CN107602678A - A kind of Bt Pesticidal toxins of Acetamiprid coupling and preparation method and application - Google Patents
A kind of Bt Pesticidal toxins of Acetamiprid coupling and preparation method and application Download PDFInfo
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- 239000003053 toxin Substances 0.000 title claims abstract description 40
- 231100000765 toxin Toxicity 0.000 title claims abstract description 40
- 230000000361 pesticidal effect Effects 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 238000010168 coupling process Methods 0.000 title claims abstract description 21
- WCXDHFDTOYPNIE-RIYZIHGNSA-N (E)-acetamiprid Chemical compound N#C/N=C(\C)N(C)CC1=CC=C(Cl)N=C1 WCXDHFDTOYPNIE-RIYZIHGNSA-N 0.000 title claims abstract description 20
- 230000008878 coupling Effects 0.000 title claims abstract description 20
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 33
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 33
- 230000000749 insecticidal effect Effects 0.000 claims abstract description 32
- 238000005859 coupling reaction Methods 0.000 claims abstract description 22
- -1 carboxyl Acetamiprid Chemical compound 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 9
- 239000000417 fungicide Substances 0.000 claims abstract description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 19
- 239000007853 buffer solution Substances 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 9
- 101100275683 Bacillus thuringiensis subsp. kurstaki cry2Ab gene Proteins 0.000 claims description 7
- 230000004913 activation Effects 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 7
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- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 5
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- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 5
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 5
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 5
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 4
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 4
- DKIDEFUBRARXTE-UHFFFAOYSA-N 3-mercaptopropanoic acid Chemical compound OC(=O)CCS DKIDEFUBRARXTE-UHFFFAOYSA-N 0.000 claims description 3
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
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- 244000025254 Cannabis sativa Species 0.000 description 1
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- Peptides Or Proteins (AREA)
Abstract
The present invention provides a kind of Bt Pesticidal toxins of Acetamiprid coupling, it is in Bt insecticidal proteins Cry2Ab, the new coupling type Pesticidal toxins bonded together to form with carboxyl Acetamiprid under coupling agent EDC, NHS collective effect, the Bt Pesticidal toxins have stronger virulence, it has more preferable insecticidal effect to diamondback moth, there is more preferable application prospect to preventing and treating agricultural pests, can be as the agricultural fungicides for diamondback moth；And the preparation method of the Bt Pesticidal toxins is disclosed simultaneously, the preparation method process is simple and convenient, workable.
Description
【Technical field】
The invention belongs to Agricultural pest control field, and in particular to a kind of Bt Pesticidal toxins of Acetamiprid coupling and its preparation
Method and application.
【Background technology】
In recent years, the fast development of agricultural sciences, pesticide control play a certain degree of facilitation.Agricultural chemicals can be effective
The harmful organisms such as agricultural disease, worm, grass, mouse are prevented and treated, ensure that this important step of agriculture increase harvest.But the production of disease drug resistance
It is raw, it is the problem that chemical pesticide or biological pesticide are not avoided that；Insect is exactly to overcome agricultural chemicals position to the agricultural chemicals resistance to the action of a drug
Point effect, is allowed to fail, is passivated.In actual applications, agricultural chemicals rotation administration, mixed pesticide are applied, increase agricultural chemicals to insect
The diversity of contact area, the sensitive strain for cultivating insect, increase ecological diversity to increase insect etc., substantially makes agriculture
Medicine action site keeps enough sensitiveness, seeks to overcome the means of resistance.Bioconjugation is to utilize Bioconjugation technology by two
Kind is toxin conjugated to form new toxin, and sensitiveness to keeping agricultural chemicals site etc. has great importance.
Dipel (Bacilus thuringiensis, abbreviation Bt) is the current maximum of output in the world, using most
For extensive a kind of microbial insecticide, its crystalline protein is to more than 570 Species of Lepidopterous Insect Pests and Diptera, Hymenoptera and coleoptera
Etc. tens of kinds insects have insecticidal toxicity.It is generally believed that insect is after feeding crystalline protein, by the midgut proteinase of itself by its
Digest and combine, go forward side by side with the acceptor in midgut epithelial cell brush edge film (BBMVs) again for the toxin protein of activity, toxin protein
In one step insertion film, hole or ion channel are formed, causes Ion leakage, Gut wall epithelial cells is destroyed, oozes enteron aisle Dissolve things inside
Enter haemocoele, cause septicemia, cause insect death；But the resistance to the action of a drug of insect causes the preventive effect of existing Bt toxin drastically to decline,
Therefore, Bt toxin is transformed, there is provided a kind of to have the Bt toxin compared with strong virus force be that practitioner institute is highly desirable.
