CN107267632B - The new small peaceful mite of Pasteur is to the molecular labeling of avermectin resistance and its application and detection method - Google Patents

Abstract

The invention discloses a molecular marker of Neoseiusius pastereii's resistance to abamectin, the nucleotide sequence of which is shown in SEQ ID No.3, and primers 4F/4R for amplifying the marker; also disclosed The application of molecular markers in the classification of Neoseius pastereii to abamectin-sensitive and resistant strains; also disclosed the detection method of molecular markers of Neoseius pastereii's resistance to avermectin: first extract The total DNA of Neoseiius pasteurii to be tested is amplified by PCR with primers 4F/4R using the total DNA as a template to obtain a NbGluCl gene fragment containing a mutation region, and the obtained gene fragment is combined with the gene fragment shown in SEQ ID No.3 Sequence comparison of the molecular markers of Neoseius pasteurii's resistance to abamectin can determine whether the strain is sensitive or resistant to avermectin. The marker has strong specificity and high stability, and can analyze whether the sample to be tested is a sensitive strain or a resistant strain to abamectin.

Description

Molecular markers and their application and detection of resistance to abamectin in Neoseius pasterei method

technical field

The invention belongs to the technical field of molecular biology and relates to a molecular marker, in particular to a molecular marker for the resistance of Neoseiusius pasteri to abamectin and its application and detection method.

Background technique

Neoseiidi genus Acarina, Parasitica, Gamasinae, Phytoseiidae, and Neoseiidi genus, because of its short development period, low mortality rate, high oviposition rate, and spreading ability Strong advantages are considered to be one of the best biological control natural enemies. Neoseius pasteurii is a polyphagous predatory mite. In addition to preying on spider mites and thrips, it can also prey on aphids, psyllids, whiteflies, scale insects, springtails, tarsal mites, filamentous fungi and mosquitoes. larvae etc.

Abamectin is a new biological insecticide and acaricide, and it is a widely used pesticide preparation. It is mainly used to control Panonychus citrus in citrus orchards. The laboratory research shows that avermectin also has strong toxicity to Neoseiius pasteurii, and the LC ₅₀ value of indoor bioassay is 50.03mg/L, so it is easy to cause neoseiius pasteurii during the application process. A large number of seid mites died, and chemical control and biological control could not be coordinated.

The resistant Neoseiius pastereii was obtained through indoor screening, and after being released into the orchard, the predatory mite can effectively cope with the pesticide stress, which is very practical for the establishment of field populations of Neoseius pastereius and the comprehensive control of harmful mites value. At present, the resistance of mites to abamectin is generally believed to be caused by the gene mutation of its target glutamate-gated chloride channel gene (NbGluCl).

Contents of the invention

According to the blank in the field of classification of Neoseius pastereii to abamectin sensitive and resistant strains, the invention provides a molecular marker of Neoseius pastereii's resistance to avermectin.

Another object of the present invention is to provide the application of the molecular marker in the classification of the abamectin-sensitive and resistant strains of Neoseius pastereii.

The technical scheme adopted by the present invention to solve the technical problem: a molecular marker for the resistance of Neoseiusius pasteri to abamectin, the nucleotide sequence of the molecular marker is shown in SEQ ID No.3.

The present invention also provides a specific forward and reverse primer for the molecular marker of Neoseius pastereii's resistance to abamectin, and the primer sequence is as follows:

4F: 5'-AAACGAAACGGGCTACTGT-3';

4R: 5'-CTTCTGCGTGGACTCGTTA-3'.

The present invention also provides a method for detecting molecular markers of Neoseiius pasteurii's resistance to abamectin: firstly extract the total DNA of a single head of Neoseiius pasteurii to be tested, and use the total DNA as a template to use the right The specific forward and reverse primers in claim 3 are amplified by PCR to obtain the NbGluCl gene fragment comprising the mutation region, and the obtained gene fragment is combined with the molecule of Neoseius pasteurii in claim 1 to be resistant to abamectin Sequence comparison of markers can determine whether the strain is sensitive or resistant to abamectin.

