CN111057713A - CRISPR/Cas9 vector applicable to erwinia bacterium FS110 and construction method and application thereof - Google Patents

CRISPR/Cas9 vector applicable to erwinia bacterium FS110 and construction method and application thereof Download PDF

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CN111057713A
CN111057713A CN201911311250.6A CN201911311250A CN111057713A CN 111057713 A CN111057713 A CN 111057713A CN 201911311250 A CN201911311250 A CN 201911311250A CN 111057713 A CN111057713 A CN 111057713A
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叶伟
黄自磊
章卫民
李赛妮
朱牧孜
孔亚丽
刘桃妹
刘珊
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Guangdong Detection Center of Microbiology of Guangdong Institute of Microbiology
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Abstract

The invention discloses a CRISPR/Cas9 vector applicable to Erysian FS110, and a construction method and application thereof. The invention firstly utilizes CRISPR/Cas9 technology to construct a recombinant Edwardsiella FS110 strain with a knocked-out gliotoxin biosynthetic gene, and establishes a CRISPR/Cas9 gene knock-out system suitable for deep-sea fungus Edwardsiella FS110, thereby promoting the genetic engineering transformation of the Edwardsiella FS110 and laying a molecular biological foundation for the elucidation of the biosynthesis mechanism of the Edwardsiella FS110 gliotoxin and the obtainment of more gliotoxin derivatives with obvious biological activity.

Description

CRISPR/Cas9 vector applicable to erwinia bacterium FS110 and construction method and application thereof
Technical Field
The invention belongs to the technical field of biochemistry and molecular biology, and particularly relates to a CRISPR/Cas9 vector applicable to Erysian FS110, and a construction method and application thereof.
Background
The deep sea fungus Edwardsiella (Dicchotomyces cejpii) FS110 is an ascomycete from deep sea, and can generate a large amount of novel gliotoxins with anti-tumor activity, about 30 gliotoxin compounds are separated from the deep sea fungus Edwardsiella FS110 in the early stage, and the deep sea fungus Edwardsiella FS110 also comprises gliotoxin dimer compounds with rare structures, wherein most gliotoxin compounds have better anti-tumor activity and activity of resisting α -glucosidase, so the deep sea fungus Edwardsiella FS110 has the prospect of being developed into a drug lead compound.
CRISPR/Cas9 is a technology for specific DNA modification of targeted genes by sgRNA mediated Cas9 nuclease. The two nuclease activity areas of RuVC1 and HNH make the gene have endonuclease activity, and the target gene can be cut under the guide of gRNA. The CRISPR/Cas9 system has wide application in genome editing of eukaryotic cells such as mammalian cells, stem cells and plants due to the advantages of high gene knockout efficiency, simple construction, low cost and the like, and the CRISPR/Cas9 system is less in filamentous fungi due to the lack of corresponding vectors, so that a report of gene knockout on deep-sea fungi by using the CRISPR/Cas9 system is not seen.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a CRISPR/Cas9 vector suitable for deep-sea fungus Edwardsiella FS110 and a construction method and application thereof.
The first purpose of the invention is to provide a construction method of CRISPR/Cas9 vector suitable for erwinia bacterium FS110, which comprises the following steps:
a. performing PCR amplification by using the genomic DNA of the erd bacteria FS110 as a template and using primers 5SrRNA-promoter-F and 5SrRNA-promoter-R as primers to obtain a fragment 1 of a 5SrRNA promoter containing a BglII enzyme cutting site, wherein the nucleotide sequence of the fragment 1 is shown as SEQ ID NO. 1;
b. carrying out PCR amplification by taking an artificially synthesized sgRNA sequence and a Terminator sequence thereof as templates and taking a primer 5SrRNA-sgRNA-F, sgRNA-Terminator-R as a primer to obtain a fragment 2 containing a PacI enzyme cutting site, sgRNA and a Terminator thereof, wherein the nucleotide sequence of the fragment 2 is shown as SEQ ID NO. 2;
c. mixing the fragment 1 obtained in the step a and the fragment 2 obtained in the step b as a template, and carrying out fusion PCR to obtain a 5SrRNA promoter-targeting sequence-sgRNA-sgRNA terminator fragment;
d. and c, carrying out PacI and BglII double enzyme digestion on the pFC332 plasmid and the 5SrRNA promoter-targeting sequence-sgRNA-sgRNA terminator fragment obtained in the step c respectively, and connecting the enzyme-digested fragments by using T4 DNA ligase to obtain a CRISPR/Cas9 vector pFC332-sgRNA suitable for the Edwardsiella FS 110.
