CN104845976B - Liriomyza sativae odor binding protein and application thereof - Google Patents
Liriomyza sativae odor binding protein and application thereof Download PDFInfo
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Abstract
The invention relates to a separated liriomyza sativae odor binding protein gene, an encoded protein and application thereof, wherein the protein can be combined with odor substances including 4-ethylbenzaldehyde, chamomile blue, 3, 4-dimethylacetophenone and p-ethylacetophenone, and the combination of the protein and the odor substances can be further used for preventing and controlling liriomyza sativae.
Description
Technical Field
The invention relates to the technical field of insect biotechnology, in particular to a novel odor binding protein of liriomyza sativae and application thereof.
Background
Liriomyza sativae Blanchard is a omnivorous pest, harming many host plants. Currently, the use of chemical pesticides remains an important control tool. However, the high resistance of the liriomyza pests to chemical insecticides directly led to the occurrence of the 2009- "Ducowpea" event (http:// english. peoplesaily. com. cn/90001/90776/90882) in Hainan province in China. How to continuously and effectively control the harm of the liriomyza sativae is a serious problem currently faced.
With the development of molecular biology, control by modulating chemosensory effects of target pests on host plants is a potentially effective approach. The selectivity of liriomyza sativae to hosts is mainly determined by the aversion response of adults to their host volatiles. The host plant volatile matter has obvious attraction and avoidance effects on actions such as foraging and oviposition of the liriomyza sativae. Therefore, the research on the chemosensory system of the liriomyza sativae is particularly important. The chemical sensing systems of insects mainly include olfactory and gustatory sensing systems. The olfactory sensory system is mainly present in insect olfactory receptors, and the odorant binding protein is the first type of olfactory protein that the odorant molecule contacts after entering the insect receptors, and understanding the nature and function of the odorant binding protein is crucial for understanding the olfactory sensory system of insects.
Disclosure of Invention
According to the method, the odor binding protein gene is cloned according to the transcription group result of the liriomyza sativae, the expression spectrum of the gene in the insect body is analyzed by utilizing fluorescent quantitative PCR (quantitative PCR), meanwhile, the odor binding protein is obtained through prokaryotic expression and purification, and the binding property of the main host volatile matter of the liriomyza sativae is analyzed by utilizing a fluorescent binding technology.
The invention provides the following technical scheme:
the invention provides an isolated liriomyza sativae odor binding protein gene, wherein the structure of the gene is shown as SEQ ID No. 1.
The invention also provides an isolated liriomyza sativae odor binding protein, wherein the structure of the protein is shown as SEQ ID No. 2.
The invention also provides a primer, wherein the primer can specifically amplify the Liriomyza sativae odor binding protein gene, and the sequence of the primer is shown as SEQ ID No.15 and SEQ ID No. 16.
The invention also provides the application of the isolated liriomyza sativae odor binding protein, wherein the protein is combined with odor substances.
The invention also provides application of the odorant combined with the liriomyza sativae odorant binding protein in controlling liriomyza sativae.
The present invention also provides a method for controlling liriomyza sativae, wherein the liriomyza sativae is controlled with an odorant bound to the above-mentioned liriomyza sativae odorant binding protein.
Preferably, the odorant is 2, 4-dimethylstyrene, 4-ethylbenzaldehyde, azulene, 3, 4-dimethylacetophenone, p-ethylacetophenone, methyl isonicotinate, naphthalene, 1, 4-cineole, o-isopropylbenzene, terpinolene, butyl acetate, benzaldehyde, more preferably, the odorant is 4-ethylbenzaldehyde, azulene, 3, 4-dimethylacetophenone, p-ethylacetophenone.
Preferably, the odorant is combined with the insect catching device and/or the reagent, and the liriomyza sativae is attracted to the insect catching device and/or the reagent by the odorant, so that the control is performed.
The invention obtains a purified liriomyza sativae odor binding protein by gene cloning and prokaryotic expression methods, provides materials for researching the properties and functions of the liriomyza sativae odor binding protein, detects the binding spectrum of the protein and host plant volatiles by a fluorescence binding method, and screens 12 odor molecules bound with the odor binding protein. The molecular level provides a theoretical basis for the avoidance response of the liriomyza sativae to different host volatile matters, and also provides a new idea and way for controlling the liriomyza sativae.
