CN111704660A - Bemisia tabaci CREB transcription factor and method for detecting the resistance or sensitivity of Bemisia tabaci to imidacloprid - Google Patents

Abstract

The invention belongs to the field of biotechnology, and specifically discloses a Bemisia tabaci CREB transcription factor, an encoded gene, a recombinant vector, a recombinant cell line and a recombinant strain. Further, the present invention discloses a method for detecting the resistance or sensitivity of Bemisia tabaci to imidacloprid, which detects the phosphorylation level of the amino acid sequence 111 of the amino acid sequence encoded by Bemisia tabaci CREB gene, and the used method for Bemisia tabaci. Phosphorylated antibody specific for position 111 of lice CREB. The invention can quickly detect the resistance or sensitivity of B. tabaci to imidacloprid, or the drug resistance level of B. tabaci in the field to imidacloprid.

Description

Bemisia tabaci CREB transcription factor and its detection of imidacloprid resistance or susceptibility in Bemisia tabaci Methods

technical field

The invention belongs to the field of biotechnology, and particularly relates to a Bemisia tabaci CREB transcription factor, an encoded gene and 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 technique

Bemisia tabaci (Gennadius) is a worldwide agricultural pest, and its damage mainly includes direct sucking of plant sap; transmission of plant virus; secretion of honeydew leading to sooty disease. The control of Bemisia tabaci is mainly based on chemical control. Because Bemisia tabaci is a small piercing and sucking insect, the entire developmental period includes egg stage, nymph stage and adult stage. Traditional contact insecticides such as bifenthrin, Avi Bacteriocin can effectively kill the adults of Bemisia tabaci, but cannot effectively kill the eggs and nymphs on the underside of leaves. Neonicotinoids such as imidacloprid, thiamethoxam, etc. have good plant systemic properties. By spraying and irrigating crops, they can not only kill B. tabaci adults, but also efficiently kill B. tabaci nymphs. The drug of choice for suction pest control. However, with the extensive and long-term use of pesticides, drug resistance has gradually formed. The research on the mechanism of drug resistance is an important guarantee for the rational use of pesticides, and is also an indispensable part of agricultural safe and efficient production.

The neonicotinoid insecticide is a new type of insecticide with high efficiency, low toxicity, environmental friendliness and strong selectivity. Its mechanism of action is to selectively inhibit the important part of the nervous system of pests. nAChRs), compete with the neurotransmitter acetylcholine to bind nAChRs, thereby blocking the pest neurotransmission, causing paralysis and death of the pest muscle cells. Imidacloprid is the first generation of neonicotinoid compounds, developed by Bayer in the 1980s and promoted to the world. In 2014, the global pesticide market survey showed that neonicotinoid pesticides accounted for more than 25% of the global pesticide market share, and the use of imidacloprid reached 33.5% of that of neonicotinoid pesticides, becoming a piercing-sucking pest The global drug of choice for prevention and treatment.

Due to the widespread and long-term use of neonicotinoid insecticides, pests have gradually formed resistance to the insecticides, and Bemisia tabaci is no exception. For example, the United States, Spain and other countries have found that B. tabaci have serious resistance to the first-generation neonicotinoid insecticide imidacloprid through field monitoring. Luo et al. detected imidacloprid resistance levels for the first time in my country, and found that geographic populations such as Beijing and Xinjiang are relatively sensitive to imidacloprid, while Zhejiang, Jiangsu and Hubei regions have developed medium-to-high resistance. Subsequently, Wang et al. found a population with moderate resistance in Shanghai, and found a population of Bemisia tabaci with extremely high resistance to imidacloprid through field monitoring in Jiangsu. 1470 times, indicating that the region has developed severe drug resistance. The applicant conducted field monitoring in 2011, and found a population of Bemisia tabaci with high resistance to imidacloprid in the Nankou area of Beijing, and found a resistant population of Bemisia tabaci in higher latitudes.

