Method for detecting frequency of early resistant allele of Asiatic corn borer to Bt corn and application

Abstract

The invention relates to a method for detecting the frequency of early resistant alleles of Asiatic corn borers to Bt corn and application of the method, and belongs to the technical field of biology. The method comprises the following steps: firstly, preparing a Bt protein diagnostic dose, and respectively preparing purified proteins Cry1Ab and Cry1F into a Bt protein diagnostic reagent; performing F1-generation hybridization on the resistant population cultured indoors and the population collected in the field; determining the death number of newly hatched larvae of each single female family under the diagnostic dose concentration; according to the fact that target pests with the corrected survival rate reaching 50% under the diagnostic dosage serve as resistant individuals, the number of resistant families of F1-generation newly hatched larvae hybridized with the resistant population under the Cry1Ab protein diagnostic dosage of 93 ng / cm < 2 > and the Cry1F protein diagnostic dosage of 197 ng / cm < 2 > is counted, and the frequency of resistant allelic genes is calculated. The method can be used for rapidly detecting the early rare resistance allele frequency in the field population, has the characteristics of high sensitivity, short period and the like, can be used for systematically monitoring the change of the inter-annual resistance allele frequency of the field population for a long time, and provides technical support for early warning of resistance, resistance treatment and the like.

Description

Technical Field

[0001] The invention relates to a method for detecting the frequency of early resistance alleles of Asian corn borer to Bt corn and its application, belonging to the field of biotechnology. Background Art

[0002] As a new generation of insect-resistant crops, transgenic insect-resistant corn began to be commercially planted in the United States in 1996 and was quickly promoted to major corn-producing countries such as Brazil. The global area of ​​Bt protein crops planted increased from 1 million hectares in 1996 to 109 million hectares in 2019. Transgenic insect-resistant corn effectively controls pests such as Ostrinia nublilalis, the European corn borer, and Spodoptera frugiperda, the fall armyworm. The key to the sustainable planting of transgenic insect-resistant crops is the resistance of target pests. The "high dose / refuge" strategy is the main strategy for managing pest resistance to Bt crops. Its basic principles are: ① Insect-resistant crops must express high doses of insecticidal proteins, which can kill almost all resistant heterozygous individuals RS or all sensitive individuals SS; ② The initial frequency of resistance genes in the target pest population is at a very low level; ③ Adult insects from insect-resistant crop plots and non-insect-resistant crop plots (refuges) can randomly mix and mate in the field. Among them, the frequency of the initial resistance alleles of insects to Bt crops is at a low level, generally required to be less than 0.001. Population genetics theory predicts that the initial resistance allele frequency is closely related to the formation of resistance. The frequency of any initial resistance gene can help assess the risk of resistance, so the resistance allele frequency is an important parameter. The detection and monitoring of the resistance allele frequency of target pests is the basis for implementing resistance management.

[0003] Although genetically modified insect-resistant corn has not yet been commercially planted in my country, some transformants have obtained safety certificates for production and application. For example, Beijing Da Bei Nong Biotechnology Co., Ltd.'s insect-resistant and herbicide-resistant corn DBN9936, which has Cry1Ab and epsps genes transferred into it. The Cry1Ab gene usually confers insect resistance to corn, which can have toxic effects on specific pests, reduce pest damage to corn, reduce pest losses, and help improve corn yield and quality. The epsps gene makes corn resistant to herbicides. When using specific herbicides for field weeding, the corn plants are not affected and can more effectively control weed growth. The DBN9936 transformant obtained safety certificates for production and application in the northern spring corn area and the Huanghuaihai summer corn area, the southern corn area, the southwestern corn area, and the northwestern corn area in 2019 and 2020. In addition, Hangzhou Ruifeng Biotechnology Co., Ltd. has transformed the Cry1Ab / Cry2Aj and G10evo-epsps genes into the insect-resistant and herbicide-resistant corn "Ruifeng 125". The Cry1Ab / Cry2Aj genes give Ruifeng 125 corn insect resistance, and the G10evo-epsps gene makes corn resistant to herbicides, so that specific herbicides can be used for weeding in field management without causing harm to corn plants. Ruifeng 125 obtained a safety certificate for production and application in the northern spring corn area in 2020, and a safety certificate for production and application in the Huanghuaihai summer corn area and the northwestern corn area in 2021. In addition, multiple transformation events such as the expression of Cry1F are nearing completion, among which the Asian corn borer is one of the main target pests of Cry1Ab and Cry1F. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a method for detecting the frequency of early resistance alleles of Asian corn borer to Bt corn and its application.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for detecting the frequency of early resistance alleles of Asian corn borer to Bt corn comprises the following steps:

