CN1322123C - Recombinant serine protease and fungicide containing the same - Google Patents
Recombinant serine protease and fungicide containing the same Download PDFInfo
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Abstract
The present invention relates to a recombination serine proteinase which comprises fusion protein formed by fusing serine stretch protein which comes from fungus and chitin binding domain of chitinase, which comes from insects, and a separated nucleotide sequence code coming from gene and code which come from serine stretch protein of fungus and gene which comes from chitin binding domain of chitinase of insects. The recombination serine proteinase obtained by the present invention can be enriched on the body wall of insects so as to increase the speed of insect body wall degradation and improve the characteristic of insecticidal fungus toxicity.
Description
Technical field
The present invention relates to a kind of new fusion rotein, relate in particular to the recombinant protein of the serine stretch protein enzyme sequence of the chitin binding domain that contains insect chitinase and fungi.
The invention still further relates to the nucleotide sequence and the aminoacid sequence of the above-mentioned recombinant protein of coding.
The present invention relates to express above-mentioned Recombinant Protein Expression carrier.
The invention still further relates to the disinsection fungal agent that contains above-mentioned recombinant protein.
Background technology
Under the physical environment, insect population quantity is controlled, and gives the credit to popular on a large scale (Chanley, The Mycota IV Environmental and MicrobialRelationships, 1997, the PP 185-201) of disinsection fungal disease to a great extent.Disinsection fungal is as the biological prevention and control agent (Feng Mingguang that has been widely used in China, America and Europe and Africa; " plant protection 21 century prospect and the 3rd the youth of the nation plant protection scientific worker scientific seminar collection of thesis "; 1998); but also have simultaneously shortcomings such as knocking down long and preventive effect instability of insect time, these drawbacks limit disinsection fungal use widely.Therefore, the molecule mechanism that the research disinsection fungal infects, significant to the transformation of disinsection fungal.
Similar with plant pathogenic fungi, disinsection fungal mainly is the synergy by mycelia mechanical pressure and lytic enzyme, directly penetrate insect body wall so that cause infect morbific.Multiple lytic enzymes such as disinsection fungal extracellular proteinase, chitinase and esterase in this course, proteolytic enzyme and the chitinase (Charnley that plays an important role wherein, Fungal spore disease initiation in plants and animals, 1991, pp267-287), its corresponding gene or is cloned in succession.
The further investigation pathogenic fungi infects morbific molecule mechanism, excavates new Disease-causing gene, is the focus that people study always.Yet,, be the complex system of a controlled by multiple genes because the disinsection fungal pathogenesis is very complicated.So,, make slow progress although a plurality of in the world research group all is devoted to seek some new virulence factors.So, can utilize the modern biotechnology means, more existing virulence factors are carried out molecular modification, acquisition vigor or pathogenic stronger virulence factor? as mentioned above, proteolytic enzyme is the important virulence factor of disinsection fungal, can improve the ability that proteolytic enzyme decomposes insect body wall, thereby improve the virulence of disinsection fungal by strengthening or improveing its active or relevant enzymatic property
Present result of study shows that the function of the chitin binding domain in the chitinase has two: one is that chitinase is combined with chitin; Another one is to destroy between the crystal chitin to play the hydrogen bond of stabilization, thereby helps the effect of chitinase.A large amount of chitinases that studies show that the disappearance chitin binding domain does not change the hydrolysis ability of soluble substrate, but the chitinous hydrolysis ability of crystal is but reduced, and fungistatic effect has also reduced (Taira T et al., Biosci.Biotechno1.Biochem, 2002,66 (5): 970-977).On the other hand, on the chitinase that does not have chitin binding domain, add chitin binding domain and can increase the concentration of chitinase, thereby improve the chitinous ability of decomposing in substrate surface.On the wooden now mould chitinase gene Chit42 in the Kazakhstan that does not have the chitin calmodulin binding domain CaM, add chitin calmodulin binding domain CaM or cellulose binding domain and can both significantly improve this hybrid enzyme and be attached to chitinous ability in the fungal cell wall, and strengthened degrade chitin speed (
Et al., FEMS Microbiology Letters, 2001,198:57-63).
In the body wall structure of insect, the chitin fibril is wrapped up by protein.If have chitin binding domain in the molecular structure of proteolytic enzyme, just might improve proteolytic enzyme to containing the decomposition rate of chitin-protein complex.But analysis revealed, in the proteolytic enzyme only from proteolytic enzyme Sp22D (the Gorman MJ et al. of anopheles (Anopheles gambiae), Gene, 2000,251:9-17), replace protease A prIV (the Katsushiro M et al. of sporangium (Alteromonassp.) O-7 bacterial strain, Journal of Bacteriology, 2002,184:1865-1872) with from proteolytic enzyme C (the Sidhu SSet al. of streptomyces griseus (Streptomyces griseus), The Journal of biological chemistry, 1994,269:20167-20171) and the serine protease of two derivations of streptomyces coelicolor (Streptomyces coelicolor) have chitin binding domain.Katsushiro M (Journal of Bacteriology, 2002,184:1865-1872) wait the people that the proteinase gene AprIV that replaces sporangium (Alteromonas sp.) O-7 bacterial strain has been carried out disappearance research.Result of study shows, when with shrimp shell (not removing protein) as substrate, the protein that complete AprIV discharges is 3 times of AprIV Δ C (having lacked the AprIV of chitin binding domain).When handle the shrimp shell of handling through AprIV and AprIV Δ C with chitinase, detected chitinase activity is apparently higher than AprIV Δ C in the shrimp shell that AprIV handled.This result shows, when the proteolytic enzyme with chitin binding domain is not removed proteinic shrimp shell in effect, can increase the speed of degraded substrate protein white matter and increase the effect of chitinase.But from the serine protease that the insect entomogenous fungi is cloned, all find no at present the existence of chitin binding domain, the report that the serine protease in entomogenous fungi source and the chitin binding domain of insect chitinase are merged is not mutually arranged yet.
Summary of the invention
One object of the present invention is to provide a kind of recombinant serine protease, the chitin binding domain that it contains the serine stretch protein that derives from fungi and derives from the chitinase of insect has the characteristic that improves degraded insect body wall activity and improve the disinsection fungal virulence.
Another object of the present invention is to provide the nucleotide sequence and the aminoacid sequence of the above-mentioned recombinant protein of coding.
A further object of the present invention is to provide the expression vector that contains above-mentioned nucleotide sequence.
A further object of the present invention is to provide the disinsection fungal that contains recombinant protein of the present invention.
According to an aspect of the present invention, a kind of recombinant serine protease contains a fusion rotein, and the chitin binding domain of the serine stretch protein that described fusion rotein origin comes from fungi and the chitinase that derives from insect merges and forms.In a specific embodiments of the present invention, the chitin binding domain (hereinafter to be referred as SwChBD) of the serine protease (hereinafter to be referred as CDEP-1) that described fusion rotein origin comes from beauveria bassiana (Beauveria bassiana) and the chitinase that derives from silkworm (Bombyx mori) merges and forms.
According to a further aspect in the invention, the nucleotide sequence of code book invention recombinant serine protease contains the gene of chitin binding domain that gene and coding that coding derives from the serine stretch protein of fungi derive from the chitinase of insect.In a specific embodiments of the present invention, adopt Protocols in Molecular Biology to make up a recombinant nucleotide sequence, it contains the gene of chitin binding domain of gene and the chitinase of coding silkworm of the serine protease of coding beauveria bassiana (Beauveria bassiana), and it has nucleotide sequence and the aminoacid sequence shown in SEQ ID NO.2 shown in SEQID NO.1.
Further, the signal peptide sequence of one section serine protease be can when making up recombinant nucleotide sequence, insert, the structure and the proteic expression of subsequent recombination carrier are beneficial to.The recombinant serine protease sequence that contains signal peptide sequence is shown in SEQ ID NO.3, and its amino acid sequence coded is shown in SEQ ID NO.2.
In accordance with a further aspect of the present invention, made up recombinant expression vector, the recombinant nucleotide sequence that the present invention is made up changes suitable plasmid vector over to, transform appropriate host again and can obtain recombinant expression vector of the present invention, preferred plasmid vector can be for being suitable for the plasmid vector of yeast expression, preferred host can be yeast host, more preferably pichia spp (Pichia pastoris).
