinant plasmid was transformed into E.coli, which was cultured on a Mackanka agar medium containing 100. mu.g/mL of ampicillin.
Further: placing at 16 ℃ for connecting overnight; the next day, the recombinant plasmid was transformed into E.coli DH5a, and the cells were plated on 100. mu.g/mL ampicillin Mackanka agar medium and cultured overnight at 37 ℃.
Preferably: the enzyme digestion system in the step (4) is as follows: 25 μ L of plasmid, 5 μ L of 10 XNEB cutSmart Buffer, 20U/. mu.L HindIII-HF 1 μ L, 20U/. mu.L Kpn I-HF1 μ L, plus ddH2O to 50 mu L, and water bath at 37 ℃ for 3 h.
Further, the enzyme digestion product is subjected to 1% agarose gel electrophoresis, and sgRNA-Cas9(5.7kb) and linearized pCAMBIA1301 are recovered after gel cutting;
the ligation reaction system is as follows: mu.L of digested sgRNA-Cas9, 3. mu.L of digested p CAMBIA1301, 1. mu.L of 10 XT 4 ligase buffer, 1. mu. L T4 DNA ligase (400U/. mu.L) (NEB, New England Biolabs, USA), made up to 10. mu.L with water. Ligation was carried out overnight at 16 ℃. The following day, the ligation product was transformed into E.coli DH5a, spread on LB medium containing 50. mu.g/mL kanamycin, and cultured overnight at 37 ℃.
Preferably: the steps (2) and (4) also comprise colony PCR identification of positive clones, and plasmid extraction after correct sequencing, and the specific steps are as follows:
a: with M13F: 5'-TGTAAAACGACGGCCAGT-3', SEQ ID NO.3 and Bo GASA-R primers to perform colony PCR to identify positive clones;
b: selecting positive clones from PCR products, culturing with LB culture solution of 100 mug/mL ampicillin, and sequencing bacterial solution;
c: the clone with correct sequencing is shake-cultured by LB culture solution of 50 mug/mL kanamycin, and plasmid is extracted by a plasmid extraction kit and stored at-20 ℃ for later use.
Further, the colony PCR reaction system is as follows: mu. M M13F 1. mu.L, 10. mu.M Bo GASA-R1. mu.L, 12.5. mu.L 2 XQuick Taq HS DyeMix, 10.5. mu.L ddH2O；
The reaction system is as follows: pre-denaturation at 98 deg.C for 3min, denaturation at 94 deg.C for 30s, annealing at 53 deg.C for 30s, and extension at 72 deg.C for 20s, and circulating for 30 times, and extension at 72 deg.C for 5 min;
selecting positive clones from the PCR products through a 1% agarose gel electrophoresis result, performing shake culture at 37 ℃ overnight by using LB culture solution of 100 mu g/mL ampicillin, and sequencing bacterial solution; clones with correct sequencing were shake-cultured overnight at 37 ℃ in LB medium with 50. mu.g/mL kanamycin, plasmids were extracted with the axygen plasmid extraction kit, and stored at-20 ℃ for further use.
Preferably: the preparation method of the bacterial liquid in the step (5) comprises the following steps:
1) transferring the plasmid obtained in the step (4) into agrobacterium, performing PCR (polymerase chain reaction) identification on positive clone after culture, and shaking the culture solution for later use;
2) and taking the agrobacterium tumefaciens single colony subjected to streak culture, inoculating and shaking culture, centrifugally collecting bacteria, and suspending by using an eluent.
