CN108504672A - Ralstonia solanacearum N477 extracellular proteins PHD and its encoding gene and application - Google Patents
Ralstonia solanacearum N477 extracellular proteins PHD and its encoding gene and application Download PDFInfo
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- CN108504672A CN108504672A CN201810294230.1A CN201810294230A CN108504672A CN 108504672 A CN108504672 A CN 108504672A CN 201810294230 A CN201810294230 A CN 201810294230A CN 108504672 A CN108504672 A CN 108504672A
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Abstract
The invention discloses Ralstonia solanacearum N477 extracellular proteins PHD and its encoding gene and applications.Ralstonia solanacearum N477 extracellular protein PHD, amino acid sequence is as shown in SEQ ID NO.1, and encoding gene nucleotide sequence is as shown in SEQ ID NO.2.The application of PHD or its encoding gene in the biogenic pesticide for preparing resistance to bacterial wilt.Design primer expands PHD coding region sequences using genomic DNA as template, and product is connected to protein expression vector pET30a (+) carrier, then converts and carries out protein expression into e. coli bl21 (DE3), with ni-sepharose purification PHD albumen.Greenhouse test the results show that the albumen can also evoking tobacco resisting tobacco mosaic virus infect, preventive effect 56.88%.
Description
Technical field
The invention belongs to genetic engineering fields, are related to Ralstonia solanacearum N477 extracellular proteins PHD (PopW Harpin domain)
And its encoding gene and application.
Background technology
The hrp gene clusters of most of gramnegative bacterium can cause allergic reaction on non-host plant, and
It is shown on host plant pathogenic.The product of hrp genes plays regulating and controlling effect in its expression and secretion process.Such as
HrpZ the and Ralstonia solanacearum's of the HrpN of Erwinia amylovora, Pseudomona syringae
Harpin albumen similar PopA is accredited out, they can secrete system secretion and cause non-post to extracellular by three types
Allergic reaction occurs for main plant, is that one kind is rich in glycine, serine, lacks cysteine and a kind of heat-resisting albumen,
There is no similitude on amino acid sequence.
Bacterial wilt is a kind of destructive soil-borne disease, is that harm in the world is maximum, distribution is most wide, cause damages most serious
One of plant disease, there is no effectively chemical pesticide and other prevention and treatment methods so far.Therefore bacterial wilt is referred to as plant
" cancer ".In recent years, northern China protection ground area obviously expands, and southern vegetable production under protection expands, therefore in part
The occurrence and development of regional bacterial wilt, or even become crushing disaster, cause CR Critical economic loss.
Ralstonia solanacearum can cause hundreds of crops to be wilted in worldwide, can infect a section more than 200 more than 50
Kind plant, wherein plant of Solanaceae is aggrieved most heavy, including tomato, potato, tobacco, peanut, banana etc..Germ passes through host root
Wound infection enters xylem, is then diffused rapidly to entire plant.Its pathogenesis is that Ralstonia solanacearum can when growing in the catheter
A large amount of exocellular polysaccharide is generated, the Water Transportation in plant can be influenced and hinder, is especially easy to petiole knot and leaflet
Smaller aperture due vessel perforation plate results in blockage, thus causes plant withered.In type strain GMI1000, three extracellular proteins
It is accredited out, is PopA1, PopB and PopC respectively.PopA1 is a kind of to secrete system secretion to extracellular by three types
Albumen, while lacking the amino acid of the ends N- 92.The derivative PopA3 of PopA1 is also from being grown in the barren culture of nutrition
It is purified in the supernatant of base.PopA1 and PopA3 is responsible for the allergic reaction of petunia.PopA is located at Ralstonia solanacearum
Hrp gene clusters upstream 2kb, expression are regulated and controled by hrpB genes.Arlat groups are found that new egg from the genome of Ralstonia solanacearum
White PopB and PopC, PopB albumen are made of 173 amino acid, containing there are two nuclear localization signals；1024 amino acid of PopC long,
Wherein contain 22 leucine zippers.PopB, popC and popA are to be regulated and controled simultaneously by hrpB.Under Congo red induction,
They have found that PopB and PopC are by Hrp system secretions to extracellular.
Invention content
The purpose of the present invention is the above-mentioned deficiencies for the prior art, provide Ralstonia solanacearum N477 extracellular proteins PHD.
It is a further object of the present invention to provide Ralstonia solanacearum N477 extracellular protein PHD encoding genes.
It is yet another object of the invention to provide the applications of Ralstonia solanacearum N477 extracellular protein PHD encoding genes.
