CN103014159B - Molecular detection method for Cry1Ac toxin non-recessive resistance cadherin cytomere deletion mutation of cotton bollworm - Google Patents

Abstract

The invention discloses a molecular detection method for the non-recessive resistance cadherin cytoplasmic region deletion mutation of the cotton bollworm to Cry1Ac toxin. The detection method is divided into three main steps, the first step: extract the genomic DNA of the cotton bollworm samples; the second step: perform PCR amplification of specific cadherin cytoplasmic region fragments; the third step: carry out the PCR product agar Sugar gel electrophoresis detection, according to the electrophoretic pattern, can accurately identify the genotype, and use this to determine the frequency of resistance genes in different populations. The method of the present invention does not need to raise test insects, saves labor and resources, and can quickly and accurately detect the genotype of cotton bollworm. It only takes a few hours for a single test insect, and it only takes a few weeks to collect and identify the frequency of resistant individuals in field populations. The identification results can provide correct guidance for effective resistance control of cotton bollworm in time.

Description

Molecular detection method of non-recessive resistance to Cry1Ac toxin in cotton bollworm cadherin cytoplasmic domain deletion mutation

technical field

The invention relates to biotechnology, and relates to a molecular detection method for non-recessive resistance cadherin cytoplasmic region deletion mutation of Cry1Ac toxin in cotton bollworm.

technical background

Transgenic Bt cotton expressing Cry1Ac toxin protein has been popularized and applied in my country since 1997. At present, it has been planted on a large scale in the cotton areas of the Yellow River Basin and the Yangtze River Basin, accounting for about 75% of the national cotton planting area on average. Due to the high-intensity selection pressure of large-scale promotion and application of transgenic Bt cotton, its target pest cotton bollworm (Helicoverpa armigera) has developed resistance to Cry1Ac toxin, which is the biggest threat to the use of Bt cotton. The current research shows that the mutation of the intestinal parietal cell cadherin gene in the cotton bollworm is the main reason for the resistance. Cadherin is a kind of Ca2 ⁺ -dependent glycoprotein that plays a decisive role in cell adhesion and participates in various cellular activities. It is composed of 12 repeats of extracellular region, transmembrane region and smaller intracellular region . The gene mutations found in the past are all located in the extracellular region of cadherin. The insertion or fragment deletion of the transposon leads to the premature termination of cadherin translation and the loss of the toxin binding region, thereby producing resistance to Cry1Ac. This function Resistance conferred by lossy mutations is usually recessive.

In 2009, a total of 230 field cotton bollworms in Xiajin, Shandong were detected by F1 single-pair detection method, and 67 individuals carrying resistance genes were detected. According to the principle of F1 detection (Zhang et al., Diverse genetic basis of field-evolved resistance to Bt cotton in cotton bollworm from China. PNAS, 2012, 109(26): 10275-10280), these resistant individuals were compared with the indoor resistant strain SCD- r1 single-pair hybridization (Yang et al., Introgression of adisrupted cadherin gene enables sustainable Helicoverpa armigera to obtain resistance to Bacillus thuringiensis toxin Cry1Ac.Bulletin of Entomological Research, 200,99(2):175-181), the _F1 generation was diagnosed with a dose (1μg /cm ² Cry1Ac) treated surviving individuals should contain two cadherin alleles, one from the SCD-r1 strain (r ₁ ) (Yang et al., Identification and molecular detection of a deletion mutation responsible for a truncated casherin of Helicoverpa armigera.Insect Biochemistry and Molecular Biology, 2006, 36(9):735-740), and another from a field individual (r _x ). The resistance of the SCD-r1 strain is caused by the cadherin mutant gene r ₁ , which lacks most of the extracellular region and the entire transmembrane region and cytoplasmic region, so it can be selectively cloned by 4 sets of _specific primers The cadherin gene r _x of resistant individuals in the field (Zhao et al., Diverse cadherin mutations conferring resistance to toxin Cry1Ac in Helicoverpa arimgera., 2010, 40(2):113-118). The cadherin gene sequences of 20 field-resistant individuals were cloned, and a new cadherin mutant gene r ₁₅ was identified. Sequencing results showed that exon 32 located in the cytoplasmic region of cadherin was missing 165 bases, resulting in the deletion of 55 amino acids encoded by it. This mutation in the cytoplasmic region of cadherin can significantly reduce the toxicity of Cry1Ac, thereby lead to resistance. (See attached drawing 1, see attached drawing 2).

