CN116769786A - Migratory locust smell binding protein LmOBP11 gene and encoding protein and application thereof - Google Patents
Migratory locust smell binding protein LmOBP11 gene and encoding protein and application thereof Download PDFInfo
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Abstract
The invention discloses a migratory locust smell binding protein LmOBP11 gene and a coding protein and application thereof. The nucleotide sequence of the LmOBP11 gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO.2; the LmOBP11 gene can regulate the immunity of insects, can reduce the immunity of the migratory locust to the metarhizium anisopliae by activating the expression of the LmOBP11 gene, and can better prevent and control the migratory locust by particularly mixing the phenethyl alcohol and the metarhizium anisopliae, thereby improving the protection of crops.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a migratory locust smell binding protein LmOBP11 gene, and a coding protein and application thereof.
Background
Insects rely on smell to find food, habitat, spouse, etc. The olfactory recognition process of insects is considered to modulate the behavioral response of insects by binding a class of compounds highly expressed in the olfactory tissues of insects to related proteins, particularly odor-binding proteins (odorant binding proteins, OBPs) and chemosensory proteins (chemosensory proteins, CSPs), recognizing small molecular compounds such as lipids, alcohols, fatty acids, etc., which enter the olfactory lymph and then carrying them to olfactory receptors (odorant receptors, ORs). However, more and more researches find that some olfactory proteins are widely expressed in insect immune tissues including blood cells and fat bodies besides being highly expressed in olfactory tissues, and infection of pathogenic microorganisms and colonization of symbiotic bacteria can cause significant differential expression of insect olfactory related proteins. This suggests that insect olfactory proteins may play an important role in immune regulation in addition to play a role in behavioral regulation.
The entomopathogenic fungi has important application potential in green prevention and control of pests, but has the defects of low insecticidal speed, unstable prevention effect and the like. The slow insecticidal speed and unstable control effect are influenced by various factors such as fungus toxicity, environmental factors, host defense system and the like. In one aspect, some insects are believed to be able to modulate their evasion behavior by recognizing volatile compounds released by fungi. Based on the binding capacity of the insect odor binding protein and the fungal compound, the insect odor binding protein can be used for screening key compounds for regulating the insect repellent behavior, so that the insect repellent is developed. On the other hand, by studying the role of olfactory proteins in regulating the immunological interaction of insects with pathogenic fungi, it is expected to develop a highly potent fungal pesticide acting on pest olfactory proteins. The migratory locust (Locusta migratoria) is an important crop pest, and once the migratory locust disaster occurs, a large amount of migratory locust can swallow the cereal field, so that agricultural products are completely destroyed, and serious economic loss is caused, so that food is in shortage. Metarrhizium anisopliae (M.anitopliae) is a broad-spectrum entomopathogenic bacteria, and is counted to have more than 200 host insects, and can parasitize golden tortoise shells, weevils, golden worms, lepidopteran pest larvae, hemipteran stink bugs and the like. The metarhizium anisopliae is harmless to human and livestock, safe to natural enemy insects and free from environmental pollution. However, the metarhizium anisopliae also has the defects of low insecticidal speed, unstable control effect and the like, and has important significance for enhancing the virulence of metarhizium anisopliae to migratory locust. The method for obtaining the migratory locust to avoid and efficiently kill the insect fungi is a long-acting and efficient method for resisting the insect pests by regulating the interaction mechanism of the insect and the pathogenic fungi based on the insect olfactory protein. The invention is based on the novel application of pest olfactory protein (locusta migratory locust LmOBP11 protein) for regulating the interaction of insects (locusta migratory locust) and pathogenic fungi (metarhizium anisopliae) to obtain olfactory protein, and a method for enhancing the toxicity of metarhizium anisopliae to the locusta migratory locust is obtained.
Disclosure of Invention
In view of the above, it is an object of the present invention to provide a migratory locust odor binding protein LmOBP11 gene, characterized in that the nucleotide sequence of the LmOBP11 gene has a sequence similarity of 90% or more with SEQ ID NO. 1.
Preferably, the base sequence of the LmOBP11 gene is identical to the sequence of SEQ ID NO. 1.
The second object of the present invention is to provide the protein coded by the migratory locust smell binding protein LmOBP11 gene, which is characterized in that the amino acid sequence of the protein of the LmOBP11 gene has the sequence similarity of more than 80% with that of SEQ ID NO. 2.