In accordance with a further aspect of the present invention, a kind of disinsection fungal agent is provided, it contains recombinant serine protease of the present invention and disinsection fungal, and wherein disinsection fungal can be beauveria bassiana (Beauveria bassiana), Metarhizium anisopliae (Metarhizium anisopliae), yellowish green green muscardine fungus (Metarhizium flvoviride), muscardine (Beauveria brongniartii), paecilomyces fumosoroseus (Paecilomycesfumosoroseus), Tang Pusen is by hair spore (Hirsutella thompsonii), powder Paecilomyces varioti (Paecilomycesfarinosus) etc.
In accordance with a further aspect of the present invention, provide a kind of disinsection fungal to transform bacterial strain, obtain to express the disinsection fungal conversion bacterial strain of recombinant protein of the present invention with the plasmid vector conversion disinsection fungal of the present invention's structure, in a specific embodiments of the present invention, made up beauveria bassiana and transformed bacterial strain.
The recombinant serine protease that the present invention obtained can obtain enrichment at insect body wall, thereby improves the speed of degraded insect body wall.Synergism proves that the serine protease that adds reorganization in beauveria bassiana can obviously improve the virulence of beauveria bassiana.
The beauveria bassiana of commentaries on classics recombinant serine protease gene provided by the invention transforms bacterial strain.The virulence analytical results proves that the virulence of transformant contrast is significantly improved.
Below, in conjunction with the accompanying drawings,, describe the present invention in detail by description to better embodiment of the present invention.
Brief description of drawings
Fig. 1 is used for the plasmid map of yeast expression, A wherein, and B is respectively the plasmid map of pPIC9K-CDEP, pPIC9K-CDEP-SwChBD;
Fig. 2 is for changeing beauveria bassiana plasmid map, wherein A:pUC-bar ∷ gfp-CDEP; B:pUC-bar ∷ gfp-CDEP-SwChBD;
Fig. 3 is the PCR product in CDEP-SwChBD Yeast expression carrier of the present invention and the beauveria bassiana conversion carrier set-up procedure and the partially digested collection of illustrative plates of cloning vector;
A wherein, the SwChBD of pcr amplification; B, the CDEP-1 of pcr amplification (1 does not have signal peptide, and 2 have signal peptide); C, EcoRI and NotI double digestion pPIC9K-CDEP-SwChBD; D, EcoRI and HindIII double digestion pUC-bar ∷ gfp-CDEP-SwChBD; Marker among the A is the 50bp DNA Ladder of MBI company; B, C, the marker among the D are the λ/Eco91115 of MBI company;
Fig. 4 is that the SDS-PAGE of CDEP-1 and CDEP-SwChBD analyzes;
M wherein, the small molecular weight protein standard; 1, CDEP-SwChBD; 2, CDEP-1;
It is that substrate is measured protein concn and the chitinase activity that discharges after the protease treatment that Fig. 5 is sloughed off with silkworm; A, protein concn; B, the chitinase activity behind the adding protease treatment 2h, adds chitinase again and handles 2h earlier; C, the chitinase Bbchit1 individual curing substrate of beauveria bassiana; 1, after the proteolytic enzyme CDEP-1 of beauveria bassiana handles substrate, add chitinase and handle; 2, CDEP-SwChBD adds the chitinase processing after handling substrate again; 3, after the proteolytic enzyme CDEP-1 of beauveria bassiana (adding final concentration is the PMSF arrestin enzymic activity of 1mmol/L) handles substrate, add chitinase and handle; 4, CDEP-SwChBD (adding final concentration is the PMSF arrestin enzymic activity of 1mmol/L) adds the chitinase processing after handling substrate again.This result shows that proteolytic enzyme and hybridization proteolytic enzyme all can improve the activity of chitinase, but the amplitude that hybridization proteolytic enzyme improves is apparently higher than wild-type protease.
Fig. 6 expressed proteins enzyme joins the virulence analysis of beauveria bassiana to aphid as additive;
C wherein, 0.05% Tween-80; Bb0062,10
7The beauveria bassiana spore of individual/mL; CDEP-1, concentration is 10
7Having added final concentration in the beauveria bassiana spore of individual/mL is the CDEP-1 of 0.16 μ mol/L; CDEP-SwChBD, concentration is 10
7Having added final concentration in the beauveria bassiana spore of individual/mL is the CDEP-SwChBD of 0.16 μ mol/L.
Fig. 7 expressed proteins enzyme joins the virulence analysis of beauveria bassiana to cabbage caterpillar as additive;
C wherein, 0.05% Tween-80; Bb0062,10
7The beauveria bassiana spore of individual/mL; CDEP-1, concentration is 10
7Having added final concentration in the beauveria bassiana spore of individual/mL is the CDEP-1 of 0.16 μ mol/L; CDEP-SwChBD, concentration is 10
7Having added final concentration in the beauveria bassiana spore of individual/mL is the CDEP-SwChBD of 0.16 μ mol/L.
The embodiment of invention
The used experiment material of the present invention is commercially available purchase product if no special instructions.
[embodiment 1]
1, the clone of chitin binding domain
The template of amplification is that the cDNA (the reverse transcription test kit is a TaKaRa company product) of the mRNA reverse transcription in four silkworm in age (silkworm and mulberry institute of Agricultural University Of Southwest provides) periods of casting off a skin is template.
According to the silkworm chitinase (GenBank accession number: sequences Design primer AB052914):
P1：5’-ACT?AGT?CAC?AAC?CAC?CAC?CAC?CGT?G-3’
P2：5’-GCG?GCC?GCC?CGG?GTT?ACG?AAC?ATT?CCG?GTC?TGT-3’
Pfu archaeal dna polymerase (ancient cooking vessel state company product) with high-fidelity carries out pcr amplification, and the pcr amplification condition is as follows: 94 ℃ of 5min; 94 ℃ of 30sec, 58 ℃, 30sec, 72 ℃ of 1min, 30 circulations; The Taq archaeal dna polymerase that adds 0.7 unit extends 20min again.Pcr amplification obtained about 340bp in conjunction with domain gene (sequence is shown in SEQ ID NO.4), and introduce corresponding restriction enzyme site.
The PCR product is after gel reclaims test kit (Amersham company product) purifying, and A-T clones into pMD-T carrier (TaKaRa company product).With the checking of SpeI/NotI double digestion, the positive colony of checking is sequence verification (Shanghai Bo Ya company) again, the plasmid called after pMD-SwChBD that checks order correct.
2, the clone of serine protease gene
With cicada slough inductive beauveria bassiana cDNA library is template, according to the sequence of beauveria bassiana serine protease gene CDEP-1 (GenBank accession number: AY040532) design primer:
P3：5’-GAA?TTC?GTT?GAG?CCT?GCT?CCT?CTC?ATC-3’
P4：5’-GCG?GCC?GCC?CGG?GTT?AGG?TGG?CGC?CTT?AAA?TGC?C-3’
P5：5’-TCT?AGA?GT?GGC?GCC?TTA?AAT?GCC-3’
P6：5’-GAA?TTC?ATG?CGT?CTA?TCA?ATC?ATT?GCT-3’
1) amplification is used for the serine protease gene (signal peptide of no proteolytic enzyme itself) of yeast expression
Increase with primer P3 and P4 primer.Pfu archaeal dna polymerase (ancient cooking vessel state company product) with high-fidelity carries out pcr amplification, and the pcr amplification condition is as follows: 94 ℃ of 5min; 94 ℃ of 30sec, 58 ℃, 30sec, 72 ℃ of 2min, 30 circulations; The Taq archaeal dna polymerase that adds 0.7 unit extends 20min again.Pcr amplification has obtained the serine protease gene of 1086bp, and introduces corresponding restriction enzyme site (sequence is shown in SEQID NO.5).
The PCR product is after gel reclaims test kit (Amersham company product) purifying, and A-T clones into pMD-T carrier (TaKaRa company product).With the checking of EcoRI/NotI double digestion, the positive colony of checking is sequence verification (Shanghai Bo Ya company) again, the plasmid called after pMD-CDEP that checks order correct.
2) the amplification serine protease gene (signal peptide of no proteolytic enzyme itself) that is used for yeast expression and can merges mutually with chitin binding domain
Increase with primer P3 and P5 primer.Pfu archaeal dna polymerase (ancient cooking vessel state company product) with high-fidelity carries out pcr amplification, and the pcr amplification condition is as follows: 94 ℃ of 5min; 94 ℃ of 30sec, 58 ℃, 30sec, 72 ℃ of 2min, 30 circulations; The Taq archaeal dna polymerase that adds 0.7 unit extends 20min again.Pcr amplification has obtained the proteinase gene of about 1086bp, and introduces corresponding restriction enzyme site (sequence is shown in SEQ IDNO.6).