The mechanisms by which pests develop resistance to insecticides mainly include target resistance caused by reduced sensitivity to insecticide targets and detoxification resistance caused by overexpression of detoxification enzymes or enhanced enzyme activity. Target insensitivity is an important reason why pests develop high levels of resistance to pesticides. For example, the field aphid nAChR receptor β subunit R81T point mutation significantly reduces its binding ability to imidacloprid, resulting in high resistance. In addition, the resistance formation mechanism of rice pest N. lugens to imidacloprid includes the conservation of nAChR at the 151 position of the α1 and α3 subunits Mutation of tyrosine (Y) to serine (S) of N. lugens reduced the binding ability of the nAChR receptor of N. lugens to imidacloprid, thus providing direct evidence for resistance caused by reduced target sensitivity. Recent research reports indicate that nAChR receptor α8 is also associated with imidacloprid resistance in N. lugens.

Compared with target resistance, detoxification resistance is more prevalent, especially the low-to-moderate resistance-forming mechanism. There are many detoxification enzymes related to physiological functions and metabolism in pests, such as three common detoxification enzymes: multifunctional oxidase (Cytochrome P450-dependent Monooxygenases, P450), glutathione transferase (GlutathioneS-transferases, GST) And carboxylesterase (Carboxleseterases, CarE). When pests come into contact with pesticides, they will form an emergency response in the body. By mobilizing the detoxification enzymes in the body, detoxification and metabolism are carried out to a certain extent, and then these substances that are toxic to the body are eliminated from the body. The most widely studied detoxification resistance is the mechanism of cytochrome P450 detoxification metabolism.

Cytochrome P450-mediated insecticide resistance is very common. Insecticides entering the body of pests first undergo hydroxylation by cytochrome P450 to form more water-soluble substances, and then degrade or excrete them under the action of other enzymes. A series of studies on the mechanism of insecticide resistance have shown that P450 plays an important role in the formation of insect resistance mechanisms. For example, in Drosophila, it was found that overexpression of CYP6G1 is related to DDT resistance, and the resistance of Anopheles gambiae to DDT is related to CYP6Z1. Gene expression was up-regulated. The resistance to deltamethrin in the red millet was due to the overexpression of CYP6BQ9 in the brain. The resistance of brown planthopper to imidacloprid was related to the up-regulation of CYP6AY1 and CYP6ER1 expression. The resistance of cotton bollworm to fenvalerate was due to CYP337B3. Fusion leads to, .

The molecular mechanism of insecticide resistance caused by cytochrome P450 is mainly achieved in two ways. One is to increase the expression of P450, thereby accumulating P450 protein in the body, and then detoxify and metabolize exogenous substances; It is through the change of the P450 gene or the enzyme structure itself that its detoxification activity is enhanced, and finally resistance is formed. The increase in the expression of P450 is also a research hotspot, and there are many related reports every year that the resistance of pests is related to the expression of P450, but its molecular regulation mechanism is less studied. The regulation mechanism of P450 at the transcriptional level is mainly Involves cis-regulators and trans-regulators. For example, the overexpression of DDT resistance-related gene CYP6G1 in Drosophila is related to the insertion of the retrotransposon Accord into the 5' untranslated region, and the 5' flanking region upstream of the pyrethrin resistance gene CYP6A2 in Drosophila contains a complete Cap "n" The collar C ( CncC ) / Muscle aponeurosis Fibromatosis ( Maf ) binding site acts as a transcriptional activator. CYP6BQ9 , which is resistant to deltamethrin in the red millet, can also be activated and expressed by the transcription factor CncC / Maf . Potato beetle Genes that degrade foreign substances (multiple P450 genes) can also be regulated by the transcription factor CncC / Maf .

