CN108913697A

CN108913697A - Mature peptide and the application of fit reconstruction B family's killing gene and its coding

Info

Publication number: CN108913697A
Application number: CN201810868007.3A
Authority: CN
Inventors: 刘泽文; 黄立鑫; 于娜; 王照英; 张懿熙
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2018-08-02
Filing date: 2018-08-02
Publication date: 2018-11-30
Anticipated expiration: 2038-08-02
Also published as: CN108913697B

Abstract

The invention discloses mature peptide and the applications of a kind of fit reconstruction B family's killing gene and its coding, and the nucleotide sequence of fit reconstruction B family killing gene is as shown in SEQ ID NO.1-3；The amino acid of the mature peptide of fit reconstruction B family's killing gene coding is as shown in SEQ ID NO.7-9.The present invention utilizes bioinformatic analysis method, gene in fit reconstruction transcript profile database is screened, obtain the candidate gene of a kind of similar fit reconstruction toxin, this is gene constructed into prokaryotic expression carrier pET-32a (+), it can be obtained the insecticidal peptide of gene coding, the insecticidal peptide is as completely new killing gene resource, with the insecticidal mechanism for being different from Bt toxin, to the biologically active new type disinsection genetic resources of expansion, it reduces existing Bt toxin and existing all kinds of security risks is widely used, the use for reducing insecticide has important science and realistic meaning.

Description

Mature peptide and the application of fit reconstruction B family's killing gene and its coding

Technical field

The invention belongs to genetic engineerings and field of biological control, are related to a kind of killing gene and its coding albumen, specifically relate to And mature peptide and the application of a kind of fit reconstruction B family's killing gene and its coding.

Background technique

The killing gene for being now widely used in control of insect is mainly the gene of bacillus thuringiensis generation Bt toxin, Bt Toxin shows high desinsection specificity to agricultural pests such as Lepidoptera, Diptera, coleopteras.Therefore Bt toxin gene is turned In the important crops that gene is planted extensively to cotton, corn, tobacco etc..The genetically modified crops of anti insect gene are carried in agricultural pests Prevention and treatment aspect plays an important role.However, single anti insect gene increases pest constantly to the resistance of Bt toxin Add.Various insects group produces resistance to Bt protein formulation and Bt genetically modified crops in a natural environment, such as bollworm, pickles Moth and striped rice borer etc. (Yi Xiaoli etc., insect is to Bt toxin resistance progress [J], Jiangsu's agriculture science, the 7th phase in 2014). Therefore, the novel anti insect gene or protein of developing another high-efficiency environment friendly can increase selection and reduction to anti insect gene The development of 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.2013,58:475-496.).Spider toxin can be made Receptor is associated with G-protein for a variety of channels of insect cell membrane and receptor, such as ion channel, neural ligand gated channel. Therefore all there is pest-resistant effect to various insects.But it focuses mostly on to the research of spider toxin concentrate weight in latrodectus mactans at present The mechanism of action for wanting toxin is few to the direct test and comparison of its insecticidal action.To sum up, the existing research to spider toxin can't Meet the selection demand of anti insect gene.Therefore a kind of spider toxin with insecticidal effect is needed, which can pass through molecule The method great expression of genetic engineering is expressed in crop by transgenic technology, to achieve the effect that pest-resistant.

Summary of the invention

Goal of the invention：In view of the problems of the existing technology, the present invention provides a kind of fit reconstruction B family desinsection base Cause, can get the mature peptide of gene coding by biological means, which has as completely new killing gene resource Neurotoxicity acts on insect ion channel, has the insecticidal mechanism different from Bt toxin, biologically active new to expanding Type killing gene resource reduces existing Bt toxin and existing all kinds of security risks is widely used, and the use for reducing insecticide has Important science and realistic meaning.

Invention additionally discloses the mature peptide of fit reconstruction B family's killing gene coding and applications.

Technical solution：To achieve the goals above, a kind of fit reconstruction B family as described in the present invention killing gene, The nucleotide sequence of the killing gene is as shown in SEQ ID NO.1-3.

The insecticidal proteins of fit reconstruction B of the present invention family's killing gene coding, the insecticidal proteins amino Acid sequence is as shown in SEQ ID NO.4-6.

