CN108998456B

CN108998456B - Pseudoleopard pseudoannulata D family insecticidal gene, and coded mature peptide and application thereof

Info

Publication number: CN108998456B
Application number: CN201810868231.2A
Authority: CN
Inventors: 刘泽文; 黄立鑫; 张懿熙; 鲍海波; 王照英
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2018-08-02
Filing date: 2018-08-02
Publication date: 2022-02-22
Anticipated expiration: 2038-08-02
Also published as: CN108998456A

Abstract

The invention discloses a parnapus pseudoannulatus family D insecticidal gene, a coded mature peptide and application thereof, wherein the nucleotide sequence of the parnapus pseudoannulatus family D insecticidal gene is shown in SEQ ID NO. 1-5; amino acids of mature peptide coded by the pesticidal gene of the pseudoannulatella D family are shown in SEQ ID NO. 11-15. 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

Pseudoleopard pseudoannulata D family insecticidal gene, and coded mature peptide and application thereof

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 D family 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 D 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 D family insecticidal gene of the pseudoannulatella and application thereof.

The technical scheme is as follows: in order to achieve the purpose, the insecticidal gene of the pardosa pseudoannulata D family is shown in SEQ ID NO. 1-5.

The insecticidal protein coded by the D family insecticidal gene of the pardosa pseudoannulata is shown in SEQ ID NO.6-10 in amino acid sequence.

The amino acid sequence of the mature peptide coded by the D family insecticidal gene of the pardosa pseudoannulata is shown in SEQ ID NO. 11-15.

The pseudoleopard spider D family insecticidal gene encoding the mature peptide is shown in SEQ ID NO. 16-20.

The recombinant plasmid containing the D family insecticidal gene of the pardosa pseudoannulata is provided.

The invention relates to application of a mature peptide coded by a D family insecticidal gene of a pardosa pseudoannulata in crop pest control.

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 family D insecticidal gene.

The invention relates to application of a pesticide containing a mature peptide coded by a pseudoleopard spider D 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 Pseudorhabdomina D family insecticidal gene through the Pseudorhabdoviridae, can obtain a mature peptide coded by the gene as an insecticidal peptide through a biological means according to the gene, and the insecticidal peptide has neurotoxicity as a brand-new insecticidal gene resource, acts on an insect ion channel, has an insecticidal mechanism different from Bt toxin, namely the Bt toxin acts on an insect peritrophic membrane, and has important scientific and practical significance for expanding the novel insecticidal gene resource with bioactivity, 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-07;

FIG. 2 is a graph showing mortality of Laodelphax striatellus at various time points after injection of CK and recombinant toxin PPTX-07;

FIG. 3 is a graph of mortality of Sogatella furcifera at various time points after injection of CK and recombinant toxin PPTX-07.

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-07, the base sequence of the PPTX-07 is shown as SEQ ID NO.1, the amino acid sequence of the coded protein is shown as SEQ ID NO.6, predicting signal peptide, propeptide and mature peptide of the pseudoringworm spider toxin through SpiderP (http:// www.arachnoserver.org/spiderP. html), carrying out codon optimization on the mature peptide sequence according to codon preference of escherichia coli, designing the PPTX-07 toxin gene sequence for expressing in the escherichia coli to be shown as SEQ ID NO.16, and synthesizing the designed gene by an Invitrogen company, and completing sequencing in Nanjing Kingsry company, cloning the synthesized gene into an escherichia coli expression vector pET-32a (+), constructing pET-32a (+) -PPTX-07 recombinant plasmid containing a target gene, and introducing a BanH I and EcoR I enzyme cutting site into the toxin gene.

Inducible expression of recombinant plasmid pET-32a (+) -PPTX-07

The pET-32a (+) -PPTX-07 recombinant plasmid is transformed into Escherichia coli BL21 strain, after growing for 16h in an LB plate containing carbenicillin, different positive bacterial plaques are picked 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 h. 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-07 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; centrifuging at 4 deg.C and 18000rpm for 10min after bacteria breaking, collecting supernatant, removing precipitate, and using the broken solution 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-07

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-07 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-07

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 9.85mg/L, the purified protein is mature peptide, namely recombinant toxin PPTX-07, and the amino acid sequence of the purified protein is shown as SEQ ID NO. 11.