The resistance mechanism of B. tabaci to neonicotinoid insecticides has not yet been reported on target mutations. Current research mainly focuses on the mechanism of detoxification enzyme resistance. Among them, Israeli scholar Karunker et al. found that the overexpression of CYP6CM1 gene is extremely related to imidacloprid resistance. , protein expression experiments show that this gene can not only hydroxylate on the 5th position of imidazoline ring of imidacloprid to reduce the toxicity of imidacloprid, but also metabolize other neonicotinoid insecticides such as Clothianidin and thiacloprid (Thiacloprid). The latest research reports show that CYP303 , CYP6CX3 and CYP4C64 and glutathione transferase may also be involved in the formation of B. tabaci resistance to neonicotinoid insecticides. However, the research on the involvement of B. tabaci-related transcription factors in drug resistance is still in a blank state.

SUMMARY OF THE INVENTION

In view of the problems existing in the prior art, the inventors collected B. tabaci in the field in the early stage to obtain imidacloprid-resistant populations, obtained imidacloprid-resistant and sensitive populations with the same genetic background through forward and reverse selection indoors, and then carried out fluorescence quantification. PCR analysis showed that CREB was over-expressed in the resistant population, and the protein was over-expressed in the resistant population by Western Blot detection. Further analysis found that the 111 S amino acid of the CREB protein of Bemisia tabaci can be phosphorylated. By analyzing the imidacloprid-resistant strains of B. tabaci, it was found that the phosphorylation level of the S111 site of CREB protein was highly expressed in imidacloprid-resistant populations. The phosphorylation level of the locus is overexpressed in the field resistant population, and the results will be helpful for the detection and early warning of the protein level in the imidacloprid-resistant population of B. tabaci.

Based on the above research results, the present invention first provides a Bemisia tabaci CREB transcription factor, characterized in that its amino acid sequence is shown in SEQ ID NO:2.

Further provide the encoding gene of CREB transcription factor, preferably its nucleotide sequence is as shown in SEQ ID NO:1.

The recombinant vector, recombinant cell line, and recombinant strain of the encoding gene are further provided.

Another aspect of the present invention provides a method for detecting the resistance or susceptibility of B. tabaci to imidacloprid, which is characterized by detecting the phosphorylation level at position 111 of the amino acid sequence encoded by the Bemisia tabaci CREB gene.

In a specific embodiment, the specific steps of the detection method are as follows: the phosphorylation level is detected by Western Blot with a specific phosphorylation antibody against the 111th site of CREB of Bemisia tabaci. In the detection results, the detection of phosphorylation at the specific site of CREB (the position of S111) indicates resistance, otherwise there is no resistance.

The present invention also provides a specific phosphorylation antibody against the 111th site of CREB of Bemisia tabaci. It further provides a kit for detecting the resistance or susceptibility of Bemisia tabaci to imidacloprid, characterized in that it includes the antibody. More preferably, it further includes Bemisia tabaci protein extraction reagents, and reagents required for Western Blot.

Through in-depth research in the present invention, it is finally found that the specific phosphorylation of the specific site of CREB transcription factor, that is, the specific phosphorylation of the 111th site is related to the resistance or sensitivity of B. tabaci to imidacloprid. The specific phosphorylation antibody of the site, thus realizing the method for detecting the resistance or sensitivity of Bemisia tabaci to imidacloprid, the detection result is accurate, the method is quick to operate, and has the value of popularization and application, and for the resistance of Bemisia tabaci to imidacloprid formation is also important.

Description of drawings

Fig. 1 Phylogenetic tree of Bemisia tabaci CREB;

Figure 2. CREB of Bemisia tabaci is overexpressed in imidacloprid-resistant populations;

Figure 3 Sequence analysis of the upstream promoter of B. tabaci CYP6CM1;

Figure 4 CREB promotes CYP6CM1 gene expression;

Fig. 5 The effect of CREB gene of B. tabaci on imidacloprid resistance;

Figure 6 Detection of CREB phosphorylation sites in Bemisia tabaci;

Fig. 7 Field application of CREB phosphorylation sites in B. tabaci.

Detailed ways

The research process and examples of the present invention are described below through specific examples, but do not constitute a limitation of the present invention.

Among them, in the embodiment, the experiment was mainly performed on the imidacloprid-resistant populations and the indoor sensitive populations of Bemisia tabaci in the field. The resistance of all populations to imidacloprid is shown in Table 1.

