CN103484468A - Diamondback moth peptidoglycan recognition protein, preparation method and application thereof - Google Patents
Diamondback moth peptidoglycan recognition protein, preparation method and application thereof Download PDFInfo
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Abstract
The present invention discloses diamondback moth peptidoglycan recognition protein, a preparation method and an application thereof, wherein the nucleotide sequence of the diamondback moth peptidoglycan recognition protein gene is represented by SEQIDNO:1-2, and semi-quantitative RT-PCR is adopted to detect temporal and spatial expression of diamondback moth PGRP-S1 in the body. According to the present invention, isaria fumosorosea is adopted to treat diamondback moth larva, quantitative RT-PCR is adopted to detect an immune recognition effect of the diamondback moth PGRP-SA gene on fungi paecilomyces fumosoroseus isaria, and the PGRP-S1 is the innate immune recognition receptor of diamondback moth, activates innate immunity, regulates antibacterial peptide expression, and provides a new target for prevention and control of diamondback moth from immunology.
Description
Technical field
The present invention relates to field of molecular microbiology, particularly, relate to a kind of small cabbage moth peptidoglycan recognition protein and preparation method thereof and application.
Background technology
Small cabbage moth is the cresss such as the worldwide insect that migrates, main harm wild cabbage, purple cabbage, broccoli, a kind of sedge dish, leaf mustard, Cauliflower, Chinese cabbage, rape, radish.It possesses typical insect evolution advantage, and as body is little, fecundity is strong, and ecological adaptation power is strong etc., has most importantly produced extremely strong resistance, at present Multiple Pesticides has been produced to resistance, as Imidacloprid, and Chlorpyrifos 94, BT, Avrmectin etc.The year nineties in last century, the small cabbage moth wildness was faced in many places, pasted medical help.Owing to occurring, area is large, harm time long, the control difficulty, and small cabbage moth replaces cabbage caterpillar gradually and becomes No. 1 insect of vegetables, and bring about great losses every year to agriculture production.Along with the development of biotechnology, will search out the agricultural chemicals new target drone of more control small cabbage moth.
The macromole that the mixed polysaccharide that peptidoglycan (peptidoglycan) alternately is connected with-acetylmuramic acid by N-Acetyl-D-glucosamine forms from the different peptide cross connections that form.Peptidoglycan is the main component of many bacteria cell walls, and especially, in gram positive bacterium, its contained peptidoglycan accounts for 50 ~ 80% of cell walls dry weight.
Peptidoglycan recognition protein (peptidoglycan recognition proteins, PGRPs) is a kind of innate immune molecule, extensively is present in vertebrates, can the specific recognition peptidoglycan.Peptidoglycan recognition protein is invaded the peptidoglycan of pathogenetic bacteria by identification, and then activates the immune signal path such as Toll and Imd and mediate phagolysis, therefore in the middle of the process of opposing cause of disease invasion, plays an important role.Structurally; peptidoglycan recognition protein contains an acylase (amidase) structure at the C of its sequence end, and this structure approximately has 165 amino acid, all demonstrates the conservative property of height from the insect to the Mammals; but, outside the PGRP structural domain, aminoacid sequence differs greatly.This structure has lactamase activity in some peptidoglycan recognition protein, this activity can the bacterium for degrading cell walls in the amido linkage of peptidoglycan, cause bacterium death, so this class peptidoglycan recognition protein has sterilizing ability.
Research about peptidoglycan identification receptor in insect starts from 1996 the earliest, Yoshida H etc. find from silkworm hemolymph and purifying has obtained one to have signal peptide, iso-electric point be 6.5 and the molecular weight albumen that is 19 kD, and by its called after PGRP, it is also the small-sized peptidoglycan recognition protein that organic sphere obtains the earliest from insect, under the condition existed without calcium ion, this albumen energy binding peptide glycan and containing the bacterium of peptidoglycan.According to structure, peptidoglycan recognition protein can be divided into two hypotypes, comprises elongated and short type.The PGRP-L of insect mainly is expressed in hemocyte, and PGRP-S is present in hemolymph, epidermis and internal organ, and a small amount of expression is also arranged in hemocyte, wherein some PGRPs is constitutive expression, as PGRP-SA/SD/SB, it is inducible expressions that some PGRPs are also arranged, as PGRP-LC.And for the peptidoglycan recognition protein of small cabbage moth and recognition mechanism thereof so far without relevant report.