Drawings
FIG. 1 is a schematic diagram of 5 hydrophobic regions of an OBP protein
FIG. 2 shows the OBP gene expression levels of different tissues of male and female Liriomyza sativae adults
FIG. 3 shows the expression level of OBP in different developmental stages of liriomyza sativae
FIG. 4 construction of recombinant plasmid vector
FIG. 5 PCR amplification electrophoresis detection map (A) and cleavage of recombinant OBP plasmid (B) by electrophoresis
FIG. 6 SDA-PAGE analysis (A) and Western blot detection (B) of recombinant OBP protein expression products
FIG. 7 fluorescence characteristics of OBP combined with 1-NPN
FIG. 8 binding curves of fluorescent probe 1-NPN and OBP and Scatchard equation
Detailed Description
The technical solution of the present invention is further described with reference to the following specific examples. It will be understood that the specific embodiments described herein are shown by way of example and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
Example 1 sequencing of the Whole Length Gene of the odor binding protein OBP of Liriomyza sativae
Materials and methods
(1) Extraction of total RNA of Liriomyza sativae adults
According to (EASYspin Plus tissue/cell RNA rapid extraction kit) using instructions:
1) liquid nitrogen grinding and homogenizing: freezing the collected polypide and tissues with liquid nitrogen, fully grinding in a grinder, adding 350ul of tissue lysate, violently shaking for 20s, and transferring into a 1.5ml centrifuge tube. The lysate (sheared DNA) was aspirated through a 1ml tip.
2) After homogenization the lysate was centrifuged at 13000rpm for 3min and the whole lysate supernatant was applied to a DNA clean-up column (clean-up column placed in the collection tube).
3) Centrifuge immediately at 13000rpm for 60s, and retain filtrate (RNA in filtrate).
4) Accurately estimating the volume of the filtrate by using a micropipette, adding 70% ethanol with the same volume, and immediately sucking and uniformly mixing.
5) The mixture was immediately added to an adsorption column RA, centrifuged at 13000rpm for 30s and the waste liquid discarded.
6) Adding 700ul deproteinized solution RW1, standing at room temperature for 1min, centrifuging at 12000rpm for 30s, and discarding the waste liquid.
7) 500ul of the rinsing solution RW was added and centrifuged at 12000rpm for 30 seconds, and the waste liquid was discarded. 500ul of the rinsing solution RW was added and repeated.
8) And (4) putting the adsorption column RA back into the empty collection pipe, centrifuging at 13000rpm for 2min, and removing the rinsing liquid as much as possible so as to prevent residual ethanol in the rinsing liquid from inhibiting downstream reaction.
9) The adsorption column RA was removed and placed in an RNase free centrifuge tube, 30ul of RNase free water was added to the middle of the adsorption membrane according to the expected RNA yield (previously heated in a 70-90 ℃ water bath), and the membrane was left at room temperature for 1min and centrifuged at 12000rpm for 1 min.
(2) First Strand of synthetic cDNA
The reaction system is as follows: MgCl2(25mM)4 ul; reverse transcription 10 × Buffer 2 ul; dNTPMixture (10mM)2 ul; 0.5ul of recombined RNase lnhibitor (40U/ul); AMVReverse transfer (23U/ul)0.5 ul; oligo (dT) Primer 1 ul; total RNA 2ul, plus ddH20 to 20 ul. The reaction conditions are as follows: 45min at 42 ℃ and 5min at 95 ℃ and storing at 0-5 ℃ for later use.
(3) Amplification of intermediate fragment of OBP Gene
TABLE 1 primers used in the experiments
RT-PCR reaction system:
reaction procedure:
(4) amplification of the 3' end of the Liriomyza sativae OBP gene
Reverse transcription system:
reaction conditions are as follows:
42℃ 60min
70℃ 15min
-20℃ Hold
outer PCR reaction system:
reaction conditions are as follows:
(5) amplification of the 5' end of the liriomyza sativae OBP gene
Dephosphorizing treatment (CIAP)
Removing phosphoric acid reaction liquid:
reacting at 50 ℃ for 1 h.