Table 1 Determination of the resistance level of Bemisia tabaci to imidacloprid

Among them, N is the number of B. tabaci in each biological analysis; df is the degree of freedom; FL is the _Fiducial limit _;

Embodiment 1, CREB gene full-length verification

Based on the B. tabaci genome annotation information and transcriptome annotation, primers were designed to clone the CREB gene sequence. The primer sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4 (F: ATGGACGGGATGGTGGAGG; R: TCAATCTGTTTTTTGCTGGCAATAGAGC), and post-translation comparison The amino acid sequence differences and phylogeny of imidacloprid-resistant strains were analyzed using genomic DNA information to analyze the gene structure of the CREB gene in B. tabaci.

The results showed that the CREB gene of B. tabaci had a full length of 849 bases (the sequence is shown in SEQ ID NO: 1), encoding 282 amino acids (the amino acid sequence is shown in SEQ ID NO: 2), and at the end there is a CREB family gene. A typical characteristic DNA-binding region (leucine zipper structure), and further phylogenetic tree analysis showed that this gene belongs to a typical CREB family (Figure 1).

Embodiment 2

(1) Detection of CERB gene transcription level expression in resistant strains

The CREB gene real-time PCR primers (F-ACTCAAGGCAGTCTCCAAACCC; RTTTCTGCTCCGCCTAAATCGTT) were designed (the amplification efficiency was 99%), and the expression levels of adult worms of the resistant strains were detected at the transcription level, and the differences between the resistant and susceptible strains were compared and analyzed. Extracting RNA samples of two groups of adult worms, then reverse transcribing them into cDNA templates, and performing the reaction in a fluorescence quantitative PCR reaction system including the specific primers described in step 1;

(1) The extraction steps of B. tabaci adult RNA are as follows:

①Take 30 adults of Bemisia tabaci from the sensitive and resistant populations, and freeze them in liquid nitrogen for future use;

②Put the adult worms into a glass homogenizer, and add 1 ml of Trizol reagent to the homogenizer to fully homogenize. After homogenization, pour the liquid into a 1.5ml centrifuge tube and leave it at room temperature for 5 minutes;

③ Add 0.2 ml of chloroform to the centrifuge tube, shake vigorously for 15 s, and then place at room temperature for 3 min;

④ Centrifuge at 12,000 rpm for 15 min at 4°C;

⑤ Transfer the supernatant to a new centrifuge tube, add 0.5 ml isopropanol, gently mix the liquid in the tube, and let it stand at room temperature for 10 min;

⑥ Centrifuge at 12,000 rpm for 10 min at 4°C;

⑦ Discard the supernatant, then add 1 ml of 75% ethanol, gently wash the pellet, centrifuge at 4°C, 7500 rpm for 5 min, and discard the supernatant;

⑧After the RNA precipitation is naturally dried, add about 30µl of DEPC water to dissolve, store on ice, measure the OD ₂₆₀ /OD ₂₈₀ value, and detect the bands by electrophoresis. Select qualified RNA samples for the following reverse transcription experiments or store at -80°C for later use.

(2) Synthesize cDNA template by reverse transcription:

The following is an example of the PrimeScript ^® RT kit from TaKaRa, Japan. The experimental process is as follows:

① Thaw the components of the kit and mix by centrifugation before use;

②Removal reaction of genomic DNA:

③Reverse transcription reaction (on ice):

(2) Detection of CERB protein expression in allergy-resistant strains

Analyze the CREB gene characteristics of Bemisia tabaci, select the relevant epitope to synthesize polypeptide (TTPKTPKKITFDTN), and send the company to synthesize rabbit polyclonal antibody. After verifying that the antibody is available, detect the difference in CREB protein levels in imidacloprid-resistant and sensitive strains

1. Collect protein samples

Western and IP cell lysate was used to lyse 200 B. tabaci adult samples of allergy-resistant strains, and protein samples were obtained for protein quantification.

2. Electrophoresis

To prepare an SDS-PAGE gel, add an appropriate amount of concentrated SDS-PAGE protein loading buffer to the collected protein samples.

