CN107557476A - It is a kind of to analyze method of the thuricade-1 to target worm hypoactivity reason - Google Patents
It is a kind of to analyze method of the thuricade-1 to target worm hypoactivity reason Download PDFInfo
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- CN107557476A CN107557476A CN201710741934.4A CN201710741934A CN107557476A CN 107557476 A CN107557476 A CN 107557476A CN 201710741934 A CN201710741934 A CN 201710741934A CN 107557476 A CN107557476 A CN 107557476A
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- factor
- desinsection
- hemolymph
- thuricade
- hypoactivity
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Abstract
Method of the thuricade-1 to target worm hypoactivity reason is analyzed the invention discloses a kind of, healthy target worm some of the thuricade-1 to its hypoactivity is chosen, gathers target worm hemolymph；The bacillus thuringiensis preparation as insecticiding factor is fixed on molecular scaffold；Hemolymph is added to and is fixed with the molecular scaffold of the desinsection factor, the composition that can be combined in hemolymph with the desinsection factor is combined with the desinsection factor, and so as to be indirectly fixed on molecular scaffold, and the composition that can not be combined with the desinsection factor will not then be fixed；The composition combined in hemolymph with the desinsection factor is analyzed and identified, finds out the reason for thuricade-1 is to target worm hypoactivity, finds overcome approach accordingly accordingly.The present invention can seek to overcome existing production strain to provide foundation to the method for target worm hypoactivity for thuricade-1 manufacturer.
Description
Technical field
The invention belongs to thuricade-1 analysis technical field, and in particular to one kind analysis thuricade-1 pair
The method of target worm hypoactivity reason.
Background technology
China is the few agricultural production big country in arable land that has a large population, and the species of crop is more, and the species of harmful organism is also more.
The yield for improving unit cultivated area is the major measure for solving food problem, and agricultural chemicals is protecting crops from harmful organism
Played an important role in terms of harm, the resistance for improving crops and the agriculture volume increase of promotion.Although chemical pesticide does harm in preventing and treating
Significant economic benefit is obtained on worm, but over time, its drawback (harm to health, to beneficial organism
Killing, the pollution to environment and food and pest resistance to insecticide sharp increase etc.) become increasingly conspicuous, thus the application of biological insecticides and day
It is all to increase.
Thuricade-1 is microbial insecticide the most successful so far, has been widely used in agricultural sanitary insect pest
Preventing and treating.But still having many important agricultural sanitary insect pests, existing thuricade-1 is low to its preventive effect.It is each
The expenses for prevention and control of kind important pests is annual all hundreds of millions of, and the market space is very huge.Nevertheless, producer generally will not be easily
Production strain is changed, though its reason is a lack of corresponding effective strain or has its fermenting property of efficient bacterium bad or existing
Equipment can not meet its fermentation etc. with condition.Therefore, the reason for thuricade-1 is to target pest hypoactivity is analyzed,
And then seek corresponding conquering method, it is significant for thuricade-1 industry.
The blood of insect is the lymph sample liquid body of body cavity internal circulation flow, frequently referred to hemolymph, and its content is with worm kind and worm
State difference and change it is very big, typically constitute from the 15%~75% of body volume.Insect hemolymph with mammalian and
The dual-use function of lymph, there is transport and mechanical function, storage and metabolic function, defence and immunologic function.Hemolymph conduct
Insect defends the important place with being immunized, and important angle should be played in " fight " of target pest and thuricade-1
Color, but relevant report is had no at present, also thuricade-1 is analyzed to target worm hypoactivity reason not over hemolymph
Method.
The content of the invention
It is an object of the invention in place of overcome the deficiencies in the prior art, there is provided one kind analysis thuricade-1 pair
The method of target worm hypoactivity reason.
