CN108949771B - Pseudoleopard pardalus C family insecticidal gene, and coded mature peptide and application thereof - Google Patents
Pseudoleopard pardalus C family insecticidal gene, and coded mature peptide and application thereof Download PDFInfo
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- CN108949771B CN108949771B CN201810868357.XA CN201810868357A CN108949771B CN 108949771 B CN108949771 B CN 108949771B CN 201810868357 A CN201810868357 A CN 201810868357A CN 108949771 B CN108949771 B CN 108949771B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- 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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- 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
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Abstract
The invention discloses a pseudorhabdomina C family insecticidal gene, a coded mature peptide thereof and application, wherein the nucleotide sequence of the pseudorhabdomina C family insecticidal gene is shown in SEQ ID NO. 1-3; amino acids of mature peptides coded by the insecticidal gene of the pardosa pseudoannulata family C are shown in SEQ ID NO. 7-9. The invention utilizes a bioinformatics analysis method to screen genes in a pardosa mimicus transcriptome database to obtain a similar candidate gene of the pardosa mimicus toxin, constructs the gene into a prokaryotic expression vector pET-32a (+), and can obtain the insecticidal peptide coded by the gene.
Description
Technical Field
The invention belongs to the field of genetic engineering and biological control, relates to a pesticidal gene and a coding protein thereof, and particularly relates to a pardosa pseudoannulata family C pesticidal gene, a coded mature peptide thereof and application thereof.
Background
The insecticidal gene widely used for pest control at present is mainly a gene of Bt toxin generated by bacillus thuringiensis, and the Bt toxin shows extremely high insecticidal specificity to agricultural pests such as lepidoptera, diptera, coleoptera and the like. Therefore, the Bt toxin gene is genetically modified to important crops such as cotton, corn, tobacco and the like which are widely planted. Transgenic crops carrying insect-resistant genes play an important role in the control of agricultural pests. However, the long-term use of a single insect-resistant gene has led to an increasing resistance of pests to Bt toxins. Various insect populations develop resistance to Bt protein preparations and Bt transgenic crops in natural environments, such as cotton bollworm, diamond back moth, chilo suppressalis, and the like (dawn-li et al, progress in insect resistance to Bt toxins [ J ], Jiangsu agricultural science, 2014, stage 7). Therefore, the development of another novel insect-resistant gene or protein which is highly efficient and environmentally friendly can increase the selection of the insect-resistant gene and reduce the development of resistance.
Spider toxins have received much attention as potential pesticides due to their chemical diversity and broad spectrum (G.F. King, et al, Spider-Venom peptides: structures, pharmacology, and potential for control of insect pests, Annu.Rev.Entomol.2013,58: 475-496.). Spider toxins are capable of acting on a variety of channels and receptors in insect cell membranes, such as ion channels, nerve ligand gated channels, and G protein-associated receptors. Therefore, the insecticidal composition has an insect-resistant effect on various insects. However, the research on spider toxins at present mostly focuses on the action mechanism of important toxins in black widow spiders, and direct tests on the insecticidal action of the toxins are less. In conclusion, the existing researches on spider toxins cannot meet the selection requirement of insect-resistant genes. Therefore, there is a need for a pesticidal spider toxin that can be expressed in large quantities by molecular genetic engineering methods or in crops by transgenic technology to achieve an anti-pest effect.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a pardosa pseudorhabdomina C family insecticidal gene, mature peptide coded by the gene can be obtained by a biological means, and the mature peptide is used as a brand new insecticidal gene resource, has neurotoxicity, acts on insect ion channels, has an insecticidal mechanism different from Bt toxin, and has important scientific and practical significance for expanding novel insecticidal gene resources with biological activity, reducing various safety risks existing in the wide use of the existing Bt toxin and reducing the use of an insecticide.
The invention also discloses a mature peptide coded by the C family insecticidal gene of the pseudoannulatex and application thereof.
The technical scheme is as follows: in order to achieve the purpose, the insecticidal gene of the pardosa pseudoannulata family C is shown in SEQ ID NO. 1-3.
The insecticidal protein coded by the insecticidal gene of the pardosa pseudoannulata family C is shown in SEQ ID NO. 4-6.