The mature peptide of fit reconstruction B of the present invention family's killing gene coding, the maturation peptide amino acid sequence As shown in SEQ ID NO.7-9.

Encode the fit reconstruction B family killing gene of mature peptide of the present invention, the nucleotide sequence of the gene As shown in SEQ ID NO.10-12.

Recombinant plasmid containing fit reconstruction B of the present invention family killing gene.

The mature peptide of fit reconstruction B of the present invention family's killing gene coding answering in crop pests prevention and treatment With.

Further, application of the mature peptide in crop pests brown paddy plant hopper, small brown rice planthopper, white backed planthopper prevention and treatment.

The insecticide of mature peptide containing fit reconstruction B of the present invention family's killing gene coding.

The insecticide of the mature peptide of the family's killing gene of B containing fit reconstruction coding of the present invention is in crop pests Application in prevention and treatment.

The present invention refers to the toxin gene of the spiders such as black widow, using bioinformatic analysis method, to fit reconstruction Gene in transcript profile database is screened, and is obtained the candidate gene of a kind of similar fit reconstruction toxin, is then utilized Polymerase chain reaction (PCR) and the sequencing of Sanger method, obtain the complete sequence of one of gene.This is gene constructed to original It in nuclear expression carrier pET-32a (+), is expressed through prokaryotic system, the mature peptide that can be obtained gene coding is insecticidal peptide.System The standby period is short, and amino acid sequence is small, is suitble to external large-scale production, meanwhile, the insecticidal peptide as completely new killing gene resource, With the insecticidal mechanism for being different from Bt toxin, to biologically active new type disinsection genetic resources are expanded, existing Bt poison is reduced Existing all kinds of security risks are widely used in element, and the use for reducing insecticide has important science and realistic meaning.

Beneficial effect：Compared with prior art, the invention has the advantages that：

The fit reconstruction that the present invention uses is for a long time food, toxin main function with insect as agricultural pests natural enemy It is comparatively safe to vertebrate in insect.The present invention screens completely new fit reconstruction B family desinsection by fit reconstruction Gene, can get the mature peptide that the gene encodes by biological means according to the gene is insecticidal peptide, the insecticidal peptide conduct Completely new killing gene resource has neurotoxicity, acts on insect ion channel, has the insecticidal mechanism different from Bt toxin It is wide to reduce existing Bt toxin to biologically active new type disinsection genetic resources are expanded in insect funnel for Bt detoxifying function General to use existing all kinds of security risks, the use for reducing insecticide has important science and realistic meaning.

The present invention predicts the mature peptide of fit reconstruction toxin, and success construction of expression vector, optimizes inductive condition, makes egg White maximum production, while optimized purification condition reduce loss of proteins.In addition, the vivoexpression system that the present invention uses is efficient And yield is high, obtained insecticidal peptide purity is high, and has greater activity.Recombinant toxin desinsection with higher prepared by the present invention Activity.

Detailed description of the invention

After Fig. 1 is injection CK and recombinant toxin PPTX-04, the death rate schematic diagram of brown paddy plant hopper in different time points；

After Fig. 2 is injection CK and recombinant toxin PPTX-04, the death rate schematic diagram of small brown rice planthopper in different time points；

After Fig. 3 is injection CK and recombinant toxin PPTX-04, the death rate diagram of white backed planthopper in different time points.

Specific embodiment

Below in conjunction with drawings and examples, the invention will be further described.

Designed reagent and culture medium prescription in embodiment：

(1) LB liquid medium：

10g tryptone is added in 900ml distilled water, 5g yeast extract and 5g NaCl are stirred and evenly mixed, with double It steams water and is settled to 1L, be placed in high-pressure sterilizing pot, 121 DEG C, sterilize 20min, and cooling is placed on 4 DEG C of preservations.

(2) LB solid medium：

10g tryptone, 5g yeast extract are added in 900ml distilled water, 5g NaCl and 15g agar stirs mixed It is even, it is settled to 1L with 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 The carbenicillin of 100mg/L, a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, cooling are placed on 4 DEG C of preservations.

(3) transferring film buffer：

Glycine 2.9g, Tris base 0.8g, SDS 0.37g, methanol 200ml are added distilled water and are 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：

The Tris-HCl of 1M is diluted to 20mM.