The PCR system is: 25 μL of PCR Master Mix, 2 μL of forward and reverse primers, 50-100 ng of DNA template of the sample to be tested, and supplemented with ddH ₂ O to 50 μL.

The procedure of the PCR amplification is: pre-denaturation at 94°C for 3 min; then denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 1 min, 35 cycles; and finally extension at 72°C for 5 min.

The beneficial effects of the present invention are:

1) The present invention locates the molecular marker of Neoseius pastereii's resistance to abamectin. The marker has strong specificity and high stability, and through the analysis of the molecular marker of the Neoseiidus pasteurii sample to be tested, it can be known whether the sample to be tested is a sensitive strain or a resistant strain to abamectin. Therefore, the drug resistance of predatory mites can be monitored at any time, which is beneficial to the biological control strategy of "killing insects with insects", and is of great significance for reducing the use of chemical pesticides, environmental protection, and sustainable development of orchards.

2) The present invention provides the specific forward and reverse primers of the molecular markers of Neoseiius pasteruii resistance to abamectin, and also provides an optimized PCR system and amplification program, which can successfully and rapidly obtain the abamectin-resistant A 499bp DNA band was amplified from the mite, which is the sequence closely related to the resistance of Neoseius mite to abamectin. The method is simple and quick to operate, with strong detection specificity and sensitivity high.

Description of drawings

Figure 1 shows the SNP analysis of NbGluCl gene in the resistant and sensitive strains of Neoseiius pasteurii, SS: sensitive strain; RS: resistant strain.

Figure 2 shows the difference analysis of the amino acid sequences corresponding to the NbGluCl gene in the resistant and sensitive strains of Neoseius pasterii, SS: sensitive strain; RS: resistant strain.

Detailed ways

Example 1 Clone and SNP Analysis of Glutamate-gated Chloride Channel Gene of Neoseiusius mite Resistance and Sensitivity Strains

1. Materials

1.1 Source of tested mite

Mite source tested

Sensitive strain: Neoseiius _pasteruii was collected from the lemon leaves around the orchard of the Citrus Research Institute of the Chinese Academy of Agricultural Sciences. After collection, it was reared indoors for multiple generations without contact with pesticides. At lower levels, there was no apparent resistance to abamectin.

Resistant strain: using Neoseius pastereius sensitive strain as the raw material for resistance screening, after multiple generations of screening, the LC ₅₀ of Neoseius pastereii to abamectin increased from 50.03mg/L to 20572.3mg/L L, the resistant population increased by 411.2 times compared with the sensitive population. After that, it will be treated with chemicals at regular intervals depending on its population density to ensure that its resistance will not decline.

The above-mentioned Neoseius pastereii are known materials, which are preserved in our laboratory and can be distributed to the public for verification tests.

1.2 Reagents

The reagent Kit is a reverse transcription kit (TaKaRa Company, Japan), a DNA purification and recovery kit (Tiangen Company, China), a pMD-19T vector kit (TaKaRa Company, Japan), and DH5α competent cells (Tiangen Company, Japan). China), PCR Master Mix (Tiangen, China), Trizol (Invitrogen, USA).

2. Method

2.1 Extraction of total RNA from resistant and sensitive strains of Neoseius pasterii

Total RNA was extracted from the resistant and sensitive strains of Neoseius pastereius using Trizol reagent, respectively.

2.2 Synthesis of cDNA

The total RNA obtained above was used The RT reagent Kit reverse transcription kit was used to reverse transcribe into cDNA. The method steps were carried out according to the kit instructions. The reaction system was 20 μL, and the reaction conditions were: 37°C, 15min; 85°C, 5sec.

2.3 Cloning of the glutamate-gated chloride ion channel gene and designed primers

Through manual search, the glutamate-gated chloride channel gene NbGluCl was found in the transcriptome database of Neoseius pasterii.

