CN110564860B - Mutation site of a Bemisia tabaci resistance gene to thiamethoxam - Google Patents

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

Mutation site of a Bemisia tabaci resistance gene to thiamethoxam

Background technique

Bemisia tabaci (Gennadius) is a worldwide agricultural pest, which brings great harm to production. Since the 1980s, they have been successively distributed in more than 90 countries and regions on all continents except Antarctica, and there are more than 500 species of resident plants in 74 families. In China, this pest became an important economic crop pest in my country in the late 1990s. The main damage species is the B biotype, which is called "super pest" due to its strong invasiveness and great harm; It was first discovered by our laboratory in the flower market in Yunnan in 2009, and then quickly spread to all parts of the country. At present, these two biotypes of Bemisia tabaci are the most harmful biotypes on crops such as vegetables and flowers in my country. Bemisia tabaci not only pierce and suck plant sap directly, causing the plant to become weak and dry, but also transmit plant virus diseases. The harm caused by Bemisia tabaci as a virus vector even made the whole shed tomato harvest in some areas impossible, which brought extremely serious harm to survival. In view of the seriousness of direct and indirect damage of Bemisia tabaci, the control of Bemisia tabaci in agricultural production has reached an urgent stage.

Bemisia tabaci is mainly controlled by chemical control. Excessive use of insecticides has resulted in different degrees of resistance of Bemisia tabaci to various control drugs. Neonicotinoids are the most resistant. Thiamethoxam is a second-generation neonicotinoid insecticide with a new structure. Since it entered the Chinese market in 2000, it has been the first choice for the control of Bemisia tabaci. The resistance of Bemisia tabaci to neonicotinoid insecticides was first discovered in the horticultural production area of southern Spain, and soon after, nearly 1,000-fold thiamethoxam-resistant populations of Bemisia tabaci were found in Israel and Spain. The resistant population had significant cross-resistance to other nicotinic agents. In recent years, there have also been reports of Bemisia tabaci developing resistance to neonicotinoid drugs in most provinces in my country, including Beijing, Jiangsu, Zhejiang, Xinjiang, and Hubei. B. tabaci are distributed and harmed in most areas from east to Shanghai, and the harmful biotypes are mainly Q type and B type, and the drug resistance is generally high. Therefore, drug resistance is one of the main reasons for B. tabaci outbreaks and difficult to control.

Pest resistance management relies on the elucidation of pest resistance mechanisms, but the mechanisms of B. tabaci resistance to neonicotinoid insecticides are still "little understood". Regarding the mechanism of action of neonicotinoid insecticides, it is generally believed that they mainly act as agonists of post-synaptic nicotinic acetylcholine receptors (nAChRs), acting on the central nervous system of insects and causing twitching, paralysis and death of insects. Therefore, studies on the resistance mechanism of neonicotinoid insecticides generally focus on nAChRs. For example, nAChRs mutations and neonicotinoid insecticides have been found in green peach aphid, fruit flies, rice planthoppers, malaria mosquitoes, and honeybees. drug resistance. In addition to target insensitivity, the resistance mechanism of insects to neonicotinoid insecticides is also related to metabolic mechanisms. For example, cytochrome P450 oxidase gene-mediated metabolic detoxification was recently found in the green peach aphid, housefly, and brown planthopper. associated with thiamethoxam resistance. In Bemisia tabaci, there is no report that the mutation of nAChRs is related to the resistance to neonicotinoid insecticides, but a cytochrome P450 gene (CYP6CM1) was found to be related to the resistance to imidacloprid by means of biochemical and molecular biology methods. .

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a mutation site of the thiamethoxam resistance gene P450 gene CYP4C64 of Bemisia tabaci, and a method for identifying whether the thiamethoxam resistance gene P450 gene CYP4C64 of Bemisia tabaci is mutated. There is no specific mutation site for the resistance detection of lice to the neonicotinoid insecticide thiamethoxam. The present invention detects the single base site mutation in the 5'UTR region of the B. tabaci resistance gene CYP4C64. The resistance level of B. tabaci in the field to thiamethoxam fills the gap in this field, and provides a theoretical basis for the rapid detection of B. tabaci resistance to thiamethoxam in the field.

Therefore, the present invention provides the following technical solutions:

A PCR primer pair for identifying whether the thiamethoxam resistance gene P450 gene CYP4C64 of Bemisia tabaci is mutated, the nucleotide sequence of the primer pair is as follows:

CYP4C64 Mutation Identification-F: ATCGATCGGAAATTCGGGGCTGA

CYP4C64 mutation identification-R: TTTGATGGAATTTTTTCCCGGTCATGC.