Preferably, the protein amino acid sequence of the LmOBP11 gene is identical to the sequence of SEQ ID NO. 2.
Because of more migratory grasshopper species, the sequences of the genes in different migratory grasshoppers are not completely identical, so that the gene sequences with high similarity (more than 90% and 80% of similarity respectively) with the sequence of SEQ ID NO.1 and the sequence of SEQ ID NO.2 are the genes.
The invention further aims to provide application of the migratory locust smell binding protein LmOBP11 gene in regulating insect immunity. The LmOBP11 gene has the nucleotide sequence with the sequence similarity of more than 90% with SEQ ID NO.1 and the amino acid sequence with the sequence similarity of more than 80% with SEQ ID NO.2, and has the function of regulating the immunity of insects.
Preferably, the LmOBP11 gene is applied to reducing the immunity of the migratory locust to the metarhizium anisopliae. The base sequence of LmOBP11 gene is identical to SEQ ID NO.1, and the amino acid sequence is identical to SEQ ID NO. 2.
Preferably, the LmOBP11 gene is mixed with phenethyl alcohol (PEA) in Metarrhizium anisopliae used in applications for reducing the immunity of Metarrhizium anisopliae to Metarrhizium anisopliae. By adding phenethyl alcohol, the concentration of phenethyl alcohol is even as low as 0.000001mol/L, the expression of the locusts LmOBP11 gene can be induced, so that the expression of immune effect genes (safensin) is reduced, and finally the immunity of the locusts to the metarhizium anisopliae is reduced, thereby preventing and controlling the locusts.
Preferably, the concentration of the phenethyl alcohol used is less than 1mol/L, so that the normal growth of the metarhizium anisopliae is not affected.
The fourth object of the present invention is to provide a method for combating migratory locust, which achieves the effect of combating by using a mixture of phenethyl alcohol and metarhizium anisopliae in the place where it is needed to combat. The mixture of phenethyl alcohol and metarhizium anisopliae can be sprayed on crops, and can also be added into soil or prepared into other preparations so as to defend migratory locust.
Preferably, the concentration of the phenethyl alcohol is less than 1mol/L, so that the normal growth of the metarhizium anisopliae is not affected. The method for spraying the mixture of the phenethyl alcohol and the metarhizium anisopliae is simple and high in repeatability, can be carried out regularly, and can effectively resist migratory locust. The migratory locust has obvious avoidance behavior on the phenethyl alcohol, the minimum avoidance concentration is 0.01mol/L, when the migratory locust is specifically used, the concentration of the phenethyl alcohol is less than 1mol/L, if the concentration of the phenethyl alcohol is high (0.01-1 mol/L), the LmOBP11 gene is combined with the phenethyl alcohol to lead the migratory locust to avoid, crops are protected, the low concentration (< 0.01 mol/L) can still induce the expression of the LmOBP11 gene of the migratory locust, thereby reducing the immunity of the migratory locust to the metarhizium anisopliae and having higher mortality.
The migratory locust smell binding protein LmOBP11 gene has a sequence shown as SEQ ID NO.1 or SEQ ID NO.2, and the protein of the gene is used for binding smell molecule phenethyl alcohol so as to generate evasion, and the phenethyl alcohol can activate the expression of the gene, and the expression of the gene can reduce the expression of the fensin gene so as to reduce the immunocompetence of the migratory locust on the metarhizium anisopliae, improve the resistance of the migratory locust, improve the protection of crops and reduce the loss of the crops.
Drawings
FIG. 1 is a diagram showing RNAi efficiency analysis of LmOBP11 gene of the present invention;
FIG. 2 is a diagram showing expression and purification of the LmOBP11 protein of the present invention;
FIG. 3 is a graph showing the binding analysis of LmOBP11 protein to odor molecules according to the present invention;
FIG. 4 is a graph showing the electrophysiological response of migratory locust to phenethyl alcohol and a graph showing the evasion analysis;
FIG. 5 is a graph showing the analysis of the resistance of LmOBP11 silencing migratory locust to metarhizium anisopliae infection;
FIG. 6 is a graph showing the effect of LmOBP11 silencing on the expression of immune effector (defensin) genes in different migratory locust tissues after fungal infection according to the present invention;
FIG. 7 is a graph showing the effect of phenethyl alcohol on LmOBP11 gene expression and the effect of phenethyl alcohol and M.anitopliae alone or in combination on migratory locust survival according to the present invention.