The mature peptide coded by the C-family insecticidal gene of the pardosa pseudoannulata is shown in SEQ ID NO. 7-9.
The pardosphaeus pseudoannulata C family insecticidal gene encoding the mature peptide is shown in SEQ ID NO. 10-12.
The recombinant plasmid containing the pesticidal gene of the pseudoleopard spider family C provided by the invention.
The mature peptide coded by the C family insecticidal gene of the pardosa pseudoannulata is applied to prevention and control of crop pests.
Further, the mature peptide is applied to prevention and control of crop pests such as brown planthopper, gray planthopper and sogatella furcifera.
The invention relates to an insecticide containing a mature peptide coded by a pseudoleopard spider C family insecticidal gene.
The invention relates to application of a pesticide containing a mature peptide coded by a pseudoleopard C family insecticidal gene in crop pest control.
The invention refers to toxin genes of spiders such as black widow, screens genes in a pseudoorbicularis phalaenopsis transcriptome database by using a bioinformatics analysis method to obtain a similar candidate gene of the pseudoorbicularis phalaenopsis toxin, and then obtains a complete sequence of one of the genes by using Polymerase Chain Reaction (PCR) and Sanger sequencing. The gene is constructed into a prokaryotic expression vector pET-32a (+), and the mature peptide coded by the gene is obtained through prokaryotic system expression, namely the insecticidal peptide. The insecticidal peptide has short preparation period and small amino acid sequence, is suitable for in-vitro large-scale production, has an insecticidal mechanism different from that of Bt toxin as a brand-new insecticidal gene resource, and has important scientific and practical significance for expanding novel insecticidal gene resources with biological activity, reducing various safety risks existing in the wide use of the existing Bt toxin and reducing the use of insecticides.
Has the advantages that: compared with the prior art, the invention has the following advantages:
the leopard pseudoannulata adopted by the invention is used as a natural enemy of agricultural pests, and the insects are eaten for a long time, and the toxins of the leopard pseudoannulata mainly act on the insects and are relatively safe to vertebrates. The invention screens a brand-new parvalla A family insecticidal gene, can obtain a mature peptide coded by the gene as an insecticidal peptide by a biological means according to the gene, has neurotoxicity as a brand-new insecticidal gene resource, acts on an insect ion channel, has an insecticidal mechanism different from Bt toxin, and has important scientific and practical significance for expanding novel insecticidal gene resources with biological activity, reducing various safety risks existing in the wide use of the existing Bt toxin and reducing the use of an insecticide.
The invention predicts the mature peptide of the pardosphaera annulata toxin, successfully constructs an expression vector, optimizes induction conditions, maximizes protein yield, optimizes purification conditions and reduces protein loss. In addition, the in vitro expression system adopted by the invention has high efficiency and high yield, and the obtained insecticidal peptide has high purity and higher activity. The recombinant toxin prepared by the invention has higher insecticidal activity.
Drawings
FIG. 1 is a graph showing mortality of Nilaparvata lugens at different time points after injection of CK and the recombinant toxin PPTX-01;
FIG. 2 is a graph showing mortality of Laodelphax striatellus at various time points after injection of CK and the recombinant toxin PPTX-01;
FIG. 3 is a graph of mortality of Sogatella furcifera at various time points after injection of CK and the recombinant toxin PPTX-01.
Detailed Description
The invention is further illustrated by the following figures and examples.
The reagents and media formulations designed in the examples:
(1) LB liquid medium:
adding 10g tryptone, 5g yeast extract and 5g NaCl into 900ml double distilled water, stirring, mixing, fixing volume to 1L with double distilled water, placing in an autoclave, sterilizing at 121 deg.C for 20min, cooling, and storing at 4 deg.C.
(2) LB solid medium:
adding 10g of tryptone, 5g of yeast extract, 5g of NaCl and 15g of agar into 900ml of double distilled water, uniformly stirring, fixing the volume to 1L by using the double distilled water, placing the mixture into an autoclave, sterilizing the mixture for 20min at 121 ℃, cooling the mixture to 50 ℃, adding carbenicillin with the final concentration of 100mg/L, pouring the mixture into a plate, cooling the plate, and storing the cooled plate at 4 ℃.
