CN104845976A - Vegetable leafminer odorant-binding protein and application thereof - Google Patents
Vegetable leafminer odorant-binding protein and application thereof Download PDFInfo
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Abstract
The invention relates to a separated vegetable leafminer odorant-binding protein, its encoding protein and an application thereof. The protein can bind to odors matters of 4-ethyl benzaldehyde, azulene, 3,4-dimethyl acetophenone and p-ethylacetophenone, and combination of the protein and the odors matters can be used for preventing and treating vegetable leafminer.
Description
Technical field
The present invention relates to insect biological technical field, be specifically related to new odorant binding protein (OBP) and the application thereof of Americal rice leaf miner.
Background technology
Americal rice leaf miner (Liriomyza sativae Blanchard) is a kind of polyphagy insect, endangers multiple host plant.At present, chemical insecticide is used to be still important means of prevention.But Liriomyza insect is to the resistance generation that directly results in China Hainan Province 2009-2010 " malicious cowpea " event (http://english.peopledaily.com.cn/90001/90776/90882) of chemical insecticide.How continuously and effectively controlling causing harm of Americal rice leaf miner, is the severe problem of current of facing.
Along with molecular biological development, be a kind of potential effective ways by regulating target pest to prevent and treat the chemoreception of host plant.Americal rice leaf miner is mainly decided by the reaction of walking quickly and keeping away of adult to its host's volatile matter the selectivity of host.Host plant volatile has the looking for food of Americal rice leaf miner, the behavior such as to lay eggs lures and avoidance effect significantly.Therefore, particularly important to the research of Americal rice leaf miner chemosensing system.The chemosensing system of insect mainly comprises sense of smell and taste perception system.Nasal receptor system is mainly present in insect olfactory receptor, odorant binding protein (OBP) is the first kind sense of smell albumen contacted after scent molecule enters insect susceptor, and character and the function of understanding odorant binding protein (OBP) are most important to the nasal receptor system understanding insect.
Summary of the invention
The present invention has cloned odorant binding protein (OBP) gene according to Americal rice leaf miner transcript profile result, quantitative fluorescent PCR (quantitative PCR) is utilized to analyze the express spectra of gene in polypide, obtain odorant binding protein (OBP) by prokaryotic expression, purifying, application fluorescence combination technology analyzes the binding characteristic of Americal rice leaf miner main host plants volatile matter simultaneously.
The invention provides following technical scheme:
The invention provides a kind of Americal rice leaf miner odorant binding protein (OBP) gene of separation, wherein, the structure of described gene is as shown in SEQ ID No.1.
The present invention also provides a kind of Americal rice leaf miner odorant binding protein (OBP) of separation, and wherein, the structure of described albumen is as shown in SEQ ID No.2.
The present invention also provides a kind of primer, and the specific amplification Americal rice leaf miner odorant binding protein (OBP) gene of wherein said primer energy, the sequence of described primer is as shown in SEQ ID No.15 and 16.
The present invention also provides a kind of application of Americal rice leaf miner odorant binding protein (OBP) of above-mentioned separation, and wherein, described albumen and odoring substance combine.
The odoring substance that the present invention also provides a kind of and above-mentioned Americal rice leaf miner odorant binding protein (OBP) to be combined is preventing and treating the application in Americal rice leaf miner.
The present invention also provides a kind of prevention and controls of Americal rice leaf miner, wherein, prevents and treats Americal rice leaf miner with the odoring substance be combined with above-mentioned Americal rice leaf miner odorant binding protein (OBP).
Preferably, described odoring substance is 2,4-dimethyl styrene, 4-ethylbenzene formaldehyde, azulene, 3,4-dimethyl acetophenone, p-ethylacetophenone, iso methyl nicotinate, naphthalene, 1,4-cineole, o-isopropyl benzene, terpinolene, N-BUTYL ACETATE, phenyl aldehyde, preferred, described odoring substance is 4-ethylbenzene formaldehyde, azulene, 3,4-dimethyl acetophenones, p-ethylacetophenone.
Preferably, above-mentioned odoring substance is combined with pest-catching device and/or Reagent evaluation, utilizes odoring substance Americal rice leaf miner to be attracted to pest-catching device and/or reagent place, thus prevents and treats.