[0007] (1) Configuration of Bt protein diagnostic reagents

[0008] The purified proteins Cry1Ab and Cry1F were prepared into 4.65 μg / mL and 9.85 μg / mL protein solutions respectively using a CAPS buffer solution with a concentration of 50 mmol / L and a pH of 10.5, thereby obtaining Bt protein diagnostic reagents of Cry1Ab and Cry1F, which were stored in a refrigerator for future use;

[0009] (2) Test insect populations

[0010] Sources of indoor resistant and sensitive strains: Asian corn borers collected from regular corn ears in the field were divided into three parts. One part was added with Cry1Ab protein diagnostic reagents to the surface of artificial feed by smearing method, and then the newly hatched larvae of Asian corn borers were connected for continuous feeding to cultivate Cry1Ab resistant strains; the second part was added with Cry1F protein diagnostic reagents to the surface of artificial feed by smearing method, and then the newly hatched larvae of Asian corn borers were connected for continuous feeding to cultivate Cry1F resistant strains; the third part was directly cultured with artificial feed, and corn borers that were not exposed to chemical pesticides and Bt insecticidal proteins during the period were cultivated as sensitive strains;

[0011] Collection of Asian corn borer populations in the field: Egg masses or larvae of Asian corn borer were collected from corn fields. The collected larvae were raised indoors with artificial feed until they became adults. All larvae and adults in the field were cultured in an incubator with a temperature of 27°C ± 1°C, a humidity of 70% to 80%, and a photoperiod of 16h / 8h. The field populations were not exposed to any chemical pesticides or Bt insecticidal proteins during the breeding process.

[0012] (3) F1 hybridization between indoor resistant population and field population

[0013] Male moths from the Asian corn borer population raised to adults in the field are paired with female moths of the resistant strain screened indoors, or female moths from the Asian corn borer population in the field are paired with male moths of the resistant strain screened indoors, and after pairing, they are placed in a plastic cup, covered with gauze on the top, and a cotton ball soaked in honey water is placed in the plastic cup to provide nutrition, a single female family is established, and egg masses produced by each family are collected and hatched, and the hatched F1 generation newly hatched larvae are used for bioassay;

[0014] (4) Bioassay

[0015] Squeeze the artificial feed into the 24-well insect culture plate with a squeeze bottle, squeeze 1.5-2mL of artificial feed into each small hole, use a pipette to take 40μl of Cry1Ab protein diagnostic reagent with a concentration of 4.65μg / mL and Cry1F protein diagnostic reagent with a concentration of 9.85μg / mL, add them to the small holes respectively, shake well, and then place the insect culture plate at room temperature to dry, inoculate the newly hatched F1 larvae produced by each single female family into the culture plate, inoculate one larva into each hole, cover the lid and tie it tightly, use artificial feed without adding protein as the control, and culture all the culture plates at a temperature of 27℃±1℃, a humidity of 70%~80%, and a photoperiod of 16h / 8h; record the survival of the single female family larvae in the test group and the control group on the 7th day after inoculation;

[0016] (5) Calculation of resistance allele frequency and 95% confidence interval

[0017] According to the criterion that the family-corrected survival rate of target pests reaches 50% under the diagnostic dose of Bt protein, the survival rate of newly hatched larvae of F1 generation after hybridization with resistant population at the diagnostic dose of Cry1Ab protein 93ng / cm 2 、Cry1F protein diagnostic dose 197ng / cm 2 Number of resistant families: R software was used to calculate the resistance gene frequency and 95% confidence interval of the experimental data to obtain the resistance allele frequency.

[0018] In the method for detecting the frequency of early resistance alleles of the Asian corn borer to Bt corn, the resistance multiple of the Cry1Ab resistance strain in the test is 280 times, and the resistance multiple of the Cry1F resistance strain is 300 times.

