CN111704660B - Bemisia tabaci CREB transcription factor and method for detecting resistance or sensitivity of Bemisia tabaci to imidacloprid - Google Patents
Bemisia tabaci CREB transcription factor and method for detecting resistance or sensitivity of Bemisia tabaci to imidacloprid Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly discloses bemisia tabaci CREB transcription factor, and a coding gene, a recombinant vector, a recombinant cell line and a recombinant strain. Further, the invention discloses a method for detecting the resistance or sensitivity of the bemisia tabaci to imidacloprid, which detects the phosphorylation level of the 111 th site of an amino acid sequence coded by the CREB gene of the bemisia tabaci, and a specific phosphorylation antibody of the 111 th site of the CREB gene of the bemisia tabaci. The method can be used for rapidly detecting the resistance or sensitivity of the bemisia tabaci to the imidacloprid or the drug resistance level of the field bemisia tabaci to the imidacloprid.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to Bemisia tabaci CREB transcription factor, a coding gene, a recombinant vector, a recombinant cell line, a recombinant strain and a method for detecting the resistance or sensitivity of Bemisia tabaci to imidacloprid.
Background
Bemisia tabaci (Fr.) KuntzeBemisia tabaci (genoplus) is a worldwide agricultural pest whose hazards mainly include direct piercing and sucking of plant juices; transmitting a plant virus; honeydew secretion causes sooty mould and the like. The control of the bemisia tabaci is mainly chemical control, the bemisia tabaci belongs to piercing-sucking small insects, the whole development period comprises an egg stage, a nymph stage and an adult stage, and the traditional contact insecticide such as bifenthrin, abamectin and the like can well kill the adult bemisia tabaci and can not effectively kill eggs and nymphs on the back sides of leaves. The neonicotinoid insecticides such as imidacloprid, thiamethoxam and the like have good plant systemic property, can kill both bemisia tabaci adults and bemisia tabaci nymphs at high efficiency by spraying and irrigating crops, and are the first-choice drugs for preventing and controlling piercing-sucking pests. However, with the wide and long-term use of pesticides, the drug resistance is gradually formed, the research on the drug resistance mechanism is an important guarantee for reasonably using the pesticides, and the drug resistance mechanism is also an indispensable important component for safe and efficient agricultural production.
The neonicotinoid insecticide is a novel insecticide with high efficiency, low toxicity, environmental friendliness and strong selectivity, and the action mechanism is to selectively inhibit Nicotinic Acetylcholine Receptors (nAChRs) which are important components of the nervous system of pests, and competitively combine the nAChRs with neurotransmitter Acetylcholine, so that the nerve transmission of the pests is blocked, and muscle cells of the pests are paralyzed and die. Imidacloprid (Imidacloprid) is the first generation of neonicotinoid compounds developed by the bayer company and popularized worldwide in the 80 s of the 20 th century. Global pesticide market survey in 2014 shows that the neonicotinoid pesticide has more than 25% of the global pesticide market share, wherein the usage amount of imidacloprid reaches 33.5% of the neonicotinoid pesticide, and the neonicotinoid pesticide becomes a global first-choice drug for controlling piercing-sucking pests.
Because the neonicotinoid insecticides are widely used for a long time, the pests gradually develop the drug resistance, and the bemisia tabaci is no exception. For example, in the United states, Spain and other countries have found, through field monitoring, that Bemisia tabaci has developed severe resistance to the first generation of the neonicotinoid insecticide imidacloprid. Luo et al first detected imidacloprid resistance level in China, found that geographical species such as Beijing and Xinjiang are sensitive to imidacloprid, while Zhejiang, Jiangsu and Hubei regions have medium and high resistance. Later, Wang et al found moderately resistant populations in Shanghai areas, found a very high imidacloprid resistant Bemisia tabaci population in Jiangsu areas through field monitoring, with a resistance multiple of up to 1840, and the thiamethoxam resistance multiple of up to 1470, indicating that severe resistance had developed in the areas. The applicant carries out field monitoring in 2011, discovers a tobacco whitefly imidacloprid high-resistance population in the southern area of Beijing, and discovers a tobacco whitefly resistant population in the higher latitude area.