Heat at 100°C or in a boiling water bath for 3-5 minutes to fully denature the protein.

After cooling to room temperature, the protein samples can be directly loaded into the loading wells of the SDS-PAGE gel. In order to observe the effect of electrophoresis and transmembrane transfer, and to judge the molecular weight of the protein, it is best to use a pre-stained protein molecular weight standard.

3. Transfer

A standard wet transfer device from Bio-Rad was used, the transfer current was set to 400 mA, and the transfer time was 60 minutes. It is also possible to transfer the membrane overnight at 15-20 mA. The effect of membrane transfer can be observed by the pre-stained protein molecular weight standard used. Usually, the 1-2 bands with the largest molecular weight are difficult to transfer to the membrane.

4. Blocking

Immediately after transferring the membrane, place the protein membrane in the pre-prepared Western washing solution and rinse for 1-2 minutes to wash off the transfer solution on the membrane. Use a dropper to suck up the washing solution, add Western blocking solution, shake slowly on a shaker, and block at room temperature for 60 minutes. For some antibodies with high background, block overnight at 4°C.

5. Primary antibody incubation

CREB primary antibody was diluted 1:10000 with Western Primary Antibody Diluent. Use a dropper to suck up the blocking solution, immediately add the diluted primary antibody, and incubate for one hour at room temperature or 4°C with gentle shaking on a side-swing shaker. If the primary antibody does not work well for one hour, you can incubate overnight at 4°C with gentle shaking.

6. Secondary antibody incubation

The horseradish peroxidase (HRP)-conjugated secondary antibody was diluted 1:2000 with Western Secondary Antibody Diluent. Use a dropper to suck up the washing solution, immediately add the diluted secondary antibody, and incubate for one hour at room temperature or 4°C with gentle shaking on a side-swing shaker.

7. Detection of proteins

Use BeyoECL Plus for ECL-like reagents to detect proteins.

The results showed that the mRNA and protein levels of CREB gene in imidacloprid-resistant (IMR) and sensitive lines (IMS) of B. tabaci were detected by qPCR and WB, and it was found that the gene was overexpressed by 2.9 times in resistant lines, and Protein levels were also significantly overexpressed (Figure 2).

Embodiment 3. Dual-luciferase reporter gene detection system

The upstream promoter sequence of the CYP6CM1 gene (sequence shown in SEQ ID NO: 5) was analyzed in Drosophila S2 cells using the Dual-Luciferase® ReporterAssay System (# E1980, Promega), and in S2 Transcription factor proteins were overexpressed in cells, and the activation and regulation of transcription factors on the upstream region of CYP6CM1 gene was analyzed.

The Drosophila S2 cell line was donated to the laboratory of Chen Dahua, Institute of Zoology, Chinese Academy of Sciences. S2 cells were cultured in a large petri dish with a diameter of 15 cm, using HyClone SFX medium, and placed in a constant temperature incubator without CO ₂ at a culture temperature of 27 °C.

S2 cells were transfected with liposome Lipofectamine 2000 (1ug/uL). Before transfection, cells in good growth state were taken, and the density reached 1.5~2.0× ¹⁰⁶ cells after dilution. Pipette an appropriate amount into the cells to be transfected. In the plate, shake and mix well, let stand for 24 hours, remove the medium, add serum-free medium, and let it stand at room temperature for 1 hour. During this period, prepare the transfection solution:

Transfection Solution A: 200 μL of serum-free medium was added with an appropriate amount of plasmid, and mixed gently;

Transfection solution B: 200 μL of serum-free medium was added with an appropriate amount of Lipofectamine 2000, gently shaken and mixed, and left at room temperature for 5 minutes;

Mix transfection solution A and transfection solution B, shake gently, leave at room temperature for 20 minutes, add the mixture dropwise to the cell culture plate, place it in an incubator, and replace with fresh medium after 6 hours, and after 24-48 hours Cells were lysed for detection.

