CN108586587A - A kind of artificial tuberculosis yersinia genus insecticidal proteins and its encoding gene and application - Google Patents
A kind of artificial tuberculosis yersinia genus insecticidal proteins and its encoding gene and application Download PDFInfo
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- CN108586587A CN108586587A CN201810451847.XA CN201810451847A CN108586587A CN 108586587 A CN108586587 A CN 108586587A CN 201810451847 A CN201810451847 A CN 201810451847A CN 108586587 A CN108586587 A CN 108586587A
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Abstract
The invention discloses a kind of artificial tuberculosis yersinia genus insecticidal proteins and its encoding gene and applications, belong to genetic engineering and biological pesticide technical field, and the insecticidal proteins are by SEQ ID NO:The protein that amino acid sequence shown in 1 forms, or in SEQ ID NO:It is substituted, lacks or is inserted into that one or several amino acid sequences are obtained has same active protein in amino acid sequence shown in 1.The present invention also provides the encoding gene of the insecticidal proteins, expression vector, engineering bacteria, the present invention also provides class yersinia pseudotuberculosis insecticidal proteins isolation and purification methods.Application of the encoding gene of application and the albumen of the albumen in insecticide in converting plant.The present invention provides a kind of encoding genes of insecticidal proteins；The insecticidal proteins of the present invention can be used for killing the lepidoptera pests such as greater wax moth, bollworm, beet armyworm and Ostrinia furnacalis, improve the killing ability of genetically modified crops.
Description
Technical field
The invention belongs to genetic engineerings and biological pesticide technical field, are related to a kind of artificial tuberculosis yersinia genus insecticidal proteins
And its encoding gene and application, specifically, being related to deriving from yersinia pseudotuberculosis (Yersinia
Pseudotuberculosis toxic protein YpkA303 and its gene) and the application in biological control.
Background technology
One of the Main Agricultural disaster in China is diseases and pests of agronomic crop, it has, and type is more, influences big and breaks out often
The features such as causing disaster, occurrence scope and severity often result in heavy losses to China's agricultural production.According to statistics, China it is annual because
Pest and disease damage loss grain is up to 50,000,000,000 jin, 18,000,000 tons of all kinds of industrial crops.China's crops common disease and pest has：Corn borer,
Bollworm, locust, greater wax moth, beet armyworm etc..Currently, the various insect pests of prevention crop depend on making for chemical pesticide
With.It is long-term largely to use pesticide, on the one hand, to cause Some Insects to produce certain drug resistance or resistance, cause real in production
The dosage for trampling middle pesticide is increasing year by year, or replaces the stronger completely new pesticide of toxicity, meanwhile, forcing pesticide research, person must not
New pesticide is not developed.On the other hand, long-term excessively to use chemical pesticide, to natural ecological environment by different degrees of pollution and
The destruction of unrepairable.Therefore, find it is completely new, safe and reliable, meet sustainable development requirement, environmentally friendly biology
Means of prevention has become the important scientific problems in current agricultural research and production.Using the means of biotechnology, find anti-
Worm gene, cultivating anti-pest GM crop becomes as the great direction in current various countries' agricultural production research.
Now, exploitation is most ripe, and most popular Plant Extrinsic Anti-insect Genes are derived from bacillus thuringiensis
(Bacillus thuringiensis, Bt) 2 class anti insect genes, are divided into according to its toxic activity：It is produced when from sporulation
Raw parasporal crystal toxin, i.e. insecticidal crystal protein (insecticidal crystalline protein, ICP), including crystalline substance
Body toxin (crystalline toxin, Cry) and cell cracking toxin (cytolytic toxin, Cyt).Wherein, ICP be by
Cry genes and Cyt gene codes, there is strong toxicity to sensitive insect, and it is non-toxic to higher mammal and people.But these turn base
Because anti-pest crop uses CrylA class insecticidal protein genes, the unification of insecticidal protein gene, large area anti-using acceleration pest more
Property generation, constrain turn CrylA class anti-pest GM crops apply the time limit.In addition, Bt desinsections are widely used before more number
There are different degrees of cross resistances for gene.Therefore, it is necessary to excavate new killing gene, find new, higher virulence and with
The new gene of the insecticidal protein gene no interactions resistance used is applied in agricultural production.But it yet there are no report
From the toxic protein and its gene of yersinia pseudotuberculosis.