Further, step 1) transferring the plasmid obtained in step (4) into agrobacterium GV3101 by using a freeze-thaw method, carrying out shake culture on the transformed agrobacterium in an antibiotic-free LB culture solution at 28 ℃ for 2-3 h, coating the transformed agrobacterium on an LB solid culture medium plate containing 50 mug/mL rifampicin and 50 mug/mL cana, placing the plate on an LB solid culture medium plate at 28 ℃ for carrying out inversion culture for 48h, carrying out PCR (polymerase chain reaction) to identify positive clone, and shaking the strain with an LB culture solution containing 50 mug/mL rifampicin and 50 mug/mL cana for later use;
2) taking a single agrobacterium colony subjected to streak culture, inoculating the single agrobacterium colony to an LB liquid culture medium containing 50 mu g/mL kanamycin and 50 mu g/mL rifampicin, and performing shake culture overnight at 28 ℃ and 200 rpm; centrifuging the Agrobacterium liquid at 6500rpm and 20 deg.C for 8min the next day, collecting bacteria by three times, and suspending (OD 5.8) with eluent (MS +20g/L sucrose)600Value 0.3).
Preferably: step (5), explant: fully sterilizing common head cabbage seeds, inoculating the seeds to a germination culture medium, and culturing for 5 days under the illumination condition; cutting sterile seedling cotyledon with stalk as explant after 5d, inoculating to pre-culture medium, and pre-culturing for 3d under illumination condition;
infection in step (5): taking the cotyledon with stalk which is pre-cultured for 3d, and infecting the cotyledon with stalk by using the suspended bacterial liquid; inoculating on a co-culture medium, after dark culture for 3d, inoculating the explant on a differentiation medium, and culturing under the illumination condition; and transferring the selected strong resistant bud into a rooting culture medium to grow into a complete plant, and hardening the seedling.
Further, explant:
firstly, sowing seeds, namely, sterilizing a common head cabbage material with 'sweet taste 55' (selected by vegetable research institute of agricultural academy of sciences of Jiangsu province, 2015, identified by crop variety identification of Jiangsu province, identification number: Suzhan cabbage 201511) by 70% alcohol for 1min, then, dropwise adding a tween into a 7% sodium hypochlorite solution for sterilizing for 15min, continuously shaking the mixture to fully sterilize the seeds, washing the seeds with sterile water for three times, putting the seeds on filter paper for sucking dry, sowing the seeds on a germination culture medium (1/2MS +30g/L sucrose +7g/L agar powder pH5.8), and culturing the seeds for 5d under the condition of illumination of 25 ℃ and 16 h;
② explant preculture, cutting the cotyledon with stalk of the aseptic seedling after seeding culture for 5d as explant, placing in preculture medium (MS +30g/L sucrose +7g/L agar powder +2.0 mg/L6-BA +0.05mg/L NAA +7.5mg/L AgNO)3pH5.8), and culturing at 25 deg.C in dark for 1d, and then at 25 deg.C for 2d under 16h light.
Infection: taking the stem-carrying cotyledon pre-cultured for 3d, infecting for 1min with the suspended bacterial liquid, placing on absorbent paper, sucking dry bacterial liquid, and then inoculating to the pre-culture medium (MS +30g/L sucrose +7g/L agar powder +2.0 mg/L6-BA +0.05mg/L NAA +7.5mg/L AgNO)3pH5.8), carrying out co-culture, carrying out dark culture for 3d at 25 ℃, inoculating the explant to a differentiation culture medium (MS +2.0 mg/L6-BA +0.06mg/L NAA +7g/L agar powder +30g/L sucrose +200mg/L timentin +5mg/L hygromycin) after 3d, and carrying out culture under the illumination condition of 25 ℃ for 16 h;
rooting, namely selecting strong resistant buds to be transferred into a rooting culture medium (MS +0.3mg/L NAA +200mg/L timentin +5mg/L hygromycin +30g/L cane sugar +7g/L agar powder pH5.8) for rooting culture, and culturing at the temperature of 25 ℃ for 16h under the illumination condition;
hardening seedlings, opening bottles to train for 24-48 h after rooting of transformed seedlings, carefully washing off a culture medium at roots, inserting the washed culture medium into a moist seedling medium (Danish Topyl peat soil seedling medium, imported original package, peat soil particle size specification of 0-6 mm, water absorption of 80%, pH of 5.5-6), covering and moisturizing by using a plastic film for 1-2 d, then uncovering, and carrying out normal cultivation and management.