The invention also provides a CRISPR/Cas9 vector pFC332-sgRNA which is constructed by the construction method and is suitable for the Edwardsiella FS 110.
The invention also provides a fungus containing the CRISPR/Cas9 vector pFC332-sgRNA applicable to the Edwardsiella FS 110.
The invention also provides application of the CRISPR/Cas9 vector pFC332-sgRNA suitable for the Edwardsiella FS110 in fungal gene knock-out.
Preferably, the fungus is erdinia sp.fs 110 or erdinia sp.fs 140.
Preferably, the fungus is erdinia sp.fs 110.
Preferably, the application comprises the following steps:
a. searching a target site in a gene to be knocked out, correspondingly designing a target primer, annealing to form a double chain, and performing enzyme digestion to connect the double chain to a vector pFC332-sgRNA to obtain a target knocking out recombinant vector;
b. introducing the targeted knockout recombinant vector into an Edwardsiella FS110 protoplast by a protoplast-mediated method, screening by a hygromycin-resistant PDA plate, selecting a positive clone genome DNA to verify the introduction of the recombinant vector, and verifying the knockout of a target gene by Cruiser enzyme cutting and sequencing.
Preferably, the application is targeted knockout of gliotoxin biosynthesis gene GliG, and comprises the following steps:
designing targeted primers of gliG-F1 and gliG-R1 aiming at the gene gliG, and carrying out phosphorylation and annealing reaction by utilizing T4 PNK enzyme to form a gliG-F1/gliG-R1 double chain; the nucleotide sequence of the gliG-F1 is shown in SEQ ID NO.3, and the nucleotide sequence of the gliG-R1 is shown in SEQ ID NO. 4;
carrying out PCR amplification by using the constructed vector pFC332-sgRNA as a template and a primer BglII-F, PacI-R as a primer, carrying out BglII and PacI double enzyme digestion on the PCR product and a gliG-F1/gliG-R1 double strand, recovering enzyme digestion product glue, carrying out ligation and transformation by using T4Ligase, and constructing to obtain a G1-pFC332-sgRNA plasmid, namely the recombinant vector for targeted knockout of gliotoxin biosynthesis gene GliG.
Preferably, the application is targeted knockout of gliotoxin biosynthesis gene GliO, and comprises the following steps:
designing targeted primers of gliO-F1 and gliO-R1 aiming at gene gliO, carrying out phosphorylation by utilizing T4 PNK enzyme and carrying out annealing reaction to form a gliO-F1/gliO-R1 double chain; the nucleotide sequence of the gliO-F1 is shown in SEQ ID NO.5, and the nucleotide sequence of the gliO-R1 is shown in SEQ ID NO. 6;
carrying out PCR amplification by using the constructed vector pFC332-sgRNA as a template and a primer BglII-F, PacI-R as a primer, carrying out BglII and PacI double enzyme digestion on the PCR product and a gliO-F1/gliO-R1 double strand, recovering enzyme digestion product glue, carrying out ligation and transformation by using T4Ligase, and constructing to obtain an O1-pFC332-sgRNA plasmid, namely the recombinant vector for targeted knockout of gliotoxin biosynthesis gene GliO.
The CRISPR/Cas9 vector suitable for the deep-sea fungus Edwardsiella sp FS110 is constructed, the gliotoxin biosynthesis genes are knocked out, a molecular biological basis is laid for analyzing the biological mechanism of the FS110 gliotoxin in the later stage, and the development and utilization of gliotoxin compounds are promoted.