(7) combination buffer：

20mM sodium phosphate, 0.5M sodium chloride, 10mM imidazoles, distilled water are settled to 1L, pH7.4.

(8) elution buffer：

20mM sodium phosphate, 0.5M sodium chloride, 500mM imidazoles, distilled water are settled to 1L, pH7.4.

Embodiment 1

The preparation of toxin

The design and building of spider toxin gene

Fit reconstruction picks up from Nanjing Pukou District paddy field, and is taken indoors with after brown paddy plant hopper raising 90 days Poison gland simultaneously carries out transcript profile sequencing, according to annotation as a result, fit reconstruction toxin gene is filtered out, according to the number of its cysteine Amount, arrangement mode, structural domain prediction and sequence analysis, obtain the candidate gene of a kind of fit reconstruction toxin, and choose wherein One kind, i.e. PPTX-04, base sequence as shown in SEQ ID NO.1, coding protein amino acid sequence such as SEQ Shown in ID NO.4, pass through SpiderP (http://www.arachnoserver.org/spiderP.html) predict its signal Its mature peptide sequence is carried out codon optimization according to e. coli codon Preference, designs use by peptide, propetide and mature peptide In the PPTX-04 toxin gene sequence in expression in escherichia coli as shown in SEQ ID NO.10, design the gene of completion by The synthesis of Invitrogen company, and complete to be sequenced in Nanjing Jin Sirui company, by the gene cloning of synthesis to Bacillus coli expression In carrier pET-32a (+), pET-32a (+)-PPTX-04 recombinant plasmid containing target gene is constructed, which draws Enter BanH I and EcoR I restriction enzyme site.

The inducing expression of recombinant plasmid pET-32a (+)-PPTX-04

By pET-32a (+)-PPTX-04 recombinant plasmid transformed into Escherichia coli BL21 bacterial strain, containing After growing 16h in the LB plate of carbenicillin, the different positive bacterial plaque of picking is added separately to green containing 100mg/L carboxylic benzyl In the LB liquid medium of mycin, 250r/min in constant-temperature table, 37 DEG C, 12h.By the bacterium solution after culture according to 1: 100 body Product ratio, is inoculated into the LB liquid medium containing 100mg/L carbenicillin, 200r/min, 37 DEG C, cultivates 4h, at this time bacterium The OD value of liquid is 0.5-0.7；The IPTG of final concentration of 0.4mmol/L is added in pET-32a (+)-PPTX-04 recon, 200r/min, 37 DEG C, inducing expression culture 5h；Bacterium solution 4ml after taking expression, 4 DEG C, 12000rpm is centrifuged 5min, abandons supernatant, Tris-HCl, the pH=7.4 of 1.5ml 20mM are added in bacterial sediment, carries out carrying out ultrasonic bacteria breaking after resuspension, intensity 12%, 10min, 5s is run, 5s is suspended；4 DEG C, 18000rpm after broken bacterium, it is centrifuged 10min, takes supernatant, abandons precipitating, liquid is crushed and is examined for recombinant toxin It surveys.

Western Blot detection

The broken liquid for taking 50 μ l adds the beta -mercaptoethanol of the Loading Buffer and 2 μ l of 17 μ l, and 100 DEG C are boiled 5min, Add the mixed liquor of 20 μ l to SDS- polyacrylamide gel, and the standard protein sample of 10 μ l is added, 50V, 50min, then 100V is straight To sample-loading buffer to gel bottom；After electrophoresis, enter in transferring film buffer after gel is taken out, and pvdf membrane is impregnated It is transferred to transferring film buffer after methanol 1min, carries out transferring film, 100mA, 50min later；It after transferring film, takes the film out, PBST Wash 3 times, each 5min, confining liquid close 2h, 37 DEG C, 80r/min；It being taken the film out after closing, PBST is washed 3 times, each 10min, Be transferred to be added primary antibody confining liquid (1: 1000), be incubated for 2h, 37 DEG C, 80r/min；After primary antibody is incubated for, take the film out, PBST Wash 3 times, each 10min, be transferred to be added secondary antibody confining liquid (1: 2000), be incubated for 1.5h, 37 DEG C, 80r/min；Secondary antibody is incubated for knot Shu Hou, PBST are washed 3 times, each 10min, and luminescent solution is added, is protected from light 3min, after filter paper blots surface liquid, chemiluminescence detection.