Preferably: and (6) gene editing plant detection: taking leaf tissue of a gene editing plant, extracting genome DNA, and performing amplification reaction by using a forward primer: 5'-TATGATGCAGTGAAAGTTCA-3', SEQ ID NO.4 and reverse primer 5'-TTTGATTGCCGGGGACGTCG-3', SEQ ID NO.5 amplify the target sequence; and (3) recovering a PCR product, connecting the recovered fragment to pMD18-T, converting the recovered fragment into Escherichia coli competent DH5a, culturing, picking a single colony, and sequencing.
Further, the amplification system is: 2 XKOD FXNEO Buffer 25. mu.L, 2mM dNTPs 5. mu.L, 10. mu.M forward primer 2. mu.L, 10. mu.M reverse primer 2. mu.L, 1. mu.L common head cabbage genomic DNA template, 14.5. mu.L ddH2O，0.5μL KOD FX NEO(Toyobo)；
The PCR reaction system is as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 30s, extension at 72 ℃ for 30s, and extension at 72 ℃ for 5min after 30 cycles;
the PCR product was subjected to 1% agarose gel electrophoresis and then gel-cut for recovery, the recovered fragment was ligated to pMD18-T and transformed into E.coli competent DH5a, the transformed solution was smeared on LB medium of 100. mu.g/mL ampicillin and cultured overnight at 37 ℃, a single colony was picked up, and then shaken turbid at 37 ℃ with LB medium of 100. mu.g/mL ampicillin and sequenced.
The invention also provides a gene editing plant obtained by any one of the methods.
According to the technical scheme, compared with the prior art, the method for directionally editing the common head cabbage gene by using the CRISPR/Cas9 and the application thereof are disclosed, the obtained technical effects provide a regeneration system and a genetic transformation method of the common head cabbage, a set of complete gene editing technical system is established on the basis, and the method has important significance for development of reverse genetics research and gene function analysis of horticultural plants.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic drawing of a portion of the psgR-Cas9-At body provided by the present invention.
FIG. 2 is a schematic diagram of the differentiation culture provided by the present invention, wherein A: aseptic seedlings; b: pre-culturing; c: co-culturing; d: performing differentiation culture; e: resistant buds; f: and (6) rooting.
FIG. 3 is a schematic diagram of gene-editing plant sequencing provided by the invention. Wherein WT represents the sequencing result of the BoGASA gene for the 'sweet 55' common head cabbage without gene editing; -22 represents a deletion of 22 bases from one of the DNA duplexes of the BoGASA gene; +1 represents 1 more bases on the other strand of the DNA duplex of BoGASA.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention discloses a method for directionally editing common head cabbage genes by using CRISPR/Cas9 and application thereof.
The raw materials and the kit related in the embodiment are obtained from commercial channels, do not have requirements on brands of the raw materials and the kit, and meet the test requirements, for example, an MS culture medium comprises the following components in 1L:
NH4HO3 33000mg、KNO3 38000mg、CaCl2.2H2O 8800mg、KH2PO4 3400mg、MgSO4.7H2O 7400mg、FeSO4.7H2O 560mg、Na2EDTA 2H2O 460mg、H3BO3 1240 mg、MnSO4.H2O 4460mg、ZnSO4.7H2O 1720mg、Na2MoO4.2H2O 50mg、CuSO4.5H2O 5mg、CoCl2.6H2o5 mg, KI 166mg, inositol 20000mg, glycine 400mg, IVB nicotinic acid 100mg, vitamin B120 mg, vitamin B6100 mg and the balance of sterile water.
1/2MS Medium, calculated as 1L, NH4HO316500mg, and the rest is the same as MS.
The methods not mentioned are all routine laboratory test methods and are not described in detail here.
