WO2022166433A1 - Nat1基因作为筛选标记在卵菌遗传转化中的应用 - Google Patents
Nat1基因作为筛选标记在卵菌遗传转化中的应用 Download PDFInfo
- Publication number
- WO2022166433A1 WO2022166433A1 PCT/CN2021/139229 CN2021139229W WO2022166433A1 WO 2022166433 A1 WO2022166433 A1 WO 2022166433A1 CN 2021139229 W CN2021139229 W CN 2021139229W WO 2022166433 A1 WO2022166433 A1 WO 2022166433A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- gene
- screening
- oomycete
- transformants
- nat1
- Prior art date
Links
- 241000233654 Oomycetes Species 0.000 title claims abstract description 80
- 238000012216 screening Methods 0.000 title claims abstract description 64
- 239000003550 marker Substances 0.000 title claims abstract description 43
- 230000009466 transformation Effects 0.000 title claims abstract description 33
- 230000002068 genetic effect Effects 0.000 title claims abstract description 16
- 101100459439 Caenorhabditis elegans nac-2 gene Proteins 0.000 title 1
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 72
- 101150082943 NAT1 gene Proteins 0.000 claims abstract description 62
- NRAUADCLPJTGSF-ZPGVOIKOSA-N [(2r,3s,4r,5r,6r)-6-[[(3as,7r,7as)-7-hydroxy-4-oxo-1,3a,5,6,7,7a-hexahydroimidazo[4,5-c]pyridin-2-yl]amino]-5-[[(3s)-3,6-diaminohexanoyl]amino]-4-hydroxy-2-(hydroxymethyl)oxan-3-yl] carbamate Chemical compound NCCC[C@H](N)CC(=O)N[C@@H]1[C@@H](O)[C@H](OC(N)=O)[C@@H](CO)O[C@H]1\N=C/1N[C@H](C(=O)NC[C@H]2O)[C@@H]2N\1 NRAUADCLPJTGSF-ZPGVOIKOSA-N 0.000 claims abstract description 31
- 241000948155 Phytophthora sojae Species 0.000 claims abstract description 23
- 239000013598 vector Substances 0.000 claims description 37
- 210000001938 protoplast Anatomy 0.000 claims description 20
- 230000014509 gene expression Effects 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 16
- 230000000295 complement effect Effects 0.000 claims description 15
- 239000002773 nucleotide Substances 0.000 claims description 12
- 125000003729 nucleotide group Chemical group 0.000 claims description 12
- 239000007787 solid Substances 0.000 claims description 12
- 244000005700 microbiome Species 0.000 claims description 8
- 239000012620 biological material Substances 0.000 claims description 7
- 210000004027 cell Anatomy 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 7
- 230000009261 transgenic effect Effects 0.000 claims description 6
- 238000012546 transfer Methods 0.000 claims description 4
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 claims description 3
- 102000003960 Ligases Human genes 0.000 claims description 3
- 108090000364 Ligases Proteins 0.000 claims description 3
- 102000018120 Recombinases Human genes 0.000 claims description 3
- 108010091086 Recombinases Proteins 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 230000001404 mediated effect Effects 0.000 claims description 3
- 238000011161 development Methods 0.000 abstract description 5
- 238000003209 gene knockout Methods 0.000 abstract description 4
- 101100293261 Mus musculus Naa15 gene Proteins 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 9
- 229930195725 Mannitol Natural products 0.000 description 9
- 239000000594 mannitol Substances 0.000 description 9
- 235000010355 mannitol Nutrition 0.000 description 9
- 241000233614 Phytophthora Species 0.000 description 8
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 229920001223 polyethylene glycol Polymers 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000013518 transcription Methods 0.000 description 6
- 230000035897 transcription Effects 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 235000010582 Pisum sativum Nutrition 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000007739 pm medium Substances 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- 101100507655 Canis lupus familiaris HSPA1 gene Proteins 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- 241000233866 Fungi Species 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- 101710202061 N-acetyltransferase Proteins 0.000 description 3
- 241000233616 Phytophthora capsici Species 0.000 description 3
- 241000219843 Pisum Species 0.000 description 3
- 238000012271 agricultural production Methods 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 235000013601 eggs Nutrition 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 108091033409 CRISPR Proteins 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 102000008300 Mutant Proteins Human genes 0.000 description 2
- 108010021466 Mutant Proteins Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 241001443921 Phytophthora litchii Species 0.000 description 2
- 240000004713 Pisum sativum Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 108091036078 conserved sequence Proteins 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000004744 fabric Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 239000000417 fungicide Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 239000000575 pesticide Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 102100040623 60S ribosomal protein L41 Human genes 0.000 description 1
- 241000919511 Albugo Species 0.000 description 1
- 206010002961 Aplasia Diseases 0.000 description 1
- 102100038108 Arylamine N-acetyltransferase 1 Human genes 0.000 description 1
- 229920002498 Beta-glucan Polymers 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 101000674326 Homo sapiens 60S ribosomal protein L41 Proteins 0.000 description 1
- 101150062179 II gene Proteins 0.000 description 1
- 102000004317 Lyases Human genes 0.000 description 1
- 108090000856 Lyases Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010064998 N-acetyltransferase 1 Proteins 0.000 description 1
- 241000233679 Peronosporaceae Species 0.000 description 1
- 229920002565 Polyethylene Glycol 400 Polymers 0.000 description 1
- 241000233639 Pythium Species 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000010310 bacterial transformation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000010216 calcium carbonate Nutrition 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000000855 fungicidal effect Effects 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- JLFNLZLINWHATN-UHFFFAOYSA-N pentaethylene glycol Chemical compound OCCOCCOCCOCCOCCO JLFNLZLINWHATN-UHFFFAOYSA-N 0.000 description 1
- 244000000003 plant pathogen Species 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229960002920 sorbitol Drugs 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 235000015192 vegetable juice Nutrition 0.000 description 1
- 229910001868 water Inorganic materials 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
Definitions
- the invention relates to the technical field of genetic engineering, in particular to the application of Nat1 gene as a screening marker in the genetic transformation of oomycetes.
- Oomycetes is a kind of eukaryotic organism, and at least 1800 species of oomycetes have been found, most of which are pathogenic bacteria of plants, animals and other organisms, causing huge economic losses to human beings.
- Plant pathogenic oomycetes mainly include Downy mildews, Phytophthora, Pythium and Albugo. Compared with fungi, oomycetes have some unique physiological and biochemical characteristics, such as: the hyphae have no or few septa, the vegetative body is diploid, and the main component of the cell wall is cellulose.
- Phytophthora refers to a class of microorganisms belonging to the phylum Oomycota, most of which are plant pathogens. Diseases caused by Phytophthora cause devastating damage to many crops and plants and are called "plant blights". At present, the serious harm of oomycete disease to agricultural production has gradually attracted worldwide attention. The continuous release of oomycete genome sequence information has greatly promoted the genetic research of oomycetes, prompting researchers to develop new genetic manipulation tools to knock out and complement target genes in pathogenic oomycetes and identify their functions.
- patent CN108373497A discloses a fluthiazopyridine resistance gene and its application as oomycete transformation screening markers, which can cause egg
- the coding gene of the mutant protein that produces more than 500 times resistance to fluthiazopyridone is used as a screening marker for the screening of transformants during the transformation of Phytophthora sojae
- Patent CN108060173A discloses a carboxylic acid amide fungicide resistance gene as an egg
- the application of the bacterial transformation screening marker, the encoding gene of the mutant protein that can cause the oomycete to produce more than 100 times resistance to carboxylic acid amide fungicides is used as the screening marker for transformant screening during the transformation of Phytophthora capsici.
