CN107488665A - The insecticidal peptide and application of killing gene PPTX-3a and its coding - Google Patents
The insecticidal peptide and application of killing gene PPTX-3a and its coding Download PDFInfo
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- CN107488665A CN107488665A CN201710695548.6A CN201710695548A CN107488665A CN 107488665 A CN107488665 A CN 107488665A CN 201710695548 A CN201710695548 A CN 201710695548A CN 107488665 A CN107488665 A CN 107488665A
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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
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
- C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
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Abstract
The present invention discloses a kind of the killing gene PPTX 3a from fit reconstruction and its coded insect-killing peptide and application, the killing gene nucleotide sequence from fit reconstruction is as shown in SEQ ID NO.1, the desinsection peptide amino acid sequence of the gene code is as shown in SEQ ID NO.2, and its ripe peptide sequence is as shown in SEQ ID NO.3.The insecticidal peptide can express through prokaryotic system, and short preparation period, amino acid sequence is small, be adapted to external large-scale production, can reduce existing Bt toxin and widely use existing security risk, reducing the use of insecticide has important science and realistic meaning.
Description
The present invention relates to genetic engineering and field of biological control, particularly a kind of killing gene and its coding for technical field
Insecticidal peptide and application.
The killing gene that background technology is now widely used in control of insect is mainly that bacillus thuringiensis produces Bt toxin
Gene, Bt toxin shows high desinsection specificity to agricultural pests such as Lepidoptera, Diptera, coleopteras.Therefore Bt poison
In the important crops that plain gene is planted extensively by transgenosis to cotton, corn, tobacco etc..Carry the genetically modified crops of anti insect gene
Played an important role in terms of agricultural insect pests control.However, the long-term use of single anti insect gene makes insect to Bt toxin
Resistance is continuously increased.Various insects colony generates resistance under natural environment to Bt protein formulations and Bt genetically modified crops, such as
Bollworm, diamondback moth and striped rice borer etc. (Yi Xiaoli etc., insect is to Bt toxin resistances progress [J], Jiangsu's agriculture science,
7th phase in 2014).Therefore, developing the new anti insect gene or protein of another high-efficiency environment friendly can increase to pest-resistant base
The selection of cause and the development for reducing resistance.
Spider toxin is received significant attention because of its Chemical Diversity and broad spectrum activity as potential insecticide
(G.F.King, et al., Spider-Venom peptides：Structure, pharmacology, and potential
For control of insect pests, Annu.Rev.Entomol.2013,58：475-96.).Spider toxin can be made
For a variety of passages and acceptor of insect cell membrane, such as ion channel, neural ligand gated channel associates acceptor with G-protein.
Therefore all there is pest-resistant effect to various insects.But the research to spider toxin at present focuses mostly on concentrates weight in latrodectus mactans
The mechanism of action of toxin is wanted, it is few to the direct test and comparison of its insecticidal action.
To sum up, the existing research to spider toxin is not met by the selection demand of anti insect gene.Therefore a kind of tool is needed
There is the spider toxin of insecticidal effect, the toxin by the method great expression of molecular gene engineering or can pass through transgenic technology
Expressed in crop, so as to reach pest-resistant effect.
The content of the invention purpose of the present invention is the above-mentioned deficiency for prior art, there is provided one kind comes from fit reconstruction
Killing gene PPTX-3a, its nucleotide sequence is as shown in SEQ ID NO.1.
It is a further object of the present invention to provide it is a kind of come from fit reconstruction killing gene PPTX-3a coding insecticidal peptide,
Its amino acid sequence is as shown in SEQ ID NO.2, its ripe peptide sequence such as SEQ ID NO.3.
It is another object of the present invention to the insecticidal peptide of the killing gene and its coding of the present invention for coming from fit reconstruction
Application in terms of crop pests preventing and treating.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of killing gene PPTX-3a for coming from fit reconstruction, its nucleotide sequence is as shown in SEQ ID NO.1.
