CN110564860B - Mutant site of tobacco whitefly resistant gene to thiamethoxam - Google Patents
Mutant site of tobacco whitefly resistant gene to thiamethoxam Download PDFInfo
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- CN110564860B CN110564860B CN201910528417.8A CN201910528417A CN110564860B CN 110564860 B CN110564860 B CN 110564860B CN 201910528417 A CN201910528417 A CN 201910528417A CN 110564860 B CN110564860 B CN 110564860B
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- bemisia tabaci
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Abstract
The invention discloses a P450 gene for identifying a thiamethoxam resistance gene of bemisia tabaciCYP4C64Mutant PCR primer pair and P450 gene for identifying thiamethoxam resistance gene of bemisia tabaciCYP4C64And (c) whether mutation is performed. The nucleotide sequences of the primer pairs are as follows:CYP4C64mutation identification-F: ATCGATCGGAAATTCGGGCTGA,CYP4C64identification of mutations-R: TTTGATGGAATTTTTCCCGGTCATGC. No specific mutation site exists in the resistance detection of the bemisia tabaci to the neonicotinoid insecticide thiamethoxam, and the invention uses the resistance gene of the bemisia tabaciCYP4C64The single base site mutation in the 5' UTR region is used for detecting the resistance level of the tobacco whitefly to the thiamethoxam in the field, so that the blank of the field is filled, and a theoretical basis is provided for the rapid detection of the drug resistance of the tobacco whitefly to the thiamethoxam in the field.
Description
Background
Bemisia tabaci (Gennadius) is a worldwide agricultural pest and brings great harm to production. After the 80's of the 20 th century, the plants were distributed in more than 90 countries and regions around the world in continents other than Antarctic, and host as many as 500 species of 74 families. In China, the pest becomes an important economic crop pest in China in the late 90 s, the main pest species is B biotype, and the pest is also covered by 'super pest' due to strong invasiveness and high harmfulness; the Q biological bemisia tabaci was discovered in 2003 by the laboratory in the flower market in Yunnan, and then rapidly spread all over the country, and at present, the two biological bemisia tabaci are the most harmful biotypes on crops such as vegetables and flowers in China. The bemisia tabaci not only directly stabs and absorbs plant juice to cause plant weakness and withering, but also spreads plant virus diseases. The harm caused by the bemisia tabaci as a toxin-transmitting medium even prevents tomatoes in the whole shed in part of areas from being harvested, and extremely serious harm is brought to survival. In view of the severity of direct and indirect damage to bemisia tabaci, the control of bemisia tabaci has reached an irreparable stage in agricultural production.
The bemisia tabaci is mainly subjected to chemical control, and due to excessive use of the pesticide, the bemisia tabaci has different degrees of resistance to various control medicines, and particularly, the neonicotinoid pesticide which is longest in use and has the widest application range is strongest in the control of the bemisia tabaci. Thiamethoxam is a neonicotinoid insecticide with a second-generation brand-new structure, and is always the first choice for controlling bemisia tabaci after entering the Chinese market in 2000. The first discovery that bemisia tabaci is resistant to neonicotinoid insecticides was in horticultural growing areas in the south of spain, and shortly thereafter approximately 1000 times more resistant populations of bemisia tabaci thiamethoxam were found in succession in israel and spain, and the highly resistant populations had significant cross-resistance to other nicotinic agents. In recent years, the resistance of bemisia tabaci to neonicotinoid agents is reported in most nationwide provinces such as Beijing, jiangsu, zhejiang, xinjiang and Hubei in China, most areas from north to Heilongjiang, south to Hainan, west to Qinghai and east to Shanghai in China are distributed and damaged by bemisia tabaci, the types of harmful organisms are mainly Q type and B type, and the drug resistance is generally high. Therefore, the drug resistance is one of the main reasons causing the big outbreak of the bemisia tabaci and difficult treatment.
