CN110294794A - A kind of Rhodopseudomonas palustris GroEL albumen and its preparation method and application - Google Patents
A kind of Rhodopseudomonas palustris GroEL albumen and its preparation method and application Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
-
- 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
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Abstract
The invention discloses a kind of Rhodopseudomonas palustris GroEL albumen and its preparation method and application, belong to biological pesticide technical field；The Rhodopseudomonas palustris GroEL albumen prokaryotic expression and is obtained by protein purification in Escherichia coli by Rhodopseudomonas palustris GROEL gene；The Rhodopseudomonas palustris is Rhodopseudomonas palustris Rhodopseudomonas pal ustris LY-6 bacterial strain, is stored in China typical culture collection center, and deposit number is CCTCC NO:M2014525, and the deposit date is on October 29th, 2014.There is no in existing biological pesticide effectively carry out Pyricularia oryzae prevention and treatment aiming at the problem that, the present invention provides a kind of Rhodopseudomonas palustris GroEL albumen and its preparation method and application, it can effectively inhibit Pyricularia oryzae.
Description
Technical field
The invention belongs to biological pesticide technical fields, specifically, be related to a kind of Rhodopseudomonas palustris GroEL albumen and
Preparation method and application.
Background technique
Molecular chaperones are a kind of unstable conformations that can identify other oroteins, and by being effectively combined and releasing therewith
It puts, promotes its stable functional protein.Currently, molecular chaperones are divided into according to the size of its sequence, structure and function
Hsp100 (Heat shock protein 100), Hsp90, Hsp70, Hsp60 and sHsp (small heat shock protein) five are big
Class.Hsp60 is also known as 60 albumen of chaperonin, and GroEL is named as in prokaryotes, which is homotype oligomeric complex, by
14 identical molecular mass (57ku) subunits form bicyclic cylindrical-shaped structure, and 7 subunits form a ring, and two rings are to lean against
The form of back combines, and forms two cavitys.It is trans- that GroEL combines polypeptide to form ternary with the help of auxiliary molecular chaperones GroES
Complex, GroES are released in trans- ring, in conjunction with other GroEL molecules, after peptide substrate is separated, in conjunction with
Polypeptide is released to particular space, completes segmentation there and folds.This series of process shows that Chaperonin GroEL exists
It plays a significant role in the folding and assembling of new polypeptide chain and the renaturation process of albuminate, and the folding of partially protein
Folded stringent dependence GroEL, cannot be substituted by other molecular chaperones.
GroEL in addition to playing a significant role in protein folding, interaction between microorganism and host and
Certain effect is also functioned in terms of plant resistance to environment stress.Brood cell's bacillus fusiformis is the pathogen for causing diarrhea and colitis, in Mice Body
Inside being immunized after the GroEL albumen of the pathogen can significantly reduce pathogen and colonizes mouse is intracorporal, show that GroEL is improved
The immunocompetence of mouse.Kanchan et al. has found, after the bacillus anthracis GroEL albumen of mouse internal injection recombination, can improve
Immunogene lgG1, lgG2a and lgG2b content in Mice Body, and significantly improve survival energy of the mouse after bacillus anthracis infects
Power.GroEL is immune in other hosts induction, improves and also obtains similar result to the research of pathogenetic bacteria infection ability.Currently,
In addition on animal, GroEL also has been reported that Genes For Plant Tolerance disease pest ability is improved.The study found that the GroEL of Bemisia tabaci can with it is more
A virion combines that it is interfered to cause a disease, and after the GroEL that Bemisia tabaci is expressed in tobacco, can significantly reduce tomato yellowing
The generation of leaf curl viral disease (TYLCV) and Cucumber Mosaic Virus (CMV).Xenorhabdus nematophilus secretes a kind of and large intestine bar
Bacterium GroEL homologous XnGroEL albumen, growth and development stops after bollworm eats the albumen, further in tobacco gene group
After expressing XnGroEL albumen, the cotton bollworm larvae death rate can be made to reach 100%, reduce plant insect pest 55-70%.This is a series of
Result of study show GroEL as an inhibiting factor, can inhibit pathogen and insect colonize and growth and development.Mesh
Before, GroEL does not appear in the newspapers to the inhibition of disease fungus also.