Field monitoring at multiple sites over the years showed that although the resistance level of cotton bollworm to Bt cotton did not increase significantly, the frequency of resistance genes to Cry1Ac toxin in the field increased year by year, especially the frequency of newly discovered non-recessive resistance genes increased significantly. At present, the main resistance monitoring techniques include: dose logarithm-death probability value method (resistance multiple method), F1 detection method, F2 detection method, diagnostic dose detection method and molecular detection method. However, the above four resistance monitoring methods have obvious shortcomings in the monitoring of the resistance of cotton bollworm to Bt cotton, such as the resistance gene type cannot be detected, it is difficult to determine the appropriate diagnostic dose, and the corresponding resistant strains must be available indoors, which is costly. , takes a long time, etc., resulting in inaccurate and delayed resistance monitoring, which seriously affects the development and implementation of subsequent effective resistance management. The molecular detection method can directly detect the resistance genotype, with high accuracy, no requirement for the state of the sample to be tested, no requirement for the dominant or recessive resistance gene, and has flexibility and stability.

Contents of the invention

The frequency of non-recessive resistance genes in field cotton bollworms due to the deletion mutation of cadherin cytoplasmic region increased significantly, and there were three different types at the genomic DNA level. The object of the present invention is to be beneficial to the PCR method, according to the characteristics of the deletion mutation sequence of the cytoplasmic region of cadherin, establish a fast and accurate molecular detection method, and clarify the non-recessive resistance gene frequency caused by this type of mutation, and to the cotton bollworm. Bt cotton resistance monitoring and management provide guidance and have practical value.

DNA molecular detection method of three genotypes of cadherin cytoplasmic region deletion mutations of non-recessive resistance to Cry1Ac toxin in cotton bollworm, designing specific primers, and using PCR technology to detect individual cadherin cytoplasm in cotton bollworm populations Whether there is a deletion of 55 amino acids in exon 32 of the intracellular region due to three different insertions or deletions.

The DNA molecular detection method of three genotypes of the cadherin cytoplasmic region deletion mutation of non-recessive resistance to Cry1Ac toxin in cotton bollworm of the present invention preferably comprises the following three steps:

The first step: extract the genomic DNA of cotton bollworm larvae or adults;

Second step: Utilize primer A shown in SEQ ID NO.1 and primer B shown in SEQ ID NO.2, PCR amplifies the cotton bollworm genomic DNA that last step extracts;

The third step: Perform 1% agarose gel electrophoresis on the product obtained by PCR in the previous step, and accurately identify the deletion mutation of the cadherin cytoplasmic region of the individual cotton bollworm and its specific type according to the electrophoresis pattern, and distinguish the resistant homozygote, Resistant heterozygotes and susceptible individuals.

The r _15-1 mutation is a 1459bp fragment inserted into exon 32, and the size of the PCR amplification product obtained is about 2500bp. FJ997310.1) has 95% similarity in the 3' non-coding region. If the electrophoresis results only show this band, it is a homozygous resistance to the mRNA deletion mutation caused by the insertion of genomic DNA in the intracellular region of cadherin. If there are two bands of 2500bp and 1000bp in the electrophoresis, it is a resistant heterozygote (as shown in Figure 3, lane 1 and lane 2). The r _15-2 mutant allele lacks 82 bp of DNA sequence on exon 32, and the resulting amplified product is 750 bp. If the electrophoresis results only show this band, it is a deletion of genomic DNA in the intracellular region of cadherin And the resistance homozygote caused by deletion mutation of exon 32 of mRNA, if there are two bands of 750bp and 1000bp in electrophoresis, it is a resistance heterozygote (as shown in Figure 3, lane 3 and lane 4). The r _15-3 mutation is formed by the insertion of a fragment of about 4000 bp on exon 32, and the amplified product is about 5000 bp. If the electrophoresis results only show this band, it is caused by the insertion of genomic DNA in the intracellular region of cadherin If the resistance homozygote for the mRNA deletion mutation of the gene has 5000 bp and two bands in the electrophoresis, it is a resistance heterozygote (see Figure 3, lane 5 and lane 6). If the PCR amplified fragment is only 1000bp (as shown in Figure 3, lane 7), it is a sensitive individual.