[0019] The formulas for calculating corrected survival rate, resistance gene frequency, and 95% confidence interval are:

[0020]

[0021] Where: s is the number of families with a corrected survival rate of 50% at the diagnostic dose, n is the number of samples tested, E(q) is the frequency of the resistance allele, Var(q) is the variance of the frequency of the resistance allele, and 95% (CI) is the 95% confidence interval.

[0022] The method of the present invention is used in monitoring the frequency of early resistance alleles of Asian corn borer to Bt corn. Beneficial effects of the present invention:

[0023] The method for detecting the frequency of early resistance alleles of Asian corn borer to Bt corn comprises the following steps: firstly, preparing a Bt protein diagnostic dose, respectively preparing purified proteins Cry1Ab and Cry1F into Bt protein diagnostic reagents; hybridizing an indoor resistant population with a field population F1 generation; determining the number of deaths of newly hatched larvae of each single female family at the diagnostic dose concentration; taking the target pests with a corrected survival rate of 50% at the diagnostic dose as the resistant individuals, and counting the number of newly hatched larvae of the F1 generation after hybridization with the resistant population at the Cry1Ab protein diagnostic dose of 93ng / cm 2 、Cry1F protein diagnostic dose 197ng / cm 2 The number of resistant families was counted and the resistance allele frequency was calculated.

[0024] Compared with the conventional dose-mortality bioassay method, the single diagnostic dose resistance detection method and the F2 generation method, the method of the present invention hybridizes the indoor resistance population with the field population for F1 generation testing, can quickly detect the early rare resistance allele frequency in the field population, has the characteristics of high sensitivity and short cycle, can carry out long-term systematic monitoring, understand the changes in the resistance allele frequency of the field population between years, and provide technical support for early warning of resistance and delay of resistance.

[0025] The experiment showed that according to the criterion that 50% of the surviving families of target pests are resistant individuals under different diagnostic doses of Bt protein, the corn borers collected in Dehui City, Jilin Province, Gongzhuling City, Jilin Province, Gucheng County, Hebei Province and Xinxiang City, Henan Province in 2023 and the corn borers collected in Gucheng, Hebei Province and Xinxiang, Henan Province in 2024 were crossed with resistant populations. The newly hatched larvae of the F1 generation were resistant to Cry1Ab protein at a diagnostic dose of 93ng / cm 2 ) and Cry1F protein diagnostic dose (197ng / cm 2 ) had no resistant families, and the resistance allele frequencies were 0.001762 and 0.0045237, respectively, and the resistance allele frequencies were at a low level. DETAILED DESCRIPTION

[0026] The specific implementation modes of the present invention are further described in detail below in conjunction with the embodiments.

[0027] In the early stage, we used the dose-mortality bioassay method to establish the sensitivity baseline of 15 populations of Ostrinia furnacalis to Cry1Ac, Cry1Ab and Cry1F in the summer corn region of Huanghuaihai from 2015 to 2016. The results showed that there was no significant difference between years and regions. Therefore, the test data of 15 populations were combined to calculate the LC of Cry1Ab. 99 and its 95% confidence interval is 93ng / cm 2 、(63ng / cm 2 ~180ng / cm 2 );Calculate the LC of Cry1Ac 99 and its 95% confidence interval is 45 ng / cm 2 (26ng / cm 2 ~105ng / cm 2 );Calculate the LC of Cry1F 99 and its 95% confidence interval is 197ng / cm 2 (89ng / cm 2 ~752ng / cm 2 ); then verified LC 99 , and the mortality rates at the upper and lower limits of the 95% confidence interval. Finally, the diagnostic doses of Cry1Ab, Cry1Ac, and Cry1F were determined to be Cry1Ab: 93 ng / cm 2 、Cry1Ac:45ng / cm 2 、Cry1F:197ng / cm 2 , and a method for detecting and monitoring the frequency of early resistance alleles in the F2 generation + diagnostic dosage determination was established.

[0028] The initial resistance allele frequencies of Asian corn borer to Cry1Ab, Cry1Ac and Cry1F in the field measured in 2018 were 0.002 (95% FL 0.000283-0.006484), 0.001 (95% FL 0.000030-0.004295) and 0.001 (95% FL 0.000030-0.00042950), respectively. Since 2015, two resistant lines of corn borer have been screened and cultivated indoors, of which the resistance to Cry1Ab is 280 times and the resistance to Cry1F is 300 times.