Rice blast is one of important disease of rice, and the disease is widely distributed, and there are about more than 80 countries and regions hairs in the whole world
It is sick this, also there are generation in China South And North Rice Regions.Plant disease epidemic area, the general underproduction 10%~20%, up to 40% when serious
~50%, especially even No kernels or seeds are gathered, as in a year of scarcity for heavy field.Prevention and treatment for rice blast, at present or based on chemical prevention, but by
Enhance in drug resistance and find that applications of pesticide pole is not scientific and reasonable not in time, applications of pesticide dosage and number severely exceed, and draw
Play the significant problems such as environmental pollution and agricultural product quality and safety.Therefore, pesticide dosage and green composite prevention and control rice how to be reduced
Seasonal febrile diseases are still the major issue faced in current China's Rice Production.
Biological control is nontoxic with its, it is harmless, pollution-free, be not likely to produce drug resistance and it is efficient the advantages that, in plant pest
It is increasingly valued by people in prevention and treatment.Had been achieved in terms of the biological control research of rice blast at present it is certain into
Exhibition.In microbial source biological pesticide, have now been found that the microorganisms such as bacillus, fungus Trichoderma and its metabolite can have
The nutrient growth of the inhibition Pyricularia oryzae of effect.
Summary of the invention
1, it to solve the problems, such as
Aiming at the problem that there is no effectively progress Pyricularia oryzae prevention and treatment in existing biological pesticide, it is red that the present invention provides a kind of marsh
Pseudomonad GroEL albumen and its preparation method and application, it can effectively inhibit Pyricularia oryzae.
2, technical solution
To solve the above problems, the present invention adopts the following technical scheme that.
A kind of Rhodopseudomonas palustris GroEL albumen, the Rhodopseudomonas palustris GroEL albumen are red false single by marsh
Born of the same parents' bacterium GROEL gene expression obtains.
Preferably, the Rhodopseudomonas palustris is Rhodopseudomonas palustris Rhodopseudomonas palustris
LY-6 is stored in China typical culture collection center, and deposit number is CCTCC NO:M2014525, and the deposit date is 2014
On October 29, in.
A kind of preparation method of Rhodopseudomonas palustris GroEL albumen,
The following steps are included:
The Rhodopseudomonas palustris GroEL albumen is by Rhodopseudomonas palustris GROEL gene in Escherichia coli Central Plains
Nuclear expression is simultaneously obtained by protein purification.
Preferably, the step of prokaryotic expression are as follows:
(1) nucleotide sequence of Rhodopseudomonas palustris GROEL gene carrier construction: is connected to drug containing resistant gene
Plasmid on；
(2) Escherichia coli convert: the plasmid after building in step (1) being imported in expression competent cell, is coated on and contains
It is cultivated on the LB plate of drug resistance；
(3) conversion bacterial strain spread cultivation with it is protein induced: the single colonie turned out in step (2) is chosen and continues to cultivate to stabilization
Phase；Then it spreads cultivation, culture to logarithmic growth phase is added IPTG, induces the expression of destination protein.
Preferably, the plasmid of the drug containing resistant gene is PET32a；The LB plate of the drug containing resistance is
The LB plate of the resistance of benzyl containing ammonia.
Preferably, the culture medium of the LB plate of the resistance of benzyl containing ammonia, formula are as follows: yeast extract 5g/L, peptone
10g/L, NaCl 5g/L, ammonia benzyl 100mg/L, agar 15g/L.
Preferably, the final concentration of 1M of the IPTG；The temperature of the induction is 25 DEG C.
Preferably, the step of protein purification are as follows: the bacterium solution after will be protein induced in step (3) carries out centrifugation and column
Purification process.
Preferably, the column purification processing is to cross column purification albumen using His label.
A kind of Rhodopseudomonas palustris GroEL albumen is inhibiting the application in Pyricularia oryzae.