Three pairs of primers as shown in Table 1 were designed to amplify the NbGluCl gene. The reaction system was: PCR Master Mix 25 μL, ddH ₂ O 18 μL, forward and reverse primers 2 μL each, and the cDNA template obtained above 3 μL. Reaction conditions: pre-denaturation at 94°C for 3 min, followed by denaturation at 94°C for 30 sec, annealing at 57°C for 30 sec, extension at 72°C for 1 min, a total of 35 cycles, and finally extension at 72°C for 5 min.

Table 1 NbGluCl gene amplification primers

The PCR products were detected by electrophoresis on 1% agarose gel (120V, the electrophoresis buffer was 1% TAE), stained with ethidium bromide (EB), photographed, and the amplification patterns were compared and analyzed. Cut out the target band under ultraviolet light, use the DNA purification and recovery kit to recover the target fragment, and connect the recovered target fragment to the PMD19-T carrier at a molar ratio of 3:1 at 16°C overnight; the connection solution is transformed into DH5α Competent Cells, spread the transformed bacteria on LB solid medium containing Amp+IPTG+X-Gal, culture overnight for 12-16 hours, and finally pick white single colonies, and send the bacterial solution to Huada Sequencing Company after PCR verification sequencing.

The cloned sequences were spliced and assembled using DNAMAN5.2.2 (Lynon Biosoft) software. The DNA sequences were compared by Blast using the online website NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Finally, the full-length NbGluCl gene (respectively SEQ ID NO.1 and SEQ ID NO.2) of the 1689bp NbGluCl gene sensitive and resistant to abamectin-sensitive strains of Neoseius pastereii was obtained.

Example 2 Obtaining of molecular markers of Neoseiius pasteurii's resistance to abamectin

The sequence comparison software BioXM2.6 was used to compare the target genes of the sensitive strain and the resistant strain. The comparison results are shown in Figure 1. It was found that there was a mutation base at the 1202 position, and the G base mutation of the sensitive system was resistant The A base of the strain. At the same time, the mutation of the base eventually led to the change of the amino acid sequence. As shown in Figure 2, the base fragment was translated into an amino acid fragment using the BioXM2.6 software, and the amino acid mutation was found. The glycine (G) mutation of the sensitive strain at the 401st position Aspartic acid (D) is the resistant line.

According to the location of the resistance mutation site, the resistance marker of Neoseiusius pasterei was carried out:

1) Primer design: select the nucleotide sequences near the upstream of site 912 and the downstream of site 1392 of the target gene, and use Primer Premier 5 primer design software to design a primer pair 4F/4R that can quickly clone the region where the mutation site is located , the sequence is 4F: 5'-AAACGAAACGGGCTACTGT-3'; 4R: 5'-CTTCTGCGTGGACTCGTTA-3', the 499bp base containing all the mutation regions can be cloned-that is, the resistance of Neoseiusius pasteruii to abamectin The molecular marker, the nucleotide sequence of the molecular marker is shown in SEQ ID No.3. PCR amplification reaction system: PCRMaster Mix 25 μL, ddH ₂ O 18 μL, 4F/4R specific positive and negative primers 2 μL each, sample DNA template to be tested 3 μL. PCR reaction conditions: pre-denaturation at 94°C for 3 min, then denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 1 min, a total of 35 cycles, and finally extension at 72°C for 5 min.

2) Mutation frequency detection: Crossbreed 100 unmated female Neoseius mite-sensitive strains (SS♀) and 100 male Neoseiidi mite-resistant strains (RR♂) to obtain hybrid F2 generation group. In the experimental group, F2 was treated with 500mg/L avermectin, and the surviving individuals were used as experimental materials; in the control group, F2 generations were treated with water, and the surviving individuals were used as experimental materials. Take 30 Neoseius mite from the experimental group and the control group respectively, and use Tissue Ex-Amp PCRKit (ABM Company) and the above-mentioned specific primers 4F/4R to carry out PCR amplification on a single Neoseius mite, The genotype (G/G, G/D, D/D) and mutation frequency of each mite were detected. The target fragment of the PCR product was recovered and sent to a sequencing company for sequencing. The results are shown in Table 2. The frequency of mutation of glycine (Gly) to aspartic acid (Asp) in the F2 generation experimental group was 96.70%, indicating that after avermectin treatment, survival Most of the descended individuals had Asp mutation sites, while the frequency of Asp mutations in the clear water control group was 48.3%, which was consistent with the trait segregation ratio. The above monitoring results can show that G401D is linked with the resistance traits of Neoseiius pasteurii.