(3) And (3) membrane transfer buffer solution:
2.9g of glycine, 0.8g of Tris base, 0.37g of SDS and 200ml of methanol, and adding double distilled water to the solution to reach the constant volume of 1L.
(4)PBST：
Tween-20 was added to PBS at a volume of 0.05%.
(5) Sealing liquid:
1% skimmed milk powder was added to PBST.
(6)Tris-HCl：
Tris-HCl 1M was diluted to 20 mM.
(7) Binding buffer:
20mM sodium phosphate, 0.5M sodium chloride, 10mM imidazole and double distilled water are added to a constant volume of 1L, and the pH value is 7.4.
(8) Elution buffer:
20mM sodium phosphate, 0.5M sodium chloride, 500mM imidazole and double distilled water are added to a constant volume of 1L, and the pH value is 7.4.
Example 1
Preparation of toxins
Design and construction of spider toxin genes
The method comprises the steps of collecting the pseudoringworm spider from a rice field in Pukou area of Nanjing city, Jiangsu province, taking poison glands and carrying out transcriptome sequencing after feeding brown planthopper indoors for 90 days, screening the pseudoringworm spider toxin gene according to annotation results, obtaining a class of candidate genes of the pseudoringworm spider toxin according to the quantity, arrangement mode, structural domain prediction and sequence analysis of cysteine of the pseudoringworm spider toxin, selecting one of the candidate genes, namely PPTX-01, the base sequence of the pseudoringworm spider toxin is shown as SEQ ID NO.1, the amino acid sequence of the encoded protein is shown as SEQ ID NO.4, predicting signal peptide, propeptide and mature peptide of the pseudoringworm spider toxin through SpiderP (http:// www.arachnoserver.org/iderP. html), carrying out codon optimization on the mature peptide sequence according to the codon preference of escherichia coli, designing the PPTX-01 toxin gene sequence for expressing in the escherichia coli to be shown as SEQ ID NO.10, and synthesizing the designed gene by Invitrogen company, and completing sequencing in Nanjing Kingsry company, cloning the synthesized gene into an escherichia coli expression vector pET-32a (+), and constructing a pET-32a (+) -PPTX-01 recombinant plasmid containing a target gene, wherein the toxin gene is introduced into enzyme cutting sites of BanH I and EcoR I.
Inducible expression of recombinant plasmid pET-32a (+) -PPTX-01
The pET-32a (+) -PPTX-02 recombinant plasmid is transformed into Escherichia coli BL21 strain, after the Escherichia coli BL21 strain grows in an LB plate containing carbenicillin for 16 hours, different positive bacterial plaques are picked up and respectively added into an LB liquid culture medium containing 100mg/L of carbenicillin, and the mixture is put into a constant temperature shaker for 250r/min, 37 ℃ and 12 hours. Inoculating the cultured bacterial liquid into LB liquid culture medium containing 100mg/L carbenicillin according to the volume ratio of 1: 100, culturing at 37 deg.C for 4h at 200r/min, wherein the OD value of the bacterial liquid is 0.5-0.7; adding IPTG with the final concentration of 0.4mmol/L into the pET-32a (+) -PPTX-01 recombinant, carrying out induced expression culture at 200r/min and 37 ℃ for 5 h; taking 4ml of the expressed bacterial liquid, centrifuging at 4 ℃ and 12000rpm for 5min, removing supernatant, adding 1.5ml of 20mM Tris-HCl into the bacterial precipitation, wherein the pH value is 7.4, carrying out ultrasonic bacteria breaking after heavy suspension, carrying out the ultrasonic bacteria breaking with the intensity of 12 percent, carrying out the ultrasonic bacteria breaking for 10min, running for 5s, and pausing for 5 s; and (4) after the bacteria are broken, centrifuging at 18000rpm for 10min, taking the supernatant, and discarding the precipitate for recombinant toxin detection.