The method of the present invention may also include judging the frequency of the resistance gene within the population based on the result of the third step.

Beneficial effects: the present invention is based on the basis of previous indoor research, and has developed a genomic DNA molecular detection technology for deletion mutations in the cytoplasmic region of cadherin, which has (1) fast, accurate and simultaneous detection of mutant genomes in the cytoplasmic region of cadherin Three different types at the DNA level, (2) determine the genotype of the mutated individual, calculate the individual frequency of the non-recessive resistance gene related to the mutation, (3) the whole process takes 5-8 hours, etc.

Compared with traditional resistance monitoring techniques, this method eliminates the need to raise field test insects to an appropriate age, conduct bioassays to determine resistance levels, and then conduct genetic crosses with existing resistant and sensitive lines in the laboratory to determine the level of resistance. Determine the dominant and recessive levels of resistance, and finally carry out genotype identification. If it takes several months or even longer to complete the above work, the actual guiding significance of the results to production is seriously lagging behind. However, the method of the present invention does not need to raise test insects, saves labor and resources, and can quickly and accurately detect the non-recessive resistance cadherin cytoplasmic region deletion mutation of cotton bollworm to Cry1Ac toxin, as well as the variation type of specific DNA. It only takes a few hours. If a representative test insect population in the field is collected and identified, it only takes a few weeks to obtain the frequency of resistance genes in the population, and the identification results can provide correct guidance for effective resistance management of cotton bollworm in time.

Description of drawings

Figure 1 Comparison of the amino acid sequences of the cadherin gene of the cotton bollworm SCD strain and the r15 allele. The same sequence is indicated by a dot, and the horizontal arrow indicates the predicted starting point of the domain. SIG, signal peptide region; EC, cadherin repeat region; MPR, proximal membrane region; TM, transmembrane region; CYT, cytoplasmic region. Black boxes indicate missing fragments. SCD-cadherin gene sequence GenBank accession number: JN836544.1, r15 sequence GenBank accession number: JN898956.1.

Fig. 2 Schematic diagram of the structure of cadherin in cotton bollworm.

A: Cotton bollworm cadherin structure, which is divided into extracellular region, transmembrane region and intracellular region, in which the extracellular region includes signal peptide region (SIG), 11 cadherin repeat subregions (1-11) and Proximal membrane region (MPR).

B: The structure of the cadherin genome of the cotton bollworm.

The deletion mutation in the intracellular region was due to the insertion of a 1459bp or 4000bp DNA fragment or the deletion of an 82bp DNA fragment on the 33rd exon of its genomic DNA, resulting in a deletion of 55 amino acids in the intracellular region.

Fig. 3 is a diagram of PCR amplification electrophoresis results of Example 1, which is used to verify the deletion mutation of the cytoplasmic region of cadherin in cotton bollworm and the corresponding genomic DNA mutation type.

Different lanes represent genotypes with different mutations, and each lane represents 1:r _15-1 r _15-1 ;2:r _15-1 s;3:r _15-2 r _15-2 ;4:r _{15- 2} s;5:r _15-3 r _15-3 ;6:r _15-3 s;7:ss;M:DNA Ladder. The r _15-1 mutation amplified fragment was 2500bp, the r _15-2 mutation amplified fragment was 750bp, the r _15-3 mutation amplified fragment was 5000bp, and the ss amplified fragment was 1000bp.

Figure 4 is a diagram of PCR amplification electrophoresis results of Example 2, which is used to determine the deletion mutation of the cytoplasmic region of cadherin and the corresponding genomic DNA mutation type in the field population of cotton bollworm.

Different lanes represent genotypes with different mutations, and each lane represents 1,2:r _15-2 r _15-2 , which is a resistant homozygote; 3,4,5:r _15-2 s, which is a resistant heterozygote Zygote 6, 7: ss, is a sensitive individual; M: DNA Ladder.

Detailed ways

Example 1

Step 1: extraction of genomic DNA from single cotton bollworm larvae or adults.