Table 2 The frequency of genotype mutations in the F2 generation of Neoseius pakistan

sequence listing

<110> Southwest University

<120> Molecular marker and its application and detection method of Neoseius pastereii's resistance to abamectin

<160> 11

<210> 1

<211> 1689

<212>DNA

<213> Artificial sequence

<220>

<223> Glutamate-gated Chloride Channel Gene of Neoseius pastereii Sensitive to Abamectin

<400> 1

atgttgcaag acgtatacaa caaccaccac gaaaacagcc ttagctgcaa cgaacacgac 60

actacaagca gcagcagtag cagctgcgcc agcggcagca gtagcagcgg aagacgtcag 120

tcagaaaccc gagcagacaa taaacttcca gttttccgaa atgaacaatc tacggcggag 180

aggagaacgg agttgccgag aagaacattc accttggcca tcgtggctgc tatcgcagtt 240

ctcagcgtcc cggtggtcgg cgttgaagct cggcccgcag accacggag tcagatcgcc 300

catcagttta tgaactatac agataagcag atcctggatt atctcgttaa caacagcaga 360

tacgactaca ggaacagacc cgaagaccac acaagggtga atgtcagcgt cttattgctc 420

agcatgtcct caccggatga atccagtttg aaatacgaga tcgagtttct gctgatccaa 480

aaatggatcg atcagcgtct ggcgtatcac gaaaccggct cgaatcattc gcatctgaat 540

ggccttctac acgaagggaa aatttggaag cccgacatct acttcatcaa gcacggtctc 600

gaagatgaaa ctaaggagca cgaggagtgc accgagtatt gtggtcaagg agagtttaag 660

actccactca atccggttca tatgagcctt cgtctttatc ccaatggcac tgtcgtctac 720

acgatgagaa gacacatgac cctggtctgt caggggaacc tacaaatatt cccattcgac 780

aacccgaagt gcccattcgc cgtcgagtcg atgtcgtatg aggagagcca gctcaagatt 840

gactggtccc aggaagagga aaacatcacg agggcgtcct cacttcgtgc gctaaatgcc 900

tatctcgcca aaaacgaaac gggctactgt gacaagaggc acacgtggcg gggcaattat 960

tcctgcctga gagttctgct cgtattcacg agagacaagt cattctacat ttcaacggtg 1020

ttcgtacccg gcatcgtgct tgtgacctct tcattcatct ctttttggct tgatatcaac 1080

gccgtacccg cccgagtaat gatcggagta acaaccatgt tgaatttttg tacaaccacc 1140

aactcattcc ggtcgacact ccccgtcgtc tcgaacctga cggcaatgaa tctgtgggac 1200

ggcgtatgca tgtttttcat ctacgcatca atgctggaat ttgtaatcgt gaactacctc 1260

taccgtaacc tgggccagca tactcgtcac agaagataca gttacgcgtc ttcggtggcc 1320

tcaaagaatc atccacaagc aacgccactt cttcaacaga acaacaataa acacaatgtc 1380

tatgttaagg gtaacgagtc cacgcagaag acggaaacca cgacgttcgt cgtggacccg 1440

cctgaagaag aacaggaccg gggtttcggt cgatttcgaa ctgtggcaat gctattcgtc 1500

tggcttcgga accccacaaga gttctcaggg cggtttcgaa tgcccaagcc acgcctcgcg 1560

aaagcaatcg actacttctc acgaacagtg ttccccgtgttcttcggagt gttcactttt 1620

ggcttcttcc tcaattatgc tgtcatcggt ccgctgtctg aagaaaactg gagacttatt 1680

gaatattaa 1689

<210> 2

<211> 1689

<212>DNA

<213> Artificial sequence

<223> Glutamate-gated Chloride Channel Gene of Neoseius pastereii to Abamectin-resistant Strain