Western Blot detection
Taking 50 mu l of the crushing liquid, adding 17 mu l of Loading Buffer and 2 mu l of beta-mercaptoethanol, boiling for 5min at 100 ℃, adding 20 mu l of the mixed liquid to SDS-polyacrylamide gel, adding 10 mu l of protein standard sample, 50V for 50min, and then adding 100V till the sample Loading Buffer solution reaches the bottom of the gel; after electrophoresis is finished, taking out the gel, putting the gel into a membrane transferring buffer solution, soaking the PVDF membrane in methanol for 1min, transferring the PVDF membrane into the membrane transferring buffer solution, and then transferring the PVDF membrane into a membrane, wherein the mA is 100mA and the time is 50 min; after the membrane is transferred, taking out the membrane, washing the membrane for 3 times by PBST (PBST), wherein each time is 5min, sealing the membrane for 2h by sealing liquid, and the temperature is 37 ℃ and 80 r/min; taking out the membrane after sealing, washing for 3 times by PBST (PBST), 10min each time, transferring into sealing solution (1: 1000) added with primary antibody, incubating for 2h at 37 ℃ and 80 r/min; after the primary antibody incubation is finished, taking out the membrane, washing for 3 times by PBST (PBST), transferring into a blocking solution (1: 2000) added with a secondary antibody each time for incubation for 1.5h at 37 ℃ at 80 r/min; after the incubation of the secondary antibody is finished, PBST is washed for 3 times, each time is 10min, luminescent liquid is added, the mixture is protected from light for 3min, and after surface liquid is absorbed by filter paper, chemiluminescence detection is carried out.
Large-scale inducible expression of recombinant plasmid pET-32a (+) -PPTX-01
Inoculating the cultured bacterial liquid into 100ml LB liquid culture medium containing 100mg/L carbenicillin according to the volume ratio of 1: 100, wherein the volume of the culture medium cannot exceed 20% of the volume of the container, 200r/min, 37 ℃, and culturing for 4h, wherein the OD value of the bacterial liquid is 0.5-0.7; adding IPTG with the final concentration of 0.4mmol/L into the pET-32a (+) -PPTX-01 recombinant, carrying out induced expression culture at 200r/min and 37 ℃ for 5 h; centrifuging the expressed bacterial solution at 4 ℃ and 12000rpm for 15min, removing the supernatant, adding 20% of Tris-HCl with the volume of 20% of the culture medium into the bacterial precipitation, wherein the pH is 7.4, carrying out ultrasonic bacteria breaking after heavy suspension, the intensity is 12%, the speed is 30min, the operation is carried out for 5s, and the suspension is carried out for 5 s; centrifuging at 4 deg.C and 18000rpm for 30min after breaking, collecting supernatant, removing precipitate, and separating and purifying the recombinant toxin from the crude protein solution after breaking. .
Purification of recombinant toxin PPTX-01
The crude protein solution after the mass induction expression and bacterial disruption was filtered through a 0.22 μm filter membrane and purified by using an AKTA avant full-automatic protein isolation and purification system and a HisTrpTM HP nickel column (5 ml). Firstly, 5 column volumes of binding buffer solution are used for balancing the nickel column, the flow rate is 5ml/min, the sample loading flow rate is 1ml/min, after sample loading, 6 column volumes of binding buffer solution are used for washing the column, the flow rate is 1ml/min, and finally 3 column volumes of elution buffer solution are used for eluting the target protein, and the flow rate is 5 ml/min. The yield of the purified protein is about 7.85mg/L, the purified protein is mature peptide, namely recombinant toxin PPTX-01, and the amino acid sequence of the purified protein is shown as SEQ ID NO. 7.
Example 2
The method for designing and constructing the spider toxin gene in the embodiment 1 is adopted to prepare the insecticidal gene SEQ ID NO. 2-3: PPTX-22, PPTX-44; corresponding recombinant plasmids are constructed by the same method and are respectively induced to express in large quantities, and recombinant toxins coded by different genes are obtained after purification.
