CN117286181B - Efficient targeted mutagenesis gene editing system for CRISPR/Cas9 mediated tetraploid patchouli - Google Patents
Efficient targeted mutagenesis gene editing system for CRISPR/Cas9 mediated tetraploid patchouli Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
Abstract
A gene editing system for efficient targeted mutagenesis of tetraploid patchouli mediated by CRISPR/Cas9 relates to application of a genome editing system mediated by CRISPR/Cas9 in targeted mutagenesis of tetraploid patchouli, and belongs to the technical field of plant genetic engineering. The agrobacterium-mediated genetic transformation method for transforming the PcUbi-pEGCas9 gene editing recombinant vector comprises the following steps: agrobacterium transformation, preparation of infection solution, preculture, infection, co-culture, bacterial washing, screening of resistant callus, differentiation screening and rooting culture. The CRISPR/Cas9 mediated genome editing system provided by the invention has high-efficiency targeted mutation capability in the tetraploid cultivated species patchouli, is rich in mutation types and perfect in technology, provides good technical support for molecular breeding of patchouli and research of functional genome, and can also be widely applied to improving effective active ingredients in patchouli as a gene manipulation tool.
Description
Technical Field
The invention relates to application of a CRISPR/Cas9 mediated genome editing system in targeted mutagenesis of tetraploid patchouli, belonging to the field of plant genetic engineering.
Background
Patchouli Pogostemon cablin (Blanco) benth belongs to genus of family Labiatae, and is native to southeast Asian such as Philippines, malaysia, vietnam, etc. Herba Agastaches is used as the main material of various Chinese medicinal materials, and has effects of eliminating turbid pathogen with aromatics, relieving exterior syndrome and relieving summer heat. In addition, patchouli oil extracted by the patchouli oil is widely applied to medicine and light industry, and can be used for preparing various ointments, pills, cosmetic skin care products, perfume fixatives, pesticides and the like.
The patchouli is rarely flowering, and even if the patchouli is flowering, the patchouli is difficult to bear fruits, and asexual propagation is usually carried out in a cutting manner in cultivation. Asexual propagation reduces genetic diversity of patchouli, making it impossible to develop new germplasm and variety by traditional crossbreeding. In addition, long-term asexual propagation causes germplasm resource degradation, so that medicinal material quality is unstable, and resistance to diseases and insect pests is reduced. For example, the perennial incidence rate of bacterial wilt is 10% -20%, and the serious land block death rate is as high as 90%, which has become a main obstacle for patchouli cultivation. No highly resistant material is reported in the current germplasm. Therefore, improvement of quality and disease resistance of patchouli through molecular genetic improvement has become urgent. The patchouli plant is rich in patchouli essential oil, mainly comprises patchouli ketone, patchouli alcohol and the like, and has an inhibiting effect on agrobacterium-mediated genetic transformation, so that the genetic transformation system of the patchouli is not mature. In addition, the patchouli cultivar is a tetraploid hybrid of the compensatory aneuploidy, and the genetic background is complex, so that the molecular biological research and genetic improvement are relatively backward.
CRISPR/Cas9 mediated genome editing technology has great potential in genetic improvement, gene function verification, plant metabolic network regulation and new germplasm creation, has been widely used for targeted improvement of quality, stress resistance and other traits of varieties on many crops, but its editing ability depends on sgrnas and Cas9 proteins. Therefore, there is a need to optimize the conditions of use in a particular species to achieve efficient genome editing. Phytoene desaturase (Phytoene desaturase, PDS) is a commonly used visual marker gene that is commonly used to test the efficiency of gene editing tools in plants, where disruption of function can result in plants exhibiting a albino phenotype. However, genetic application of the patchouli genome efficient gene editing system is not reported at present. Therefore, constructing a CRISPR/Cas 9-based efficient genome editing system for genetic improvement, gene function research and active ingredient regulation of patchouli becomes a technical problem to be solved urgently.
Disclosure of Invention
The invention aims to solve the defects of a genetic transformation system and a gene editing tool in the current patchouli research, and provides a method for targeted knockout of five copies of patchouli PatPDS in tetraploid patchouli by a high-efficiency CRISPR/Cas9 genome editing system, which can be used for quality breeding and gene function research of patchouli.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
an agrobacterium-mediated genetic transformation method for transforming a PcUbi-pEGCas9 gene editing recombinant vector comprises the following steps:
(1) Agrobacterium transformation
Transforming the PcUbi-pEGCas9 plasmid into competent cells of Agrobacterium tumefaciens GV 3101;
the PcUbi-pEGCas9 plasmid comprises the following gene fragments: 35S promoter CaMV 35S from cauliflower mosaic virus, herbicide basta resistance gene BlpR, terminator CaMV poly (A), ubiquitin promoter PcUbi from parsley, translation enhancer TMVΩ, kozak sequence for enhancing eukaryotic gene translation efficiency, endonuclease gene Cas9, nuclear localization signal element SV40 NLS, terminator E9 terminator, arabidopsis AtU6-1 promoter, expression cassette sgRNA1 comprising PatPDS gene editing target site T1, arabidopsis AtU6-29 promoter, expression cassette sgRNA2 comprising PatPDS gene editing target site T2;
the sequence 5'-3' of the expression cassette sgRNA1 is:
AGAAATCTCAAAATTCCGGCAGAACAATTTTGAATCTCGATCCGTAGAAACGAGACG
GTCATTGTTTTAGTTCCACCACGATTATATTTGAAATTTACGTGAGTGTGAGTGAGA
CTTGCATAAGAAAATAAAATCTTTAGTTGGGAAAAAATTCAATAATATAAATGGGCTT
GAGAAGGAAGCGAGGGATAGGCCTTTTTCTAAAATAGGCCCATTTAAGCTATTAACA
ATCTTCAAAAGTACCACAGCGCTTAGGTAAAGAAAGCAGCTGAGTTTATATATGGTT
AGAGACGAAGTAGTGATTGATTCCAGCTGCGCGTGCTTTGTTTTAGAGCTAGAAATA
GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC；
the sequence 5'-3' of the expression cassette sgRNA2 is:
AAAATATCAGAGATCTCTTACAGTTAGTTTCGTTCTTAATCCAAACTACTGCAGCCT
GACAGACAAATGAGGATGCAAACAATTTTAAAGTTTATCTAACGCTAGCTGTTTTGT
TTCTTCTCTCTGGTGCACCAACGACGGCGTTTTCTCAATCATAAAGAGGCTTGTTTT
ACTTAAGGCCAATAATGTTGATGGATCGAAAGAAGAGGGCTTTTAATAAACGAGCCC
GTTTAAGCTGTAAACGATGTCAAAAACATCCCACATCGTTCAGTTGAAAATAGTAGC
TCTGTTTATATATTGGTAGAGTCGACTAAGAGATTGTGTTATCAAGCTCTGGTCGGT
TTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC；
(2) Preparation of dyeing liquor
Inoculating the transformed agrobacterium into 1mL of YEB liquid culture medium added with 50mg/L kanamycin, 25mg/L rifampicin and 50mg/L gentamicin, and shaking overnight in a shaking table at 28 ℃ and 200rpm for culture;
Transferring all cultures into 100mL of YEB liquid culture medium containing 50mg/L kanamycin, 25mg/L rifampicin and 50mg/L gentamicin, culturing until OD600 reaches 0.6-0.8, centrifuging at 5000rpm for 5min, and collecting agrobacterium cells; the precipitated cells were resuspended in an equal volume of infection medium and incubated for 2h to activate toxic genes for infection transformation;
(3) Pre-culture
Transferring the induced embryogenic callus to a preculture medium, and culturing for 2d at 25 ℃ in a dark way;
(4) Infestation of the human body
Transferring the pre-cultured embryogenic callus into an infection culture medium, placing 100 explants per 50mL of the infection culture medium in a 90r/min shaking table, and shaking for 20-30 min at 28 ℃ to enable the pre-cultured explants to be in full contact with the transformed agrobacterium;
(5) Co-cultivation
After infection, sucking the bacterial liquid on the infected embryogenic callus with sterile filter paper, transferring the bacterial liquid into a co-culture medium, and culturing for 2-3 d under the dark culture condition at 25 ℃;
(6) Bacteria washing
Washing the callus which forms plaque in the co-culture process with sterile water containing 300mg/L cephalosporin and 200mg/L timentin for 2-3 times, and then sucking the water on the surface with sterile filter paper;
(7) Resistant callus screening
Transferring the calli to a resistant callus screening culture medium, culturing in dark at 25 ℃ once every 2 weeks, and continuously carrying out 2-3 times of subculture to screen the resistant calli;
(8) Differentiation screening
Transferring the resistant callus to a differentiation screening culture medium, inducing the differentiation of the adventitious buds at 28 ℃ under 16/8h of photoperiod, and inducing the adventitious buds once every 2 weeks;
(9) Rooting culture
When the buds grow to 2-3 cm, transferring the buds into a rooting induction culture medium to induce rooting.