Compared with the prior art, the invention has the following beneficial effects:
at present, the gene knockout of the fungi is generally carried out by adopting a Cre/loxp homologous recombination method, but the defects of low homologous recombination efficiency, complex vector construction and the like exist, so that the gene knockout of the filamentous fungi is slow in progress, and the genetic operation modification and the discovery of novel secondary metabolites of the filamentous fungi are seriously hindered. The CRISPR/Cas9 gene knockout system has the advantages of simple vector construction, relatively high gene knockout efficiency and the like, and can effectively promote the genetic engineering transformation of filamentous fungi, thereby exploring more lead compounds with biological activity.
The Erythrophyte (Dichroomyes cejpii) FS110 of the present invention is disclosed in the literature: the molecular identification and anti-phytopathogen and cytotoxic activity research of 23 marine fungi in Yanumalan, Chenyu Chan, Lihaohua, Yidefender and the report of biotechnology 2014,8: 132-. The applicant also holds that the present invention is provided to the public within 20 years from the date of filing.
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FIG. 1 is a pFC332-sgRNA plasmid map and validation; wherein, the picture A is pFC332-sgRNA plasmid map, and the picture B is recombinant vector construction verification map.
FIG. 2 is a verification diagram of introduction of recombinant vector G1-pFC332-sgRNA or O1-pFC332-sgRNA into Edwardsiella FS 110; wherein A is a transformant on hygromycin-resistant PDA plates; b is recombinant vector introduction verification.
FIG. 3 shows the gene knockout verification of Edwardsiella FS110 gliotoxin biosynthesis.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1: construction of targeting gliotoxin biosynthesis gene knockout vector
On the basis of pFC332 plasmid, PacI and BglII sites are subjected to double digestion on pFC332, and a sequence is inserted: the 5SrRNA promoter-targeting sequence-sgRNA terminator (comprising the 5SrRNA promoter suitable for use in erwinia FS110, the sgRNA targeting sequence backbone gene that functions in yeast cells and its terminator) enables the plasmid to stably express Cas9 protein and transcribe sgRNA simultaneously after transformation into protoplasts (the plasmid is designated pFC 332-sgRNA).
Primers designed for constructing the vector are shown in table 1, and a pFC332-sgRNA vector is constructed by using an enzyme digestion connection mode. The construction method is specifically as follows:
TABLE 1 primer sequences
Figure BDA0002324587460000041
Performing PCR amplification by using Erysiphe FS110 genome DNA as a template and using primers 5SrRNA-promoter-F and 5SrRNA-promoter-R as front and rear primers to obtain a fragment 1 (the nucleotide sequence of which is shown as SEQ ID NO. 1) of a 5SrRNA promoter containing a BglII enzyme cutting site; carrying out PCR amplification by taking the artificially synthesized sgRNA and a Terminator sequence thereof as templates and taking a primer 5SrRNA-sgRNA-F, sgRNA-Terminator-R as a front primer and a rear primer to obtain a fragment 2 (the nucleotide sequence of which is shown in SEQ ID NO. 2) containing a PacI enzyme cutting site, the sgRNA and a Terminator thereof; using the fragments 1 and 2 prepared above as mixed templates, fusion PCR was performed using Prime STAR MAX (TAKARA, japan) high fidelity premix to obtain 5SrRNA promoter-targeting sequence-sgRNA terminator fragments. PacI and BglII double enzyme digestion is carried out on pFC332 plasmid, PacI and BglII double enzyme digestion is carried out on the obtained 5SrRNA promoter-targeting sequence-sgRNA-sgRNA-terminator fragment, the fragments after enzyme digestion are connected by T4 DNA ligase, so that a CRISPR/Cas9 vector suitable for Eriger FS110 is obtained and named as pFC332-sgRNA, and the vector map is shown in figure 1A.