Invention content
The purpose of the present invention is to provide a kind of artificial tuberculosis yersinia genus insecticidal proteins and its encoding gene and applications, are
A kind of novel insecticidal proteins and its gene with broad-spectrum insecticidal activity.
Its specific technical solution is：
A kind of artificial tuberculosis yersinia genus insecticidal proteins, by SEQ ID NO:The albumen that amino acid sequence shown in 1 forms
Matter, or in SEQ ID NO:One or several amino acid sequences are substituted, lacked or are inserted into amino acid sequence shown in 1 to be obtained
What is obtained has same active protein.
A kind of encoding gene of artificial tuberculosis yersinia genus insecticidal proteins, nucleotide sequence such as SEQ ID NO:Shown in 2.
Application of the artificial tuberculosis yersinia genus insecticidal proteins of the present invention in insecticide preparation process.
Encoding gene the answering in insecticide preparation process of artificial tuberculosis yersinia genus insecticidal proteins of the present invention
With.
A kind of induction of artificial tuberculosis yersinia genus insecticidal proteins of the present invention and purification process, include the following steps：
1) it activates：The protein expression strain e. coli bl21 (DE3) (pET28a-YpkA303) that will have been built, chooses list
Bacterium colony is inoculated in containing in appropriate Km 5mL LB liquid mediums, is cultivated to plateau in 220rpm, 37 DEG C of shaking tables；
2) switching expands culture：Bacterium is transferred in the 500mL LB liquid mediums containing appropriate Km in 1% ratio
In the triangular flask of 1L, 37 DEG C, 180rpm shakes culture；OD600 is detected, until when 0.6, shaking table temperature is adjusted to 26 DEG C, and by eventually
Derivant IPTG is added in concentration 0.5mM, starts to induce；It is incubated overnight, after about 16-18 hours, induction finishes；
3) bacterium is received：Thalline is collected at 4 DEG C with refrigerated centrifuge, 8000rpm 10 minutes, repeats this step, until collecting
To the thalline in all bacterium solutions, Binding buffer (25mM Tris-Cl, 150mM NaCl, 30mM are then used
Imidazole) washing thalline is twice；
4) it is crushed thalline：Appropriate Binding buffer are added, thalline is suspended, through cryogenic high pressure by fully shaking
After cell crushing instrument squeezing is crushed thalline, it is placed in the progress abundant suspended bacterias of ultrasound Binding buffer in sonicator
Body, broken with ultrasonic cell disruption instrument under the conditions of 4 DEG C, power 200W, work 4s, interval 5s, recurring number 99, directly
To super clear, the 10000rpm by bacterium solution, centrifuges 30 minutes, supernatant is transferred in clean triangular flask at 4 DEG C；
5) upper prop：Supernatant in previous step is all added in batches in prior activated Ni-NTA affinity purifications column,
Until whole liquid are flowed down by pillar；
6) it washs：With Washing buffer (25mM Tris-Cl, 150mM NaCl, 100mM imidazole) by column
Son is washed twice；
7) it elutes：It is eluted with Elution buffer (25mM Tris-Cl, 150mM NaCl, 250mM imidazole),
And eluent is collected in the EP pipes that 1.5mL is pre-chilled in advance every time, is positioned on ice.
8) it dialyses：Well-done bag filter is cleaned up with deionized water, the protein eluate being collected into is injected and is dialysed
In bag, both ends are clipped with clip, be put into dialyzate (10mM Tris-Cl, 100mM NaCl) (DTT is added in dialyzate, with
Prevent albumen from being aoxidized), dialysed overnight at 4 DEG C；
9) it dispenses：Protein solution is dispensed, -80 DEG C of preservations；Run SDS-PAGE glue come detect albumen purifying situation and into
Row insecticidal activity detects.