A kind of prokaryotic vector containing people source of the present invention killing gene.
The insecticidal peptide of fit reconstruction killing gene coding is derived from as described herein in terms of crop pests preventing and treating
Using.
A kind of insecticide containing the insecticidal peptide for being derived from fit reconstruction killing gene coding as described herein.
The toxin gene of the latrodectus mactans that present invention reference has been found that etc., it is right using bioinformatic analysis method
Gene in fit reconstruction transcript profile database (disclosure) is screened, and obtains candidate's base of fit reconstruction toxin
Cause, then it is sequenced using polymerase chain reaction (PCR) and Sanger methods, obtains the complete sequence of the gene.By the gene structure
It is built in prokaryotic expression carrier pET-32a (+), is expressed through prokaryotic system, you can obtains the insecticidal peptide of the gene code.Prepare week
Phase is short, and amino acid sequence is small, is adapted to external large-scale production, meanwhile, the insecticidal peptide has as brand-new killing gene resource
Different from the insecticidal mechanism of Bt toxin, to expanding the new type disinsection genetic resources with bioactivity, it is wide to reduce existing Bt toxin
All kinds of security risks existing for general use, reducing the use of insecticide has important science and realistic meaning.
Brief description of the drawings：
After Fig. 1 is injection CK and recombinant toxin PPTX-3a, the death rate of the brown paddy plant hopper in different time points.
After Fig. 2 is injection CK and recombinant toxin PPTX-3a, the death rate of the small brown rice planthopper in different time points.
After Fig. 3 is injection CK and recombinant toxin PPTX-3a, the death rate of the white backed planthopper in different time points.
Embodiment：
Designed reagent and culture medium prescription in embodiment：
(1) LB fluid nutrient mediums：
10g tryptones, 5g yeast extracts and 5gNaCl are added in 900ml distilled waters, is stirred and evenly mixed, with double steamings
Water is settled to 1L, is placed in high-pressure sterilizing pot, 121 DEG C, and sterilized 20min, and 4 DEG C of preservations are placed in after cooling.
(2) LB solid mediums：
10g tryptones are added in 900ml distilled waters, 5g yeast extracts, 5gNaCl and 15g agar, are stirred mixed
It is even, 1L is settled to distilled water, is placed in high-pressure sterilizing pot, 121 DEG C, sterilize 20min, final concentration of to 50 DEG C of additions after cooling
100mg/L carbenicillin, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, 4 DEG C of preservations are placed in after cooling.
(3) transferring film buffer solution：
Glycine 2.9g, Tris base 5.8g, SDS 0.37g, methanol 200ml, add distilled water and be settled to 1L.
(4)PBST：
The Tween-20 of 0.05% volume is added in PBS.
(5) confining liquid：
1% skimmed milk power is added in PBST.
(6)Tris-HCl：
1M Tris-HCl is diluted to 20mM.
(7) combination buffer：
20mM sodium phosphates, 0.5M sodium chloride, 10mM imidazoles, distilled water are settled to 1L, pH7.4.
(8) elution buffer：
20mM sodium phosphates, 0.5M sodium chloride, 500mM imidazoles, distilled water are settled to 1L, pH7.4.
Embodiment 1
The preparation of toxin
The design and structure of spider toxin gene
Gene in fit reconstruction transcript profile database (disclosure) is screened, obtains fit reconstruction toxin
Candidate gene PPTX-3a, by its ripe peptide sequence according to e. coli codon Preference carry out codon optimization, design
For the PPTX-3a gene orders in expression in escherichia coli.The gene that design is completed is synthesized by Invitrogen companies, and
Nanjing Jin Sirui companies complete sequencing, by the gene cloning of synthesis into coli expression carrier pET-32a (+), construct
PET-32a (+)-PPTX-3a recombinant plasmids containing target gene, the toxin gene introduce BanH I and EcoR I digestions position
Point.