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. 2-4: PPTX-13, PPTX-28, PPTX-38 and PPTX-39; 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-07 of the Pseudorhapontidae is SEQ ID NO.1 as follows:

atgatgtctttgaaaatgcaagcgatgttgttggttgtaggcttgattacattcatagcagttcacgctgaagaagatataagtgaaaccgtagaatcagaacgatcgtgcgctaaggaataccaaagatgtgattggaacaacaaaccctgttgcgataatatttcttgcgtgtgtagcttgataggaaccaactgtgaatgtaagaaaggtatcatacgtactataaaagactggttcagtggaaaa

the nucleotide sequence of the pesticidal gene PPTX-13 of the Pseudorhapontidae is SEQ ID NO.2 as follows:

atgatgagagcctcaactgtattcggattatgtgcaattgctgttttgctgcttacgatcccagacgtttctggtgaagatgagatcaattctcaaaatgctccagaagaacgtggatactgtgctgagaacggcattcgatgtgacgatattcactgctgcactggtctaaaatgcaaatgcaacgccagcggttacaactgtgtttgcaggaagaag

the nucleotide sequence of the pesticidal gene PPTX-28 of the Pseudorhapontidae is SEQ ID NO.3 as follows:

atgggccgttggatttttgccattttccttggaattactcttttgactcaagtgcttcttccatcagcatatatggccacttcagacgcagatacgcctgccgtggatgactatgcagatgtcgctcgcttgttatactttgctcggaagagaagttgtattaagcgtggatcgagctgcgatcatcggccaaatgactgctgtgacaactcttcctgtcgatgcaatttgtggggaactaattgccgctgccaacgaatgggcctcttccagaagtggggcaaa

the nucleotide sequence of the pesticidal gene PPTX-38 of the Pseudorhapontidae is SEQ ID NO.4 as follows:

atgaaatacttgctgttagcgacagttgttgtattcctgtttgtgcaagctctggcggcagtgccatacccaccacctcttgatcgtaacctctcagatgactacaatgaaaatgtggacatgtacatgaaagcggacaagcgtgcttgcattcgcaggggaggtggatgtgacggaaaacccaatgactgctgccccaactcatcatgcaggtgcaacctctggggaaccaattgcagatgcgaacgtgcaggtctcttccagcaatggggcaaa

the nucleotide sequence of the pesticidal gene PPTX-39 of the Pseudorhapontidae is SEQ ID NO.5 as follows:

atgaaatactttctaaaacctttggttgtgacggtcctcctattcttatgctatgctgtcatgactaatgcttacgtaatgcgtgacagttctctggacagctaccaggaaccgaactatgaagctctccgccagtacctcctgtcgacacggaaaaggagctgcattcgacgaggtagcagctgcgatcaccgaccaagtgactgctgtttcaactcatcctgcagatgcaacttgtggggaaccaactgcagatgccagagggctggtctctttcagaaatggggaaag

the amino acid sequence of the pesticidal gene PPTX-07 of the Pseudorhapontidae is SEQ ID NO.6 as follows:

MMSLKMQAMLLVVGLITFIAVHAEEDISETVESERSCAKEYQRCDWNNKPCCDNISCVCSLIGTNCECKKGIIRTIKDWFSGK

the amino acid sequence of the pesticidal gene PPTX-13 of the Pseudorhapontidae is SEQ ID NO.7 as follows:

MMRASTVFGLCAIAVLLLTIPDVSGEDEINSQNAPEERGYCAENGIRCDDIHCCTGLKCKCNASGYNCVCRKK

the amino acid sequence of the pesticidal gene PPTX-28 of the Pseudorhapontidae is SEQ ID NO.8 as follows:

MGRWIFAIFLGITLLTQVLLPSAYMATSDADTPAVDDYADVARLLYFARKRSCIKRGSSCDHRPNDCCDNSSCRCNLWGTNCRCQRMGLFQKWGK

the amino acid sequence of the pesticidal gene PPTX-38 of the Pseudorhapontidae is SEQ ID NO.9 as follows:

MKYLLLATVVVFLFVQALAAVPYPPPLDRNLSDDYNENVDMYMKADKRACIRRGGGCDGKPNDCCPNSSCRCNLWGTNCRCERAGLFQQWGK

the amino acid sequence of the pesticidal gene PPTX-39 of the Pseudorhapontidae is SEQ ID NO.10 as follows:

MKYFLKPLVVTVLLFLCYAVMTNAYVMRDSSLDSYQEPNYEALRQYLLSTRKRSCIRRGSSCDHRPSDCCFNSSCRCNLWGTNCRCQRAGLFQKWGK

the mature peptide sequence of the pesticidal gene PPTX-07 of the Pseudorhapontidae is SEQ ID NO.11 as follows:

ERSCAKEYQRCDWNNKPCCDNISCVCSLIGTNCECKKGIIRTIKDWFSGK

the mature peptide sequence of the pesticidal gene PPTX-13 of the Pseudorhapontidae is SEQ ID NO.12 as follows:

RGYCAENGIRCDDIHCCTGLKCKCNASGYNCVCRKK

the mature peptide sequence of the pesticidal gene PPTX-28 of the Pseudorhapontidae is SEQ ID NO.13 as follows:

KRSCIKRGSSCDHRPNDCCDNSSCRCNLWGTNCRCQRMGLFQKWGK

the mature peptide sequence of the pesticidal gene PPTX-38 of the Pseudorhapontidae is SEQ ID NO.14 as follows:

KRACIRRGGGCDGKPNDCCPNSSCRCNLWGTNCRCERAGLFQQWGK

the mature peptide sequence of the pesticidal gene PPTX-39 of the Pseudorhapontidae is SEQ ID NO.15 as follows:

KRSCIRRGSSCDHRPSDCCFNSSCRCNLWGTNCRCQRAGLFQKWGK

the gene sequence of the mature peptide of the pesticidal gene PPTX-07 of the Pseudorhabdospider is SEQ ID NO.16 as follows:

gaacgatcgtgcgctaaggaataccaaagatgtgattggaacaacaaaccctgttgcgataatatttcttgcgtgtgtagcttgataggaaccaactgtgaatgtaagaaaggtatcatacgtactataaaagactggttcagtggaaaa

the gene sequence of the mature peptide of the pesticidal gene PPTX-13 of the Pseudorhabdospider is SEQ ID NO.17 as follows:

cgtggatactgtgctgagaacggcattcgatgtgacgatattcactgctgcactggtctaaaatgcaaatgcaacgccagcggttacaactgtgtttgcaggaagaag

the gene sequence of the mature peptide of the pesticidal gene PPTX-28 of the Pseudorhabdospider is SEQ ID NO.18 as follows:

aagagaagttgtattaagcgtggatcgagctgcgatcatcggccaaatgactgctgtgacaactcttcctgtcgatgcaatttgtggggaactaattgccgctgccaacgaatgggcctcttccagaagtggggcaaa

the gene sequence of the mature peptide of the pesticidal gene PPTX-38 of the Pseudorhabdospider is SEQ ID NO.19 as follows:

aagcgtgcttgcattcgcaggggaggtggatgtgacggaaaacccaatgactgctgccccaactcatcatgcaggtgcaacctctggggaaccaattgcagatgcgaacgtgcaggtctcttccagcaatggggcaaa

the gene sequence of the mature peptide of the pesticidal gene PPTX-39 of the Pseudorhabdospider is SEQ ID NO.20 as follows:

aaaaggagctgcattcgacgaggtagcagctgcgatcaccgaccaagtgactgctgtttcaactcatcctgcagatgcaacttgtggggaaccaactgcagatgccagagggctggtctctttcagaaatggggaaag

test example 1

Bioassay process and method

The insecticidal activity of the recombinant toxin PPTX-07 on 3 rice planthoppers is measured by adopting a microinjection method, 5-year-old nymphs are selected for test insects, each treatment is carried out for 3 times, and 30 heads are selected for each timeTesting insects, before injection, using CO for testing insects₂And (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-07. FIGS. 1, 2 and 3 show the death of 3 rice planthoppers at different time points after recombinant toxin PPTX-07 is injected, and the recombinant toxin PPTX-07 has excellent insecticidal activity on the 3 rice planthoppers, so that the mature peptide of the insecticidal gene PPTX-07 of the pseudoringworm leopard spider obtained by the invention is 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-13, PPTX-28, PPTX-38 and PPTX-39 have similar functions to the recombinant toxin PPTX-07.