- the purpose of the present invention is to provide the application of Nat1 gene as a screening marker in the genetic transformation of oomycetes, which solves the problem that no new screening markers are available in the existing oomycete gene complementation experiments;
- the selection marker of oomycete has high screening efficiency for oomycete transformants.
- the main technical scheme adopted in the present invention includes:
- the present invention provides the application of the Nat1 gene as a selection marker in the genetic transformation of oomycetes, and the nucleotide sequence of the Nat1 gene is shown in SEQ ID NO: 1.
- the Nat1 gene in the present invention is a nourseothricin resistance gene, namely nourseothricin N-acetyltransferase gene 1, nourseothricin N-acetyl transferase 1, or Nat1 gene for short.
- the amino acid sequence of Nourseothricin N-acetyltransferase (NAT) is shown in SEQ ID NO: 2.
- the oomycete is Phytophthora sojae.
- the present invention uses Phytophthora sojae as a test strain to describe the application mode and application effect of Nat1 gene in the application of oomycete genetic transformation.
- Nat1 gene as a selection marker, it can also be applied to the genetic transformation of other oomycetes such as Phytophthora capsici, Peronophythoralitchii, etc., which all belong to the protection scope of the present invention.
- the screening is the screening of oomycete gene complement mutants of knockout transformants obtained by screening with other selectable marker genes, or for oomycete knockout mutants obtained by screening with other selectable marker genes Transformation screening for re-knockout of other genes in transformants.
- selectable marker genes may be the NPT II genes commonly used in the prior art, but other selectable marker genes developed with the development of research are not excluded.
- the new selection marker provided by the present invention makes up for the deficiency of the single selection marker gene in the prior art. But whether it is used alone as a selectable marker or used in combination with other selectable markers to conduct more complex genetic engineering experiments (multi-step knockout or complementation experiments, etc.), it all falls within the protection scope of the present invention.
- the "screening of oomycete gene aplasia mutants of knockout transformants” comprises the following steps:
- S3 utilize the method mediated by PEG to transfer the complementing vector into the oomycete protoplast to obtain a transformant
- the transformants are placed in a medium containing nourseothricin for culturing, and the complement transformants are obtained by screening.
- the method for preparing the oomycete with the target gene knocked out is the prior art, which is not limited here.
- the NPTII gene is used as the selection marker, and the transformants are screened with Geneticin G418, and the target gene of the oomycete is replaced with the exogenous gene NPTII, and the target gene is knocked out eggs. bacteria (ie, the transformants in which the gene of interest has been knocked out).
- step S1 the construction method of the complementing vector specifically includes:
- the pTOR vector linked to the Nat1 gene was linearized using ClaI and EcoRI endonucleases, and the target gene was linked to the pTOR vector using T4 ligase.
- the step S4 specifically includes:
- the transformants were placed in PM solid medium containing nourseothricin, and cultured in the dark at 25°C for 3 to 4 days for the first screening;
- V8 solid medium containing nourseothricin was covered on the PM solid medium with mycelial growth, and cultured at 25°C in the dark for 3 to 4 days for the second screening;
- the regenerated strains are transferred to the V8 solid medium containing nourseothricin, and the third screening is performed to obtain complementary transformants.
- the nourseothricin was in the PM solid medium.
- the concentration is preferably 30 to 50 ⁇ g/mL.
- the present invention provides the application of the biological material related to the Nat1 gene in the genetic transformation of oomycetes, wherein the biological material is any one of the following:
- the nucleotide sequence of the Nat1 gene is shown in SEQ ID NO: 1.
- the expression cassette refers to a DNA sequence capable of expressing the Nat1 gene in a host cell, and the DNA sequence may include not only a promoter for initiating the transcription of the Nat1 gene, but also a terminator for terminating the transcription of the Nat1 gene.
- the expression cassette may also include a target gene, and the target gene and the Nat1 gene use different promoters and terminators, respectively.
- the promoter that initiates transcription of Nat1 gene is Hsp70 promoter, and its nucleotide sequence is shown in SEQ ID NO: 3; the terminator that terminates Nat1 gene transcription is Hsp70 terminator, and its nucleotide sequence is shown in SEQ ID NO: 4 Show.
- the promoter that initiates transcription of the target gene is Ham34 promoter, and its nucleotide sequence is shown in SEQ ID NO: 5; the terminator that terminates the transcription of the target gene is Ham34 terminator, and its nucleotide sequence is shown in SEQ ID NO: 6.
- the promoter and terminator can also select commonly used promoters and terminators in oomycetes such as Ham34 promoter HJV, Ham34 terminator HJV, 5'P.sojae RPL41 promoter, 5'P.sojae RPL41terminator, etc. It can function in bacteria for gene expression.
- the recombinant vector may contain a DNA molecule encoding the Nat1 gene.
- a recombinant vector containing the Nat1 gene or the Nat1 gene expression cassette can be constructed by using an existing common molecular cloning vector or an oomycete expression vector, such as, but not limited to, pTOR vector and pYF515 vector.
- the recombinant microorganism can be yeast, bacteria and fungi.
- the oomycete is Phytophthora sojae.
- the invention provides a screening method for oomycete genetic transformation, which comprises the following steps:
- the nucleotide sequence of the Nat1 gene is shown in SEQ ID NO: 1;
- the oomycete is Phytophthora sojae.
- the present invention uses Phytophthora sojae as the test strain to describe the transformation and screening effects in the screening method for the genetic transformation of oomycetes.
- using the Nat1 gene as a selection marker can also be applied to the genetic transformation of other oomycetes such as Phytophthora capsici and Peronophythoralitchii, which all belong to the protection scope of the present invention.
- the present invention discovers for the first time an antibiotic, Nourseothricin (NTC), which can be used as a screening marker for oomycete transformation.
- NTC Nourseothricin
- the screening efficiency of transformants can reach more than 90%, which can greatly reduce the workload.
- the development of the selection marker solves the problem of a single selection marker during oomycete transformation, and provides a strong guarantee for the work of gene complementation and multiple gene knockout during oomycete transformation.
- the calculation method of screening efficiency is as follows: the transformants screened by the medium containing nourseothricin are "candidate transformants", and the “candidate transformants” are further extracted by DNA, RNA and other methods to verify the "transformants that successfully express the target gene”.
- the screening efficiency is the ratio of "transformants that successfully express the target gene” to "candidate transformants”.
- Figure 1 shows the colony growth of the Avh109 gene complementing transformants obtained by using the Nat1 gene as a selection marker; in the figure, from left to right, the first plate and the second plate are wild-type Phytophthora sojae (WT), The third plate and the fourth plate are Avh109 gene complement transformants;
- WT wild-type Phytophthora sojae
- Figure 2 shows the PCR verification results of the Avh109 gene complemented transformants obtained by using the Nat1 gene as a selection marker; in the figure, M: Marker, WT: wild-type strain, lanes 1 and 2: Avh109 gene complemented transformants; in the figure , PsACT is a conserved gene in Phytophthora sojae, as the experimental control group;
- Figure 3 shows the results of PCR verification of the expression of the Avh109 gene and the Nat1 gene in the Avh109 gene complement transformants obtained by using the Nat1 gene as a selection marker; in the figure, M: Marker, WT: wild-type strain, KO (knockout): Avh109 knockout strain, complemented lines: Avh109 complement transformants (15 candidate transformants).