【The content of the invention】
The technical problems to be solved by the invention are Bt Pesticidal toxins and its preparation side for providing a kind of Acetamiprid coupling
Method and application.
The present invention is that solve above-mentioned technical problem by the following technical programs：
A kind of Bt Pesticidal toxins of Acetamiprid coupling, the chemical structural formula of the Bt Pesticidal toxins are as follows：
The preparation method of the Bt Pesticidal toxins of above-mentioned Acetamiprid coupling is disclosed simultaneously, and the concrete operations of the preparation method are such as
Under：By 1:1.5:1.5 mol ratio weighs carboxyl Acetamiprid, EDC and NHS respectively, and is dissolved in DMSO solvents, with to carboxyl pyridine
The carboxyl of worm amidine is activated, the carboxyl Acetamiprid after must activating, stand-by；The configuration of Bt Cry2Ab insecticidal proteins is taken to contain 5mmol/L
The Na of Bt Cry2Ab insecticidal proteins2CO3/ NaHCO3Buffer solution, i.e. Bt Cry2Ab insecticidal proteins solution, it is stand-by；Measure respectively
Carboxyl Acetamiprid and the Bt Cry2Ab insecticidal proteins solution after the activation prepared, stir 1 hour and carry out coupling reaction,
Obtain Bt Pesticidal toxins.
Further, the preparation process of the Bt Cry2Ab insecticidal proteins is as follows：
(1) cry2Ab genes are converted to colibacillus engineering to obtain containing cry2Ab genes by thermal shock method
Colibacillus engineering, it is seeded on LB solid mediums, LB solid mediums is placed in incubated at 30 DEG C afterwards
24h carries out actication of culture；
(2) the colibacillus engineering strain after activation is inoculated in LB fluid nutrient mediums, is placed in shaking flask 37 DEG C
Fermented and cultured, speed setting 180r/min, treat bacterium solution OD600After reaching 0.5,25 DEG C are adjusted to, 180r/min continues
Cultivate 24h；Then zymotic fluid obtained by fermented and cultured is placed in 4 DEG C, centrifuges 10min under the conditions of 10000r/min, after centrifugation terminates
Precipitation is taken to be resuspended in Na2CO3Lysate, the ultrasonic degradation 30min at 4 DEG C, 4 DEG C of solution, 10000r/min centrifugations after cracking
30min, take supernatant；
(3) supernatant obtained by step (2) is crossed into IDA-Ni affinity columns, and impurity elimination egg is first removed using Ni50 buffer solutions
In vain, afterwards using Ni500 buffer solutions elution destination protein；Elution gained destination protein then obtains Bt through desalting column PD-10 desalinations
Cry2Ab insecticidal proteins, Bt Cry2Ab insecticidal proteins are placed at -20 DEG C and preserved, it is standby.
Further, the formula of the Ni50 buffer solutions is NaCl 300mM, NaH2PO450mM, imidazoles 50mM；Ni500
The formula of buffer solution is NaCl 300mM, NaH2PO450mM, imidazoles 500mM.
Further, the carboxyl Acetamiprid preparation process is：By mercaptopropionic acid, potassium hydroxide and Acetamiprid in mass ratio
1:1:2 ratio is dissolved in organic solvent DMSO, and reacts 2h under 100 DEG C of constant temperature；Reaction terminates rear question response system cooling
To room temperature, pour into the clean beaker for filling redistilled water, use 12molL-1The pH of reaction solution is adjusted to 3 by HC1；Then it is anti-
Answer liquid repeatedly to be extracted with dichloromethane, collect organic phase；Then organic phase is subjected to the crude product that is concentrated under reduced pressure to obtain, crude product is through silicon
Plastic column chromatography, which separates, produces the carboxyl Acetamiprid.
The present invention is it is also disclosed that application of the Bt Pesticidal toxins as agricultural fungicides.
The beneficial effects of the present invention are：A kind of Bt Pesticidal toxins of Acetamiprid coupling are provided, the Bt Pesticidal toxins have
Stronger virulence, it has more preferable insecticidal effect to diamondback moth, can be used as agricultural fungicides, i.e., to preventing and treating agricultural pests tool
There is more preferable application prospect；And the preparation method of the Bt Pesticidal toxins is disclosed simultaneously, the preparation method process is simple and convenient, can
Strong operability.