And (3) knocking out a targeting site of the gliG and gliO functional genes, wherein the targeting site is positioned near the initiation codon and has a sequence of about 20 bp. According to gliotoxin biosynthesis genes GliG and GliO in the Edwardsiella FS110, a target site gliG1 and primers gliG-F1 and gliG-R1 corresponding to the GliG gene are selected and designed, and phosphorylation and annealing reaction are carried out by utilizing T4 PNK enzyme to form a gliG-F1/gliG-R1 double chain; selecting and designing a targeted site gliO1 and primers gliO-F1 and gliO-R1 corresponding to the targeted site gliO1 for the gliO gene, and carrying out phosphorylation and annealing reaction by using T4 PNK enzyme to form a gliO-F1/gliO-R1 double strand; the reaction conditions were as follows: 30min at 37 ℃ and 20min at 65 ℃; then 2.5. mu.L of 1M sodium chloride solution is added into the reaction system, the temperature is 95 ℃ for 5min, and then the temperature is slowly reduced to 4 ℃.
Performing PCR amplification by using the constructed recombinant vector pFC332-sgRNA as a template and a primer BglII-F, PacI-R as front and back primers, performing FD BglII enzyme and FD PacI enzyme double digestion on the PCR product and gliG-F1/gliG-R1 double strands, recovering enzyme digestion product glue, performing ligation and transformation by using T4Ligase, constructing a G1-pFC332-sgRNA plasmid, and performing sequencing verification.
Performing PCR amplification by using a constructed recombinant vector pFC332-sgRNA as a template and a primer BglII-F, PacI-R as front and rear primers, performing FD BglII enzyme and FD PacI enzyme double digestion on a PCR product and gliO-F1/gliO-R1 double strands, recovering enzyme digestion product glue, performing ligation and transformation by using T4Ligase, constructing an O1-pFC332-sgRNA plasmid, and performing sequencing verification.
Sequencing verification confirms that the pFC332-sgRNA vector, the G1-pFC332-sgRNA plasmid and the O1-pFC332-sgRNA plasmid are successfully constructed by the method respectively. The bands of PCR products for each fragment are shown in FIG. 1B (the verification primers used were gliG F1, gliG R1; gliO F1, and gliO R1, respectively).
Example 2
Knocking out biosynthesis genes of Edwardsiella FS110 gliotoxin:
the method for introducing the exogenous gene into the Edwardsiella FS110 protoplast comprises the following steps:
(1) the prepared erwinia amylovora FS110 protoplast (the specific preparation method refers to the patent number 201510540618.1 of the inventor and the patent named as the erwinia amylovora FS110 protoplast and the preparation method and the transformation method thereof) (1 x 108mL) and 2.5-5 mu g of G1-pFC332-sgRNA plasmid or O1-pFC332-sgRNA plasmid are respectively mixed evenly and placed on ice for 5min, then 200 mu L of PEG4000 with the volume fraction of 30% is added, the mixture is placed at 30 ℃ for 15min, then 400 mu L of PEG4000 is added, the mixture is placed at 30 ℃ for 15min, then 1.2mLW5 solution is added to terminate the reaction, and finally 4mL of WI buffer solution is added and placed on a shaker at 30 ℃ for overnight culture at low speed;
(2) cooling the melted TB3 solid culture medium to room temperature, taking 20mL each time, gently mixing with the overnight culture solution in the step (1), adding hygromycin with the final concentration of 200 mug/mL, uniformly coating the mixture, and culturing at 30 ℃ for 5 d;
(3) after a small mycelium grows out, picking a fungus colony to be transferred to a PDA culture medium containing hygromycin with the final concentration of 200 mug/mL, and then screening;
(4) and (3) preserving the positive clone hyphae growing on the hygromycin PDA plate in the step (3) (namely, picking part of hyphae to transfer to a new hygromycin PDA plate), then placing the rest fungus hyphae into a sterile EP tube, adding liquid nitrogen to fully grind, then immediately placing in a water bath kettle at 100 ℃ for 5min, then placing in liquid nitrogen for 1min, repeating the process for 3 times, finally adding 50 mu L of ultrapure water to dissolve, centrifuging at the maximum rotating speed for 5min, and taking the supernatant (namely, total genome DNA) to be placed at-20 ℃ for preservation. Designing hygromycin gene front and back primers hph-F and hph-R (see table 1) to amplify the hygromycin resistance gene sequence to verify whether the introduction is successful; on the basis of successful introduction, sequences of targeted sites of gliG and gliO are amplified by using designed pre-and-post targeted gene primers (gliG F1, gliG R1; gliO F1 and gliOR1), annealed and treated by Cruiser Enzyme (Gill Biotech Co., Ltd. of Jiangsu Jirui), and then subjected to agarose gel electrophoresis to verify the gene editing efficiency.