The present invention also provides a method for identifying whether the thiamethoxam resistance gene P450 gene CYP4C64 of Bemisia tabaci is mutated, the method comprising the following steps:

(1) Extracting the total RNA of Bemisia tabaci;

(2) using the CYP4C64 mutation identification primers to carry out PCR;

(3) ligation conversion sequencing identification after PCR product recovery;

(4) The ratio of A and T at position 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 Q-type Bemisia tabaci.

At the same time, the present invention also provides a method for reducing the drug resistance of Bemisia tabaci to thiamethoxam. The method performs RNAi interference on Bemisia tabaci-resistant strains to reduce the expression of P450 gene CYP4C64.

At the same time, the present invention also provides the mutation site of the thiamethoxam resistance gene P450 gene CYP4C64 of Bemisia tabaci, the mutation site is located in the 5'UTR region, and the sequence obtained by sequencing the mutation primer is used to detect the 32-bit A or T. The resistance level to thiamethoxam was detected by counting the frequency of the site A, which was mainly A base in thiamethoxam resistance, but mainly appeared in the form of T base in sensitive populations.

The invention has the following beneficial effects: the invention detects the resistance level of B. tabaci in the field to thiamethoxam through the single-base site mutation in the 5'UTR region of the Bemisia tabaci resistance gene CYP4C64, which makes up for the shortage in this field. blank, which provides a theoretical basis for the rapid detection of thiamethoxam resistance of B. tabaci in the field. Through the application of the invention, scientific research can quickly detect the drug resistance level of Bemisia tabaci in the field to thiamethoxam, thereby providing technical support for rational use of pesticides.

Description of drawings

Fig. 1 Expression of CYP4C64 gene in thiamethoxam-resistant and sensitive lines of B. tabaci.

Figure 2 The resistance level of B. tabaci to thiamethoxam was significantly decreased after CYP4C64 gene RNAi.

Fig. 3 Comparison of 5'UTR regions of thiamethoxam-resistant strains of CYP4C64.

Figure 4. Schematic diagram of the mutation of the 5'UTR region of the thiamethoxam-resistant strain CYP4C64.

Detailed ways

Test insects

The thiamethoxam-susceptible B. tabaci Qstrain (THQS), the thiamethoxam-susceptible B. tabaci Qstrain (THQS), is a relatively sensitive population obtained by reverse selection of the thiamethoxam-resistant population THQR in the indoor greenhouse. Zhong pepper #4; Capsicum annuum L.) has been reared so far without any pesticide application. The thiamethoxam-resistant population THQR was collected in Hangzhou, Zhejiang in 2011, and has been kept indoors for selection. Another thiamethoxam-resistant population, NK, was collected in the Nankou area of Beijing in 2018 and tested immediately after collection.

test reagent

RNA extraction reagent Trizol: purchased from Life Technologies Corporation

RACE kit SMART ^TM RACE cDNA Amplification kit: purchased from Clontech

RT reagent Kit(REAL TIME)：购自日本TaKaRa公司cDNA Reverse Transcription Kit

RT reagent Kit (REAL TIME): purchased from Japan TaKaRa Company

Fluorescence quantitative PCR kit SuperReal PreMix Plus (SYBR Green): purchased from Beijing Tiangen Biochemical Technology Co., Ltd.

dsRNA synthesis kit T7RiboMAX Express RNAi system: purchased from promega

DNA molecular weight standard, proteinase K, X-Gal and IPTG: purchased from Beijing Tiangen Biochemical Technology Co., Ltd.;

benzyl penicillin; yeast extract and tryptone: purchased from Amresco Co., Ltd., USA;

PCR primers, Goldview and agarose: synthesized and purchased from Beijing Qingke Xinye Biotechnology Co., Ltd.;

Agarose gel running buffer TBE configuration:

High concentration stock solution 5×TBE: Tris base 54.0g, boric acid 27.5g, 0.5M Ethylene Diamine Tetraacetic Acid (EDTA) 20mL, use a pH meter to finely adjust the pH to 8.0, add ddH2O to make up to 1000mL ; The working solution is 0.5×TBE after 10 times dilution of 5×TBE stock solution;

PCR EsTaq mixed solution with 6×Loading Buffer: purchased from Beijing Kangwei Century Biotechnology Co., Ltd.;

Top10 competent cells; PCR product purification kit, pEASY-T1 vector: purchased from Quanzhijin Beijing Company;

LB medium configuration:

Tryptone 10g, yeast extract 5g, NaCl 10g, add 950mL of clean distilled water, adjust the pH to 7.0 with 1.0mol/L NaOH, set the volume to 1L, sterilize by high pressure moist heat at 115°C for 20 minutes, set aside;

Other common laboratory chemical reagents (analytical grade) are commercially available.

main instrument

Fluorescence quantitative PCR instrument: American ABI7500;

PCR instruments: Bio-Rad S1000 and Bio-Rad C1000;

Electrophoresis tank and water bath: Beijing Liuyi Instrument Factory;

High-speed centrifuge: German Sigma 3K15;

Nucleic acid electrophoresis system and gel imaging system (Gel Doc EQ): Bio-Rad, USA;

Ultrapure water meter: ZMQ55VOTI Mini Q pure water meter;

Pipette for molecular experiments: Eppendorf, Germany;

Ultra-clean workbench: Suzhou Purification Technology Co., Ltd.