【Brief description of the drawings】
The invention will be further described in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is the collection of illustrative plates that the SDS-PAGE of embodiment 4 is detected in the present invention.
【Embodiment】
For a better understanding of the present invention, further illustrated with reference to embodiment and application examples in the explanation present invention
Hold, but these embodiments and application examples are only scopes that is exemplary, being not intended to limit the invention.
Embodiment 1
The preparation of carboxyl Acetamiprid
Acetamiprid (2.26g) is dissolved with 15mL DMSO in three-necked flask, and adds KOH (1.13g), separately under agitation
The β-mercaptopropionic acid (1.13g) of 10mL DMSO dissolvings is added dropwise with constant pressure funnel, is gradually heating to 100 DEG C afterwards, reacts 2h；Instead
Question response system is cooled to room temperature after should terminating, and pours into the clean beaker for filling redistilled water, uses 12molL-1HC1 will
The pH of reaction solution is adjusted to 3；Then reaction solution is repeatedly extracted with dichloromethane, collects organic phase, and organic phase is concentrated under reduced pressure slightly to produce
Thing, crude on silica gel column chromatography for separation obtain carboxyl Acetamiprid.
The structure of carboxyl Acetamiprid is identified by MS and NMR technology：
13C NMR(100MHz,DMSO)：δ173.05,157.91,157.43,149.18,136.70, 128.16,
122.19,118.16,50.47,38.01,34.59,25.10,19.52；
HR-ESI-MS:C13H15N4O2S([M-H]-):291.0975。
So that it is determined that the chemical constitution of the carboxyl Acetamiprid, then reactive chemistry formula involved by the embodiment is：
Embodiment 2
The insecticidal proteins of thuringiensis are the preparation of Bt Cry2Ab insecticidal proteins
(1) cry2Ab genes are converted to colibacillus engineering to obtain containing cry2Ab genes by thermal shock method
Colibacillus engineering, the colibacillus engineering for containing cry2Ab genes is seeded on LB solid mediums afterwards, will
LB solid mediums are placed in incubated 24h at 30 DEG C and carry out actication of culture；
(2) the colibacillus engineering strain after activation is inoculated in LB fluid nutrient mediums, is placed in shaking flask 37 DEG C
Fermented and cultured, speed setting 180r/min, treat bacterium solution OD600After reaching 0.5,25 DEG C are adjusted to, 180r/min continues
Cultivate 24h；Then zymotic fluid obtained by fermented and cultured is placed in 4 DEG C, centrifuges 10min under the conditions of 10000r/min, after centrifugation terminates
Precipitation is taken to be resuspended in Na2CO3Lysate, the ultrasonic degradation 30min at 4 DEG C, 4 DEG C of solution, 10000r/min centrifugations after cracking
30min, take supernatant；
(3) supernatant obtained by step (2) is crossed into IDA-Ni affinity columns, first using Ni50 buffer solutions (NaCl 300mM,
NaH2PO450mM, imidazoles 50mM) remove foreigh protein removing；Ni500 buffer solutions (NaCl 300mM, NaH are used afterwards2PO450mM, miaow
Azoles 500mM) elution destination protein；Elution gained destination protein then obtains Bt Cry2Ab desinsection eggs through desalting column PD-10 desalinations
In vain, Bt Cry2Ab insecticidal proteins are placed at -20 DEG C and preserved, it is standby.
Embodiment 3
The preparation of Bt Pesticidal toxins
Example 1 prepares the carboxyl Acetamiprid (0.5mmol) of gained, is dissolved with 1mL DMSO, adds EDC
(0.75mmol) and NHS (0.75mmol), is stirred at room temperature 2h, the carboxyl of carboxyl Acetamiprid is activated, the carboxylic after must activating
Base Acetamiprid, it is stand-by；Example 2 prepares the Bt Cry2Ab insecticidal proteins configuration Cry2Ab desinsections of Bt containing 5mmol/L of gained
The Na of albumen2CO3/NaHCO3Buffer solution, i.e. Bt Cry2Ab insecticidal proteins solution, it is stand-by；Afterwards under the conditions of 4 DEG C, by 100 μ L
Carboxyl Acetamiprid after activation adds the Bt Cry2Ab insecticidal proteins solution 1mL, stirs 1 hour and carries out coupling reaction, obtains
Bt Pesticidal toxins.