The present invention obtains a kind of Americal rice leaf miner odorant binding protein (OBP) of purifying by the method for gene clone and prokaryotic expression, for the character and function of studying the odorant binding protein (OBP) that America spot is dived provide material, and have detected the bind profile of this albumen and host plant volatile by the method that fluorescence combines, filter out combine with odorant binding protein (OBP) of the present invention 12 in scent molecule.From molecule aspect for Americal rice leaf miner provides theoretical foundation to the reaction of walking quickly and keeping away that different host's volatile matter is made, also for control Americal rice leaf miner provides new thinking and approach.
Accompanying drawing explanation
Fig. 1 is 5 water repellent region schematic diagram of OBP albumen
Fig. 2 is Americal rice leaf miner female male imago different tissues OBP gene expression amount
Fig. 3 Americal rice leaf miner different developmental phases OBP expression amount
Fig. 4 recombinant plasmid vector design of graphics
Fig. 5 pcr amplification electrophoresis detection figure (A) and restructuring OBP plasmid enzyme restriction (B) electrophoretic analysis
Fig. 6 recombinate OBP protein expressioning product SDA-PAGE analyze (A) and western blot detection (B)
The fluorescent characteristics that Fig. 7 OBP and 1-NPN combines
The binding curve of Fig. 8 fluorescent probe 1-NPN and OBP and Scathard equation
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is further described.Be understandable that, particular implementation described here represents by way of example, and it is not as limitation of the present invention.When not deviating from the scope of the invention, principal character of the present invention may be used for various embodiment.One skilled in the art will appreciate that and maybe can confirm, only use normal experiment, many equivalents can be applied in particular step described herein.These equivalent places of being considered within the scope of the present invention, and cover by claim.
Embodiment 1 Americal rice leaf miner odorant binding protein (OBP) OBP full-length gene checks order
Materials and methods
(1) Adult of Liriomyza Sativae Blanchard total serum IgE is extracted
Carry out according to (EASYspin Plus tissue/cell RNA rapid extraction test kit) operation instruction:
1) liquid nitrogen grinding homogenate: by the polypide of collection and each tissue juice chilled nitrogen, fully grind in beveller, add 350ul Tissue lysates, thermal agitation 20s, proceed in 1.5ml centrifuge tube.1ml rifle head lashes lysate (shearing DNA).
2) the centrifugal 3min of lysate 13000rpm after homogenate, is all added to DNA and removes (removing post is placed in collection tube) on post by lysate supernatant.
3) the centrifugal 60s of 13000rpm at once, retains filtered liquid (RNA is in filtered solution).
4) more accurately estimate filtered solution volume with micropipet, add isopyknic 70% ethanol, inhale immediately and play mixing.
5) added by mixture at once in an adsorption column RA, the centrifugal 30s of 13000rpm, discards waste liquid.
6) add 700ul protein liquid removal RW1, room temperature places the centrifugal 30s of 1min, 12000rpm, discards waste liquid.
7) add 500ul rinsing liquid RW, the centrifugal 30s of 12000rpm, discards waste liquid.Add 500ul rinsing liquid RW, come again.
8) put back in sky collection tube by adsorption column RA, the centrifugal 2min of 13000rpm, removes rinsing liquid as far as possible, in order to avoid residual ethanol suppresses downstream reaction in rinsing liquid.
9) adsorption column RA is taken out, put into a RNase free centrifuge tube, add 30ul RNase free water (heating in 70-90 DEG C of water-bath in advance) according to expection RNA output in the middle part of adsorption film, room temperature places the centrifugal 1min of 1min, 12000rpm.
(2) cDNA first chain is synthesized
Reaction system is: MgCl2 (25mM) 4ul; Reverse Transcriptio 10 × Buffer 2ul; DNTP Mixture (10mM) 2ul; Recombinant Rnasin Ribonuc leaselnhibitor (40U/ul) 0.5ul; AMV Reverse Transcription (23U/ul) 0.5ul; Oligo (dT) Primer 1ul; Total serum IgE 2ul, adds ddH20 to 20ul.Reaction conditions is: 42 DEG C of 45min, 95 DEG C of 5min, be stored in 0-5 DEG C for subsequent use.