The mechanisms by which pests develop resistance to pesticides include mainly target resistance due to reduced sensitivity of the pesticide target and detoxification resistance due to overexpression of detoxification enzymes or enhanced enzymatic activity. Target insensitivity is an important cause of high levels of resistance of pests to pesticides. For example, the point mutation of a beta subunit R81T of a nAChR receptor of field aphids obviously reduces the binding capacity of the nAChR receptor to imidacloprid, which results in high resistance, and in addition, the resistance forming mechanism of rice pest brown planthopper to imidacloprid comprises that tyrosine (Y) conserved at the alpha 1 and alpha 3 subunit 151 positions of nAChR is mutated into serine (S), which reduces the binding capacity of the nAChR receptor of brown planthopper and imidacloprid, thereby providing direct evidence for the resistance caused by the reduction of target sensitivity. Recent research reports indicate that nAChR receptor α 8 is also associated with resistance of brown planthoppers to imidacloprid.
Detoxification resistance is more prevalent than target resistance, particularly with a medium to low degree of resistance formation mechanisms. Many detoxifying enzymes related to physiological functions and metabolism exist in the body of pests, such as three common detoxifying enzyme systems: multifunctional oxidases (Cytochrome P450-dependent Monooxygenes, P450), Glutathione transferases (GST) and carboxylesterases (CarE). After the pests contact the insecticide, an emergency reaction is formed in vivo, detoxification and metabolism are carried out to a certain extent by mobilizing detoxification enzyme systems in vivo, and then substances which are harmful to the body are discharged out of the body. The most widely studied detoxification resistance is the cytochrome P450 detoxification metabolic mechanism.
Cytochrome P450 mediated pest resistance is quite commonIn the pest body, the pesticide is firstly hydroxylated by cytochrome P450 to form a substance with stronger water solubility, and then degraded or eliminated from the body under the action of other enzymes. A series of insecticide resistance mechanism researches show that P450 plays an important role in the formation of insect resistance mechanism, for example, the researches in fruit fly show thatCYP6G1Overexpression is associated with DDT resistance, and Anopheles gambiae is resistant to DDTCYP6Z1The up-regulation of gene expression is related, and the resistance of the tribolium castaneum to the deltamethrin is caused by over-expression in brainCYP6BQ9Formation, resistance of Nilaparvata lugens to ImidaclopridCYP6AY1AndCYP6ER1expression is up-regulated, and the resistance of bollworm to fenvalerate is due toCYP337B3The fusion results in.
The molecular mechanism of resistance of pests to pesticides caused by cytochrome P450 is mainly realized through two forms, one is that P450 protein is accumulated in vivo by increasing the expression level of P450, and then exogenous substances are detoxified and metabolized; secondly, the detoxification activity of the polypeptide is enhanced through the change of a P450 gene or an enzyme structure, and finally resistance is formed. The increase of the expression level of P450 is the hot spot of research nowadays, and there are many reports related to each year that the drug resistance of pests is related to the expression level of P450, but the molecular regulation mechanism is less researched, and the regulation mechanism of P450 at the transcription level mainly relates to cis-regulatory factor and trans-regulatory factor. Genes involved in the development of resistance to DDT, for example in DrosophilaCYP6G1Overexpression is associated with the insertion of the retrotransposon Accord into the 5' untranslated region, the pyrethrin resistance gene of DrosophilaCYP6A2The upstream 5' flanking region contains the entire Cap "n" tubular C: (CncC) /Muscle aponeurosis Fibromatosis (Maf) Binding sites, acting as transcriptional activators, are resistant to deltamethrinCYP6BQ9Can also be transcribed byCncC/MafActivating expression, the gene (a plurality of P450 genes) for degrading exogenous substances by the potato beetles can also be used as a transcription factorCncC/MafRegulating expression.
No target mutation has been reported on the resistance mechanism of the bemisia tabaci to the neonicotinoid insecticides, and the research is mainly focused on the resistance mechanism of the detoxification enzymeKarunker et al, color train discoveredCYP6CM1The overexpression of the gene is extremely related to the resistance of imidacloprid, and protein expression experiments show that the gene not only can be hydroxylated at the 5 th position of an imidazoline ring of the imidacloprid to reduce the toxicity of the imidacloprid, but also can metabolize other neonicotinoid insecticides such as Clothianidin (Clothianidin) and Thiacloprid (Thiacloprid). The latest research report shows thatCYP303, CYP6CX3AndCYP4C64and glutathione transferase may also be involved in the development of resistance of bemisia tabaci to neonicotinoid insecticides. The research that the transcription factor related to the bemisia tabaci participates in drug resistance is still blank.