The PCR product is after gel reclaims test kit (Amersham company product) purifying, and A-T clones into pMD-T carrier (TaKaRa company product).With the checking of EcoRI/SpeI double digestion, the positive colony of checking is sequence verification (Shanghai Bo Ya company) again, the plasmid called after pMD-hCDEP that checks order correct.
3) amplification is used for the serine protease gene (signal peptide that comprises serine protease) that beauveria bassiana is expressed
Increase with primer P6 and P4 primer.Pfu archaeal dna polymerase (ancient cooking vessel state company product) with high-fidelity carries out pcr amplification, and the pcr amplification condition is as follows: 94 ℃ of 5min; 94 ℃ of 30sec, 58 ℃, 30sec, 72 ℃ of 2min, 30 circulations; The Taq archaeal dna polymerase that adds 0.7 unit extends 20min again.Pcr amplification has obtained the proteinase gene of about 1140bp, and introduces corresponding restriction enzyme site (sequence is shown in SEQ IDNO.7).
The PCR product is after gel reclaims test kit (Amersham company product) purifying, and A-T clones into pMD-T carrier (TaKaRa company product).With the checking of EcoRI/SpeI double digestion, the positive colony of checking is sequence verification (Shanghai Bo Ya company) again, the plasmid called after pMD-spCDEP that checks order correct.
4) the amplification serine protease gene (signal peptide that comprises serine protease) that is used for that beauveria bassiana is expressed and can merges mutually with chitin binding domain
Increase with primer P6 and P5 primer.Pfu archaeal dna polymerase (ancient cooking vessel state company product) with high-fidelity carries out pcr amplification, and the pcr amplification condition is as follows: 94 ℃ of 5min; 94 ℃ of 30sec, 58 ℃, 30sec, 72 ℃ of 2min, 30 circulations; The Taq archaeal dna polymerase that adds 0.7 unit extends 20min again.Pcr amplification has obtained the proteinase gene of about 1140bp, and introduces corresponding restriction enzyme site (sequence is shown in SEQ IDNO.8).
The PCR product is after gel reclaims test kit (Amersham company product) purifying, and A-T clones into pMD-T carrier (TaKaRa company product).With the checking of EcoRI/SpeI double digestion, the positive colony of checking is sequence verification (Shanghai Bo Ya company) again, the plasmid called after pMD-hspCDEP that checks order correct.
The structure of CDEP-1, CDEP-SwChBD Yeast expression carrier and yeast conversion
[embodiment 2]
1, the structure of CDEP-1 Yeast expression carrier
Cut pMD-CDEP with the EcoRI/NotI enzyme, reclaim the external source fragment of about 1kb, be cloned into pPIC9K (Invitrogen company) carrier with same enzyme digestion.Cut checking with the EcoRI/NotI enzyme, positive colony called after pPIC9K-CDEP, its plasmid structure iron see Figure 1A (aminoacid sequence of expression product is shown in SEQ ID NO.9).
2, the structure of CDEP-SwChBD Yeast expression carrier
Cut pMD-hCDEP with the EcoRI/XbaI enzyme, reclaim the external source fragment of about 1kb, cut pMD-SwChBD with the SpeI/NotI enzyme, reclaim the external source fragment of 340bp, two fragments of above recovery are cloned simultaneously pPIC9K (Invitrogen company) carrier of into cutting with the EcoRI/NotI enzyme.The EcoRI/NotI enzyme is cut the positive colony called after pPIC9K-CDEP-SwChBD of evaluation, and its plasmid structure iron is seen Figure 1B, the aminoacid sequence of expression product such as SEQ ID NO.2.
3, the conversion and the abduction delivering of CDEP-1, CDEP-SwChBD Yeast expression carrier
Extract above-mentioned plasmid, electricity transforms pichia spp.
Be cultured to OD600 among picking transformant and the BMGY and be about at 3 o'clock, 4 ℃ of centrifugal 5min of following 1500g collect thalline, are resuspended among the BMMY, OD600 is reached be about 1.Add the methanol induction of final concentration 1% every 24h.After inducing 72h, 4 ℃ of centrifugal 10min of following 8000g.Collect supernatant liquor, SDS-PAGE analyzes.The result shows that expression product is present in the supernatant liquor.
The prescription of substratum BMGY/BMMY:
BMGY: every liter contains 13.4g YNB, 0.0004g vitamin H, 10g yeast powder, 20g peptone, the potassiumphosphate of 100mmol/L pH6.0, the glycerine of 10ml
BMMY: every liter contains 13.4g YNB, 0.0004g vitamin H, 10g yeast powder, 20g peptone, the potassiumphosphate of 100mmol/L pH6.0, the methyl alcohol of 5ml
4, the purifying of tunning
With 4 ℃ of centrifugal 10min of following 8000g of supernatant liquor of abduction delivering, supernatant liquor concentrates 6 times with PEG 20,000 in dialysis tubing (molecular weight cut-off 14,000 dalton, BBI company product).With the potassium phosphate buffer balance Hiload26/60 post of 20mmol/LpH6.0, go up sample 5mL earlier at every turn, use the level pad wash-out, flow velocity 3mL/min, elutriant is collected step by step, gets expression product.With substrate Suc-(Ala) 2-Pro-Phe-NA (Sigma company) detection serine protease is arranged.The CDEP of purifying and the SDS-PAGE result of CDEP-SwChBD are as shown in Figure 4.
The structure of CDEP-1, CDEP-SwChBD beauveria bassiana conversion carrier
[embodiment 3]
1, the structure of CDEP-1 beauveria bassiana conversion carrier and conversion
With EcoRI/SmaI double digestion pMD-spCDEP, reclaim the fragment of 1140bp, with SmaI/HindIII double digestion pUC-bar ∷ gfp, reclaim the Trp terminator of 750bp, with EcoRI/HindIII double digestion pUC-bar ∷ gfp, reclaim the carrier sequence of 10000bp.Three fragments of above-mentioned recovery are connected, be transformed among the intestinal bacteria Dh5 α.Verify with EcoRI/HindIII.Enzyme is cut the positive colony called after pUC-bar ∷ gfp-CDEP of evaluation, and its plasmid structure iron is seen Fig. 2 A.Extract the plasmid of positive colony, (beauveria bassiana (Beauveriabassiana) Bb0062 separates the cabbage caterpillar (Pierisrape) of self-infection with transforming goal beauveria bassiana Bb0062 bacterial strain after the HindIII linearizing, be stored in Agricultural University Of Southwest's biotechnology center, also can obtain from known approach, for example CCTCC AF93297 or separate from other approach obtains).
2, the structure of CDEP-SwChBD beauveria bassiana conversion carrier
With EcoRI/SmaI double digestion pMD-hspCDEP, reclaim the fragment of 1500bp,, reclaim the Trp terminator of 750bp with SmaI/HindIII double digestion pUC-bar ∷ gfp, with EcoRI/HindIII double digestion pUC-bar ∷ gfp, reclaim the carrier sequence of about 10000bp.Three fragments of above-mentioned recovery are connected, be transformed among the intestinal bacteria Dh5 α.Verify with EcoRI/HindIII.Enzyme is cut the positive colony called after pUC-bar ∷ gfp-CDEP-SwChBD of evaluation, and its plasmid structure iron is seen Fig. 2 B (nucleotide sequence such as SEQID NO.3, the aminoacid sequence of expression product is shown in SEQ ID NO.2).Extract the plasmid of positive colony, with transforming goal beauveria bassiana (Beauveria bassiana) Bb0062 bacterial strain after the HindIII linearizing.
The enzyme of CDEP-SwChBD Yeast expression carrier and beauveria bassiana expression vector establishment is cut qualification result as shown in Figure 3.
[embodiment 4] CDEP-1 and CDEP-SwChBD are to the Degradation of silkworm body wall
Get the 1.5ml centrifuge tube, accurately weighing 10mg substrate (silkworm body wall) adds the washing of 500 μ l100mmol/L potassium phosphate buffers (pH 6.0), 12000rpm, and 5min abandons supernatant.Handle by the following method:
C, the Bbchit1 individual curing substrate of beauveria bassiana; 1, after the proteolytic enzyme CDEP-1 of beauveria bassiana handles substrate, add chitinase and handle; 2, CDEP-SwChBD adds the chitinase processing after handling substrate again.3, after the proteolytic enzyme CDEP-1 of beauveria bassiana (adding final concentration is the PMSF arrestin enzymic activity of 1mmol/L) handles substrate, add chitinase and handle; 4, CDEP-SwChBD (adding final concentration is the PMSF arrestin enzymic activity of 1mmol/L) adds the chitinase processing after handling substrate again.This result shows that proteolytic enzyme and hybridization proteolytic enzyme all can improve the activity of chitinase, but the amplitude that hybridization proteolytic enzyme improves is apparently higher than wild-type protease.