Recombinant plasmid pET-32a (+)-PPTX-3a induced expression
The different positive bacterial plaque of picking in LB flat boards, is added separately to the LB liquid containing 100mg/L carbenicillins
In culture medium, 250r/min in constant-temperature table, 37 DEG C, 12h.By the bacterium solution after culture according to 1: 100 ratio, be inoculated into containing
In the LB fluid nutrient mediums of 100mg/L carbenicillins, 200r/min, 37 DEG C, 4h is cultivated, now the OD values of bacterium solution are 0.5-
0.7；Final concentration of 0.4mmol/L IPTG, 200r/min, 37 DEG C is added in pET-32a (+)-PPTX-3a recons, is lured
Lead expression culture 5h；The bacterium solution 4ml after expression is taken, 4 DEG C, 12000rpm centrifugation 5min, supernatant is abandoned, is added in bacterial sediment
1.5ml 20mM Tris-HCl, pH=7.4, carrying out ultrasonic bacteria breaking is carried out after resuspension, intensity 12%, 10min, runs 5s, suspends 5s；
4 DEG C, 18000rpm after broken bacterium, 10min is centrifuged, supernatant is taken, abandons precipitation.
Western Blot are detected
50 μ l broken liquid is taken, adds 17 μ l Loading Buffer and 2 μ l beta -mercaptoethanol, 100 DEG C are boiled 5min,
Add 20 μ l mixed liquor to SDS- polyacrylamide gels, and add 10 μ l standard protein sample, 50V, 50min, then 100V is straight
To sample-loading buffer to gel bottom；After electrophoresis terminates, enter after gel is taken out in transferring film buffer solution, and pvdf membrane is soaked
Transferring film buffer solution is transferred to after methanol 1min, carries out transferring film, 100mA, 50min afterwards；After transferring film terminates, take the film out, PBST
Wash 3 times, each 5min, confining liquid closing 2h, 37 DEG C, 80r/min；Being taken the film out after closing, PBST is washed 3 times, each 10min,
Be transferred to add primary antibody confining liquid (1: 1000), be incubated 2h, 37 DEG C, 80r/min；After primary antibody incubation terminates, take the film out, PBST
Wash 3 times, each 10min, be transferred to add secondary antibody confining liquid (1: 2000), be incubated 1.5h, 37 DEG C, 80r/min；Secondary antibody is incubated knot
Shu Hou, PBST are washed 3 times, each 10min, addition luminescent solution, lucifuge 3min, after filter paper blots surface liquid, chemiluminescence detection.
Recombinant plasmid pET-32a (+)-PPTX-3a a large amount of induced expressions
By the bacterium solution after culture according to 1: 100 ratio, the LB liquid that 100ml contains 100mg/L carbenicillins is inoculated into
In body culture medium, the volume of culture medium cultivates 4h no more than 20%, 200r/min, 37 DEG C of container volume, now bacterium solution
OD values are 0.5-0.7；Final concentration of 0.4mmol/L IPTG, 200r/ is added in pET-32a (+)-PPTX-3a recons
Min, 37 DEG C, induced expression culture 5h；The bacterium solution after expression is taken, 4 DEG C, 12000rpm centrifugation 15min, supernatant is abandoned, is sunk in thalline
The 20mM of 20% culture volume Tris-HCl, pH=7.4 are added in shallow lake, carries out carrying out ultrasonic bacteria breaking after resuspension, intensity 12%,
30min, 5s is run, suspend 5s；4 DEG C, 18000rpm after broken bacterium, 30min is centrifuged, supernatant is taken, abandons precipitation.
Recombinant toxin PPTX-3a purifying
A large amount of induced expressions are broken to 0.22 μm of membrane filtration of the crude protein liquid after bacterium, it is full-automatic using AKTA avant
Protein separation system and HisTrapTMHP nickel post (5ml) is purified.First put down with the combination buffer of 5 column volumes
Weighed nickel post, flow velocity 5ml/min, loading flow velocity 1ml/min, and pillar, flow velocity are cleaned with the combination buffer of 6 column volumes after loading
1ml/min, finally elute destination protein, flow velocity 5ml/min with the elution buffer of 3 column volumes.Protein yield after purification
About 0.85mg/L.