Example 1
1, primer design:
using the sequence of common head cabbage BoGASA gene (Genbank accession number XM-013767612.1) as an initial sequence, and CRISPR Primer Designer software (https:// onlinestriberry. wiley. com/doi/epdf/10.1111/jipb.12295) is used to design primers, and the sequences of the primers are as follows:
BoGASA-F：5’-GATTGCTTCCTCACCTCACACAGAA-3’，SEQ ID NO.1；
BoGASA-R：5’-AAACTTCTGTGTGAGGTGAGGAAGC-3’，SEQ ID NO.2；
wherein, the forward primer (BoGASA-F) is 20 bases (excluding NGG) before the bases of the GATT + G + BoGASA gene NGG; the reverse primer (BoGASA-R) is the reverse complementary sequence + C20 bases (excluding NGG) before NGG base in AAAC + BoGASA gene.
Example 2
2, gene editing vector construction process:
2.1) primer pairing.
mu.L of 100. mu.M forward primer, 1. mu.L of 100. mu.M reverse primer, 1. mu.L of 10 XT 4 DNA ligase buffer, 6.5. mu.L of ddH2O, 0.5. mu. L T4 Polynucleotide kinase (10U/. mu.L) (NEB, New England Biolabs, USA) 10. mu.L of the reaction solution was prepared, followed by 30min at 37 ℃ and 5min at 95 ℃Reacting, and finally cooling to 25 ℃ at the speed of 0.2 ℃/s. ddH for reaction products2Diluting with O by 250 times for later use.
2.2) shaking of the culture broth containing 100. mu.g/mL ampicillin in LB medium containing psgR-Cas9-At (see FIG. 1 for a schematic of the vector body part, see the full sequence of the vector: liu W, Zhu X, Lei M, et al (2015) A failed procedure for CRISPR/Cas9-media gene editing in Arabidopsis thaliana [ J]Scientific bulletin (English edition), 60(15): 1332-1347.) Escherichia coli. The next day the plasmid was extracted and digested with BbsI at 37 ℃ for 2 h. The enzyme digestion system is as follows: 20 μ L of ps gR-Cas9-At, 1 μ L of BbsI (10U/. mu.L) (NEB, New England Biolabs, USA), 1 μ L of bovine small intestine alkaline phosphatase (10U/. mu.L) (NEB, New England Biolabs, USA), 4 μ L of 10 XNEBuffer 2.1, 14 μ L of ddH2And O. And (3) carrying out electrophoresis on the enzyme digestion product through 1% agarose gel, cutting the gel and recovering.
2.3) ligation. mu.L of the recovered product of step 2.2, 1. mu.L of the reaction product of step 2.1, 1. mu.L of 10 XT 4 DNA ligase buffer, 1. mu. L T4 DNA ligase (400U/. mu.L) (NEB, New England Biolabs, USA), 6. mu.L of ddH2O, and ligated overnight at 16 ℃. The following day, the ligation product was transformed into E.coli DH5a, and spread on 100. mu.g/mL ampicillin Mackanka agar medium and cultured overnight at 37 ℃.
2.4) colony PCR. Positive clones were identified by colony PCR using M13F (5'-TGTAAAACGACGGCCAGT-3', SEQ ID NO.3) and BoGASA-R as primers. The reaction system is as follows: mu. L M13F (10. mu.M), 1. mu.L of BoGASA-R (10. mu.M), 12.5. mu.L of 2 XQuick Taq HS DyeMix (Toyobo), 10.5. mu.L of ddH2O。
The PCR reaction system is as follows: pre-denaturation at 98 deg.C for 3min, denaturation at 94 deg.C for 30s, annealing at 53 deg.C for 30s, and extension at 72 deg.C for 20s, and circulating for 30 times, and extension at 72 deg.C for 5 min. The PCR product was subjected to 1% agarose gel electrophoresis to select positive clones, which were shake-cultured overnight at 37 ℃ in LB medium containing 100. mu.g/mL ampicillin.
Meanwhile, 50. mu.g/mL of LB culture solution containing kanamycin was used to shake culture Escherichia coli containing pCAMBIA1301 plasmid at 37 ℃. The plasmid was extracted with the axygen plasmid extraction kit and stored at-20 ℃ for future use.