Disclosure of Invention
Aiming at the problems in the prior art, the inventor carries out field bemisia tabaci collection in the early stage to obtain an imidacloprid high-resistance population, obtains imidacloprid resistance and sensitive populations with consistent genetic background through positive and negative selection in a room, then, fluorescent quantitative PCR analysis is carried out to find that CREB is over expressed in the resistant population, and the protein is found to be over-expressed in a resistant population through Westernon Blot detection, and further analysis shows that the 111 th S amino acid of the Bemisia tabaci CREB protein can be phosphorylated, by making a phosphorylation antibody of the site, analyzing the anti-sensitive strain of the bemisia tabaci imidacloprid, finding that the phosphorylation level of the CREB protein S111 site is highly expressed in the imidacloprid resistant population, and field verification is carried out on the imidacloprid resistant population by collecting field bemisia tabaci, the phosphorylation level of the site is found to be over-expressed in the field resistant population, and the research result is favorable for protein level detection and early warning of the imidacloprid resistant population of the bemisia tabaci.
Based on the research results, the invention firstly provides the bemisia tabaci CREB transcription factor which is characterized in that the amino acid sequence is shown as SEQ ID NO. 2.
Further provides a coding gene of the CREB transcription factor, and preferably the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1.
Further provides the recombinant vector, the recombinant cell line and the recombinant strain of the coding gene.
In another aspect, the invention provides a method for detecting the resistance or sensitivity of bemisia tabaci to imidacloprid, which is characterized by detecting the phosphorylation level of the 111 th site of an amino acid sequence coded by a bemisia tabaci CREB gene.
In a specific embodiment, the detection method comprises the following specific steps: phosphorylation level detection was performed using a western Blot with specific phosphorylation antibody against the 111 th site of bemisia tabaci CREB. In the test results, the detection of phosphorylation of CREB at a specific site (position of S111) indicates resistance, and conversely, no resistance.
The invention also provides a specific phosphorylation antibody aiming at the 111 th site of the bemisia tabaci CREB. Still further, a kit for detecting the resistance or sensitivity of bemisia tabaci to imidacloprid is provided, which is characterized by comprising the antibody. More preferably, the kit further comprises a bemisia tabaci protein extraction reagent and a reagent required by Westernon Blot.
According to the invention, through intensive research, the correlation between the specific phosphorylation of the CREB transcription factor at the 111 th site and the resistance or sensitivity of bemisia tabaci to imidacloprid is finally discovered, so that the method for detecting the resistance or sensitivity of the bemisia tabaci to the imidacloprid is realized by preparing the specific phosphorylation antibody aiming at the 111 th site of the bemisia tabaci CREB, the detection result is accurate, the method is rapid in operation, and the method has popularization and application values and also has important significance for the formation of the resistance of the bemisia tabaci to the imidacloprid.
Drawings
FIG. 1 Bemisia tabaci CREB phylogenetic tree;
FIG. 2 Bemisia tabaci CREB was overexpressed in the imidacloprid resistant population;
FIG. 3 sequence analysis of the Bemisia tabaci CYP6CM1 upstream promoter;
FIG. 4 CREB promotes CYP6CM1 gene expression;
FIG. 5 Effect of Bemisia tabaci CREB gene on Imidacloprid resistance;
FIG. 6 Bemisia tabaci CREB phosphorylation site assay;
figure 7 bemisia tabaci CREB phosphorylation sites field application.
Detailed Description
The following examples illustrate the procedures and embodiments of the present invention, but are not intended to limit the invention thereto.
In the embodiment, experiments are mainly carried out on the imidacloprid resistant population and the indoor sensitive population by using field bemisia tabaci, and the imidacloprid resistant situations of all the populations are shown in table 1.
TABLE 1 Bemisia tabaci resistance level assay for Imidacloprid
Wherein N is the number of b.tabaci in each bioassay; df is the degree of freedom; FL is Fiducial limit; RR (resistance ratio), LC of sample bacteria50LC of IMS bacteria50。
The first embodiment,CREBGene full-Length verification
Designing primer clone on the basis of Bemisia tabaci genome annotation information and transcriptome annotationCREBGene sequence, primer sequence is shown as SEQ ID NO 3 and SEQ ID NO 4 (F: ATGGACGGGATGGTGGAGG; R: TCAATCTGTTTTTTGCTGGCAATAGAGC), amino acid sequence difference and phylogenetic condition of imidacloprid anti-allergic strain are compared after translation, gene structure analysis is carried out by utilizing genome DNA information, and the method is clearCREBThe in vivo characteristics of the gene in bemisia tabaci.