Further, the composition and content of the culture medium involved in the transformation process are as follows:
pre-culture medium: MS+30g/L sucrose+5.5 g/L agar, pH 5.8;
infection medium: MS+30g/L sucrose+200 μm acetosyringone, pH 5.8;
co-culture medium: MS+2.0 mg/L6-BA+0.2 mg/L NAA+4.625 g/L2-morpholinoethanesulfonic acid+200 μm acetosyringone+30 g/L sucrose+5.5 g/L agar, pH 5.8;
resistant callus screening media: MS+2.0 mg/L6-BA+0.2 mg/L NAA+0.64 g/L2-morpholinoethanesulfonic acid+30 g/L sucrose+300 mg/L cephalosporin+200 mg/L timentin+5.0 mg/L basta+5.5g/L agar, pH 5.8;
differentiation screening medium: MS+1.0 mg/L6-BA+0.2 mg/L NAA+0.64 g/L2-morpholinoethanesulfonic acid+30 g/L sucrose+300 mg/L cephalosporin+200 mg/L timentin+5.0 mg/L basta+5.5g/L agar, pH 5.8;
Induction rooting culture medium: MS+0.2mg/L NAA+0.64 g/L2-morpholinoethanesulfonic acid+30 g/L sucrose+150 mg/L cephalosporin+100 mg/L timentin+5.0 mg/L basta+5.5g/L agar, pH 5.8.
Further, the agrobacterium transformation of step (1) is specifically as follows:
(1) Thawing Agrobacterium tumefaciens GV3101 competent cells on ice, adding 1 μg of PcUbi-pEGCas9 plasmid, mixing well, and standing on ice for 5min;
(2) Rapidly cooling in liquid nitrogen for 5min, rapidly transferring into 37deg.C water bath for 5min, and rapidly taking out ice bath for 5min;
(3) Adding 1mL of YEB liquid culture medium without antibiotics, and culturing for 2-3 h at 28 ℃ and 220 r/min;
(4) Centrifuging at 5000r/min for 1min, collecting thallus, sucking the supernatant to 900 μL, and reserving 100 μL;
(5) Re-suspending the bacterial cells and coating the bacterial cells on a YEP solid plate containing 25 mug/mL rifampicin, 50 mug/mL kanamycin and 50 mug/mL gentamicin, and inversely culturing the bacterial cells for 48 to 72 hours at the temperature of 28 ℃;
(6) Single colony PCR detection is selected, correct single colony is inoculated into liquid YEB culture medium containing 25 mug/mL rifampicin, 50 mug/mL kanamycin and 50 mug/mL gentamicin, and the culture is carried out at 28 ℃ and 220r/min, 50% glycerol with equal volume sterilization is added, and the strain is preserved at-80 ℃ for standby.
Further, the preparation method of the PcUbi-pEGCas9 plasmid comprises the following steps:
(S1) nucleotide sequence characterization and amplification of tetraploid Pogostemon cablin PatPDS Gene
Amplifying the PatPDS alleles of five copies of tetraploid patchouli by PCR by using the genomic DNA of the tetraploid patchouli as a template and adopting a primer combination PatPDS-F/PatPDS-R;
the sequence 5'-3' of PatPDS-F is ATGATGTCTCAATTTGGGCAC;
the sequence 5'-3' of the PatPDS-R is TTAGGCGTAGCTTGCCTCT;
detecting the PCR product by using 1% agarose gel electrophoresis, and purifying and recovering the PCR product by using an agarose gel recovery kit;
connecting the purified and recovered PCR product to a pCE2_TA-Blunt-Zero vector through a TOPO cloning kit to obtain a recombinant pCE2_TA-Blunt-Zero vector; transforming the recombinant pCE2_TA-Blunt-Zero vector into an escherichia coli competent cell DH5 alpha by adopting a heat shock method, screening by ampicillin and kanamycin, and selecting a positive monoclonal to sequence to obtain a nucleotide sequence of five copies of the tetraploid patchouli PatPDS gene;
(S2) construction of PatPDS Gene-editing recombinant vector (S2.1) screening of target sites of Gene-editing recombinant vector
According to the nucleotide sequences of five copies of the tetraploid patchouli PatPDS gene, selecting two target sites near the 5' -end conserved region of the PatPDS allele, namely a target site T1 positioned in a first exon and a target site T2 positioned in a second exon; the sequence 5'-3' of the target site T1 is ATTCCAGCTGCGCGTGCTTTTGG;
The sequence 5'-3' of the target site T2 is CCACGACCAGAGCTTG ATAACAC;
(S2.2) first round PCR amplification
AtU6-1 promoter was amplified by primer set U-F/AtU6-1-R using pU6 as a template, atU-29 promoter by primer set U-F/AtU 6-29-R;
using sgRNA as a template, connecting a target site T1 to the gRNA through a primer combination gRT/gRNA-R to obtain gRNA1, and connecting a target site T2 to the gRNA through a primer combination gRT/gRNA-R to obtain gRNA2;
the primer sequences for the first round of PCR amplification were as follows:
the sequence 5'-3' of the U-F is CTCCGTTTTACCTGTGGAATCG;
the sequence 5'-3' of the AtU6-1-R is AAAGCACGCGCAGCTGGAATCaatcactacttcgtct;
the sequence 5'-3' of the AtU6-29-R is CGACCAGAGCTTGATAACACaatctcttagtcgact;
the sequence 5'-3' of gRT1 is ATTCCAGCTGCGCGTGCTTTgttttagagctagaaat;
the sequence 5'-3' of gRT2 is TGTTATCAAGCTCTGGTCGgttttagagctagaaat;
the sequence 5'-3' of the gRNA-R is CGGAGGAAAATTCCATCCAC;
(S2.3) second round PCR amplification
Amplifying the expression cassette sgRNA1 containing the AtU6-1 promoter and the gRNA1 in the step (S2.2) by overlap PCR using AtU-1 and the gRNA1 as templates and adopting a primer combination Pps-1/Pgs-1;
amplifying the expression cassette sgRNA2 containing the AtU6-29 promoter and the gRNA2 in the step (S2.2) by overlap PCR using AtU-29 and the gRNA2 as templates and adopting a primer combination Pps-2/Pgs-2;
The primer sequences for the second round of PCR amplification were as follows:
the sequence 5'-3' of the Pps-1 is TTCAGAggtctcTaccgACTAGTATGGAATCGGCAGCAAAGG;
the sequence 5'-3' of Pgs-1 is AGCGTGggtctcGtcagggTCCATCCACTCCAAGCTC;
the sequence 5'-3' of the Pps-2 is TTCAGAggtctcTctgacacTGGAATCGGCAGCAAAGG;
the sequence 5'-3' of Pgs-2 is AGCGTGggtctcGctcgACGCGTATCCATCCACTCCAAGCTC;
the second round of PCR products were detected by 1% agarose gel electrophoresis, and the expression cassette sgRNA1 and the expression cassette sgRNA2 were purified and recovered by agarose gel recovery kit;
(S2.4) ligating the expression cassette sgRNA1 and the expression cassette sgRNA2 with the pEGCas9-PcUBI-B vector;
(S2.5) screening and identification of Gene editing recombinant vector Positive clones
The ligation product obtained in step (S2.4) was transformed into E.coli competent cells DH 5. Alpha. And colony PCR detection was performed using the primer combination E9Ter-F/SP-R, with the following primer sequences:
the sequence 5'-3' of the E9Ter-F is TGGATTTGTAGTTGAGTATGAA;
the sequence 5'-3' of the SP-R is TGCAATAACTTCGTATAGGC;
sequencing the monoclonal which is detected to be positive, comparing the nucleotide sequence obtained by sequencing with the nucleotide sequence of the expression cassette sgRNA1 and the nucleotide sequence of the expression cassette sgRNA2, culturing a single colony of the recombinant vector pEGCas9-PcUBI-B containing the expression cassette sgRNA1 and the expression cassette sgRNA2, and extracting a plasmid for later use, wherein the plasmid is PcUbi-pEGCas9.
Further, the method comprises the steps of,
in step (S1), the PCR reaction conditions are: denaturation at 98℃for 10s; annealing at 55 ℃ for 15s; extending at 72 ℃ for 60s; amplifying for 30 cycles; final extension for 10min at 72 ℃;
in step (S2.2), the first round PCR reaction conditions are: pre-denaturation at 95 ℃ for 2min; denaturation at 95℃for 20s; annealing at 55 ℃ for 20s; extending at 72 ℃ for 20s; amplifying for 20 cycles; final extension for 5min at 72 ℃;
in step (S2.3), the second round PCR reaction conditions were: pre-denaturation at 95 ℃ for 2min; denaturation at 95℃for 20s; annealing at 55 ℃ for 30s; extending at 72 ℃ for 30s; amplifying for 20 cycles; final extension for 5min at 72 ℃;
in the step (S2.4), the Golden Gate method is adopted to realize connection; the conditions of the enzyme digestion and enzyme ligation reaction are as follows: 37 ℃ for 5min;20 ℃ for 5min;15 cycles;
in the step (S2.5), a heat shock conversion method is adopted to realize conversion; the PCR reaction conditions were: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s; annealing at 55 ℃ for 30s; extending at 72 ℃ for 60s; amplifying for 30 cycles; and finally, the temperature is 72 ℃ and the extension is 5min.