3, beneficial effect
Compared with the prior art, the invention has the benefit that
(1) the Rhodopseudomonas palustris GroEL albumen described in is by Rhodopseudomonas palustris GROEL gene for the first time in large intestine bar
It prokaryotic expression and is obtained by protein purification in bacterium, simple production process, incubation time are short, at low cost；
(2) the Rhodopseudomonas palustris GroEL albumen finds that rice blast pathogen conidiospore attachment can be effectively suppressed for the first time
The formation of born of the same parents, and can also effectively inhibit the pathogenic of Pyricularia oryzae, and have it is environmentally friendly, nontoxic to people and animals, to crop
The features such as no phytotoxicity, application simple and convenient, at low cost.
Detailed description of the invention
Fig. 1 is the SDS-PAGE adhesive tape band of GroEL albumen in embodiment 1；
Fig. 2 is influence of the GroEL albumen to rice blast pathogen conidiospore note fields rate in embodiment 2；
Fig. 3 is influence of the GroEL albumen to rice blast bacterium pathogenicity in embodiment 2；
Fig. 4 is that FabA albumen, BamA albumen and GroEL albumen respectively adhere to rice blast pathogen conidiospore in embodiment 3
The influence of born of the same parents' formation rate.
Specific embodiment
The present invention is further described below combined with specific embodiments below.
Material employed in following embodiment and instrument are commercially available.Rhodopseudomonas palustris described herein
GroEL albumen (is originated from Chaperonin GroEL, base-pair 1641bp, molecular weight of albumen by Rhodopseudomonas palustris GROEL gene
60. 2kD) expression obtains, and the Rhodopseudomonas palustris is Rhodopseudomonas palustris Rhodopseudomonas
Palustris LY- 6, is stored in China typical culture collection center, and deposit number is CCTCC NO:M2014525, preservation
Date is on October 29th, 2014.
Embodiment 1
The preparation method of Rhodopseudomonas palustris GroEL albumen,
The following steps are included:
(1) Rhodopseudomonas palustris GROEL gene prokaryotic expression in Escherichia coli
Carrier construction: the nucleotide sequence of Rhodopseudomonas palustris GROEL gene is connected to containing ampicillin resistance
On PET32a plasmid；
Escherichia coli conversion: the plasmid after constructing in previous step is imported in expression competent cell BL21, is coated on
It is cultivated on the LB plate of the resistance of benzyl containing ammonia；The culture medium of the LB plate of the resistance of benzyl containing ammonia, formula are as follows: yeast mentions
Take object 5g/L, peptone 10g/L, NaCl 5g/L, ammonia benzyl 100mg/L, agar 15g/L, pH=7.0；
Conversion bacterial strain spread cultivation with it is protein induced: the single colonie that will be turned out in previous step accesses in 120mL conical flask,
37 DEG C were cultivated to stationary phase；Then the bacterium solution in stationary phase is inoculated into another conical flask and is spread cultivation, inoculum concentration is total
The 5% of volume, condition of culture are 37 DEG C, pH=7.0, and the IPTG of final concentration of 1M is added to logarithmic growth phase in culture, 25 DEG C
Induce the expression of destination protein；
(2) purifying of Rhodopseudomonas palustris GroEL albumen
Bacterium solution after will be protein induced in previous step is centrifuged, so that bacterium broken wall, it is pure to cross column followed by His label
Change albumen, and by SDS-PAGE glue verifying purpose protein expression, as shown in Figure 1, GroEL albumen is able to effective expression and pure
Change.