The nucleotide sequence of the pesticidal gene PPTX-01 of the Pseudorhapontidae is SEQ ID NO.1 as follows:
atgattaagtacgtggtcatctcggctctcttggttgttgccgtgtattcattgacgattgacgaagagattgaagatgcattattagacgaggaacgcgaagaattaaaccctgaagaagaaagaaggattgccttaccacctggtgccgtgtgcaacggtcacaagagcgactgccagtgtttcggagccaaatacaagtgcagctgtccattcctttggcgtttcagaaggtcggccaggtgtcactgcaagaagggatgggcctggaccgcactcaagaaacgatcctgccgtaacagataccaatggagcggt
the nucleotide sequence of the pesticidal gene PPTX-22 of the Pseudorhapontidae is SEQ ID NO.2 as follows:
atgattaaattcattttgatctctgctttactggttgctgcggtctattcatttgctgttgaaaatgatgaagctgtaccccaggatgcagaacaggaagtaatgccagaagaagccaggagcttacccccaggagctgaatgcgatggcgacaagcctgactgccagtgttacggaaaatggcacaagtgtggctgtccttggtttggggggaaatgcacctgccagaagggaatgaaattcacctgcatccagaagctctcgtgccccaacaagggcgaatggggcctcgactggaggagtgaagaatccgaaagaagtccttgc
the nucleotide sequence of the pesticidal gene PPTX-44 of the Pseudorhapontidae is SEQ ID NO.3 as follows:
atgaagaatattcttgctttggtgttcatttgtctccttggtgcatcgactgccttcaacttagatgacacggatgaagcttcagatgaatttgcggtagaatcagcacaagaagctccagaacaagcccgcaggacgtgcttgcagactggctctgaatgcgatggcagcaaagacgactgccagtgctgtggggcatacgtcgagtgcaagtgtcccttcggcatcaactggcccagcgtcctaggaccctgcaagtgcaccatggaccacttgggaacctgcctggctaagcagaagtgccccaacaagcaggagtggggcggcggaaactgcaaatcccccaaaaagccacgacgaggg
the amino acid sequence of the pesticidal gene PPTX-01 of the Pseudorhapontidae is SEQ ID NO.4 as follows:
MIKYVVISALLVVAVYSLTIDEEIEDALLDEEREELNPEEERRIALPPGAVCNGHKSDCQCFGAKYKCSCPFLWRFRRSARCHCKKGWAWTALKKRSCRNRYQWSG
the amino acid sequence of the pesticidal gene PPTX-22 of the Pseudorhapontidae is SEQ ID NO.5 as follows:
MIKFILISALLVAAVYSFAVENDEAVPQDAEQEVMPEEARSLPPGAECDGDKPDCQCYGKWHKCGCPWFGGKCTCQKGMKFTCIQKLSCPNKGEWGLDWRSEESERSPC
the amino acid sequence of the pesticidal gene PPTX-44 of the Pseudorhapontidae is SEQ ID NO.6 as follows:
MKNILALVFICLLGASTAFNLDDTDEASDEFAVESAQEAPEQARRTCLQTGSECDGSKDDCQCCGAYVECKCPFGINWPSVLGPCKCTMDHLGTCLAKQKCPNKQEWGGGNCKSPKKPRRG
the mature peptide sequence of the pesticidal gene PPTX-01 of the Pseudorhapontidae is SEQ ID NO.7 as follows:
RIALPPGAVCNGHKSDCQCFGAKYKCSCPFLWRFRRSARCHCKKGWAWTALKKRSCRNRYQWSG
the mature peptide sequence of the pesticidal gene PPTX-22 of the Pseudorhapontidae is SEQ ID NO.8 as follows:
SLPPGAECDGDKPDCQCYGKWHKCGCPWFGGKCTCQKGMKFTCIQKLSCPNKGEWGLDWRSEESERSPC
the mature peptide sequence of the pesticidal gene PPTX-44 of the Pseudorhapontidae is SEQ ID NO.9 as follows:
RTCLQTGSECDGSKDDCQCCGAYVECKCPFGINWPSVLGPCKCTMDHLGTCLAKQKCPNKQEWGGGNCKSPKKPRRG
the gene sequence of the mature peptide of the pesticidal gene PPTX-01 of the Pseudorhabdospider is SEQ ID NO.10 as follows:
aggattgccttaccacctggtgccgtgtgcaacggtcacaagagcgactgccagtgtttcggagccaaatacaagtgcagctgtccattcctttggcgtttcagaaggtcggccaggtgtcactgcaagaagggatgggcctggaccgcactcaagaaacgatcctgccgtaacagataccaatggagcggt
the gene sequence of the mature peptide of the pesticidal gene PPTX-22 of the Pseudorhabdospider is SEQ ID NO.11 as follows:
agcttacccccaggagctgaatgcgatggcgacaagcctgactgccagtgttacggaaaatggcacaagtgtggctgtccttggtttggggggaaatgcacctgccagaagggaatgaaattcacctgcatccagaagctctcgtgccccaacaagggcgaatggggcctcgactggaggagtgaagaatccgaaagaagtccttgc