The technical solution adopted for the present invention to solve the technical problems is：
A kind of thuricade-1 of analyzing to the method for target worm hypoactivity reason, including：
1) healthy target worm some of the thuricade-1 to its hypoactivity is chosen, gathers target worm hemolymph；
2) the bacillus thuringiensis preparation as insecticiding factor is fixed on molecular scaffold：Weigh 0.18~0.22g Sepharose
It is added in the desinsection factor solutions that concentration is 0.8~1.2mg/mL, 100~200rpm is incubated 4~6h at room temperature；After the completion of incubation
9~11mg Glycine are added, continues 5~7h of incubation and is closed；After the completion of closing, the molecule of the desinsection factor is fixed
Support；
3) it is 7~9 according to the mass ratio of hemolymph and the molecular scaffold for being fixed with the desinsection factor:1 ratio, takes hemolymph
It is added to and is fixed with the molecular scaffold of the desinsection factor, 0~5 DEG C of 0.5~1.5h of incubation after mixing, during incubation, in hemolymph
The composition that can be combined with the desinsection factor is combined with the desinsection factor, so as to be indirectly fixed on molecular scaffold, and can not be with desinsection
The composition that the factor combines will not then be fixed；Then 1500~2500rpm centrifuges 25~35s at 0~5 DEG C, isolates blood strangury
The composition that Palestine and China can not be combined with the desinsection factor；
4) molecular scaffold that step 3) obtains is handled, made in hemolymph with the composition that the desinsection factor is combined from molecule
Discharged on support；
5) composition combined in the hemolymph obtained to step 4) with the desinsection factor analyzes and identifies：Carry out SDS-PAGE
Electrophoresis and coomassie brilliant blue staining, analyzed with the binding constituents to albumen, polypeptide, and use LC-MS analyzer pair
Albumen, component polypeptides carry out structural analysis identification；Enter row agarose gel electrophoresis and EB dyeing, to be combined into nucleic acid
Analyzed, and sequencing identification is carried out to nucleic acid constituents using nucleic acid sequencing instrument.
In one embodiment：The target worm is the instar larvae of beet armyworm 4.
In one embodiment：The desinsection factor is at least one of Cry7Ab4 albumen or Cry1Ab1 albumen.
In one embodiment：In the step 1), target worm is before hemolymph is gathered, prior to indoor feeding：Use man-made feeds
Raise in program-controlled manual climate box, 25 DEG C ± 1 DEG C of temperature, relative humidity 60 ± 5%, photoperiod L:D=16:8, intensity of illumination
≥6000Lux。
In one embodiment：In the step 1), the method for collection target worm hemolymph includes：Catch take the normal target worm of growth in
Place processing 10min on ice, target worm head gently clamped with eye scissors, be suspended from above sterile chamber, with another eye scissors in
Osculum is cut off at abdominal foot, the hemolymph for collecting target worm is instilled in sterile chamber.
In one embodiment：In the step 2), the Sepharose for weighing 0.2g is added to the desinsection factor that concentration is 1mg/mL
In solution, 150rpm is incubated 5h at room temperature；10mg Glycine are added after the completion of incubation, continues incubation 6h and is closed.
In one embodiment：In the step 3), the mass ratio of molecular scaffold of the hemolymph with being fixed with the desinsection factor is 8:
1；Hemolymph is added to be fixed with the molecular scaffold of the desinsection factor after 4 DEG C be incubated 1h；Then 2000rpm is centrifuged at 4 DEG C
30s, isolate the composition that can not be combined in hemolymph with the desinsection factor.
In one embodiment：In the step 4), the processing method to molecular scaffold is heating.
Compared with background technology, it has the following advantages that the technical program：
In hemolymph of the present invention by identifying target pest whether containing can with the desinsection of thuricade-1 because
The molecule that son combines, can be thuricade-1 to analyze the reason for thuricade-1 is to target worm hypoactivity
Manufacturer overcomes existing production strain to provide foundation and thinking to target worm hypoactivity.
Brief description of the drawings
The invention will be further described with reference to the accompanying drawings and examples.
A kind of analysis thuricade-1 that Fig. 1 is the present invention is illustrated to the flow of the method for target worm hypoactivity reason
Figure, selection target pest (target worm) → collection target worm hemolymph → is embodied respectively and consolidates the desinsection factor of thuricade-1
Be scheduled on molecular scaffold, make in hemolymph to combine the composition of the desinsection factor and other compositions separate → utilize analyzer
What the composition that the desinsection factor is combined in device identification hemolymph is, finds original of the thuricade-1 to target worm hypoactivity
Cause.
