CN107474121A - A kind of Bt Pesticidal toxins of AVM coupling and its application - Google Patents
A kind of Bt Pesticidal toxins of AVM coupling and its application Download PDFInfo
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- CN107474121A CN107474121A CN201710860848.5A CN201710860848A CN107474121A CN 107474121 A CN107474121 A CN 107474121A CN 201710860848 A CN201710860848 A CN 201710860848A CN 107474121 A CN107474121 A CN 107474121A
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- avm
- cry2ab
- pesticidal toxins
- succinoyl
- insecticidal proteins
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- 108700012359 toxins Proteins 0.000 title claims abstract description 42
- 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 30
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 13
- 230000008878 coupling Effects 0.000 title claims abstract description 11
- 238000010168 coupling process Methods 0.000 title claims abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 32
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 32
- 230000000749 insecticidal effect Effects 0.000 claims abstract description 30
- 238000002360 preparation method Methods 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 8
- 239000000417 fungicide Substances 0.000 claims abstract description 5
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 239000007853 buffer solution Substances 0.000 claims description 12
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- 150000002460 imidazoles Chemical class 0.000 claims description 8
- 101100275683 Bacillus thuringiensis subsp. kurstaki cry2Ab gene Proteins 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 7
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 7
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 229940014800 succinic anhydride Drugs 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 claims description 6
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 5
- 238000004587 chromatography analysis Methods 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 5
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 5
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 5
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 4
- 231100000614 poison Toxicity 0.000 claims description 4
- 239000002574 poison Substances 0.000 claims description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 4
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 230000015556 catabolic process Effects 0.000 claims description 3
- 238000005336 cracking Methods 0.000 claims description 3
- 238000006731 degradation reaction Methods 0.000 claims description 3
- 238000010612 desalination reaction Methods 0.000 claims description 3
- 238000011033 desalting Methods 0.000 claims description 3
- 239000006166 lysate Substances 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 230000035939 shock Effects 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 3
- 230000009466 transformation Effects 0.000 claims description 3
- 238000004440 column chromatography Methods 0.000 claims description 2
- 230000003139 buffering effect Effects 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000011010 flushing procedure Methods 0.000 claims 1
- 210000002429 large intestine Anatomy 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- 229940086542 triethylamine Drugs 0.000 claims 1
- 241000500437 Plutella xylostella Species 0.000 abstract description 12
- 241000607479 Yersinia pestis Species 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 230000001018 virulence Effects 0.000 abstract description 3
- 230000008569 process Effects 0.000 abstract description 2
- -1 succinoyl Chemical group 0.000 abstract description 2
- 239000007822 coupling agent Substances 0.000 abstract 1
- 241000238631 Hexapoda Species 0.000 description 10
- 239000003905 agrochemical Substances 0.000 description 6
- 235000013601 eggs Nutrition 0.000 description 6
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 4
- 239000000575 pesticide Substances 0.000 description 4
- 241000500441 Plutellidae Species 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 239000005660 Abamectin Substances 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000305071 Enterobacterales Species 0.000 description 2
- CGQCWMIAEPEHNQ-UHFFFAOYSA-N Vanillylmandelic acid Chemical compound COC1=CC(C(O)C(O)=O)=CC=C1O CGQCWMIAEPEHNQ-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 241000724266 Broad bean mottle virus Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 241000254173 Coleoptera Species 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000257303 Hymenoptera Species 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- RRZXIRBKKLTSOM-XPNPUAGNSA-N avermectin B1a Chemical compound C1=C[C@H](C)[C@@H]([C@@H](C)CC)O[C@]11O[C@H](C\C=C(C)\[C@@H](O[C@@H]2O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](OC)C3)[C@@H](OC)C2)[C@@H](C)\C=C\C=C/2[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\2)O)C[C@H]4C1 RRZXIRBKKLTSOM-XPNPUAGNSA-N 0.000 description 1
- 150000003851 azoles Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000021110 pickles Nutrition 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 150000003222 pyridines Chemical class 0.000 description 1
- 208000013223 septicemia Diseases 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 235000017550 sodium carbonate Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- 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
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides a kind of Bt Pesticidal toxins of AVM coupling, it is that the Pesticidal toxins are bonded together to form under coupling agent EDC, NHS collective effect in the Bt insecticidal proteins O succinoyl AVMs of Cry2Ab and 4 "; 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 process of the Bt Pesticidal toxins 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 AVM coupling and its with
Using.