A large amount of inducing expressions of recombinant plasmid pET-32a (+)-PPTX-04

By the bacterium solution after culture according to 1: 100 volume ratio, it is inoculated into the LB that 100ml contains 100mg/L carbenicillin In fluid nutrient medium, the volume of culture medium cultivates 4h, at this time bacterium solution no more than 20%, 200r/min, 37 DEG C of container volume OD value be 0.5-0.7；The IPTG, 200r/ of final concentration of 0.4mmol/L are added in pET-32a (+)-PPTX-04 recon Min, 37 DEG C, inducing expression culture 5h；Bacterium solution after taking expression, 4 DEG C, 12000rpm is centrifuged 15min, abandons supernatant, heavy in thallus The Tris-HCl of the 20mM of 20% culture volume, pH=7.4 are added in shallow lake, carries out carrying out ultrasonic bacteria breaking after resuspension, intensity 12%, 30min runs 5s, suspends 5s；4 DEG C, 18000rpm after broken bacterium, it is centrifuged 30min, takes supernatant, abandons precipitating, crude protein liquid after being crushed For isolating and purifying recombinant toxin.

The purifying of recombinant toxin PPTX-04

0.22 μm of membrane filtration of crude protein liquid after a large amount of inducing expressions to be broken to bacterium, it is full-automatic using AKTA avant Protein separation system and HisTrapTM HP nickel column (5ml) are purified.It is first flat with the combination buffer of 5 column volumes Weigh nickel column, flow velocity 5ml/min, loading flow velocity 1ml/min, cleans pillar, flow velocity with the combination buffer of 6 column volumes after loading 1ml/min finally elutes destination protein, flow velocity 5ml/min with the elution buffer of 3 column volumes.Protein yield after purification About 7.85mg/L, albumen after purification are mature peptide, that is, recombinant toxin PPTX-04, amino acid sequence such as SEQ ID NO.7 It is shown.

Embodiment 2

Using the design and construction method of spider toxin gene in embodiment 1, the killing gene of fit reconstruction is prepared SEQ ID NO.2-3：PPTX-20,PPTX-21；And it is constructed by identical method corresponding recombinant plasmid and big respectively Inducing expression is measured, by the recombinant toxin for obtaining different genes coding after purification.

The nucleotides sequence of the killing gene PPTX-04 of fit reconstruction is classified as SEQ ID NO.1, as follows：

atgaaattcgcagttgttctacttttttccctggttgtacttgcagttgcaagtgaatttgtggaggaagatataag agatattgaagaagaacttccagagcaacagaggggttgcgctgatcttcgggaaccatgtacagacgactgcagct gctgtggaagtgaaggagtttgcaactgtaaccatccccgtaaacctggttgcttctgcaaaagggctggacctctt gaaaaaatagcgaagaaatttaagaattgtggcaag

The nucleotides sequence of the killing gene PPTX-20 of fit reconstruction is classified as SEQ ID NO.2, as follows：

atgaaatacacaatagttctgctgttttcgttggtcttgcttgttgttgcaagcgaatcggttgaagatactaatag agaggattttccagaacaacaaagagcctgtgctggacctagagaaccatgtacaaaaggcgatgattgtagttgct gtggagatcgaggaaagtgcgactgtaactggcagggaaaaccaggctgctattgcatgacagccatgtttttaaca ggaattaagaagttatttgaatgtcgaatcggg

The nucleotides sequence of the killing gene PPTX-21 of fit reconstruction is classified as SEQ ID NO.3, as follows：

atgaaactcgcaatattcctggtgttttctttgattgtgcttgtcgttgcaagcgagtccatggaagaaaatataaa tgatgatcttccggagcaagaaagggcgtgtgccgatctcaaccagaaatgcacagatgactgcagttgctgtggag aaagaggaaagtgcgactgtaactggcccagcaaaccgggatgctactgcatgagaggaggacccatcgatctcatc gccaagaagtttaaatgc

The amino acid sequence of the killing gene PPTX-04 of fit reconstruction is SEQ ID NO.4, as follows：