Compared with prior art, beneficial effects of the present invention：
The insecticidal protein gene YpkA303 of the present invention derives from yersinia pseudotuberculosis (Yersinia
Pseudotuberculosis), with it has been reported that and the Bt insecticidal protein genes and luminous bacillus desinsection that are widely used at present
Gene TccC does not have homology, is a kind of completely new insecticidal proteins and gene.
Description of the drawings
Fig. 1 is YpkA303 gene PCR amplifications；
Fig. 2 is the small induction result of YpkA303 recombinant plasmids；
Fig. 3 is YPKA303 recombinant protein purification results；
Fig. 4 is YPKA303 bacterium colonies PCR；
Fig. 5 is YPKA303 double digestion genetic fragments；
Fig. 6 is YpkA303 recombinant vectors.
Specific implementation mode
Technical scheme of the present invention is described in more detail with reference to specific embodiment.
The structure of YpkA303 inducible expression carriers
New gene YpkA303 is obtained, using special primer from wild type yersinia pseudotuberculosis (Yersinia
Pseudotuberculosis YPIII) amplification YpkA303 primer sequences are as follows in genomic DNA
Forward primer YpkA303-F:5'-CGCGGATCCATGAAACCGTTAGATCAGAATATTA-3'
Reverse primer YpkA303-R:5'-CCGCTCGAGTTAACATCCTCCGCCACCTTTTGAG-3'
(1) amplification of YpkA303 genes
1 YpkA303 gene PCR amplification systems of table
PCR programs：
After PCR product is subjected to electrophoresis detection
(2) YpkA303 gene PCRs amplified production double digestion：
2 YpkA303PCR product double digestions reaction system (150 μ L) of table
After centrifuging mixing, it is put into 37 DEG C of water-baths, digestion is overnight (14-16hours)；15 μ L Loading are added
Buffer (10 ×), 100 DEG C of heating 3min, terminates enzyme activity；A small amount of digestion products are taken to be detected into row agarose gel electrophoresis；
(3) recycling of YpkA303PCR endonuclease bamhis (with reference to Tiangeng plasmid QIAquick Gel Extraction Kit specification)
1) column equilibration：CB2 is put into preprepared collecting pipe, 500 μ L BL is drawn with liquid-transfering gun, is added to suction
In attached column CB2, stand 5min, 12,000rpm, centrifuge 1min, discard waste liquid in pipe；
2) it after the PC solution mixings for adding equimultiple to submit YpkA303PCR digestion products, is fully transferred in adsorption column CB2,
5min is stood, 12,000rpm centrifugation 1min discard waste liquid in pipe；
3) it is added in 600 μ l PW to adsorption column, 12,000rpm, centrifuges 1min, discard waste liquid in pipe；
4) step 5 is repeated；
5) 12,000rpm, centrifugation 3min, by adsorption column in ventilation, is dried to nothing with drying liquid remaining in pillar
Ethyl alcohol smell；
6) adsorption column is put on 1.5mL EP, the centre position into recovery column, it is hanging that 80 μ L of sterilizing ddH2O have been added (
Preheated in 55 DEG C), 12,000rpm, 2min is centrifuged, to improve organic efficiency, can be repeated once；
7) in -20 DEG C of storages.