Summary of the invention
The purpose of this invention is to provide a kind of small cabbage moth peptidoglycan recognition protein.
The preparation method who the purpose of this invention is to provide a kind of small cabbage moth peptidoglycan recognition protein.
Another object of the present invention is to provide a kind of application of small cabbage moth identification albumen.
The present invention's above-mentioned purpose that is achieved by the following technical programs:
A kind of small cabbage moth peptidoglycan recognition protein PGRP-S1 gene, its nucleotide sequence is as shown in SEQ ID NO:1～2.
A kind of small cabbage moth peptidoglycan recognition protein PGRP-S1, its aminoacid sequence is as shown in SEQ ID NO 3.
The primer of pair for amplification small cabbage moth peptidoglycan recognition protein PGRP-S1 gene, the nucleotide sequence of primer is as shown in SEQ ID NO:11～12.
A kind of recombinant expression vector, inserted the nucleotide sequence of SEQ ID NO:1～2 described small cabbage moth peptidoglycan recognition protein PGRP-S1 genes by the multiple clone site of the carrier that sets out.
A kind of recombinant bacterial strain, described bacterial strain contains small cabbage moth peptidoglycan recognition protein PGRP-S1 gene.Recombinant bacterial strain is by recombinant expression vector transformation receptor strain construction gained as above.
The cloning process of small cabbage moth peptidoglycan recognition protein PGRP-S1 gene comprises the steps: that (1) is according to the PGRP Unigene sequence of transcribing group order-checking gained as mentioned above, design respectively 3 ' RCAE primer and 5 ' RCAE primer (2) is processed diamondback moth larvae with gram-positive microorganism, extracting RNA, reverse transcription becomes RACE cDNA, through Download pcr amplification repeatedly, obtains total length.
Measure the spatial and temporal expression situation of small cabbage moth peptidoglycan recognition protein PGRP-S1 gene in its body as mentioned above, purpose is in order to identify the expression pattern of PGRP-S1 gene in the small cabbage moth body.
Small cabbage moth glycan identification albumen PGRP-S1 gene pairs fungi paecilomyces fumosoroseus as above has immune identification action.
Beneficial effect of the present invention:
The present invention clones and obtains peptidoglycan recognition protein PGRP-S1 gene first from Important Agricultural insect small cabbage moth.And carried out the detection of transcriptional level to its spatial and temporal expression pattern with to fungi fungi paecilomyces fumosoroseus, for the harm that utilizes paecilomyces fumosoroseus to prevent and treat small cabbage moth on producing lays the foundation, tool has very great significance.
The accompanying drawing explanation
Fig. 1. 3'-RACE PCR result; M:DL2000; The 3' end amplification of 1:Px PGRP-S1 sequence, about 400bp.
Fig. 2. 5'-RACE PCR result; M:DL2000; The 5' end amplification of 1:Px PGRP-S1 sequence, about 400bp.
Fig. 3. small cabbage moth PGRP-S1 full length gene cDNA sequence; M:DL2000; The full length cDNA sequence of 1:Px PGRP-S1 gene order, 822bp.
Fig. 4. the sequence map of small cabbage moth total length; Full length cDNA sequence, 5 ' UTR:1-36bp; ORF:37-756b; 3 ' UTR:757-822bp.