MKFAVVLLFSLVVLAVASEFVEEDIRDIEEELPEQQRGCADLREPCTDDCSCCGSEGVCNCNHPRKPGC FCKRAGPLEKIAKKFKNCGK

The amino acid sequence of the killing gene PPTX-20 of fit reconstruction is SEQ ID NO.5, as follows：

MKYTIVLLFSLVLLVVASESVEDTNREDFPEQQRACAGPREPCTKGDDCSCCGDRGKCDCNWQGKPGCY CMTAMFLTGIKKLFECRIG

The amino acid sequence of the killing gene PPTX-21 of fit reconstruction is SEQ ID NO.6, as follows：

MKLAIFLVFSLIVLVVASESMEENINDDLPEQERACADLNQKCTDDCSCCGERGKCDCNWPSKPGCYCM RGGPIDLIAKKFKC

The mature peptide sequence of the killing gene PPTX-04 of fit reconstruction is SEQ ID NO.7, as follows：

CADLREPCTDDCSCCGSEGVCNCNHPRKPGCFCKRAGPLEKIAKKFKNCGK

The mature peptide sequence of the killing gene PPTX-20 of fit reconstruction is SEQ ID NO.8, as follows：

CAGPREPCTKGDDCSCCGDRGKCDCNWQGKPGCYCMTAMFLTGIKKLFECRIG

The mature peptide sequence of the killing gene PPTX-21 of fit reconstruction is SEQ ID NO.9, as follows：

CADLNQKCTDDCSCCGERGKCDCNWPSKPGCYCMRGGPIDLIAKKFKC

The gene order of the mature peptide of the killing gene PPTX-04 of fit reconstruction is SEQ ID NO.10, as follows：

tgcgctgatcttcgggaaccatgtacagacgactgcagctgctgtggaagtgaaggagtttgcaactgtaaccatcc ccgtaaacctggttgcttctgcaaaagggctggacctcttgaaaaaatagcgaagaaatttaagaattgtggcaag

The gene order of the mature peptide of the killing gene PPTX-20 of fit reconstruction is SEQ ID NO.11, as follows：

tgtgctggacctagagaaccatgtacaaaaggcgatgattgtagttgctgtggagatcgaggaaagtgcgactgtaa ctggcagggaaaaccaggctgctattgcatgacagccatgtttttaacaggaattaagaagttatttgaatgtcgaa tcggg

The gene order of the mature peptide of the killing gene PPTX-21 of fit reconstruction is SEQ ID NO.12, as follows：

tgtgccgatctcaaccagaaatgcacagatgactgcagttgctgtggagaaagaggaaagtgcgactgtaactggcc cagcaaaccgggatgctactgcatgagaggaggacccatcgatctcatcgccaagaagtttaaatgc

Test example 1

The raw process and method surveyed

Recombinant toxin PPTX-04 is determined to the insecticidal activity of 3 kinds of planthoppers using microinjection, test worm chose for 5 ages Nymph, 3 repetitions of each processing are each to repeat to choose 30 test worms, before injection, first test worm CO₂Anesthesia, every test worm note Recombinant toxin 20nl is penetrated, injection site is the first and second plastron corias, and after injection, 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, reduce the injury to test worm.It is real It tests and is divided into control group and experimental group, control group injects the PBS of equivalent, pH=7.4, and experimental group injects recombinant toxin PPTX-04. Fig. 1,2 and 3 are respectively the death condition of 3 kinds of planthoppers in different time points after injecting recombinant toxin PPTX-04, it can be seen that The insecticidal activity that the toxin PPTX-04 of recombination has excellent insecticidal activity excellent 3 kinds of planthoppers, it was demonstrated that the present invention obtained The mature peptide of the killing gene PPTX-04 of fit reconstruction is effective insecticidal peptide, can be used for preparing insecticide and is applying Crop pests for example brown paddy plant hopper, small brown rice planthopper, white backed planthopper etc. prevention and treatment, of the invention other recombinant toxins PPTX-20, PPTX-21 It is similar with recombinant toxin PPTX-04 function.