- Phytophthora is far from fungi in classification. Phytophthora belongs to Oomycetes and Oomycetes. The main components of its cell wall are ⁇ -glucan and cellulose. Diploid, while fungi belong to the fungal phylum, the cell wall is mainly composed of chitin, and the nucleus is haploid. Therefore, most antibiotics available for fungal strain screening cannot be applied to oomycetes. Nourseothricin is an antibiotic that inhibits protein synthesis. It can effectively inhibit the growth of many prokaryotes and is also used to inhibit the growth of eukaryotes, but it has not been used in agricultural production.
- the inventors unexpectedly discovered that the Nat1 gene can be used as a screening marker to screen oomycete strains by using nourseothricin, and the screening method is simple, convenient and reproducible. invention.
- test materials used in the examples of the present invention are all conventional test materials in the art, and can be purchased through commercial channels. in:
- Enzyme lysis buffer Lsing enzyme 0.15g, Celluse 0.06g, 0.8M mannitol 10mL, ddH 2 O 8mL, 0.5M KCl 800 ⁇ L, 0.5M MES 800 ⁇ L, 0.5M CaCl 2 400 ⁇ L.
- PEG400 6g 40% PEG solution (prepared for current use): PEG400 6g, 0.8M mannitol 3.75mL, 0.5M CaCl2 3mL, H2O 3mL.
- W5 solution KCl 0.186g, CaCl 2 -H 2 O 9.2g, NaCl 4.5g, glucose 15.6g, ddH 2 O to 500mL.
- 0.8M mannitol mannitol 145.76g, ddH 2 O to make the volume to 1L.
- CaCl 2 -H 2 O CaCl 2 -H 2 O 14.7g, ddH 2 O to 200mL.
- KCl KCl 7.5g, ddH2O to 200mL.
- MMg solution 18.22 g of Mannitol, 0.76 g of MgCl 2 -6H 2 O, 2 mL of 0.5M MES, and ddH 2 O to make the volume to 250 mL.
- Nutrient pea culture medium put 60g of peas into a conical flask, add an appropriate amount of deionized water, sterilize at 121°C for 15min, take the filtrate, and add the following reagents in sequence: KH 2 PO 4 0.5g, K 2 HPO 4 ⁇ 2H 2 O 0.65g, KNO 3 1.5g, MgSO 4 0.25g, CaCl 2 0.04g, CaCO 3 1.0g, D-sorbitol 2.5g, D-glucose 2.5g, YeastExtract 1g, fully dissolved, centrifuged at 4000g for 10min, fixed Volume to 500mL, 121 °C, 15min sterilization.
- PM solid medium put 60g of peas into a conical flask, add an appropriate amount of deionized water, sterilize at 121°C for 15min, take the filtrate, add 45.5g of Mannitol, fully dissolve, centrifuge at 4000g for 10min, make up to 500mL, add 1.0% agar, mix well, sterilize at 121°C for 15min.
- V8 solid medium add 0.2% CaCO3 powder to V8 vegetable juice (purchased ) , dissolve fully, centrifuge at 5000rpm for 20min, take the supernatant, add 9 times the volume of the supernatant to dilute with deionized water, add 1.5%-2.0 % agar, mix well, sterilize at 121°C for 15min.
- Nat1-F 5'-ACACAAGGGCCCGTTTCGCATGGGTACCACTCTTGACG-3' (SEQ ID NO: 7);
- Nat1-R 5'-TTCGAACCCCAGAGTCCCGCTTAGGGGCAGGGCATGCT-3' (SEQ ID NO: 8).
- G Avh109-F 5'-CCATCGATATGCGTCTCCAGTATGCCG-3' (SEQ ID NO: 10);
- G Avh109-R 5'-GGAATTCATCAGCGGTTTGTCGCC-3' (SEQ ID NO: 11).
- the pTOR vector was linearized with linearized vector primers (pTOR-F and pTOR-R), the Nat1 gene was connected to the pTOR vector by homologous recombinase, and the construction status of the vector was detected by sequencing.
- the pTOR vector connected with the Nat1 gene was linearized with ClaI and EcoRI endonucleases, the Avh109 gene was connected with the pTOR vector with T4 ligase, and the construction status of the vector was detected by sequencing.
- PTOR-F 5'-GCGGGACTCTGGGGTTCG-3' (SEQ ID NO: 12);
- PTOR-R 5'-GCGAAACGGGGCCCTTGT-3' (SEQ ID NO: 13).
- the pTOR vector contains oomycete universal promoter and terminator, wherein Hsp70 promoter (SEQ ID NO: 3) and Hsp70 terminator (SEQ ID NO: 4) regulate the expression of Nat1 gene, Ham34promoter (SEQ ID NO: 5) and Ham34terminator (SEQ ID NO: 5) ID NO: 6) regulates Avh109 gene expression.
- Phytophthora sojae P6497 wild type strain was activated on Nutrient Pea Medium (NPB) plates and cultivated in the dark at 25°C (activated hyphae were used within one week). It should be noted that the wild-type strain of Phytophthora sojae P6497 is only the material used in the examples of the present invention. In fact, when applying the selection marker of the present invention, any commercially available oomycete strain can be used.
- the Avh109 gene knockout transformant was obtained, and the Avh109 gene was replaced by the exogenous gene NPT II.
- the preparation method is the existing common gene knockout technology, for example, you can refer to the article Yufeng, Fang, Linkai, et al. Efficient Genome Editing in the Oomycete Phytophthora sojae Using CRISPR/Cas9 [J]. Current Protocols in Microbiology, 2017. for preparation.
- the ultra-clean workbench was sterilized by ultraviolet light for 30 minutes, and the lyase and 40% PEG were weighed in a sterilized 50mL beaker (weighing operation was strictly standardized to reduce pollution).
- transforming plasmid that is, a complementing vector containing Nat1 gene and Avh109 gene.
- the 15 candidate transformants screened in Example 1 were transferred to V8 medium containing 50 ⁇ g/mL nourseothricin to observe whether they could grow again, with wild-type Phytophthora sojae (WT) as the control.
- WT wild-type Phytophthora sojae
- the screened transformants can grow normally on the V8 medium containing 50 ⁇ g/mL nourseothricin (only 2 candidate transformants are shown in Figure 1, and the remaining 13 candidate transformants are The growth conditions were the same); while the control strain could not grow on the V8 medium containing 50 ⁇ g/mL Nourseothricin, but could grow normally on the V8 medium without Nourseothricin.
- RNA extraction kits were used to extract the RNA of Avh109 knockout strain (knockout, KO), wild strain (WT) and 15 apoplectic transformants (candidate transformants) strains. After DNA was removed, cDNA was obtained by reverse transcription. The recorded cDNA was used as a template, and the expression of the target gene Avh109 was verified by RT-PCR. The results are shown in Figure 3. The results in Figure 3 show that there is no Avh109 gene expression in the knockout transformant (KO), while the wild strain (WT) and the 15 complemented transformants screened have successfully expressed the Avh109 gene. After three parallel tests, the screening efficiency can reach more than 90%.