【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 application for providing a kind of AVM coupling.
The present invention is that solve above-mentioned technical problem by the following technical programs：A kind of Bt desinsections poison of AVM coupling
Element, the concrete operations of its preparation method are as follows：By 1:1.5:1.5 mol ratio weighs 4 "-O-succinoyl Avermectins respectively
Element, EDC and NHS, and be dissolved in DMSO solvents, so as to be activated with the carboxyl to 4 "-O-succinoyl AVMs, obtain
4 "-O-succinoyl AVMs after to activation, it is stand-by；Bt Cry2Ab insecticidal proteins are taken to prepare Bt containing 5mmol/L
The Na2CO3/NaHCO3 buffer solutions of Cry2Ab insecticidal proteins, i.e. Bt Cry2Ab insecticidal proteins solution, it is stand-by；Preparation is measured respectively
Good-O-succinoyl the AVMs of activation 4 " and the Bt Cry2Ab insecticidal proteins solution, stirring carry out being coupled instead for 1 hour
Should, 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 the big of cry2Ab genes by thermal shock method
Enterobacteria engineering bacteria, it is seeded to afterwards on LB solid mediums, LB solid mediums is placed in incubated 24h at 30 DEG C
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, 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 4 "-O-succinoyl AVM preparation process is：By mole mass ratio 1:3:6 weigh Ah
Rhzomorph, tert-butyl chloro-silicane and imidazoles are tieed up, is dissolved in tetrahydrofuran, reaction 2 hours is stirred at room temperature, so as to Avermectin
The 5-OH of element is protected；AVM after fetching protection is dissolved in dichloromethane, and adds DMAP, three second
Amine and succinic anhydride, and AVM after protecting, DMAP, the mol ratio of triethylamine and succinic anhydride are 1:4:
8:16, lucifuge water-bath afterwards is flowed back 3 hours, and carboxylated transformation is carried out to-the OH of AVM 4 ", and product, post layer are extracted with ether
Analyse separation product；Take the AVM after chromatography to be dissolved in methanol, add p-methyl benzenesulfonic acid, and after chromatography Ah
The mol ratio for tieing up rhzomorph and p-methyl benzenesulfonic acid is 1:6, reaction is stirred at room temperature and is deprotected within 30 minutes, then is extracted using ethyl acetate
Product is taken, last TLC separation obtains 4 "-O-succinoyl AVMs.
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 AVM coupling, Bt Pesticidal toxins tool are provided
There is 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
With more preferable application prospect；And the preparation method of the Bt Pesticidal toxins is disclosed simultaneously, the preparation method process is simple and convenient,
Operability.
【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.And it should be noted that
Involved culture medium is existing routinely culture medium in the case of without specified otherwise in the present invention.
Embodiment 1
The preparation of 4 "-O-succinoyl AVMs
By mole mass ratio 1:3:6 weigh AVM, tert-butyl chloro-silicane and imidazoles, and are dissolved in tetrahydrochysene furan
Mutter, reaction 2 hours is stirred at room temperature, so as to be protected to the 5-OH of AVM；AVM after fetching protection is dissolved in
In dichloromethane, and DMAP is added, triethylamine and succinic anhydride, and AVM, 4- diformazan ammonia after protection
The mol ratio of yl pyridines, triethylamine and succinic anhydride is 1:4:8:16, lucifuge water-bath afterwards flow back 3 hours, to AVM 4 "-
OH carries out carboxylated transformation, and product, column chromatography for separation product are extracted with ether；The AVM after chromatography is taken to be dissolved in first
In alcohol, p-methyl benzenesulfonic acid is added, and AVM after chromatography and the mol ratio of p-methyl benzenesulfonic acid are 1:6, it is stirred at room temperature
Reaction is deprotected for 30 minutes, then adopts and product is extracted with ethyl acetate, and last TLC separation obtains 4 "-O-
Succinoyl AVMs.