Embodiment 4
SDS-PAGE detects coupling effect
The coupling effect of coupling reaction process in embodiment 3, testing result (Fig. 1 as shown in Figure 1 are detected by SDS-PAGE
In, M：marker；10:Sampled after coupling 10min；20：Sampled after coupling 20min；30：Sampled after coupling 30min；40：Coupling
Sampled after 40min；50：Sampled after coupling 50min；C：Bt Cry2Ab insecticidal proteins before coupling)；As seen from Figure 1, in idol
Obvious coupling, front and rear point of coupling occur with the carboxyl Acetamiprid after activation for Bt Cry2Ab albumen after connection starts 50min
Son amount increase 1Kd or so, that is, be coupled successfully.
Application examples 1
The insecticidal activity analysis of Bt Pesticidal toxins
(1) preparation of diamondback moth
The pickles chrysalis of indoor population is gathered, is sprouted wings, collects Adult worms producting eggs, an Eggs of Diamondback Moth is collected within every 24 hours, receives
The Eggs of Diamondback Moth of same batch of collection is raised in case LC in growth cabinet with similarity condition50Measure, the condition of the growth cabinet
For：25 DEG C of temperature, relative humidity 70%, photoperiod 16：8(L： D).
(2) verification method and result
The Bt that gained is prepared using 24 hole plate feeding methods measure Bt Cry2Ab insecticidal proteins, Acetamiprid and embodiment 3 is killed
Worm poison element is to the death rate of diamondback moth second instar larvae, while it (is respectively one of control group and right to set experimental group and two control groups
According to group two).Specific experiment method is experimental group：Bt Pesticidal toxins are diluted, obtain the Bt Pesticidal toxins of various concentrations
Solution；Then in an aseptic environment, the feed for drawing 1mL is sub-packed in 24 orifice plates simultaneously naturally dry, draws 100 μ L respectively afterwards
The Bt Pesticidal toxins solution of the various concentrations configured is simultaneously uniformly coated on feed surface, with 100 μ L Na2CO3/ NaHCO3
As blank control, each group is carried out dries buffer solution under mark, nature；Two age diamondback moths are inoculated into 24 orifice plates (per hole 5-7
Only), each hole of concentration gradient 4 repeats, after 48 hours measure diamondback moth the death rate (worm it is dead to touch diamondback moth with writing brush
It is motionless to be defined), draw LC50.In one of control group, unique difference with experimental group be using Bt Cry2Ab insecticidal proteins as
Effector.The two of control group, unique difference with experimental group is to be used as effector using Acetamiprid.Result of the test see the table below
1。
The insecticidal effect of the Bt Pesticidal toxins of table 1
Analyzed, drawn via table 1 and using the regression model of SPSS softwares：One of control group is that Bt Cry2Ab are killed
Worm albumen is 23.825 μ g/mL to the LC50 of two age diamondback moths, and experimental group is Bt Pesticidal toxins of the present invention to two age diamondback moths
LC50 is 9.881 μ g/mL, and insecticidal toxicity probably improves 2.41 times；LC50 of two Acetamiprids of control group to two age diamondback moths
For 276.53 μ g/mL.This result shows that Bt Pesticidal toxins of the present invention can actually improve the insecticidal toxicity to diamondback moth.
In summary, the present invention has stronger virulence, Bt desinsections of the present invention by coupling and the Bt Pesticidal toxins formed
Toxin has more preferable insecticidal effect to diamondback moth, and in other words, it can be used as agricultural fungicides to be used to murder diamondback moth, right
Preventing and treating agricultural pests have more preferable application prospect.
Claims (6)
- A kind of 1. Bt Pesticidal toxins of Acetamiprid coupling, it is characterised in that：The structural formula of the Bt Pesticidal toxins is as follows：
- 2. the preparation method for the Bt Pesticidal toxins that Acetamiprid described in a kind of claim 1 is coupled, it is characterised in that：The preparation method Concrete operations it is as follows：By 1:1.5:1.5 mol ratio weighs carboxyl Acetamiprid, EDC and NHS respectively, and is dissolved in DMSO solvents It is interior, activated with the carboxyl to carboxyl Acetamiprid, the carboxyl Acetamiprid after must activating is stand-by；Take Bt Cry2Ab insecticidal proteins Configure the Na of the Cry2Ab insecticidal proteins of Bt containing 5mmol/L2CO3/NaHCO3Buffer solution, i.e. Bt Cry2Ab insecticidal proteins solution, It is stand-by；The carboxyl Acetamiprid after the activation prepared and the Bt Cry2Ab insecticidal proteins solution are measured respectively, are stirred 1 hour Coupling reaction is carried out, obtains Bt Pesticidal toxins.