Fig. 5. small cabbage moth PGRP-S1 is at the expression amount of different developmental phases; Actin: the small cabbage moth housekeeping gene is as internal reference, at its different developmental phases stably express; Egg: ovum; The 1s:1 instar larvae; The 2s:2 instar larvae; The 3s:3 instar larvae; The 4s:4 instar larvae; Pp: prepupal period; P: pupa time first day; Adult: emergence first day adult.
Fig. 6. the expression amount of 4 small cabbage moth PGRP-S1 in age in different tissues; Actin: the small cabbage moth housekeeping gene is as internal reference, stably express in the different tissues in its body; Mg: middle intestines; Mt: Malpighian tube; He: hemolymph; Fb: fatty body; Ep: epidermis.
Fig. 7. the paecilomyces fumosoroseus spore suspension is processed the timetable expression patterns of PGRP-S1 after 4 age diamondback moth larvaes, and X-coordinate means to inject 10 of 8 μ L
5spore/mL paecilomyces fumosoroseus spore suspension, the expression amount of the PGRP-S1 of small cabbage moth after 0h, 3h, 6h, 12h, 18h, 24h and 36h successively; Ordinate zou means the expression multiple of the relative reference gene actin of small cabbage moth PGRP-S1.Error bar means the standard deviation (p<=0.05) of 3 independent experiments.
Embodiment
Further describe the present invention below in conjunction with the drawings and specific embodiments.Unless stated otherwise, the reagent adopted in embodiment and method are conventional reagent and the method for using in this area.
The clone of embodiment 1 small cabbage moth peptidoglycan recognition protein (PGRP-S1) gene
S1. the laboratory rearing small cabbage moth (
plutella xylostella) larva, paecilomyces fumosoroseus (
paecilomyces Fumosoroseus) as supplying the examination bacterial classification.
S2. small cabbage moth is transcribed the mensuration of group sequence: collect 1 age of small cabbage moth to 4 instar larvaes, prepupa, pupa, adult, extract after total RNA and utilized the RNA-seq technical measurement it transcribes the group sequence, the extracting method of total RNA operates with reference to Trizol (Invitrogen, USA) test kit specification sheets.Measured altogether 34,522,812 clean reads, the reads mean length is 90bp, obtains 3,107,053,080 base.Finally be assembled into 107710 Unigene, length range is 200 ~ 3000bp.COG, GO, KEGG annotation and analyze after obtain altogether the Unigene of 68984 higher annotation confidence levels of tool, 725 of the Unigenes directly related with Insect immunity, the Unigene1 bar of PGRP-S1 wherein, its sequence is 318 bp, as shown in SEQ ID NO:4.
S3. according to the Unigene design 3 ' end of PGRP-S1 and the special primer of 5 ' end RACE end rapid amplifying reaction, 3 ' RACE primer sequence is: RACE31:5 '-GCGCACAGTCCATGCGCAACCTTCAGA-3'(is as shown in SEQ ID NO:5), RACE32:5'-TTGAACGATAAGGGATGGGATTTGCC-3'(is as shown in SEQ ID NO:6), 5 ' RACE primer is that RACE51:5'-AAGAGTTGTAGCCGTAAGTGTGAGCC-3'(is as shown in SEQ ID NO:7), RACE52:5 '-ACGCGGCCGTCGTTGCCGATCACG-3'(is as shown in SEQ ID NO:8).3 ' end and 5 ' end RACE match and carry out the reaction of RACE end rapid amplifying with UPMs respectively, and the UPMs primer sequence is: UPM-L:5 '-CTAATACGACTCACTATAGGGCAAGCAG TGGTATCAACGCAGAGT-3 ' (as shown in SEQ ID NO:9) and UPM-S:5 '-CTAATACGAC TCACTATAGGGC-3 ' (as shown in SEQ ID NO:10).
S4.cDNA the first chain is synthetic:
At first, extract the total RNA of small cabbage moth: the Escherichia coli bacteria liquid that is 0.8～1.0 by the OD value stimulates small cabbage moth 4 instar larvaes, carries out inducing of peptidoglycan recognition protein.Get the small cabbage moth of 0.5～1g after 6 hours induce, grind in liquid nitrogen, by the Trizol method, extract the total RNA of small cabbage moth; The extracting method of total RNA operates with reference to Trizol (Invitrogen, USA) test kit specification sheets.
