CN109258693A - Application of the recombinant chitinase at desinsection or antibacterial aspect - Google Patents
Application of the recombinant chitinase at desinsection or antibacterial aspect Download PDFInfo
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- CN109258693A CN109258693A CN201811162807.XA CN201811162807A CN109258693A CN 109258693 A CN109258693 A CN 109258693A CN 201811162807 A CN201811162807 A CN 201811162807A CN 109258693 A CN109258693 A CN 109258693A
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- chitinase
- chtii
- desinsection
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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
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- Microbiology (AREA)
- Biotechnology (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Agronomy & Crop Science (AREA)
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Abstract
The present invention discloses a kind of recombinant chitinase in the application of desinsection or antibacterial aspect, and the recombinant chitinase is only present in the insect, the recombinant chitinase ChtII-B4C1 of Multidomain that has；It has been experimentally confirmed its new role in terms of biological pest control, wherein, there is insecticidal action to Frankliniella occidentalis, it has additionally been experimentally confirmed it and good bacteriostatic activity is shown to capsicum anthrax, tomato early epidemic, Curvularia etc., show that chitinase ChtII-B4C1 has the activity of antibacterial desinsection, plays a significant role and application value in terms of agriculture field is applied to biological pest control.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of recombinant chitinase in insect source is in desinsection or antibacterial
The application of aspect.
Background technique
Chitin (chitin) is the straight chain line being polymerized by β-N-acetylglucosamine by β-(1,4) glycosidic bond
Property high polymer, is fungal cell wall, the main component of insect cuticle.It is that content is only second to the natural of cellulose in nature
Polysaccharide, and at least 10,000,000,000 tons of annual output (microorganism chitinase progress [J], Agriculture of Anhui science, 2010,38:11685-
11688)。
Chitinase (chitinase) is the enzyme of chitin of can degrading, and is widely distributed in using chitin as carbon source
Microorganism and using chitin as the fungi of constituent, in shellfish and insect.Furthermore in higher plant, high dynamic
There is also chitinases in object.The study found that chitinase has the function of preventing and treating pest and disease damage.It can be thin by hydrolysis fungi
Chitin in cell wall inhibits growth (application [J] of the fungal source chitinase in plant resistance to fungal disease, the plant of fungi
Protection, 2017,43:29-35)；It can inhibit the normal hair of insect by the chitin in degradation insect midgut funnel
Educate, improve pest to the sensibility of insecticide (insect chitinase and its application [J] in plant protection, insect knowledge,
2000,37:314-317)；It can play the role of preventing and treating nematode (microorganism chitin by the chitin in degradation line eggs
Application [J] of the enzyme in fields such as plant nematode diseases prevention and treatments, Exploitation of Agriculture in Heilongjiang science, 2015,11:153-155).Therefore, several
Fourth matter enzyme is widely used in field of biological control, and for disease and pest control, (application study of chitinase agriculturally is general
Condition [J], agriculture in south, 2017,11:41-43).
In recent years, with the research to proteomics, discovery insect is responsible for degrading in the ecdysial fluid of epidermis, and there are one
Kind has the chitinase ChtII of multiple chitin binding structural domains and catalyst structure domain.Further study show that it contains three
The truncated protein ChtII-B4C1 of a chitin binding structural domain and a chitinase catalyst structure domain has very high hydrolysis
The activity of chitin, being of great significance for development and utilization chitin resource, (a kind of Multidomain inscribe in insect source is several
Fourth matter enzyme, its relevant biomaterial and its application [P], China, 201610803226,2016.09.06), but related chitin
Research of the enzyme ChtII-B4C1 in terms of desinsection and bacteriostatic activity still belongs to blank.
Summary of the invention
The invention discloses the truncated protein ChtII-B4C1 of chitinase ChtII to have new desinsection and bacteriostatic activity,
Pest management is carried out using it to be of great significance.
Firstly, the present invention discloses a kind of application of the recombinant chitinase from insect at desinsection or antibacterial aspect, institute
Recombinant chitinase is stated containing there are three chitin binding structural domain and a chitinase catalyst structure domains.Specifically, described heavy
Group chitinase is the Multidomain endochitinase ChtII-B4C1 in insect source.
