CN106282218A - A kind of method of nif gene nifDK gene knockout - Google Patents

A kind of method of nif gene nifDK gene knockout Download PDF

Info

Publication number
CN106282218A
CN106282218A CN201610835340.5A CN201610835340A CN106282218A CN 106282218 A CN106282218 A CN 106282218A CN 201610835340 A CN201610835340 A CN 201610835340A CN 106282218 A CN106282218 A CN 106282218A
Authority
CN
China
Prior art keywords
nifdk
gene
pcr
fragment
amplification
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201610835340.5A
Other languages
Chinese (zh)
Inventor
甄涛
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Microbiology of Heilongjiang Academy of Sciences
Original Assignee
Institute of Microbiology of Heilongjiang Academy of Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Microbiology of Heilongjiang Academy of Sciences filed Critical Institute of Microbiology of Heilongjiang Academy of Sciences
Priority to CN201610835340.5A priority Critical patent/CN106282218A/en
Publication of CN106282218A publication Critical patent/CN106282218A/en
Pending legal-status Critical Current

Links

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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

A kind of method that the invention discloses nif gene nifDK gene knockout, its step is as follows: one, the nifDK gene upstream and downstream fragment in PCR amplification Slow-growing Soybean rhizobia, respectively nifDK AB and nifDK CD；Two, carrying out fusion DNA vaccine with the PCR primer of nifDK AB and nifDK CD for template, PCR expands nifDK AD；Three, nifDK AD is connected with carrier pK18mobsacB, digestion verification, nifDK AD is proceeded in E.coli DH5 α；Four, by donor bacterium nifDK AD, auxiliary bacterium pRK2013, recipient bacterium triparental mating；Five, recon is screened for the first time；Six, programmed screening recon；Seven, PCR checking.The present invention uses the method for fusion DNA vaccine, will merge fragment and be connected with plasmid pK18mobsacB, and pass through KmRResistance screening and sacB compel to bring it about double crossing at the screening pressure of 10% sucrose, after 2 homologous recombination, use PCR method screening mutant, improve screening success rate.