Detailed Description
The present invention will be described in detail below with reference to examples and drawings, which are for illustrative purposes only and are not limiting to the scope of application of the present invention. The present invention is not limited to the following embodiments or examples, and modifications and variations made without departing from the spirit of the present invention are intended to be included in the scope of the present invention.
Experimental example 1: cloning and bioinformatic identification of the migratory locust odor binding protein LmOBP11 Gene
Total RNA of the migratory locust was extracted according to Trizol reagent (Takara, japan) and reverse transcribed into cDNA using Reverse transcription kit (Thermofisher, USA). The cDNA of migratory locust is used as a template, and the ORF of the LmOBP11 gene is amplified by PCR. The primer pairs for PCR amplification are as follows:
forward primer: 5'-TCGCCTACCGTCGCCACCAT-3'
Reverse primer: 5'-CAACGTATTCTTCAAATTCTCTTGA-3'
The PCR amplification reaction system is 2000 ng/. Mu.L cDNA template 1. Mu.L, 2 Xgold plate mix (green) 25. Mu.L, 10. Mu.M forward primer 2. Mu.L, 10. Mu.M reverse primer 2. Mu.L, RNase Free ddH2O supplemented to 50. Mu.L; the reaction procedure is 98 ℃ for 3min;98℃10s,57℃15s,72℃30s,35 cycles; 72℃for 5min and 4 ℃.
The fragment obtained by PCR amplification is subcloned into pUC19-T vector and sequenced to obtain the locusta migratory odor binding protein LmOBP11 gene sequence, namely SEQ ID NO.1, the corresponding LmOBP11 protein sequence is SEQ ID NO.2, the LmOBP11 protein is a protein composed of 155 amino acids, the protein molecular weight is 16.43kDa, the isoelectric point is 4.56, and the protein contains a signal peptide 20aa.
Experimental example 2: rnai silencing of the LmOBP11 gene from migratory locust
dsRNA gene silencing target sequences of LmOBP11 were designed based on specificity and RNAi efficiency analysis, and dsRNA primer sequences 5'-AGCGATGTAAAAGCCTGCAT-3',5'-AGCAGTTGGCAACGATGACT-3' were designed. dsRNA of LmOBP11/GFP was synthesized using MEGAscript transcription kit (Ambion, austin, TX, USA). Briefly, dsRNA templates were amplified by PCR and dsRNA was purified using a gel purification kit. And (3) carrying out in vitro transcription and reverse transcription on the dsRNA template and a corresponding reverse transcription reagent to obtain the dsRNA. The dsRNA product was precipitated using LiCl solution. The DNA template in the product was digested with DNase and phenol/chloroform extraction was performed to purify the target product. The obtained dsRNA concentration was calculated by a NanoVue Plus spectrophotometer (GE Healthcare Life Sciences, little chanfont, uk). mu.L of dsRNA solution (300 ng/. Mu.L) was injected into the blood lymph of migratory locust. To test the knockdown efficiency of dsRNA, five migratory grasses were randomly selected 3 days and 7 days after dsRNA injection, respectively, and their tentacles were dissected to analyze the expression level of the target gene compared to the control treatment. As shown in fig. 1, RNAi efficiency results for LmOBP11 in GFP dsRNA treatment and LmOBP11 dsrna+m.anitopliae (metarhizium anisopliae) -treated migratory locust were: the transcript level of LmOBP11 was reduced to about 10% of the control group 3 days and 7 days after injection, indicating that the LmOBP11 gene was effectively silenced.
Experimental example 3: lmOBP11 is used for identifying migratory locust and avoiding related smell substances
1. Expression and purification of recombinant LmOBP11 protein
LmOBP11 open reading frame will be cloned into pET32a vector (Novagen, darmstadt, germany) after removal of the corresponding signal peptide sequence and transformed into E.coli BL21 strain. The backbone vector of the recombinant vector is not particularly limited, and expression vectors well known in the art may be used. Single positive colonies grown on LB agar plates (100. Mu.g/mL, ampicillin) were selected and grown in 5mL LB liquid medium (100. Mu.g/mL, ampicillin) with shaking at 220rpm overnight at 37 ℃. The cell culture was inoculated in 500mL LB/ampicillin liquid medium until the OD (600 nm) reached 0.6 to 0.8. Then, 0.5mM IPTG was added to the culture and the mixture was shaken overnight at 220rpm/min at 11℃to induce protein expression. Cell pellets were collected by centrifugation at 3,000rpm and resuspended in HEPES buffer (10 mM HEPES, 100mM NaCl, pH 7.5). After sonication and centrifugation at 14,000rpm and 4 ℃ for 30 minutes, the supernatant and pellet were collected, respectively. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that the recombinant protein was soluble (FIG. 2 (A)). The protein was purified using His-trap affinity column (Cobalt Chelating Resin, G-Biosciences, USA). Bound protein was eluted with HEPES buffer containing increasing imidazole from 50mM to 500 mM. After electrophoretic analysis, the solutions containing the recombinant proteins were combined and dialyzed 3 times against 3L HEPES buffer at 4℃overnight. His-tag antibodies (Thermofisher, USA) were used to perform western blots to further confirm the size of the recombinant protein, if the protein size matches the theoretical value as shown in FIG. 2 (B).