2.5) enzyme cutting, connection and transformation. Taking the product of step 2.4The plasmid was digested with Hind III and Kpn I in the following manner: mu.L of plasmid, 5. mu.L of 10 XNEB CutSmart Buffer, 1. mu.L of HindIII-HF (20U/. mu.L) (NEB, New England Biolabs, USA), 1. mu.L of Kpn I-HF (20U/. mu.L) (NEB, New England Biolabs, USA), plus ddH2O to 50. mu.L. Water bath at 37 ℃ for 3 h. After the enzyme digestion product is subjected to 1% agarose gel electrophoresis, the gel is run and cut to recover sgRNA-Cas9(5.7kb) and linearized pCAMBIA 1301.
The ligation reaction system is as follows: mu.L of digested sgRNA-Cas9, 3. mu.L of digested pCAMBIA1301, 1. mu.L of 10x T4 ligase buffer, 1. mu. L T4 DNA ligase (400U/. mu.L) (NEB, New England Biolabs, USA), made up to 10. mu.L with water. Ligation was carried out overnight at 16 ℃. The following day, the ligation product was transformed into E.coli DH5a, spread on LB medium containing 50. mu.g/mL kanamycin, and cultured overnight at 37 ℃.
2.6) colony PCR. Positive clones were identified by colony PCR using M13F (5'-TGTAAAACGACGGCCAGT-3', SEQ ID NO.3) and BoGASA-R as primers. The reaction system is as follows: mu. L M13F (10. mu.M), 1. mu.L of BoGASA-R (10. mu.M), 12.5. mu.L of 2 XQuick Taq HS DyeMix (Toyobo), 10.5. mu.L of ddH2O。
The PCR reaction system is as follows: pre-denaturation at 98 deg.C for 3min, denaturation at 94 deg.C for 30s, annealing at 53 deg.C for 30s, and extension at 72 deg.C for 20s, and circulating for 30 times, and extension at 72 deg.C for 5 min. The PCR product was subjected to 1% agarose gel electrophoresis to select positive clones, which were shake-cultured overnight at 37 ℃ in LB medium of 100. mu.g/mL ampicillin, and the bacterial solution was sequenced. The correctly sequenced clones were shake-cultured overnight at 37 ℃ in LB medium with 50. mu.g/mL kanamycin. The plasmid was extracted with the axygen plasmid extraction kit and stored at-20 ℃ for future use.
2.7) Agrobacterium transformation. Transferring the plasmid obtained in the step 2.6 into agrobacterium GV3101 by a freeze-thaw method, carrying out shake culture on the transformed agrobacterium in an antibiotic-free LB culture solution at 28 ℃ for 2-3 h, coating the transformed agrobacterium on an LB solid culture medium plate containing 50 mug/mL rifampicin and 50 mug/mL cana, and carrying out inverted culture at 28 ℃ for 48 h. Positive clones were identified by PCR, and 50. mu.g/mL rifampicin + 50. mu.g/mL kanamycin was shaken in LB medium for further use.
Example 3
3 culturing and genetic transformation of common head cabbage explant (each step is schematically shown in figure 2, wherein A is aseptic seedling, B is pre-culture, C is co-culture, D is differentiation culture, E is resistant bud, and F is rooting).
3.1) explant preparation:
seeding: sterilizing the common head cabbage material 'sweet 55' with 70% alcohol for 1min, then adding a drop of Tween into 7% sodium hypochlorite solution for 15min, continuously shaking to fully sterilize the seeds, washing with sterile water for three times, placing on filter paper for blotting, sowing in a germination culture medium (1/2MS +30g/L sucrose +7g/L agar powder pH5.8), and culturing for 5d at 25 ℃ under 16h illumination.
Preculture of explants: cutting the cotyledon with stalk of the aseptic seedling after seeding culture for 5 days as explant, and horizontally placing in a pre-culture medium (MS +30g/L sucrose +7g/L agar powder +2.0 mg/L6-BA +0.05mg/L NAA +7.5mg/L AgNO)3pH5.8), and culturing at 25 deg.C in dark for 1d, and then at 25 deg.C for 2d under 16h light.