Ralstonia solanacearum N477 extracellular protein PHD, amino acid sequence is as shown in SEQ ID NO.1.
The encoding gene of the Ralstonia solanacearum N477 extracellular proteins PHD.
The encoding gene preferred nucleotide sequence of the Ralstonia solanacearum N477 extracellular proteins PHD is as shown in SEQ ID NO.2.
The recombinant expression carrier of encoding gene containing the Ralstonia solanacearum N477 extracellular proteins PHD.
The genetic engineering bacterium of encoding gene containing the Ralstonia solanacearum N477 extracellular proteins PHD.
Applications of the Ralstonia solanacearum N477 extracellular proteins PHD in the biogenic pesticide for preparing resistance to bacterial wilt.
Encoding gene the answering in the biogenic pesticide for preparing resistance to bacterial wilt of the Ralstonia solanacearum N477 extracellular proteins PHD
With.
Application of the recombinant expression carrier in the biogenic pesticide for preparing resistance to bacterial wilt.
Application of the genetic engineering bacterium in the biogenic pesticide for preparing resistance to bacterial wilt.
The genetic engineering application of encoding gene containing the Ralstonia solanacearum N477 extracellular proteins PHD, including：
1) clone of PHD genes
Ralstonia solanacearum N477 is incubated at YGPA culture mediums under the conditions of 30 DEG C, waits for strain growth to logarithmic phase, according to bacterium base
Because group extracts kit operating instruction extracts Ralstonia solanacearum genomic DNA；
The primer of the encoding gene of design amplification PHD uses high-fidelity pfu polymeric enzymatic amplifications using genomic DNA as template
The coding region sequence of PHD genes, amplification program are：94 DEG C of pre-degeneration 4min, 94 DEG C of denaturation 30s, 58 DEG C of renaturation 30s, 72 DEG C are prolonged
1min is stretched, after 30 recycle, 72 DEG C of 10min, product gel extraction；The primer of the encoding gene of the amplification PHD, which is selected from, to be drawn
Object 1：1 sense primer of primer is as shown in SEQ ID NO.3, and 1 downstream primer of primer is as shown in SEQ ID NO.4；
2) expression and purifying of PHD albumen
It is connected to protein expression vector pET30a after being connected to intermediate carrier digestion or direct enzyme cutting after product gel extraction
(+) carrier, sequencing identification, obtains recombinant vector pET-PHD, then converts and carries out albumen table into e. coli bl21 (DE3)
It reaches, the positive colony screened is inoculated into the test tube containing LB liquid medium, 37 DEG C, 180rpm overnight incubations, second
It presses 1:100 ratio is inoculated into 10ml LB, and according to above-mentioned condition culture 3h, isopropyl-β-D-thiogalactose is added
Glycosides, makes its final concentration of 1mM, after Fiber differentiation 2h, 4 DEG C, 6000rpm, centrifuges 10min, collects thalline, be suspended in 20mM
The stupid methanesulfonyl fluoride of final concentration of 1mM is added in Tris-HCl, pH 8.0, is then carried out to thalline on sonicator
Broken, when bacterium solution, which becomes, to be clarified, supernatant is drawn in centrifugation, carries out polyacrylamide gel electrophoresis detection, ni-sepharose purification PHD eggs are used in combination
In vain, concrete operations are carried out with reference to High-Affinity Ni-IDA Resin operational manuals, obtain purifying PHD.
Advantageous effect
The PHD that the present inventor obtains is a kind of heat-resisting harpin albumen, can cause tobacco that allergic reaction occurs.
PopW is that inactive pectase homologue passes through although can also HR be caused to react present invention discover that is played a decisive role is
PHD, PHD can more effectively inhibit infecting for the anti-TMV of tobacco, and with obvious effects better than PopW.
3, the PHD processing tobaccos that the present invention obtains can resist infecting for tobacco mosaic virus (TMV), and resistance with evoking tobacco
It is not limited only to the blade of PHD processing, also includes surrounding blade, and along with the expression of PR-1 genes during this.It says
Bright PHD can be with evoking tobacco system resume.This illustrates the value that PHD has exploitation into biogenic pesticide.
Description of the drawings
Fig. 1 .PHD protein expression results.
M is albumen marker (SM0431, MBI), and 1 is 1%BSA, and 2 be PopW clonal expressions as a result, 3 be PHD clone's tables
Up to result.