(4) expression plasmid pET28a extractions (slightly being changed with reference to Tiangeng plasmid extraction kit method)
1) in adsorption column CP3, with pipettor plus BL, the 12000rpm of 500 μ L, 1min is centrifuged, waste liquid is discarded；
2) 5mL is inoculated into containing Km's from 3 monoclonals of each picking on the e. coli tg1 tablet of the expression vector containing pET28a
In LB liquid medium, 37 DEG C, 220r shaking tables are incubated overnight；
3) thalline were collected by centrifugation：It will be incubated overnight and express unloaded bacterium solution TG1 3Ml, 12000rpm, centrifugation containing pET28a
2min is collected into 1.5mL EP pipes, adds 250 μ L solution P1, thorough suspension thalline；Add 250 μ L solution P2, spins upside down 4-6 times
(soft), makes thalline fully crack, and pays attention to avoiding cracking excessive；Add 350 μ L solution P3, spins upside down immediately 4-6 times (light
It is soft), until there is white flock precipitate, ice bath 5min, 12000rpm centrifuge 20min；It shifts in supernatant to adsorption column,
12000rpm centrifuges 1min, is repeated once, discards waste liquid；Add 500 μ L PD, 12000rpm, centrifuges 2min, discard waste liquid；Add
Enter 600 μ LPW in adsorption column, 2000rpm centrifuges 1min, is repeated once；Adsorption column is placed back in collecting pipe,
12000rpm centrifuges 2min；By adsorption column in ventilation, dry to no ethyl alcohol smell；It is new that adsorption column is placed in sterilization treatment
1.5mL EP pipe, is vacantly added 100 μ L of sterilizing ddH2O (in 55 DEG C of preheatings) to recovery column centre position, and 12,000rpm, from
Heart 2min, is repeated once；- 20 DEG C of storages.
(5) double digestion of expression plasmid pET28a
Each reactant is added by following system
3 pET28a plasmid double digestions reaction system (200 μ L) of table
It after centrifuging mixing, is put into 37 DEG C of incubators, digestion overnight；It is mixed that 20 μ L Binding Buffer (10 ×) are added
It is even, enzyme activity is terminated, product is subjected to agarose electrophoresis detection and analysis；Target fragment is recycled.
(6) structure of recombinant expression carrier：
1) it presses following reaction system and each reactant is added
4 carrier segments linked system of table
16 DEG C of connection instrument are placed in, connection is overnight.
2) heat-shock transformed (overall process is carried out on ice) takes out TG1 competent cells；It is in super-clean bench that connection product is complete
It is complete that competent cell is added, it is sure not to blow and beat；Ice bath half an hour, 42 DEG C of heat shocks 90 seconds are put immediately on ice, ice bath 3-5 minutes；Add
Enter 800 μ l LB culture mediums, (37 DEG C of preheating nonreactives), 100rpm, 37 DEG C of shaking tables, recovery 40-55 minutes；4000rpm, centrifugation
3min abandons supernatant.Precipitation is resuspended, coated plate, is incubated overnight by 37 DEG C.
3) screening and identification of bacterium colony PCR positive colonies
The bacterium colony PCR preliminary screenings of 5 recombinant plasmid transformed TG1 of table
It is that each reactive component is added according to above-mentioned PCR reaction systems, the appropriate monoclonal of picking on the tablet containing transformed clone,
It is added in PCR pipe；Carry out PCR programs；
PCR programs：
After PCR product is subjected to electrophoresis detection；Filter out correct positive colony；Picking positive colony, in liquid 5Ml
In LB culture mediums, 37 DEG C, 120rpm shaking tables are incubated overnight.Carry out a small amount of double digestion verifications；And to the positive colony of digestion verification
Song Shanghai Sheng Gong biotech firms are sequenced, and are filtered out the positive colony plasmid of sequencing result mutation, are saved backup.
(7) expression cloning is obtained：
Positive gram of 1 μ L are added in L21 (DE3) competent cell for taking out -80 DEG C of preservations well prepared in advance in super-clean bench
Grand plasmid, gently after mixing；Ice bath half an hour, 42 DEG C of heat shocks 90 seconds are put immediately on ice, ice bath 3-5 minutes；800 μ l are added
LB culture mediums, (37 DEG C of preheating nonreactives), 100rpm, 37 DEG C of shaking tables, recovery 60-90 minutes；Take 50-100 μ L bacterium solution coated plates, 37
DEG C, it is incubated overnight, obtains the single bacterium colony of recombinant expression clone.
