Method for judging high-dose expression of transgenic insect-resistant cotton Bt protein

Abstract

The invention belongs to the technical field of agricultural biology, discloses a method for judging high-dose expression of transgenic insect-resistant cotton Bt protein, and aims to judge the high-dose expression of the transgenic insect-resistant cotton Bt protein. The method comprises the following steps: collecting leaves at a seedling stage, leaves at a bud stage, small buds at the bud stage, leaves at a boll stage and small bolls at the boll stage of cotton at proper time, determining the Bt protein content of each tissue of the insect-resistant cotton by using an ELISA method, and calculating an average value; respectively collecting leaves of cotton in a second-generation cotton bollworm vigorous generation period, a third-generation cotton bollworm vigorous generation period and a fourth-generation cotton bollworm vigorous generation period for bioassay, and calculating an average value of corrected death rates of second-generation to fourth-generation bioassay cotton bollworm larvae. And judging by combining the protein and a bioassay result, and when the average value of the expression quantity of the Bt protein is greater than 450 ng / g fresh weight and the average value of the corrected death rate of larvae is greater than 60%, judging that the transgenic insect-resistant cotton Bt protein is in high-dose expression.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a method for determining high-dose expression of Bt protein in transgenic insect-resistant cotton. Background Art

[0002] Cotton is an important cash crop and the main source of natural fiber. It plays an important role in the national economy. However, cotton production is vulnerable to pests, which causes huge losses. Cotton bollworm is the most harmful pest on cotton. When the damage is serious, it may even cause a total crop failure. In the 1990s, with the promotion of transgenic technology, transgenic insect-resistant cotton gradually became the mainstream. Transgenic insect-resistant cotton can effectively control cotton bollworm, greatly reduce the use of pesticides, solve the problem of cotton bollworm resistance, and produce great economic, ecological and social benefits.

[0003] In order to ensure the sustainable and healthy development of transgenic insect-resistant cotton and delay the resistance of cotton bollworm to transgenic insect-resistant cotton, high-dose expression of Bt protein in insect-resistant cotton has become an important strategy for resistance management. At present, the method of high-dose expression of Bt protein in insect-resistant cotton mainly relies on enzyme-linked immunosorbent assay (ELISA) to determine the Bt protein content. Summary of the invention

[0004] The purpose of the present invention is to determine whether a transgenic insect-resistant cotton Bt protein is expressed in high doses. The present invention provides a method for determining whether a transgenic insect-resistant cotton Bt protein is expressed in high doses.

[0005] The technical solution proposed by the present invention is:

[0006] A method for determining whether a transgenic insect-resistant cotton Bt protein is expressed at a high dose, wherein the transgenic insect-resistant cotton Bt protein is determined to be expressed at a high dose when the transgenic insect-resistant cotton satisfies the following conditions at the same time:

[0007] 1) The average Bt protein expression in cotton tissue is greater than 450 ng / g (fresh weight);

[0008] 2) The average corrected mortality rate of larvae in the bioassay results of cotton leaves against target pests was greater than 60%.

[0009] The cotton tissue is one or more of cotton leaves at the seedling stage, leaves at the bud stage, budlets at the bud stage, leaves at the boll stage, and bolllets at the boll stage.

[0010] The expression level of Bt protein in cotton tissue is determined by ELISA method.

[0011] The cotton leaves are collected from cotton sown at an appropriate time and managed normally during the peak period of occurrence of the second generation of target pests, the peak period of occurrence of the third generation of target pests, and the peak period of occurrence of the fourth generation of target pests.

[0012] The target pest is cotton bollworm.

[0013] The specific operation of the program is as follows:

[0014] (1) Leaves at the seedling stage, leaves at the bud stage, budlets at the bud stage, leaves at the boll stage, and bolllets at the boll stage were collected at appropriate times. All samples were crushed into powder at low temperature using a Retsch MM400 tissue crusher (Retsch GmbH, Haan, Germany), and then stored in a -80°C refrigerator. The Bt protein content in each tissue of the insect-resistant cotton was determined using the ELISA method, and the average value was calculated.

[0015] (2) The kit used in the ELISA method is the QualiPlate Kits for Cry1Ab / Cry1Ac from ENVIR LOGIX. According to the instructions, 0.4 g of tissue powder was weighed and added to 4 mL of extract PBST (PBS with a pH of 7.4 and a concentration of 0.01 mol / L + 0.55% Tween 20), and placed on a shaker at 4°C overnight; centrifuged at 12000 r / min for 15 min, the supernatant was taken, and diluted with an appropriate amount of extract; the Cry1Ab standard was diluted in a gradient manner, and 50 μL was added to each well of a 96-well ELISA plate. After adding enzyme-conjugate Cry1Ab, 50 μL of sample, negative control and standard were added respectively, the ELISA plate was sealed with a sealing film, gently shaken for 20-30 seconds, and placed on a shaker (200 r / min) for 1-2 hours; after discarding the liquid, 200 μL of washing solution was added to each well, and the plate was washed repeatedly for 3 times and then dried with absorbent paper; 100 μL of color substrate was added to each well, the ELISA plate was sealed with a sealing film, covered with tin foil to avoid light, gently shaken for 20-30 seconds, and placed at room temperature for 15-30 minutes; 100 μL of stop solution was added to each well to terminate the reaction, and the plate was analyzed using an Infinite M200 Pro ELISA reader (Tecan, Switzerland) measured the OD value at a wavelength of 450 nm.