Embodiment 2
Rhodopseudomonas palustris GroEL albumen is inhibiting the application in Pyricularia oryzae
(1) Rhodopseudomonas palustris GroEL albumen is inhibiting the application on rice blast pathogen conidiospore note fields
(final concentration is respectively 0 μ g/ml, 50 μ g/ml, 1 to Rhodopseudomonas palustris GroEL albumen prepared by selection example 1
00 μ g/ml, 200 μ g/ml, 500 μ g/ml), it is added to rice blast pathogen conidiospore liquid (1 × 105A/ml) in mixed
It closes, then takes 30 μ l to be added drop-wise in hydrophobic membrane, each group (corresponds to CK group, 50 μ g/ml groups, 100 μ g/ml groups, 200 μ g/ml
Group, 500 μ g/ml groups) 3 repetitions are set, microexamination and its note fields rate is counted after 8 hours, evaluates and respectively inhibit effect
Fruit；
As shown in Figure 2, the results showed that adhere to after the Rhodopseudomonas palustris GroEL albumen processing of final concentration of 500ug/mL
There were significant differences for the formation rate and CK group of born of the same parents and other processing groups, shows that GroEL albumen has conspicuousness to the formation of appresorium
Inhibitory effect, have a good application prospect.
(2) application of the Rhodopseudomonas palustris GroEL albumen in rice blast bacterium pathogenicity
Rhodopseudomonas palustris GroEL albumen (final concentration of 500 μ g/mL) prepared by selection example 1, is added to containing matter
Measure the rice blast pathogen conidiospore liquid (1 × 10 that score is 0.2% gelatin5A/ml) in and spore suspension is made, use larynx
Spore suspension is equably sprayed on 2 weeks or so healthy rice leafs of growth by sprayer, then by 28 DEG C of rice immigration,
In dark, high humidity (RH > 95%) container after culture for 24 hours, it is placed in alternation of light and darkness (each 12h) and the container of high humidity and continues to train
It supports, setting CK is compareed and (replaced ATSP2 albumen with the water of isodose) and PBS control (is replaced with the PBS buffer solution of isodose
GroEL albumen), each processing sets 3 repetitions, observes result after 7d.
As shown in figure 3, can obviously observe that marsh is red from rice leaf after CK control is handled respectively with PBS control
The typical scab of pseudomonad, and typical Lesion number is more, and after the processing of GroEL albumen, corresponding scab is significantly smaller,
And quantity is few, it is pathogenic on rice that this shows that the processing of GroEL albumen can significantly inhibit Pyricularia oryzae.
Embodiment 3
Referring in embodiment 2, " Rhodopseudomonas palustris CroEL albumen is inhibiting rice blast pathogen conidiospore note fields
On application ", after prokaryotic expression, respectively select concentration be 500ug/mL3- hydroxyl acyl dehydratase FabA, outer membrane protein group
Dress factor B amA and GroEL albumen is tested, its note fields rate is counted.Wherein 3- hydroxyl acyl dehydratase FabA
From 3- hydroxyl acyl group-[acyl catenin] dehydratase, base-pair 525bp, molecular weight of albumen 18.8kD；Wherein outer membrane protein
It assembles factor B amA and is originated from the outer membrane protein assembling factor, base-pair 2454bp, molecular weight of albumen 93.5kD.
As shown in figure 4, rice blast pathogen conidiospore note fields rate only reaches 75% or so after the processing of GroEL albumen,
Substantially less than CK compare (* * represents extremely significant difference), and FabA albumen and BamA albumen processing after with CK contrast ratio indifference；
Compared with FabA albumen and BamA albumen, test result shows there was only GroEL egg in three kinds of albumen in Rhodopseudomonas palustris
It is white that effect is significantly inhibited to Pyricularia oryzae.
The above content is specific embodiment is combined, invention is further described in detail, and it cannot be said that the present invention is specific
Implementation is only limited to these instructions, and for those of ordinary skill in the art to which the present invention belongs, is not departing from the present invention
Design under the premise of, several simple deduction or replace can also be made, all shall be regarded as belonging to the power submitted of the present invention
The protection scope that sharp claim determines.
Claims (10)
1. a kind of Rhodopseudomonas palustris GroEL albumen, it is characterised in that: the Rhodopseudomonas palustris GroEL albumen by
Rhodopseudomonas palustris GROEL gene expression obtains.
2. Rhodopseudomonas palustris GroEL albumen according to claim 1, it is characterised in that: the marsh is red false single
Born of the same parents bacterium is Rhodopseudomonas palustris Rhodopseudomonas palustris LY-6 bacterial strain, is stored in Chinese Typical Representative culture
Collection, deposit number are CCTCC NO:M2014525, and the deposit date is on October 29th, 2014.