- 3. the preparation method for the Bt Pesticidal toxins being coupled according to a kind of Acetamiprid of claim 2, it is characterised in that：The Bt The preparation process of Cry2Ab insecticidal proteins is as follows：(1) cry2Ab genes are converted to colibacillus engineering to obtain the large intestine bar containing cry2Ab genes by thermal shock method Bacterium engineering bacteria, it is seeded to afterwards on LB solid mediums, LB solid mediums are placed in into incubated 24h at 30 DEG C is carried out Actication of culture；(2) the colibacillus engineering strain after activation is inoculated in LB fluid nutrient mediums, is placed in 37 DEG C of fermentations in shaking flask Culture, speed setting 180r/min, treats bacterium solution OD600After reaching 0.5,25 DEG C are adjusted to, 180r/min continues to cultivate 24h；Then zymotic fluid obtained by fermented and cultured is placed in 4 DEG C, centrifuges 10min under the conditions of 10000r/min, it is heavy that centrifugation takes after terminating Shallow lake is resuspended in Na2CO3Lysate, the ultrasonic degradation 30min at 4 DEG C, 4 DEG C of solution, 10000r/min centrifugation 30min after cracking, Take supernatant；(3) supernatant obtained by step (2) is crossed into IDA-Ni affinity columns, and foreigh protein removing is first removed using Ni50 buffer solutions, after Destination protein is eluted using Ni500 buffer solutions；Elution gained destination protein then obtains Bt Cry2Ab through desalting column PD-10 desalinations Insecticidal proteins, Bt Cry2Ab insecticidal proteins are placed at -20 DEG C and preserved, it is standby.
- A kind of 4. preparation method of the Bt Pesticidal toxins of Acetamiprid coupling according to claim 3, it is characterised in that：It is described The formula of Ni50 buffer solutions is NaCl 300mM, NaH2PO450mM, imidazoles 50mM；The formula of Ni500 buffer solutions is NaCl 300mM、NaH2PO450mM, imidazoles 500mM.
- A kind of 5. preparation method of the Bt Pesticidal toxins of Acetamiprid coupling according to claim 2, it is characterised in that：It is described Carboxyl Acetamiprid preparation process is：By mercaptopropionic acid, potassium hydroxide and Acetamiprid in mass ratio 1:1:2 ratio is dissolved in organic molten In agent DMSO, and 2h is reacted under 100 DEG C of constant temperature；Reaction terminates rear question response system and is cooled to room temperature, pours into and fills secondary steaming In the clean beaker of distilled water, 12molL is used-1The pH of reaction solution is adjusted to 3 by HC1；Then reaction solution dichloromethane repeatedly extracts Take, collect organic phase；Then organic phase is subjected to the crude product that is concentrated under reduced pressure to obtain, crude on silica gel column chromatography for separation produces described Carboxyl Acetamiprid.
- 6. the application for the Bt Pesticidal toxins that Acetamiprid described in a kind of claim 1 is coupled, it is characterised in that：The Bt desinsections poison Application of the element as agricultural fungicides.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710859987.6A CN107602678B (en) | 2017-09-21 | 2017-09-21 | Acetamiprid-coupled Bt insecticidal toxin and preparation method and application thereof |
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| CN201710859987.6A CN107602678B (en) | 2017-09-21 | 2017-09-21 | Acetamiprid-coupled Bt insecticidal toxin and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109061158A (en) * | 2018-09-21 | 2018-12-21 | 中国烟草总公司郑州烟草研究院 | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting Acetamiprid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109061158A (en) * | 2018-09-21 | 2018-12-21 | 中国烟草总公司郑州烟草研究院 | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting Acetamiprid |
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Effective date of registration: 20231217 Address after: 350000 pudang village, Fuzhou suburb, Fujian Province Patentee after: Institute of Resources, Environment, and Soil Fertilizers, Fujian Academy of Agricultural Sciences Address before: 350000 No. 54, 247, Fuzhou, Fujian Patentee before: AGRICULTURAL BIORESOURCES INSTITUTE OF FUJIAN ACADEMY OF AGRICULTURAL SCIENCES |