the gene sequence of the mature peptide of the pesticidal gene PPTX-44 of the Pseudorhabdospider is SEQ ID NO.12 as follows:
aggacgtgcttgcagactggctctgaatgcgatggcagcaaagacgactgccagtgctgtggggcatacgtcgagtgcaagtgtcccttcggcatcaactggcccagcgtcctaggaccctgcaagtgcaccatggaccacttgggaacctgcctggctaagcagaagtgccccaacaagcaggagtggggcggcggaaactgcaaatcccccaaaaagccacgacgaggg
test example 1
Bioassay process and method
The insecticidal activity of the recombinant toxin PPTX-01 on 3 rice planthoppers is measured by adopting a microinjection method, nymphs of 5 years old are selected for the test insects, each treatment is carried out for 3 times, 30 test insects are selected for each time, and before injection, the test insects are treated by CO2And (3) anaesthetizing, injecting 20nl of recombinant toxin into each test insect, wherein the injection part is a first chest plate internode membrane and a second chest plate internode membrane, after the injection is finished, placing the test insects in a disposable cup filled with rice seedlings, fixing the rice seedlings by 2% of agar, and reducing the damage to the test insects in the whole experiment process. The experiment group is divided into a control group and an experiment group, wherein the control group is injected with PBS with the same amount and the pH value of 7.4, and the experiment group is injected with the recombinant toxin PPTX-01. FIGS. 1, 2 and 3 show the death of 3 rice planthoppers at different time points after recombinant toxin PPTX-01 is injected, and the recombinant toxin PPTX-01 has excellent insecticidal activity on the 3 rice planthoppers, so that the mature peptide of the insecticidal gene PPTX-01 of the pseudoringworm leopard spider is proved to be an effective insecticidal peptide and can be used for preparing insecticides for preventing and treating crop pests such as brown planthopper, laodelphax striatellus, sogatella furcifera and the like, and other recombinant toxins PPTX-22 and PPTX-44 have similar functions to the recombinant toxin PPTX-01.
Sequence listing
<110> Nanjing university of agriculture
<120> Pseudoleopard C family insecticidal gene, coded mature peptide thereof and application
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 318
<212> DNA
<213> insecticidal gene PPTX-01 of Pholiota annulata (PPTX-01)
<400> 1
atgattaagt acgtggtcat ctcggctctc ttggttgttg ccgtgtattc attgacgatt 60
gacgaagaga ttgaagatgc attattagac gaggaacgcg aagaattaaa ccctgaagaa 120
gaaagaagga ttgccttacc acctggtgcc gtgtgcaacg gtcacaagag cgactgccag 180
tgtttcggag ccaaatacaa gtgcagctgt ccattccttt ggcgtttcag aaggtcggcc 240
aggtgtcact gcaagaaggg atgggcctgg accgcactca agaaacgatc ctgccgtaac 300
agataccaat ggagcggt 318
<210> 2
<211> 327
<212> DNA
<213> insecticidal gene PPTX-22 of Pholiota leopard (PPTX-22)
<400> 2
atgattaaat tcattttgat ctctgcttta ctggttgctg cggtctattc atttgctgtt 60
gaaaatgatg aagctgtacc ccaggatgca gaacaggaag taatgccaga agaagccagg 120
agcttacccc caggagctga atgcgatggc gacaagcctg actgccagtg ttacggaaaa 180