(1) Obtain the larvae of cotton bollworm confirmed to be resistant homozygous, resistant heterozygous and sensitive individuals through biological assay methods and genetic crossing with sensitive strains respectively.

(2) Extract larval genomic DNA using a commercially available DNA extraction kit. This method uses the AxyPrep Genomic DNA Mini Kit from Ai Sijin Biotechnology (Hangzhou) Co., Ltd.

(3) Take 30 mg of abdominal tissue of 3-4 instar larvae or adults of cotton bollworm, add 350 mL of 0.01mol/LPH7.4 phosphate buffer solution and 0.9 μL RNaseA (50 mg/mL) solution into a 1.5 mL centrifuge tube, and grind evenly . Collect 350μL tissue homogenate in a 2mL centrifuge tube.

(4) Add 150 μL of lysate (Buffer C-L) and 20 μL of proteinase K (15 mg/mL) solution, vortex for 1 minute, centrifuge briefly, and place it in a 56-degree water bath for 10 minutes.

(5) Add 350 μL of protein removal solution (Buffer P-D), vortex for 30 seconds, and centrifuge at 12,000×g for 10 minutes.

(6) Put the DNA preparation tube into another 2mL centrifuge tube, transfer the supernatant in (5) to the preparation tube, and centrifuge at 12,000×g for 1 minute.

(7) Discard the filtrate, put the preparation tube back into the original 2mL centrifuge tube, add 500 μL of washing solution (Buffer W ₁ ), and centrifuge at 12,000×g for 1 minute.

(8) Discard the filtrate, put the preparation tube back into the original 2mL centrifuge tube, add 700μL desalted solution (Buffer W ₂ ), and centrifuge at 12,000×g for 1 minute.

(9) Discard the filtrate, put the preparation tube back into the original 2mL centrifuge tube, and centrifuge at 12,000×g for 1 minute.

(10) Discard the filtrate, put the preparation tube into another clean 1.5mL centrifuge tube, add 100-200μL of 2.5mmol/L Tris-HCL solution to the center of the preparation tube membrane, and centrifuge at 12,000×g for 1 minute to obtain a genomic DNA solution , Save at -20 degrees for later use.

In the second step, the PCR amplification of the cadherin-specific gene fragment is performed on the genomic DNA template extracted in the first step.

(1) According to the sequence characteristics of the deletion of exon 32 in the cytoplasmic region of cadherin of cotton bollworm, specific PCR primers were designed, which can simultaneously detect three different insertions or deletions at the genomic DNA level through amplification.

The sequence of the upstream primer A is: TGATTGTTGTTGTGTGCTGCTTATTGTG (SEQ ID NO.1)

The sequence of the downstream primer B is: CGATCAGGTCTGAGTCGTATGAC (SEQ ID NO.2)

(2) Set up a PCR reaction system with a total reaction volume of 25 μL in a 0.2 mL PCR tube.

(3) The PCR reaction conditions are: pre-denaturation at 94°C for 3 minutes, denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, extension at 72°C for 5 minutes and 30 seconds, 30 cycles, and finally extension at 72°C for 10 minutes. After the reaction, the reaction product was stored at 4°C for subsequent agarose gel electric pulse detection.

In the third step, the PCR product obtained in the second step is subjected to agarose gel electric pulse detection.

(1) Prepare 1% (g/mL) agarose gel.

(2) Take 10 μL of the PCR amplification product for electrophoresis at a steady flow of 80 mA, and observe under ultraviolet light after 1.5 hours.

From the results of electrophoresis, it can be seen that the pair of PCR primers amplified three types for the deletion mutation of 55 amino acids in the intracellular region of the cadherin gene of cotton bollworm, which can be identified simultaneously according to the size of the electrophoresis band, and can be distinguished at one time Resistant homozygotes, resistant heterozygotes and sensitive individuals were obtained, and the results are shown in Figure 3.

Three mutations caused by insertion or deletion of DNA lead to deletion of exon 32 at the mRNA level of the cytoplasmic region of cadherin.