Moist heat autoclave: Sanyo, Japan;

Desktop Refrigerated Shaker (THZ-C-1): Taicang Experimental Equipment Factory.

Example 1: Bemisia tabaci genomic DNA, RNA extraction and cDNA synthesis

Bemisia tabaci genomic DNA was extracted using TaKaRa MiniBEST Universal Genomic DNAExtraction Kit Ver.5.0. The steps were referred to the operating instructions. The obtained DNA was stored at -20°C.

The Triol method was used to extract the total RNA of B. tabaci. The specific steps were as follows: 50 B. tabaci adults with good living conditions were taken, and the age was as consistent as possible, and the male-to-male ratio was close to 1:1. Bemisia tabaci were immediately frozen in liquid nitrogen to prevent RNA degradation. Put the Bemisia tabaci samples into a 1 mL homogenizer that was sterilized at 180 °C for 6 h in an ultra-clean bench, add 1 mL of Trizol to it to fully homogenize, and then carefully transfer it to a 1.5 mL RNA-specific centrifuge tube and keep it at room temperature. Leave on for 5 minutes. Add 200 μl of chloroform, shake vigorously with a vortex shaker for 30 s, stand at room temperature for 5 minutes, centrifuge at low temperature, and centrifuge at 12,000 rpm at 4° C. for 15 minutes. After centrifugation, take out the centrifuge tube carefully, gently aspirate 400 μl of the supernatant without touching the protein layer in the middle, transfer the supernatant to another clean centrifuge tube, add an equal volume of pre-cooled isopropanol, gently Gently invert and mix. After cooling on ice for 10 minutes, RNA was precipitated, and then centrifuged at 12,000rpm for 10 minutes at 4°C. Carefully aspirate the supernatant, add 1 mL of 70% absolute ethanol for rinsing, and centrifuge at 8,000rpm for 5 minutes at 4°C. Carefully aspirate ethanol, dry in an ultra-clean bench for 5 minutes, add an appropriate amount of pre-cooled DEPC water to dissolve, measure the ratio and content of OD260/OD280, and perform denaturing gel electrophoresis to detect RNA quality.

RT reagent Kit进行cDNA合成。Using the kit from TaKaRa company

RT reagent Kit for cDNA synthesis.

1) Genomic DNA removal

To the PCR tube dedicated to RNA, carefully add 5×gDNA Eraser Buffer 2.0 μL; gDNA Eraser 1.0 μL; Total RNA 1.0 μg (convert the volume to be added according to the concentration of extracted RNA); RNase Free ddH ₂ O supplemented to 10 μL, and quickly put into the PCR machine at 42°C for 2 minutes.

2) cDNA synthesis

Add 5×PrimeScript Buffer 2 4.0 μL; PrimeScript RTEnzyme Mix 1.0 μL; RT Prime Mix 1.0 μL; RNase Free ddH ₂ O 4.0 μL to the PCR tube from which DNA has been removed, and then quickly put it into the PCR machine, 37°C for 15 minutes RNA was reverse transcribed at 85°C for 5 seconds to inactivate reverse transcriptase, and then stored at -20°C for future use (-80°C can be stored for a long time).

Example 2: Fluorescence quantitative PCR analysis

Bemisia tabaci in the field are severely resistant to the neonicotinoid insecticide thiamethoxam, but the resistance mechanism is still unclear. The inventors found that the CYP4C64 gene was overexpressed in the resistant population screened in the laboratory through preliminary research. Field population, and the expression of CYP4C64 gene was analyzed by fluorescence quantitative PCR to verify whether this gene is involved in the formation of drug resistance of B. tabaci in the field. Primer 2 in Table 1 was used to analyze the expression of CYP4C64 gene, primers 2 and 3 in Table 1 were selected as the internal reference gene, and the expression of CYP4C64 gene was determined by calculating the relative expression level. The SuperRealPreMixPlus kit of Tiangen Company was used for qPCR detection. The reaction system and reaction procedure are shown in Table 2. Each reactant was added according to the instructions, and then the primers were detected on ABI7500. The qRT-PCR data was calculated by the 2- ^ΔΔCt method. differences in gene expression.