Sequence listing

<110> Nanjing university of agriculture

<120> Pseudoleopard spider D family insecticidal gene, coded mature peptide thereof and application

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 249

<212> DNA

<213> insecticidal gene PPTX-07 of Pholiota annulata (PPTX-07)

<400> 1

atgatgtctt tgaaaatgca agcgatgttg ttggttgtag gcttgattac attcatagca 60

gttcacgctg aagaagatat aagtgaaacc gtagaatcag aacgatcgtg cgctaaggaa 120

taccaaagat gtgattggaa caacaaaccc tgttgcgata atatttcttg cgtgtgtagc 180

ttgataggaa ccaactgtga atgtaagaaa ggtatcatac gtactataaa agactggttc 240

agtggaaaa 249

<210> 2

<211> 219

<212> DNA

<213> insecticidal gene PPTX-13 of Pholiota annulata (PPTX-13)

<400> 2

atgatgagag cctcaactgt attcggatta tgtgcaattg ctgttttgct gcttacgatc 60

ccagacgttt ctggtgaaga tgagatcaat tctcaaaatg ctccagaaga acgtggatac 120

tgtgctgaga acggcattcg atgtgacgat attcactgct gcactggtct aaaatgcaaa 180

tgcaacgcca gcggttacaa ctgtgtttgc aggaagaag 219

<210> 3

<211> 285

<212> DNA

<213> insecticidal gene PPTX-28 of Pholiota leopard (PPTX-28)

<400> 3

atgggccgtt ggatttttgc cattttcctt ggaattactc ttttgactca agtgcttctt 60

ccatcagcat atatggccac ttcagacgca gatacgcctg ccgtggatga ctatgcagat 120

gtcgctcgct tgttatactt tgctcggaag agaagttgta ttaagcgtgg atcgagctgc 180

gatcatcggc caaatgactg ctgtgacaac tcttcctgtc gatgcaattt gtggggaact 240

aattgccgct gccaacgaat gggcctcttc cagaagtggg gcaaa 285

<210> 4

<211> 276

<212> DNA

<213> insecticidal gene PPTX-38(PPTX-38) of Pseudorhabdospider

<400> 4

atgaaatact tgctgttagc gacagttgtt gtattcctgt ttgtgcaagc tctggcggca 60

gtgccatacc caccacctct tgatcgtaac ctctcagatg actacaatga aaatgtggac 120

atgtacatga aagcggacaa gcgtgcttgc attcgcaggg gaggtggatg tgacggaaaa 180

cccaatgact gctgccccaa ctcatcatgc aggtgcaacc tctggggaac caattgcaga 240

tgcgaacgtg caggtctctt ccagcaatgg ggcaaa 276

<210> 5

<211> 291

<212> DNA

<213> insecticidal gene PPTX-39 of Pholiota leopard (PPTX-39)

<400> 5

atgaaatact ttctaaaacc tttggttgtg acggtcctcc tattcttatg ctatgctgtc 60

atgactaatg cttacgtaat gcgtgacagt tctctggaca gctaccagga accgaactat 120

gaagctctcc gccagtacct cctgtcgaca cggaaaagga gctgcattcg acgaggtagc 180

agctgcgatc accgaccaag tgactgctgt ttcaactcat cctgcagatg caacttgtgg 240

ggaaccaact gcagatgcca gagggctggt ctctttcaga aatggggaaa g 291

<210> 6

<211> 83

<212> PRT

<213> insecticidal gene PPTX-07 of Pholiota annulata (PPTX-07)

<400> 6

Met Met Ser Leu Lys Met Gln Ala Met Leu Leu Val Val Gly Leu Ile

1 5 10 15

Thr Phe Ile Ala Val His Ala Glu Glu Asp Ile Ser Glu Thr Val Glu

20 25 30

Ser Glu Arg Ser Cys Ala Lys Glu Tyr Gln Arg Cys Asp Trp Asn Asn

35 40 45

Lys Pro Cys Cys Asp Asn Ile Ser Cys Val Cys Ser Leu Ile Gly Thr

50 55 60

Asn Cys Glu Cys Lys Lys Gly Ile Ile Arg Thr Ile Lys Asp Trp Phe

65 70 75 80

Ser Gly Lys

<210> 20

<211> 73

<212> PRT

<213> insecticidal gene PPTX-13 of Pholiota annulata (PPTX-13)