The r _15-1 mutation is a 1459bp fragment inserted into exon 32, and the size of the PCR amplification product obtained is about 2500bp. The 3' UTR of FJ997310.1) has 95% similarity. The electrophoresis results of the resistance homozygote only show this band (as shown in Figure 3, lane 1), and the resistance heterozygote electrophoresis has two 2500bp and 1000bp Bands (as shown in Figure 3, Lane 2). The r _15-2 mutant allele lacks 82bp of DNA sequence on exon 32, and the amplified product is 750bp (as shown in Figure 3, lane 3), and the electrophoresis results of resistant homozygotes only show this band , There are two bands of 750bp and 1000bp in the electrophoresis of resistant heterozygotes (as shown in Figure 3, lane 4). The r _15-3 mutation is formed by inserting a fragment of about 4000bp on exon 32, and the amplified product is about 5000bp (as shown in Figure 3, lane 5). There are two bands of 5000bp and 1000bp in the electrophoresis of sex heterozygotes (see Figure 3, lane 6). The PCR amplified fragment of the sensitive individual is only 1000bp (as shown in Figure 3, lane 7).

Example 2

The following describes the preferred technical scheme of the present invention for the detection method of cadherin intracellular region deletion mutation gene frequency in the cotton bollworm population

Step 1: extraction of genomic DNA from single cotton bollworm larvae or adults.

(1) Collect cotton bollworm larvae directly from the field or use high-pressure mercury lamps to trap cotton bollworm adults, soak the obtained test insects in 95% ethanol and bring them back to the laboratory, and randomly select 100 cotton bollworm larvae or adult individuals for gene Frequency detection.

(2) Use a commercially available DNA extraction kit to extract genomic DNA from larvae or adults. This method uses the AxyPrep Genomic DNA Mini Kit from Ai Sijin Biotechnology (Hangzhou) Co., Ltd.

(3) Take 30 mg of abdominal tissue of 3-4 instar larvae or adults of cotton bollworm, add 350 mL of 0.01mol/LPH7.4 phosphate buffer solution and 0.9 μL RNaseA (50 mg/mL) solution into a 1.5 mL centrifuge tube, and grind evenly . Collect 350μL tissue homogenate in a 2mL centrifuge tube.

(4) Add 150 μL of lysate (Buffer C-L) and 20 μL of proteinase K (15 mg/mL) solution, vortex for 1 minute, centrifuge briefly, and place it in a 56-degree water bath for 10 minutes.

(5) Add 350 μL of protein removal solution (Buffer P-D), vortex for 30 seconds, and centrifuge at 12,000×g for 10 minutes.

(6) Put the DNA preparation tube into another 2mL centrifuge tube, transfer the supernatant in (5) to the preparation tube, and centrifuge at 12,000×g for 1 minute.

(7) Discard the filtrate, put the preparation tube back into the original 2mL centrifuge tube, add 500 μL of washing solution (Buffer W ₁ ), and centrifuge at 12,000×g for 1 minute.

(8) Discard the filtrate, put the preparation tube back into the original 2mL centrifuge tube, add 700μL desalted solution (Buffer W ₂ ), and centrifuge at 12,000×g for 1 minute.

(9) Discard the filtrate, put the preparation tube back into the original 2mL centrifuge tube, and centrifuge at 12,000×g for 1 minute.

(10) Discard the filtrate, put the preparation tube into another clean 1.5mL centrifuge tube, add 100-200μL of 2.5mmol/L Tris-HCL solution to the center of the preparation tube membrane, and centrifuge at 12,000×g for 1 minute to obtain a genomic DNA solution , Save at -20 degrees for later use.

In the second step, the PCR amplification of the cadherin-specific gene fragment is performed on the genomic DNA template extracted in the first step.

(1) According to the sequence characteristics of the deletion of exon 32 in the cytoplasmic region of cadherin of cotton bollworm, specific PCR primers were designed, which can simultaneously detect three different insertions or deletions at the genomic DNA level through amplification.

The sequence of the upstream primer A is: TGATTGTTGTTGTGTGCTGCTTATTGTG (SEQ ID NO.1)

The sequence of the downstream primer B is: CGATCAGGTCTGAGTCGTATGAC (SEQ ID NO.2)

(2) Set up a PCR reaction system with a total reaction volume of 25 μL in a 0.2 mL PCR tube.