The Promega-Dual-Luciferase® Reporter Assay System was used to detect the expression of the target gene promoter gene. In order to eliminate the differences between cells and the formation of the transfection process, dual reporter genes were used, namely pGL4.10 and pGL4. 73. The firefly fluorescence emitted by the former is used to detect the promoter activity, and the latter contains the Renilla gene, and the fluorescence is mainly used to correct the background difference.

Transfected cells 24~48h for dual luciferase assay:

Prepare 10×PBS buffer solution (2g KH ₂ PO ₄ , 11.5g Na ₂ HPO ₄ , 2g KCl, 80g NaCl) stock solution, and dilute 10 times to the working concentration during use.

According to the instructions of the Promega-Dual-Luciferase® Reporter Assay System kit, prepare 1×Passive Lysis Buffer PLB lysate: pipette an appropriate amount of 5×PLB into a clean centrifuge tube, add 4 times the volume of ddH ₂ O, shake and mix Evenly, the lysate is used and prepared immediately;

Prepare 1×Stop & Glo® Substrate working solution at the same time: add a certain amount of 50×Stop & Glo® Substrate to Stop & Glo® Buffer to make it 1 times the working concentration, these two reagents should be fully melted at room temperature before use;

Take out the cell culture plate to be transfected from the incubator, gently remove the cells, gently wash the cells with 1×PBS, and discard the washing solution;

Add a certain amount of 1×PLB to the culture plate, place it on a shaker and shake it slowly at 100rpm for 15min to lyse the cells;

Pipette 20 μL of cell lysate into a 96-well plate specially designed for fluorescence detection of the Modulus II microplate multi-function detector for luciferase detection.

The results showed that the upstream promoter region of the CYP6CM1 gene was first explored, and a significantly up-regulated promoter region was found in the upstream -920 to -939 region (Figure 3). Furthermore, by overexpressing CREB protein in Drosophila S2 cells, it was found that this protein has the activity of enhancing the expression of the imidacloprid resistance gene CYP6CM1 in B. tabaci (5.98 times), indicating that this gene has the effect of promoting the imidacloprid resistance of B. tabaci ( Figure 4).

Embodiment four, Bemisia tabaci RNA interference

Design dsRNA primers according to the needs of the CREB gene:

F: TAATACGACTCACTATAGGGAAGCGGTGACCCTTTATCCT;

R: TAATACGACTCACTATAGGG TTGACGACTCCACTTTGCAG

After PCR sequencing verification, T7 RiboMAX Express RNAi system and protocols (#P1700, Promega) kit was used to synthesize dsRNA of CREB transcription factor in large quantities. The RNAi experiment of Bemisia tabaci by feeding method was used to observe the effect of CYP6CM1 gene expression and the change of imidacloprid resistance level in Bemisia tabaci after silencing CREB transcription factor.

First, prepare dsRNA and exogenous control gene dsGFP, and prepare the experimental concentration by using Bemisia tabaci nutrient solution. Take a small piece of parafilm, stretch the first layer as thin as possible, cover one end of the glass tube, then add 100 μL of the above prepared nutrient solution, and then take a small piece of parafilm and stretch it and cover it on the droplet , try to eliminate air bubbles, and make a small bag containing nutrient solution and dsRNA formed by double-layer parafilm for feeding by Bemisia tabaci. Finally, place the other open end of the glass tube on the leaf with Bemisia tabaci, tap the leaf to make Bemisia tabaci fly into the tube, then carefully plug the black cotton plug, and wrap the light-shielding tube cover. It was placed in an incubator with the feeding pouch facing the light source, and the cultivation conditions were 14:10 light and 80% humidity (higher humidity can prevent the nutrient solution from volatilizing).

The results showed that through the RNA interference experiment of CREB gene in B. tabaci, it was found that the expression of CYP6CM1 gene was significantly reduced after the gene was silenced, and the resistance level of B. tabaci to imidacloprid was significantly reduced (Figure 5).