Biomaterial preservation information
N477 bacterial strains, Classification And Nomenclature are Solanaceae Ralstonia Pseudomonas Ralstonia solanacearum, in January, 2018
It is preserved within 22nd China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1, Institute of Microorganism, Academia Sinica, culture presevation number are CGMCC NO.15253.
Specific implementation mode
Embodiment 1
(1) clone of PopW gene orders
Ralstonia solanacearum (Ralstonia solanacearum) N477 is incubated at YGPA culture mediums (0.5% under the conditions of 30 DEG C
Peptone, 0.5% yeast extract powder, 1% glucose and 1.5% agar), wait for that strain growth to logarithmic phase, extracts genomic DNA
(Axyprep Bacterial Genomic DNA kit, Hangzhou).According to pattern bacterium Ralstonia solanacearum GMI1000 whole genome sequences
Design amplimer：
Sense primer F1：ATGTCCATCCAGATTGATCGC(SEQ ID NO.4)
Downstream primer R1：GCCCGAGTAGGCCTTGTAG(SEQ ID NO.5)
Using N477 genomic DNAs template, PCR amplification is carried out, PCR programs are as follows：94 DEG C of pre-degeneration 4min, 94 DEG C of denaturation
30s, 60 DEG C of renaturation 1min, 72 DEG C of extension 1min, after 30 recycle, 72 DEG C of 10min are expanded with 1% agarose electrophoresis detection PCR
Increase production object 1143bp, is recycled according to Agarose Gel DNA Purification Kit (TaKaRa, Dalian) handbook.
The 4 μ l of product being recovered to are connected on carrier pMD19-T, after reacting 16h at 16 DEG C, with heat shock method conversion Escherichia coli impression
State DH5 α, picking positive colony are sequenced, and sequencing result shows the complete coding of Ralstonia solanacearum N477 extracellular protein genes PopW
Area is 1143bp (SEQ ID NO.3).
(2) PopW protein expressions
According to the complete encoding sequence of Ralstonia solanacearum N477 extracellular protein PopW genes, enzyme enzyme site is distinguished at design both ends
Primer：
Sense primer F2：CGCATATGTCCATCCAGATTGATCGC Nde I(SEQ ID NO.6)
Downstream primer R2：GCAAGCTTGCCCGAGTAGGCCTTGTAG Hind III(SEQ ID NO.7)
Using the genomic DNA of N477 as template, the coding region sequence of PCR amplification popW genes is carried out, amplification program is：94
DEG C pre-degeneration 4min, 94 DEG C of denaturation 30s, 60 DEG C of renaturation 1min, 72 DEG C of extension 1min, after 30 cycles, 72 DEG C of 10min, product
Size is 1143bp, gel extraction to product end add A after be connected on carrier pMD19-T, utilize Nde I and Hind
III digestion site is cloned into protein expression vector pET30a (+) carrier, and sequencing identification obtains recombinant plasmid pET-PopW,
Then it converts and carries out protein expression into BL21 (DE3), by the cultured e. coli bl21 containing above-mentioned recombinant plasmid
(DE3) thalline were collected by centrifugation, is suspended in 20mM Tris-HCl (pH 8.0), and the protease that final concentration of 1mM is added inhibits
Agent PMSF, progress albumen is broken on sonicator, then extracts albumen, carries out polyacrylamide gel electrophoresis detection,
With ni-sepharose purification PopW albumen.
(3) clone of PHD
According to the complete encoding sequence of Ralstonia solanacearum N477 extracellular protein PopW genes, amplimer is designed：
Sense primer F3：GGTCGGCTCGGGCGGCTT(SEQ ID NO.8)
Downstream primer R3：TCCATCCAGATTGATCGCCCG(SEQ ID NO.9)
Using N477 genomic DNAs template, PCR amplification is carried out, PCR programs are as follows：94 DEG C of pre-degeneration 4min, 94 DEG C of denaturation
30s, 58 DEG C of renaturation 30s, 72 DEG C of extension 1min, after 30 recycle, 72 DEG C of 10min are expanded with 1% agarose electrophoresis detection PCR
Increase production object 454bp, is recycled according to Agarose Gel DNA Purification Kit (TaKaRa, Dalian) handbook.