CDNA the first chain is synthetic: according to the RACE of CLONTECH company test kit specification sheets, carry out: the total RNA of 10 μ g adds reaction solution 20 μ L, 42 ℃ of reaction 90min.72 ℃ of reaction 10min, termination reaction.Reaction solution forms: 50 mmole Repone K, 3 mmole magnesium chlorides, 10 mmole Tris-HCL pH8.3,1 mmole DTT, 5 micromole d NTP, 25U RNA enzyme inhibitors, 8U AMV reversed transcriptive enzyme.
S5. 3 ' RACE and 5 ' RACE reaction: cDNA the first chain that the step S4 reverse transcription of take obtains is template, and the primer shown in S3 carries out the PCR reaction, and chain polymerization enzyme reaction (PCR) reagent and condition are as follows:
At first by following reagent mix together
3 '-RACE in, forward primer and reverse primer be respectively 3 '-RACE(is as shown in SEQ ID NO:5 and SEQ ID NO:6) and UPMs primer (as shown in SEQ ID NO:9 and SEQ ID NO:10); 5 '-RACE in, forward primer and reverse primer be respectively 5 '-RACE(is as shown in SEQ ID NO:7 and SEQ ID NO:8) and UPMs primer (as shown in SEQ ID NO:9 and SEQ ID NO:10).
The PCR reaction conditions is: then 94 ℃ of sex change 5min at first enter following circulation: 94 ℃ of 30s, and 65 ℃ of 30 s, 72 ℃ of 50s, carry out 35 circulations altogether, and last 72 ℃ are extended 10min, and result is as Fig. 1 and 2.
Reaction product purifying: utilize OMEGA BIO-TEK company product (Gel Extraction Kit) operation steps to be undertaken by product description.By serving extra large invitrogen company after the product cloning of purifying, checked order.
S7. the acquisition of small cabbage moth peptidoglycan recognition protein (PGRP-S1) full length gene cDNA
According to the RACE sequencing result, design pair of primers ORFF and ORFR, primer sequence is as follows:
ORFF 5 '-GGGGAGTGTTAAAACCGCACCATA-3 ' (as shown in SEQ ID NO:11).
ORFR 5 '-TCACTTAGTAAGATCGTAAATCTG-3 ' (as shown in SEQ ID NO:12).
The cDNA of small cabbage moth 4 instar larvaes of take is template, and reaction conditions is as follows, and described mixed solution is composed as follows:
Mixed solution, at 94 ℃ of denaturation 5min, is then entered to following circulation: 94 ℃, 40 seconds, 56 ℃, 40 seconds, 72 ℃, 50 seconds, carry out altogether 35 circulations, last 72 ℃ are extended 7 minutes, the complete rearmounted 4 ℃ of termination reactions that increase, result is as shown in Figure 3.
Reaction product purifying: utilize OMEGA BIO-TEK company product (Gel Extraction Kit) operation steps to be undertaken by product description.By serving extra large invitrogen company after the product cloning of purifying, checked order.Institute's calling sequence and gene pool sequence are compared.
The sequencing result analysis is found: the full length sequence of small cabbage moth peptidoglycan recognition protein (PGRP-S1) gene that the clone obtains is that 822bp(is as shown in SEQ ID NO:1), the ORF of small cabbage moth peptidoglycan recognition protein (PGRP-S1) gene is 720bp, and the ORF sequence is as shown in SEQ ID NO:2; 239 the amino acid whose albumen of encoding, coding protein sequence is as shown in SEQ ID NO:3.
Embodiment 2 small cabbage moth peptidoglycan recognition protein PGRP-S1 spatial and temporal expression patterns
S1. collect the small cabbage moth of different development durations, comprise ovum, 1 instar larvae; 2 instar larvaes; 3 instar larvaes; 4 instar larvaes; Prepupal period; Pupa time first day; Emergence first day adult.