The structure of 4 "-O-succinoyl AVMs is identified by MS and NMR technology：
1H NMR (400MHz, CDCl3) δ 4.78 (d, J=3.1Hz, 1H), 4.68 (br, 2H), 3.36 (s, 3H), 2.69
(m,4H),1.87(s,3H)；
13C NMR(101MHz,CDCl3)δ176.55,173.73,171.60,139.51,137.99,137.85,
136.30,135.17,127.71,124.77,120.42,118.33,118.04,98.33,95.80,94.99,82.02,
80.71,80.36,79.23,76.60,75.61,74.91,68.45,68.38,67.68,67.18,66.45,56.93,
56.54,45.74,40.45,39.73,36.51,35.15,35.00,34.48,34.22,30.57,29.07,28.80,
27.49,23.42,20.69,20.23,19.90,18.37,17.32,16.37,15.10,12.96,12.04；
ESI-MS(m/z):995.50[M+Na]+(C52H76O17, Exact Mass:972.50825, Mol.Wt.:
973.14964)。
So that it is determined that the chemical constitution of the 4 "-O-succinoyl AVMs, its chemical constitution are as follows：
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 the big of cry2Ab genes by thermal shock method
Enterobacteria engineering bacteria, the colibacillus engineering for containing cry2Ab genes is seeded on LB solid mediums afterwards, by LB
Solid medium is placed in incubated 24h at 30 DEG C and 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, 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 4 "-O-succinoyl AVMs (0.5mmol) of gained, is dissolved with 1mL DMSO, and
EDC (0.75mmol) and NHS (0.75mmol) is added, 2h is stirred at room temperature, with the carboxyl to 4 "-O-succinoyl AVMs
Activated, 4 "-O-succinoyl AVMs after being activated are stand-by；Example 2 prepares the Bt Cry2Ab of gained
Insecticidal proteins configure the Na of the Cry2Ab insecticidal proteins of Bt containing 5mmol/L2CO3/NaHCO3Buffer solution, i.e. Bt Cry2Ab desinsections egg
White solution, it is stand-by；Afterwards under the conditions of 4 DEG C, 4 "-O-succinoyl AVMs after 100 μ L are activated add the Bt
In Cry2Ab insecticidal proteins solution 1mL, stir 1 hour and carry out coupling reaction, obtain Bt Pesticidal toxins.
It should be noted that AVM, which is one kind, has sterilization, desinsection, mite killing, the ten of eelworm-killing activity hexa-atomic big rings
Lactone compound；Using AVM and Bt it is toxin conjugated after, improve the adhesion of Bt toxin and acceptor, improve killing for Bt toxin
Worm poison power, so as to administer insect to resistance of Bt toxin etc..
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 Pesticidal toxins of gained are prepared with embodiment 3 using 24 hole plate feeding methods measure Bt Cry2Ab insecticidal proteins
To the death rate of diamondback moth second instar larvae, experimental group and control group are set.Specific experiment method is experimental group：By Bt desinsections poison
Element is diluted, and obtains the Bt Pesticidal toxins solution of various concentrations；Then in an aseptic environment, the feed for drawing 1mL is sub-packed in
In 24 orifice plates and naturally dry, the Bt Pesticidal toxins solution of the various concentrations that 100 μ L have been configured and uniform is drawn respectively afterwards
Feed surface is coated on, with 100 μ L Na2CO3/NaHCO3Buffer solution as blank control, carry out under mark, nature by each group
Dry；Two age diamondback moths are inoculated into 24 orifice plates () per hole 5-7 only, and each hole of concentration gradient 4 repeats, diamondback moth after 48 hours measure
The death rate (worm dead diamondback moth is motionless to be defined to be touched with writing brush), draw LC50.It is unique with experimental group in control group
Difference is to be used as effector using Bt Cry2Ab insecticidal proteins.