Embodiment
Present disclosure is illustrated below by embodiment：
A kind of thuricade-1 of analyzing to the method for target worm hypoactivity reason, including：
1) healthy target worm some of the thuricade-1 to its hypoactivity is chosen, gathers target worm hemolymph：This reality
Among applying example, the target worm of selection is the instar larvae of beet armyworm 4；Specifically,
Indoor feeding health target worm：Raised using man-made feeds in program-controlled manual climate box, 25 DEG C ± 1 DEG C of temperature, phase
To humidity 60 ± 5%, photoperiod 16:8(L:D), intensity of illumination >=6000Lux；
Catch and take the normal target borer population ten of growth, in placing processing 10min on ice, target worm head is gently clamped with eye scissors
Portion, it is suspended from above the 1.5mL EP pipes after sterilizing, osculum is cut off at abdominal foot with another eye scissors, allows the hemolymph of target worm to drip
Enter in EP pipes.The hemolymph of 30 beet armyworms is collected altogether, is sub-packed in 3 EP pipes, is put it into -80 DEG C of refrigerators and frozen
Deposit standby；
2) the bacillus thuringiensis preparation as insecticiding factor is fixed on molecular scaffold：Among the present embodiment, the molecule branch of selection
Frame is the Sepharose of GE companies, and the desinsection factor is Cry7Ab4 albumen；Specifically,
The Sepharose for weighing 0.2g is added in the Cry7Ab4 protein solutions that concentration is 1mg/mL, under room temperature (25 DEG C)
150rpm is incubated 5h；10mg Glycine are added after the completion of incubation, continues to be incubated 6h, closes in Sepharose and be not associated with
The group of Cry7Ab4 albumen；After the completion of closing, the molecular scaffold of the desinsection factor is fixed；
3) it is 8 according to the mass ratio of hemolymph and the molecular scaffold for being fixed with the desinsection factor:1 ratio, takes hemolymph to add
To being fixed with the molecular scaffold of the desinsection factor, 4 DEG C of incubation 1h after mixing, can be with the desinsection factor in hemolymph during incubation
With reference to composition combined with the desinsection factor, so as to being indirectly fixed on molecular scaffold, and can not be combined with the desinsection factor into
Divide and will not then be fixed；Then under low temperature (4 DEG C) low speed (2000rpm) it is of short duration centrifugation (30s), after centrifugation, molecular scaffold with
And be incorporated in the composition that can be combined in the hemolymph on molecular scaffold with the desinsection factor and be located at lower floor, can not be with desinsection in hemolymph
The composition that the factor combines is located at upper strata, separates levels, and it is the composition that can not be combined in hemolymph with the desinsection factor to remove upper strata；
4) lower floor obtained to step 3), i.e. molecular scaffold carry out 60~100 DEG C heating, make in hemolymph with desinsection
The composition that the factor combines discharges from molecular scaffold；
5) composition combined in the hemolymph obtained to step 4) with the desinsection factor analyzes and identifies, specifically,
Protein sample-loading buffer is added to the composition combined in the hemolymph of step 4) release with the desinsection factor, carried out
SDS-PAGE electrophoresis and coomassie brilliant blue staining, display albumen, polypeptide binding constituents and analyzed, and using liquid matter connection
Albumen, component polypeptides are identified with analyzer；
Nucleic acid sample-loading buffer is added to the composition combined in the hemolymph of step 4) release with the desinsection factor, carries out agar
Sugared gel electrophoresis and EB dyeing, the binding constituents of show nucleic acid class are simultaneously analyzed, and using nucleic acid sequencing instrument to nucleic acid into
Divide and identified.
According to the albumen, polypeptide, nucleic acid constituents identified, original of the thuricade-1 to target worm hypoactivity is found out
Cause, find overcome approach accordingly accordingly.
Among another embodiment of the present invention, for beet armyworm, according to the method described above for desinsection factor Cry1Ab1 eggs
After being analyzed in vain, the composition combined with desinsection factor Cry1Ab1 is have found, qualification result is storage protein.
In addition, can not be combined in the upper strata that can also be centrifuged out to step 3), i.e. hemolymph with the desinsection factor
Composition carry out similar analysis, to further determine that the reason for thuricade-1 is to target worm hypoactivity.
It is described above, only present pre-ferred embodiments, therefore the scope that the present invention is implemented can not be limited according to this, i.e., according to
The equivalent changes and modifications that the scope of the claims of the present invention and description are made, all should still it belong in the range of the present invention covers.