Further, in step (S1), the primer PatPDS-F and the primer PatPDS-R are each treated with ddH 2 O is dissolved into working solution, and a PCR reaction system is as follows: 2X PrimeStar Max Premix was added at 25. Mu.L, the primer PatPDS-F was added at 1. Mu.L, the primer PatPDS-R was added at 1. Mu.L, the patchouli DNA was added at 1. Mu.L, and ddH 2 O is added to 50 mu L;
in step (S2.2), the first round of PCR amplificationRespectively with ddH 2 O is dissolved into working solution, and the first round of PCR reaction system is as follows: the addition amount of 2 XPform Master Mix was 5. Mu.L, the addition amount of the primer combination working solution was 0.4. Mu.L, the addition amount of the template DNA was 0.1. Mu.L, and ddH 2 O is added to 10 mu L;
in step (S2.3), gRNA1 and gRNA2 are treated with ddH, respectively 2 O is diluted 10 times, and 0.5 mu L of each O is taken as a reaction template of the second round of PCR respectively;
the second round of PCR reaction for amplifying the expression cassette sgRNA1 was as follows: the addition amount of 2 XPFU Master Mix was 25. Mu.L, the addition amount of the primer set Pps-1/Pgs-1 working solution was 2. Mu.L, the addition amount of the first round PCR product AtU-1 promoter after 10-fold dilution was 0.5. Mu.L, the addition amount of the first round PCR product gRNA1 after 10-fold dilution was 0.5. Mu.L, and ddH 2 O is added to 50 mu L;
the second round of PCR reaction for amplifying the expression cassette sgRNA2 was as follows: the addition amount of 2 XPFU Master Mix was 25. Mu.L, the addition amount of the primer set Pps-2/Pgs-2 working solution was 2. Mu.L, the addition amount of the first round PCR product AtU-29 promoter after 10-fold dilution was 0.5. Mu.L, the addition amount of the first round PCR product gRNA2 after 10-fold dilution was 0.5. Mu.L, and ddH 2 O is added to 50 mu L;
in step (S2.4), the ligation reaction system of the expression cassette sgRNA1 and the expression cassette sgRNA2 with the pEGCas9-PcUBI-B vector is as follows: 10 XCutSmart Buffer 1.5. Mu.L, 10mM ATP 1.5. Mu.L, pEGCas 9-PcUBI-B100 ng/. Mu.L, expression cassette sgRNA1 30 ng/. Mu.L, expression cassette sgRNA2 30 ng/. Mu.L, bsa I-HF 10U/. Mu.L, T4 DNA ligase 400U/. Mu.L, ddH 0.1. Mu.L 2 O was made up to 15. Mu.L.
Further, in the step (S1), the specific method for analyzing the nucleotide sequence characteristics of the tetraploid patchouli PatPDS gene is as follows: the nucleotide coding sequence KC854409.1 of the tetraploid patchouli PatPDS gene is obtained on the NCBI database, the tetraploid patchouli PatPDS gene KC854409.1 is taken as a reference sequence, and the reference genome of the tetraploid patchouli is analyzed, so that five copies of the tetraploid patchouli PatPDS gene exist in the tetraploid patchouli genome and are respectively positioned on four chromosomes A05, A06, B05 and B06 and the scaffold 89 and are respectively named Pat_A05G005500, pat_B05G005800, pat_A06G005300, pat_B06G005200 and Pat_U0021200.
Further, in the step (S1), the genome DNA of the tetraploid patchouli is extracted by adopting a CTAB method, a tetraploid patchouli reference genome is obtained in a BIG Data Center database, and a conserved specific primer PatPDS-F and a primer PatPDS-R are designed according to the nucleotide sequences of five PatPDS alleles in the tetraploid patchouli reference genome.
Further, in step (S2.1), the on-line tool http:// crispor.tefor.net/, in order to reduce the off-target efficiency of the PatPDS allele knockout, a conserved segment of five copies of the PatPDS gene is selected, and the target site for PatPDS gene editing is designed according to the GC content and the PAM sequence.
Further, the specific method for transforming the ligation product obtained in the step (S2.4) into E.coli competent cells DH 5. Alpha. Is as follows: adding 2-5 mu L of the connection product into 100 mu L of escherichia coli competent cells DH5 alpha, mixing, and carrying out ice bath for 30min; rapidly placing in a constant-temperature water bath kettle at 42 ℃, carrying out heat shock for 90s, and carrying out ice bath for 2min; adding 700 mu L of LB liquid medium and uniformly mixing; shaking culture is carried out for 45-60 min at 37 ℃ and 200 rpm; the bacterial liquid is evenly coated on LB solid medium containing 50mg/L kanamycin, and the solid medium is placed in a constant temperature incubator at 37 ℃ for overnight culture.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a high-efficiency stable genetic transformation system, and proves the high efficiency of the designed CRISPR/Cas9 gene editing system in tetraploid patchouli targeted mutation. The genetic transformation system and the editing system have wide application prospects in aspects of genome editing, genetic improvement, gene function research and the like of patchouli.
Drawings
FIG. 1 is a graph of replication events and treeing analysis of patchouli PatPDS alleles. m1 and m2 represent alternative splicing of the PatPDS gene, PDS protein sequence from Scutelaria baicalensis (KC 417313.1) as outer population.
FIG. 2 is a PatPDS gene editing target site design. Boxes represent exons of the coding region of the PatPDS gene, with T1 being located at the first exon and T2 being located at the second exon. SNP sites near T2 (sites 391, 399 and 400) were used for PatPDS allele typing.
FIG. 3 is a schematic diagram of the construction of a multiplex gene editing recombinant vector PcUbi-pEGCas 9. LB refers to the left boundary, RB refers to the right boundary; atU6-1 and AtU6-29 refer to the Arabidopsis U6 promoter; sgRNA1 is an expression cassette comprising the PatPDS gene editing target site T1; sgRNA2 is an expression cassette comprising the PatPDS gene editing target site T2; pcUbi refers to the sequence from the parsley ubiquitin promoter, TMV.OMEGA.refers to the translational enhancer, kozak refers to the Kozak sequence used to enhance the translational efficiency of eukaryotic genes, cas9 refers to the Cas9 nuclease, SV40 NLS refers to the nuclear localization signal element, E9 terminator; caMV 35S refers to the 35S promoter from cauliflower mosaic virus, blpR refers to the herbicide basta resistance gene, and CaMV poly (A) refers to the terminator.
FIG. 4 shows the regeneration system of Pogostemon cablin. I, inducing callus by using buds; II, embryogenic callus; III, adventitious bud induction; IV, inducing rooting.
FIG. 5 shows the growth of wild-type calli of Pogostemon cablin in medium of different basta concentration.
FIG. 6 is an Agrobacterium-mediated patchouli gene editing system. I. Co-culturing after infection of the callus by agrobacterium; II. Basta-screened resistant calli; III, albino seedlings; IV, the culture time is prolonged, so that part of albino seedlings are dwarfed and die. Genetic transformation of PcUbi-pEGCas9 produced three phenotypes, normal, chimeric and albino.
FIG. 7 identifies positive transgenic plants for PCR amplification of Cas9 gene. PcUbi-pEGCas9 vector plasmid was used as positive control (+), wild type DNA as negative control (WT), ddH 2 O served as a blank (-).
FIG. 8 is a peak plot of target sites T1 and T2 sequencing. Taking Line11 as an example, no overlapping peak appears at the target site T1, and overlapping peaks appear near the target site T2, indicating that the PcUbi-pEGCas9 system induces a mutation at the target site T2.
FIG. 9 is a graph showing the efficiency and mutation type of the PcUbi-pEGCas9 system mediated patchouli PatPDS allele targeted editing.
FIG. 10 shows PcUbi-pEGCas9 system mediated long fragment mutation of the patchouli PatPDS gene.
Detailed Description
The technical scheme of the invention is further described below through examples.
Example 1
(1) Nucleotide sequence characterization and amplification of PatPDS genes
The nucleotide coding sequence (KC 854409.1) of the patchouli PatPDS gene was obtained on the NCBI database (https:// www.ncbi.nlm.nih.gov /).
The tetraploid patchouli reference genome (Shen et al 2022) was analyzed with the tetraploid patchouli PatPDS gene KC854409.1 as reference sequence, and it was found that there were five copies of the patchouli genome, located on the four chromosomes a05, a06, B05 and B06 and on the scaffold 89, respectively, and in the BIG Data Center database, the five copies were named pat_a05g005500, pat_b05g005800, pat_a06g005300, pat_b06G005200 and pat_u0021200.
Sequence alignment analysis found that the coding region between the PatPDS alleles was more conserved, including 14 exons and 13 introns. Wherein the Pat_B06G005200 and Pat_U0021200 genes are highly conserved, differing only by 1 base at 199 upstream of the start codon. The PatPDS gene underwent a replication event in the tetraploid pogostemon cablin, and the PatPDS allele had 9 alternative splices (fig. 1).
Extracting genome DNA of a patchouli tetraploid cultivar by adopting a CTAB method (Li et al, 2013), obtaining a tetraploid patchouli reference genome in a BIG Data Center database, and designing a conserved specific primer PatPDS-F and a primer PatPDS-R according to the nucleotide sequences of five PatPDS alleles in the tetraploid patchouli reference genome;
The primer PatPDS-F has a sequence of ATGATGTCTCAATTTGGGCAC at 5 '-3';
the sequence 5'-3' of the primer PatPDS-R is TTAGGCGTAGCTTGCCTCT;
the primers PatPDS-F and PatPDS-R were each subjected to ddH 2 O was dissolved in 10 μm working fluid and the PatPDS allele of tetraploid Pogostemon cablin was amplified using high fidelity PrimeStar Max DNA polymerase. The PCR reaction condition is 98 ℃, and the denaturation is carried out for 10s; annealing at 55 ℃ for 15s; extending at 72 ℃ for 60s; amplifying for 30 cycles; and finally, the temperature is 72 ℃ and the extension is 10min. The PCR reaction system is shown in Table 1.