2. Binding experiments of recombinant LmOBP11 protein on odor molecules
The binding of LmOBP11 protein to odor molecules (i.e., 6 Metarrhizium anisopliae volatile compounds: 2-ethoxyethanol, ethylene glycol acetate, phenethyl alcohol, 2-n-hexyl acid, n-hexadecanoic acid, and 2, 4-di-tert-butylphenol) was detected using recombinant LmOBP11 protein and performed using fluorescent competitive binding. The fluorescence competitive binding experiment is used for pre-determining the applicability analysis of the LmOBP11 and the fluorescent probe 1-NPN, and the saturation effect exists in the fluorescence values of the LmOBP11 and the 1-NPN along with the increase of the concentration of the 1-NPN, so that the LmOBP11 has a single binding site and can be used for the determination of the fluorescence competitive binding force, and the binding constant K1-NPN=11.62 mu M of the LmOBP11 and the 1-NPN. Competition binding experiments with 1-NPN as fluorescent probe for ligand-odor molecules showed that LmOBP11 binds most strongly to 2-ethoxyethanol (ki=3.62 μm), followed by ethylene glycol acetate (ki=7.23 μm) and phenethyl alcohol (ki=9.37 μm), 2-n-hexyl acid (ki=10.76 μm), n-hexadecanoic acid (ki=15.37 μm), and the weakest binding capacity to 2, 4-di-tert-butylphenol (ki=20.58 μm) (fig. 3).
3. Equipped locust p-phenethyl alcohol recognition capability experiment based on antenna electrophysiological analysis
The migratory locust antenna was cut off and inserted into a glass capillary with two silver electrodes (recording electrode and reference electrode) and the EAG response was analyzed by the EAG system (Syntech, netherlands). The amplified electrophysiological signal was recorded by a Syntech PC-based signal processing system (Kirchzarten, germany). In the dose effect experiments, five different concentrations of phenethyl alcohol (10 -4 、10 -3 、10 -2 0.1 and 1 mol/L), each dose was tested at each antenna every 1 minute for five times. In each group, at least 5 migratory grasses were analyzed for their different tentacles, respectively. The results showed that LmOBP11 silences migratory locust against different concentrations (10 -4 The electrophysiological response capacity to mol/L-0.1 mol/L) of 2-phenylethanol was significantly reduced, but the electrophysiological response to 1mol/L phenylethanol was not significantly different from that of the control (FIG. 4A).
4. Experiment of migratory locust avoidance behavior
Each closed square cage contained 15 migratory grasses, and a dish of PEA+wheat bran (1 mL of ethanol-dissolved 0.01mol/L PEA solution was mixed with 1.5g of wheat bran) and a dish of 1mL of ethanol+1.5 g of wheat bran were placed on the diagonal line of the cage, respectively, and after eating for 12 hours in the dark, the food consumption was calculated, respectively. The results show that the migratory locust of the control group has obvious avoidance behavior on the phenethyl alcohol, the minimum avoidance concentration is 0.01mol/L, and the avoidance capability of LmOBP11 for silencing the migratory locust on the phenethyl alcohol is obviously reduced compared with that of the control group (figure 4B).
Example 4: role of LmOBP11 in modulating the immune response of migratory locust
1. LmOBP11 RNAi can improve survival rate of migratory locust under metarhizium anisopliae infection
LmOBP11 RNAi migratory locust was inoculated with Metarrhizium anisopliae spores 3 days after dsRNA injection, and survival rates were calculated every 12 hours. The results showed that without inoculating the metarhizium anisopliae spores, lmOBP11 RNAi did not affect the survival rate of migratory locust (GFP RNAi as control) (fig. 5A), whereas inoculating metarhizium anisopliae spores significantly improved the survival rate of migratory locust in LmOBP11 RNAi group (fig. 5A) and prolonged the half-lethal time by about 0.5 days (fig. 5B) compared to metarhizium anisopliae infection group alone.