Claims (8)
1. a kind of analyze method of the thuricade-1 to target worm hypoactivity reason, it is characterised in that：Including：
1) healthy target worm some of the thuricade-1 to its hypoactivity is chosen, gathers target worm hemolymph；
2) the bacillus thuringiensis preparation as insecticiding factor is fixed on molecular scaffold：The Sepharose for weighing 0.18~0.22g is added to
Concentration is in 0.8~1.2mg/mL desinsection factor solutions, and 100~200rpm is incubated 4~6h at room temperature；Added after the completion of incubation
9~11mg Glycine, continue 5~7h of incubation and closed；After the completion of closing, the molecule branch of the desinsection factor is fixed
Frame；
3) it is 7~9 according to the mass ratio of hemolymph and the molecular scaffold for being fixed with the desinsection factor:1 ratio, takes hemolymph to be added to
It is fixed with the molecular scaffold of the desinsection factor, 0~5 DEG C of 0.5~1.5h of incubation after mixing；Then 1500 at 0~5 DEG C~
2500rpm centrifuges 25~35s, isolates the composition that can not be combined in hemolymph with the desinsection factor；
4) molecular scaffold that step 3) obtains is handled, made in hemolymph with the composition that the desinsection factor is combined from molecular scaffold
Upper release；
5) composition combined in the hemolymph obtained to step 4) with the desinsection factor analyzes and identifies：Carry out SDS-PAGE electrophoresis
And coomassie brilliant blue staining, analyzed with the binding constituents to albumen, polypeptide, and albumen, component polypeptides are tied
Structure analyzes and identifies；Enter row agarose gel electrophoresis and EB dyeing, analyzed with the binding constituents to nucleic acid, and to nucleic acid
Composition carries out sequencing identification.
2. analysis thuricade-1 according to claim 1 exists to the method for target worm hypoactivity reason, its feature
In：The target worm is the instar larvae of beet armyworm 4.
3. analysis thuricade-1 according to claim 1 exists to the method for target worm hypoactivity reason, its feature
In：The desinsection factor is at least one of Cry7Ab4 albumen or Cry1Ab1 albumen.
4. analysis thuricade-1 according to claim 1 exists to the method for target worm hypoactivity reason, its feature
In：In the step 1), target worm is before hemolymph is gathered, prior to indoor feeding：Raised using man-made feeds in program-controlled manual gas
Wait in case, 25 DEG C ± 1 DEG C of temperature, relative humidity 60 ± 5%, photoperiod L:D=16:8, intensity of illumination >=6000Lux.
5. analysis thuricade-1 according to claim 1 exists to the method for target worm hypoactivity reason, its feature
In：In the step 1), the method for collection target worm hemolymph includes：Catch and take the normal target worm of growth to be handled in placement on ice
10min, target worm head is gently clamped with eye scissors, be suspended from above sterile chamber, cut off with another eye scissors at abdominal foot small
Mouthful, the hemolymph for collecting target worm is instilled in sterile chamber.
6. analysis thuricade-1 according to claim 1 exists to the method for target worm hypoactivity reason, its feature
In：In the step 2), the Sepharose for weighing 0.2g is added in the desinsection factor solutions that concentration is 1mg/mL, at room temperature
150rpm is incubated 5h；10mg Glycine are added after the completion of incubation, continues incubation 6h and is closed.
7. analysis thuricade-1 according to claim 1 exists to the method for target worm hypoactivity reason, its feature
In：In the step 3), the mass ratio of molecular scaffold of the hemolymph with being fixed with the desinsection factor is 8:1；Hemolymph is added to fixation
After having on the molecular scaffold of the desinsection factor 1h is incubated at 4 DEG C；Then 2000rpm centrifuges 30s at 4 DEG C, isolates in hemolymph
The composition that can not be combined with the desinsection factor.
8. analysis thuricade-1 according to claim 1 exists to the method for target worm hypoactivity reason, its feature
In：In the step 4), the processing method to molecular scaffold is heating.
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| CN105994304A (en) * | 2016-05-09 | 2016-10-12 | 华东理工大学 | Application of novel insect cell immunosuppressor |
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2017
- 2017-08-25 CN CN201710741934.4A patent/CN107557476A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010008767A1 (en) * | 1994-01-25 | 2001-07-19 | Stephen C. Trowell | Agents and method for identifying insects |
| CA2277546A1 (en) * | 1997-01-10 | 1998-07-16 | Agricultural Genetic Engineering Research Institute (Ageri) | Bacillus thuringiensis isolates with broad spectrum activity |
| CN101743251A (en) * | 2007-03-26 | 2010-06-16 | 联邦科学技术研究组织 | Peptides with anitfungal activity |
| CN105683211A (en) * | 2013-10-28 | 2016-06-15 | 拜斯科医疗有限公司 | Novel polypeptides |
| CN104988092A (en) * | 2015-06-25 | 2015-10-21 | 河南科技大学 | Bacillus thuringiensis, preparation method and application |
| CN105669842A (en) * | 2016-02-25 | 2016-06-15 | 华侨大学 | Non-receptor binding protein composition of insecticidal crystal protein as well as extracting method and application of non-receptor binding protein composition |
| CN105994304A (en) * | 2016-05-09 | 2016-10-12 | 华东理工大学 | Application of novel insect cell immunosuppressor |
Non-Patent Citations (7)
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Application publication date: 20180109 |