3.2) preparing bacterial liquid: a single colony of the streaked Agrobacterium was inoculated into LB liquid medium containing 50. mu.g/mL kanamycin and 50. mu.g/mL rifampicin and shake-cultured overnight at 28 ℃ and 200 rpm. Centrifuging the Agrobacterium strain liquid at 6500rpm and 20 deg.C for 8min the next day, collecting bacteria by centrifugation three times, and suspending (OD 5.8) with eluent (MS +20g/L sucrose pH5.8)600Value 0.3).
3.3) Co-culture and differentiation culture: taking the stem-carrying cotyledon pre-cultured for 3d, infecting for 1min with the suspended bacterial liquid, placing on absorbent paper, sucking dry bacterial liquid, and then inoculating to the pre-culture medium (MS +30g/L sucrose +7g/L agar powder +2.0 mg/L6-BA +0.05mg/L NAA +7.5mg/L AgNO)3pH5.8), culturing in dark for 3d at 25 ℃, inoculating the explant to a differentiation medium (MS +2.0 mg/L6-BA +0.06mg/L NAA +30g/L sucrose +7g/L agar powder +200mg/L timentin +5mg/L hygromycin) after 3d, and culturing at 25 ℃ for 16h under illumination.
3.4) rooting: the selected resistant bud with strong growth is transferred into a rooting culture medium (MS +0.3mg/L NAA +200mg/L timentin +5mg/L hygromycin +30g/L sucrose +7g/L agar powder pH5.8) for rooting culture, and the rooting culture is carried out at the temperature of 25 ℃ for 16h under the illumination condition.
3.5) hardening seedlings: opening a bottle to train for 24-48 h after rooting of the transformed seedling, carefully washing off a culture medium at the root, inserting the seedling into a moist seedling substrate (Danish brand Torpu peat soil seedling substrate, imported raw package, peat soil particle size specification of 0-6 mm, water absorption of 80%, pH 5.5-6), covering and moisturizing by using a plastic film for 1-2 d, then uncovering, and carrying out normal cultivation and management.
3.6) statistics of conversion: and after differential culture, counting the selected robust resistant buds for about 4-6 weeks. Per dish resistant bud/explant × 100%, the average conversion of the brassica napus CRISPR-Cas9 vector was 5.84%, see table 1 for details.
TABLE 1 cabbage genetic transformation statistics
Culture dish Explant Resistant bud Conversion rate% Culture dish Explant Resistant bud Conversion rate%
M1 41 2 4.88 M11 40 1 2.50
M2 45 3 6.67 M12 37 3 8.11
M3 46 3 6.52 M13 38 2 5.26
M4 42 2 4.76 M14 31 1 3.23
M5 28 1 3.57 M15 19 0 0.00
M6 21 2 9.52 M16 42 4 9.52
M7 35 3 8.57 M17 25 1 4.00
M8 45 2 4.44 M18 19 2 10.53
M9 36 1 2.78 M19 27 3 11.11
M10 42 2 4.76 M20 33 2 6.06
Mean value of 38.1 2.1 5.65 Mean value of 31.1 1.9 6.03
Example 4
4. Gene editing plant detection
Taking leaf tissue of a gene editing plant, extracting genome DNA by using a Kangwei plant genome DNA kit, and amplifying a target sequence by using a forward primer (5'-TATGATGCAGTGAAAGTTCA-3', SEQ ID NO.4) and a reverse primer (5'-TTTGATTGCCGGGGACGTCG-3', SEQ ID NO.5), wherein the amplification system is as follows: 2 XKOD FXNEO Buffer 25. mu.L, 2mM dNTPs 5. mu.L, 10. mu.M forward primer 2. mu.L, 10. mu.M reverse primer 2. mu.L, 1. mu.L common head cabbage genomic DNA template, 14.5. mu.L ddH2O，0.5μL KOD FX NEO(Toyobo)。
The PCR reaction system is as follows: pre-denaturation at 95 deg.C for 3min, denaturation at 94 deg.C for 30s, annealing at 53 deg.C for 30s, and extension at 72 deg.C for 30s, and circulating for 30 times, and extension at 72 deg.C for 5 min. Carrying out gel cutting and recovery on a PCR product after 1% agarose gel electrophoresis, connecting a recovered fragment to pMD18-T, converting Escherichia coli competent DH5a, smearing a conversion solution on an LB culture medium of 100 mu g/mL ampicillin for overnight culture at 37 ℃, picking a single colony, shaking the LB culture medium of 100 mu g/mL ampicillin at 37 ℃ for turbidity, and then carrying out sequencing, wherein the sequencing result is shown in figure 3, wherein WT represents the sequencing result of the BoGASA gene of the kohlrabi 'sweet 55' which is not subjected to gene editing; after the BoGASA genes are edited by using a CRISPR/Cas9 technology, mutant plants have biallelic mutation, and the sequencing result of the biallelic mutation of one mutant plant is-22/+ 1; -22 represents a deletion of 22 bases from one of the DNA duplexes of the BoGASA gene; +1 represents 1 more bases on the other strand of the DNA duplex of BoGASA.