The induction and purifying of YpkA303 albumen
1) it activates：The protein expression strain e. coli bl21 (DE3) (pET28a-YpkA303) that will have been built, chooses list
Bacterium colony is inoculated in containing in appropriate Km 5mL LB liquid mediums, is cultivated to plateau in 220rpm, 37 DEG C of shaking tables；
2) switching expands culture：Bacterium is transferred in the 500mL LB liquid mediums containing appropriate Km in 1% ratio
In the triangular flask of 1L, 37 DEG C, 180rpm shakes culture；OD600 is detected, until when 0.6, shaking table temperature is adjusted to 26 DEG C, and by eventually
Derivant IPTG is added in concentration 0.5mM, starts to induce；It is incubated overnight, after about 16-18 hours, induction finishes；
3) bacterium is received：Thalline is collected at 4 DEG C with refrigerated centrifuge, 8000rpm 10 minutes, repeats this step, until collecting
To the thalline in all bacterium solutions, Binding buffer (25mM Tris-Cl, 150mM NaCl, 30mM are then used
Imidazole) washing thalline is twice；
4) it is crushed thalline：Appropriate Binding buffer are added, thalline is suspended, through cryogenic high pressure by fully shaking
After cell crushing instrument squeezing is crushed thalline, it is placed in the progress abundant suspended bacterias of ultrasound Binding buffer in sonicator
Body, broken with ultrasonic cell disruption instrument under the conditions of 4 DEG C, power 200W, work 4s, interval 5s, recurring number 99, directly
To super clear, the 10000rpm by bacterium solution, centrifuges 30 minutes, supernatant is transferred in clean triangular flask at 4 DEG C；
5) upper prop：Supernatant in previous step is all added in batches in prior activated Ni-NTA affinity purifications column,
Until whole liquid are flowed down by pillar；
6) it washs：With Washing buffer (25mM Tris-Cl, 150mM NaCl, 100mM imidazole) by column
Son is washed twice；
7) it elutes：It is eluted with Elution buffer (25mM Tris-Cl, 150mM NaCl, 250mM imidazole),
And eluent is collected in the EP pipes that 1.5mL is pre-chilled in advance every time, is positioned on ice.
8) it dialyses：Well-done bag filter is cleaned up with deionized water, the protein eluate being collected into is injected and is dialysed
In bag, both ends are clipped with clip, be put into dialyzate (10mM Tris-Cl, 100mM NaCl) (DTT is added in dialyzate, with
Prevent albumen from being aoxidized), dialysed overnight at 4 DEG C；
9) it dispenses：Protein solution is dispensed, -80 DEG C of preservations；Run SDS-PAGE glue come detect albumen purifying situation and into
Row insecticidal activity detects；
The insect bioassay of YpkA303 purifying proteins：
In order to detect the insecticidal activity of the insecticidal proteins YpkA303 obtained through prokaryotic expression, to greater wax moth, bollworm, sweet tea
The method that dish noctuid and corn borer are used uniformly hemocoel injection.Using micro syringe from the right abdominal foot of the second couple of 5 ages big larva
A concentration of 0.00 μ g/g of 50 μ L of injection injection, 5.00 μ g/g, 10.00 μ g/g, 15.00 μ g/g, 20.00 μ g/g, 30.00 μ respectively
In g/g, 40.00 μ g/g protein solutions to haemocoele, control group injects 50 μ L insect physiological saline, and the larva after injection is positioned over
In 37 DEG C of insulating boxs, every group takes similar 30 larvas of weight, records body weight, and experiment every time repeats 3 times.It is observed every 2h
Larva state, interior death condition, statistical result simultaneously carry out data processing to record with SPSS Statistics softwares for 24 hours.
YpkA303 purifying protein desinsection data analyses LC50 the results detailed in Table 6.