- Example 1 shows that the positive transformants (candidate transformants) screened in Example 1 are the transformants whose Avh109 gene is complemented successfully (the transformants that successfully express the target gene), and through statistics, the Nat1 gene is used as Phytophthora sojae.
- Gene complement mutant selection marker by using nourseothricin for screening, the gene complement strain can be effectively screened, which makes up for the lack of selectable marker genes in the genetic transformation of oomycetes, and the screening efficiency can reach more than 90% .
- the calculation method of screening efficiency is as follows: the transformants screened by the medium containing nourseothricin are "candidate transformants", and the “candidate transformants” are further extracted by DNA, RNA and other methods to verify the "transformants that successfully express the target gene”.
- the screening efficiency is the ratio of "transformants successfully expressing the target gene” to "candidate transformants”.
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
一种Nat1基因作为筛选标记在卵菌遗传转化中的应用。通过将可引起卵菌对诺尔丝菌素抗性的编码基因Nat1作为筛选标记，用于大豆疫霉转化时转化子的筛选。该筛选标记的开发解决了卵菌转化时筛选标记单一的问题，为卵菌转化时基因回补、基因多敲除等方面的工作提供了有力保障。
Description
本发明涉及基因工程技术领域，具体涉及Nat1基因作为筛选标记在卵菌遗传转化中的应用。
卵菌(Oomycetes)是一种真核生物，目前已发现至少1800种卵菌，大部分卵菌是植物、动物以及其他生物的病原菌，给人类造成了巨大的经济损失。植物病原卵菌主要包括霜霉(Downy mildews)、疫霉(Phytophthora)、腐霉(Pythium)和白锈病(Albugo)。与真菌相比，卵菌具有一些独特的生理生化特征，例如：菌丝没有或有很少的隔膜，营养体为二倍体，细胞壁的主要成分是纤维素等。
疫霉菌(Phytophthora)是指隶属于卵菌门(Oomycota)下的一类微生物，多数是植物病原菌。疫霉菌引起的病害给许多农作物和植物造成毁灭性危害，称为“植物疫病”。目前，卵菌病害对农业生产的严重危害逐渐引起全世界的关注。不断释放的卵菌基因组序列信息，极大促进了卵菌的遗传研究，促使研究者开发新的遗传学操作工具，在病原卵菌中敲除和回补目的基因并鉴定其功能。
目前卵菌中可利用的筛选标记非常有限，唯一可靠的筛选标记是NPT II基因，其通过卵菌通用强启动子ham34启动编码的蛋白可使得卵菌对遗传霉素(G418)的抗性，从而使转化子被筛选出来。但是NPT II用于敲除体系时，因携带该标记的转化子具有G418抗性而不能被再次利用进行回补实验，导致对敲除转化子进行基因回补实验存在着没有新的筛选标记可用的问题。
对于新型卵菌转化筛选标记的开发，现有技术中有少量报道，例如：专利CN108373497A公开了一种氟噻唑吡乙酮抗性基因及其作为卵菌转化筛选 标记的应用，其将可引起卵菌对氟噻唑吡乙酮产生500倍以上抗性的突变蛋白的编码基因作为筛选标记，用于大豆疫霉菌转化时转化子的筛选；专利CN108060173A公开了羧酸酰胺类杀菌剂抗性基因作为卵菌转化筛选标记的应用，其将可引起卵菌对羧酸酰胺类杀菌剂产生100倍以上抗性的突变蛋白的编码基因作为筛选标记，用于辣椒疫霉菌转化时的转化子筛选。
虽然羧酸酰胺类化合物与氟噻唑吡乙酮都是防治卵菌病害的农药，但是，农业生产中大量使用该两种农药会造成菌株产生抗药性。因此，上述两种新的卵菌转化筛选标记在实际应该过程中可能会存在抗药性的问题。
因此，开发新型卵菌转化筛选标记，对于卵菌基因功能的研究具有重要意义。
发明内容
针对上述现有技术，本发明的目的是提供Nat1基因作为筛选标记在卵菌遗传转化中的应用，解决了现有卵菌基因回补实验中没有新的筛选标记可用的问题；而且，本发明的筛选标记对卵菌转化子具有较高的筛选效率。
为实现上述目的，本发明采用的主要技术方案包括：
第一方面，本发明提供了Nat1基因作为筛选标记在卵菌遗传转化中的应用，所述Nat1基因的核苷酸序列如SEQ ID NO：1所示。
本发明所述Nat1基因为诺尔丝菌素抗性基因，即诺尔丝菌素N-乙酰转移酶基因1，nourseothricin N-acetyl transferase 1，简称Nat1基因。诺尔丝菌素N-乙酰转移酶(NAT)的氨基酸序列如SEQ ID NO：2所示。
作为本发明进一步的实施方式，所述卵菌为大豆疫霉菌(Phytophthora sojae)。
需要说明的是，本发明以大豆疫霉菌(Phytophthora sojae)作为试验菌株对Nat1基因在卵菌遗传转化应用中的应用方式和应用效果进行说明。但以 Nat1基因作为筛选标记，也可以应用于辣椒疫霉(Phytophthora capsici)、荔枝霜疫霉(Peronophythoralitchii)等其他卵菌的遗传转化中，均属于本发明的保护范围。
作为本发明进一步的实施方式，所述筛选为以其他筛选标记基因筛选获得的敲除转化子的卵菌基因回补突变体的筛选，或用于以其他筛选标记基因筛选获得的卵菌敲除转化子中其他基因的再敲除的转化筛选。
需要说明的是，所述其他筛选标记基因可为现有技术中常用的NPT II基因，但并不排除随着研究发展而开发的其他筛选标记基因。本发明所提供的新的筛选标记弥补了现有技术中筛选标记基因单一的不足。但无论是其单独作为筛选标记，还是与其他筛选标记组合使用，以进行更为复杂的基因工程实验(多步敲除或回补实验等)，均属于本发明的保护范围。
作为本发明进一步的实施方式，所述“敲除转化子的卵菌基因回补突变体的筛选”包括以下步骤：
S1、构建包含Nat1基因和目的基因的回补载体；
S2、以目的基因被敲除的卵菌为实验材料，制备得到卵菌原生质体；
S3、利用PEG介导的方法向所述卵菌原生质体中转入所述回补载体，得到转化子；
S4、将所述转化子置于含有诺尔丝菌素的培养基中进行培养，筛选获得回补转化子。
需要说明的是，上述步骤中，目的基因被敲除的卵菌的制备方法为现有技术，在此不作限制。例如，在本发明具体实施方式中，是以NPTⅡ基因作为筛选标记，以遗传霉素G418进行转化子的筛选，将卵菌的目的基因替换为外源基因NPTⅡ，获得目的基因被敲除的卵菌(即目的基因被敲除的转化子)。
作为本发明进一步的实施方式，在步骤S1中，所述回补载体的构建方法具体包括：
将pTOR载体线性化，利用同源重组酶将Nat1基因连接于pTOR载体；
将连接Nat1基因的pTOR载体使用ClaI、EcoRI内切酶将载体线性化，利用T4连接酶将目的基因连接于pTOR载体。
作为本发明进一步的实施方式，所述步骤S4具体包括：
将所述转化子置于含有诺尔丝菌素的PM固体培养基，25℃黑暗培养3～4天，进行第一次筛选；
在有菌丝长出的PM固体培养基上覆盖一层含有诺尔丝菌素的V8固体培养基，于25℃黑暗中培养3～4天，进行第二次筛选；
待菌株长出后，将再生的菌株转接到含有诺尔丝菌素的V8固体培养基上，进行第三次筛选，获得回补转化子。
作为本发明进一步的实施方式，为保证转化子筛选效率及转化子的阳性率，在第一次、第二次和第三次筛选转化子时，所述诺尔丝菌素在PM固体培养基中的浓度优选为30～50μg/mL。