TABLE 1 PCR reaction System
| Reaction components | Addition amount (mu L) |
| 2×PrimeStar Max Premix | 25 |
| PatPDS-F | 1 |
| PatPDS-R | 1 |
| Pogostemon cablin DNA | 1 |
| ddH 2 O | Make up to 50 mu L |
Detecting the PCR product by using 1% agarose gel electrophoresis, and purifying and recovering the PCR product by using an agarose gel recovery kit;
connecting the purified and recovered PCR product to a pCE2_TA-Blunt-Zero vector through a TOPO cloning kit to obtain a recombinant pCE2_TA-Blunt-Zero vector; transforming the recombinant pCE2_TA-Blunt-Zero vector into an escherichia coli competent cell DH5 alpha by adopting a heat shock method, screening by ampicillin and kanamycin, and selecting a positive monoclonal to sequence to obtain a nucleotide sequence of five copies of the tetraploid patchouli PatPDS gene;
(2) Construction of PatPDS Gene editing recombinant vector
(2.1) screening of target sites of Gene editing recombinant vectors
According to the nucleotide sequences of five copies of the tetraploid patchouli PatPDS gene, an online tool (http:// crispor.tefor.net /) is used to select the conserved sections of the five copies of the PatPDS gene, and according to GC content and PAM sequence, the target site for editing the PatPDS gene is designed. To reduce the off-target efficiency of the PatPDS allele knockout, two target sites near the 5' conserved region of the PatPDS allele were selected, target site T1 at the first exon and target site T2 at the second exon, respectively (fig. 2);
the sequence 5'-3' of the target site T1 is ATTCCAGCTGCGCGTGCTTTTGG;
the sequence 5'-3' of the target site T2 is CCACGACCAGAGCTTG ATAACAC.
Since the nucleotide sequences of five copies of the PatPDS gene are relatively conserved, SNP polymorphism sites (391, 399 and 400 sites) near the target site T2 are selected according to sequence characteristics for subsequent SNP genotyping, so as to count the knockout efficiency and editing type among the five copies of the gene (FIG. 2).
(2.2) first round PCR amplification of U6 promoter and gRNA
Primers amplified by the first round PCR were individually subjected to ddH 2 O is dissolved into working solution with the diameter of 10 mu m;
AtU6-1 promoter was amplified by primer set U-F/AtU6-1-R using pU6 as a template, atU-29 promoter by primer set U-F/AtU 6-29-R;
Target site T1 was ligated to gRNA by primer combination gRT/gRNA-R using sgRNA as template to give gRNA1, and primer combination gRT2 +. g RNA-R connects target site T2 to gRNA to obtain gRNA2;
the primer sequences for the first round of PCR amplification were as follows:
the sequence 5'-3' of the U-F is CTCCGTTTTACCTGTGGAATCG;
the sequence 5'-3' of the AtU6-1-R is AAAGCACGCGCAGCTGGAATCaatcactacttcgtct;
the sequence '-3' of AtU6-29-R is CGACCAGAGCTTGATAACACaatctcttagtcgact;
the sequence 5'-3' of gRT1 is ATTCCAGCTGCGCGTGCTTTgttttagagctagaaat;
the sequence 5'-3' of gRT2 is TGTTATCAAGCTCTGGTCGgttttagagctagaaat;
the sequence 5'-3' of the gRNA-R is CGGAGGAAAATTCCATCCAC;
PCR reaction conditions: pre-denaturation at 95 ℃ for 2min; denaturation at 95℃for 20s; annealing at 55 ℃ for 20s; extending at 72 ℃ for 20s; amplifying for 20 cycles; and finally, the temperature is 72 ℃ and the extension is 5min. The first round PCR reaction is shown in Table 2.
TABLE 2 reaction System for first round PCR amplification
| Reaction components | Addition amount (mu L) |
| 2×Pfu MasterMix | 5 |
| Primer combination working solution | 0.4 |
| Template DNA | 0.1 |
| ddH 2 O | Make up to 10 mu L |
The product sequence of the first round of PCR amplification is as follows:
the sequence 5'-3' of the AtU-1 promoter is as follows:
the sequence 5'-3' of the AtU6-29 promoter is as follows:
AAAATATCAGAGATCTCTTACAGTTAGTTTCGTTCTTAATCCAAACTACTGCAGCCTGACAGACAAATGAGGATGCAAACAATTTTAAAGTTTATCTAACGCTAGCTGTTTTGTTTCTTCTCTCTGGTGCACCAACGACGGCGTTTTCTCAATCATAAAGAGGCTTGTTTTACTTAAGGCCAATAATGTTGATGGATCGAAAGAAGAGGGCTTTTAATAAACGAGCCCGTTTAAGCTGTAAACGATGTCAAAAACATCCCACATCGTTCAGTTGAAAATAGTAGCTCTGTTTATATATTGGTAGAGTCGACTAAGAGATTG；
The sequence 5'-3' of the gRNA1 is:
ATTCCAGCTGCGCGTGCTTTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC；
the sequence 5'-3' of the gRNA2 is:
TGTTATCAAGCTCTGGTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC；
(2.3) second round PCR amplification of sgRNA expression cassette
gRNA1 and gRNA2 were subjected to ddH 2 O was diluted 10-fold, and 0.5. Mu.L of each was used as a reaction template for the second PCR.
Amplifying the expression cassette sgRNA1 containing the AtU6-1 promoter and the gRNA1 in the step (2.2) by overlap PCR using AtU-1 and the gRNA1 as templates and using a primer combination Pps-1/Pgs-1;
amplifying the expression cassette sgRNA2 containing the AtU6-29 promoter and the gRNA2 in the step (2.2) by overlap PCR using AtU-29 and the gRNA2 as templates and adopting a primer combination Pps-2/Pgs-2;
the primer sequences for the second round of PCR amplification were as follows:
the sequence 5'-3' of the Pps-1 is TTCAGAggtctcTaccgACTAGTATGGAATCGGCAGCAAAGG;
the sequence 5'-3' of Pgs-1 is AGCGTGggtctcGtcagggTCCATCCACTCCAAGCTC;
the sequence 5'-3' of the Pps-2 is TTCAGAggtctcTctgacacTGGAATCGGCAGCAAAGG;
the sequence 5'-3' of Pgs-2 is AGCGTGggtctcGctcgACGCGTATCCATCCACTCCAAGCTC;
PCR reaction conditions: pre-denaturation at 95 ℃ for 2min; denaturation at 95℃for 20s; annealing at 55 ℃ for 30s; extending at 72 ℃ for 30s; amplifying for 20 cycles; and finally, the temperature is 72 ℃ and the extension is 5min.
The second round of PCR reaction for amplifying expression cassette sgRNA1 and expression cassette sgRNA2 is shown in tables 3 and 4,
TABLE 3 second round PCR reaction System for amplifying expression cassette sgRNA1
| Reaction components | Addition amount (mu L) |
| 2×Pfu Master Mix | 25 |
| Primer combination Pps-1/Pgs-1 working solution | 2 |
| First round PCR product AtU-1 promoter (10-fold dilution) | 0.5 |
| First round PCR product gRNA1 (10-fold dilution) | 0.5 |
| ddH 2 O | Make up to 50 mu L |
TABLE 4 second round PCR reaction System for amplifying expression cassette sgRNA2
The second round of PCR products were detected by 1% agarose gel electrophoresis, and the expression cassette sgRNA1 and the expression cassette sgRNA2 were purified and recovered by agarose gel recovery kit;
the expression cassette sgRNA1 and the expression cassette sgRNA2 have the sequences as follows:
the sequence 5'-3' of the expression cassette sgRNA1 is:
the sequence 5'-3' of the expression cassette sgRNA2 is:
(2.4) ligation of the expression cassette sgRNA1 and the expression cassette sgRNA2 with pEGCas9-PcUBI-B vector using Golden Gate method, ligation reaction system is shown in Table 5. The conditions of the enzyme digestion and enzyme ligation reaction are as follows: 37 ℃ for 5min;20 ℃ for 5min;15 cycles. The pEGCas9-PcUBI-B vector was purchased from Wuhan Aidi Biotechnology Co.
TABLE 5 ligation reaction System of expression cassette sgRNA1 and expression cassette sgRNA2 with pEGCas9-PcUBI-B vector
| Reaction components | Addition amount (mu L) |
| 10×CutSmart Buffer | 1.5 |
| 10mM ATP | 1.5 |
| pEGCas9PcUBI-B(100ng/μL) | 1 |
| sgRNA1 expression cassette (30 ng/. Mu.L) | 1 |
| sgRNA2 expression cassette (30 ng/. Mu.L) | 1 |
| BsaI-HF(10U/μl) | 1 |
| T4 DNA ligase(400U/μL) | 0.1 |
| ddH 2 O | Make up to 15 mu L |
(2.5) screening and identification of Gene editing recombinant vector Positive clones
The ligation product obtained in the step (2.4) is transformed into competent cells DH5 alpha of the escherichia coli by adopting a heat shock transformation method, and the specific steps are as follows: adding 2-5 mu L of the connection product into 100 mu L of escherichia coli competent cells DH5 alpha, mixing, and carrying out ice bath for 30min; rapidly placing in a constant-temperature water bath kettle at 42 ℃, carrying out heat shock for 90s, and carrying out ice bath for 2min; adding 700 mu L of LB liquid medium and uniformly mixing; shaking culture is carried out for 45-60 min at 37 ℃ and 200 rpm; the bacterial liquid is evenly coated on LB solid medium containing 50mg/L kanamycin, and the solid medium is placed in a constant temperature incubator at 37 ℃ for overnight culture.