The result shows that the full length of the Bemisia tabaci CREB gene is 849 bases (the sequence is shown as SEQ ID NO: 1), the gene codes 282 amino acids (the amino acid sequence is shown as SEQ ID NO: 2), the terminal has a typical characteristic DNA binding region (leucine zipper structure) of a CREB family gene, and the gene belongs to a typical CREB family through evolutionary tree analysis (shown as figure 1).
Example two
(1) In the anti-allergic strainCERBGene transcript level expression detection
Design ofCREBGene fluorescent quantitative PCR primer (F-ACTCAAGGCAGTCTCCAAACCC; R TTTCTGCTCCGCCTAAATCGTT) (amplification efficiency 99%), respectively detecting expression quantity of anti-allergic strain adult at transcription level, and simultaneously comparing and analyzingDifferences between resistant and sensitive lines. Extracting RNA samples of two groups of adults, then carrying out reverse transcription to obtain a cDNA template, and carrying out reaction in a fluorescent quantitative PCR reaction system comprising the specific primer in the step 1;
(1) the extraction steps of the RNA of the bemisia tabaci adults are as follows:
firstly, taking 30 heads of sensitive and resistant population bemisia tabaci adults, and freezing in liquid nitrogen for later use;
secondly, putting the adults into a glass homogenizer, adding l ml of Trizol reagent into the homogenizer for sufficient homogenization, pouring the liquid into a 1.5ml centrifugal tube after homogenization, and standing for 5min at room temperature;
③ adding 0.2 ml of chloroform into the centrifugal tube, violently shaking for 15 s, and then standing for 3 min at room temperature;
fourthly, centrifuging for 15min at the temperature of 4 ℃ at 12,000 rpm;
fifthly, sucking the supernatant fluid and transferring the supernatant fluid into a new centrifuge tube, adding 0.5 ml of isopropanol, gently mixing the liquid in the tube uniformly, and standing for 10 min at room temperature;
sixthly, centrifuging for 10 min at the temperature of 4 ℃ at the speed of 12,000 rpm;
seventhly, discarding the supernatant, then adding l ml of 75% ethanol, gently washing the precipitate, centrifuging at 4 ℃ and 7500 rpm for 5min, and discarding the supernatant;
after the red RNA precipitation is naturally dried, about 30 mul DEPC water is added for dissolving, the mixture is stored on ice, and OD is measured260/OD280The band is detected by electrophoresis, and the RNA sample meeting the conditions is selected to carry out the following reverse transcription experiment or stored at-80 ℃ for later use.
(2) Reverse transcription synthesis of cDNA template:
next, the genome-deleted PrimeScript was manufactured by TaKaRa, Japan®The RT kit is taken as an example, and the experimental process is as follows:
unfreezing and centrifuging each component of the kit and uniformly mixing the components before use;
② removal reaction of genome DNA:
③ reverse transcription reaction (on ice):
(2) in the anti-allergic strainCERBProtein level expression assay
Analysis of Bemisia tabaciCREBSelecting related epitope synthetic polypeptide (T-T-P-K-T-P-K-K-K-I-T-F-D-T-N), sending to company to synthesize rabbit polyclonal antibody, verifying that the antibody can be used for detecting imidacloprid resistance and sensitive strainsCREBDifferences in protein levels
1. Collecting protein samples
And (3) cracking 200 adult bemisia tabaci samples of the anti-allergy strains by using Western and IP cell lysates, performing protein quantification after obtaining protein samples, and finally, balancing the sample loading amount to 20 micrograms of total protein and storing at low temperature for later use.
2. Electrophoresis
SDS-PAGE gels are prepared, and a proper amount of concentrated SDS-PAGE protein loading buffer is added into the collected protein samples.
Heating at 100 deg.C or boiling water bath for 3-5 min to fully denature protein.
And cooling to room temperature, and directly loading the protein sample into the SDS-PAGE gel sample loading hole. In order to facilitate observation of the electrophoretic effect and the transmembrane effect and judgment of the molecular weight of the protein, a prestained protein molecular weight standard is preferably used.