Table 6：YpkA303 albumen is to greater wax moth, bollworm, beet armyworm, corn borer insecticidal activity assay result
| YpkA303 | LC50μg/g | 95%limiteds |
| Greater wax moth | 10.00 | 20.00 |
| Bollworm | 10.00 | 20.00 |
| Beet armyworm | 5.00 | 10.00 |
| Corn borer | 15.00 | 30.00 |
The foregoing is only a preferred embodiment of the present invention, protection scope of the present invention is without being limited thereto, it is any ripe
Those skilled in the art are known in the technical scope of present disclosure, the letter for the technical solution that can be become apparent to
Altered or equivalence replacement are each fallen in protection scope of the present invention.
Sequence table
<110>Xibei Univ. of Agricultural ＆ Forest Science ＆ Technology
<120>A kind of artificial tuberculosis yersinia genus insecticidal proteins and its encoding gene and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 81
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Met Lys Pro Leu Asp Gln Asn Ile Thr Tyr Gly Gln Ala Arg Gly Tyr
1 5 10 15
Glu Gln Phe Tyr Ile Asp Glu Phe Gly Thr Lys Thr Gly Val Arg Gly
20 25 30
Asp Asp Ile Ser Pro Met Asn Arg Gly Asn Lys Ile Asn Ser Tyr Asp
35 40 45
Ser Asn Ser Thr Thr Arg Asp Pro Ile Arg Gln Gln Tyr Phe Asp Asp
50 55 60
Ala Tyr Asp Ser Lys Lys Ser Gly Thr Ser Ser Lys Gly Gly Gly Gly
65 70 75 80
Cys
<210> 2
<211> 246
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
atgaaaccgt tagatcagaa tattacatat ggtcaagctc gtggctatga gcagttttat 60
attgatgagt ttggaaccaa aactggggtg aggggagatg atatatcacc aatgaatcga 120
ggaaataaaa taaactccta cgatagtaat agtacaactc gtgatccaat aagacagcaa 180
tattttgatg atgcttatga tagtaagaaa agtggtacga gctcaaaagg tggcggagga 240
tgttaa 246
<210> 3
<211> 34
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
cgcggatcca tgaaaccgtt agatcagaat atta 34
<210> 4
<211> 34
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ccgctcgagt taacatcctc cgccaccttt tgag 34
Claims (5)
1. a kind of artificial tuberculosis yersinia genus insecticidal proteins, which is characterized in that by SEQ ID NO:Amino acid sequence group shown in 1
At protein, or in SEQ ID NO:One or several amino acid are substituted, lack or are inserted into amino acid sequence shown in 1
What sequence was obtained has same active protein.
2. a kind of encoding gene of artificial tuberculosis yersinia genus insecticidal proteins described in claim 1, which is characterized in that its nucleosides
Acid sequence such as SEQ ID NO:Shown in 2.
3. application of the artificial tuberculosis yersinia genus insecticidal proteins in insecticide preparation process described in claim 1.
4. the encoding gene of artificial tuberculosis yersinia genus insecticidal proteins answering in insecticide preparation process described in claim 2
With.