S2. dissect 4 instar larvaes, collect respectively the immunity organ of fatty body, hemocyte, epidermis, middle intestines and Malpighian tube.
S3. will collect the small cabbage moth of the different length of time and different tissues, add liquid nitrogen, and use the RNAiso Plus of TAKARA to extract reagent, be operated to specifications, obtain respectively total RNA of the different length of time and different tissues.
S4. according to the RACE of the CLONTECH company test kit specification sheets cDNA in the synthetic different length of times, once add following reagent: the total RNA of 10 microgram adds 42 ℃ of reactions of reaction solution 20 microlitre 90 minutes.72 ℃ of 10 minutes termination reactions.Reaction solution forms: 50 mmole Repone K, 3 mmole magnesium chlorides, 10 mmole Tris-Hcl pH8.3,1 mmole DTT, 5 micromole d NTP, 25 RNA of unit enzyme inhibitorss, 8 AMV of unit reversed transcriptive enzymes.
The housekeeping gene actin of S5. usining in the small cabbage moth body, as reference gene, designs respectively sxemiquantitative primer and the sxemiquantitative Actin primer of PGRP-S1, and sxemiquantitative Actin primer sequence is:
Actin-F 5 '-ATGTGCGACGACGACGTAGCCGCC-3 ' (as shown in SEQ ID NO:13).
Actin-R 5 '-CAGGGCGACGTAGCAGAGCTTCTC-3 ' (as shown in SEQ ID NO:14).
The sxemiquantitative primer of PGRP-S1 is:
RT-PCRF:5'-GCACCATAGGCAAAATATGTCTT-3'(is as shown in SEQ ID NO:15).
RT-PCRR:5'-CAGCAGTATTAAATTCACTTAGT-3'(is as shown in SEQ ID NO:16).
S6. the spatial and temporal expression pattern of PGRP-S1 in the small cabbage moth body of having utilized sxemiquantitative RT-PCR to detect, result is as Fig. 5 and 6.As can be known from Fig. 5 and Fig. 6: the internal reference actin gene (AB282645) of small cabbage moth at it without development duration stably express all; And notable difference appears in the expression amount of immunity identification gene PGRP-S1.PGRP-S1 expresses faint in 2 ages and adult stage, in conjunction with raising the small cabbage moth experience, the small cabbage moth that we analyzed for 2 ages in conform and in a large number feed excessively between section, the physique sensitivity, the expression amount of PGRP-S1 is relatively less; Know that by Fig. 5 PGRP-S1 mainly is expressed in fatty body, and the fatty body that its body of the small cabbage moth in adult stage contains reduces much than larva, so the expression amount of PGRP-S1 also seldom; PGRP-S1 4 ages, prepupa and pupa time expression amount more, in fact, this and small cabbage moth section between these three growths has very strong resistibility and is undivided.
Embodiment 3 small cabbage moth PGRP-S1 to paecilomyces fumosoroseus (
p.fumosoroseus) recognition effect
S1. get 4 age diamondback moth larvae, the spore of rose dark brown Isaria is collected and is diluted to 10 with sterilized water
5spore/mL, inject small cabbage moth with microsyringe, and every about 8uL of injection gets respectively 6 small cabbage moths when 0h, 3h, 6h, 12h, 18h, 24h and 36h.Extract total RNA, after reverse transcription cDNA, take that it detects the expression that rose dark brown Isaria infects the PGRP-S1 of rear different time points as template, experiment repeats 3 times.
S2. take small cabbage moth β-Actin gene (AB282645) is reference gene, according to small cabbage moth PGRP-S1 and β-Actin sequences Design fluorescence quantification PCR primer.The quantitative primer sequence of PGRP-S1 is:
QF:5'-TCATCAACCACTCCGTGTCCCCC-3'(is as shown in SEQ ID NO:17).