[0029] On the premise of existing resistant strains and diagnostic doses, a method was developed to use indoor resistant strains and field wild populations for F1 hybridization, and to detect the frequency of early rare resistance alleles in combination with diagnostic doses. The principle is: the recessive genotype of resistance of each female moth is (rr). If the male moth is heterozygous (rs), for the resistance gene r, the gene is the same as the female moth's resistance gene r, then half of its offspring will be resistant individuals, and the survival rate will be at least 50% under the diagnostic dose; if the male moth is a sensitive homozygous (ss), hybridized with a resistant female (rr), the offspring will all be (sr), there will be no homozygous (rr), and the survival rate will be 0 under the diagnostic dose.

[0030] Example 1. A method for detecting and monitoring the frequency of early resistance alleles of Asian corn borer to Bt corn 1. Materials and methods

[0031] 1.1Bt protein diagnostic dose configuration

[0032] The Cry1Ab and Cry1F proteins tested were purified proteins. Cry1Ab and Cry1F were prepared into 4.65μg / mL and 9.85μg / mL purified protein solutions respectively using 50mmol / L, pH10.5 CAPS buffer. The prepared Cry1Ab and Cry1F purified protein solutions were stored in a -80℃ refrigerator to prevent protein degradation.

[0033] 1.2 Test insect populations

[0034] Source of indoor resistant and sensitive strains: Asian corn borers were collected from regular corn ears at the Modern Agricultural Science and Technology Experimental Demonstration Base of Henan Academy of Agricultural Sciences (35°13′N, 113°42′E) in 2015 and divided into three parts. One part was added with Cry1Ab protein on the surface of artificial feed by feed smearing method indoors, and newly hatched larvae were inoculated to cultivate resistant strains; the other part was added with Cry1F protein on the surface of artificial feed by feed smearing method indoors, and newly hatched larvae were inoculated to cultivate resistant strains; the other part was raised indoors without any contact with any chemical pesticides and Bt insecticidal proteins, and raised with artificial feed to cultivate as sensitive strains. The LC values ​​of Cry1Ab and Cry1F resistant strains and sensitive strains to Cry1Ab and Cry1F were determined respectively. 50 Value, resistance multiple = LC of resistant strain 50 Value / Sensitive strain LC 50 The results showed that the resistance multiples to Cry1Ab resistance were 280 times, and the resistance multiples to Cry1F resistance were 300 times (Table 1, Table 2).

[0035] Table 1 LC of resistant and sensitive strains to Cry1Ab 50 value

[0036]

[0037] Table 2 LC of resistant and sensitive strains to Cry1F 50 value

[0038]

[0039] Collection of field Asian corn borer populations: In August 2023, corn borer larvae or egg masses were collected from Yuanyang County, Henan Province, Gucheng County, Hebei Province, Gongzhuling City, Jilin Province, and Dehui City, Jilin Province; in August 2024, corn borer larvae were collected from Yuanyang County, Henan Province and Gucheng County, Hebei Province, and placed in an incubator with a temperature of 27℃±1℃, a humidity of 70%-80%, and a photoperiod of L / D=16h / 8h. They were raised indoors with artificial feed until they became adults. The main components of the artificial feed were soybean flour, wheat germ, and yeast (see the feed formula of ZL 2018 1 0667004.3), waiting to establish hybridization with resistant populations. These field populations were not exposed to any chemical pesticides and Bt insecticidal proteins during the breeding process. The information on insect source collection is shown in Table 3.

[0040] Table 3 Collection information of Asian corn borer population in the field

[0041]

[0042] 1.3 F1 hybridization between indoor resistant population and field population

[0043] After the collected field Asian corn borer was reared to adult, each male moth (unmated) was paired with a female moth (unmated) of a resistant strain screened indoors, or each female moth was paired with a male moth of a resistant strain screened indoors, and after pairing, they were placed in a 250 mL plastic cup, covered with gauze on the top, and the plastic cup was provided with a cotton ball soaked in 10% honey water to provide nutrition, and a single female family was established. The rearing conditions were: a temperature of 27°C ± 1°C, a humidity of 70% to 80%, and a light cycle of L / / D = 16h / / 8h in an incubator, and the egg masses produced by each family were collected and allowed to hatch, and the hatched F1 generation newly hatched larvae (<24h) were used for bioassay. Single female families were established with resistant strains and sensitive strains as positive controls and negative controls, respectively.