Colony PCR detection was performed using the primer set E9Ter-F/SP-R, with the following primer sequences:
the sequence 5'-3' of the E9Ter-F is TGGATTTGTAGTTGAGTATGAA;
the sequence 5'-3' of the SP-R is TGCAATAACTTCGTATAGGC;
PCR reaction conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s; annealing at 55 ℃ for 30s; extending at 72 ℃ for 60s; amplifying for 30 cycles; and finally, the temperature is 72 ℃ and the extension is 5min.
And (3) sequencing the monoclonal which is detected to be positive, comparing the nucleotide sequence obtained by sequencing with the nucleotide sequence of the expression cassette sgRNA1 and the nucleotide sequence of the expression cassette sgRNA2, culturing a single colony of the recombinant vector pEGCas9-PcUBI-B containing the expression cassette sgRNA1 and the expression cassette sgRNA2, and extracting a plasmid for later use, wherein the plasmid is PcUbi-pEGCas9 (figure 3).
EXAMPLE 2 establishment of regeneration System of Pogostemon cablin
Plant regeneration systems are a prerequisite for genetic transformation and development of genetic manipulation tools. Although it has been reported that transgenic plants of patchouli are obtained using leaves as explants, the transformation process is largely inefficient due to the inhibitory effect of patchouli essential oil accumulated in the leaves on agrobacterium infection.
In this example, the culture medium involved in the patchouli regeneration system is as follows:
callus induction medium: MS+2.0 mg/L6-BA+0.2 mg/L NAA+30g/L sucrose+5.5 g/L agar powder, pH5.8;
differentiation medium: MS+1.0 mg/L6-BA+0.2 mg/L NAA+0.64 g/L2-morpholinoethanesulfonic acid+30 g/L sucrose+5.5 g/L agar powder, pH5.8;
rooting medium: MS+0.2mg/L NAA+0.64 g/L2-morpholinoethanesulfonic acid+30 g/L sucrose+5.5 g/L agar powder, pH5.8.
The young terminal buds of patchouli are used as explants, and seedlings are regenerated through the processes of callus induction, differentiation culture, rooting culture and the like, and the specific steps are as follows:
(1) Cutting young terminal buds (low in patchouli essential oil) of healthy patchouli seedlings as explants induced by callus;
(2) Soaking the explant with 75% ethanol for 30s, then soaking in 0.2% mercuric chloride for 5min, and shaking for several times;
(3) Taking out the explant, and placing the explant in sterile water for cleaning for 3-4 times;
(4) Placing the sterilized buds into a callus induction culture medium, culturing at 28 ℃ in a dark place, and changing the culture medium every 20 d;
(5) After two months of induction culture, the induced embryogenic callus is transferred into a differentiation culture medium, and adventitious bud differentiation is induced under the conditions of 28 ℃ and 16/8h of bright and dark photoperiod;
(6) And (3) subculturing every 20d, and transferring the buds to a rooting medium to induce rooting when the buds grow to 2-3 cm.
Under the regeneration culture system of this example, the induction rate and differentiation rate of patchouli callus reached 85% and 100% respectively, indicating that the optimized hormone combination was suitable for patchouli genetic transformation (fig. 4).
Example 3 sensitivity analysis of Pogostemon cablin callus to basta
Appropriate concentrations of basta are important for screening resistant calli to obtain transgenic plants. To determine the optimal basta concentration for screening positive transgenes, a basta sensitivity experiment was performed based on the patchouli regeneration system established in example 2, as follows:
(1) Respectively preparing callus induction culture mediums containing basta with different concentrations, wherein the concentration gradient is as follows: 0mg/L, 0.1mg/L, 0.3mg/L, 0.5mg/L, 1.0mg/L, 3.0mg/L, 5.0mg/L, 8.0mg/L and 10mg/L;
(2) Transferring the embryogenic callus induced in example 2 to callus induction medium containing different concentrations of basta, and culturing in the dark at 28 ℃;
(3) The medium was changed every 2 weeks, and the optimum screening concentration of basta was evaluated according to the growth state and browning frequency of embryogenic callus.
After 4 weeks of continuous culture, the results showed that the frequency of callus browning gradually increased with increasing basta concentration. When the basta concentration is more than 8.0mg/L, all the calli are brown and die; most calli brown to death at 5mg/L basta, indicating that 5mg/L basta is the optimal concentration for screening transgenic resistant calli (FIG. 5).
EXAMPLE 4 transformation of Agrobacterium with PcUbi-pEGCas9 Gene editing recombinant vector
The plasmid PcUbi-pEGCas9 constructed in example 2 was transformed into competent cells of Agrobacterium tumefaciens GV3101 by freeze thawing, and the procedure was as follows:
(1) Thawing Agrobacterium tumefaciens GV3101 competent cells on ice, adding 1 μg of PcUbi-pEGCas9 plasmid, mixing well, and standing on ice for 5min;
(2) Rapidly cooling in liquid nitrogen for 5min, rapidly transferring into 37deg.C water bath for 5min, and rapidly taking out ice bath for 5min;
(3) Adding 1mL of YEB liquid culture medium without antibiotics, and culturing for 2-3 h at 28 ℃ and 220 r/min;
(4) Centrifuging at 5000r/min for 1min, collecting thallus, sucking the supernatant to 900 μL, and reserving 100 μL;
(5) Re-suspending the bacterial cells and coating the bacterial cells on a YEP solid plate containing 25 mug/mL rifampicin, 50 mug/mL kanamycin and 50 mug/mL gentamicin, and inversely culturing the bacterial cells for 48 to 72 hours at the temperature of 28 ℃;
(6) Single colony PCR detection is selected, correct single colony is inoculated into liquid YEB culture medium containing 25 mug/mL rifampicin, 50 mug/mL kanamycin and 50 mug/mL gentamicin, and the culture is carried out at 28 ℃ and 220r/min, 50% glycerol with equal volume sterilization is added, and the strain is preserved at-80 ℃ for standby.
EXAMPLE 5 Agrobacterium-mediated genetic transformation of Pogostemon cablin
In this example, the following media were used during genetic transformation of Pogostemon cablin:
pre-culture medium: MS+30g/L sucrose+5.5 g/L agar, pH 5.8;
infection medium: MS+30g/L sucrose+200 μm acetosyringone, pH 5.8;
co-culture medium: MS+2.0 mg/L6-BA+0.2 mg/L NAA+4.625 g/L2-morpholinoethanesulfonic acid+200 μm acetosyringone+30 g/L sucrose+5.5 g/L agar, pH 5.8;
resistant callus screening media: MS+2.0 mg/L6-BA+0.2 mg/L NAA+0.64 g/L2-morpholinoethanesulfonic acid+30 g/L sucrose+300 mg/L cephalosporin+200 mg/L timentin+5.0 mg/L basta+5.5g/L agar, pH 5.8;
Differentiation screening medium: MS+1.0 mg/L6-BA+0.2 mg/L NAA+0.64 g/L2-morpholinoethanesulfonic acid+30 g/L sucrose+300 mg/L cephalosporin+200 mg/L timentin+5.0 mg/L basta+5.5g/L agar, pH 5.8;
induction rooting culture medium: MS+0.2mg/L NAA+0.64 g/L2-morpholinoethanesulfonic acid+30 g/L sucrose+150 mg/L cephalosporin+100 mg/L timentin+5.0 mg/L basta+5.5g/L agar, pH 5.8.
An agrobacterium-mediated genetic transformation method for transforming a PcUbi-pEGCas9 gene editing recombinant vector comprises the following steps:
(1) Agrobacterium transformation
Specifically in example 4;
(2) Preparation of dyeing liquor
The strain stored in example 4 was inoculated into 1mL of YEB liquid medium added with 50mg/L kanamycin, 25mg/L rifampicin and 50mg/L gentamicin, and cultured overnight with shaking in a shaker at 28℃and 200 rpm; transferring all cultures into 100mL of YEB liquid culture medium containing 50mg/L kanamycin, 25mg/L rifampicin and 50mg/L gentamicin, culturing until OD600 reaches 0.6-0.8, centrifuging at 5000rpm for 5min, and collecting agrobacterium cells; the precipitated cells were resuspended in an equal volume of infection medium and incubated for 2h to activate toxic genes for infection transformation;
(3) Pre-culture
Transferring the embryogenic callus induced in example 2 to a preculture medium, and culturing in the dark at 25 ℃ for 2d for agrobacterium infection; (4) Infestation of the human body
Transferring the pre-cultured embryogenic callus into a prepared infection culture medium, placing 100 explants per 50mL of infection liquid (100 explants are added into each 50mL of infection liquid) into a 90r/min shaking table, and shaking for 20-30 min at 28 ℃ to enable the pre-cultured explants to be in full contact with the transformed agrobacterium;
(5) Co-cultivation
After infection, sucking the bacterial liquid on the surface of the callus by using sterile filter paper, transferring the bacterial liquid into a co-culture medium, and co-culturing for 2-3 d under the dark culture condition at 25 ℃;
(6) Bacteria washing
Washing the callus which forms plaque in the co-culture process with sterile water containing 300mg/L cephalosporin and 200mg/L timentin for 2-3 times, and then sucking the water on the surface with sterile filter paper;
(7) Resistant callus screening
Transferring the callus to a resistant callus screening culture medium, culturing in dark at 25 ℃ and carrying out subculture once every 2 weeks, and continuously carrying out subculture for 2-3 times to screen the resistant callus;
(8) Differentiation screening
Transferring the resistant callus to a differentiation screening culture medium, inducing the differentiation of the adventitious buds at 28 ℃ under 16/8h of photoperiod, and inducing the adventitious buds once every 2 weeks;
(9) Rooting culture
When the buds grow to 2-3 cm, transferring the buds into a rooting induction culture medium to induce rooting.