(3) amplification of OBP gene intermediate segment
The primer in table 1 test
RT-PCR reaction system:
Response procedures:
(4) Americal rice leaf miner OBP gene 3 ' end amplification
Reverse transcription system:
Reaction conditions:
42℃ 60min
70℃ 15min
-20℃ Hold
Outer PCR reaction system:
Reaction conditions:
(5) Americal rice leaf miner OBP gene 5 ' end amplification
Dephosphorylation process (CIAP)
Dephosphorization acid-respons liquid:
50 DEG C of reaction 1h.
In above-mentioned reaction solution, add the 3M CH3COONa (pH5.2) of 20ul, after the RNaseFree dH20 of 130ul, fully mix.
Add the phenol/chloroform/primary isoamyl alcohol (25: 24: 1) of 200ul, fully the centrifugal 5min of 13000rpm room temperature after mixing, by upper water phase transition in new Microtube.
Add the chloroform of 200ul, fully the centrifugal 5min of 13000rpm room temperature after mixing, by upper water phase transition in new Miorotube.
Add Homogeneous phase mixing after the NA Carrier of 2ul.
Add the Virahol of 200ul, fully after mixing, cooled on ice 10min, 13000rpm 4 DEG C of centrifugal 20min, abandon supernatant.
Add 70% cold ethanol (the RNase Free dH20 prepares) rinsing of 500ul, 13000rpm 4 DEG C of centrifugal 5min, dry after abandoning supernatant.
Add the RNase Free dH20 dissolution precipitation of 7ul, obtain CIAP-treated RNA.
" remove cap " and react (TAP)
The reaction solution that " removes cap ":
37 DEG C of reaction 1h.
This reaction solution is CIAP/TAP-treated RNA.Get 5ul for 5 ' RACE Adaptor ligation, remaining 5ul is stored in-80 DEG C.
The connection of 5 ' RACE Adaptor.
CIAP/TAP-treated RNA 5ul
5′RACE Adaptor(15uM) 1ul
RNase Free dH20 4ul
Place 2min on ice after 65 DEG C of insulation 5min, then add
16 DEG C of reaction 1h.
In above-mentioned reaction solution, add the 3M CH3COONa (pH5.2) of 20ul, after the RNase FreedH20 of 140ul, fully mix.
Add the phenol/chloroform/primary isoamyl alcohol (25: 24: 1) of 200ul, fully the centrifugal 5min of 13000rpm room temperature after mixing, by upper water phase transition in new Microtube.
Add the chloroform of 200ul, fully the centrifugal 5min of 13000rpm room temperature after mixing, by upper water phase transition in new Microtube.
Add Homogeneous phase mixing after the NA Carrier of 2ul.
Add the Virahol of 200ul, fully after mixing, cooled on ice 10min, 13000rpm 4 DEG C, from 20min, abandons supernatant.
Add 70% cold ethanol (the RNase Free dH20 prepares) rinsing of 500ul, 13000rpm 4 DEG C of centrifugal 5min, dry after abandoning supernatant.
Add the RNase Free dH20 dissolution precipitation of 6ul, obtain Ligated RNA.
Reverse transcription reaction.
Reverse transcription system:
Reaction conditions:
30℃ 10min
42℃ 1h
70℃ 15min
Outer PCR reacts
Outer PCR reaction solution:
Reaction conditions:
Inner PCR reacts
Inner PCR reaction system:
Reaction conditions:
Target fragment reclaims through cutting glue, connection carrier, order-checking after transforming.
Americal rice leaf miner odorant binding protein (OBP) OBP full-length gene (644bp) is obtained, wherein open reading frame (ORF) 469bp by RACE technology.By bioinformatics method, its base sequence (SEQ IDNo.1) and aminoacid sequence (SEQ ID No.2) are analyzed, the hydrophilic and hydrophobic of the aminoacid sequence of Americal rice leaf miner OBP obtains according to Kyte J and Doolittle (1982) (http://web.expasy.org/protscale/) algorithm, hydrophobic on the occasion of expression, negative value represents hydrophilic.Analyze and find that it has 5 obvious water repellent regions (shown in Figure 1A-E), the hydrophobic pocket of these 5 water repellent regions possibility constitutive protein inside, relevant in conjunction with hydrophobic scent molecule with it.