The 4 μ l of product being recovered to are connected on carrier pMD19-T, after reacting 16h at 16 DEG C, with heat shock method conversion Escherichia coli impression
State DH5 α, picking positive colony are sequenced, and sequencing result shows that the complete coding region of N477 extracellular protein genes PHD is
454bp。
(4) PHD protein expressions
According to the complete encoding sequence of Ralstonia solanacearum N477 extracellular protein PopW genes, enzyme enzyme site is distinguished at design both ends
Primer：
Sense primer F4：ATAAGCTTGGTCGGCTCGGGCGGCTT Hind III(SEQ ID NO.10)
Downstream primer R4：GCCATATGTCCATCCAGATTGATCGCCCG Nde I(SEQ ID NO.11)
Using N477 genomic DNAs template, the coding region sequence of PCR amplification PHD genes, amplification program is：94 DEG C of pre- changes
Property 4min, 30s, 58 DEG C of renaturation 30s, 72 DEG C of extension 1min of 94 DEG C of denaturation, after 30 cycles, 72 DEG C of 10min, primer size is
454bp is cloned into intermediate carrier pMD19-T, is further cloned into using the Nde I and Hind III digestions site of introducing
Protein expression vector pET30a (+) carrier, sequencing identification, obtains recombinant plasmid pET-PHD, then converts into BL21 (DE3)
Protein expression is carried out, thalline were collected by centrifugation by the cultured e. coli bl21 (DE3) containing above-mentioned recombinant plasmid, is hanged
Float on 20mM Tris-HCl (pH 8.0), the protease inhibitors PMSF of final concentration of 1mM is added, on sonicator
It is broken to carry out albumen, then extracts albumen, carries out polyacrylamide gel electrophoresis detection and obtains egg with ni-sepharose purification PHD albumen
White a concentration of 25mg/LW (Fig. 1).
Embodiment 2
Respectively with the PopW soluble proteins of above-mentioned acquisition and PHD albumen, tobacco leaf is sprayed on spray-on process, with without
The aqua sterilisa and empty carrier of PHD albumen are handled as a contrast.After two days, using the method for frictional inoculation respectively to being jetted through PHD
Blade and its above and below two panels blade inoculation tobacco mosaic virus (TMV), each handle 3 repetitions, every time repeat 12 plants of cigarettes
Careless seedling.We observe the number of scab after 4 days, and preventive effect is calculated by the quantity of scab.Control effect=[(control blade
Withered spot number on upper withered spot number-processing blade)/compare withered spot number on blade] x 100%.The results show that and the blank that does not handle
Control is compared, and scab number significantly reduces on PopW soluble proteins and the processed blades of PHD, and preventive effect is respectively up to 45% He
56.88% (table 1) illustrates that PopW albumen and PHD can cause the system resistant of tobacco, but PHD can more effectively inhibit tobacco anti-
TMV's infects, to effectively inhibit infecting for tobacco mosaic virus (TMV).
Biocontrol effects of 1 PHD of table to tobacco mosaic virus disease
Note：The withered spot number on every sick leaf is recorded after handling 4 days, calculates withered spot inhibiting rate, as albumen control effect.
Above example shows the new extracellular protein PHD of a kind of Ralstonia solanacearum N477 cloned in the present invention, it has Harpin
Feature, include the composition of amino acid, thermal stability and migrates the characteristics such as slower, PHD at protease-sensitive on SDS-PAGE
HR can be caused to react with PopW.The PHD of the present invention handles tobacco, can resist infecting for tobacco mosaic virus (TMV) with evoking tobacco,
And resistance is not limited only to the blade of PHD processing, also includes surrounding blade, and along with PR-1 genes during this
Expression.Illustrate that PHD can illustrate that PHD can develop into biogenic pesticide with evoking tobacco system resume.