Pest resistance management relies on the elucidation of pest resistance mechanisms, but currently, little is known about the resistance mechanism of bemisia tabaci against neonicotinoid insecticides. With regard to the mechanism of action of neonicotinoid insecticides, it is widely believed that they act primarily as agonists of postsynaptic nicotinic acetylcholine receptors (nAChRs) on the central nervous system of insects to cause them to die by convulsions and paralysis. Therefore, research on the mechanism of resistance to neonicotinoid insecticides has generally focused on nAChRs, a mutation of which has been found to be associated with neonicotinoid insecticide resistance in insects such as myzus persicae, drosophila melanogaster, rice planthopper, anopheles mosquitoes, and bees. In addition to target insensitivity, the resistance mechanism of insects to neonicotinoid insecticides is also related to metabolic mechanisms, such as the recent discovery that cytochrome P450 oxidase gene-mediated metabolic detoxification is related to thiamethoxam resistance in all of myzus persicae, musca domestica, and nilaparvata lugens. In bemisia tabaci, however, no report has been made that mutations in nAChRs are associated with resistance to neonicotinoid insecticides, but a cytochrome P450 gene (CYP 6CM 1) and imidacloprid resistance have been discovered through biochemical and molecular biology means.
Disclosure of Invention
The invention aims to solve the technical problems of providing a mutant site of Bemisia tabaci for a thiamethoxam resistant gene P450 gene CYP4C64 and a method for identifying whether the Bemisia tabaci for the thiamethoxam resistant gene P450 gene CYP4C64 is mutated, wherein the Bemisia tabaci has no specific mutant site for the resistance detection of neonicotinoid insecticide thiamethoxam, and the invention detects the resistance level of the Bemisia tabaci to thiamethoxam in the field by the mutation of a single base site in the 5' UTR region of the Bemisia tabaci resistant gene CYP4C64, makes up the blank in the field and provides a theoretical basis for the rapid detection of the drug resistance of the Bemisia tabaci in the field to thiamethoxam.
Therefore, the invention provides the following technical scheme:
a PCR primer pair for identifying whether a P450 gene CYP4C64 of a thiamethoxam resistance gene of bemisia tabaci is mutated or not is disclosed, and the nucleotide sequence of the primer pair is as follows:
CYP4C64 mutation identification-F: ATCGATCGGAAATTCGGGGCTGA
CYP4C64 mutation identification-R: TTTGATGGAATTTTTCCCGGTCATGC.
The invention also provides a method for identifying whether the tobacco whitefly has mutation on the thiamethoxam resistance gene P450 gene CYP4C64, which comprises the following steps:
(1) Extracting total RNA of bemisia tabaci;
(2) Carrying out PCR by using the CYP4C64 mutation identification primer; (ii) a
(3) After the PCR product is recovered, connecting, converting, sequencing and identifying;
(4) The ratio of A and T at positions 32 in the sequence was analyzed to identify the level of resistance to thiamethoxam.
The primer pair or the method, wherein the bemisia tabaci is a type Q bemisia tabaci.
Meanwhile, the invention also provides a method for reducing the drug resistance of bemisia tabaci to thiamethoxam, and the method performs RNAi interference on the resistant strain of the bemisia tabaci and reduces the expression of the P450 gene CYP4C 64.
Meanwhile, the invention also provides a mutation site of the tobacco whitefly to thiamethoxam resistance gene P450 gene CYP4C64, wherein the mutation site is located in a 5' UTR region, a sequence obtained after sequencing of a mutation primer is used for detecting the ratio of 32A or T, and the resistance level to thiamethoxam is detected by counting the frequency of the site A, wherein the site is mainly A base in thiamethoxam resistance and mainly appears in a T base form in a sensitive population.
The invention has the following beneficial effects: according to the invention, the single base site mutation in the 5' UTR region of the bemisia tabaci resistance gene CYP4C64 is used for detecting the resistance level of the field bemisia tabaci to thiamethoxam, so that the blank of the field is made up, and a theoretical basis is provided for the rapid detection of the drug resistance of the field bemisia tabaci to thiamethoxam. By applying the method, the resistance level of the tobacco whitefly to the thiamethoxam in the field is rapidly detected through scientific research, so that technical support is provided for reasonable use of the insecticide.
Drawings
FIG. 1 shows the expression level of CYP4C64 gene in resistant and sensitive strains of Bemisia tabaci to thiamethoxam.
FIG. 2 shows that the level of resistance of Bemisia tabaci to thiamethoxam is reduced remarkably after RNAi of CYP4C64 gene.
FIG. 3 comparison of the thiamethoxam resistant strain CYP4C 64' UTR region.
FIG. 4 shows the regional mutation of the CYP4C 64' UTR region of the thiamethoxam resistant strain.