tggcacaagt gtggctgtcc ttggtttggg gggaaatgca cctgccagaa gggaatgaaa 240
ttcacctgca tccagaagct ctcgtgcccc aacaagggcg aatggggcct cgactggagg 300
agtgaagaat ccgaaagaag tccttgc 327
<210> 3
<211> 363
<212> DNA
<213> insecticidal gene PPTX-44 of Pholiota leopard (PPTX-44)
<400> 3
atgaagaata ttcttgcttt ggtgttcatt tgtctccttg gtgcatcgac tgccttcaac 60
ttagatgaca cggatgaagc ttcagatgaa tttgcggtag aatcagcaca agaagctcca 120
gaacaagccc gcaggacgtg cttgcagact ggctctgaat gcgatggcag caaagacgac 180
tgccagtgct gtggggcata cgtcgagtgc aagtgtccct tcggcatcaa ctggcccagc 240
gtcctaggac cctgcaagtg caccatggac cacttgggaa cctgcctggc taagcagaag 300
tgccccaaca agcaggagtg gggcggcgga aactgcaaat cccccaaaaa gccacgacga 360
ggg 363
<210> 4
<211> 106
<212> PRT
<213> insecticidal gene PPTX-01 of Pholiota annulata (PPTX-01)
<400> 4
Met Ile Lys Tyr Val Val Ile Ser Ala Leu Leu Val Val Ala Val Tyr
1 5 10 15
Ser Leu Thr Ile Asp Glu Glu Ile Glu Asp Ala Leu Leu Asp Glu Glu
20 25 30
Arg Glu Glu Leu Asn Pro Glu Glu Glu Arg Arg Ile Ala Leu Pro Pro
35 40 45
Gly Ala Val Cys Asn Gly His Lys Ser Asp Cys Gln Cys Phe Gly Ala
50 55 60
Lys Tyr Lys Cys Ser Cys Pro Phe Leu Trp Arg Phe Arg Arg Ser Ala
65 70 75 80
Arg Cys His Cys Lys Lys Gly Trp Ala Trp Thr Ala Leu Lys Lys Arg
85 90 95
Ser Cys Arg Asn Arg Tyr Gln Trp Ser Gly
100 105
<210> 5
<211> 109
<212> PRT
<213> insecticidal gene PPTX-22 of Pholiota leopard (PPTX-22)
<400> 5
Met Ile Lys Phe Ile Leu Ile Ser Ala Leu Leu Val Ala Ala Val Tyr
1 5 10 15
Ser Phe Ala Val Glu Asn Asp Glu Ala Val Pro Gln Asp Ala Glu Gln
20 25 30
Glu Val Met Pro Glu Glu Ala Arg Ser Leu Pro Pro Gly Ala Glu Cys
35 40 45
Asp Gly Asp Lys Pro Asp Cys Gln Cys Tyr Gly Lys Trp His Lys Cys
50 55 60
Gly Cys Pro Trp Phe Gly Gly Lys Cys Thr Cys Gln Lys Gly Met Lys
65 70 75 80
Phe Thr Cys Ile Gln Lys Leu Ser Cys Pro Asn Lys Gly Glu Trp Gly
85 90 95
Leu Asp Trp Arg Ser Glu Glu Ser Glu Arg Ser Pro Cys
100 105
<210> 6
<211> 121
<212> PRT
<213> insecticidal gene PPTX-44 of Pholiota leopard (PPTX-44)
<400> 6
Met Lys Asn Ile Leu Ala Leu Val Phe Ile Cys Leu Leu Gly Ala Ser
1 5 10 15
Thr Ala Phe Asn Leu Asp Asp Thr Asp Glu Ala Ser Asp Glu Phe Ala
20 25 30
Val Glu Ser Ala Gln Glu Ala Pro Glu Gln Ala Arg Arg Thr Cys Leu
35 40 45
Gln Thr Gly Ser Glu Cys Asp Gly Ser Lys Asp Asp Cys Gln Cys Cys
50 55 60
Gly Ala Tyr Val Glu Cys Lys Cys Pro Phe Gly Ile Asn Trp Pro Ser
65 70 75 80
Val Leu Gly Pro Cys Lys Cys Thr Met Asp His Leu Gly Thr Cys Leu
85 90 95
Ala Lys Gln Lys Cys Pro Asn Lys Gln Glu Trp Gly Gly Gly Asn Cys
100 105 110
Lys Ser Pro Lys Lys Pro Arg Arg Gly
115 120
<210> 7
<211> 64
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Arg Ile Ala Leu Pro Pro Gly Ala Val Cys Asn Gly His Lys Ser Asp
1 5 10 15
Cys Gln Cys Phe Gly Ala Lys Tyr Lys Cys Ser Cys Pro Phe Leu Trp
20 25 30
Arg Phe Arg Arg Ser Ala Arg Cys His Cys Lys Lys Gly Trp Ala Trp
35 40 45
Thr Ala Leu Lys Lys Arg Ser Cys Arg Asn Arg Tyr Gln Trp Ser Gly
50 55 60
<210> 8
<211> 69
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Ser Leu Pro Pro Gly Ala Glu Cys Asp Gly Asp Lys Pro Asp Cys Gln
1 5 10 15
Cys Tyr Gly Lys Trp His Lys Cys Gly Cys Pro Trp Phe Gly Gly Lys
20 25 30