QR:5'-AAGAGTTGTAGCCGTAAGTGTGAG-3'(is as shown in SEQ ID NO:18).
The sequence of the quantitative primer of Actin is:
Actin QF 5'-ATGGTCGGTATGGGACAGAA 3'(is as shown in SEQ ID NO:19).
Actin QR 5'-AGGTGTGGTGCCAGATCTTC 3'(is as shown in SEQ ID NO:20).
Adopt SYBR Green dye method to carry out the relative quantification detection to the timetable expression patterns of small cabbage moth PGRP-S1 gene, result as shown in Figure 7.As can be seen from Figure 7: to 10 of small cabbage moth injection in 4 ages 8ul
5spore/mL rose dark brown cluster spore diluent, the expression amount of PGRP-S1 is obvious variation tendency along with time lapse shows.With regard to the whole period, two small peaks appear in the relative expression quantity of PGRP-S1, after being for the first time injection spore liquid 3h, relative expression quantity reaches 40 times, 6h, 12h are reduced to the level of contrast 0h afterwards, this may be because in injection process, and the trace that stress immune cause the PGRP-S1 expression amount of small cabbage moth raises; After 12h, PGRP-S1 starts great expression and identifies fungal spore, at the 24h expression amount, up to 110 times, causes the immune pathway reaction of small cabbage moth, and then kills spore, recovers control level after 36h.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
SEQUENCE LISTING
<110 > Agricultural University Of South China
<120 > a kind of small cabbage moth peptidoglycan recognition protein and preparation method thereof and application
<130>
<160> 20
<170> PatentIn version 3.3
<210> 1
<211> 822
<212> DNA
<213 > PGRP-S1 full length gene sequence
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acatggggag tgttaaaacc gcaccatagg caaaatatgt cttcacgtac cgaggttccg 60
ttttggacgt ccgtgggaca gagtctaagg aactcgagtc gaacagagaa gatcttttgc 120
tgtgtatgct ggaccatcat actcggggct gtcgcagcca ttgtttactt actcgccttc 180
agacaacagg agtctccatc caacgtttgg aacataaccc gtgccatgtg gctcggcgcg 240
gacatcgccg gagacccggt gaagtaccgg cccttgaagc tggtcatcat caaccactcc 300
gtgtcccccg agtgcaggag cttggaaggc tgcgcacagt ccatgcgcaa ccttcagaac 360
ttcttcttga acgataaggg atgggatttg ccttacaact tcgtgatcgg caacgacggc 420
cgcgtgtacg aaggaagagg ctgggatcgc gagggggctc acacttacgg ctacaactct 480
tgcagcttgg gcgtcggatt tataggcgac taccggccag gcttcggcaa taccgtgccg 540
acatcgctgc aaatggaaag atttaaggag ctgatgcagt acggagtcct tatgggctac 600
ctagaccctg aatacgcggt ggtgggcgcc agtgacctgc agaccagcgc cagccccggc 660
gacaacctgc tcaaacaaat gaaggccggg agtcattaca accaggataa atatcgcaat 720
atgacgtgcg ctcagattta cgatcttact aagtgaattt aatactgtcg caataaatga 780
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acccgtgcca tgtggctcgg cgcggacatc gccggagacc cggtgaagta ccggcccttg 240
aagctggtca tcatcaacca ctccgtgtcc cccgagtgca ggagcttgga aggctgcgca 300
cagtccatgc gcaaccttca gaacttcttc ttgaacgata agggatggga tttgccttac 360
aacttcgtga tcggcaacga cggccgcgtg tacgaaggaa gaggctggga tcgcgagggg 420
gctcacactt acggctacaa ctcttgcagc ttgggcgtcg gatttatagg cgactaccgg 480
ccaggcttcg gcaataccgt gccgacatcg ctgcaaatgg aaagatttaa ggagctgatg 540
cagtacggag tccttatggg ctacctagac cctgaatacg cggtggtggg cgccagtgac 600
ctgcagacca gcgccagccc cggcgacaac ctgctcaaac aaatgaaggc cgggagtcat 660
tacaaccagg ataaatatcg caatatgacg tgcgctcaga tttacgatct tactaagtga 720
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Met Ser Ser Arg Thr Glu Val Pro Phe Trp Thr Ser Val Gly Gln Ser