To the reaction mixture were added 20ul of 3M CH3COONa (pH5.2) and 130ul of RNase Free dH20, followed by thorough mixing.
200ul phenol/chloroform/isoamyl alcohol (25: 24: 1) was added thereto, mixed well and centrifuged at 13000rpm for 5min at room temperature, and the upper aqueous phase was transferred to a new Microtube.
200ul of chloroform was added, mixed well and centrifuged at 13000rpm for 5min at room temperature, and the upper aqueous phase was transferred to a new Miorotube.
2ul of NA Carrier was added and mixed well.
Adding 200ul isopropanol, mixing, cooling on ice for 10min, centrifuging at 13000rpm at 4 deg.C for 20min, and discarding the supernatant.
The mixture was rinsed with 500ul of 70% cold ethanol (prepared in RNase Free dH 20), centrifuged at 13000rpm at 4 ℃ for 5min, and the supernatant was discarded and dried.
7ul of RNase Free dH20 was added to dissolve the precipitate to obtain CIAP-treated RNA.
"decapping" reaction (TAP)
Reaction solution for "decapping":
the reaction was carried out at 37 ℃ for 1 h.
The reaction solution was CIAP/TAP-treated RNA. 5ul was used for 5' RACE Adaptor ligation, and the remaining 5ul was stored at-80 ℃.
Ligation of 5' RACE Adaptor.
CIAP/TAP-treated RNA 5ul
5′RACE Adaptor(15uM) 1ul
RNase Free dH20 4ul
Keeping the temperature at 65 deg.C for 5min, standing on ice for 2min, and adding
Reacting at 16 ℃ for 1 h.
20ul of 3M CH3COONa (pH5.2) and 140ul of RNase Free dH20 were added to the reaction mixture, followed by thorough mixing.
200ul phenol/chloroform/isoamyl alcohol (25: 24: 1) was added thereto, mixed well and centrifuged at 13000rpm for 5min at room temperature, and the upper aqueous phase was transferred to a new Microtube.
200ul of chloroform was added thereto, and after thoroughly mixing, the mixture was centrifuged at 13000rpm for 5min at room temperature, and the upper aqueous phase was transferred to a new Microtube.
2ul of NA Carrier was added and mixed well.
Adding 200ul isopropanol, mixing, cooling on ice for 10min, separating at 13000rpm at 4 deg.C for 20min, and discarding the supernatant.
The mixture was rinsed with 500ul of 70% cold ethanol (prepared in RNase Free dH 20), centrifuged at 13000rpm at 4 ℃ for 5min, and the supernatant was discarded and dried.
6ul of RNase Free dH20 was added to dissolve the precipitate to obtain Ligated RNA.
And (5) reverse transcription reaction.
Reverse transcription system:
reaction conditions are as follows:
30℃ 10min
42℃ 1h
70℃ 15min
outer PCR reaction
Outer PCR reaction solution:
reaction conditions are as follows:
inner PCR reaction
Inner PCR reaction System:
reaction conditions are as follows:
and (3) cutting the target fragment, recovering, connecting with a vector, converting and sequencing.
The liriomyza sativae odorant binding protein OBP full-length gene (644bp) was obtained by RACE technique, in which the Open Reading Frame (ORF) is 469 bp. The base sequence (SEQ ID No.1) and the amino acid sequence (SEQ ID No.2) of the OBP were analyzed by bioinformatics, and the hydrophilicity and hydrophobicity of the amino acid sequence of the OBP were obtained according to Kyte J and Doolittle (1982) (http:// web. expasy. org/protscale /) algorithms, with positive values indicating hydrophobicity and negative values indicating hydrophilicity. Analysis found that it had 5 distinct hydrophobic regions (shown in fig. 1A-E), which 5 hydrophobic regions probably constituted the hydrophobic cavity inside the protein, associated with its binding to hydrophobic odour molecules.