Cys Thr Cys Gln Lys Gly Met Lys Phe Thr Cys Ile Gln Lys Leu Ser
35 40 45
Cys Pro Asn Lys Gly Glu Trp Gly Leu Asp Trp Arg Ser Glu Glu Ser
50 55 60
Glu Arg Ser Pro Cys
65
<210> 9
<211> 77
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Arg Thr Cys Leu Gln Thr Gly Ser Glu Cys Asp Gly Ser Lys Asp Asp
1 5 10 15
Cys Gln Cys Cys Gly Ala Tyr Val Glu Cys Lys Cys Pro Phe Gly Ile
20 25 30
Asn Trp Pro Ser Val Leu Gly Pro Cys Lys Cys Thr Met Asp His Leu
35 40 45
Gly Thr Cys Leu Ala Lys Gln Lys Cys Pro Asn Lys Gln Glu Trp Gly
50 55 60
Gly Gly Asn Cys Lys Ser Pro Lys Lys Pro Arg Arg Gly
65 70 75
<210> 10
<211> 192
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
aggattgcct taccacctgg tgccgtgtgc aacggtcaca agagcgactg ccagtgtttc 60
ggagccaaat acaagtgcag ctgtccattc ctttggcgtt tcagaaggtc ggccaggtgt 120
cactgcaaga agggatgggc ctggaccgca ctcaagaaac gatcctgccg taacagatac 180
caatggagcg gt 192
<210> 11
<211> 207
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
agcttacccc caggagctga atgcgatggc gacaagcctg actgccagtg ttacggaaaa 60
tggcacaagt gtggctgtcc ttggtttggg gggaaatgca cctgccagaa gggaatgaaa 120
ttcacctgca tccagaagct ctcgtgcccc aacaagggcg aatggggcct cgactggagg 180
agtgaagaat ccgaaagaag tccttgc 207
<210> 12
<211> 231
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
aggacgtgct tgcagactgg ctctgaatgc gatggcagca aagacgactg ccagtgctgt 60
ggggcatacg tcgagtgcaa gtgtcccttc ggcatcaact ggcccagcgt cctaggaccc 120
tgcaagtgca ccatggacca cttgggaacc tgcctggcta agcagaagtg ccccaacaag 180
caggagtggg gcggcggaaa ctgcaaatcc cccaaaaagc cacgacgagg g 231
Claims (8)
1. A pardosa pseudoannulata family C insecticidal gene is characterized in that the nucleotide sequences of the insecticidal genes are respectively shown in any one of SEQ ID NO. 1-3.
2. An insecticidal protein encoded by the insecticidal gene of the Araneus parva family C according to claim 1, wherein the amino acid sequences of the insecticidal protein are respectively represented by any one of SEQ ID NO. 4-6.
3. A mature peptide coded by the pesticidal gene of the C family of pardosa according to claim 1, wherein the amino acid sequence of the mature peptide is as shown in any one of SEQ ID NOs.7-9.
4. A pardosa pseudonana C family insecticidal gene encoding the mature peptide according to claim 3, wherein the nucleotide sequences of the genes are respectively as shown in any one of SEQ ID NO. 10-12.
5. A recombinant plasmid containing the pseudoleopard spider family C insecticidal gene according to claim 4.
6. The application of the mature peptide coded by the pseudoleopard spider C family insecticidal gene according to claim 3 in prevention and treatment of crop pests, namely brown planthopper, gray planthopper and sogatella furcifera.
7. An insecticide comprising a mature peptide encoded by the pseudoleopard spider family C insecticidal gene of claim 3.
8. The application of the insecticide containing the mature peptide coded by the pseudoleopard spider C family insecticidal gene according to claim 7 in controlling crop pests brown planthopper, gray planthopper and sogatella furcifera.
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