3. Transfer film (Transfer)
A Bio-Rad standard wet type membrane transfer apparatus was used, and a membrane transfer current was set to 400mA and a membrane transfer time was set to 60 minutes. The membrane can also be rotated overnight at 15-20 mA. The effect of membrane transfer can be observed by using molecular weight standard of prestained protein, and usually 1-2 bands with maximum molecular weight are difficult to transfer to the membrane completely.
4. Seal (Blocking)
And immediately placing the protein membrane into a prepared Western washing solution after membrane transfer, and rinsing for 1-2 minutes to wash off the membrane transfer solution on the membrane. The washing solution was aspirated off with a dropper or the like, and Western blocking solution was added thereto, followed by slow shaking on a shaker and blocking at room temperature for 60 minutes. For some antibodies with higher background, blocking can be performed overnight at 4 ℃.
5. Primary antibody incubation (Primary antibody incubation)
According to the following steps of 1: 10000 ratio CREB primary antibody was diluted with Western primary antibody diluent. The blocking solution is sucked up by a dropper and the like, the diluted primary antibody is immediately added, and the incubation is carried out for one hour by slowly shaking on a side-swinging table at room temperature or 4 ℃. If the primary antibody is not effective after one hour incubation, the incubation can be carried out overnight at 4 ℃ with slow shaking.
6. Second antibody incubation (second antibody incubation)
Horseradish peroxidase (HRP) -labeled secondary antibody was diluted with Western secondary antibody dilution at a ratio of 1: 2000. After the washing solution was aspirated off by a dropper or the like, the diluted secondary antibody was immediately added thereto, and incubated on a side-shaking table at room temperature or 4 ℃ for one hour with slow shaking.
7. Protein Detection (Detection of proteins)
ECL-like reagents were performed using BeyoECL Plus to detect proteins.
The results showed that the CREB gene was overexpressed by 2.9-fold in the resistant strains and the protein level was also significantly overexpressed by examining the expression levels of mRNA and protein levels of the CREB gene in the bemisia tabaci imidacloprid resistant (IMR) and sensitive strains (IMS) by qPCR and WB (see fig. 2).
EXAMPLE III Dual-luciferase reporter assay System
The analysis was performed in Drosophila S2 cells using the Dual-Luciferase Reporter gene detection System (Dual-Luciferase Reporter Assay System, # E1980, Promega)CYP6CM1Upstream promoter sequence of gene (sequence shown as SEQ ID NO: 5), and overexpression of transcription factor protein in S2 cell, analysis of transcription factor pairCYP6CM1Activation regulation of the upstream region of the gene.
The Drosophila S2 cell line is donated to Chenda Hua laboratory of animal research institute of Chinese academy of sciences, and has cell source and macrophage-like cells in late stage of Drosophila embryo. S2 cells were cultured in a 15 cm-diameter large dish using Hyclone SFX medium and placed in a CO-free place2The culture temperature in the constant temperature incubator is 27 ℃.
S2 cell transfection is carried out by using Lipofectamine 2000 (1 ug/uL), cells with good growth state are taken before transfection, and the density reaches 1.5-2.0 multiplied by 10 after dilution6And (2) sucking a proper amount of the cell culture plate to be transfected, shaking, uniformly mixing, standing and culturing for 24 hours, removing the culture medium, adding a serum-free culture medium, standing for 1 hour at room temperature, and preparing a transfection solution during the period:
and (3) transferring dye liquor A: adding a proper amount of plasmid into 200 mu L of serum-free culture medium, and gently mixing uniformly;
and (3) transferring dye liquor B: adding appropriate amount of Lipofectamine 2000 into 200 μ L of serum-free culture medium, shaking gently, mixing, and standing at room temperature for 5 min;
and mixing the transfection solution A and the transfection solution B, shaking up gently, standing at room temperature for 20min, dropwise adding the mixed solution into a cell culture plate, placing the cell culture plate in an incubator for culture, replacing a fresh culture medium after 6h, and cracking cells for detection after 24-48 h.
A Promega-Dual-Luciferase vector Reporter Assay System is used for detecting the expression condition of target gene promoter genes, in order to eliminate differences formed among cells and in the transfection process, double Reporter genes are used, namely pGL4.10 and pGL4.73 respectively, firefly fluorescence emitted by the Promega-Dual-Luciferase vector promoter System is used for detecting the activity of the promoter, and the firefly fluorescence emitted by the Promega-Dual-Luciferase vector promoter System is mainly used for correcting background differences.