The results show that the target gene of the kohlrabi can be effectively edited according to the method described in the specification.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
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Nanjing Rihua agricultural science and technology Co., Ltd
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Claims (10)

1. A method for targeted editing of common head cabbage genes using CRISPR/Cas9, characterized in that the application of the BoGASA gene in the gene editing of common head cabbage comprises the following steps:
(1) designing and pairing primers: the designed forward and reverse primers and ddH2Preparing reaction liquid by using O, ligase and polynucleotide kinase, and carrying out pairing treatment to obtain a reaction product;
wherein the forward primer sequence is BoGASA-F: 5'-GATTGCTTCCTCACCTCACACAGAA-3', SEQ ID NO. 1;
the reverse primer sequence is BoGASA-R: 5'-AAACTTCTGTGTGAGGTGAGGAAGC-3', SEQ ID NO. 2;
(2) and (3) plasmid digestion: digesting and recovering the psgR-Cas9-At plasmid to obtain a linear plasmid;
(3) connecting: connecting the reaction product obtained in the step (1) and the linear plasmid obtained in the step (2) to obtain a recombinant plasmid;
(4) enzyme digestion, connection and transformation: performing double enzyme digestion on the recombinant plasmid obtained in the step (3) by adopting Hind III and Kpn I to obtain a sgRNA-Cas9 fragment, and connecting the sgRNA-Cas9 fragment with the linearized pCAMBIA1301 to obtain pCAMBIA1301-sgRNA-Cas 9;
(5) genetic transformation: infecting the explant by bacterial liquid containing the plasmid pCAMBIA1301-sgRNA-Cas9 in the step (4) and culturing to obtain a complete plant;
(6) and (3) gene editing plant detection: extracting and editing plant leaf DNA as a template, and carrying out PCR amplification and sequence detection.
2. The method for directionally editing common head cabbage genes by using CRISPR/Cas9 according to claim 1, wherein the step (1) is specifically as follows: mu.L of 100. mu.M forward primer, 1. mu.L of 100. mu.M reverse primer, 1. mu.L of 10 XT 4 DNA ligase buffer, 6.5. mu.L of ddH2O, 0.5 mu L of 10U/mu L T4 polynucleotide kinase, preparing 10 mu L reaction solution, cooling to 25 ℃ at 37 ℃ for 30min and 95 ℃ for 5min, and then cooling to 25 ℃ at 0.2 ℃/s; ddH for reaction products2Diluting with O for later use.