Detailed Description
Test insects
The method comprises the following steps of A, breeding a field sensitive population (THQS) of the type Q Bemisia tabaci thiamethoxam-stable B.tabaci Q strain, carrying out indoor reverse elimination on the field thiamethoxam resistant population THQR to obtain a relatively sensitive population, and feeding pepper (pepper # 4. Thiamethoxam resistant population THQR is collected in Hangzhou areas of Zhejiang in 2011, and indoor feeding is carried out until now, and thiamethoxam is used for selection. Another thiamethoxam resistant population NK was collected in the southern area of Beijing in 2018 and tested immediately after collection.
Test reagent
RNA extraction reagent Trizol: available from Life Technologies Corporation
RACE kit SMART TM RACE cDNA Amplification kit: from Clontech Inc
cDNA reverse transcription kit
RT reagent Kit (REAL TIME): purchased from TaKaRa, japan
Fluorescent quantitative PCR kit, supra PreMix Plus (SYBR Green): purchased from Beijing Tiangen Biochemical technology Ltd
dsRNA synthesis kit T7RiboMAX Express RNAi system: purchased from Promega corporation
DNA molecular weight standards, proteinase K, X-Gal and IPTG: purchased from Beijing Tiangen Biochemical technology Ltd;
benzyl penicillin; yeast extract and tryptone: purchased from Amresco ltd, usa;
PCR primers, goldview and agarose: synthesized and purchased from Beijing optimalist Biotechnology Ltd;
agarose gel electrophoresis buffer TBE preparation:
concentrated stock solution 5 × TBE: tris base 54.0g, boric Acid 27.5g,0.5M Ethylene Diamine Tetraacetic Acid (EDTA) 20mL, finely adjusting pH to 8.0 with a pH meter, adding ddH2O to reach 1000mL; the working solution is 0.5 times TBE after being diluted by 10 times by the 5 times TBE storage solution;
PCR EsTaq mix with 6 × Loading Buffer: purchased from Beijing kang, a century Biotechnology Ltd;
top10 competent cells; PCR product purification kit, pEASY-T1 vector: purchased from general-purpose jinbeijing corporation;
LB culture medium configuration:
10g of tryptone, 5g of yeast extract and 10g of NaCl, adding 950mL of clean distilled water, adjusting the pH value to 7.0 by using 1.0mol/L of NaOH, fixing the volume to 1L, and performing high-pressure moist heat sterilization at 115 ℃ for 20 minutes for later use;
other laboratory reagents (analytical grade) are commercially available.
Main instrument
Fluorescent quantitative PCR instrument: ABI7500 in usa;
PCR instrument: bio-Rad S1000 and Bio-Rad C1000;
electrophoresis tank and water bath: six instrument factories in Beijing;
a high-speed centrifuge: germany Sigma 3K15;
nucleic acid electrophoresis system and Gel imaging system (Gel Doc EQ): bio-Rad, USA;
ultra-pure water instrument: ZMQ55VOTI Mini Q pure water instrument;
pipette for molecular experiments: eppendorf, germany;
superclean bench: suzhou purification technology, inc.
Moist heat autoclave: sanyo corporation, japan;
table type freezer shaker (THZ-C-1): taicang City laboratory plant.
Example 1: tobacco whitefly genome DNA, RNA extraction and cDNA synthesis
Extracting the genome DNA of the bemisia tabaci by adopting a TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 Kit, referring to the operation instruction in the steps, and storing the obtained DNA at-20 ℃.