Description

A kind of method of nif gene nifDK gene knockout
Technical field
The invention belongs to biological technical field, a kind of method relating to nif gene nifDK gene knockout.
Background technology
In the experimentation of gene knockout, generally use fusion DNA vaccine technology to be connected laggard row filter with r plasmid, adopt Screening efficiency can be caused low with single r plasmid screening, be difficult to find recon, final the failure of an experiment.
Summary of the invention
In order to solve to build the technical problem of Slow-growing Soybean rhizobia mutant, the invention provides a kind of nif gene The method of nifDK gene knockout.
It is an object of the invention to be achieved through the following technical solutions:
A kind of method of nif gene nifDK gene knockout, comprises the following steps:
One, PCR amplification Slow-growing Soybean rhizobia in nifDK gene upstream and downstream fragment, respectively nifDK-AB and nifDK-CD；
Two, carrying out fusion DNA vaccine with the PCR primer of nifDK-AB and nifDK-CD for template, PCR expands nifDK-AD；
Three, nifDK-AD is connected with carrier pK18mobsacB, digestion verification, nifDK-AD is proceeded to E.coli DH5 α In；
Four, by donor bacterium nifDK-AD, auxiliary bacterium pRK2013, recipient bacterium HW-05 triparental mating；
Five, homologous recombination for the first time: by KmRFlat board on grow, do not grow at 10% sucrose plate, for the first time sieve Select recon；
Six, homologous recombination for the second time: by KmRFlat board on do not grow, 10% sucrose plate grow, second time sieve Select recon；
Seven, PCR checking, determines that the recon of screening has been inserted in the genome of Slow-growing Soybean rhizobia, and base Because group does not contains wild strain.
The present invention uses the method for fusion DNA vaccine, will merge fragment and be connected with plasmid pK18mobsacB, and pass through KmRResistance is sieved Choosing and sacB compel to bring it about double crossing at the screening pressure of 10% sucrose, after 2 homologous recombination, use the screening of PCR method prominent Mutant, improves screening success rate.
Accompanying drawing explanation
Fig. 1 is screening recon flow chart；
Fig. 2 is extracting genome DNA result, M:DNA Marker DL10000,1:HW-05 genomic DNA, 2:HH103 Genomic DNA；
Fig. 3 is nifDK gene PCR amplification, M:DNA Marker DL10000,1:USDA110nifDK gene PCR Amplified production, 2:HW-05nifDK gene PCR amplified production；
Fig. 4 is nifDK-AB fragment PCR amplification, M:DNA Marker DL10000, and 1:nifDK-AB fragment PCR expands Volume increase thing；
Fig. 5 is nifDK-CD fragment PCR amplification, M:DNA Marker DL10000,1-2:nifDK-CD fragment PCR Amplified production；
Fig. 6 is nifDK-AD fragment PCR amplification, M:DNA Marker DL10000, and 1:nifDK-AD fragment PCR expands Volume increase thing；
Fig. 7 is to extract result, M:DNA Marker DL10000,1:nifDK-after nifDK-AD/pK18mobsacB converts AD/pK18mobsacB converts after connecting and extracts result；
Fig. 8 is clone's enzyme action qualification result, M:DNA Marker DL10000,1: clone's EcoR I and BamH I double digestion produces Thing；
Fig. 9 is recombinant screen, M:DNA Marker DL10000,1-2:nifDK fragment pcr amplification product.
Detailed description of the invention
Below in conjunction with the accompanying drawings technical scheme is further described, but is not limited thereto, every to this Inventive technique scheme is modified or equivalent, without deviating from the spirit and scope of technical solution of the present invention, all should contain In protection scope of the present invention.
As a example by Slow-growing Soybean rhizobia HW-05, the method for the nif gene nifDK gene knockout that the present invention provides is such as Under:
One, test method
1, the separation of HW-05
Choose a big and full root nodule, cut (band portion root) with shears, root nodule is placed in clear water immersion 4～ 5min, washes away impurity, then with HgCl with 0.1% after 95% soak with ethanol 5min2Surface sterilizing 5min, then rushes with sterilized water Wash 10 times.In the case of sterile working, rule on YMA medium after single root nodule is clipped broken, train in the incubator of 28 DEG C After supporting 3～5d, a little thalline of picking observes the features such as its form, size, transparency, viscosity, color, gloss and standard bacteria pair Ratio, chooses in medium slant by the thalline of acquisition, continues to cultivate and observes, if bacterium colony is without exception, then line dilution divides repeatedly From, until purification.
2, the clone of nifDK gene in HW-05
With the genomic DNA of Slow-growing Soybean rhizobia HW-05 as template, use homologous sequence cloning, design nifDK The specific primer of gene, use PCR method amplifying target genes nifDK full length DNA reaction after, reclaim PCR primer with PMD18-T Vector Kit is attached and converts to E.coli DH5 α.Send and survey with Shanghai biological engineering company limited Sequence.
3, the nifDK-AB fragment in PCR amplification HW-05
(1) PCR pipe is sequentially added into following reagent, is primer amplification nifDK-AB with nifDK-A (F) and nifDK-B (R) Fragment, amplification system is shown in Table 1:
Table 1
(2) amplified reaction parameter is shown in Table 2:
Table 2
(3) take 5 μ l product, add 1 μ L6 × loading buffer, 1% agarose gel electrophoresis detection.
4, the nifDK-CD fragment in PCR amplification HW-05
(1) PCR pipe is sequentially added into following reagent, is primer amplification nifDK-CD with nifDK-C (F) and nifDK-D (R) Fragment, amplification system is shown in Table 3:
Table 3
(2) amplified reaction parameter is shown in Table 4:
Table 4
(3) take 5 μ l product, add 1 μ l 6 × loading buffer, 1% agarose gel electrophoresis detection.
5, nifDK-AB fragment and nifDK-CD segment composition
(1) PCR pipe is sequentially added into following reagent, with nifDK-A (F) and nifDK-D (R) as primer, nifDK-AB 1 μ L and nifDK-CD 1 μ l is template amplification nifDK-AD fragment, and amplification system is shown in Table 5:
Table 5
(2) amplified reaction parameter is shown in Table 6:
Table 6
(3) take 5 μ L product, add 1 μ L6 × loading buffer, 1% agarose gel electrophoresis detection.
6, purified product is connected with pK18mobsacB
PCR primer nifDK-AD of purification is connected overnight with carrier pK18mobsacB at 16 DEG C, reaction system such as table 7:
Table 7
7, enzyme action is identified
Restricted enzyme identifies the plasmid extracted.According to pK18mobsacB carrier restriction enzyme mapping, select EcoRI and The positive recombiant plasmid of BamHI double digestion screening, reaction condition is 37 DEG C of water-bath 4h.Reaction takes 3 μ L digestion products after terminating respectively Carry out 0.8% agarose gel electrophoresis detection, observe enzyme action result, it is determined whether have exogenous sequences and the Insert Fragment size to be No correctly, thus tentatively determine whether recombinant clone.
Double digestion system (20 μ l) is shown in Table 8:
Table 8
8, nifDK-AD proceeds to HW-05 recombinant screen
By twice homologous recombination, 10% sucrose and KmRScreen laggard performing PCR detection positive colony, react at 20 μ LPCR In system, as template with 1 μ L plasmid DNA (through 20 times of dilutions), add corresponding PCR reacted constituent, carry out PCR amplification, electrophoresis Detect whether to expand specific band, to determine recon.Screening recon flow chart is as shown in Figure 1.
Two, experimental result
1, HW-05 total DNA extraction
Total DNA extraction result, obtains genomic DNA according to Total DNA extraction method, and result is shown in Fig. 2.
2, nifDK fragment in PCR amplification HW-05
Result is shown in Fig. 3.
3, nifDK-AB fragment in PCR amplification HW-05
PCR amplification nifDK-AB fragment products size, about at 600bp, is consistent with expection size, and result is shown in Fig. 4.
4, nifDK-CD fragment in PCR amplification HW-05
PCR amplification nifDK-CD fragment products size, about at 600bp, is consistent with expection size, and result is shown in Fig. 5.
5, nifDK-AD fragment in PCR amplification HW-05
The AB fragment of nifDK upstream region of gene and the CD fragment in downstream carry out fusion DNA vaccine amplification, and amplification obtains about 1200bp Purpose band, result is shown in Fig. 6.
6, nifDK-AD fragment is connected to pK18mobsacB carrier and qualification
I and the BamH I two kind of restriction endonuclease of EcoR selecting nifDK-AD fragment and pK18mobsacB carrier to have, carries out enzyme Convert after cutting connection, extract plasmid, occur that band, result are shown in Fig. 7 at about 7kb.
Selecting EcoR I and BamH I enzyme action to verify occur that two bands respectively may be about 1kb and 6kb, result is shown in Fig. 8.
7, nifDK-AD proceeds to HW-05 recombinant screen
By nifDK-AD fragment after triparental mating proceeds to HW-05, PCR expands nifDK fragment, and band is about 1200bp For recon, result is shown in Fig. 9.