The specific method comprises the following steps:
① obtaining hybrid DNA, namely designing primers (gliG F1, gliG R1; gliO F1, gliOR1) near the target site to ensure that the band amplified by PCR is at 300-600bp) as much as possible, incubating for 3min at 98 ℃, then cooling to room temperature, and successfully hybridizing;
② verification of direct Enzyme digestion hybridization fragment of Cruiser Enzyme, taking 2-5 μ L of PCR product, 1 μ L of Cruiser Enzyme, 2 μ L of 5x Cruiser buffer, adding water to 10 μ L, incubating for 20min at 45 ℃, immediately reducing to 4 ℃, finally adding 2 μ L of 6 × stop buffer for agarose gel electrophoresis verification, observing agarose gel electrophoresis lane, if 2 bands appear, indicating successful knockout.
Verifying that the G1-pFC332-sgRNA plasmid or the O1-pFC332-sgRNA plasmid is successfully introduced into the Edwardsiella FS110 protoplast, extracting a genome as a template, and amplifying by using primers hph-F and hph-R to obtain 2 positive clones of the gliG knockout vector and 1 positive clone of the gliO gene knockout vector (figure 2).
The successfully introduced genome of G1-1, G1-3 and O1-2 bacteria is used as a template, upstream and downstream primers (gliG F1, gliG R1; gliO F1 and gliO R1) of the knock-out fragment are used for carrying out PCR, PCR product gel is recovered, the temperature is 95 ℃ for 4min, annealing is slowly carried out to 4 ℃, agarose gel electrophoresis verification is carried out, two bands (shown in figure 3) appear in the thermally denatured-annealed fragment, G1-1 and O1-2, and TA cloning is used for sequencing verification, so that the target gene is found to have base deletion. The successful knockout of the gliG gene in G1-2 and the successful knockout of the gliO gene in O1-4 were confirmed.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Sequence listing
<110> Guangdong province institute for microbiology (Guangdong province center for microbiological analysis and detection)
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Claims (8)

1. A construction method of CRISPR/Cas9 vector suitable for Erysian FS110 is characterized by comprising the following steps:
a. performing PCR amplification by using the genomic DNA of the erd bacteria FS110 as a template and using primers 5SrRNA-promoter-F and 5SrRNA-promoter-R as primers to obtain a fragment 1 of a 5SrRNA promoter containing a BglII enzyme cutting site, wherein the nucleotide sequence of the fragment 1 is shown as SEQ ID NO. 1;
b. carrying out PCR amplification by taking an artificially synthesized sgRNA sequence and a Terminator sequence thereof as templates and taking a primer 5SrRNA-sgRNA-F, sgRNA-Terminator-R as a primer to obtain a fragment 2 containing a PacI enzyme cutting site, sgRNA and a Terminator thereof, wherein the nucleotide sequence of the fragment 2 is shown as SEQ ID NO. 2;
c. mixing the fragment 1 obtained in the step a and the fragment 2 obtained in the step b as a template, and carrying out fusion PCR to obtain a 5SrRNA promoter-targeting sequence-sgRNA-sgRNA terminator fragment;
d. and c, carrying out PacI and BglII double enzyme digestion on the pFC332 plasmid and the 5SrRNA promoter-targeting sequence-sgRNA-sgRNA terminator fragment obtained in the step c respectively, and connecting the enzyme-digested fragments by using T4 DNA ligase to obtain a CRISPR/Cas9 vector pFC332-sgRNA suitable for the Edwardsiella FS 110.