<400> 2

atgttgcaag acgtatacaa caaccaccac gaaaacagcc ttagctgcaa cgaacacgac 60

actacaagca gcagcagtag cagctgcgcc agcggcagca gtagcagcgg aagacgtcag 120

tcagaaaccc gagcagacaa taaacttcca gttttccgaa atgaacaatc tacggcggag 180

aggagaacgg agttgccgag aagaacattc accttggcca tcgtggctgc tatcgcagtt 240

ctcagcgtcc cggtggtcgg cgttgaagct cggcccgcag accacggag tcagatcgcc 300

catcagttta tgaactatac agataagcag atcctggatt atctcgttaa caacagcaga 360

tacgactaca ggaacagacc cgaagaccac acaagggtga atgtcagcgt cttattgctc 420

agcatgtcct caccggatga atccagtttg aaatacgaga tcgagtttct gctgatccaa 480

aaatggatcg atcagcgtct ggcgtatcac gaaaccggct cgaatcattc gcatctgaat 540

ggccttctac acgaagggaa aatttggaag cccgacatct acttcatcaa gcacggtctc 600

gaagatgaaa ctaaggagca cgaggagtgc accgagtatt gtggtcaagg agagtttaag 660

actccactca atccggttca tatgagcctt cgtctttatc ccaatggcac tgtcgtctac 720

acgatgagaa gacacatgac cctggtctgt caggggaacc tacaaatatt cccattcgac 780

aacccgaagt gcccattcgc cgtcgagtcg atgtcgtatg aggagagcca gctcaagatt 840

gactggtccc aggaagagga aaacatcacg agggcgtcct cacttcgtgc gctaaatgcc 900

tatctcgcca aaaacgaaac gggctactgt gacaagaggc acacgtggcg gggcaattat 960

tcctgcctga gagttctgct cgtattcacg agagacaagt cattctacat ttcaacggtg 1020

ttcgtacccg gcatcgtgct tgtgacctct tcattcatct ctttttggct tgatatcaac 1080

gccgtacccg cccgagtaat gatcggagta acaaccatgt tgaatttttg tacaaccacc 1140

aactcattcc ggtcgacact ccccgtcgtc tcgaacctga cggcaatgaa tctgtgggac 1200

gacgtatgca tgtttttcat ctacgcatca atgctggaat ttgtaatcgt gaactacctc 1260

taccgtaacc tgggccagca tactcgtcac agaagataca gttacgcgtc ttcggtggcc 1320

tcaaagaatc atccacaagc aacgccactt cttcaacaga acaacaataa acacaatgtc 1380

tatgttaagg gtaacgagtc cacgcagaag acggaaacca cgacgttcgt cgtggacccg 1440

cctgaagaag aacaggaccg gggtttcggt cgatttcgaa ctgtggcaat gctattcgtc 1500

tggcttcgga accccacaaga gttctcaggg cggtttcgaa tgcccaagcc acgcctcgcg 1560

aaagcaatcg actacttctc acgaacagtg ttccccgtgttcttcggagt gttcactttt 1620

ggcttcttcc tcaattatgc tgtcatcggt ccgctgtctg aagaaaactg gagacttatt 1680

gaatattaa 1689

<210>3

<211> 499

<212>DNA

<213> Artificial sequence

<223> Molecular markers of Neoseius pastereii's resistance to abamectin

<400> 3

aaacgaaacg ggctactgtg acaagaggca cacgtggcgg ggcaattatt cctgcctgag 60

agttctgctc gtattcacga gagacaagtc attctacatt tcaacggtgt tcgtacccgg 120

catcgtgctt gtgacctctt cattcatctc tttttggctt gatatcaacg ccgtacccgc 180

ccgagtaatg atcggagtaa caaccatgtt gaatttttgt acaaccacca actcattccg 240

gtcgacactc cccgtcgtct cgaacctgac ggcaatgaat ctgtgggacg acgtatgcat 300

gtttttcatc tacgcatcaa tgctggaatt tgtaatcgtg aactacctct accgtaacct 360

gggccagcat actcgtcaca gaagatacag ttacgcgtct tcggtggcct caaagaatca 420

tccacaagca acgccacttc ttcaacagaa caacaataaa cacaatgtct atgttaaggg 480

taacgagtcc acgcagaag 499

<210>4

<211> 21

<212>DNA

<213> Artificial sequence

<223> 1F

<400> 4