Claims (9)

1. the method for a nif gene nifDK gene knockout, it is characterised in that described method step is as follows:
One, the nifDK gene upstream and downstream fragment in PCR amplification Slow-growing Soybean rhizobia, respectively nifDK-AB and nifDK- CD；
Two, carrying out fusion DNA vaccine with the PCR primer of nifDK-AB and nifDK-CD for template, PCR expands nifDK-AD；
Three, nifDK-AD is connected with carrier pK18mobsacB, digestion verification, nifDK-AD is proceeded in E.coli DH5 α；
Four, by donor bacterium nifDK-AD, auxiliary bacterium pRK2013, recipient bacterium triparental mating；
Five, homologous recombination for the first time: by KmRFlat board on grow, do not grow at 10% sucrose plate, for the first time screening weight Group；
Six, homologous recombination for the second time: by KmRFlat board on do not grow, 10% sucrose plate grow, programmed screening weight Group；
Seven, PCR checking, determines that the recon of screening has been inserted in the genome of Slow-growing Soybean rhizobia, and genome In do not contain wild strain.
The method of nif gene nifDK gene knockout the most according to claim 1, it is characterised in that in described step one, The amplification system of amplification nifDK-AB fragment is as follows:
The method of nif gene nifDK gene knockout the most according to claim 1, it is characterised in that in described step one, The amplified reaction parameter of amplification nifDK-AB fragment is as follows:
The method of nif gene nifDK gene knockout the most according to claim 1, it is characterised in that in described step one, The amplification system of amplification nifDK-CD fragment is as follows:
The method of nif gene nifDK gene knockout the most according to claim 1, it is characterised in that in described step one, The amplified reaction parameter of amplification nifDK-CD fragment is as follows:
The method of nif gene nifDK gene knockout the most according to claim 1, it is characterised in that in described step 2, The amplification system of PCR amplification nifDK-AD is as follows:
The method of nif gene nifDK gene knockout the most according to claim 1, it is characterised in that in described step 2, The amplified reaction parameter of PCR amplification nifDK-AD is as follows:
The method of nif gene nifDK gene knockout the most according to claim 1, it is characterised in that in described step 3, The reaction system that nifDK-AD is connected with carrier pK18mobsacB is as follows:
。
The method of nif gene nifDK gene knockout the most according to claim 1, it is characterised in that in described step 3, Enzyme action system is as follows:
CN201610835340.5A 2016-09-20 2016-09-20 A kind of method of nif gene nifDK gene knockout Pending CN106282218A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610835340.5A CN106282218A (en) 2016-09-20 2016-09-20 A kind of method of nif gene nifDK gene knockout