Table 1 CYP4C64 gene fluorescent quantitative PCR primers

F, forward primer; R, reverse primer

Table 2 qRT-PCR reaction system and reaction program

Result analysis

The samples of B. tabaci were collected in the field, and two resistant lines were obtained, and the resistance folds reached a high resistance level.

table 3

The expression of CYP4C64 gene in thiamethoxam-resistant and sensitive lines was analyzed by fluorescence quantitative PCR, and it was found that the gene was overexpressed in the two resistant lines, as shown in Figure 1, the expression level of CYP4C64 gene was significant in the resistant lines Compared with the sensitive lines, the P450 gene CYP4C64 was 18-fold overexpressed in the resistant lines by comparing the Q-type B. tabaci resistant line (THR) and the sensitive line (THS) raised indoors.

Example 3: RNAi experiments in B. tabaci resistant lines

RNAi experiments were performed in resistant lines. Due to the small size of B. tabaci, RNAi by microinjection will cause high mortality, so this experiment adopts the feeding method to carry out the RNA interference experiment of B. tabaci.

1) Preparation of feeding nutrient solution

The feeding method for RNAi requires an environment outside the living plant that can maintain the normal growth of B. tabaci. Through exploration, it was found that the nutrient solution formed by mixing 30% sucrose and 5% yeast extract can maintain tobacco powder in vitro. Lice survive. First, prepare 30% sucrose solution and 5% yeast extract. After fully dissolving, use a 0.22 μm bacterial filter for filtration to prevent the growth of microorganisms during the experiment. High temperature and high pressure sterilization is not allowed, because sucrose gelatinizes at high temperature Toxic substances are generated, which is not conducive to the feeding of Bemisia tabaci.

2) Experimental setup

Prepare a transparent glass tube with a length of 50 mm and an inner diameter of 20 mm, a tube sleeve made of a sealing film, a black cotton plug and a shading cloth that can cover the above glass tube, and a lighted incubator that can control temperature and humidity. Glass tubes are used to place B. tabaci for in vitro experiments; Parafilm is used to make a feeding pouch containing nutrient solution at one end of the glass tube for B. tabaci to feed; black cotton plugs and tube sleeves are used to create a dark In the environment, there is only a light source in the direction of the feeding pouch. Using the phototropism of B. tabaci, B. tabaci quickly attaches to the direction of the feeding pouch for feeding. Otherwise, B. tabaci will adhere to other positions of the glass tube for a long time and cause experiments. uniformity is not good.

3) Bemisia tabaci feeding

First, dsRNA and exogenous control gene dsGFP were prepared, and the experimental concentration was 0.5 μg/μL using Bemisia tabaci nutrient solution. Take a small piece of parafilm, stretch the first layer as thin as possible, and cover one end of the glass tube, then add 200 μL of the above-prepared nutrient solution, and then take a small piece of parafilm and stretch it to cover 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. Placed in an incubator, the direction of the feeding pouch is facing the light source, and the cultivation conditions are 14:10 light and 80% humidity (higher humidity can prevent the nutrient solution from volatilizing). The above method was used to carry out the RNAi experiment of CYP4C64 gene in B. tabaci in the THQR population. The experimental results are shown in Figure 2. Knocking down the expression of this gene can significantly reduce the drug resistance of B. tabaci to thiamethoxam.

Example 4: Comparative analysis of B. tabaci CYP4C64 gene sequence

The primer 1 in Table 1 was used to further compare and analyze the CYP4C64 gene sequence, and it was found that there is a relatively stable mutation site in the 5'UTR region of the gene in the thiamethoxam-resistant population, which is located in the 5'UTR region of the CYP4C64 gene. 32 locus, which is mainly A base in thiamethoxam resistance, but mainly appears in the form of T base in sensitive populations. resistance levels of oxazine, see Figures 3 and 4 for experimental results.

Claims (2)

1. the application of the PCR primer pair of detecting the mutation site of CYP4C64 gene in detecting the resistance level of Q-type Bemisia tabaci population to thiamethoxam, it is characterized in that, described mutation site is in the 32nd base of sequence ATCGATCGGAAATTCGGGGCTGACGGACACGACAAACTGTTTGACGGACGGACGAGGTGTTTTAGTTTCTCGGGGTCAGCCTTGCCGTGCGGGCCGGATC The base is A or T; the total DNA of B. tabaci is extracted, PCR is carried out using the PCR primer pair, and the PCR product is recovered for sequencing and identification, and the ratio of A or T in the mutation site is detected. The frequency of A in the site was used to detect the resistance level of the Q-type B. tabaci population to thiamethoxam; the mutation site mainly appeared in the form of A base in the thiamethoxam-resistant Q-type B. tabaci population, so The mutation site mainly occurs in the form of T base in the thiamethoxam-sensitive Q-type whitefly population. 2. application according to claim 1 is characterized in that: the nucleotide sequence of described PCR primer pair is as follows: CYP4C64 Mutation Identification-F: ATCGATCGGAAATTCGGGGCTGA, CYP4C64 mutation identification-R: TTTGATGGAATTTTTTCCCGGTCATGC.

Images