[0044] Assume that the recessive resistance genotype of each female moth is (rr). If the male moth is a heterozygote (rs), for the resistance gene r, it is located at the same locus as the female moth's resistance gene r, and half of its offspring will be resistant individuals, and the survival rate will reach 50% at the diagnostic dose; if the male moth is a sensitive homozygote (ss) and is hybridized with a resistant female (rr), all offspring will be (sr), without homozygotes (rr), and no family will survive at the diagnostic dose.

[0045] 1.4 Bioassay

[0046] The method for preparing artificial feed refers to the method of patent: ZL 2018 1 0667004.3: weigh the ingredients of component A, heat the water to 40-50°C, then add wheat germ, soybean flour, yeast powder, milk powder, and white sugar, stir evenly to obtain component A; add methyl paraben and sorbic acid to agar, stir while adding water to obtain a mixture, heat the obtained mixture while stirring until all the ingredients are dissolved to obtain component B; when the temperature of component B is cooled to 50-60°C, add component A to component B and stir evenly, finally add ascorbic acid, multivitamins and formaldehyde and continue stirring. The specific feed formula is shown in Table 4.

[0047] When the prepared artificial feed is cooled to 50℃ (not solidified), squeeze it into a 24-hole insect culture plate (each hole has a diameter of 16mm and a height of 13mm) with a squeeze bottle. Squeeze 1.5-2mL into each hole. The hole diameter is 1.6cm. After the feed solidifies, a surface area of ​​2cm is formed. 2 Use a pipette to take 40 μl of 4.65 μg / mL Cry1Ab and 9.85 μg / mL Cry1F protein solutions, add them to the small holes, shake them from left to right and from front to back to make the protein cover the surface of the feed. The added Cry1Ab protein is (40 μl × 4.65 μg / mL) 186 ng, and the Cry1F protein is (40 μl × 9.85 μg / mL) 394 ng, finally forming Cry1Ab 93 ng / cm 2、Cry1F197ng / cm 2 Diagnostic dose concentrations of feed surface area were determined, with only one protein added to each well.

[0048] Place the insect culture plates with added protein at room temperature. After drying, inoculate the newly hatched larvae of the F1 generation produced by each single-female family into the culture plates, one larvae per hole, cover the lid and tie it tightly. Set up 3 replicates for each treatment, with 48 larvae in each replicate. Use artificial feed without added protein as the control. Place all culture plates at (27±1)℃, humidity of 70% to 80%, and light L / D=16h / 8h for breeding.

[0049] On the 7th day, check and record the number of dead larvae in each single-female family. Use a brush to touch the insect body. Those that cannot crawl normally are considered dead. Larvae that have not reached the second instar are also considered dead.

[0050] Table 4 Composition of artificial diet for Asian corn borer

[0051]

[0052] 1.5 Data processing

[0053] The mortality rate and corrected survival rate were calculated using the following formulas. If the corrected survival rate of any family reached 50%, the family was considered to be a resistant individual. The resistance gene frequency and 95% confidence interval were calculated using R software.

[0054]

[0055] Where: s is the number of families with a 50% corrected survival rate at the diagnostic dose, n is the number of samples tested, E(q) is the frequency of the resistance allele, Var(q) is the variance of the frequency of the resistance allele, and 95% (CI) is the 95% confidence interval. When the lower limit of the 95% (CI) confidence interval is calculated to be a negative number, the value is set to 0.000.

[0056] 2. Results and Analysis

[0057] 2.1 Corrected survival rates of indoor resistant and sensitive populations at diagnostic doses of Cry1Ab and Cry1F

[0058] Analysis of the survival of different larvae on the 7th day after feeding showed that the Cry1Ab-resistant population was 2 The corrected survival rate of the sensitive population was 0%; the Cry1F-resistant population was 197ng / cm 2The corrected survival rate under artificial diet conditions was 81.11±1.11%, while the corrected survival rate of sensitive populations was 0%; the survival rates of the three strains under normal artificial diet conditions were all above 90%, indicating that the working system was normal (Table 5).

[0059] Table 5 Survival rate of resistant and sensitive populations at diagnostic doses (%)

[0060]

[0061] 2.2 Resistance allele frequencies of resistant and sensitive strains at different Bt protein diagnostic doses

[0062] The number of positive control pairs of Cry1Ab resistant strains was 43, and 26 pairs were successful; the diagnostic dose of Cry1Ab protein (93ng / cm 2 ) feed, there were 26 families with 50% corrected survival of 7-day-old larvae, the corrected survival rate of the surviving families was over 83%, the surviving larvae reached the 3rd instar, and the frequency of resistance allele was 0.811018.