By the agrobacterium-mediated transformation protocol of this example, pcUbi-pEGCas9 gene editing recombinant vector was transformed into patchouli embryogenic callus, and regenerated plants showed three phenotypes of normal, chimeric and albino seedlings, with albino and chimeric phenotypes of callus pieces accounting for 38.67% and 15.47%, respectively.
EXAMPLE 6 identification of Positive transgenic plants
In this example, the positive transgenic plant identification process was as follows:
since somatic variations often lead to low probability albino seedlings during plant tissue culture, 43 patchouli transformed plants exhibiting albino phenotype and 12 patchouli transformed plants having chimeric phenotype were randomly selected for transgene validation.
The genomic DNA of tetraploid patchouli is extracted by adopting a CTAB method (Li et al, 2013), a specific primer Cas9-F and a primer Cas9-R are designed according to the nucleotide sequence of Cas9 protein, and the Cas9 gene in the positive transgenic tetraploid patchouli is amplified by PCR.
The sequence 5'-3' of Cas9-F is CAAGTCTGTTAAGGAACTTCTCGG;
the sequence 5'-3' of the Cas9-R is GATGATGTTCTCTGCCTGTTCCC.
The primer Cas9-F and the primer Cas9-R are respectively subjected to ddH 2 O was dissolved into 10 μm working solution and the Cas9 gene was amplified using 2 xTaq MasterMix polymerase. The PCR reaction uses PcUbi-pEGCas9 plasmid as a template as a positive control, uses the DNA of wild patchouli as a template as a negative control, and uses sterile water as a template as a blank control. The PCR reaction conditions were: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s; annealing at 55 ℃ for 30s; extending at 72 ℃ for 30s; amplifying for 30 cycles; the final extension was carried out at 72℃for 10min, and the PCR reaction system was shown in Table 6. The PCR products were detected by 1% agarose gel electrophoresis.
TABLE 6 identification of positive transgenic plants PCR reaction System
| Reaction components | Addition amount (mu L) |
| 2×Taq MasterMix | 12.5 |
| Cas9-F(10μm) | 1 |
| Cas9-R(10μm) | 1 |
| Stencil plate | 1 |
| ddH 2 O | Make up to 25. Mu.L |
The detection results (fig. 7) showed that Cas9 gene was not detected in 4 albino plants and 3 chimeric plants, and the remaining 48 plants were confirmed as transgenic plants with a conversion efficiency of 87.27%.
Example 7PatPDS Gene target site mutation type analysis (7.1) PatPDS Gene target site mutation detection
In example 6, 43 patchouli transformed plants exhibiting albino phenotype and 12 patchouli transformed plants having chimeric phenotype were randomly selected and also used for PatPDS gene target site mutation detection.
Specific primers PDS-dF and PDS-dR are designed on two sides of a target site T1 and a target site T2, PCR amplification is carried out, and single plants with overlapping peaks generated by the target sites are screened.
The PDS-dF sequence is GGCAAATGAATGGCTTATCCTT at 5 '-3';
the PDS-dR sequence is 5'-3' GCTAAACCTGTAAGAAACCCAAC.
The PCR reaction conditions were: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s; annealing at 60 ℃ for 30s; extending at 72 ℃ for 30s; amplifying for 30 cycles; the final extension was carried out at 72℃for 10min, and the PCR reaction system was shown in Table 7. The PCR products were detected by 1% agarose gel electrophoresis and the products were sequenced.
TABLE 7PatPDS Gene target site PCR reaction System
As the detection result data of the 58 patchouli transformed plants are too much, only the detection result of the plant with the number of Line11 is provided as shown in FIG. 8, the sequencing peak diagram shows that all transgenic plants have overlapping peaks at the target site T2, and the target site T1 does not show overlapping peaks, which indicates that the AtU-1 promoter may not drive the expression of sgRNA in patchouli or the efficiency of the target site T1 is low.
(7.2) verification of target editing efficiency of PatPDS allele in Pogostemon cablin by PcUbi-pEGCas9 System PCR amplification was performed on 48 transgenic plants and 1 wild type plant using primer combinations read1-PE250F-A/read2-PE250R, read1-PE250F-B/read2-PE250R, read1-PE250F-C/read2-PE250R, read1-PE250F-D/read2-PE250R and read1-PE250F-E/read2-PE250R flanking the target site T2, and high throughput sequencing was performed on PCR products, pooled for every 5 individual plants.
The forward primer read1-PE250F-A, read1-PE250F-B, read1-PE250F-C, read1-PE250F-D, read1-PE250F-E was introduced into the tag sequence to identify each individual, and genotyping was performed based on the SNP polymorphism in example 1.
The sequence 5'-3' of the read1-PE250F-A is:
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTGCATGGAAGAACTCTACTAATG ATAT；
the sequence 5'-3' of the read1-PE250F-B is:
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGTTGCGGAAGAACTCTACTAATG ATAT；
The sequence 5'-3' of the read1-PE250F-C is as follows:
the sequence 5'-3' of the read1-PE250F-D is:
the sequence 5'-3' of the read1-PE250F-E is as follows:
the sequence 5'-3' of the read2-PE250R is:
the PCR reaction conditions were: pre-denaturation at 95 ℃ for 3min; denaturation at 95℃for 15s; annealing at 50 ℃ for 15s; extending at 72 ℃ for 30s; amplifying for 30 cycles; the final extension was carried out at 72℃for 10min, and the PCR reaction system was shown in Table 8. The PCR products were detected by 1% agarose gel electrophoresis and recovered by agarose gel recovery kit gel cutting, and the recovered products were sequenced on an Illumina PE250 sequencing platform.
TABLE 8 PCR amplification reaction System for detecting target site T2 mutation based on PE250 high throughput sequencing
| Reaction components | Addition amount (mu L) |
| 5×EVOBuffer(with 10mM MgCl 2 ) | 10 |
| dNTP Mix(10mM each) | 1 |
| Primer set (10 μm) | 4 |
| Phanta EVO HS Super-Fidelity DNA Polymerase(1U/μL) | 1 |
| Template DNA | 1 |
| ddH2O | Make up to 50 mu L |
Comparing the sequencing results with wild type, the editing type between alleles was quantified by detecting PatPDS allele-specific SNP sites, and the results (fig. 9) showed that all PatPDS alleles of 2 plants (4.17%) had been completely mutated, 3 alleles of 3 plants (6.25%) had been completely edited, 2 alleles of 5 plants (10.42%) had been completely knocked out, and only 1 allele of 13 plants (27.08%) had been completely knocked out.
As a result of SNP genotyping, the results (FIG. 9) showed that the editing efficiency of the Pat_A05G005500 gene was 41.67%, the editing frequencies of the Pat_B05G005800 and Pat_A06G005300 genes were 25% and 12.5%, respectively, and the editing frequencies of the Pat_B06G005200 and Pat_U0021200 genes were only 6.25%.
The result (FIG. 9) shows that the most common mutation type of Pat_A05G005500 is 5bp nucleotide deletion (29.09%), the most common mutation type of Pat_B05G005800 is 3bp nucleotide deletion (32.64%), and the most common mutation types of Pat_A06G005300 and Pat_B06G005200/Pat_U0021200 are 2bp nucleotide deletion, accounting for 28.23% and 44.83%, respectively.
(7.3) verification of PcUbi-pEGCas9 System-mediated Long fragment mutation
In order to detect the long fragment mutation mediated by the PcUbi-pEGCas9 system in transgenic plants, PCR amplification was performed using high fidelity PrimeStar Max DNA polymerase with flanking primers PDS-dF/PDS-dR at target site T1 and target site T2, the PCR reaction conditions are shown in step (7.1), and the PCR reaction system is shown in Table 7. The PCR products were detected by 1% agarose gel electrophoresis and purified and recovered by agarose gel recovery kit.
The PCR products recovered by purification are connected to pCE2_TA-Blunt-Zero vector through TOPO cloning kit, E.coli competent cells DH5 alpha are transformed by heat shock method, and after ampicillin and kanamycin screening, at least 10 single clones are selected for each transgenic plant for sequencing. The nucleotide sequences obtained by sequencing were aligned with the nucleotide sequences of the wild-type PatPDS alleles to decode the mutation type and frequency.
The sequencing results (FIG. 10) further confirm the PcUbi-pEGCas9 system mediated long fragment editing event by Sanger sequencing, detecting large fragment deletions of 16bp, 46bp and 92bp in transgenic lines Line 5, line 10 and Line 11, respectively.