C.400 μ l100mmol/L potassium phosphate buffer (pH6.0);
1.300 μ LCDEP-1 (0.8 μ mol/L)+100 μ L 100mmol/L potassium phosphate buffer (pH6.0);
2.300 μ L CDEP-SwChBD (0.8 μ mol/L+100 μ L 100mmol/L potassium phosphate buffer (pH 6.0))
3.300 μ LCDEP-1 (0.8 μ mol/L)+4 μ L 100mmol/L PMSF+100 μ L 100 mmol/L potassium phosphate buffers (pH 6.0)
4.300 μ L CDEP-SwChBD (0.8 μ mol/L) b+4 μ l 100mmol/L PMSF+100 μ L 100mmol/L potassium phosphate buffer (pH6.0)
Under 28 ℃, 200rpm oscillation treatment 2h.The centrifugal 5min of 12000rpm, the Braford method is measured protein concn in the supernatant (deduction adds proteinic amount), the results are shown in accompanying drawing 5A.Abandon supernatant, add chitinase Bbchit1 (concentration 0.4 μ mol/L) and the 4 μ L 100mmol/L PMSF of 350 μ L 100mmol/L potassium phosphate buffers (pH 6.0), 100 μ L; 28 ℃, 200rpm, 2h; 12000rpm, 5min surveys the chitinase activity, sees accompanying drawing 5B.The result shows that the protein that CDEP-SwChBD processing insect body wall discharges is 3.14 times of CDEP-1.And CDEP-SwChBD handled the detected chitinase activity of substrate and compares with CDEP-1 and increased by 35.7%.These results show that all CDEP-SwChBD compares with CDEP-1, has strengthened the degradation capability to insect body wall.
[embodiment 5] CDEP-1 and CDEP-SwChBD are to the influence of beauveria bassiana virulence
Used black peach aphid is this laboratory rearing in following experiment, and cabbage caterpillar is by being grown on the wild cabbage of field.
1. with the black peach aphid examination worm
The spore of 0.05%Tween-80 suspension wild-type beauveria bassiana, vortex, four layers of lens wiping paper filter, and 0.85%NaCl is resuspended, regulates spore final concentration to 1 * 10
7Individual/mL.Proteolytic enzyme CDEP-1 that adds and the final concentration of CDEP-SwChBD are 0.16 μ mol/L.The spore suspension for preparing is sprayed directly on to polypide with spray tower.With 0.05%Tween-80 is blank, analyzes the influence of hybridization proteolytic enzyme to wild-type beauveria bassiana virulence.
The results are shown in Figure 6.Statistics shows: the end is infected mortality ratio and is respectively 31.4%, 37.6% and 54.3% after adding proteolytic enzyme, added wild-type protease and hybridizing the wild-type beauveria bassiana spore processing black peach aphid of proteolytic enzyme.Illustrate that hybridization proteolytic enzyme CDEP-SwChBD has tangible insecticidal synergistic effect to the wild-type beauveria bassiana.
2. cabbage caterpillar is the examination worm
0.05% aseptic Tween-80 collects the spore of wild-type beauveria bassiana, vortex, and four layers of lens wiping paper filter, and centrifugal, 0.85%NaCl is resuspended, regulates spore final concentration 1 * 10
6, 5 * 10
6, 1 * 10
7, 5 * 10
7, 1 * 10
8, the final concentration of the proteolytic enzyme of interpolation is 0.16 μ mol/L.The spore suspension for preparing is sprayed directly on to polypide with spray tower.With 0.05%Tween-80 is blank, analyzes hybridization proteolytic enzyme CDEP-SwChBD to the parasiticidal synergism of wild-type beauveria bassiana.
The results are shown in Figure 7.Statistics shows: the LD50 that has added the wild-type beauveria bassiana of wild-type protease and hybridization proteolytic enzyme CDEP-SwChBD is respectively 1.3 * 10
7Individual/mL and 1.0 * 10
7Individual/mL, and the LD50=2.5 of wild-type beauveria bassiana * 10
7Individual/mL.The result shows that the LD50 of the wild-type beauveria bassiana spore processing cabbage caterpillar of having added hybridization proteolytic enzyme CDEP-SwChBD obviously descends.
The virulence analysis of [embodiment 6] hybridization proteolytic enzyme beauveria bassiana transformant
0.05% aseptic Tween-80 collects the spore of proteolytic enzyme (comprising wild-type and recombinant type) beauveria bassiana transformant, vortex, and four layers of lens wiping paper filter, and centrifugal, aseptic 0.85%NaCl is resuspended, regulates spore final concentration 1 * 10
7Individual/mL.The spore suspension for preparing is sprayed directly on to polypide (cabbage caterpillar) with spray tower.With 0.05%Tween-80 is blank, with the negative contrast of the spore of wild-type beauveria bassiana, analyzes the virulence of transformant.The result shows: compare with the proteolytic enzyme transformant of wild-type, the median lethal time of hybridization proteolytic enzyme transformant has shortened 24%, 28% and 33% respectively, and this result shows that transformed recombinant serine protease can significantly improve the virulence of beauveria bassiana.
By above result as seen, the present invention has successfully obtained the disinsection fungal bacterial strain of recombinant serine protease and conversion.
Above detailed description of the present invention does not limit the present invention, and those skilled in the art can make various changes and distortion according to the present invention, and only otherwise break away from spirit of the present invention, these changes and distortion all should belong to the scope of claims.
05P101407.ST25
SEQUENCE?LISTING
＜110〉Southwestern University
＜120〉recombinant serine protease and contain its disinsection fungal agent