第二方面，本发明提供了与Nat1基因相关的生物材料在卵菌遗传转化中的应用，其特征在于，所述生物材料为以下任意一种：
A)含有Nat1基因的表达盒；
B)含有Nat1基因的重组载体、或含有A)所述表达盒的重组载体；
C)含有Nat1基因的重组微生物、或含有A)所述表达盒的重组微生物、或含有B)所述重组载体的重组微生物；
D)含有Nat1基因的转基因卵菌细胞系、或含有A)所述表达盒的转基因卵菌细胞系、或含有B)所述重组载体的转基因卵菌细胞系；
所述Nat1基因的核苷酸序列如SEQ ID NO：1所示。
上述生物材料中，A)所述表达盒是指能够在宿主细胞中表达Nat1基因的DNA序列，该DNA序列不但可包括启动Nat1基因转录的启动子，还可包括终止Nat1基因转录的终止子。所述表达盒还可包括目的基因，所述目的基因和所述Nat1基因分别用不同的启动子和终止子。
其中，启动Nat1基因转录的启动子为Hsp70 promoter，其核苷酸序列如 SEQ ID NO：3所示；终止Nat1基因转录的终止子为Hsp70 terminator，其核苷酸序列如SEQ ID NO：4所示。启动目的基因转录的启动子为Ham34 promoter，其核苷酸序列如SEQ ID NO：5所示；终止目的基因转录的终止子为Ham34 terminator，其核苷酸序列如SEQ ID NO：6所示。
当然，所述启动子和终止子还可以选用Ham34 promoter HJV、Ham34 terminator HJV，5'P.sojae RPL41 promoter，5'P.sojae RPL41terminator等卵菌中常用的启动子和终止子，只要能在卵菌中发挥功能用于基因表达即可。
上述生物材料中，B)所述重组载体可含有编码Nat1基因的DNA分子。可用现有的常见的分子克隆载体或卵菌表达载体构建含有Nat1基因或所述Nat1基因表达盒的重组载体，例如但不限于，pTOR载体、pYF515载体。
上述生物材料中，C)所述重组微生物可为酵母、细菌和真菌。
作为本发明进一步的实施方式，所述卵菌为大豆疫霉菌(Phytophthora sojae)。
第三方面，本发明提供了一种卵菌遗传转化的筛选方法，其包括以下步骤：
以Nat1基因作为筛选标记进行卵菌转化子的筛选；
所述Nat1基因的核苷酸序列如SEQ ID NO：1所示；
所述卵菌为大豆疫霉菌(Phytophthora sojae)。
需要说明的是，本发明以大豆疫霉菌(Phytophthora sojae)作为试验菌株对卵菌遗传转化的筛选方法中的转化及筛选效果进行说明。但以Nat1基因作为筛选标记也可以应用于辣椒疫霉(Phytophthora capsici)、荔枝霜疫霉(Peronophythoralitchii)等其他卵菌的遗传转化中，均属于本发明的保护范围。
与现有技术相比，本发明的有益效果如下：
本发明首次发现了一种可作为卵菌转化筛选标记的抗生素—诺尔丝菌素(Nourseothricin，NTC)，通过将可引起卵菌对诺尔丝菌素抗性的编码基因Nat1作为筛选标记，用于卵菌转化时转化子的筛选，转化子筛选效率可以达到90％ 以上，可极大的减少工作量。该筛选标记的开发解决了卵菌转化时筛选标记单一的问题，为卵菌转化时基因回补、基因多敲除等方面的工作提供了有力保障。
筛选效率的计算方式为：通过含有诺尔丝菌素的培养基筛选后的转化子为“候选转化子”，对“候选转化子”进一步提取DNA、RNA等方式验证确定“成功表达目的基因的转化子”，筛选效率是“成功表达目的基因的转化子”占“候选转化子”的比率。
上述发明内容相关记载是本申请技术方案的概述，为了让本领域普通技术人员能够更清楚地了解本申请的技术方案，进而可以依据说明书的文字及附图记载的内容予以实施，并且为了让本申请的上述目的及其它目的、特征和优点能够更易于理解，以下结合本申请的具体实施方式及附图进行说明。
附图仅用于示出本申请具体实施方式以及其他相关内容的原理、实现方式、应用、特点以及效果等，并不能认为是对本申请的限制。
在说明书附图中：
图1示出了以Nat1基因作为筛选标记获得Avh109基因回补转化子的菌落生长情况；图中，从左往右数，第一平板和第二平板为野生型大豆疫霉菌(WT)，第三平板和第四平板为Avh109基因回补转化子；
图2示出了以Nat1基因作为筛选标记获得Avh109基因回补转化子的PCR验证结果；图中，M：Marker，WT：野生型菌株，泳道1、2：Avh109基因回补转化子；图中，PsACT为大豆疫霉菌中的保守基因，作为试验对照组；
图3示出了以Nat1基因作为筛选标记获得的Avh109基因回补转化子中Avh109基因及Nat1基因表达情况的PCR验证结果；图中，M：Marker，WT：野生型菌株，KO(knockout)：Avh109基因敲除菌株，complemented lines： Avh109基因回补转化子(15个候补转化子)。
应该指出，以下详细说明都是例示性的，旨在对本申请提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义；本文中对相关术语的使用只是为了描述具体的实施例，而不是旨在限制本申请。
疫霉菌在分类上与真菌相去甚远，疫霉菌属于卵菌门、卵菌纲，其细胞壁主要成分为β-葡聚糖和纤维素，菌丝是无隔多核的菌丝体，且细胞核为二倍体，而真菌属于真菌门，细胞壁成分主要为几丁质，细胞核为单倍体。因此，大多数可用于真菌菌株筛选的抗生素都不能够应用于卵菌。诺尔丝菌素是一种抑制蛋白质合成的抗生素，能广泛地有效地抑制许多原核生物生长，并也被用于抑制真核生物的生长，但目前还未应用于农业生产。
在实现本发明的过程中，发明人意外的发现，将Nat1基因作为筛选标记，利用诺尔丝菌素能够进行卵菌菌株的筛选，并且筛选方法简单方便、可重复性好，由此提出了本发明。
为了使得本领域技术人员能够更加清楚地了解本申请的技术方案，以下结合具体的实施例详细说明本申请的技术方案。
除非另有说明，本发明实施例中所用的试验材料均为本领域常规的试验材料，均可通过商业渠道购买得到。其中：
酶解缓冲液：Lsing enzyme 0.15g、Celluse 0.06g、0.8M mannitol 10mL、ddH
2O8mL、0.5M KCl 800μL、0.5M MES 800μL、0.5M CaCl
2 400μL。
40％PEG溶液(现用现配)：PEG400 6g、0.8M mannitol 3.75mL、0.5M CaCl
2 3mL、H
2O 3mL。
W5 solution：KCl 0.186g、CaCl
2-H
2O 9.2g、NaCl 4.5g、glucose 15.6g，ddH
2O定容至500mL。
0.8M mannitol：mannitol 145.76g，ddH
2O定容至1L。
0.5M CaCl
2-H
2O：CaCl
2-H
2O 14.7g，ddH
2O定容至200mL。
0.5M KCl：KCl 7.5g，ddH
2O定容至200mL。
MMg solution：Mannitol 18.22g、MgCl
2-6H
2O 0.76g、0.5M MES 2mL，ddH
2O定容至250mL。
营养豌豆培养基(NPB)：取60g豌豆放入锥形瓶中，加入适量去离子水，121℃，15min灭菌，取滤液，依次加入以下试剂：KH
2PO
4 0.5g，K
2HPO
4·2H
2O 0.65g，KNO
3 1.5g，MgSO
4 0.25g，CaCl
2 0.04g，CaCO
3 1.0g，D-sorbitol 2.5g，D-glucose 2.5g，YeastExtract 1g，充分溶解，4000g离心10min，定容到500mL，121℃，15min灭菌。
PM固体培养基：取60g豌豆放入锥形瓶中，加入适量去离子水，121℃，15min灭菌，取滤液，加入45.5g Mannitol，充分溶解，4000g离心10min，定容到500mL，加入加1.0％的琼脂，混合均匀，121℃，15min灭菌。
V8固体培养基：向V8蔬菜汁(购买)中加入0.2％的CaCO
3粉末，充分溶解，5000rpm离心20min，取上清液，加上清液9倍体积去离子水稀释，加入1.5％-2.0％的琼脂，混合均匀，121℃，15min灭菌。
实施例1 筛选标记Nat1基因在大豆疫霉基因回补中的应用
1、克隆Nat1基因与目的基因(Avh109基因)片段：
利用下述引物Nat1-F和Nat1-R克隆Nat1基因片段，克隆得到的Nat1基因片段的核苷酸序列如SEQ ID NO：1所示。
Nat1-F:5’-ACACAAGGGCCCCGTTTCGCATGGGTACCACTCTTGACG-3’(SEQ ID NO：7)；