atgttgcaag acgtatacaa c 21

<210>5

<211> 21

<212>DNA

<213> Artificial sequence

<223> 1R

<400> 5

cttgatgaag tagatgtcggg g 21

<210>6

<211> 20

<212>DNA

<213> Artificial sequence

<223> 2F

<400> 6

cattcgcatc tgaatggcct 20

<210>7

<211> 20

<212>DNA

<213> Artificial sequence

<223> 2R

<400> 7

atggttgtta ctccgatcat 20

<210>8

<211> 20

<212>DNA

<213> Artificial sequence

<223> 3F

<400> 8

gagacaagtc attctacatt 20

<210>9

<211> 20

<212>DNA

<213> Artificial sequence

<223> 3R

<400> 9

atattcaata agtctccagt 20

<210>10

<211> 19

<212>DNA

<213> Artificial sequence

<223> 4F

<400> 10

aaacgaaacg ggctactgt 19

<210>11

<211> 19

<212>DNA

<213> Artificial sequence

<223> 4R

<400> 11

cttctgcgtg gactcgtta 19

Claims (6)

1. new molecular labeling of the small peaceful mite to avermectin resistance of Pasteur, the nucleotide sequence of the molecular labeling such as SEQ ID Shown in No.3.

2. molecular labeling described in claim 1 is in the new small peaceful mite of Pasteur to avermectin sensitive strain and the non-diagnostic mesh of resistant strain Classification in application.

3. detecting forward and reverse primer of molecular labeling described in claim 1, it is characterised in that: the primer sequence is as follows:

4F:5 '-AAACGAAACGGGCTACTGT- 3 '；

4R:5 '-CTTCTGCGTGGACTCGTTA-3 '.

4. a kind of new small peaceful mite of Pasteur exists to the non-diagnostic purpose detection method of the molecular labeling of avermectin resistance, feature In: the total DNA of the new small peaceful mite of Pasteur to be measured is first extracted, is carried out by forward and reverse primer in template claim 3 of total DNA PCR amplification is obtained comprising sudden change regionNbGluClGenetic fragment, by the Pasteur in obtained genetic fragment and claim 1 New small peaceful mite carries out sequence alignment to the molecular labeling of avermectin resistance, that is, can determine whether that the strain is the sensitivity to avermectin Strain or resistant strain.

5. the new small peaceful mite of Pasteur according to claim 4 detects the non-diagnostic purpose of the molecular labeling of avermectin resistance Method, it is characterised in that: the reaction system of the PCR amplification are as follows: PCR Master Mix 25 μ L, forward and reverse each 2 μ L of primer, Sample to be tested 50~100ng of DNA profiling supplements ddH₂O to 50 μ L.

6. the new small peaceful mite of Pasteur according to claim 4 or 5 is to the non-diagnostic purpose of the molecular labeling of avermectin resistance Detection method, it is characterised in that: the program of the PCR amplification are as follows: 94 DEG C of initial denaturation 3min；Then 94 DEG C of denaturation 30sec, 58 DEG C Anneal 30sec, 72 DEG C of 1 min of extension, 35 circulations；Last 72 DEG C of extensions 5min.