Example 2 tissue expression of OBP in Liriomyza sativae
Materials and methods
Collecting female and male Liriomyza sativae adults, different tissues of the head, the chest, the abdomen and the feet and insect bodies in different development stages, freezing by liquid nitrogen, and storing at-80 ℃ for later use.
Extracting total RNA of each part and different development stages of the female and male imagoes of the liriomyza sativae, and synthesizing a first chain of cDNA. The method is as above
Primers for fluorescent quantitative PCR were designed and synthesized:
the gene specific primers of the odor binding protein of the liriomyza sativae are as follows:
YG-3S：5′-CCGACAAGTCAGACGGCAAAAA-3′(SEQ ID No.11)
YG-3A: 5'-TACGACGGGGCACAAACAAAAG-3' (SEQ ID No.12) amplified fragment length is 234bp
The specific primers of the β -actin gene as the internal reference are as follows:
actin-F3：5′-GCCCAAAGCAAAAGAGGT-3′(SEQ ID No.13)
actin-R3: 5'-GGAGCGACACGGAGTTCA-3' (SEQ ID No.14) amplified fragment length is 188bp
And (3) carrying out PCR experiments by taking the cDNA as a template, detecting PCR results by electrophoresis, and verifying whether the primer has specificity.
Fluorescent quantitative PCR
Taking cDNA of male and female imagoes of liriomyza sativae in different tissues and different development stages, and setting 3 repeats for each treatment
Reaction system:
reaction conditions are as follows:
after the reaction is finished, the amplification curve and the melting curve of the fluorescent quantitative PCR reaction are confirmed.
β -actin is used as an internal reference gene and passes through 2-ΔΔctThe method carries out relative quantitative analysis on OBP gene expression quantities of different tissues and different development stages of female and male imagoes of the liriomyza sativae, the difference significance of PCR results is analyzed by a single-factor variance analysis method of SPSS19.0 statistical software, the significance determination is tested by a Duncan's new repolarization method, and the significance test level is that P is less than 0.05.
The tissue expression specificity of OBP in the Liriomyza sativae was analyzed by fluorescent quantitative PCR. The results show that: the OBP gene is expressed in the head, the chest, the abdomen and the feet of the American Musca maculata, the head expression level is far higher than that of other tissues, compared with the head tissue (the expression level is regarded as 1) of the female, the relative expression levels of the chest, the abdomen and the foot tissues of the female are respectively 0.08, 0.06 and 0.12, the expression levels of the head, the chest, the abdomen and the foot tissues of the male are respectively 1.3, 0.1, 0.08 and 0.14, and the expression levels of all the tissues of the male are higher than that of the corresponding parts of the female (figure 2). The OBP gene also exists in the body of the worm at different development stages, and compared with the female adult (the expression quantity is regarded as 1), the OBP gene also has expression quantities of 0.15, 0.2, 0, 05, 0.04, 0.01 and 1.8 in 1-year, 2-year, 3-year larvae, preputia, pupae and male worm respectively. (see FIG. 3).
EXAMPLE 3 preparation of OBP recombinant proteins
Materials and methods
Design of primers for expressing OBP protein of liriomyza sativae
OBP-O-S：5′-GATATCGAAGAGTGGAAACGCAAGGAAT-3′(SEQ ID No.15)
OBP-O-A：5′-CTCGAGTTATTCTTCTTTCTTTTGGGCT-3' (SEQ ID No.16) (underlined in italics as cleavage site, EcoRV, Xho I cleavage site, respectively)
After PCR amplification, gel cutting, recovery and purification, overnight connection with a vector to obtain recombinant OBP, transformation into Escherichia coli competent cells Trans1-T1, electrophoresis detection of PCR amplification, and sequencing. After the sequencing is correct, the DNA is transformed into an Escherichia coli BL21(DE3) competent cell for prokaryotic expression.