3. The method for directionally editing the common head cabbage gene by using the CRISPR/Cas9 as claimed in claim 2, wherein the enzyme in step (2) is BbsI, and the enzyme system is as follows: 20 μ L of psgR-Cas9-At, 10U/. mu.L of BbsI 1 μ L, 10U/. mu.L of bovine small intestine alkaline phosphatase 1 μ L, 4 μ L of 10 XNEBuffer 2.1, 14 μ L of ddH2O。
4. The method for directionally editing common head cabbage genes by using CRISPR/Cas9 according to claim 3, wherein the step (3) is specifically as follows: mu.L of the linearized plasmid obtained in step (2), 1. mu.L of the reaction product obtained in step (1), 1. mu.L of 10 XT 4 DNA ligase buffer, 1. mu.L of 400U/. mu. L T4 DNA ligase, 6. mu.L of ddH2O, ligation was performed overnight, and the resulting recombinant plasmid was transformed into E.coli, which was cultured on a Mackanka agar medium containing 100. mu.g/mL of ampicillin.
5. The method for directionally editing common head cabbage genes by using CRISPR/Cas9 according to claim 4, wherein the enzyme cutting system in step (4) is as follows: 25 μ L of plasmid, 5 μ L of 10 XNEB cutSmart Buffer, 20U/. mu.L HindIII-HF 1 μ L, 20U/. mu.L Kpn I-HF1 μ L, plus ddH2O to 50 mu L, and water bath at 37 ℃ for 3 h.
6. The method for directionally editing common head cabbage genes by using CRISPR/Cas9 as claimed in claim 5, wherein the steps (2) and (4) further comprise colony PCR to identify positive clones, and extracting plasmids after the sequencing is correct, and the specific steps are as follows:
a: M13F: 5'-TGTAAAACGACGGCCAGT-3', SEQ ID NO.3 and BoGASA-R primers to perform colony PCR to identify positive clones;
b: selecting positive clones from PCR products, culturing with LB culture solution of 100 mug/mL ampicillin, and sequencing bacterial solution;
c: the clone with correct sequencing is shake-cultured by LB culture solution of 50 mug/mL kanamycin, and plasmid is extracted by a plasmid extraction kit and stored at-20 ℃ for later use.
7. The method for directionally editing common head cabbage genes by using CRISPR/Cas9 as claimed in claim 6, wherein the preparation method of the bacterial liquid in step (5) comprises the following steps:
1) transferring the plasmid obtained in the step (4) into agrobacterium, performing PCR (polymerase chain reaction) identification on positive clone after culture, and shaking the culture solution for later use;
2) and taking the agrobacterium tumefaciens single colony subjected to streak culture, inoculating and shaking culture, centrifugally collecting bacteria, and suspending by using an eluent.
8. The method for directionally editing common head cabbage genes by using CRISPR/Cas9 according to claim 7, wherein the CRISPR/Cas9 is characterized in that:
step (5) the explant: fully sterilizing common head cabbage seeds, inoculating the seeds to a germination culture medium, and culturing for 5 days under the illumination condition; cutting sterile seedling cotyledon with stalk as explant after 5d, inoculating to pre-culture medium, and pre-culturing for 3d under illumination condition;
infection in step (5): taking the cotyledon with stalk which is pre-cultured for 3d, and infecting the cotyledon with stalk by using the suspended bacterial liquid; inoculating on a co-culture medium, after dark culture for 3d, inoculating the explant on a differentiation medium, and culturing under the illumination condition; and transferring the selected strong resistant bud into a rooting culture medium to grow into a complete plant, and hardening the seedling.
9. The method for directionally editing a common head cabbage gene by using CRISPR/Cas9 according to claim 8, wherein the gene editing plant of step (6) detects that: taking leaf tissue of a gene editing plant, extracting genome DNA, and performing amplification reaction by using a forward primer: 5'-TATGATGCAGTGAAAGTTCA-3', SEQ ID NO.4 and reverse primer 5'-TTTGATTGCCGGGGACGTCG-3', SEQ ID NO.5 amplify the target sequence; and (3) recovering a PCR product, connecting the recovered fragment to pMD18-T, converting the recovered fragment into Escherichia coli competent DH5a, culturing and picking a single colony, shaking the bacteria and sequencing.
10. A gene-edited plant obtained by the method of any one of claims 1 to 9.
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Application publication date: 20201211