Claims (9)
1. An agrobacterium-mediated genetic transformation method for transforming a PcUbi-pEGCas9 gene editing recombinant vector, which is characterized by comprising the following steps:
(1) Agrobacterium transformation
Transforming the PcUbi-pEGCas9 plasmid into competent cells of Agrobacterium tumefaciens GV 3101;
the PcUbi-pEGCas9 plasmid comprises the following gene fragments: 35S promoter CaMV 35S from cauliflower mosaic virus, herbicide basta resistance gene BlpR, terminator CaMV poly A, ubiquitin promoter PcUbi from parsley, translation enhancer TMVΩ, kozak sequence for enhancing eukaryotic gene translation efficiency, endonuclease gene Cas9, nuclear localization signal element SV40 NLS, terminator E9 terminator, arabidopsis AtU6-1 promoter, expression cassette sgRNA1 comprising PatPDS gene editing target site T1, arabidopsis AtU6-29 promoter, expression cassette sgRNA2 comprising PatPDS gene editing target site T2;
the sequence 5'-3' of the expression cassette sgRNA1 is:
AGAAATCTCAAAATTCCGGCAGAACAATTTTGAATCTCGATCCGTAGAAACGAGACGGTCATTGTTTTAGTTCCACCACGATTATATTTGAAATTTACGTGAGTGTGAGTGAGACTTGCATAAGAAAATAAAATCTTTAGTTGGGAAAAAATTCAATAATATAAATGGGCTTGAGAAGGAAGCGAGGGATAGGCCTTTTTCTAAAATAGGCCCATTTAAGCTATTAACAATCTTCAAAAGTACCACAGCGCTTAGGTAAAGAAAGCAGCTGAGTTTATATATGGTTAGAGACGAAGTAGTGATTGATTCCAGCTGCGCGTGCTTTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC；
the sequence 5'-3' of the expression cassette sgRNA2 is:
AAAATATCAGAGATCTCTTACAGTTAGTTTCGTTCTTAATCCAAACTACTGCAGCCTGACAGACAAATGAGGATGCAAACAATTTTAAAGTTTATCTAACGCTAGCTGTTTTGTTTCTTCTCTCTGGTGCACCAACGACGGCGTTTTCTCAATCATAAAGAGGCTTGTTTTACTTAAGGCCAATAATGTTGATGGATCGAAAGAAGAGGGCTTTTAATAAACGAGCCCGTTTAAGCTGTAAACGATGTCAAAAACATCCCACATCGTTCAGTTGAAAATAGTAGCTCTGTTTATATATTGGTAGAGTCGACTAAGAGATTGTGTTATCAAGCTCTGGTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC；
(2) Preparation of dyeing liquor
Inoculating transformed Agrobacterium into YEB liquid culture medium of 1 mL added with 50 mg/L kanamycin, 25 mg/L rifampicin and 50 mg/L gentamicin, and shaking overnight at 28deg.C in a shaking table of 200 rpm for culture;
transferring all cultures to a liquid medium of 100 mL YEB containing 50 mg/L kanamycin, 25 mg/L rifampicin and 50 mg/L gentamicin, culturing until OD600 reaches 0.6-0.8, centrifuging at 5000 rpm for 5 min, and collecting agrobacterium cells;
resuspending the pelleted cells in an equal volume of infection medium, incubating 2 h to activate toxic genes for infection transformation;
(3) Pre-culture
Transferring the induced embryogenic callus to a preculture medium, and culturing at 25 ℃ in a dark state for 2 d;
(4) Infestation of the human body
Transferring the pre-cultured embryogenic callus into an infection culture medium, placing 100 explants/50 mL infection culture medium in a 90 r/min shaking table, and shaking for 20-30 min at 28 ℃ to enable the pre-cultured explants to be in full contact with the transformed agrobacterium;
(5) Co-cultivation
After infection, sucking the bacterial liquid on the infected embryogenic callus with sterile filter paper, transferring the bacterial liquid into a co-culture medium, and culturing for 2-3 d under the dark culture condition at 25 ℃;
(6) Bacteria washing
Washing the callus which forms plaque in the co-culture process with sterile water containing 300 mg/L cephalosporin and 200 mg/L timentin for 2-3 times, and then sucking the surface water with sterile filter paper;
(7) Resistant callus screening
Transferring the calli to a resistant callus screening culture medium, culturing in dark at 25 ℃ once every 2 weeks, and continuously carrying out 2-3 times of subculture to screen the resistant calli;
(8) Differentiation screening
Transferring the resistant callus to a differentiation screening culture medium, inducing the differentiation of the adventitious buds at 28 ℃ under 16/8 h bright and dark photoperiod, and inducing the adventitious buds once every 2 weeks;
(9) Rooting culture
Transferring the buds to a rooting induction culture medium to induce rooting when the buds grow to 2-3 cm;
the preparation method of the PcUbi-pEGCas9 plasmid comprises the following steps:
(S1) nucleotide sequence characterization and amplification of tetraploid Pogostemon cablin PatPDS Gene
Amplifying the PatPDS alleles of five copies of tetraploid patchouli by PCR by using the genomic DNA of the tetraploid patchouli as a template and adopting a primer combination PatPDS-F/PatPDS-R;
the sequence 5'-3' of PatPDS-F is ATGATGTCTCAATTTGGGCAC;
the sequence 5'-3' of the PatPDS-R is TTAGGCGTAGCTTGCCTCT;
Detecting the PCR product by using 1% agarose gel electrophoresis, and purifying and recovering the PCR product by using an agarose gel recovery kit;
connecting the purified and recovered PCR product to a pCE2_TA-Blunt-Zero vector through a TOPO cloning kit to obtain a recombinant pCE2_TA-Blunt-Zero vector; transforming the recombinant pCE2_TA-Blunt-Zero vector into an escherichia coli competent cell DH5 alpha by adopting a heat shock method, screening by ampicillin and kanamycin, and selecting a positive monoclonal to sequence to obtain a nucleotide sequence of five copies of the tetraploid patchouli PatPDS gene;
(S2) construction of PatPDS Gene editing recombinant vector
(S2.1) screening of target sites of Gene editing recombinant vectors
According to the nucleotide sequences of five copies of the tetraploid patchouli PatPDS gene, selecting two target sites near the 5' -end conserved region of the PatPDS allele, namely a target site T1 positioned in a first exon and a target site T2 positioned in a second exon;
the sequence 5'-3' of the target site T1 is ATTCCAGCTGCGCGTGCTTTTGG;
the sequence 5'-3' of the target site T2 is CCACGACCAGAGCTTG ATAACAC;
(S2.2) first round PCR amplification
AtU6-1 promoter was amplified by primer set U-F/AtU6-1-R using pU6 as a template, atU-29 promoter by primer set U-F/AtU 6-29-R;
Using sgRNA as a template, connecting a target site T1 to the gRNA through a primer combination gRT/gRNA-R to obtain gRNA1, and connecting a target site T2 to the gRNA through a primer combination gRT/gRNA-R to obtain gRNA2;
the primer sequences for the first round of PCR amplification were as follows:
the sequence 5'-3' of the U-F is CTCCGTTTTACCTGTGGAATCG;
the sequence 5'-3' of the AtU6-1-R is AAAGCACGCGCAGCTGGAATCaatcactacttcgtct;
the sequence 5'-3' of the AtU6-29-R is CGACCAGAGCTTGATAACACaatctcttagtcgact;
the sequence 5'-3' of gRT1 is ATTCCAGCTGCGCGTGCTTTgttttagagctagaaat;
the sequence 5'-3' of gRT2 is TGTTATCAAGCTCTGGTCGgttttagagctagaaat;
the sequence 5'-3' of the gRNA-R is CGGAGGAAAATTCCATCCAC;
(S2.3) second round PCR amplification
Amplifying the expression cassette sgRNA1 containing the AtU6-1 promoter and the gRNA1 in the step (S2.2) by overlap PCR using AtU-1 and the gRNA1 as templates and adopting a primer combination Pps-1/Pgs-1;
amplifying the expression cassette sgRNA2 containing the AtU6-29 promoter and the gRNA2 in the step (S2.2) by overlap PCR using AtU-29 and the gRNA2 as templates and adopting a primer combination Pps-2/Pgs-2;
the primer sequences for the second round of PCR amplification were as follows:
the sequence 5'-3' of the Pps-1 is TTCAGAggtctcTaccgACTAGTATGGAATCGGCAGCAAAGG;
The sequence 5'-3' of Pgs-1 is AGCGTGggtctcGtcagggTCCATCCACTCCAAGCTC;
the sequence 5'-3' of the Pps-2 is TTCAGAggtctcTctgacacTGGAATCGGCAGCAAAGG;
the sequence 5'-3' of Pgs-2 is AGCGTGggtctcGctcgACGCGTATCCATCCACTCCAAGCTC;
the second round of PCR products were detected by 1% agarose gel electrophoresis, and the expression cassette sgRNA1 and the expression cassette sgRNA2 were purified and recovered by agarose gel recovery kit;
(S2.4) ligating the expression cassette sgRNA1 and the expression cassette sgRNA2 with the pEGCas9-PcUBI-B vector;
(S2.5) screening and identification of Gene editing recombinant vector Positive clones
The ligation product obtained in step (S2.4) was transformed into E.coli competent cells DH 5. Alpha. And colony PCR detection was performed using the primer combination E9Ter-F/SP-R, with the following primer sequences:
the sequence 5'-3' of the E9Ter-F is TGGATTTGTAGTTGAGTATGAA;
the sequence 5'-3' of the SP-R is TGCAATAACTTCGTATAGGC;
sequencing the monoclonal which is detected to be positive, comparing the nucleotide sequence obtained by sequencing with the nucleotide sequence of the expression cassette sgRNA1 and the nucleotide sequence of the expression cassette sgRNA2, culturing a single colony of the recombinant vector pEGCas9-PcUBI-B containing the expression cassette sgRNA1 and the expression cassette sgRNA2, and extracting a plasmid for later use, wherein the plasmid is PcUbi-pEGCas9.