Through constructing prokaryotic expression vector (figure 4, 5), IPTG induced expression and affinity chromatography purification, OBP recombinant protein with higher purity (figure 6) is obtained, figure 6 SDA-PAGE analysis (A) and Western blot detection (B) of recombinant OBP protein expression product are obtained, wherein M: standard protein molecular weight; 1, a: uninduced pET30 cells; 2, b: induced pET30 cells; 3, c: non-induced recombinant OBP protein thalli; 4, d: induced recombinant OBP protein thallus
Example 4 binding Effect of OBP with Probe N-phenyl-1-naphthylamine
OBP has a binding effect with probe N-phenyl-1-naphthylamine (N-phenyl-I-naphthalene amine, 1-NPN) (FIG. 7). With the increase of the concentration of the 1-NPN, the endogenous fluorescence intensity of the protein is reduced, the exogenous fluorescence intensity is gradually enhanced and tends to be saturated, and the saturation value is 22 umol/L. The binding constant of I-NPN and OBP is 13.06umol/L (FIG. 8).
Example 5 binding of OBP to an odorant Compound
Five favorite host plants of liriomyza sativae, namely, bean, cowpea, lettuce, white radish and cucumber leaf volatiles, were analyzed by GC-MS to obtain 34 odorous compounds.
Binding constants of 34 odor standards to recombinant OBP protein were investigated using competitive binding experiments. In the experiment, 2ml of 1nmol/L recombinant OBP protein (dissolved in 20mmol/L pH7.4Tris-HCl) was mixed with 14 umol/L1-NPN, odor standards (dissolved in methanol) of different concentrations were added stepwise, and the concentration of the bound ligand was estimated as the fluorescence intensity at the maximum emission spectrum at 350-480nm under excitation light at 337 nm. The graphs were linearized by the Scatchard method, with the abscissa representing the concentration of bound ligand and the ordinate representing the ratio of the concentration of bound ligand to the concentration of free ligand. According to [ IC ]50]Value (concentration of competing ligand capable of replacing 50% of the 1-NPN) the dissociation concentration of competing ligand is calculated as follows: kD＝[IC50]/(I+[1-NPN]/K1-NPN) Wherein [ I-NPN]Unbound I-NPN concentration; k1-NPNThe dissociation concentration of the OBP/I-NPN compound.
The results of a binding test of 34 odor samples of odor compounds obtained by GC-MS analysis on five favorite host plants of liriomyza sativae, namely beans, cowpeas, lettuce, white radishes and cucumber leaf volatiles show that 12 odor compounds have binding reaction with OBP protein, wherein 4 odor compounds of 4-ethylbenzaldehyde, chamomile blue, 3, 4-dimethylacetophenone and p-ethylacetophenone can complete 50% replacement at the concentration of 22umol/L, the binding constants of the 4 odor compounds are 11.497, 12.383, 8.639 and 20.098umol/L respectively, and the binding capacity is relatively strong.
Table 234 odor standard compound binding characteristics
Claims (5)
1. An isolated liriomyza sativae odorant binding protein gene, the sequence of which is shown in SEQ id No. 1.
2. An isolated liriomyza sativae odorant binding protein, wherein the amino acid sequence of said protein is represented by seq id No. 2.
3. A primer, wherein the primer can specifically amplify the gene of the liriomyza sativae odor binding protein, and the sequence of the primer is shown as SEQ ID No.15 and SEQ ID No. 16.
4. Use of an odorant for controlling liriomyza sativae, wherein the odorant is bound to the liriomyza sativae odorant binding protein of claim 2, wherein the odorant is 2, 4-dimethylstyrene, 4-ethylbenzaldehyde, azure, 3, 4-dimethylacetophenone, p-ethylacetophenone, methyl isonicotinate, naphthalene, 1, 4-cineole, o-isopropylbenzene, terpinolene, butyl acetate or benzaldehyde.
5. A method for controlling liriomyza sativae, characterized in that liriomyza sativae is controlled with an odorous substance bound to the liriomyza sativae odor binding protein of claim 2, wherein the odorous substance is 2, 4-dimethylstyrene, 4-ethylbenzaldehyde, chamomile blue, 3, 4-dimethylacetophenone, p-ethylacetophenone, methyl isonicotinate, naphthalene, 1, 4-cineole, o-isopropylbenzene, terpinolene, butyl acetate or benzaldehyde.
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