Further, present invention pathogen described above includes capsicum anthrax (Colletotrichun
Gloeosporioides), tomato early epidemic (Alternaria solani) or Curvularia (Curvularia lunata).It is real
It applies and has detected the solution containing chitinase ChtII-B4C1 or preparation in example to the inhibitory effect of a variety of pathogens, wherein is right
Capsicum anthrax, tomato early epidemic, Curvularia etc. show stronger bacteriostatic activity, especially reach to the inhibiting rate of capsicum anthrax
70%.The antibacterial recombinant chitinase concentration is the protein solution of 10~30ppm.In the case where being more highly preferred to, embodiment
Used in recombinant chitinase concentration be 20ppm protein solution.
Further, chitinase ChtII-B4C1 disclosed by the invention has insecticidal activity to model insects Frankliniella occidentalis.
The recombinant chitinase concentration of the desinsection is the protein solution of 80~120ppm.
Further, the dilution of recombinant chitinase described above is the 20mM phosphate buffer of pH7.4.
Beneficial outcomes of the invention are that chitinase ChtII-B4C1 realizes control and pathogenic microorganism to pest
Inhibit, especially outstanding is during bacteriostatic experiment, and used recombinant chitinase concentration is only that the albumen of 20ppm is molten
Liquid plays a significant role in terms of biological pest control.
Detailed description of the invention
The insecticidal activity of Fig. 1 chitinase ChtII-B4C1；
The bacteriostatic activity of Fig. 2 chitinase ChtII-B4C1.
Specific embodiment
Following nonlimiting examples can with a person of ordinary skill in the art will more fully understand the present invention, but not with
Any mode limits the present invention.Experimental method used in embodiment is conventional method unless otherwise specified, used
Material, reagent unless otherwise specified, can obtain from commercial channels.
The insecticidal activity of 1. recombinant chitinase ChtII-B4C1 of embodiment
To the toxicity test of Frankliniella occidentalis: recombinant chitinase ChtII-B4C1 is dissolved in phosphate buffer (20mM
Na2HPO4, pH7.4) in, make its final concentration of 100ppm, is made into medical fluid.ChtII-B4C1 solution is filled into 1.5mL centrifuge tube,
Medical fluid is taken out after placing 4h, centrifuge tube is dried for use.The small of diameter about 2~3mm is scalded in the centrifuge tube tube bottom dried with fine needle
Hole.Fresh cabbage leaves are broken into the circular blade that diameter is 1.5cm with punch, 10s is soaked in medical fluid.Treated blade
It is sandwiched after drying with tweezers in the centrifuge tube of corresponding medical fluid, every pipe 1.It is that control carries out identical place with phosphate buffer
Reason.4 repetitions of each experimental group, every Guan Weiyi repetition.Centrifuge tube tube bottom will be passed through for the Frankliniella occidentalis of examination with vacuum pump
It scalds hole and sucks centrifuge tube.First control group post-processing, each centrifuge tube inhale 15 2 age nymphs, then cover pipe lid, sealed with sealed membrane
Hole is scalded well.It is 25 DEG C in temperature, is placed in the environment of illumination 16L: 8D.Every checking the death rate for 24 hours, polypide is touched with writing brush point
Cannot the person of creeping be considered as death.
It makes discovery from observation, with the extension of time, the test group that ChtII-B4C1 is added is not causing Ji a few days ago
The death of horse had a small amount of thrips dead at the 3rd, 4 day, showed apparent insecticidal activity to Frankliniella occidentalis at the 5th and the 6th day,
Have more than 40% thrips death (Fig. 1).
The bacteriostatic activity of 2 recombinant chitinase ChtII-B4C1 of embodiment
The suppression of recombinant chitinase ChtII-B4C1 (is carried out) referring to NY/T1156.2-2006 using growth rate method measurement
Bacterium activity.ChtII-B4C1 is dissolved in phosphate buffer (20mM Na2HPO4, pH7.4) in the final concentration of 20ppm of configuration egg
White solution.Respectively by target bacterium (cause of disease oomycetes: melon and fruit corruption mould (Pythium aphanidermatum), Phytophthora capsici
(Phytophthora capsici)；Disease fungus: rice banded sclerotial blight (Rhizoctonia solani), Curvularia
(Curvularia lunata), tomato early epidemic (Alternaria solani), tomato gray mould (Botrytis cinerea), rice
Seasonal febrile diseases bacterium (Magnaporthe grisea), capsicum anthrax (Colletotrichun gloeosporioides), watermelon are withered
(Fusarinm oxysporum f.sp.nimum) connects bacterium on PDA plate, preculture 6d at 25 ± 1 DEG C (condition of culture according to
Different target bacterium are adjusted).Then the punch for being 5mm with diameter is beaten on the same circumference close to colony edge and takes bacterium
Cake (guarantees that the same duplicate cell age for trying germ is identical), and is aseptically trained pure culture biscuits involvng inoculation to drug containing with transfer needle
Base plate center is supported, mycelia is face-down, is placed under dark and cultivates.It is compared so that the plate of equivalent phosphate buffer is added.Often
Processing is repeated 4 times measurement, measures colony diameter after cultivating 5d, finds out 4 duplicate average values, obtain the average colony of each processing
Increase diameter (i.e. average colony diameter -5mm).Calculate inhibiting rate of the medicament to target bacterium under experimental concentration.Inhibiting rate calculates
Method are as follows: the bacterium colony of (bacterium colony of control increases diameter-processing bacterium colony and increases diameter)/control increases diameter.