3. a kind of preparation method of Rhodopseudomonas palustris GroEL albumen as claimed in claim 1 or 2, it is characterised in that:
The following steps are included:
The Rhodopseudomonas palustris GroEL albumen is by Rhodopseudomonas palustris GROEL gene protokaryon table in Escherichia coli
It reaches and is obtained by protein purification.
4. the preparation method of Rhodopseudomonas palustris GroEL albumen according to claim 3, it is characterised in that:
The step of described prokaryotic expression are as follows:
(1) nucleotide sequence of Rhodopseudomonas palustris GROEL gene carrier construction: is connected to the matter of drug containing resistant gene
On grain；
(2) Escherichia coli convert: the plasmid after building in step (1) being imported in expression competent cell, drug containing is coated on
It is cultivated on the LB plate of resistance；
(3) conversion bacterial strain spread cultivation with it is protein induced: the single colonie turned out in step (2) is chosen and continues to cultivate to stationary phase；
Then it spreads cultivation, culture to logarithmic growth phase is added IPTG, induces the expression of destination protein.
5. the preparation method of Rhodopseudomonas palustris GroEL albumen according to claim 4, it is characterised in that:
The plasmid of the drug containing resistant gene is PET32a；The LB plate of the drug containing resistance is the resistance of benzyl containing ammonia
LB plate.
6. the preparation method of Rhodopseudomonas palustris GroEL albumen according to claim 5, it is characterised in that:
The culture medium of the LB plate of the resistance of benzyl containing ammonia, formula are as follows: yeast extract 5g/L, peptone 10g/L, NaCl
5g/L, ammonia benzyl 100mg/L, agar 15g/L.
7. the preparation method of Rhodopseudomonas palustris GroEL albumen according to claim 4, it is characterised in that:
The final concentration of 1M of the IPTG；The temperature of the induction is 25 DEG C.
8. the preparation method of Rhodopseudomonas palustris GroEL albumen according to claim 4, it is characterised in that:
The step of described protein purification are as follows:
Bacterium solution after will be protein induced in step (3) carries out centrifugation and column purification processing.
9. the preparation method of Rhodopseudomonas palustris GroEL albumen according to claim 8, it is characterised in that:
The column purification processing is to cross column purification albumen using His label.
10. a kind of Rhodopseudomonas palustris GroEL albumen as described in claim 1 is inhibiting the application in Pyricularia oryzae.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111363056A (en) * | 2020-03-17 | 2020-07-03 | 湖南省植物保护研究所 | Rhodopseudomonas palustris exopolysaccharide and preparation method and application thereof |
| WO2024060118A1 (en) * | 2022-09-22 | 2024-03-28 | Bened Biomedical Co., Ltd. | Method for treating sleeping disorders with groel protein |
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| CN105794853A (en) * | 2016-05-19 | 2016-07-27 | 湖南省植物保护研究所 | Application of rhodopseudomonas palustris fermentation liquor to rice blast prevention and control |
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| CN105794853A (en) * | 2016-05-19 | 2016-07-27 | 湖南省植物保护研究所 | Application of rhodopseudomonas palustris fermentation liquor to rice blast prevention and control |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111363056A (en) * | 2020-03-17 | 2020-07-03 | 湖南省植物保护研究所 | Rhodopseudomonas palustris exopolysaccharide and preparation method and application thereof |
| CN111363056B (en) * | 2020-03-17 | 2021-07-09 | 湖南省植物保护研究所 | Rhodopseudomonas palustris exopolysaccharide and preparation method and application thereof |
| US11473115B2 (en) | 2020-03-17 | 2022-10-18 | Hunan Plant Protection Institute | Exopolysaccharide from Rhodopseudomonas palustris and method for preparing and use thereof |
| WO2024060118A1 (en) * | 2022-09-22 | 2024-03-28 | Bened Biomedical Co., Ltd. | Method for treating sleeping disorders with groel protein |
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