Nat1-R：5’-TTCGAACCCCAGAGTCCCGCTTAGGGGCAGGGCATGCT-3’(SEQ ID NO：8)。
从NCBI数据库(http://www.ncbi.nlm.nih.gov/)中搜索PsAvh109基因，下载其CDS以及基因组序列，设计引物G Avh109-F和G Avh109-R，克隆得到Avh109基因片段，Avh109基因的核苷酸序列如SEQ ID NO：9所示。
G Avh109-F：5’-CCATCGATATGCGTCTCCAGTATGCCG-3’(SEQ ID NO：10)；
G Avh109-R：5’-GGAATTCATCAGCGGTTTGTCGCC-3’(SEQ ID NO：11)。
2、将目的基因(Avh109基因)与Nat1基因构建于回补载体pTOR：
用线性化载体引物(pTOR-F和pTOR-R)将pTOR载体线性化，利用同源重组酶将Nat1基因连接于pTOR载体，测序检测载体构建状况。将连接Nat1基因后的pTOR载体使用ClaI、EcoRI内切酶将载体线性化，利用T4连接酶将Avh109基因连接于pTOR载体，测序检测载体构建状况。
PTOR-F：5’-GCGGGACTCTGGGGTTCG-3’(SEQ ID NO：12)；
PTOR-R：5’-GCGAAACGGGGCCCTTGT-3’(SEQ ID NO：13)。
pTOR载体中含有卵菌通用启动子和终止子，其中Hsp70 promoter(SEQ ID NO：3)与Hsp70 terminator(SEQ ID NO：4)调控Nat1基因表达，Ham34promoter(SEQ ID NO：5)与Ham34terminator(SEQ ID NO：6)调控Avh109基因表达。
3、用PEG介导的原生质体转化系统将回补载体转化到疫霉菌原生质体，得转化子：
(1)实验材料准备：
在营养豌豆培养基(NPB)平板上活化大豆疫霉菌P6497(野生型菌株)， 在25℃下黑暗培养(活化菌丝在一周内使用)。需要说明的是，大豆疫霉野生型菌株P6497仅为本发明在实施例所使用的材料，事实上，在应用本发明所述筛选标记时，可使用任何商购途径获得的卵菌菌株。
培养3-4天后，将其切成3*3mm的菌丝块，向每个装有50mL营养豌豆培养基的250mL三角瓶中放入6个菌丝块，共培养3瓶(以保证实验过程中菌丝材料充足)，在25℃黑暗静置培养2.5d-3d。培养期间每天摇晃一次。
以上述培养得到的野生型大豆疫霉菌P6497菌丝为亲本获得Avh109基因敲除转化子，其Avh109基因被外源基因NPT II所取代。制备方法为现有的常用基因敲除技术，例如可以参考文章Yufeng,Fang,Linkai,et al.Efficient Genome Editing in the OomycetePhytophthora sojaeUsing CRISPR/Cas9[J].Current Protocols in Microbiology,2017.进行制备。
(2)原生质体制备：
①超净工作台紫外灭菌30min，用灭菌的50mL烧杯称量裂解酶和40％PEG(称量操作严格规范、减少污染)。
②用事先包扎有纱布的200mL烧杯(灭菌后)来收集菌丝(即Avh109基因敲除转化子)，将菌丝放入50mL离心管中，用镊子将菌丝放入含有40mL 0.8M Mannitol的50mL离心管漂洗一次，再用包扎有纱布的200mL烧杯收集菌丝，用镊子将菌丝移入35mL 0.8M Mannitol缓冲液的50mL离心管中，室温下混匀60rpm摇洗10min(准备封口膜)。
③配制酶解缓冲液，在灭菌的50mL烧杯中加入指定体积的各组分，用枪头搅拌充分溶解，用细菌过滤器将酶解缓冲液过滤于50mL离心管中。
④菌丝在0.8M Mannitol中漂洗，收集菌丝置于酶解缓冲液中(加入菌丝不可过多)，25℃60rpm进行酶解反应，此过程1-1.5h。
⑤配置40％PEG溶液，加入指定体积的各组分，充分溶解后用细菌过滤器过滤后冰上备用。
⑥启动低温离心机，500rpm离心3min预冷4℃。
⑦酶解结束后，用2层mira-cloth(进口的过滤布)过滤菌丝收集原生质体，显微镜观察原生质体裂解情况，将收集好的原生质体转移到50mL离心管中，4℃1500rpm离心3min。(之后的操作原生质体要始终保持低温)
⑧弃上清，加入10mL左右的W5solution轻轻重悬原生质体，加W5 solution至35mL，4℃、1500rpm离心4min，镜检原生质体质量。
⑨弃上清，加10mL左右W5solution轻轻重悬原生质体，使用血球计数板计算原生质体浓度，然后于冰上放置30min，4℃1500rpm离心4min。
⑩弃上清，加入预冷的MMg solution重悬原生质体，调整原生质体浓度为2×10
6/mL，室温放置10min。
(3)原生质体转化：
①取50mL离心管置于冰上，加入30μg转化质粒(即包含Nat1基因和Avh109基因的回补载体)。
②向离心管中加入1mL原生质体，轻轻混匀，冰上放置5-10min。
③向离心管中分3次加入1.74mL PEG溶液，轻轻混匀，冰上放置20min。
④向预冷的PM培养基中加入氨苄抗生素备用。
⑤向离心管中加入2mL步骤④中的加入氨苄抗生素后的PM培养基，轻轻混匀，冰上放置2min。
⑥向离心管中加入8mL步骤④中的加入氨苄抗生素后的PM培养基，轻轻混匀，冰上放置2min。
⑦25℃静置培养12-14h(要斜放，增大与氧气的接触面积)，得到培养物(即转化子)。
(4)对敲除转化子的疫霉菌基因回补突变体进行筛选：
①取培养物5μL于显微镜下观察原生质体再生情况，2000rpm离心5min，沉淀再生菌丝。
②弃上清，向离心管中加入5mL PM培养基，将再生菌丝重新悬浮，加入35mL含有30μg/mL诺尔丝菌素的PM培养基，混匀后倒入培养皿中，吹 干水汽，25℃黑暗培养3-4d，进行第一次筛选。
③观察培养基表面菌丝生长状况，用10mL含50μg/mL诺尔丝菌素的V8培养基覆盖并吹干水汽，于25℃黑暗中培养3-4天，进行第二次筛选。
④观察培养基表面菌丝生长状况，将再生的菌株转接到含50μg/mL诺尔丝菌素的V8培养基上，进行第三次筛选。经过三次诺尔丝菌素筛选后获得15个转化子，初步认为是回补转化子(候选转化子)，用于后续鉴定。
实施例2 疫霉菌基因回补突变体的验证：
(1)用含有诺尔丝菌素的培养基验证转化子：
把实施例1中筛选到的15个候选转化子转接到含有50μg/mL诺尔丝菌素的V8培养基上再次观察是否能够生长，以野生型大豆疫霉菌(WT)作为对照。如图1所示，筛选得到的转化子能够在含有50μg/mL诺尔丝菌素的V8培养基上正常生长(图1中仅示出了其中2个候选转化子，其余13个候选转化子的生长情况与此相同)；而对照菌株在含有50μg/mL诺尔丝菌素的V8培养基上不能够生长，在不含诺尔丝菌素的V8培养基上则可以正常生长。
(2)提取转化子的DNA用于PCR验证：
使用CTAB法提取野生型菌株及回补转化子(候选转化子)菌株的基因组，PCR扩增Nat1基因，并以野生型大豆疫霉菌(WT)作为对照，结果如图2所示。由图2可知，野生型菌株中不含有Nat1基因，而筛选得到的回补转化子基因组中含有Nat1基因(图2中，PsACT是大豆疫霉菌中的保守基因，在此作为试验对照组，目的是证明基因组提取没有问题)。
(3)提取转化子的RNA，检测基因表达情况：
使用RNA提取试剂盒提取Avh109基因敲除菌株(knockout，KO)、野 生菌株(WT)及15个回补转化子(候选转化子)菌株的RNA，去除DNA后反转录得到cDNA，以反转录得到的cDNA为模板，RT-PCR验证目的基因Avh109的表达情况，结果如图3所示。图3中结果显示，敲除转化子(KO)中没有Avh109基因表达，而野生菌株(WT)以及筛选得到的15个回补转化子(complemented lines)中均成功表达Avh109基因。经过3次平行试验检测后计算，筛选效率可达90％以上。
上述结果表明，实施例1中筛选得到的阳性转化子(候选转化子)均为Avh109基因回补成功的转化子(成功表达目的基因的转化子)，并且经统计，以Nat1基因作为大豆疫霉菌的基因回补突变体筛选标记，通过使用诺尔丝菌素进行筛选，可有效筛选到基因回补菌株，弥补了卵菌遗传转化中缺少筛选性标记基因的问题，并且筛选效率可以达到90％以上。
筛选效率的计算方式为：通过含有诺尔丝菌素的培养基筛选后的转化子为“候选转化子”，对“候选转化子”进一步提取DNA、RNA等方式验证确定“成功表达目的基因的转化子”，筛选效率是“成功表达目的基因的转化子”占“候选转化子”的比率。