<400> 20

Met Met Arg Ala Ser Thr Val Phe Gly Leu Cys Ala Ile Ala Val Leu

1 5 10 15

Leu Leu Thr Ile Pro Asp Val Ser Gly Glu Asp Glu Ile Asn Ser Gln

20 25 30

Asn Ala Pro Glu Glu Arg Gly Tyr Cys Ala Glu Asn Gly Ile Arg Cys

35 40 45

Asp Asp Ile His Cys Cys Thr Gly Leu Lys Cys Lys Cys Asn Ala Ser

50 55 60

Gly Tyr Asn Cys Val Cys Arg Lys Lys

65 70

<210> 7

<211> 95

<212> PRT

<213> insecticidal gene PPTX-28 of Pholiota leopard (PPTX-28)

<400> 7

Met Gly Arg Trp Ile Phe Ala Ile Phe Leu Gly Ile Thr Leu Leu Thr

1 5 10 15

Gln Val Leu Leu Pro Ser Ala Tyr Met Ala Thr Ser Asp Ala Asp Thr

20 25 30

Pro Ala Val Asp Asp Tyr Ala Asp Val Ala Arg Leu Leu Tyr Phe Ala

35 40 45

Arg Lys Arg Ser Cys Ile Lys Arg Gly Ser Ser Cys Asp His Arg Pro

50 55 60

Asn Asp Cys Cys Asp Asn Ser Ser Cys Arg Cys Asn Leu Trp Gly Thr

65 70 75 80

Asn Cys Arg Cys Gln Arg Met Gly Leu Phe Gln Lys Trp Gly Lys

85 90 95

<210> 8

<211> 92

<212> PRT

<213> insecticidal gene PPTX-38(PPTX-38) of Pseudorhabdospider

<400> 8

Met Lys Tyr Leu Leu Leu Ala Thr Val Val Val Phe Leu Phe Val Gln

1 5 10 15

Ala Leu Ala Ala Val Pro Tyr Pro Pro Pro Leu Asp Arg Asn Leu Ser

20 25 30

Asp Asp Tyr Asn Glu Asn Val Asp Met Tyr Met Lys Ala Asp Lys Arg

35 40 45

Ala Cys Ile Arg Arg Gly Gly Gly Cys Asp Gly Lys Pro Asn Asp Cys

50 55 60

Cys Pro Asn Ser Ser Cys Arg Cys Asn Leu Trp Gly Thr Asn Cys Arg

65 70 75 80

Cys Glu Arg Ala Gly Leu Phe Gln Gln Trp Gly Lys

85 90

<210> 9

<211> 97

<212> PRT

<213> insecticidal gene PPTX-39 of Pholiota leopard (PPTX-39)

<400> 9

Met Lys Tyr Phe Leu Lys Pro Leu Val Val Thr Val Leu Leu Phe Leu

1 5 10 15

Cys Tyr Ala Val Met Thr Asn Ala Tyr Val Met Arg Asp Ser Ser Leu

20 25 30

Asp Ser Tyr Gln Glu Pro Asn Tyr Glu Ala Leu Arg Gln Tyr Leu Leu

35 40 45

Ser Thr Arg Lys Arg Ser Cys Ile Arg Arg Gly Ser Ser Cys Asp His

50 55 60

Arg Pro Ser Asp Cys Cys Phe Asn Ser Ser Cys Arg Cys Asn Leu Trp

65 70 75 80

Gly Thr Asn Cys Arg Cys Gln Arg Ala Gly Leu Phe Gln Lys Trp Gly

85 90 95

Lys

<210> 10

<211> 50

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Glu Arg Ser Cys Ala Lys Glu Tyr Gln Arg Cys Asp Trp Asn Asn Lys

1 5 10 15

Pro Cys Cys Asp Asn Ile Ser Cys Val Cys Ser Leu Ile Gly Thr Asn

20 25 30

Cys Glu Cys Lys Lys Gly Ile Ile Arg Thr Ile Lys Asp Trp Phe Ser

35 40 45

Gly Lys

50

<210> 11

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Arg Gly Tyr Cys Ala Glu Asn Gly Ile Arg Cys Asp Asp Ile His Cys