The Triol method is adopted to extract the total RNA of the bemisia tabaci, and the specific steps are as follows: taking 50 adult bemisia tabaci in a good living state, keeping the same age as much as possible, wherein the male-female ratio is close to 1. Immediately putting the bemisia tabaci into liquid nitrogen for freezing after the bemisia tabaci is taken out, and preventing RNA degradation. The Bemisia tabaci samples were placed in a 1mL homogenizer sterilized at 180 ℃ for 6 hours in a clean bench, and 1mL Trizol was added thereto for sufficient homogenization, followed by careful transfer to a 1.5mL special RNA centrifuge tube and standing at room temperature for 5 minutes. Add 200. Mu.l chloroform, shake vigorously with a vortex shaker for 30s, stand at room temperature for 5 minutes, centrifuge at low temperature, centrifuge at 12000rpm for 15 minutes at 4 ℃. Carefully taking out the centrifuge tube after the centrifugation is finished, gently sucking 400 mul of supernatant without touching the middle protein layer, transferring the supernatant into another clean centrifuge tube, adding equal volume of precooled isopropanol, gently inverting and mixing uniformly, cooling on ice for 10 minutes, then precipitating RNA, centrifuging at 12000rpm for 10 minutes at 4 ℃, carefully sucking out the supernatant, adding 1mL of 70% absolute ethanol for rinsing, and centrifuging at 8000rpm for 5 minutes at 4 ℃. Carefully absorbing ethanol, blowing the mixture in an ultra-clean bench for 5 minutes, adding a proper amount of precooled DEPC water for dissolving, determining the ratio and content of OD260/OD280, and performing denaturing gel electrophoresis to detect the RNA quality.
Kit adopting TaKaRa company
The RT reagent Kit was used for cDNA synthesis.
1) Genomic DNA removal
Carefully add 5 × gDNA Eraser Buffer 2.0 μ L into the PCR tube dedicated to RNA; gDNA Eraser 1.0 μ L; total RNA 1.0 μ g (volume required to be added according to the concentration conversion of extracted RNA); RNase Free ddH 2 O was supplemented to 10. Mu.L, and quickly placed in a PCR apparatus at 42 ℃ for 2 minutes.
2) cDNA Synthesis
To the PCR tube from which the DNA had been removed, 5 XPrimeScript Buffer 2.0. Mu.L was added; primeScriptRT Enzyme Mix 1.0. Mu.L; RT Prime Mix 1.0. Mu.L; RNase Free ddH 2 O4.0. Mu.L, then quickly placed in a PCR instrument, subjected to RNA reverse transcription at 37 ℃ for 15 minutes, inactivated by reverse transcriptase at 85 ℃ for 5 seconds, and then stored at-20 ℃ for later use (-80 ℃ for long-term storage).
Example 2: fluorescent quantitative PCR analysis
The inventor discovers that the CYP4C64 gene is over-expressed in a resistance population screened indoors through earlier researches, and verifies whether the gene participates in the formation of the drug resistance of the bemisia tabaci in the field by acquiring the field population and carrying out fluorescent quantitative PCR analysis on the expression of the CYP4C64 gene. The primer 2 in the table 1 is adopted to analyze the CYP4C64 gene expression quantity, the primers 2 and 3 in the table 1 are selected as reference genes, and the CYP4C64 gene expression condition is determined by a method of calculating relative expression quantity. qPCR detection is carried out by using SuperReal PreMixPlus kit of Tiangen company, the reaction system and the reaction program are shown in Table 2, each reactant is added according to the instruction, then the primers are detected on ABI7500, and qRT-PCR data adopts 2 -ΔΔCt The method calculates the difference of gene expression level between resistant and sensitive lines.
TABLE 1 CYP4C64 Gene fluorescent quantitative PCR primers
F, a forward primer; r, reverse primer
TABLE 2 qRT-PCR reaction System and reaction procedure
Analysis of results
The field bemisia tabaci samples were collected to obtain two resistant strains, both of which had achieved high levels of resistance, with the results shown in table 3 below.
TABLE 3
The expression level of the CYP4C64 gene in the resistant and sensitive strains of thiamethoxam is analyzed through fluorescent quantitative PCR (polymerase chain reaction), the gene is found to be over-expressed in the two resistant strains, as shown in figure 1, the expression level of the CYP4C64 gene in the resistant strain is obviously higher than that of the sensitive strain, and the over-expression of the P450 gene CYP4C64 in the resistant strain is found to be 18 times by comparing the Q-type Bemisia tabaci resistant strain (THR) and the sensitive strain (THS) which are fed indoors.
Example 3: RNAi experiments in bemisia tabaci resistant strains
RNAi experiments were performed in resistant lines. As the bemisia tabaci is small in size, RNAi (ribonucleic acid interference) performed through microinjection can cause high mortality, the experiment adopts a feeding method to perform an RNA interference experiment of the bemisia tabaci.