<130>05P101407
<160>9
<170>PatentIn?version?3.1
<210>1
<211>1422
<212>DNA
＜213〉artificial sequence (artificial)
<220>
<221>CDS
<222>(1)..(1422)
＜223〉coding recombinant serine protease
<400>1
gtt?gag?cct?gct?cct?ctc?atc?gag?gcc?cgc?ggg?cag?acc?att?gcc?ggc 48
Val?Glu?Pro?Ala?Pro?Leu?Ile?Glu?Ala?Arg?Gly?Gln?Thr?Ile?Ala?Gly
1 5 10 15
aag?tac?att?gtc?aag?ctc?aag?gac?act?gcg?acc?att?ggt?atc?atg?gat 96
Lys?Tyr?Ile?Val?Lys?Leu?Lys?Asp?Thr?Ala?Thr?Ile?Gly?Ile?Met?Asp
20 25 30
gct?gcg?tcc?aag?gtg?ccc?aac?acc?gaa?cac?gtc?tat?gaa?aat?gtc?ctc 144
Ala?Ala?Ser?Lys?Val?Pro?Asn?Thr?Glu?His?Val?Tyr?Glu?Asn?Val?Leu
35 40 45
aag?gga?ttc?tcg?gcc?act?ctt?aac?cag?gaa?caa?ctt?gac?cgt?ctc?cgc 192
Lys?Gly?Phe?Ser?Ala?Thr?Leu?Asn?Gln?Glu?Gln?Leu?Asp?Arg?Leu?Arg
50 55 60
cac?gat?cct?gat?gtc?gag?tcc?atc?gag?cag?gat?gcc?att?gtt?agc?atc 240
His?Asp?Pro?Asp?Val?Glu?Ser?Ile?Glu?Gln?Asp?Ala?Ile?Val?Ser?Ile
65 70 75 80
aac?gcc?gtt?gtc?cgg?caa?gcc?gga?gct?ccc?tgg?ggt?cta?ggt?cgc?atc 288
Asn?Ala?Val?Val?Arg?Gln?Ala?Gly?Ala?Pro?Trp?Gly?Leu?Gly?Arg?Ile
85 90 95
tcg?cac?agg?gca?cga?ggc?gcg?acc?acg?tat?gac?tac?gac?tcg?agc?gcc 336
Ser?His?Arg?Ala?Arg?Gly?Ala?Thr?Thr?Tyr?Asp?Tyr?Asp?Ser?Ser?Ala
100 105 110
ggc?gcg?ggt?aca?tgc?gta?tat?gtc?att?gac?act?ggc?gtc?tat?gac?tct 384
Gly?Ala?Gly?Thr?Cys?Val?Tyr?Val?Ile?Asp?Thr?Gly?Val?Tyr?Asp?Ser
05P101407.ST25
115 120 125
cac?cct?gat?ttc?gaa?gga?cgt?gcc?aag?caa?atc?aaa?tcc?ttt?gtc?tct 432
His?Pro?Asp?Phe?Glu?Gly?Arg?Ala?Lys?Gln?Ile?Lys?Ser?Phe?Val?Ser
130 135 140
ggc?act?tca?gat?ggt?cac?ggc?cac?ggc?aca?cac?tgc?gcc?gga?act?att 480
Gly?Thr?Ser?Asp?Gly?His?Gly?His?Gly?Thr?His?Cys?Ala?Gly?Thr?Ile
145 150 155 160
ggc?tcc?aag?act?tac?ggc?gta?gcc?aag?aaggcg?tcc?att ttt?ggc?gtc 528
Gly?Ser?Lys?Thr?Tyr?Gly?Val?Ala?Lys?Lys?Ala?Ser?Ile?Phe?Gly?Val
165 170 175
aag?gtg?ctc?gaa?gac?agt?ggc?tcg?ggt?tcg?ctc?agc?ggc?gtc?att?gcc 576
Lys?Val?Leu?Glu?Asp?Ser?Gly?Ser?Gly?Ser?Leu?Ser?Gly?Val?Ile?Ala
180 185 190
gga?atg?gac?ttt?gtc?gct?acg?gac?cgg?aaa?tcc?cgt tca?tgc?agc?aaa 624
Gly?Met?Asp?Phe?Val?Ala?Thr?Asp?Arg?Lys?Ser?Arg?Ser?Cys?Ser?Lys
195 200 205
ggc?acc?gtc?gcc?agc?atg?tcc?ctt?ggc?ggt?ggc?tac?tcg?gcc?acc?gtg 672
Gly?Thr?Val?Ala?Ser?Met?Ser?Leu?Gly?Gly?Gly?Tyr?Ser?Ala?Thr?Val
210 215 220
aac?cag?gcc?gcc?gcg?cgt?ctg?cag?gct?tcg?ggc?gtt?ttt?gtc?gcc?gtc 720
Asn?Gln?Ala?Ala?Ala?Arg?Leu?Gln?Ala?Ser?Gly?Val?PheVal?Ala?Val
225 230 235 240
gcc?gcc?ggc?aac?gac?aat?agg?gat?gcc?gcc?cag?acc?tcg?ccc?gcc?tgg 768
Ala?Ala?Gly?Asn?Asp?Asn?Arg?Asp?Ala?Ala?Gln?Thr?Ser?Pro?Ala?Trp
245 250 255
gag?ccg?tcc?gtc?tgc?acc?gtc?gga?gct?acc?gac?tcg?tct?gac?cgc?cgc 816
Glu?Pro?Ser?Val?Cys?Thr?Val?Gly?Ala?Thr?Asp?Ser?Ser?Asp?Arg?Arg
260 265 270
tcc?agc?ttc?tcc?aac?ttt?gga?aaa?gct?gtt?gac?att?ttc?gca?cct?ggt 864
Ser?Ser?Phe?Ser?Asn?Phe?Gly?Lys?Ala?Val?Asp?Ile?Phe?Ala?Pro?Gly
275 280 285
act?ggc?att?ctg?tcg?acc?tgg?aat?aat?ggc?ggc?act?aat?acc?atc?tcg 912
Thr?Gly?Ile?Leu?Ser?Thr?Trp?Asn?Asn?Gly?Gly?Thr?Asn?Thr?Ile?Ser
290 295 300
ggc?act?tcg?atg?gcc?act?ccc?cac?att?gcc?ggt?ctc?ggt?gcc?tac?ctt 960
Gly?Thr?Ser?Met?Ala?Thr?Pro?His?Ile?Ala?Gly?Leu?Gly?Ala?Tyr?Leu
305 310 315 320
ttg?gct?ctc?ggc?aaa?ggc?act?gcc?ggc?aac?ctc?tgc?caa?act?atc?cag 1008
Leu?Ala?Leu?Gly?Lys?Gly?Thr?Ala?Gly?Asn?Leu?Cys?Gln?Thr?Ile?Gln
325 330 335
act?ctc?tcc?acc?aag?aat?gtc?ctt?act?ggc?gtt?cct?tca?ggc?acc?gtc 1056
Thr?Leu?Ser?Thr?Lys?Asn?Val?Leu?Thr?Gly?Val?Pro?Ser?Gly?Thr?Val
340 345 350
aac?tac?ctg?gca?ttt?aac?ggc?gcc?act?cta?gtc?aca?acc?acc?acc?acc 1104
Asn?Tyr?Leu?Ala?Phe?Asn?Gly?Ala?Thr?Leu?Val?Thr?Thr?Thr?Thr?Thr
355 360 365
gtg?aaa?ccg?acg?aca?aca?aga?acc?acc?gcg?agg?cca?act?act?acc?aca 1152
Val Lys?Pro?Thr?Thr?Thr?Arg?Thr?Thr?Ala?Arg?Pro?Thr?Thr?Thr?Thr
370 375 380
acg?aaa?gta?ccc?cat?ggc?acc?act?gaa?gaa?gac?ttt?gac?att?aac?gtg 1200
Thr?Lys?Val?Pro?His?Gly?Thr?Thr?Glu?Glu?Asp?Phe?Asp?Ile?Asn?Val
385 390 395 400
aga?ccg?gaa?gtc?gag?gaa?cta?ccc?acg?gaa?aac?gaa?gtc?gac?aat?gcg 1248
Arg?Pro?Glu?Val?Glu?Glu?Leu?Pro?Thr?Glu?Asn?Glu?Val?Asp?Asn?Ala
405 410 415
gat?gtg?tgt?aac?tct?gag?gac?gac?tac?ata?cca?gac?aag?aaa?gag?tgt 1296
05P101407.ST25
Asp?Val?Cys?Asn?Ser?Glu?Asp?Asp?Tyr?Ile?Pro?Asp?Lys?Lys?Glu?Cys
420 425 430
agc?aag?tat?tgg?cga?tgt?gtg?aac?ggc?gag?gga?gtt?cag?ttc?tcg?tgt 1344
Ser?Lys?Tyr?Trp?Arg?Cys?Val?Asn?Gly?Glu?Gly?Val?Gln?Phe?Ser?Cys
435 440 445
caa?ccg?ggg?aca?atc?ttc?aac?gtg?aaa?ctt?aac?gtt?tgc?gat?tgg?cct 1392
Gln?Pro?Gly?Thr?Ile?Phe?Asn?Val?Lys?Leu?Asn?Val?Cys?Asp?Trp?Pro
450 455 460
gaa?aat?aca?gac?aga?ccg?gaa?tgt tcg?taa l422
Glu?Asn?Thr?Asp?Arg?Pro?Glu?Cys?Ser
465 470
<210>2
<211>473
<212>PRT
＜213〉artificial sequence (artificial)
<400>2
Val?Glu?Pro?Ala?Pro?Leu?Ile?Glu?Ala?Arg?Gly?Gln?Thr?Ile?Ala?Gly
1 5 10 15
Lys?Tyr?Ile?Val?Lys?Leu?Lys?Asp?Thr?Ala?Thr?Ile?Gly?Ile?Met?Asp
20 25 30
Ala?Ala?Ser?Lys?Val?Pro?Asn?Thr?Glu?His?Val?Tyr?Glu?Asn?Val?Leu
35 40 45
Lys?Gly?Phe?SerAla?Thr?Leu?Asn?Gln?Glu?Gln?Leu?Asp?Arg?Leu?Arg
50 55 60
His?Asp?Pro?Asp?Val?Glu?Ser?Ile?Glu?Gln?Asp?Ala?Ile?Val?Ser?Ile
65 70 75 80
Asn?Ala?Val?Val?Arg?Gln?Ala?Gly?Ala?Pro?Trp?Gly?Leu?Gly?Arg?Ile
85 90 95
Ser?His?Arg?Ala?Arg?Gly?Ala?Thr?Thr?Tyr?Asp?Tyr?Asp?Ser?Ser?Ala
100 105 110
Gly?Ala?Gly?Thr?Cys?Val?Tyr?Val?Ile?Asp?Thr?Gly?Val?Tyr?Asp?Ser
115 120 125
His?Pro?Asp?Phe?Glu?Gly?Arg?Ala?Lys?Gln?Ile?Lys?Ser?Phe?Val?Ser
130 135 140