And (3) carrying out dual-luciferase detection on the transfected cells for 24-48 h:
10 XPBS buffer (2 g KH) was prepared2PO4，11.5g Na2HPO42g kcl, 80g nacl) mother liquor, diluted 10-fold to the working concentration at the time of use.
Preparing a 1 XPasive Lysis Buffer PLB lysate according to the Promega-Dual-Luciferase Reporter Assay System kit specification: the appropriate amount of 5 XPLB was aspirated into a clean centrifuge tube and 4 volumes of ddH were added2O, shaking slightly and mixing uniformly, and preparing the lysate in situ;
simultaneously preparing 1 × Stop & Glo Substrate working solution: adding a certain amount of 50 XStop & Glo Substrate into the Stop & Glo Buffer to ensure that the working concentration of the two reagents is 1 time, wherein the two reagents are used after being fully melted at room temperature;
taking out the cell culture plate to be transfected from the incubator, gently removing the cells, gently washing the cells with 1 × PBS, and discarding the washing solution;
adding a certain amount of 1 XPLB into the culture plate, placing the culture plate on a shaker for 15min by slow shaking at 100rpm, and carrying out cell lysis;
and (3) sucking 20 mu L of cell lysate into a 96-well plate special for the fluorescence detection of a Modulus II microporous plate type multifunctional detector, and carrying out luciferase detection.
The results showed that the upstream promoter region of CYP6CM1 gene was first explored and that a promoter region with significantly up-regulated expression was found in the upstream-920 to-939 region (FIG. 3). Further, the protein CREB is over-expressed in Drosophila S2 cells, and is found to have the effect of enhancing the expression activity (5.98 times) of the bemisia tabaci imidacloprid resistance gene CYP6CM1, indicating that the gene has the effect of promoting the bemisia tabaci to be resistant to imidacloprid (figure 4).
Example four RNA interference of Bemisia tabaci
According toCREBGenes require the design of dsRNA primers:
F：TAATACGACTCACTATAGGGAAGCGGTGACCCTTTATCCT;
R：TAATACGACTCACTATAGGG TTGACGACTCCACTTTGCAG
PCR post sequencing verification, mass synthesis using T7 RiboMAX Express RNAi system and protocols (# P1700, Promega) kitCREBdsRNA of transcription factors. RNAi experiment of Bemisia tabaci by feeding method, and silencing observationCREBBemisia tabaci post-transcription factorCYP6CM1Influence of gene expression and variation of imidacloprid resistance level.
Firstly, preparing dsRNA, preparing an exogenous control gene dsGFP, and preparing an experimental concentration by using a bemisia tabaci nutrient solution. Taking a small sealing film, stretching the first layer to be thin as much as possible, covering one end of a glass tube, adding 100 mu L of the prepared nutrient solution, taking a small sealing film, stretching the small sealing film to be proper, covering the small sealing film on liquid drops, removing air bubbles as much as possible, and manufacturing a small bag which is formed by a double-layer sealing film and contains the nutrient solution and dsRNA (double-stranded ribonucleic acid) for the bemisia tabaci to eat. Finally, the other open end of the glass tube is placed on the leaf blade attached with the bemisia tabaci, the leaf blade is lightly tapped to enable the bemisia tabaci to fly into the tube, then a black cotton plug is carefully plugged, and a shading pipe sleeve is wrapped. Placed in an incubator, the direction of the feeding pouch is towards the light source, and the culture conditions are light 14: 10, humidity 80% (higher humidity can prevent the nutrient solution from volatilizing).
The results show that the RNA interference experiment of CREB gene in bemisia tabaci shows that the CYP6CM1 gene expression level is obviously reduced after the gene is silenced, and the resistance level of the bemisia tabaci to imidacloprid is obviously reduced (figure 5).
Example five prediction, verification and application of CREB phosphorylation sites
According toCREBThe method comprises the steps of predicting a possible phosphorylation site, mutating the site, performing phosphorylation site detection by using a dual-luciferase detection system, further performing phosphorylation level detection by using Westernblot in the field bemisia tabaci imidacloprid resistant population (namely the phosphorylation level detection is performed by using Westernblot, and the method is the same as the Westernblot) in the preparation of a phosphorylation antibody of the CREB of the bemisia tabaci, and judging the drug resistance level of the bemisia tabaci in the field to the imidacloprid according to the phosphorylation level.