The killing gene PPTX-3a of fit reconstruction nucleotides sequence is classified as SEQ ID NO.1, as follows：
The killing gene PPTX-3a of fit reconstruction amino acid sequence is SEQ ID NO.2, as follows：
The killing gene PPTX-3a of fit reconstruction ripe peptide sequence is SEQ ID NO.3, as follows：
Embodiment 2
The raw process and method surveyed
Insecticidal activities of the recombinant toxin PPTX-3a to 3 kinds of planthoppers is determined using microinjection, test worm chose for 5 ages
Nymph, 3 repetitions are each handled, it is each to repeat to choose 30 test worms, before injection, first test worm is anaesthetized with CO2, every test worm note
Recombinant toxin 20nl is penetrated, injection site is the first and second plastron corias, and after injection terminates, test worm is placed on equipped with rice
In the disposable cup of seedling, rice seedlings are fixed with 2% agar, and whole experiment process is soft, reduces the injury to test worm.It is real
Test and be divided into control group and experimental group, control group injection PBS, experimental group injection recombinant toxin PPTX-3a.Fig. 1,2 and 3 are respectively
After injecting recombinant toxin PPTX-3a, death condition of 3 kinds of planthoppers in different time points, it can be seen that the toxin PPTX- of restructuring
3a has preferable insecticidal activity to 3 kinds of planthoppers.
Claims (6)
1. a kind of killing gene from fit reconstruction, its nucleotide sequence is as shown in SEQ ID NO.1.
2. the insecticidal peptide of the killing gene coding of fit reconstruction, its amino acid sequence such as SEQ are come from as claimed in claim 1
Shown in ID NO.2, its ripe peptide amino acid sequence is shown in SEQ ID NO.3.
3. a kind of contain the prokaryotic vector that fit reconstruction killing gene is come from described in claim 1.
4. application of the insecticidal peptide as claimed in claim 2 in terms of crop pests preventing and treating.
It is 5. a kind of containing having the right to want the insecticide of 2 insecticidal peptides.
A kind of 6. desinsection that modification transformation is carried out on the basis of fit reconstruction killing gene is come from described in claim 1 and is obtained
Gene and its application.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108822196A (en) * | 2018-06-06 | 2018-11-16 | 湖南师范大学 | A kind of rush blood coagulation polypeptide LGTX-F2 and its application |
| CN108912219A (en) * | 2018-08-02 | 2018-11-30 | 南京农业大学 | Mature peptide and the application of fit reconstruction F family's killing gene and its coding |
| CN108912218A (en) * | 2018-08-02 | 2018-11-30 | 南京农业大学 | Mature peptide and the application of fit reconstruction A family's killing gene and its coding |
-
2017
- 2017-08-10 CN CN201710695548.6A patent/CN107488665A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108822196A (en) * | 2018-06-06 | 2018-11-16 | 湖南师范大学 | A kind of rush blood coagulation polypeptide LGTX-F2 and its application |
| CN108822196B (en) * | 2018-06-06 | 2023-04-21 | 湖南生达生物科技有限公司 | Procoagulant polypeptide LGTX-F2 and application thereof |
| CN108912219A (en) * | 2018-08-02 | 2018-11-30 | 南京农业大学 | Mature peptide and the application of fit reconstruction F family's killing gene and its coding |
| CN108912218A (en) * | 2018-08-02 | 2018-11-30 | 南京农业大学 | Mature peptide and the application of fit reconstruction A family's killing gene and its coding |
| CN108912218B (en) * | 2018-08-02 | 2022-03-18 | 南京农业大学 | Pseudoleopard A family insecticidal gene, coded mature peptide thereof and application |
| CN108912219B (en) * | 2018-08-02 | 2022-03-25 | 南京农业大学 | Pseudoleopard pardalus F family insecticidal gene, and coded mature peptide and application thereof |
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