需要说明的是，以上所述仅为本申请的优选实施例而已，并不用于限制本申请，其中未尽详细描述的技术参数在本发明列举的参数范围内变化时，仍能够得到与上述实施例相同或相近的技术效果，仍属与本发明的保护范围。对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。
Claims (10)
- Nat1基因作为筛选标记在卵菌遗传转化中的应用，其特征在于，所述Nat1基因的核苷酸序列如SEQ ID NO：1所示。
- 根据权利要求1所述的应用，其特征在于，所述卵菌为大豆疫霉菌(Phytophthora sojae)。
- 根据权利要求1所述的应用，其特征在于，所述筛选为以其他筛选标记基因筛选获得的敲除转化子的卵菌基因回补突变体的筛选，或用于以其他筛选标记基因筛选获得的卵菌敲除转化子中其他基因的再敲除的转化筛选。
- 根据权利要求3所述的应用，其特征在于，所述“敲除转化子的卵菌基因回补突变体的筛选”包括以下步骤：S1、构建包含Nat1基因和目的基因的回补载体；S2、以目的基因被敲除的卵菌为实验材料，制备得到卵菌原生质体；S3、利用PEG介导的方法向所述卵菌原生质体中转入所述回补载体，得到转化子；S4、将所述转化子置于含有诺尔丝菌素的培养基中进行培养，筛选获得回补转化子。
- 根据权利要求4所述的应用，其特征在于，在步骤S1中，所述回补载体的构建方法具体包括：将pTOR载体线性化，利用同源重组酶将Nat1基因连接于pTOR载体；将连接Nat1基因的pTOR载体使用ClaI、EcoRI内切酶将载体线性化，利用T4连接酶将目的基因连接于pTOR载体。
- 根据权利要求4所述的应用，其特征在于，所述步骤S4具体包括：将所述转化子置于含有诺尔丝菌素的PM固体培养基，25℃黑暗培养3～4天，进行第一次筛选；在有菌丝长出的PM固体培养基上覆盖一层含有诺尔丝菌素的V8固体培养基，于25℃黑暗中培养3～4天，进行第二次筛选；待菌株长出后，将再生的菌株转接到含有诺尔丝菌素的V8固体培养基上，进行第三次筛选，获得回补转化子。
- 根据权利要求6所述的应用，其特征在于，在筛选转化子时，所述诺尔丝菌素在PM固体培养基中的浓度为30～50μg/mL。
- 与Nat1基因相关的生物材料在卵菌遗传转化中的应用，其特征在于，所述生物材料为以下任意一种：A)含有Nat1基因的表达盒；B)含有Nat1基因的重组载体、或含有A)所述表达盒的重组载体；C)含有Nat1基因的重组微生物、或含有A)所述表达盒的重组微生物、或含有B)所述重组载体的重组微生物；D)含有Nat1基因的转基因卵菌细胞系、或含有A)所述表达盒的转基因卵菌细胞系、或含有B)所述重组载体的转基因卵菌细胞系；所述Nat1基因的核苷酸序列如SEQ ID NO：1所示。
- 根据权利要求8所述的应用，其特征在于，所述卵菌为大豆疫霉菌(Phytophthora sojae)。
- 一种卵菌遗传转化的筛选方法，其特征在于，包括以下步骤：以Nat1基因作为筛选标记进行卵菌转化子的筛选；所述Nat1基因的核苷酸序列如SEQ ID NO：1所示；所述卵菌为大豆疫霉菌(Phytophthora sojae)。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US17/675,083 US20220170029A1 (en) | 2021-02-03 | 2022-02-18 | Application of nat1 gene as screening marker in genetic transformation of oomycete |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110147090.7 | 2021-02-03 | ||
| CN202110147090.7A CN112813091A (zh) | 2021-02-03 | 2021-02-03 | Nat1基因作为筛选标记在卵菌遗传转化中的应用 |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| US17/675,083 Continuation US20220170029A1 (en) | 2021-02-03 | 2022-02-18 | Application of nat1 gene as screening marker in genetic transformation of oomycete |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2022166433A1 true WO2022166433A1 (zh) | 2022-08-11 |
Family
ID=75860756
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2021/139229 WO2022166433A1 (zh) | 2021-02-03 | 2021-12-17 | Nat1基因作为筛选标记在卵菌遗传转化中的应用 |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN112813091A (zh) |
| WO (1) | WO2022166433A1 (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112813091A (zh) * | 2021-02-03 | 2021-05-18 | 山东农业大学 | Nat1基因作为筛选标记在卵菌遗传转化中的应用 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010031724A1 (en) * | 2000-02-18 | 2001-10-18 | Terry Roemer | Dominant selectable marker for gene transformation and disruption in yeasts |
| US20030167501A1 (en) * | 2001-02-23 | 2003-09-04 | Kloti Andreas S. | Novel selectable marker in plants and other organisms |
| US20160244770A1 (en) * | 2015-02-19 | 2016-08-25 | Synthetic Genomics, Inc. | High Efficiency Method for Algal Transformation |
| US20160272980A1 (en) * | 2012-11-16 | 2016-09-22 | Total Marketing Services | Method for targeted modification of algae genomes |
| CN108373497A (zh) * | 2018-03-14 | 2018-08-07 | 西北农林科技大学 | 一种用于大豆疫霉遗传转化的氟噻唑吡乙酮抗性筛选标记 |
| CN112813091A (zh) * | 2021-02-03 | 2021-05-18 | 山东农业大学 | Nat1基因作为筛选标记在卵菌遗传转化中的应用 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100783287B1 (ko) * | 2006-08-09 | 2007-12-06 | 한국생명공학연구원 | 텔로미어 전사 비활성화에 관여하는 화합물의 동정을 위한균주의 제조 |
| US9879270B2 (en) * | 2014-08-15 | 2018-01-30 | Wisconsin Alumni Research Foundation | Constructs and methods for genome editing and genetic engineering of fungi and protists |
| KR20180084135A (ko) * | 2015-12-02 | 2018-07-24 | 바스프 에스이 | 감소된 clr2 활성을 갖는 사상 진균에서 단백질을 생산하는 방법 |
-
2021
- 2021-02-03 CN CN202110147090.7A patent/CN112813091A/zh active Pending
- 2021-12-17 WO PCT/CN2021/139229 patent/WO2022166433A1/zh active Application Filing