The tissue expression of embodiment 2OBP in Americal rice leaf miner body
Materials and methods
Collect the female male imago of Americal rice leaf miner and head, chest, belly, foot different tissues, different developmental phases polypide, liquid nitrogen freezing is placed on-80 DEG C and stores for future use.
Extract each position of the female male imago of Americal rice leaf miner and different developmental phases total serum IgE, and synthesize cDNA first chain.Method is the same
Design and synthesize the primer for quantitative fluorescent PCR:
Americal rice leaf miner odorant binding protein (OBP) gene-specific primer is:
YG-3S：5′-CCGACAAGTCAGACGGCAAAAA-3′(SEQ ID No.11)
YG-3A:5 '-TACGACGGGGCACAAACAAAAG-3 ' (SEQ ID No.12) expanding fragment length is 234bp
β-actin gene-specific primer as internal reference is:
actin-F3：5′-GCCCAAAGCAAAAGAGGT-3′(SEQ ID No.13)
Actin-R3:5 '-GGAGCGACACGGAGTTCA-3 ' (SEQ ID No.14) expanding fragment length is 188bp
Be that template carries out PCR experiment, electrophoresis detection PCR result with cDNA, verify whether this primer has specificity.
Quantitative fluorescent PCR
Get the female male imago different tissues of Americal rice leaf miner, different developmental phases polypide cDNA, each process sets 3 repetitions
Reaction system:
Reaction conditions:
Reaction terminates amplification curve and the melting curve of the reaction of rear confirmation quantitative fluorescent PCR.
With β-actin for reference gene, by 2
-Δ Δ ctmethod carries out relative quantitative assay to the female male imago different tissues of Americal rice leaf miner, different developmental phases OBP gene expression amount, the one-way analysis of variance method of the significance of difference application SPSS19.0 version statistical software of PCR result is analyzed, significance test adopts the inspection of Deng Kenshi duncan's new multiple range method, and level of significance test is P < 0.05.
Quantitative fluorescent PCR is utilized to analyze the tissue expression specificity of OBP in Americal rice leaf miner body.Result shows: OBP gene all has expression in Americal rice leaf miner head, chest, abdomen, foot, head expression amount is far away higher than its hetero-organization, compared with the head tissue (expression amount regards 1 as) of female worm, the chest of female worm, the relative expression quantity in abdomen and foot tissue is respectively 0.08, the head of 0.06 and 0.12 male worm, chest, the expression amount of abdomen and foot tissue is respectively 1.3, and 0.1,0.08 and 0.14, the expression amount of male worm each several part tissue is all higher than female worm corresponding site (Fig. 2).Also there is expression in OBP gene in the polypide of different developmental phases, compared with female adult worm (expression amount regards 1 as), 1 age, 2 ages, 3 instar larvaes, prepupa, in pupa and male worm, expression amount is respectively 0.15,0.2,0,05,0.04,0.01 and 1.8, in a word, in adult, expression amount is relatively large, and male worm expression amount is also high than female worm expression amount simultaneously.(see Fig. 3).
Embodiment 3 prepares OBP recombinant protein
Materials and methods
Americal rice leaf miner OBP protein expression design of primers
OBP-O-S：5′-
GATATCGAAGAGTGGAAACGCAAGGAAT-3′(SEQ ID No.15)
OBP-O-A:5 '-
cTCGAGtTATTCTTCTTTCTTTTGGGCT-3 ' (SEQ ID No.16) (italic underlines part for restriction enzyme site, is respectively EcoRV, Xho I restriction enzyme site)
After pcr amplification, cut glue and reclaim purifying, spend the night be connected with carrier, obtain the OBP recombinated, be transformed in competent escherichia coli cell Trans1-T1, pcr amplification, through row electrophoresis detection, then checks order.Be transformed in e. coli bl21 (DE3) competent cell after order-checking is correct and carry out prokaryotic expression.