Inhibiting effect of the 1 chitinase ChtII-B4C1 of table to target bacterium
The result shows that the ChtII-B4C1 of 20ppm shows fabulous inhibitory activity to capsicum anthrax, inhibiting rate can reach
To 70% (table 1).Furthermore certain inhibitory effect is also shown to tomato early epidemic and Curvularia, illustrates that ChtII-B4C1 has
Bacteriostatic activity (Fig. 2).
Claims (7)
1. a kind of recombinant chitinase from insect is in the application of desinsection or antibacterial aspect, it is characterised in that: the recombination
Chitinase is containing there are three chitin binding structural domain and a chitinase catalyst structure domains.
2. application according to claim 1, it is characterised in that: the recombinant chitinase is ChtII-B4C1.
3. application according to claim 1, it is characterised in that: the pathogen includes capsicum anthrax
(Colletotrichun gloeosporioides), tomato early epidemic (Alternaria solani) or Curvularia
(Curvularia lunata)。
4. application according to claim 1, it is characterised in that: the antibacterial recombinant chitinase concentration be 10~
The protein solution of 30ppm.
5. application according to claim 1, it is characterised in that: the antibacterial recombinant chitinase concentration is 20ppm
Protein solution.
6. application according to claim 1, it is characterised in that: the insect of the killing is Frankliniella occidentalis.
7. application according to claim 6, it is characterised in that: the recombinant chitinase concentration of the desinsection be 80~
The protein solution of 120ppm.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811162807.XA CN109258693B (en) | 2018-09-30 | 2018-09-30 | Application of recombinant chitinase in insecticidal or bacteriostatic aspects |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811162807.XA CN109258693B (en) | 2018-09-30 | 2018-09-30 | Application of recombinant chitinase in insecticidal or bacteriostatic aspects |
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| Publication Number | Publication Date |
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| CN109258693A true CN109258693A (en) | 2019-01-25 |
| CN109258693B CN109258693B (en) | 2021-03-16 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101048626B1 (en) * | 2011-02-11 | 2011-07-12 | 한남대학교 산학협력단 | Microorganism expressing recombinant chitinase gene and uses thereof |
| WO2012063338A1 (en) * | 2010-11-10 | 2012-05-18 | 国立大学法人京都工芸繊維大学 | Protein having chitinase activity and uses thereof |
| CN102517308A (en) * | 2011-12-31 | 2012-06-27 | 太原理工大学 | Construction and application of recombinant plasmid containing chitinase gene of apple leaf miner |
| CN106350496A (en) * | 2016-09-06 | 2017-01-25 | 大连理工大学 | Multi-structural-domain endochitinase sourcing from insects, and related biological material and application of multi-structural-domain endochitinase |
-
2018
- 2018-09-30 CN CN201811162807.XA patent/CN109258693B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012063338A1 (en) * | 2010-11-10 | 2012-05-18 | 国立大学法人京都工芸繊維大学 | Protein having chitinase activity and uses thereof |
| KR101048626B1 (en) * | 2011-02-11 | 2011-07-12 | 한남대학교 산학협력단 | Microorganism expressing recombinant chitinase gene and uses thereof |
| CN102517308A (en) * | 2011-12-31 | 2012-06-27 | 太原理工大学 | Construction and application of recombinant plasmid containing chitinase gene of apple leaf miner |
| CN106350496A (en) * | 2016-09-06 | 2017-01-25 | 大连理工大学 | Multi-structural-domain endochitinase sourcing from insects, and related biological material and application of multi-structural-domain endochitinase |
Non-Patent Citations (1)
| Title |
|---|
| 李瑶 等: "昆虫几丁质酶及其在害虫防治中的应用", 《应用昆虫学报》 * |
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