2. The CRISPR/Cas9 vector pFC332-sgRNA applicable to the Edwardsiella FS110 and constructed according to the construction method of claim 1.
3. A fungus containing the CRISPR/Cas9 vector pFC332-sgRNA applicable to erworm FS110 of claim 2.
4. The use of the CRISPR/Cas9 vector pFC332-sgRNA for erwinia FS110 according to claim 2 for fungal gene knock-out.
5. The use according to claim 4, wherein the fungus is Edwardsiella FS110 or Edwardsiella FS 140.
6. Use according to claim 5, characterized in that it comprises the following steps:
a. searching a target site in a gene to be knocked out, correspondingly designing a target primer, annealing to form a double chain, and performing enzyme digestion to connect the double chain to a vector pFC332-sgRNA to obtain a target knocking out recombinant vector;
b. introducing the targeted knockout recombinant vector into an Edwardsiella FS110 protoplast by a protoplast-mediated method, screening by a hygromycin-resistant PDA plate, selecting a positive clone genome DNA to verify the introduction of the recombinant vector, and verifying the knockout of a target gene by Cruiser Enzyme cutting and sequencing.
7. The use according to claim 6, for targeted knock-out of gliotoxin biosynthesis gene GliG, comprising the following steps:
designing targeted primers of gliG-F1 and gliG-R1 aiming at the gene gliG, and carrying out phosphorylation and annealing reaction by utilizing T4 PNK enzyme to form a gliG-F1/gliG-R1 double chain; the nucleotide sequence of the gliG-F1 is shown in SEQ ID NO.3, and the nucleotide sequence of the gliG-R1 is shown in SEQ ID NO. 4;
carrying out PCR amplification by using the constructed vector pFC332-sgRNA as a template and a primer BglII-F, PacI-R as a primer, carrying out BglII and PacI double enzyme digestion on the PCR product and a gliG-F1/gliG-R1 double strand, recovering enzyme digestion product glue, carrying out ligation and transformation by using T4Ligase, and constructing to obtain a G1-pFC332-sgRNA plasmid, namely the recombinant vector for targeted knockout of gliotoxin biosynthesis gene GliG.
8. The use according to claim 6, for targeted knock-out of gliotoxin biosynthesis gene GliO, comprising the following steps:
designing targeted primers of gliO-F1 and gliO-R1 aiming at gene gliO, carrying out phosphorylation by utilizing T4 PNK enzyme and carrying out annealing reaction to form a gliO-F1/gliO-R1 double chain; the nucleotide sequence of the gliO-F1 is shown in SEQ ID NO.5, and the nucleotide sequence of the gliO-R1 is shown in SEQ ID NO. 6;
carrying out PCR amplification by taking the constructed vector pFC332-sgRNA as a template and a primer BglII-F, PacI-R as a primer, carrying out BglII and PacI double enzyme digestion on the PCR product and a gliO-F1/gliO-R1 double strand, recovering enzyme digestion product glue, carrying out ligation and transformation by utilizing T4Ligase, and constructing to obtain an O1-pFC332-sgRNA plasmid, namely the recombinant vector for targeted knockout of gliotoxin biosynthesis gene GliO.
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CN112553238B (en) * 2020-12-10 2022-06-07 广东省微生物研究所（广东省微生物分析检测中心） CRISPR/Cas9 vector applicable to coniothyrium minitans FS482 as well as construction method and application thereof
CN112608931A (en) * 2020-12-24 2021-04-06 广东省微生物研究所（广东省微生物分析检测中心） Deep-sea fungus FS140 anti-gliotoxin self-protection gene GliM and application thereof
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CN112852775B (en) * 2020-12-28 2022-03-22 广东省微生物研究所（广东省微生物分析检测中心） Novel acetyltransferase GliK of deep-sea fungi as well as coding gene and application thereof
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CN114717121A (en) * 2022-04-19 2022-07-08 中国农业科学院农产品加工研究所 Fusarium genome large fragment knockout method

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