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010031724A1 (en) * | 2000-02-18 | 2001-10-18 | Terry Roemer | Dominant selectable marker for gene transformation and disruption in yeasts |
| US20030167501A1 (en) * | 2001-02-23 | 2003-09-04 | Kloti Andreas S. | Novel selectable marker in plants and other organisms |
| US20160272980A1 (en) * | 2012-11-16 | 2016-09-22 | Total Marketing Services | Method for targeted modification of algae genomes |
| US20160244770A1 (en) * | 2015-02-19 | 2016-08-25 | Synthetic Genomics, Inc. | High Efficiency Method for Algal Transformation |
| CN108373497A (zh) * | 2018-03-14 | 2018-08-07 | 西北农林科技大学 | 一种用于大豆疫霉遗传转化的氟噻唑吡乙酮抗性筛选标记 |
| CN112813091A (zh) * | 2021-02-03 | 2021-05-18 | 山东农业大学 | Nat1基因作为筛选标记在卵菌遗传转化中的应用 |
Non-Patent Citations (1)
| Title |
|---|
| SOYER JESSICA L., ET AL: "Epigenetic Control of Effector Gene Expression in the Plant Pathogenic Fungus Leptosphaeria maculans", PLOS GENETICS, vol. 10, no. 3, 6 March 2014 (2014-03-06), pages e1004227, XP055957279, DOI: 10.1371/journal.pgen.1004227 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112813091A (zh) | 2021-05-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Rasheed et al. | A critical review: recent advancements in the use of CRISPR/Cas9 technology to enhance crops and alleviate global food crises | |
| Chen et al. | Efficient CRISPR-Cas9 gene disruption system in edible-medicinal mushroom Cordyceps militaris | |
| Chen et al. | CRISPR/Cas9-mediated efficient genome editing via blastospore-based transformation in entomopathogenic fungus Beauveria bassiana | |
| CN110268063A (zh) | 利用自动化遗传操作和菌株纯化步骤建立真菌生产菌株的方法 | |
| WO2022166433A1 (zh) | Nat1基因作为筛选标记在卵菌遗传转化中的应用 | |
| Yong et al. | MAT1-1-3, a mating type gene in the Villosiclava virens, is required for fruiting bodies and sclerotia formation, asexual development and pathogenicity | |
| Wang et al. | Protocol of Phytophthora capsici transformation using the CRISPR-Cas9 system | |
| CN104357475B (zh) | 一株金龟子绿僵菌转基因菌株及其制备方法与应用 | |
| Lu et al. | Genetic dissection of T-DNA insertional mutants reveals uncoupling of dikaryotic filamentation and virulence in sugarcane smut fungus | |
| CN104744577B (zh) | 球孢白僵菌抗菌蛋白Bbafp及其表达载体、含有其基因的转基因植物的制备方法和应用 | |
| US11236362B2 (en) | Method for direct transformation of exogenous DNA into resting spores of Penicillium amagasakiense | |
| Zhang et al. | Generation of stable transgenic rice (Oryza sativa L.) by Agrobacterium‐mediated transformation | |
| Nalam et al. | Establishment of a Fusarium graminearum infection model in Arabidopsis thaliana leaves and floral tissues | |
| US10407684B2 (en) | Method for direct transformation of exogenous DNA into resting spores of Aspergillus niger independent of mediators | |
| CN115925838A (zh) | 辣椒疫霉stt3b蛋白及其编码基因与应用 | |
| CN111118039B (zh) | 与致病力相关的灰霉菌基因Bcmet3和Bcmet16及应用 | |
| CN109868282B (zh) | 一种与致病力相关的灰霉病菌基因BcEXO70及应用 | |
| CN110055263B (zh) | 编码Bt杀虫蛋白的基因Cry1Ab-MR、其表达载体及其应用 | |
| US20220170029A1 (en) | Application of nat1 gene as screening marker in genetic transformation of oomycete | |
| Xiao et al. | Generation of yeast protoplasts by lytic actions of iron oxide magnetic nanoparticles | |
| JP5995232B2 (ja) | 麹菌細胞株の効率的融合方法 | |
| Connolly et al. | Application of the Cre/lox system to construct auxotrophic markers for quantitative genetic analyses in Fusarium graminearum | |
| CN115010795B (zh) | 辣椒疫霉调控胞外囊泡分泌关键蛋白及其编码基因与应用 | |
| CN117088948B (zh) | 辣椒疫霉调控游动孢子发育相关蛋白及其编码基因与应用 | |
| Minz et al. | Electroporation and Agrobacterium-mediated spore transformation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21924426 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 21924426 Country of ref document: EP Kind code of ref document: A1 |