Gly?Thr?Ser?Asp?Gly?His?Gly?His?Gly?Thr?His?Cys?Ala?Gly?Thr?Ile
145 150 155 160
Gly?Ser?Lys?Thr?Tyr?Gly?Val?Ala?Lys?Lys?Ala?Ser?Ile?Phe?Gly?Val
165 170 175
Lys?Val?Leu?Glu?Asp?Ser?Gly?Ser?Gly?Ser?Leu?Ser?Gly?Val?Ile?Ala
180 185 190
05P101407.ST25
Gly?Met?Asp?Phe?Val?Ala?Thr?Asp?Arg?Lys?Ser?Arg?Ser?Cys?Ser?Lys
195 200 205
Gly?Thr?Val?Ala?Ser?Met?Ser?Leu?Gly?Gly?Gly?Tyr?Ser?Ala?Thr?Val
210 215 220
Asn?Gln?Ala?Ala?Ala?Arg?Leu?Gln?Ala?Ser?Gly?Val?Phe?Val?Ala?Val
225 230 235 240
Ala?Ala?Gly?Asn?Asp?Asn?Arg?Asp?Ala?Ala?Gln?Thr?Ser?Pro?Ala?Trp
245 250 255
Glu?Pro?Ser?Val?Cys?Thr?Val?Gly?Ala?Thr?Asp?Ser?Ser?Asp?Arg?Arg
260 265 270
Ser?Ser?Phe?Ser?Asn?Phe?Gly?Lys?Ala?Val?Asp?Ile?Phe?Ala?Pro?Gly
275 280 285
Thr?Gly?Ile?Leu?Ser?Thr?Trp?Asn?Asn?Gly?Gly?Thr?Asn?Thr?Ile?Ser
290 295 300
Gly?Thr?Ser?MetAla?Thr?Pro?His?Ile?Ala?Gly?Leu?Gly?Ala?Tyr?Leu
305 310 315 320
Leu?Ala?Leu?Gly?Lys?Gly?Thr?Ala?Gly?Asn?Leu?Cys?Gln?Thr?Ile?Gln
325 330 335
Thr?Leu?Ser?Thr?Lys?Asn?Val?Leu?Thr?Gly?Val?Pro?Ser?Gly?Thr?Val
340 345 350
Asn?Tyr?Leu?Ala?Phe?Asn?Gly?Ala?Thr?Leu?Val?Thr?Thr?Thr?Thr?Thr
355 360 365
Val?Lys?Pro?Thr?Thr?Thr?Arg?Thr?Thr?Ala?Arg?Pro?Thr?Thr?Thr?Thr
370 375 380
Thr?Lys?Val?ProHis?Gly?Thr?Thr?Glu?Glu?Asp?Phe?Asp?Ile?Asn?Val
385 390 395 400
Arg?Pro?Glu?Val?Glu?Glu?Leu?Pro?Thr?Glu?Asn?Glu?Val?Asp?Asn?Ala
405 410 415
Asp?Val?Cys?Asn?Ser?GluAsp?Asp?Tyr?Ile?Pro?Asp?Lys?Lys?GluCys
420 425 430
Ser?Lys?Tyr?Trp?Arg?Cys?Val?Asn?Gly?Glu?Gly?Val?Gln?Phe?Ser?Cys
435 440 445
Gln?Pro?Gly?Thr?Ile?Phe?Asn?Val?Lys?Leu?Asn?Val?Cys?Asp?Trp?Pro
450 455 460
Glu?Asn?Thr?Asp?Arg?Pro?Glu?Cys?Ser
465 470
<210>3
05P101407.ST25
<211>1476
<212>DNA
＜213〉artificial sequence (artificial)
<220>
<221>sig_peptide
<222>(1)..(54)
＜223〉contain the recombinant serine protease sequence of signal peptide sequence
<400>3
atgcgtctat?caatcatcgc?tgccgctctt?cccctggcca?ttgcggctcc?ggtcgttgag 60
cctgctcctc?tcatcgaggc?ccgcgggcag?accattgccg?gcaagtacat?tgtcaagctc 120
aaggacactg?cgaccattgg?tatcatggat?gctgcgtcca?aggtgcccaa?caccgaacac 180
gtctatgaaa?atgtcctcaa?gggattctcg?gccactctta?accaggaaca?acttgaccgt 240
ctccgccacg?atcctgatgt?cgagtccatc?gagcaggatg?ccattgttag?catcaacgcc 300
gttgtccggc?aagccggagc?tccctggggt?ctaggtcgca?tctcgcacag?ggcacgaggc 360
gcgaccacgt?atgactacga?ctcgagcgcc?ggcgcgggta?catgcgtata?tgtcattgac 420
actggcgtct?atgactctca?ccctgatttc?gaaggacgtg?ccaagcaaat?caaatccttt 80
gtctctggca?cttcagatgg?tcacggccac?ggcacacact?gcgccggaac?tattggctcc 540
aagacttacg?gcgtagccaa?gaaggcgtcc?atttttggcg?tcaaggtgct?cgaagacagt 600
ggctcgggtt?cgctcagcgg?cgtcattgcc?ggaatggact?ttgtcgctac?ggaccggaaa 660
tcccgttcat?gcagcaaagg?caccgtcgcc?agcatgtccc?ttggcggtgg?ctactcggcc 720
accgtgaacc?aggccgccgc?gcgtctgcag?gcttcgggcg?tttttgtcgc?cgtcgccgcc 780
ggcaacgaca?atagggatgc?cgcccagacc?tcgcccgcct?gggagccgtc?cgtctgcacc 840
gtcggagcta?ccgactcgtc?tgaccgccgc?tccagcttct?ccaactttgg?aaaagctgtt 900
gacattttcg?cacctggtac?tggcattctg?tcgacctgga?ataatggcgg?cactaatacc 960
atctcgggca?cttcgatggc?cactccccac?attgccggtc?tcggtgccta?ccttttggct 1020
ctcggcaaag?gcactgccgg?caacctctgc?caaactatcc?agactctctc?caccaagaat 1080
gtccttactg?gcgttccttc?aggcaccgtc?aactacctgg?catttaacgg?cgccactcta 1140
gtcacaacca?ccaccaccgt?gaaaccgacg?acaacaagaa?ccaccgcgag?gccaactact 1200
accacaacga?aagtacccca?tggcaccact?gaagaagact?ttgacattaa?cgtgagaccg 1260
gaagtcgagg?aactacccac?ggaaaacgaa?gtcgacaatg?cggatgtgtg?taactctgag 1320
gacgactaca?taccagacaa?gaaagagtgt?agcaagtatt?ggcgatgtgt?gaacggcgag 1380
ggagttcagt?tctcgtgtca?accggggaca?atcttcaacg?tgaaacttaa?cgtttgcgat 1440
tggcctgaaa?atacagacag?accggaatgt?tcgtaa 1476
<210>4
<211>353
<212>DNA
05P101407.ST25
＜213〉silkworm (Bombyx mori)
<220>
<221>misc_feature
<222>(1)..(353)
＜223〉the silkworm chitinase is in conjunction with the territory sequence
<400>4
actagtcaca?accaccacca?ccgtgaaacc?gacgacaaca?agaaccaccg?cgaggccaac 60
tactaccaca?acgaaagtac?cccatggcac?cactgaagaa?gactttgaca?ttaacgtgag 120
accggaagtc?gaggaactac?ccacggaaaa?cgaagtcgac?aatgcggatg?tgtgtaactc 180
tgaggacgac?tacataccag?acaagaaaga?gtgtagcaag?tattggcgat?gtgtgaacgg 240
cgagggagtt?cagttctcgt?gtcaaccggg?gacaatcttc?aacgtgaaac?ttaacgtttg 300
cgattggcct?gaaaatacag?acagaccgga?atgttcgtaa?cccgggcggc?cgc 353
<210>5
<211>1105
<212>DNA
＜213〉beauveria bassiana (Beauveria bassiana)
<400>5
gaattcgttg?agcctgctcc?tctcatcgag?gcccgcgggc?agaccattgc?cggcaagtac 60
attgtcaagc?tcaaggacac?tgcgaccatt?ggtatcatgg?atgctgcgtc?caaggtgccc 120
aacaccgaac?acgtctatga?aaatgtcctc?aagggattct?cggccactct?taaccaggaa 180
caacttgacc?gtctccgcca?cgatcctgat?gtcgagtcca?tcgagcagga?tgccattgtt 240
agcatcaacg?ccgttgtccg?gcaagccgga?gctccctggg?gtctaggtcg?catctcgcac 300
agggcacgag?gcgcgaccac?gtatgactac?gactcgagcg?ccggcgcggg?tacatgcgta 360
tatgtcattg?acactggcgt?ctatgactct?caccctgatt?tcgaaggacg?tgccaagcaa 420
atcaaatcct?ttgtctctgg?cacttcagat?ggtcacggcc?acggcacaca?ctgcgccgga 480
actattggct?ccaagactta?cggcgtagcc?aagaaggcgt?ccatttttgg?cgtcaaggtg 540
ctcgaagaca?gtggctcggg?ttcgctcagc?ggcgtcattg?ccggaatgga?ctttgtcgct 600
acggaccgga?aatcccgttc?atgcagcaaa?ggcaccgtcg?ccagcatgtc?ccttggcggt 660
ggctactcgg?ccaccgtgaa?ccaggccgcc?gcgcgtctgc?aggcttcggg?cgtttttgtc 720
gccgtcgccg?ccggcaacga?caatagggat?gccgcccaga?cctcgcccgc?ctgggagccg 780
tccgtctgca?ccgtcggagc?taccgactcg?tctgaccgcc?gctccagctt?ctccaacttt 840
ggaaaagctg?ttgacatttt?cgcacctggt?actggcattc?tgtcgacctg?gaataatggc 900