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：Control group is Bt Cry2Ab desinsection eggs
The LC50 to two age diamondback moths is 0.922 μ g/cm in vain2, experimental group is LC50 of the Bt Pesticidal toxins of the present invention to two age diamondback moths
For 0.388 μ g/cm2, insecticidal toxicity probably improves 2.38 times.This result shows that Bt Pesticidal toxins of the present invention can actually carry
The high 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 (5)
- A kind of 1. Bt Pesticidal toxins of AVM coupling, it is characterised in that：The preparation method concrete operations of the Bt Pesticidal toxins It is as follows：By 1:1.5:1.5 mol ratio weighs 4 "-O-succinoyl AVMs, EDC and NHS respectively, and it is molten to be dissolved in DMSO In agent, activated with the carboxyl to 4 "-O-succinoyl AVMs, 4 "-O-succinoyl AVM hereinafters after being activated Rhzomorph, it is stand-by；Bt Cry2Ab insecticidal proteins are taken to prepare the Na of the Cry2Ab insecticidal proteins of Bt containing 5mmol/L2CO3/NaHCO3It is slow Fliud flushing, i.e. Bt Cry2Ab insecticidal proteins solution, it is stand-by；- O-succinoyl the AVMs of activation 4 " prepared are measured respectively With the Bt Cry2Ab insecticidal proteins solution, stir 1 hour and carry out coupling reaction, obtain Bt Pesticidal toxins.
- 2. the Bt Pesticidal toxins being coupled according to a kind of AVM of claim 1, it is characterised in that：The Bt Cry2Ab desinsections The preparation process of albumen 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) colibacillus engineering after activation is inoculated in LB fluid nutrient mediums, is placed in 37 DEG C of fermented and cultureds in shaking flask, Speed setting is 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, centrifugation takes precipitation weight after terminating It is suspended from Na2CO3Lysate, the ultrasonic degradation 30min at 4 DEG C, the solution after cracking take in 4 DEG C, 10000r/min centrifugation 30min 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 3. Bt Pesticidal toxins of AVM coupling according to claim 2, it is characterised in that：The Ni50 bufferings The formula of liquid is NaCl 300mM, NaH2PO450mM, imidazoles 50mM；The formula of Ni500 buffer solutions be NaCl 300mM, NaH2PO450mM, imidazoles 500mM.
- A kind of 4. Bt Pesticidal toxins of AVM coupling according to claim 1, it is characterised in that：The 4 "-O- Succinoyl AVM preparation process is：By mole mass ratio 1:3:6 weigh AVM, tert-butyl chloro-silicane And imidazoles, and tetrahydrofuran is dissolved in, reaction 2 hours is stirred at room temperature, so as to be protected to the 5-OH of AVM；Fetch protection AVM afterwards is dissolved in dichloromethane, and adds DMAP, triethylamine and succinic anhydride, and after protection AVM, DMAP, the mol ratio of triethylamine and succinic anhydride are 1:4:8:16, the backflow of lucifuge water-bath afterwards 3 Hour, carboxylated transformation is carried out to-the OH of AVM 4 ", product, column chromatography for separation product are extracted with ether；After taking chromatography AVM be dissolved in methanol, and add p-methyl benzenesulfonic acid, and AVM and mole of p-methyl benzenesulfonic acid after separating Than for 1:6, reaction is stirred at room temperature and is deprotected within 30 minutes, then adopt and product is extracted with ethyl acetate, last TLC separation Obtain-O-succinoyl the AVMs of product 4 ".
- 5. the application for the Bt Pesticidal toxins that AVM 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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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1293899A (en) * | 1999-11-02 | 2001-05-09 | 福建省农业科学院生物技术中心 | Physically multi-point coupled biological insecticide |
| CN102675411A (en) * | 2012-05-08 | 2012-09-19 | 福建省农业科学院农业生物资源研究所 | Method for preparing biotoxin |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1293899A (en) * | 1999-11-02 | 2001-05-09 | 福建省农业科学院生物技术中心 | Physically multi-point coupled biological insecticide |
| CN102675411A (en) * | 2012-05-08 | 2012-09-19 | 福建省农业科学院农业生物资源研究所 | Method for preparing biotoxin |
Non-Patent Citations (2)
| Title |
|---|
| PAN Z等: "Characterization of a new cry2Ab gene of Bacillus thuringiensis with high insecticidal activity against Plutella xylostella L.", 《WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY》 * |
| 刘波 等: "高效生物杀虫剂BtA化学耦合技术研究", 《福建农业学报》 * |
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Effective date of registration: 20231222 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 |