2. Quantitative PCR analysis of LmOBP11 silencing immune effector Gene of migratory locust (safensin)
GFP RNAi, GFP RNAi+M.anisopliae, lmOBP11 RNAi, lmOBP11 RNAi+M.anisopliae treatment was performed on migratory locust, GFP RNAi group was used as control, and quantitative PCR experiments were performed to detect the expression of immune effector gene (safenin) according to the RT-qPCR standard procedure. The reagent used for quantitative PCR was a Premix Ex TaqTM II (Tli RNaseH Plus) kit (TaKaRa, shiga, japan). The quantitative PCR instrument used was an iCycler iQ real-time PCR detection system (Bio-Rad, hercules, calif.). The reaction conditions were as follows: 95℃for 3 minutes, 95℃for 15s and 65℃for 30 s. As shown in fig. 6, the results indicate that: the expression of immune effector genes of metarhizium anisopliae infected metarhizium anisopliae is increased in different metarhizium anisopliae tissues (epidermis, blood cells, fat body and antenna), wherein the expression of the descenin in metarhizium anisopliae epidermis and fat body is higher, and the expression of the immune effector genes of LmOBP11 RNAi+metarhizium anisopliae infected group is extremely remarkable in the epidermis and fat body. The results show that LmOBP11 silencing improves the expression of the immune effector gene of the metarhizium anisopliae infected migratory locust, and is consistent with the survival rate improvement result at the 1 st point of the embodiment.
3. Phenethyl alcohol can activate LmOBP11 expression
Quantitative PCR was performed using different concentrations of PEA (0.0001 mol/L and 0.000001 mol/L) injected into the migratory locust body to examine the changes in LmOBP11 expression in different tissues of the migratory locust (tentacles, epidermis, blood cells and fat bodies), and as shown in FIG. 7A, each tissue had an elevated LmOBP11 expression, with more elevation in fat bodies. Indicating that PEA can activate LmOBP11 gene expression. Further, the expression of the dephenosin gene was examined, and the PEA treatment decreased the expression of the dephenosin gene as shown in fig. 7C.
PEA at a final concentration of 0.0001mol/L was injected in vivo alone in admixture with 5. Mu.L of M.anitopliae spore suspension, and the number of surviving migratory locust was examined daily. The survival rate of migratory locust was reduced in PEA and m.anisoplia e mixed group compared to m.anisoplia e alone treated group (fig. 7B). The combination of the metarhizium anisopliae and the phenethyl alcohol can improve the toxicity of metarhizium anisopliae, namely reduce the survival rate of migratory locust.
It should be noted that, the experimental operations related to the above experimental examples have a certain universality, so that the detailed description is not given, and the detailed description is omitted for the content of the related operations in other experimental examples or for the reference to the prior art.
Claims (10)
1. The migratory locust smell binding protein LmOBP11 gene is characterized in that the sequence similarity of the base sequence of the gene and SEQ ID NO.1 is more than 90%.
2. The migratory locust odor binding protein LmOBP11 gene according to claim 1, wherein the base sequence of the gene is identical to the sequence of SEQ ID No. 1.
3. The protein coded by the migratory locust odor binding protein LmOBP11 gene is characterized in that the amino acid sequence of the coded protein has more than 80 percent of sequence similarity with SEQ ID NO. 2.
4. A migratory locust odor binding protein LmOBP11 gene encoded protein according to claim 3, wherein the amino acid sequence of the gene is identical to the sequence of SEQ ID No. 2.
5. Application of migratory locust smell binding protein LmOBP11 gene in regulating insect immunity.
6. The use according to claim 5, wherein the LmOBP11 gene is used for reducing the immunity of migratory locust to metarhizium anisopliae.
7. The use according to claim 6, wherein the metarhizium anisopliae is mixed with phenethyl alcohol.
8. The use according to claim 7, wherein the concentration of phenethyl alcohol is less than 1mol/L.
9. A method for preventing and controlling migratory locust is characterized in that a mixture of phenethyl alcohol and metarhizium anisopliae is used in places needing prevention and control.
10. The use according to claim 9, wherein the concentration of phenethyl alcohol is less than 1mol/L and the method of use is spraying.
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