5. the induction of artificial tuberculosis yersinia genus insecticidal proteins and purification process described in a kind of claim 1, which is characterized in that packet
Include following steps：
1) it activates：Protein expression strain e. coli bl21 (DE3) pET28a-YpkA303 that will have been built, chooses single bacterium colony,
It is inoculated in containing in appropriate Km 5mL LB liquid mediums, is cultivated to plateau in 220rpm, 37 DEG C of shaking tables；
2) switching expands culture：Bacterium is transferred in the 1L of 500mL LB liquid mediums containing appropriate Km in 1% ratio
In triangular flask, 37 DEG C, 180rpm shakes culture；OD600 is detected, until when 0.6, shaking table temperature is adjusted to 26 DEG C, and press final concentration
Derivant IPTG is added in 0.5mM, starts to induce；It is incubated overnight, after 16-18 hours, induction finishes；
3) bacterium is received：Thalline is collected at 4 DEG C with refrigerated centrifuge, 8000rpm 10 minutes, repeats this step, until being collected into institute
There is the thalline in bacterium solution, then uses Binding buffer washing thallines twice；
4) it is crushed thalline：Appropriate Binding buffer are added, thalline is suspended, through cryogenic high pressure cell by fully shaking
After broken instrument squeezing is crushed thalline, the progress abundant suspension thallines of ultrasound Binding buffer in sonicator are placed in,
Broken with ultrasonic cell disruption instrument under the conditions of 4 DEG C, power 200W, work 4s, interval 5s, recurring number 99, until will
Bacterium solution is super clear, 10000rpm, centrifuges 30 minutes, supernatant is transferred in clean triangular flask at 4 DEG C；
5) upper prop：Supernatant in previous step is all added in batches in prior activated Ni-NTA affinity purifications column, until
Whole liquid are flowed down by pillar；
6) it washs：Pillar is washed twice with Washing buffer；
7) it elutes：It is eluted, and eluent is collected in the EP pipes that 1.5mL is pre-chilled in advance every time, is placed with Elution buffer
In on ice；
8) it dialyses：Well-done bag filter is cleaned up with deionized water, the protein eluate being collected into is injected in bag filter,
Both ends are clipped with clip, are put into dialyzate 10mM Tris-Cl, 100mM NaCl, DTT are added in dialyzate, to prevent albumen
It is aoxidized, dialysed overnight at 4 DEG C；
9) it dispenses：Protein solution is dispensed, -80 DEG C of preservations；SDS-PAGE glue is run to detect the purifying situation of albumen and be killed
Worm Activity determination.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111569047A (en) * | 2020-05-15 | 2020-08-25 | 西北农林科技大学 | Protein type manganese ion chelating agent, immunosuppressant and application |
| CN114921438A (en) * | 2022-05-12 | 2022-08-19 | 西北农林科技大学 | Yersinia pseudotuberculosis effector protein and application thereof |
| CN116004493A (en) * | 2022-09-21 | 2023-04-25 | 福建农林大学 | Genetically modified yersinia pestis engineering bacteria and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104311648A (en) * | 2014-10-29 | 2015-01-28 | 中国农业科学院作物科学研究所 | Insecticidal protein as well as coded gene and application of insecticidal protein |
| CN107022010A (en) * | 2017-04-06 | 2017-08-08 | 中国农业科学院植物保护研究所 | A kind of new insecticidal proteins and its nucleotide sequence |
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| CN104311648A (en) * | 2014-10-29 | 2015-01-28 | 中国农业科学院作物科学研究所 | Insecticidal protein as well as coded gene and application of insecticidal protein |
| CN107022010A (en) * | 2017-04-06 | 2017-08-08 | 中国农业科学院植物保护研究所 | A kind of new insecticidal proteins and its nucleotide sequence |
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| NONE: "Accession NO.: WP_012303460.1，hypothetical protein [Yersinia pseudotuberculosis]", 《GENBANK》 * |
| 周波 等: "《基础生物学实验教程》", 31 October 2016, 中国林业出版社 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111569047A (en) * | 2020-05-15 | 2020-08-25 | 西北农林科技大学 | Protein type manganese ion chelating agent, immunosuppressant and application |
| CN111569047B (en) * | 2020-05-15 | 2023-02-10 | 西北农林科技大学 | Protein type manganese ion chelating agent, immunosuppressant and application |
| CN114921438A (en) * | 2022-05-12 | 2022-08-19 | 西北农林科技大学 | Yersinia pseudotuberculosis effector protein and application thereof |
| CN116004493A (en) * | 2022-09-21 | 2023-04-25 | 福建农林大学 | Genetically modified yersinia pestis engineering bacteria and application thereof |
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| Publication number | Publication date |
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| CN108586587B (en) | 2020-05-05 |
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