Sequence table

<110>Agricultural University Of Nanjing

<120>Mature peptide and the application of fit reconstruction B family's killing gene and its coding

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 267

<212> DNA

<213>The killing gene PPTX-04 (PPTX-04) of fit reconstruction

<400> 1

atgaaattcg cagttgttct acttttttcc ctggttgtac ttgcagttgc aagtgaattt 60

gtggaggaag atataagaga tattgaagaa gaacttccag agcaacagag gggttgcgct 120

gatcttcggg aaccatgtac agacgactgc agctgctgtg gaagtgaagg agtttgcaac 180

tgtaaccatc cccgtaaacc tggttgcttc tgcaaaaggg ctggacctct tgaaaaaata 240

gcgaagaaat ttaagaattg tggcaag 267

<210> 2

<211> 264

<212> DNA

<213>The killing gene PPTX-20 (PPTX-20) of fit reconstruction

<400> 2

atgaaataca caatagttct gctgttttcg ttggtcttgc ttgttgttgc aagcgaatcg 60

gttgaagata ctaatagaga ggattttcca gaacaacaaa gagcctgtgc tggacctaga 120

gaaccatgta caaaaggcga tgattgtagt tgctgtggag atcgaggaaa gtgcgactgt 180

aactggcagg gaaaaccagg ctgctattgc atgacagcca tgtttttaac aggaattaag 240

aagttatttg aatgtcgaat cggg 264

<210> 3

<211> 249

<212> DNA

<213>The killing gene PPTX-21 (PPTX-21) of fit reconstruction

<400> 3

atgaaactcg caatattcct ggtgttttct ttgattgtgc ttgtcgttgc aagcgagtcc 60

atggaagaaa atataaatga tgatcttccg gagcaagaaa gggcgtgtgc cgatctcaac 120

cagaaatgca cagatgactg cagttgctgt ggagaaagag gaaagtgcga ctgtaactgg 180

cccagcaaac cgggatgcta ctgcatgaga ggaggaccca tcgatctcat cgccaagaag 240

tttaaatgc 249

<210> 4

<211> 89

<212> PRT

<213>The killing gene PPTX-04 (PPTX-04) of fit reconstruction

<400> 4

Met Lys Phe Ala Val Val Leu Leu Phe Ser Leu Val Val Leu Ala Val

1 5 10 15

Ala Ser Glu Phe Val Glu Glu Asp Ile Arg Asp Ile Glu Glu Glu Leu

20 25 30

Pro Glu Gln Gln Arg Gly Cys Ala Asp Leu Arg Glu Pro Cys Thr Asp

35 40 45

Asp Cys Ser Cys Cys Gly Ser Glu Gly Val Cys Asn Cys Asn His Pro

50 55 60

Arg Lys Pro Gly Cys Phe Cys Lys Arg Ala Gly Pro Leu Glu Lys Ile

65 70 75 80

Ala Lys Lys Phe Lys Asn Cys Gly Lys

85

<210> 5

<211> 88

<212> PRT

<213>The killing gene PPTX-20 (PPTX-20) of fit reconstruction

<400> 5

Met Lys Tyr Thr Ile Val Leu Leu Phe Ser Leu Val Leu Leu Val Val

1 5 10 15

Ala Ser Glu Ser Val Glu Asp Thr Asn Arg Glu Asp Phe Pro Glu Gln

20 25 30

Gln Arg Ala Cys Ala Gly Pro Arg Glu Pro Cys Thr Lys Gly Asp Asp

35 40 45

Cys Ser Cys Cys Gly Asp Arg Gly Lys Cys Asp Cys Asn Trp Gln Gly

50 55 60

Lys Pro Gly Cys Tyr Cys Met Thr Ala Met Phe Leu Thr Gly Ile Lys

65 70 75 80

Lys Leu Phe Glu Cys Arg Ile Gly

85

<210> 6

<211> 83

<212> PRT

<213>The killing gene PPTX-21 (PPTX-21) of fit reconstruction

<400> 6

Met Lys Leu Ala Ile Phe Leu Val Phe Ser Leu Ile Val Leu Val Val

1 5 10 15

Ala Ser Glu Ser Met Glu Glu Asn Ile Asn Asp Asp Leu Pro Glu Gln

20 25 30

Glu Arg Ala Cys Ala Asp Leu Asn Gln Lys Cys Thr Asp Asp Cys Ser

35 40 45

Cys Cys Gly Glu Arg Gly Lys Cys Asp Cys Asn Trp Pro Ser Lys Pro

50 55 60

Gly Cys Tyr Cys Met Arg Gly Gly Pro Ile Asp Leu Ile Ala Lys Lys

65 70 75 80

Phe Lys Cys

<210> 7

<211> 51

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 7

Cys Ala Asp Leu Arg Glu Pro Cys Thr Asp Asp Cys Ser Cys Cys Gly

1 5 10 15

Ser Glu Gly Val Cys Asn Cys Asn His Pro Arg Lys Pro Gly Cys Phe