[0063] The number of positive control pairs of Cry1F resistant strains was 48, and 28 pairs were successful; the diagnostic dose of Cry1F protein (197ng / cm 2 ) feed, there were 28 families with 50% larvae surviving at 7 days, the survival rate of the surviving families was over 82%, the surviving larvae reached the 3rd instar, and the frequency of resistance allele was 0.817426.

[0064] The number of negative control pairings of the sensitive strain was 225, and 176 pairs were successful. When the larvae were fed with two diagnostic doses of feed, all larvae of all families died on day 7, with a survival rate of 0. The frequency of the resistance allele was 0.002813, which was at a relatively low level (Table 6).

[0065] Table 6 Frequency of resistance alleles in resistant and sensitive populations at different Bt protein diagnostic doses

[0066]

[0067] 2.3 Resistance allele frequencies in F1 hybrids between indoor resistant populations and field populations at diagnostic doses of Cry1Ab protein

[0068] The Asian corn borer populations collected from the fields in Dehui City, Jilin Province, Gongzhuling City, Jilin Province, Gucheng County, Hebei Province, and Xinxiang City, Henan Province in 2023 were hybridized with the indoor resistant population and the field population according to the above 1.3 method to obtain the newly hatched F1 generation larvae. A total of 203 families had a diagnostic dose of Cry1Ab protein of 93ng / cm 2Bioassays were carried out under the conditions of 1:1 and 2. The corrected survival rates of larvae at 7 days were 18.75%-31.25%, the surviving larvae did not reach the second instar, no corrected survival rate of any family reached 50%, the number of resistant individuals was 0, and the frequency of resistance alleles was 0.001762.

[0069] The Asian corn borer populations collected in Gucheng County, Hebei Province and Xinxiang City, Henan Province in 2024 were hybridized with the indoor resistant populations and field populations according to the method described in 1.3 above to obtain the newly hatched F1 generation larvae. A total of 83 families had a Cry1Ab protein diagnostic dose of 93 ng / cm 2 Bioassays were conducted under the following conditions. The corrected survival rates of larvae at 7 days were 18.75%-34.37%. The surviving larvae did not reach the second instar. No corrected survival rate of any family reached 50%, that is, the number of resistant individuals was 0 and the frequency of resistance alleles was 0.004283 (Table 7).

[0070] Table 7 Frequency of resistance alleles to Cry1Ab in F1 hybrids between indoor resistant populations and field populations

[0071]

[0072] 2.4 Resistance allele frequencies in F1 hybrids between indoor resistant populations and field populations at diagnostic doses of Cry1F protein

[0073] The Asian corn borer populations collected from the fields in Dehui City, Jilin Province, Gongzhuling City, Jilin Province, Gucheng County, Hebei Province, and Xinxiang City, Henan Province in 2023 were hybridized with the indoor resistant population and the field population according to the above 1.3. A total of 191 families of F1 generation newly hatched larvae were tested. At the diagnostic dose of Cry1F protein (197ng / cm 2 ) had a corrected survival rate of 7-day-old larvae of 16.67%-34.37%, the surviving larvae did not reach the second instar, the number of resistant individuals was 0, and the frequency of the resistance allele was 0.001820.

[0074] The field populations of Asian corn borer collected in Gucheng County, Hebei Province and Xinxiang City, Henan Province in 2024 were hybridized with the indoor resistant population and the field population in 1.3 to obtain the newly hatched F1 larvae. A total of 75 families of newly hatched F1 larvae were tested. At the diagnostic dose of Cry1F protein (197 ng / cm 2 ), the corrected survival rate of 7-day larvae was 20.83%-33.33%, the surviving larvae did not reach the second instar, no family had a corrected survival rate of 50%, the number of resistant individuals was 0, and the frequency of resistance alleles was 0.004357 (see Table 8).