By analyzing a kinase activation region in the middle region of Bemisia tabaci CREB, an activation site of suspected kinase is found at the position of S111, the site is mutated (S111Y), after the site is found to be mutated, the expression level of CYP6CM1 gene is obviously reduced, and the phosphorylation level of the site is obviously improved when the site is detected in an imidacloprid resistance strain of Bemisia tabaci (figure 6). Thus, detection of phosphorylation at a specific site of CREB (position of S111) in the assay results indicates resistance, whereas no resistance is observed.
EXAMPLE six use of CREB phosphorylation levels in field populations
3 field bemisia tabaci imidacloprid resistant populations (NK, LF and YC) and a sensitive population (SY) are collected, the detection of total protein and phosphorylation levels of CREB is carried out by using Westernon Blot, the phosphorylation level of the CREB protein S111 site is found to be obviously higher than that of the imidacloprid sensitive population in the resistant population, and the phosphorylation level is consistent with the expression trend of CYP6CM1 (figure 7), which indicates that the phosphorylation level detection of the site can be used for detecting the imidacloprid resistance level of the field bemisia tabaci.
<110> vegetable and flower institute of Chinese academy of agricultural sciences
<120> Bemisia tabaci CREB transcription factor and method for detecting resistance or sensitivity of Bemisia tabaci to imidacloprid
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ATGGACGGGATGGTGGAGGAAAACGGGACAGGAAGCGGTGACCCTTTATCCTCCTCACCCAGTGCAAACACAACTAACGTCGCCACTTCAGTGCAGTCAGTTATTCAAGCCAACCAGCAATCAGTTATTCAAACTGCAACAGGGAATATTCAACCTGCTGTGCTCACTAAAGGGAACGTCATCCTAGTTAGCAAACCGAACTCTGTCATTCAAACAACTCAAGGCAGTCTCCAAACCCTCCAGGTAGTTGTAGAAGCTAATAGTGACGATAGTTTATCAGCAGAAGACGACTCAACAAGGAAACGAAGAGATATCCTCACAAGGCGGCCTTCCTATAGAAAAATTTTAAACGATTTAGGCGGAGCAGAAATAGCCGGCTGCAAAGTGGAGTCGTCAAATTCAGATTGTGATTCTAACCTTGATAGTGAATTATCTTCACATTCCTTGCCTACGCACTACCCGACAGTAATACCTGCAGGTTCATTGCAACTCTGTAGTCAAGGAGAAGGAGCTCAAGGTATTCAATCAATTACAATGACAAATGCATCATCAGGAGGGACAATAGTCCAATACGCCGGTCAAGACGGGCAATTCTTCGTACCAGGTGAAATTGTGGTGACGCAAGGATCTACCCTACCTGGAGTACCTCTAATGGCAGAGGATCAAGCAAGGAAGCGTGAACTTAGGTTATTAAAAAATAGGGAAGCAGCACGAGAGTGCAGACGAAAAAAGAAGGAATATATTAAATGTCTAGAAAATAGAGTTGCTGTACTTGAAAATCAAAATAAGGCCTTAATAGATGAGTTGAAATCGCTGAAAGAGCTCTATTGCCAGCAAAAAACAGAT
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TCAATCTGTTTTTTGCTGGCAATAGAGC
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ATGGAACTGTTGGAAATAGTTAAGTCAGCCATGGACACTCACTCGGTCCTGCTGATTTTCTTGAGTGTCATGGTTTACCTGCTCTACGTTTACCGGGACAAATTCCACTACTGGAGCAAGCGAGGCGTCCCGTGCCAAAGCCCCGCACAGAGCATCGTGCGCACCTTCCGGCTTGTCCTCCGAATGGACTCCTTCACCGACAACTTCTACGGCGTGTACAAGGCCTTCGATGGACACCCCTACGTGGGCTCTTTGGAACTTACCAAGCCTATTTTGGTCGTCCGCGACCCCGAACTTGCCAGGATCGTCCTAGTCAAGAGCTTCTCCAGCTTCTCTGGCAGATTGAAGTCACCGGACACAACATTGGATCCCCTGTCAAACCACCTTTTCACCTTAAACGGAGAGAAATGGCGGCAAGTACGTCACAAGACGGCGACAGCCTTCAGCACAGCCAAGCTGAAGAACATGTTCCACAGCCTGAAGGACTGCGCCCGGGAGATGGATGCCTACATGGAGAGAGCCATCGGTGATAAAGGAGATGTTGAATTCGATGCGCTCAAGGTTATGTCCAACTACACTCTTGAGGTCATCGGGGCTTGTGCCATGGGCATTAAGTGCGACTCCATCCACGATGAGGAAACCGAGTTTAAGAGGTTCTCCAGGGATTTCTTCAGATTTGATGCGAGGCGAATGATCTTCACTCTTTTGGATTTACTGCACCCGAAACTTCCTGTTCTTCTTAAATGGAAAGCTGTGCGACCCGAAGTTGAGAACTTTTTCAGGGAGGCCATTAAAGAGGCAGCTTCACTTAAAGAAAGCGAAGCAGCTGCCCGCACGGATTTTCTCCAAATTCTCATCGACTTCCAAAAATCTGAAAAGGCATCCAAGACTGACGCAGGAAATGATACCGAACTTGTTTTCACGGACAATATCATCGGTGGAGTGATTGGATCATTCTTCTCGGCGGGCTACGAACCTACCGCGGCGGCACTAACTTTCTGTCTATACGAGCTGGCGCGGAATCCTCAGGTTCAAGCCAAACTCCACGAGGAAATTTTAGCTGTGAAAGAAAAATTGGGTGATGACATTGAATACGAAACTTTGAAGGAATTTAAATATGCCAACCAAGTTATTGATGAGACGCTGCGACTGTACCCGGCGTCGGGGATTTTGGTGCGGACGTGCACGGAGCCTTTCAAGTTACCAGACTCGGACGTCGTCATCGAGAAAGGGACCAAGGTCTTCGTCTCCTCCTACGGCCTCCAAACGGACCCTCGATATTTTCCCGAGCCCGAAAAATTCGACCCGGAGCGCTTTTCCGAAGAGAACAAGGAAAAAATCCTCCCCGGGACCTATTTGCCTTTCGGAGACGGGCCTAGACTTTGCATAGCGATGCGACTGGCATTGATGGATGTGAAGATGATGATGGTTAGGTTGGTTTCGAAATACGAAATTCATACAACCCCCAAGACACCGAAAAAGATCACATTCGACACGAACTCATTCACGGTACAGCCTGCTGAAAAAGTATGGCTCCGCTTCCGGAGAAGGGCGTCGACGCC
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GTGAAGCTGCCTCTTTAATGGC
Claims (3)
1. A method for detecting the resistance or sensitivity of Bemisia tabaci to imidacloprid is characterized in that the phosphorylation level of the 111 th site of an amino acid sequence coded by the Bemisia tabaci CREB gene is detected, wherein the amino acid sequence coded by the Bemisia tabaci CREB gene is shown as SEQ ID NO. 2; in the detection result, if the phosphorylation at the 111 th site of the amino acid sequence coded by the Bemisia tabaci CREB gene is detected, the resistance is represented, and otherwise, the resistance is not represented.
2. The method of claim 1, wherein the phosphorylation level is measured using a western Blot.
3. The method of claim 1, wherein the detection method comprises the following specific steps: phosphorylation level detection is carried out by using Westernon Blot through a specific phosphorylation antibody aiming at 111 th site of an amino acid sequence coded by the Bemisia tabaci CREB gene.
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| CN102037933A (en) * | 2010-10-18 | 2011-05-04 | 浙江省萧山棉麻研究所 | Method for measuring virulence by feeding Bemisia tabaci Gennadius imago |
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Patent Citations (3)
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| CN102037933A (en) * | 2010-10-18 | 2011-05-04 | 浙江省萧山棉麻研究所 | Method for measuring virulence by feeding Bemisia tabaci Gennadius imago |
| WO2013182613A1 (en) * | 2012-06-08 | 2013-12-12 | Bayer Cropscience Ag | Detection system for the identification of insecticide resistance |
| CN103820446A (en) * | 2014-01-17 | 2014-05-28 | 吉林大学 | Tobacco whitefly imidacloprid resistance associated CYP6CM1v2 promoter and activity analysis |
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