Embodiment five, CREB phosphorylation site prediction, verification and its application

According to the amino acid sequence of CREB , the possible phosphorylation site was predicted, and then the site was mutated, and then the dual luciferase detection system was used to detect the phosphorylation site, and further phosphorylation of the CREB-specific site of B. tabaci was prepared. Antibodies, the phosphorylation level was detected by Westernon Blot in the imidacloprid-resistant population of B. tabaci in the field (that is, the phosphorylation level was detected by Westernblot, and the method was the same as Westernblot), and the field tobacco powder was judged by the level of phosphorylation Levels of lice resistance to imidacloprid.

By analyzing the kinase activation region in the middle region of Bemisia tabaci CREB, it was found that there was a suspected kinase activation site at the position of S111, and this site was mutated (S111Y), and it was found that after the mutation, the expression of CYP6CM1 gene was significantly reduced. , and in the imidacloprid-resistant strains of B. tabaci, it was found that the phosphorylation level of this site was significantly increased (Fig. 6). Therefore, in the detection results, the detection of phosphorylation at the specific site of CREB (the position of S111) represents resistance, otherwise there is no resistance.

Embodiment 6. Application of CREB phosphorylation level in field population

Three field B. tabaci imidacloprid-resistant populations (NK, LF, YC) and one susceptible population (SY) were collected, and Western Blot was used to detect the total protein and phosphorylation level of CREB. It was found that the phosphorylation level of CREB protein S111 was in the The resistant population was significantly higher than the imidacloprid-sensitive population, and the phosphorylation level was consistent with the expression trend of CYP6CM1 (Fig. 7), indicating that the phosphorylation level detection of this site can be used to detect the resistance level of B. tabaci to imidacloprid in the field .

<110> Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences

<120> Bemisia tabaci CREB transcription factor and its method for detecting the resistance or susceptibility of Bemisia tabaci to imidacloprid

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ATGGACGGGATGGTGGAGGAAAACGGGACAGGAAGCGGTGACCCTTTATCCTCCTCACCCAGTGCAAACACAACTAACGTCGCCACTTCAGTGCAGTCAGTTATTCAAGCCAACCAGCAATCAGTTATTCAAACTGCAACAGGGAATATTCAACCTGCTGTGCTCACTAAAGGGAACGTCATCCTAGTTAGCAAACCGAACTCTGTCATTCAAACAACTCAAGGCAGTCTCCAAACCCTCCAGGTAGTTGTAGAAGCTAATAGTGACGATAGTTTATCAGCAGAAGACGACTCAACAAGGAAACGAAGAGATATCCTCACAAGGCGGCCTTCCTATAGAAAAATTTTAAACGATTTAGGCGGAGCAGAAATAGCCGGCTGCAAAGTGGAGTCGTCAAATTCAGATTGTGATTCTAACCTTGATAGTGAATTATCTTCACATTCCTTGCCTACGCACTACCCGACAGTAATACCTGCAGGTTCATTGCAACTCTGTAGTCAAGGAGAAGGAGCTCAAGGTATTCAATCAATTACAATGACAAATGCATCATCAGGAGGGACAATAGTCCAATACGCCGGTCAAGACGGGCAATTCTTCGTACCAGGTGAAATTGTGGTGACGCAAGGATCTACCCTACCTGGAGTACCTCTAATGGCAGAGGATCAAGCAAGGAAGCGTGAACTTAGGTTATTAAAAAATAGGGAAGCAGCACGAGAGTGCAGACGAAAAAAGAAGGAATATATTAAATGTCTAGAAAATAGAGTTGCTGTACTTGAAAATCAAAATAAGGCCTTAATAGATGAGTTGAAATCGCTGAAAGAGCTCTATTGCCAGCAAAAAACAGAT