2. The agrobacterium-mediated genetic transformation method of PcUbi-pEGCas9 gene editing recombinant vector transformed according to claim 1, wherein the composition and content of the culture medium involved in the transformation process are as follows:
pre-culture medium: MS+30 g/L sucrose+5.5 g/L agar, pH 5.8;
infection medium: MS+30 g/L sucrose+200 μm acetosyringone, pH 5.8;
co-culture medium: MS+2.0 mg/L6-BA+0.2 mg/L NAA+4.625 g/L2-morpholinoethanesulfonic acid+200 μm acetosyringone+30 g/L sucrose+5.5 g/L agar, pH 5.8;
resistant callus screening media: MS+2.0 mg/L6-BA+0.2 mg/L NAA+0.64 g/L2-morpholinoethanesulfonic acid+30 g/L sucrose+300 mg/L cephalosporin+200 mg/L timentin+5.0 mg/L basta+5.5 g/L agar, pH 5.8;
differentiation screening medium: MS+1.0 mg/L6-BA+0.2 mg/L NAA+0.64 g/L2-morpholinoethanesulfonic acid+30 g/L sucrose+300 mg/L cephalosporin+200 mg/L timentin+5.0 mg/L basta+5.5 g/L agar, pH 5.8;
induction rooting culture medium: MS+0.2 mg/L NAA+0.64 g/L2-morpholinoethanesulfonic acid+30 g/L sucrose+150 mg/L cephalosporin+100 mg/L timentin+5.0 mg/L basta+5.5 g/L agar, pH 5.8.
3. The agrobacterium-mediated genetic transformation method of PcUbi-pEGCas9 gene editing recombinant vector transformed agrobacterium tumefaciens of claim 1, wherein the agrobacterium transformation of step (1) is specifically as follows:
(1) Placing the competent cells of the agrobacterium tumefaciens GV3101 on ice for melting, adding 1 mug of PcUbi-pEGCas9 plasmid, fully and uniformly mixing, and placing on ice for 5 min;
(2) Rapidly cooling in liquid nitrogen for 5 min, rapidly transferring into 37deg.C water bath for 5 min, and rapidly taking out ice bath for 5 min;
(3) Adding 1 mL of YEB liquid culture medium without antibiotics, and culturing at 28 ℃ and 220 r/min for 2-3 h;
(4) After centrifugation at 5000 r/min for 1 min, collecting thalli, sucking the supernatant to 900 [ mu ] L, and reserving 100 [ mu ] L;
(5) The cells were resuspended and spread onto YEP solid plates containing 25. Mu.g/mL rifampicin, 50. Mu.g/mL kanamycin and 50. Mu.g/mL gentamicin, and cultured upside down at 28℃for 48-72 h;
(6) Single colony PCR detection is selected, correct single colony is inoculated into liquid YEB culture medium containing 25 mug/mL rifampicin, 50 mug/mL kanamycin and 50 mug/mL gentamicin, and the culture is carried out under the conditions of 28 ℃, 220 and r/min, 50% glycerol with equal volume sterilization is added, and the strain is preserved at-80 ℃ for standby.
4. The method for genetic transformation of Agastache rugosa mediated by Agrobacterium transformed by the recombinant vector edited by PcUbi-pEGCas9 gene according to claim 1,
in step (S1), the PCR reaction conditions are: 98. denaturation at 10 c s; 55. annealing 15 s; 72. extension 60 s; amplifying for 30 cycles; 72. finally extending for 10 min at the temperature;
In step (S2.2), the first round PCR reaction conditions are: 95. pre-denaturing for 2 min at the temperature; 95. denaturation at 20℃ 20 s; annealing at 55 ℃ for 20 s; extending 20 s at 72 ℃; amplifying for 20 cycles; final extension for 5 min at 72 ℃;
in step (S2.3), the second round PCR reaction conditions were: 95. pre-denaturing for 2 min at the temperature; 95. denaturation at 20℃ 20 s; annealing at 55 ℃ for 30 s; extending 30 s at 72 ℃; amplifying for 20 cycles; final extension for 5 min at 72 ℃;
in the step (S2.4), the Golden Gate method is adopted to realize connection; the conditions of the enzyme digestion and enzyme ligation reaction are as follows: 37. at the temperature of 5 min;20 ℃ for 5 min;15 cycles;
in the step (S2.5), a heat shock conversion method is adopted to realize conversion; the PCR reaction conditions were: 95. pre-denaturing for 5 min at the temperature; 95. denaturation 30℃ 30 s; 55. annealing 30 s; 72. extension 60 s; amplifying for 30 cycles; 72. at the temperature, the final extension is carried out for 5 min.
5. The method for genetic transformation of Agastache rugosa mediated by Agrobacterium transformed by the recombinant vector edited by PcUbi-pEGCas9 gene according to claim 1,
in step (S1), the primer PatPDS-F and the primer PatPDS-R are respectively treated with ddH 2 O is dissolved into working solution, and a PCR reaction system is as follows: 2X PrimeStar Max Premix was added at 25. Mu.L, the primer PatPDS-F was added at 1. Mu.L, the primer PatPDS-R was added at 1. Mu.L, the patchouli DNA was added at 1. Mu.L, and ddH 2 O is added to 50 mu L;
in step (S2.2), the primers amplified by the first round PCR are each amplified with ddH 2 O is dissolved into working solution, and the first round of PCR reaction system is as follows: the addition amount of 2 XPFU Master Mix was 5. Mu.L, the addition amount of the primer combination working solution was 0.4. Mu.L,the amount of template DNA added was 0.1. Mu.L, ddH 2 O is added to 10 mu L;
in step (S2.3), gRNA1 and gRNA2 are treated with ddH, respectively 2 O is diluted 10 times, and 0.5 mu L of each O is taken as a reaction template of the second round of PCR respectively;
the second round of PCR reaction for amplifying the expression cassette sgRNA1 was as follows: the addition amount of 2 XPFU Master Mix was 25. Mu.L, the addition amount of the primer set Pps-1/Pgs-1 working solution was 2. Mu.L, the addition amount of the first round PCR product AtU-1 promoter after 10-fold dilution was 0.5. Mu.L, the addition amount of the first round PCR product gRNA1 after 10-fold dilution was 0.5. Mu.L, and ddH 2 O is added to 50 mu L;
the second round of PCR reaction for amplifying the expression cassette sgRNA2 was as follows: the addition amount of 2 XPFU Master Mix was 25. Mu.L, the addition amount of the primer set Pps-2/Pgs-2 working solution was 2. Mu.L, the addition amount of the first round PCR product AtU-29 promoter after 10-fold dilution was 0.5. Mu.L, the addition amount of the first round PCR product gRNA2 after 10-fold dilution was 0.5. Mu.L, and ddH 2 O is added to 50 mu L;
in step (S2.4), the ligation reaction system of the expression cassette sgRNA1 and the expression cassette sgRNA2 with the pEGCas9-PcUBI-B vector is as follows: 10 XCutSmart Buffer 1.5. Mu.L, 10 mM ATP 1.5. Mu.L, pEGCas 9-PcUBI-B100. 100 ng/. Mu.L 1. Mu.L, 30. 30 ng/. Mu.L of expression cassette sgRNA 1. Mu.L, 30. 30 ng/. Mu.L of expression cassette sgRNA2 1. Mu.L, 10. 10U/. Mu.L of Bsa I-HF 1. Mu.L, 400. 400U/. Mu.L of T4 DNA ligase 0.1. Mu.L, ddH 2 O was made up to 15. Mu.L.
6. The agrobacterium-mediated genetic transformation method of PcUbi-pEGCas9 gene editing recombinant vector according to claim 1, wherein in step (S1), the nucleotide sequence characterization of the tetraploid patchouli PatPDS gene is as follows: the nucleotide coding sequence KC854409.1 of the tetraploid patchouli PatPDS gene is obtained on the NCBI database, the tetraploid patchouli PatPDS gene KC854409.1 is taken as a reference sequence, and the reference genome of the tetraploid patchouli is analyzed, so that five copies of the tetraploid patchouli PatPDS gene exist in the tetraploid patchouli genome and are respectively positioned on four chromosomes A05, A06, B05 and B06 and the scaffold 89 and are respectively named Pat_A05G005500, pat_B05G005800, pat_A06G005300, pat_B06G005200 and Pat_U0021200.
7. The agrobacterium-mediated genetic transformation method of p-ubi-pEGCas 9 gene editing recombinant vector according to claim 1, wherein in step (S1), the genomic DNA of tetraploid patchouli is extracted by CTAB method, the tetraploid patchouli reference genome is obtained in BIG Data Center database, and conserved specific primers PatPDS-F and PatPDS-R are designed according to the nucleotide sequences of five PatPDS alleles in the tetraploid patchouli reference genome.
8. The agrobacterium-mediated genetic transformation method of p-ubi-pEGCas 9 gene editing recombinant vector according to claim 1, wherein in step (S2.1), the on-line tool http:// crispor.tefor.net/, to reduce the off-target efficiency of PatPDS allele knockout, a conserved segment of five copies of the PatPDS gene is selected, and the target site of PatPDS gene editing is designed according to GC content and PAM sequence.
9. The method for agrobacterium-mediated genetic transformation of PcUbi-pEGCas9 gene editing recombinant vector according to claim 1, wherein in step (S2.5), the ligation product obtained in step (S2.4) is transformed into escherichia coli competent cells dh5α as follows: adding 2-5 mu L of the connection product into 100 mu L of escherichia coli competent cells DH5 alpha, mixing, and carrying out ice bath for 30 min; rapidly placing in a constant-temperature water bath kettle at 42 ℃, carrying out heat shock 90 s, and carrying out ice bath for 2 min; adding 700 mu L of LB liquid medium and uniformly mixing; 37. shaking culture is carried out for 45-60 min at the temperature of 200 rpm; the bacterial liquid is evenly spread on LB solid medium containing 50 mg/L kanamycin, and the solid medium is placed in a constant temperature incubator at 37 ℃ for overnight culture.
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