By building prokaryotic expression carrier (Fig. 4,5), IPTG abduction delivering, affinitive layer purification obtain higher OBP recombinant protein (Fig. 6) Fig. 6 of purity recombinate OBP protein expressioning product SDA-PAGE analyze (A) and western blot detection (B), wherein M: standard protein molecular weight; 1, a: without the pET30 thalline of induction; 2, b: the pET30 thalline of induction; 3, c: without the restructuring OBP albumen thalline of induction; 4, d: the restructuring OBP albumen thalline of induction
Embodiment 4OBP and probe N-phenyl-1-naphthylamine in conjunction with effect
OBP and probe N-phenyl-1-naphthylamine (N-phenyl-I-naphthy lamine, 1-NPN) have in conjunction with effect (Fig. 7).Along with 1-NPN increasing concentrations, Intrinsic protein fluorescence intensity reduces, and external source fluorescence intensity strengthens gradually, and is tending towards saturated, and its saturation value is 22umol/L.I-NPN and OBP binding constant is 13.06umol/L (Fig. 8).
The combination of embodiment 5OBP and odor compound
By GC-MS analysis, 34 kinds of odor compounds are obtained to five kinds of hobby host plant Kidney beans, cowpea, romaine lettuce, white turnip and cucumber leaves volatile matters of Americal rice leaf miner.
Competitive binding experiment is utilized to study the binding constant of 34 kinds of smell standard specimens and restructuring OBP albumen.In an experiment, be that the restructuring OBP albumen (being dissolved in 20mmol/L pH7.4Tris-HCl) of 1nmol/L mixes with 14umol/L 1-NPN by 2ml concentration, progressively add the smell standard specimen (being dissolved in methyl alcohol) of different concns, under 337nm exciting light, estimate combined ligand concentra with the fluorescence intensity at emission maximum spectrum place of 350-480nm place.Be figure with the linearizing of Scatchard method, X-coordinate is the concentration of combined aglucon, and ordinate zou is the concentration of combined aglucon and the ratio of free ligand concentra.According to [IC
50] to calculate the concentration formula of dissociating of competition part as follows for value (concentration when competition aglucon can replace the 1-NPN of 50%): K
d=[IC
50]/(I+ [1-NPN]/K
1-NPN), wherein [I-NPN] is in conjunction with I-NPN concentration; K
1-NPNfor the concentration of dissociating of OBP/I-NPN mixture.
To five kinds of hobby host plant Kidney beans of Americal rice leaf miner, cowpea, romaine lettuce, white turnip and cucumber leaves volatile matter analyze 34 kinds of smell standard specimens of the odor compound obtained binding tests result by GC-MS shows 12 kinds of odor compounds and OBP albumen has association reaction, wherein 4-ethylbenzene formaldehyde, azulene, 3, 4-dimethyl acetophenone, p-ethylacetophenone 4 kinds of odor compounds can complete the replacement of 50% under 22umol/L concentration, the binding constant of these 4 kinds of odor compounds is respectively 11.497, 12.383, 8.639 and 20.098umol/L, binding ability is relatively strong.
Table 2 34 kinds of odor standards compound binding characteristics
Claims (6)
1. the Americal rice leaf miner odorant binding protein (OBP) gene be separated, it is characterized in that, the structure of described gene is as shown in SEQ ID No.1.
2. the Americal rice leaf miner odorant binding protein (OBP) be separated, it is characterized in that, the structure of described albumen is as shown in SEQ ID No.2.
3. a primer, the specific amplification Americal rice leaf miner odorant binding protein (OBP) gene of wherein said primer energy, the sequence of described primer is as shown in SEQ ID No.15 and 16.
4. an application for Americal rice leaf miner odorant binding protein (OBP) according to claim 2, is characterized in that, described albumen and odoring substance combine.
5. the application of odoring substance in control Americal rice leaf miner, it is characterized in that, described odoring substance is combined with Americal rice leaf miner odorant binding protein (OBP) according to claim 2.
6. a prevention and controls for Americal rice leaf miner, is characterized in that, prevents and treats Americal rice leaf miner with the odoring substance be combined with Americal rice leaf miner odorant binding protein (OBP) according to claim 2.
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