ggcactaata?ccatctcggg?cacttcgatg?gccactcccc?acattgccgg?tctcggtgcc 960
taccttttgg?ctctcggcaa?aggcactgcc?ggcaacctct?gccaaactat?ccagactctc 1020
tccaccaaga?atgtccttac?tggcgttcct?tcaggcaccg?tcaactacct?ggcatttaac 1080
05P101407.ST25
ggcgccacct?aacccgggcg?gccgc 1105
<210>6
<211>1094
<212>DNA
＜213〉beauveria bassiana (Beauveria bassiana)
<400>6
gaattcgttg?agcctgctcc?tctcatcgag?gcccgcgggc?agaccattgc?cggcaagtac 60
attgtcaagc?tcaaggacac?tgcgaccatt?ggtatcatgg?atgctgcgtc?caaggtgccc 120
aacaccgaac?acgtctatga?aaatgtcctc?aagggattct?cggccactct?taaccaggaa 180
caacttgacc?gtctccgcca?cgatcctgat?gtcgagtcca?tcgagcagga?tgccattgtt 240
agcatcaacgccgt?tgtccggcaagccgga?gctccctggg?gtctaggtcg?catctcgcac 300
agggcacgag?gcgcgaccac?gtatgactac?gactcgagcg?ccggcgcggg?tacatgcgta 360
tatgtcattg?acactggcgt?ctatgactct?caccctgatt?tcgaaggacg?tgccaagcaa 420
atcaaatcct?ttgtctctgg?cacttcagat?ggtcacggcc?acggcacaca?ctgcgccgga 480
actattggct?ccaagactta?cggcgtagcc?aagaaggcgt?ccatttttgg?cgtcaaggtg 540
ctcgaagaca?gtggctcggg?ttcgctcagc?ggcgtcattg?ccggaatgga?ctttgtcgct 600
acggaccgga?aatcccgt?tcatgcagcaaa?ggcaccgtcg?ccagcatgtc?ccttggcggt 660
ggctactcgg?ccaccgtgaa?ccaggccgcc?gcgcgtctgc?aggcttcggg?cgtttttgtc 720
gccgtcgccg?ccggcaacga?caatagggat?gccgcccaga?cctcgcccgc?ctgggagccg 780
tccgtctgca?ccgtcggagc?taccgactcg?tctgaccgcc?gctccagctt?ctccaacttt 840
ggaaaagctg?ttgacatttt?cgcacctggt?actggcattc?tgtcgacctg?gaataatggc 900
ggcactaata?ccatctcggg?cacttcgatg?gccactcccc?acattgccgg?tctcggtgcc 960
taccttttgg?ctctcggcaa?aggcactgcc?ggcaacctct?gccaaactat?ccagactctc 1020
tccaccaaga?atgtccttac?tggcgttcct?tcaggcaccg?tcaactacct?ggcatttaac 1080
ggcgccactc?taga 1094
<210>7
<211>1159
<212>DNA
＜213〉beauveria bassiana (Beauveria bassiana)
<400>7
gaattcatgc?gtctatcaat?catcgctgcc?gctcttcccc?tggccattgc?ggctccggtc 60
gttgagcctg?ctcctctcat?cgaggcccgc?gggcagacca?ttgccggcaa?gtacattgtc 120
aagctcaagg?acactgcgac?cattggtatc?atggatgctg?cgtccaaggt?gcccaacacc 180
gaacacgtct?atgaaaatgt?cctcaaggga?ttctcggcca?ctcttaacca?ggaacaactt 240
gaccgtctcc?gccacgatcc?tgatgtcgag?tccatcgagc?aggatgccat?tgttagcatc 300
05P101407.ST25
aacgccgttg?tccggcaagc?cggagctccc?tggggtctag?gtcgcatctc?gcacagggca 360
cgaggcgcga?ccacgtatga?ctacgactcg?agcgccggcg?cgggtacatg?cgtatatgtc 420
attgacactg?gcgtctatga?ctctcaccct?gatttcgaag?gacgtgccaa?gcaaatcaaa 480
tcctttgtct?ctggcacttc?agatggtcac?ggccacggca?cacactgcgc?cggaactatt 540
ggctccaaga?cttacggcgt?agccaagaag?gcgtccattt?ttggcgtcaa?ggtgctcgaa 600
gacagtggct?cgggttcgct?cagcggcgtc?attgccggaa?tggactttgt?cgctacggac 660
cggaaatccc?gttcatgcag?caaaggcacc?gtcgccagca?tgtcccttgg?cggtggctac 720
tcggccaccg?tgaaccaggc?cgccgcgcgt?ctgcaggctt?cgggcgtttt?tgtcgccgtc 780
gccgccggca?acgacaatag?ggatgccgcc?cagacctcgc?ccgcctggga?gccgtccgtc 840
tgcaccgtcg?gagctaccga?ctcgtctgac?cgccgctcca?gcttctccaa?ctttggaaaa 900
gctgttgaca?ttttcgcacc?tggtactggc?attctgtcga?cctggaataa?tggcggcact 960
aataccatct?cgggcacttc?gatggccact?ccccacattg?ccggtctcgg?tgcctacctt 1020
ttggctctcg?gcaaaggcac?tgccggcaac?ctctgccaaa?ctatccagac?tctctccacc 1080
aagaatgtcc?ttactggcgt?tccttcaggc?accgtcaact?acctggcatt?taacggcgcc 1140
acctaacccg?ggcggccgc 1159
<210>8
<211>1148
<212>DNA
＜213〉beauveria bassiana (Beauveria bassiana)
<400>8
gaattcatgc?gtctatcaat?catcgctgcc?gctcttcccc?tggccattgc?ggctccggtc 60
gttgagcctg?ctcctctcat?cgaggcccgc?gggcagacca?ttgccggcaa?gtacattgtc 120
aagctcaagg?acactgcgac?cattggtatc?atggatgctg?cgtccaaggt?gcccaacacc 180
gaacacgtct?atgaaaatgt?cctcaaggga?ttctcggcca?ctcttaacca?ggaacaactt 240
gaccgtctcc?gccacgatcc?tgatgtcgag?tccatcgagc?aggatgccat?tgttagcatc 300
aacgccgttg?tccggcaagc?cggagctccc?tggggtctag?gtcgcatctc?gcacagggca 360
cgaggcgcga?ccacgtatga?ctacgactcg?agcgccggcg?cgggtacatg?cgtatatgtc 420
attgacactg?gcgtctatga?ctctcaccct?gatttcgaag?gacgtgccaa?gcaaatcaaa 480
tcctttgtct?ctggcacttc?agatggtcac?ggccacggca?cacactgcgc?cggaactatt 540
ggctccaaga?cttacggcgt?agccaagaag?gcgtccattt?ttggcgtcaa?ggtgctcgaa 800
gacagtggct?cgggttcgct?cagcggcgtc?attgccggaa?tggactttgt?cgctacggac 660
cggaaatccc?gttcatgcag?caaaggcacc?gtcgccagca?tgtcccttgg?cggtggctac 720
tcggccaccg?tgaaccaggc?cgccgcgcgt?ctgcaggctt?cgggcgtttt?tgtcgccgtc 780
gccgccggca?acgacaatag?ggatgccgcc?cagacctcgc?ccgcc?tggga?gccgtccgtc 840
tgcaccgtcg?gagctaccga?ctcgtctgac?cgccgctcca?gcttctccaa?ctttggaaaa 900
gctgttgaca?ttttcgcacc?tggtactggc?attctgtcga?cctggaataa?tggcggcact 960
aataccatct?cgggcacttc?gatggccact?ccccacattg?ccggtctcgg?tgcctacctt 1020
05P101407.ST25
ttggctctcg?gcaaaggcac?tgccggcaac?ctctgccaaa?ctatccagac?tctctccacc 1080
aagaatgtcc?ttactggcgt?tccttcaggc?accgtcaact?acctggcatt?taacggcgcc 1140
actctaga 1148
<210>9
<211>361
<212>PRT
＜213〉artificial sequence (artificial)
<400>9
Val?GluPro?Ala?Pro?Leu?Ile?Glu?Ala?Arg?Gly?Gln?Thr?Ile?Ala?Gly
1 5 10 15
Lys?Tyr?Il?e?Val?Lys?Leu?Lys?Asp?Thr?Ala?Thr?Ile?Gly?Ile?MetAsp
20 25 30
Ala?Ala?Ser?Lys?Val?Pro?Asn?Thr?Glu?Hi?s?Val?Tyr?Glu?Asn?Val?Leu
35 40 45
Lys?Gly?Phe?Ser?Ala?Thr?Leu?Asn?Gln?Glu?Gln?Leu?Asp?Arg?Leu?Arg
50 55 60
His?Asp?Pro?Asp?Val?Glu?Ser?Ile?Glu?Gln?Asp?Ala?Ile?Val?Ser?Ile
65 70 75 80
Asn?Ala?Val?Val?Arg?Gln?Ala?Gly?Ala?Pro?Trp?Gly?Leu?Gly?Arg?Ile
85 90 95
Ser?His?Arg?Ala?Arg?Gly?Ala?Thr?Thr?Tyr?Asp?Tyr?Asp?Ser?Ser?Ala
100 105 110
Gly?Ala?Gly?Thr?Cys?Val?Tyr?Val?Ile?Asp?Thr?Gly?Val?Tyr?Asp?Ser
115 120 125
His?Pro?Asp?PheGlu?Gly?Arg?Ala?Lys?Gln?Ile?Lys?Ser?Phe?Val?Ser
130 135 140
Gly?Thr?Ser?Asp?Gly?His?Gly?His?Gly?Thr?His?Cys?Ala?Gly?Thr?Ile