Sequence table
<110>Agricultural University Of Nanjing
<120>Ralstonia solanacearum N477 extracellular proteins PHD and its encoding gene and application
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 147
<212> PRT
<213>Solanaceae Ralstonia Pseudomonas N477 (Ralstonia solanacearumN477)
<400> 1
Phe Gln Thr Pro Ser Thr Trp Asn His Asp Ala Gly Ser Ser Ile Asp
1 5 10 15
Thr Ser Gln Leu Gln Arg Ala Val Gln Leu Leu Asp Gln Val Leu Gln
20 25 30
Gln Leu Glu Ala Arg Lys Leu Phe Gly Asn Met Leu Asn Gln Pro Gly
35 40 45
Ala Asp Asn Ala Gly Gln Asn His Ala Gly Gly His Gly Gly Gly His
50 55 60
His Gly Gly Ser Asn Gly Phe Gly Glu Asn Gly Arg Phe Gly Ser Pro
65 70 75 80
His Ala Asn Ser Pro Ala Gln Pro Asp Leu Glu Leu Pro Ala Asn Lys
85 90 95
Pro Asn Asn Gly Lys His Asn Thr Ser Ala Ser Thr Pro Asp Thr Gln
100 105 110
Thr Ala Pro Ser Ser Thr Ser Pro Thr Thr Gly Thr Ser Pro Thr Pro
115 120 125
Thr Ser Thr Ser Ala Thr Glu Gly Lys Val Ala Tyr Gly Val Lys Pro
130 135 140
Pro Glu Pro
145
<210> 2
<211> 454
<212> DNA
<213>Solanaceae Ralstonia Pseudomonas N477 (Ralstonia solanacearumN477)
<400> 2
tttccagacg ccctcgacgt ggaatcacga tgcgggttcc agtatcgata ccagccagct 60
ccagcgcgca gtacaactgc tcgaccaggt cttgcagcag ctcgaagcgc gcaagctgtt 120
cgggaacatg ctgaaccagc cgggcgccga caacgccggc cagaaccacg ctggcggcca 180
cggcggcggt catcatggcg gctcgaacgg gttcggtgaa aacggccgct tcggttcgcc 240
gcacgccaat tcgcctgcgc agccggacct cgagctgccg gccaacaagc ccaacaacgg 300
caagcacaac acttcggctt ccacgccgga cacgcagacg gcgccgtctt ccacttcgcc 360
cacgaccggc acgtccccga cgcccacgtc cacctccgcg accgagggca aggtggccta 420
cggcgtcaag ccgcccgagc cgaagggatc ccga 454
<210> 3
<211> 1143
<212> DNA
<213>Solanaceae Ralstonia Pseudomonas N477 (Ralstonia solanacearumN477)
<400> 3
atgtccatcc agattgatcg cccgaacaac catttccaga cgccctcgac gtggaatcac 60
gatgcgggtt ccagtatcga taccagccag ctccagcgcg cagtacaact gctcgaccag 120
gtcttgcagc agctcgaagc gcgcaagctg ttcgggaaca tgctgaacca gccgggcgcc 180
gacaacgccg gccagaacca cgctggcggc cacggcggcg gtcatcatgg cggctcgaac 240
gggttcggtg aaaacggccg cttcggttcg ccgcacgcca attcgcctgc gcagccggac 300
ctcgagctgc cggccaacaa gcccaacaac ggcaagcaca acacttcggc ttccacgccg 360
gacacgcaga cggcgccgtc ttccacttcg cccacgaccg gcacgtcccc gacgcccacg 420
tccacctccg cgaccgaggg caaggtggcc tacggcgtca agccgcccga gccgaccggc 480
gtggtcgacg tcagcaagcc gatcgtcgtc aaggctggcg agacgttcga cggcggcggc 540
aagtactacc gcccgaccaa ggagatgggc gacggttcgc agaacgagca ccagaagccg 600
ctgttcattc tggagcccgg cgcgacgctc aagaacgtgc agtattccgg cggcgacggc 660
atccacatgc tgggcagcgg caagctggac cgcgtcgtca accgccaggt gggcgaggat 720
gccatcacca tcgacggcgc caagaaccac gcgcatgatg ccaagatcgc cgggatcgat 780
ccggcgtcga tccccggagg aacgcccaaa gtggagatca ccaacagcgc cttctatggc 840
gccaaggaca agctcgctca gatcaacggc gacgttgacc tgcaggtgaa aggcatgtat 900
gtcaacggcg ccggcaaggt cttccgtacc aacggtggtg atacgcagat caaggcgacg 960
gtcaacgtcc aggattcgaa tttccagaac gtgtcggagg cggttttccg gaccgattcc 1020
aagttctcga ctgcgtcgtt ctcggacgat gtgaaatcgg atgcgccctt cgatgggctg 1080
gctcccgaca agagccaagt cacaggcacc aacaaggtga gctacaaggc ctactcgggc 1140
tga 1143
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atgtccatcc agattgatcg c 21
<210> 5
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
gcccgagtag gccttgtag 19
<210> 6
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
cgcatatgtc catccagatt gatcgc 26
<210> 7
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
gcaagcttgc ccgagtaggc cttgtag 27
<210> 8
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
ggtcggctcg ggcggctt 18
<210> 9
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
tccatccaga ttgatcgccc g 21
<210> 10
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
ataagcttgg tcggctcggg cggctt 26
<210> 11
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
gccatatgtc catccagatt gatcgcccg 29
Claims (7)
1. the recombinant expression carrier of the encoding gene containing Ralstonia solanacearum N477 extracellular proteins PHD；The Ralstonia solanacearum N477 is extracellular
The encoding gene nucleotide sequence of albumen PHD is as shown in SEQ ID NO.2.