1) Preparation of nutrient solution for feeding
RNAi is carried out by a feeding method, an environment capable of maintaining normal growth of Bemisia tabaci is required to be obtained outside a living plant, and the fact that a nutrient solution formed by mixing 30% of sucrose and 5% of yeast extract can well maintain survival of the Bemisia tabaci in vitro is found through grope. Firstly, preparing 30% of sucrose solution and 5% of yeast extract, fully dissolving, and then filtering by adopting a 0.22-micron bacterial filter, so as to prevent the breeding of microorganisms in the experimental process, and not being capable of carrying out high-temperature and high-pressure sterilization, because sucrose is gelatinized at high temperature to generate toxic substances, the sucrose is not beneficial to the eating of bemisia tabaci.
2) Experimental device
Preparing a transparent glass tube with the length of 50mm and the inner diameter of 20mm, a sealing film, a pipe sleeve which is made of black cotton plug and shading cloth and can cover the glass tube, and a light incubator which can control temperature and humidity. The glass tube is used for placing bemisia tabaci to carry out in-vitro experiments; the sealing film is used for manufacturing a feeding small bag containing nutrient solution at one end of the glass tube for the bemisia tabaci to take; the black cotton plug and the pipe sleeve are used for creating a dark environment, only a light source exists in the direction of the feeding small bag, and the phototropism of the bemisia tabaci is utilized to enable the bemisia tabaci to be rapidly attached to the direction of the feeding small bag for feeding, otherwise, the bemisia tabaci can be attached to other positions of the glass tube for a long time, and the uniformity of an experiment is poor.
3) Bemisia tabaci feeding
Firstly, dsRNA is prepared, an exogenous control gene dsGFP is prepared, and the test concentration is 0.5 mu g/mu L by using bemisia tabaci nutrient solution. Taking a small piece of sealing film, stretching the first layer as thin as possible to cover one end of the glass tube, then adding 200 mu L of the prepared nutrient solution, taking a small piece of sealing film, stretching the small piece of sealing film in a proper amount, covering the small piece of sealing film on the liquid drop, removing air bubbles as far as possible, and manufacturing a small bag which is formed by double-layer sealing films and contains the nutrient solution and dsRNA for bemisia tabaci to take. 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. Placing in an incubator, wherein the feeding small bag faces to a light source, and the culture conditions are that the light is 14:10, humidity 80% (higher humidity can prevent the nutrient solution from volatilizing). RNAi experiments of CYP4C64 gene are carried out on bemisia tabaci of THQR population by applying the method, the experimental results are shown in figure 2, and knocking down the expression of the gene can obviously reduce the drug resistance of bemisia tabaci to thiamethoxam.
Example 4: comparative analysis of sequence of Bemisia tabaci CYP4C64 gene
Further comparative analysis of CYP4C64 gene sequence by using primer 1 in Table 1 shows that a stable mutation site exists in the 5' UTR region of the gene in thiamethoxam-resistant population, the site is located in 32 ' UTR region of CYP4C64 gene 5' UTR region, the site is mainly A base in thiamethoxam resistance, and is mainly in T base form in sensitive population, and the resistance level of Bemisia tabaci to thiamethoxam is judged by detecting the base form of the site, and the experimental results refer to FIGS. 3 and 4.
Claims (2)
1. Detection ofCYP4C64<xnotran> PCR Q , , ATCGATCGGAAATTCGGGGCTGACGGACACGACAAACTGTTTGACGGACGGACGAGGTGTTTTAGTTTCTCGGGGTCAGCCTTGCCGTGCGGGCCGGATC 32 A T; </xnotran> Extracting Q-type bemisia tabaci total DNA, performing PCR by using the PCR primer pair, performing sequencing identification after PCR products are recovered, detecting the ratio of A or T in the mutation site, and detecting the resistance level of Q-type bemisia tabaci species groups to thiamethoxam by counting the frequency of A in the mutation site; the mutant sites mainly appear in the form of A bases in a thiamethoxam-resistant Q-type Bemisia tabaci population, and the mutant sites mainly appear in the form of T bases in a thiamethoxam-sensitive Q-type Bemisia tabaci population.
2. Use according to claim 1, characterized in that: the nucleotide sequences of the PCR primer pair are as follows:
CYP4C64 mutation identification-F: ATCGATCGGAAATTCGGGCTGA,
CYP4C64 mutation identification-R: TTTGATGGAATTTTTCCCGGTCATGC.
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