1 5 10 15

Cys Thr Gly Leu Lys Cys Lys Cys Asn Ala Ser Gly Tyr Asn Cys Val

20 25 30

Cys Arg Lys Lys

35

<210> 12

<211> 46

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Lys Arg Ser Cys Ile Lys Arg Gly Ser Ser Cys Asp His Arg Pro Asn

1 5 10 15

Asp Cys Cys Asp Asn Ser Ser Cys Arg Cys Asn Leu Trp Gly Thr Asn

20 25 30

Cys Arg Cys Gln Arg Met Gly Leu Phe Gln Lys Trp Gly Lys

35 40 45

<210> 13

<211> 46

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Lys Arg Ala Cys Ile Arg Arg Gly Gly Gly Cys Asp Gly Lys Pro Asn

1 5 10 15

Asp Cys Cys Pro Asn Ser Ser Cys Arg Cys Asn Leu Trp Gly Thr Asn

20 25 30

Cys Arg Cys Glu Arg Ala Gly Leu Phe Gln Gln Trp Gly Lys

35 40 45

<210> 14

<211> 46

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Lys Arg Ser Cys Ile Arg Arg Gly Ser Ser Cys Asp His Arg Pro Ser

1 5 10 15

Asp Cys Cys Phe Asn Ser Ser Cys Arg Cys Asn Leu Trp Gly Thr Asn

20 25 30

Cys Arg Cys Gln Arg Ala Gly Leu Phe Gln Lys Trp Gly Lys

35 40 45

<210> 15

<211> 150

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gaacgatcgt gcgctaagga ataccaaaga tgtgattgga acaacaaacc ctgttgcgat 60

aatatttctt gcgtgtgtag cttgatagga accaactgtg aatgtaagaa aggtatcata 120

cgtactataa aagactggtt cagtggaaaa 150

<210> 16

<211> 108

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cgtggatact gtgctgagaa cggcattcga tgtgacgata ttcactgctg cactggtcta 60

aaatgcaaat gcaacgccag cggttacaac tgtgtttgca ggaagaag 108

<210> 17

<211> 138

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

aagagaagtt gtattaagcg tggatcgagc tgcgatcatc ggccaaatga ctgctgtgac 60

aactcttcct gtcgatgcaa tttgtgggga actaattgcc gctgccaacg aatgggcctc 120

ttccagaagt ggggcaaa 138

<210> 18

<211> 138

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

aagcgtgctt gcattcgcag gggaggtgga tgtgacggaa aacccaatga ctgctgcccc 60

aactcatcat gcaggtgcaa cctctgggga accaattgca gatgcgaacg tgcaggtctc 120

ttccagcaat ggggcaaa 138

<210> 19

<211> 138

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

aaaaggagct gcattcgacg aggtagcagc tgcgatcacc gaccaagtga ctgctgtttc 60

aactcatcct gcagatgcaa cttgtgggga accaactgca gatgccagag ggctggtctc 120

tttcagaaat ggggaaag 138

Claims

1. A pardosa pseudoannulata D family insecticidal gene is characterized in that the nucleotide sequence of the insecticidal gene is shown in SEQ ID NO. 1.

2. The insecticidal protein encoded by the insecticidal gene of the Pholiopsis print spider family D according to claim 1, wherein the amino acid sequence thereof is represented by SEQ ID NO. 6.

3. A mature peptide encoded by the pesticidal gene of the Pholiopsis print spider family D according to claim 1, wherein the amino acid sequence of the mature peptide is shown in SEQ ID NO. 11.

4. A pardosa pseudonans D family insecticidal gene encoding the mature peptide of claim 3, wherein the nucleotide sequence of said gene is set forth in SEQ ID NO. 16.

5. A recombinant plasmid containing the Pseudoleopard spider family D insecticidal gene according to claim 4.

6. Use of a mature peptide according to claim 3 for the control of the crop pests Nilaparvata lugens Laodelphax striatellus and Sogatella furcifera.

7. An insecticide comprising the mature peptide of claim 3.

8. Use of the insecticide of claim 7 for controlling brown planthopper, gray planthopper and sogatella furcifera by crop pests.