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610835340.5A CN106282218A (en) 2016-09-20 2016-09-20 A kind of method of nif gene nifDK gene knockout

Publications (1)

Publication Number Publication Date
CN106282218A true CN106282218A (en) 2017-01-04

Family

ID=57712843

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610835340.5A Pending CN106282218A (en) 2016-09-20 2016-09-20 A kind of method of nif gene nifDK gene knockout

Country Status (1)

Country Link
CN (1) CN106282218A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112940973A (en) * 2021-02-23 2021-06-11 黑龙江省科学院微生物研究所 High-density culture method for slow-growing soybean rhizobium

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102146415A (en) * 2010-07-16 2011-08-10 华东理工大学 Gene knockout bacterium of gluconobacter oxydans and preparation method thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102146415A (en) * 2010-07-16 2011-08-10 华东理工大学 Gene knockout bacterium of gluconobacter oxydans and preparation method thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HAHN M.等: "Insertion and deletion mutations within the nif region of Rhizobium japonicum", 《PLANT MOL BIOL.》 *
甄涛等: "基因工程菌株nifDK基因敲除", 《黑龙江科学》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112940973A (en) * 2021-02-23 2021-06-11 黑龙江省科学院微生物研究所 High-density culture method for slow-growing soybean rhizobium
CN112940973B (en) * 2021-02-23 2023-11-14 黑龙江省科学院微生物研究所 High-density culture method of slow-growing rhizobium sojae

Similar Documents

Publication Publication Date Title
Wang et al. An improved single-step cloning strategy simplifies the Agrobacterium tumefaciens-mediated transformation (ATMT)-based gene-disruption method for Verticillium dahliae
CN101875982B (en) Method for synchronously detecting multiple tobacco viruses
CN104263813B (en) For identifying Fusarinm solani or/and the primer sequence of Fusarium oxysporum, test kit and method thereof
CN101914147A (en) Plant disease resistance-related protein and coding gene and application thereof
CN104152446A (en) SSR molecular markers cosegregated from cucumber powdery mildew-resistant major QTL
CN102392080A (en) Method for identifying tomato yellow leaf curl virus resistance
CN112522300A (en) Method for cultivating broad-spectrum bacterial leaf streak resistant rice, primer and expression cassette
Adams et al. Genomic investigation of the strawberry pathogen Phytophthora fragariae indicates pathogenicity is associated with transcriptional variation in three key races
Baumgartner et al. Agrobacterium tumefaciens-mediated transformation for investigation of somatic recombination in the fungal pathogen Armillaria mellea
CN106282218A (en) A kind of method of nif gene nifDK gene knockout
CN104788549B (en) Rice Resistance characteristic of disease GAP-associated protein GAP RIR1 and its encoding gene and application
CN109234221A (en) A kind of potato virus X attenuated vaccine and the preparation method and application thereof
CN103866038B (en) For detecting tobacco to the N gene-specific primer of TMV resistance to, detection method and test kit
CN103789337A (en) Suicide vector pGMB152 for blue-white selection and application thereof
CN103966221A (en) Cloning and application of seed-root tip specific expression promoter
CN112301142A (en) Leymus speetanus anti-powdery mildew gene Pm12 molecular marker and application thereof
CN102417934B (en) Primers, TaqMan probe and kit used for detecting tomato yellow leaf curl virus
CN108866219B (en) Konjac soft rot germ specific sequence and detection primer and application thereof
CN102453085A (en) Plant stress tolerance-associated protein ZmSAPK8 and coding gene and application thereof
Krüger et al. The tropical Polyporus tricholoma (Polyporaceae)—taxonomy, phylogeny, and the development of methods to detect cryptic species
CN106434726A (en) Method for marking nodule bacteria with EYFP genes
CN105624078B (en) One plant of attenuation Salmonella choleraesuls and its preparation method and application
CN105624079B (en) It is attenuated Salmonella choleraesuls S.Choleraesuis S1210 and its preparation method and application
CN110564630A (en) aspergillus ochraceus mutant strain with ochratoxin A gene knockout function and construction and application thereof
CN107937620A (en) A kind of PCR RFLP methods for distinguishing fowl tembusu virus attenuated vaccine strain and street strain

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20170104