2. the genetic engineering bacterium of the encoding gene containing Ralstonia solanacearum N477 extracellular proteins PHD；The extracellular eggs of Ralstonia solanacearum N477
The encoding gene nucleotide sequence of white PHD is as shown in SEQ ID NO.2.
3. applications of the Ralstonia solanacearum N477 extracellular proteins PHD in the biogenic pesticide for preparing resistance to bacterial wilt；The Ralstonia solanacearum
N477 extracellular protein PHD, amino acid sequence is as shown in SEQ ID NO.1.
4. application of the encoding gene of Ralstonia solanacearum N477 extracellular proteins PHD in the biogenic pesticide for preparing resistance to bacterial wilt；It is described
Ralstonia solanacearum N477 extracellular proteins PHD encoding gene nucleotide sequence as shown in SEQ ID NO.2.
5. application of the recombinant expression carrier described in claim 1 in the biogenic pesticide for preparing resistance to bacterial wilt.
6. application of the genetic engineering bacterium described in claim 2 in the biogenic pesticide for preparing resistance to bacterial wilt.
7. the genetic engineering application of the encoding gene of Ralstonia solanacearum N477 extracellular proteins PHD, it is characterised in that including：
1) clone of PHD genes
Ralstonia solanacearum N477 is incubated at YGPA culture mediums under the conditions of 30 DEG C, waits for strain growth to logarithmic phase, according to bacterial genomes
Extracts kit operating instruction extracts Ralstonia solanacearum genomic DNA；
The primer of the encoding gene of design amplification PHD uses high-fidelity pfu polymeric enzymatic amplifications PHD using genomic DNA as template
The coding region sequence of gene, amplification program are：94 DEG C of pre-degeneration 4min, 94 DEG C of denaturation 30s, 58 DEG C of renaturation 30s, 72 DEG C extend
1min, after 30 recycle, 72 DEG C of 10min, product gel extraction；The primer of the encoding gene of the amplification PHD is selected from primer 1
Or primer 2：1 sense primer of primer is as shown in SEQ ID NO.3, and 1 downstream primer of primer is as shown in SEQ ID NO.4；On primer 2
Primer is swum as shown in SEQ ID NO.5, primer 2 downstream primer is as shown in SEQ ID NO.6；
2) expression and purifying of PHD albumen
It is connected to protein expression vector pET30a after being connected to intermediate carrier digestion or direct enzyme cutting after product gel extraction plus after A
(+) carrier, sequencing identification, obtains recombinant vector pET-popW, then converts and carries out albumen table into e. coli bl21 (DE3)
It reaches, the positive colony screened is inoculated into the test tube containing LB liquid medium, 37 DEG C, 180rpm overnight incubations, second
It presses 1:100 ratio is inoculated into 10ml LB, and according to above-mentioned condition culture 3h, isopropyl-β-D-thiogalactose is added
Glycosides, makes its final concentration of 1mM, after Fiber differentiation 2h, 4 DEG C, 6000rpm, centrifuges 10min, collects thalline, be suspended in 20mM
The stupid methanesulfonyl fluoride of final concentration of 1mM is added in Tris-HCl, pH 8.0, is then carried out to thalline on sonicator
Broken, when bacterium solution, which becomes, to be clarified, supernatant is drawn in centrifugation, carries out polyacrylamide gel electrophoresis detection, ni-sepharose purification PHD eggs are used in combination
In vain, concrete operations are carried out with reference to High-Affinity Ni-IDA Resin operational manuals, obtain purifying PHD.
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| CN112694524A (en) * | 2021-02-03 | 2021-04-23 | 浙江省农业科学院 | Anti-fusarium wilt PHD transcription factor ClPHD23, gene, expression vector, transformant and application thereof |
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| CN112048514A (en) * | 2020-08-03 | 2020-12-08 | 华南农业大学 | Phage trp574 gene and application thereof |
| CN112048514B (en) * | 2020-08-03 | 2022-08-23 | 华南农业大学 | Phage trp574 gene and application thereof |
| CN112694524A (en) * | 2021-02-03 | 2021-04-23 | 浙江省农业科学院 | Anti-fusarium wilt PHD transcription factor ClPHD23, gene, expression vector, transformant and application thereof |
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