(3) The PCR reaction conditions are: pre-denaturation at 94°C for 3 minutes, denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, extension at 72°C for 5 minutes and 30 seconds, 30 cycles, and finally extension at 72°C for 10 minutes. After the reaction, the reaction product was stored at 4°C for subsequent agarose gel electric pulse detection.

In the third step, the PCR product obtained in the second step is subjected to agarose gel electric pulse detection.

(1) Prepare 1% (g/mL) agarose gel.

(2) Take 10 μL of the PCR amplification product for electrophoresis at a steady flow of 80 mA, and observe under ultraviolet light after 1.5 hours.

From the results of electrophoresis, it can be seen that the pair of PCR primers amplified three types for the deletion mutation of 55 amino acids in the intracellular region of the cadherin gene of cotton bollworm, which can be identified simultaneously according to the size of the electrophoresis band, and can be distinguished at one time Resistance homozygotes, resistant heterozygotes and sensitive individuals were obtained, and the results are shown in Figure 4.

The deletion mutation of 55 amino acids in the intracellular region of the cotton bollworm cadherin gene detected in this field population is due to the deletion of 82 bp DNA sequence on the No. 32 exon of the allele, and the obtained amplification product is 750 bp, For the r _15-2 mutant. Swimming lanes 1 and 2 in accompanying drawing 4 are resistant homozygotes with only 750bp bands. Swimming lanes 3, 4 and 5 in accompanying drawing 4 are resistance heterozygous electrophoresis with two bands of 750bp and 1000bp. Swimming lanes 6 and 7 in accompanying drawing 4 are sensitive individuals, only 1000bp band.

The fourth step, calculation method of resistance gene frequency in field population of cotton bollworm

The calculation method of the resistance gene frequency of cadherin intracellular domain deletion mutation in the field population of cotton bollworm is as follows:

Resistance gene frequency = (number of resistant homozygous individuals × 2 + number of resistant heterozygous individuals) / (total number of detected individuals × 2).

For example, if 100 individuals in a field population are detected, the number of resistant homozygous individuals is 2, and the number of resistant heterozygous individuals is 5, then the frequency of the cadherin intracellular domain deletion mutation resistance gene in this population is:

(2×2+5)/100×2=0.045

Claims (2)

1. three kinds of genotype DNA molecular detection methods of the cadherin cytoplasmic region deletion mutantion of bollworm to the non-recessive resistance of Cry1Ac toxin, it is characterized in that designing Auele Specific Primer, adopt round pcr, detect each individual cadherin cytoplasmic region in cotton bollworm population and whether exist the insertion different due to three kinds or disappearance to cause 55 amino acid whose situations of intracellular region 32 exon disappearances; Comprise following three steps:

The first step: the genomic dna that extracts cotton bollworm larvae or adult;

Second step: utilize the primer B shown in the primer A shown in SEQ ID NO.1 and SEQ ID NO.2, the bollworm genomic dna that pcr amplification previous step is extracted;

The 3rd step: previous step PCR is obtained to product and carry out 1% agarose gel electrophoresis, according to electrophoretogram precise Identification bollworm individual its deletion mutantion of cadherin cytoplasmic region and particular type thereof, disposable resistance homozygote, resistance heterozygote and the sensitive individual distinguished: if electrophoresis result only shows 2500bp band, show that this bollworm is the resistance homozygote that cadherin intracellular region genomic dna inserts the mRNA deletion mutantion causing; If electrophoresis result shows 2500bp and two bands of 1000bp, this bollworm is resistance heterozygote; If electrophoresis result only shows 750bp band, show that this bollworm is the resistance homozygote that cadherin intracellular region genomic dna lacks mRNA the 32nd exon deletion mutantion causing; If electrophoresis result shows 750bp and two bands of 1000bp, show that this bollworm is resistance heterozygote; If electrophoresis result only shows 5000bp band, show that this bollworm is the resistance homozygote that cadherin intracellular region genomic dna inserts the mRNA deletion mutantion causing; If electrophoresis result shows 5000bp and two bands of 1000bp, show that this bollworm is resistance heterozygote; If electrophoresis result only shows 1000bp, show that this bollworm is Cry1Ac toxin sensitive individual.

2. molecular detecting method according to claim 1, is characterized in that described molecular detecting method also comprises according to the result of the 3rd step and judges resistance gene frequency in population.

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