[0075] Table 8 Frequency of resistance alleles at diagnostic doses of Cry1F protein in F1 hybrids between indoor resistant populations and field populations

[0076]

Claims

1. A method for detecting the frequency of early resistance alleles of Asian corn borer to Bt corn, characterized in that: The method comprises the following steps: (1) Preparation of Bt protein diagnostic reagent The purified proteins Cry1Ab and Cry1F were prepared into 4.65 μg / mL and 9.85 μg / mL protein solutions respectively using a CAPS buffer solution with a concentration of 50 mmol / L and a pH of 10.5, thereby obtaining Bt protein diagnostic reagents of Cry1Ab and Cry1F, which were stored in a refrigerator for future use; (2) Test insect populations Sources of indoor resistant and sensitive strains: Asian corn borers collected from regular corn ears in the field were divided into three parts. One part was added with Cry1Ab protein diagnostic reagents to the surface of artificial feed by smearing method, and then the newly hatched larvae of Asian corn borers were connected for continuous feeding to cultivate Cry1Ab resistant strains; the second part was added with Cry1F protein diagnostic reagents to the surface of artificial feed by smearing method, and then the newly hatched larvae of Asian corn borers were connected for continuous feeding to cultivate Cry1F resistant strains; the third part was directly cultured with artificial feed, and corn borers that were not exposed to chemical pesticides and Bt insecticidal proteins during the period were cultivated as sensitive strains; Collection of Asian corn borer populations in the field: Egg masses or larvae of Asian corn borer were collected from corn fields. The collected larvae were raised indoors with artificial feed until they became adults. All larvae and adults in the field were cultured in an incubator with a temperature of 27°C ± 1°C, a humidity of 70% to 80%, and a photoperiod of 16h / 8h. The field populations were not exposed to any chemical pesticides or Bt insecticidal proteins during the breeding process. (3) F1 hybridization between indoor resistant population and field population Male moths from the Asian corn borer population raised to adults in the field are paired with female moths of the resistant strain screened indoors, or female moths from the Asian corn borer population in the field are paired with male moths of the resistant strain screened indoors, and after pairing, they are placed in a plastic cup, covered with gauze on the top, and a cotton ball soaked in honey water is placed in the plastic cup to provide nutrition, a single female family is established, and egg masses produced by each family are collected and hatched, and the hatched F1 generation newly hatched larvae are used for bioassay; (4) Bioassay Squeeze the artificial feed into the 24-well insect culture plate with a squeeze bottle, squeeze 1.5-2mL of artificial feed into each small hole, use a pipette to take 40μl of Cry1Ab protein diagnostic reagent with a concentration of 4.65μg / mL and Cry1F protein diagnostic reagent with a concentration of 9.85μg / mL, add them to the small holes respectively, shake well, and then place the insect culture plate at room temperature to dry, inoculate the newly hatched F1 larvae produced by each single female family into the culture plate, inoculate one larva into each hole, cover the lid and tie it tightly, use artificial feed without adding protein as the control, and culture all the culture plates at a temperature of 27℃±1℃, a humidity of 70%~80%, and a photoperiod of 16h / 8h; record the survival of the single female family larvae in the test group and the control group on the 7th day after inoculation; (5) Calculation of resistance allele frequency and 95% confidence interval According to the criterion that the family-corrected survival rate of target pests reaches 50% under the diagnostic dose of Bt protein, the survival rate of newly hatched larvae of F1 generation after hybridization with resistant population at the diagnostic dose of Cry1Ab protein 93ng / cm 2 、Cry1F protein diagnostic dose 197ng / cm 2 Number of resistant families: R software was used to calculate the resistance gene frequency and 95% confidence interval of the experimental data to obtain the resistance allele frequency.

2. The method for detecting the frequency of early resistance alleles of Asian corn borer to Bt corn according to claim 1, characterized in that: In the experiment, the resistance multiple of Cry1Ab resistant strain was 280 times, and the resistance multiple of Cry1F resistant strain was 300 times.

3. The method for detecting the frequency of early resistance alleles of Asian corn borer to Bt corn according to claim 1, characterized in that: The formulas for calculating corrected survival rate, resistance gene frequency, and 95% confidence interval are: Where: s is the number of families with a corrected survival rate of 50% at the diagnostic dose, n is the number of samples tested, E(q) is the frequency of the resistance allele, Var(q) is the variance of the frequency of the resistance allele, and 95% (CI) is the 95% confidence interval.

4. Use of the method of claim 1 in monitoring the frequency of early resistance alleles of Asian corn borer to Bt corn.

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