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ATGGAACTGTTGGAAATAGTTAAGTCAGCCATGGACACTCACTCGGTCCTGCTGATTTTCTTGAGTGTCATGGTTTACCTGCTCTACGTTTACCGGGACAAATTCCACTACTGGAGCAAGCGAGGCGTCCCGTGCCAAAGCCCCGCACAGAGCATCGTGCGCACCTTCCGGCTTGTCCTCCGAATGGACTCCTTCACCGACAACTTCTACGGCGTGTACAAGGCCTTCGATGGACACCCCTACGTGGGCTCTTTGGAACTTACCAAGCCTATTTTGGTCGTCCGCGACCCCGAACTTGCCAGGATCGTCCTAGTCAAGAGCTTCTCCAGCTTCTCTGGCAGATTGAAGTCACCGGACACAACATTGGATCCCCTGTCAAACCACCTTTTCACCTTAAACGGAGAGAAATGGCGGCAAGTACGTCACAAGACGGCGACAGCCTTCAGCACAGCCAAGCTGAAGAACATGTTCCACAGCCTGAAGGACTGCGCCCGGGAGATGGATGCCTACATGGAGAGAGCCATCGGTGATAAAGGAGATGTTGAATTCGATGCGCTCAAGGTTATGTCCAACTACACTCTTGAGGTCATCGGGGCTTGTGCCATGGGCATTAAGTGCGACTCCATCCACGATGAGGAAACCGAGTTTAAGAGGTTCTCCAGGGATTTCTTCAGATTTGATGCGAGGCGAATGATCTTCACTCTTTTGGATTTACTGCACCCGAAACTTCCTGTTCTTCTTAAATGGAAAGCTGTGCGACCCGAAGTTGAGAACTTTTTCAGGGAGGCCATTAAAGAGGCAGCTTCACTTAAAGAAAGCGAAGCAGCTGCCCGCACGGATTTTCTCCAAATTCTCATCGACTTCCAAAAATCTGAAAAGGCATCCAAGACTGACGCAGGAAATGATACCGAACTTGTTTTCACGGACAATATCATCGGTGGAGTGATTGGATCATTCTTCTCGGCGGGCTACGAACCTACCGCGGCGGCACTAACTTTCT GTCTATACGAGCTGGCGCGGAATCCTCAGGTTCAAGCCAAACTCCACGAGGAAATTTTAGCTGTGAAAGAAAAATTGGGTGATGACATTGAATACGAAACTTTGAAGGAATTTAAATATGCCAACCAAGTTATTGATGAGACGCTGCGACTGTACCCGGCGTCGGGGATTTTGGTGCGGACGTGCACGGAGCCTTTCAAGTTACCAGACTCGGACGTCGTCATCGAGAAAGGGACCAAGGTCTTCGTCTCCTCCTACGGCCTCCAAACGGACCCTCGATATTTTCCCGAGCCCGAAAAATTCGACCCGGAGCGCTTTTCCGAAGAGAACAAGGAAAAAATCCTCCCCGGGACCTATTTGCCTTTCGGAGACGGGCCTAGACTTTGCATAGCGATGCGACTGGCATTGATGGATGTGAAGATGATGATGGTTAGGTTGGTTTCGAAATACGAAATTCATACAACCCCCAAGACACCGAAAAAGATCACATTCGACACGAACTCATTCACGGTACAGCCTGCTGAAAAAGTATGGCTCCGCTTCCGGAGAAGGGCGTCGACGCC

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Claims (9)

1. The Bemisia tabaci CREB transcription factor is characterized in that an amino acid sequence of the transcription factor is shown as SEQ ID NO. 2.

2. The CREB transcription factor coding gene as claimed in claim 1, preferably the nucleotide sequence is shown as SEQ ID NO. 1.

3. A recombinant vector, a recombinant cell line, a recombinant strain containing the coding gene of claim 2.

4. 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 a bemisia tabaci CREB gene is detected.

5. The method of claim 1, wherein the phosphorylation level is measured using a western Blot.

6. The method according to claim 4 or 5, characterized in that 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.

7. A specific phosphorylated antibody against the 111 th site of bemisia tabaci CREB.

8. A kit for detecting resistance or sensitivity to imidacloprid of bemisia tabaci comprising the antibody of claim 7.

9. The kit of claim 8, further comprising bemisia tabaci protein extraction reagents, and reagents required for western Blot.

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