1 5 10 15
Leu Arg Asn Ser Ser Arg Thr Glu Lys Ile Phe Cys Cys Val Cys Trp
20 25 30
Thr Ile Ile Leu Gly Ala Val Ala Ala Ile Val Tyr Leu Leu Ala Phe
35 40 45
Arg Gln Gln Glu Ser Pro Ser Asn Val Trp Asn Ile Thr Arg Ala Met
50 55 60
Trp Leu Gly Ala Asp Ile Ala Gly Asp Pro Val Lys Tyr Arg Pro Leu
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Lys Leu Val Ile Ile Asn His Ser Val Ser Pro Glu Cys Arg Ser Leu
85 90 95
Glu Gly Cys Ala Gln Ser Met Arg Asn Leu Gln Asn Phe Phe Leu Asn
100 105 110
Asp Lys Gly Trp Asp Leu Pro Tyr Asn Phe Val Ile Gly Asn Asp Gly
115 120 125
Arg Val Tyr Glu Gly Arg Gly Trp Asp Arg Glu Gly Ala His Thr Tyr
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Gly Tyr Asn Ser Cys Ser Leu Gly Val Gly Phe Ile Gly Asp Tyr Arg
145 150 155 160
Pro Gly Phe Gly Asn Thr Val Pro Thr Ser Leu Gln Met Glu Arg Phe
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Lys Glu Leu Met Gln Tyr Gly Val Leu Met Gly Tyr Leu Asp Pro Glu
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Tyr Ala Val Val Gly Ala Ser Asp Leu Gln Thr Ser Ala Ser Pro Gly
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Asp Asn Leu Leu Lys Gln Met Lys Ala Gly Ser His Tyr Asn Gln Asp
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Lys Tyr Arg Asn Met Thr Cys Ala Gln Ile Tyr Asp Leu Thr Lys
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agaggctggg atcgcgaggg ggctcacact tacggctaca actcttgcag cttgggcgtc 180
ggatttatag gcgactaccg gccaggcttc ggcaataccg tgccgacatc gctgcaaatg 240
gaaagattta aggagctgat gcagtacgga gtccttatgg gctacctaga ccctgaatac 300
gcggtggtgg gcgccagt 318
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ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45
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ctaatacgac tcactatagg gc 22
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ggggagtgtt aaaaccgcac cata 24
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tcacttagta agatcgtaaa tctg 24
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atgtgcgacg acgacgtagc cgcc 24
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cagggcgacg tagcagagct tctc 24
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gcaccatagg caaaatatgt ctt 23
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tcatcaacca ctccgtgtcc ccc 23
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aagagttgta gccgtaagtg tgag 24
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aggtgtggtg ccagatcttc 20
Claims (5)
1. a small cabbage moth peptidoglycan recognition protein PGRP-S1 gene, is characterized in that, its nucleotide sequence is as shown in SEQ ID NO:1～2.
2. a small cabbage moth peptidoglycan recognition protein PGRP-S1, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO 3.
3. the primer of pair for amplification small cabbage moth peptidoglycan recognition protein PGRP-S1 gene, is characterized in that, the nucleotide sequence of primer is as shown in SEQ ID NO:11～12.
4. a recombinant expression vector, is characterized in that, inserted the nucleotide sequence of the described small cabbage moth peptidoglycan recognition protein of claim 1 PGRP-S1 gene by the multiple clone site of the carrier that sets out.
5. a recombinant bacterial strain, is characterized in that, is by recombinant expression vector transformation receptor strain construction gained claimed in claim 4.
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