145 150 155 160
Gly?Ser?Lys?Thr?Tyr?Gly?Val?Ala?Lys?Lys?Ala?Ser?Ile?Phe?Gly?Val
165 170 175
Lys?Va?l?Leu?Glu?Asp?Ser?Gly?Ser?Gly?Ser?Leu?Ser?Gly?Val?IleAla
180 185 190
Gly?Met?Asp?Phe?Val?Ala?Thr?Asp?Arg?Lys?Ser?Arg?Ser?Cys?Ser?Lys
195 200 205
Gly?Thr?Val?Ala?Ser?Met?Ser?Leu?Gl?y?Gly?Gly?Tyr?Ser?Ala?Thr?Val
210 215 220
05P101407.ST25
Asn?Gln?Ala?Ala?Ala?Arg?Leu?Gln?Ala?Ser?Gly?Val?Phe?Val?Ala?Val
225 230 235 240
Ala?Ala?Gly?Asn?Asp?Asn?ArgAsp?Ala?Ala?Gln?Thr?Ser?Pro?Ala?Trp
245 250 255
Glu?Pro?Ser?Val?Cys?Thr?Val?Gly?Ala?Thr?Asp?Ser?Ser?Asp?Arg?A?rg
260 265 270
Ser?Ser?Phe?Ser?Asn?Phe?Gly?Lys?Ala?Val?Asp?Ile?Phe?Ala?Pro?Gly
275 280 285
Thr?Gly?Ile?Leu?Ser?Thr?Trp?Asn?Asn?Gly?Gly?Thr?Asn?Thr?Ile?Ser
290 295 300
Gly?Thr?Ser?Met?Ala?Thr?Pro?His?Ile?Ala?Gly?Leu?Gly?Ala?Tyr?Leu
305 310 315 320
Leu?Ala?Leu?Gly?Lys?Gly?Thr?Ala?Gly?Asn?Leu?Cys?Gln?Thr?Ile?Gln
325 330 335
Thr?Leu?Ser?Thr?Lys?Asn?Val?Leu?Thr?Gly?Val?Pro?Ser?Gly?Thr?Val
340 345 350
Asn?Tyr?Leu?Ala?Phe?Asn?Gly?Ala?Thr
355 360
Claims (13)
1, a kind of recombinant serine protease, it contains a fusion rotein, and the chitin binding domain of the serine protease that described fusion rotein origin comes from fungi and the chitinase that derives from insect merges and forms.
2, the described recombinant serine protease of claim 1, the chitin binding domain that it is characterized in that described fusion rotein origin serine protease that comes from beauveria bassiana (Beauveria bassiana) and the chitinase that derives from silkworm (Bombyxmori) merge and form.
3, the described recombinant serine protease of claim 1 is characterized in that it has the aminoacid sequence shown in the SEQ ID NO.2.
4, a kind of isolating nucleotide sequence, its coding claim 1 described recombinant serine protease containing the gene of chitin binding domain that gene and coding that coding derives from the serine protease of fungi derive from the chitinase of insect.
5, the described nucleotide sequence of claim 4 is characterized in that the gene of the serine protease that it contains coding beauveria bassiana (Beauveria bassiana) and the gene of the chitin binding domain of the chitinase of coding silkworm (Bombyx mori).
6, the described nucleotide sequence of claim 5 is characterized in that it has the sequence shown in SEQ ID NO.1.
7, the described nucleotide sequence of claim 5 is characterized in that it further contains the signal peptide sequence of a serine protease, and described nucleotide sequence has the sequence shown in the SEQ ID NO.3.
8, the recombinant expression vector that contains the described nucleotide sequence of claim 4.
9, the described recombinant expression vector of claim 8 is characterized in that described expression vector is a Yeast expression carrier.
10, contain the disinsection fungal agent of the described recombinant serine protease of claim 1.
11, the described disinsection fungal agent of claim 10 is characterized in that it contains the described recombinant serine protease and the disinsection fungal of insecticidal effective dose.
12, the disinsection fungal conversion bacterial strain that contains the described nucleotide sequence of claim 4.
13, the described disinsection fungal of claim 12 transforms bacterial strain, it is characterized in that described disinsection fungal is beauveria bassiana (Beauveria bassiana).
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| CN101880668B (en) * | 2010-05-07 | 2012-05-23 | 广西大学 | Gene for coding serpin and application thereof |
| US20130156740A1 (en) * | 2011-12-19 | 2013-06-20 | Novozymes Biologicals Holdings A/S | Bio-pestcide methods and compositions |
| CN103173467B (en) * | 2012-12-05 | 2014-08-20 | 西南大学 | Method for improving high-seepage property, oxidative stress resistance and toxicity of beauveria bassiana by utilizing genetic engineering |
| CN105441469B (en) * | 2016-01-15 | 2018-08-07 | 西南大学 | Recombinate insect moulting hormones inactivated gene Bbsp::Egt and its disinsection fungal agent |
| CN110054684B (en) * | 2019-04-20 | 2021-07-20 | 中国农业科学院植物保护研究所 | Acanthopanax migratorius serine protease inhibitor serpin-5, and coding gene and application thereof |
| CN110590411A (en) * | 2019-10-21 | 2019-12-20 | 武汉楚强生物科技有限公司 | Amino acid hydrolysate based on insect residues with viruses and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6927322B2 (en) * | 2001-03-30 | 2005-08-09 | Cornell Research Foundation, Inc. | Cabbage proteinase inhibitor gene confers resistance against plant pests |
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| US6927322B2 (en) * | 2001-03-30 | 2005-08-09 | Cornell Research Foundation, Inc. | Cabbage proteinase inhibitor gene confers resistance against plant pests |
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| Title |
|---|
| graal: a Drosophila gene coding for several mosaicserine proteases MUNIER A.I,INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 2004 * |
| graal: a Drosophila gene coding for several mosaicserine proteases MUNIER A.I,INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 2004;植物蛋白酶抑制剂在植物抗虫与抗病中的作用 卢晓风，生物化学与生物物理进展，第25卷第4期 1998 * |
| 植物蛋白酶抑制剂在植物抗虫与抗病中的作用 卢晓风，生物化学与生物物理进展，第25卷第4期 1998 * |
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