20 25 30

Cys Lys Arg Ala Gly Pro Leu Glu Lys Ile Ala Lys Lys Phe Lys Asn

35 40 45

Cys Gly Lys

50

<210> 8

<211> 53

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 8

Cys Ala Gly Pro Arg Glu Pro Cys Thr Lys Gly Asp Asp Cys Ser Cys

1 5 10 15

Cys Gly Asp Arg Gly Lys Cys Asp Cys Asn Trp Gln Gly Lys Pro Gly

20 25 30

Cys Tyr Cys Met Thr Ala Met Phe Leu Thr Gly Ile Lys Lys Leu Phe

35 40 45

Glu Cys Arg Ile Gly

50

<210> 9

<211> 48

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 9

Cys Ala Asp Leu Asn Gln Lys Cys Thr Asp Asp Cys Ser Cys Cys Gly

1 5 10 15

Glu Arg Gly Lys Cys Asp Cys Asn Trp Pro Ser Lys Pro Gly Cys Tyr

20 25 30

Cys Met Arg Gly Gly Pro Ile Asp Leu Ile Ala Lys Lys Phe Lys Cys

35 40 45

<210> 10

<211> 153

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 10

tgcgctgatc ttcgggaacc atgtacagac gactgcagct gctgtggaag tgaaggagtt 60

tgcaactgta accatccccg taaacctggt tgcttctgca aaagggctgg acctcttgaa 120

aaaatagcga agaaatttaa gaattgtggc aag 153

<210> 11

<211> 159

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 11

tgtgctggac ctagagaacc atgtacaaaa ggcgatgatt gtagttgctg tggagatcga 60

ggaaagtgcg actgtaactg gcagggaaaa ccaggctgct attgcatgac agccatgttt 120

ttaacaggaa ttaagaagtt atttgaatgt cgaatcggg 159

<210> 12

<211> 144

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 12

tgtgccgatc tcaaccagaa atgcacagat gactgcagtt gctgtggaga aagaggaaag 60

tgcgactgta actggcccag caaaccggga tgctactgca tgagaggagg acccatcgat 120

ctcatcgcca agaagtttaa atgc 144

Claims

1. a kind of fit reconstruction B family killing gene, which is characterized in that the nucleotide sequence of the killing gene such as SEQ ID Shown in NO.1-3.

2. a kind of insecticidal proteins of fit reconstruction B described in claim 1 family's killing gene coding, which is characterized in that Its amino acid sequence is as shown in SEQ ID NO.4-6.

3. a kind of mature peptide of fit reconstruction B described in claim 1 family's killing gene coding, which is characterized in that described Mature peptide amino acid sequence is as shown in SEQ ID NO.7-9.

4. a kind of fit reconstruction B family killing gene for encoding mature peptide as claimed in claim 3, which is characterized in that described The nucleotide sequence of gene is as shown in SEQ ID NO.10-12.

5. a kind of recombinant plasmid containing fit reconstruction B as claimed in claim 4 family killing gene.

6. a kind of mature peptide of fit reconstruction B as claimed in claim 3 family's killing gene coding is prevented and treated in crop pests In application.

7. application according to claim 6, which is characterized in that the mature peptide is preferably in crop pests brown paddy plant hopper, ash Application in plant hopper, white backed planthopper prevention and treatment.

8. a kind of insecticide of the mature peptide encoded containing fit reconstruction B family's killing gene as claimed in claim 3.

9. a kind of insecticide of the mature peptide of the family's killing gene of B containing fit reconstruction coding according to any one of claims 8 is in farming Application in object control of insect.