[0016] (3) Collect cotton leaves for bioassay at the peak period of the second, third and fourth generation of cotton bollworms, and calculate the average corrected mortality rate of cotton bollworm larvae in the second to fourth generation bioassays. Take 10 leaves from the upper part of each test material, inoculate each leaf with 5 newly hatched larvae of cotton bollworm, and then put them into the test box, and repeat 3 times. After inoculation, place the test box in a light incubator [(temperature of 27℃±1℃, relative humidity of 70%, photoperiod (light time: dark time) of 14:10], and check the number of dead and live insects on the 6th day after inoculation, and calculate the corrected mortality rate of cotton bollworm larvae (X t , %). Calculation formula: X t=(X1-X0) / (1-X0)×100%. In the formula, X1 is the mortality rate of treated larvae (%), and X0 is the mortality rate of control larvae (%).

[0017] (4) Combined with the protein and bioassay results, the results were judged according to the table below. When the average Bt protein expression level was greater than 450 ng / g fresh weight and the average larval corrected mortality rate was greater than 60%, it was determined that the transgenic insect-resistant cotton had a high-dose expression of Bt protein.

[0018] Bt protein expression level Average expression level of Bt protein (ng / g fresh weight) Average corrected mortality of larvae (Xt, %) Not express Y≤5 / Low expression 5＜Y≤150 / Medium Expression 150＜Y≤300 / Medium to high expression 300＜Y≤450 / High expression Y＞450 >60

[0019] The beneficial effects of the present invention are: providing the Bt protein expression amount and leaf growth test results of cotton in the whole growth period, and providing a comprehensive method for judging the high-dose expression of cotton Bt protein. DETAILED DESCRIPTION

[0020] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0021] (1) Leaves at the seedling stage, leaves at the bud stage, budlets at the bud stage, leaves at the boll stage, and bolllets at the boll stage were collected at appropriate times. All samples were crushed into powder at low temperature using a Retsch MM400 tissue crusher (Retsch GmbH, Haan, Germany), and then stored in a -80°C refrigerator. The Bt protein content in each tissue of the insect-resistant cotton was determined using the ELISA method, and the average value was calculated.

[0022] (2) The kit used in the ELISA method is the QualiPlate Kits for Cry1Ab / Cry1Ac from ENVIR LOGIX. According to the instructions, 0.4 g of tissue powder was weighed and added to 4 mL of extract PBST (PBS with a pH of 7.4 and a concentration of 0.01 mol / L + 0.55% Tween 20), and placed on a shaker at 4°C overnight; centrifuged at 12000 r / min for 15 min, the supernatant was taken, and diluted with an appropriate amount of extract; the Cry1Ab standard was diluted in a gradient manner, and 50 μL was added to each well of a 96-well ELISA plate. After adding enzyme-conjugate Cry1Ab, 50 μL of sample, negative control and standard were added respectively, the ELISA plate was sealed with a sealing film, gently shaken for 20-30 seconds, and placed on a shaker (200 r / min) for 1-2 hours; after discarding the liquid, 200 μL of washing solution was added to each well, and the plate was washed repeatedly for 3 times and then dried with absorbent paper; 100 μL of color substrate was added to each well, the ELISA plate was sealed with a sealing film, covered with tin foil to avoid light, gently shaken for 20-30 seconds, and placed at room temperature for 15-30 minutes; 100 μL of stop solution was added to each well to terminate the reaction, and the plate was analyzed using an Infinite M200 Pro ELISA reader (Tecan, Switzerland) measured the OD value at a wavelength of 450 nm.

[0023] (3) Collect cotton leaves for bioassay at the peak period of the second, third and fourth generation of cotton bollworms, and calculate the average corrected mortality rate of cotton bollworm larvae in the second to fourth generation bioassays. Take 10 leaves from the upper part of each test material, inoculate each leaf with 5 newly hatched larvae of cotton bollworm, and then put them into the test box, and repeat 3 times. After inoculation, place the test box in a light incubator [(temperature of 27℃±1℃, relative humidity of 70%, photoperiod (light time: dark time) of 14:10], and check the number of dead and live insects on the 6th day after inoculation, and calculate the corrected mortality rate of cotton bollworm larvae (X t , %). Calculation formula: X t =(X1-X0) / (1-X0)×100%. In the formula, X1 is the mortality rate of treated larvae (%), and X0 is the mortality rate of control larvae (%).

[0024] (4) Combining the protein and bioassay results, the following table is used to determine that when the average Bt protein expression level is greater than 450 ng / g fresh weight and the average larval corrected mortality rate is greater than 60%, samples 2, 4, 5, 6, 9, and 10 in the following table are determined to be high-dose expressions of Bt protein in transgenic insect-resistant cotton.

[0025]

[0026] Those skilled in the art should understand that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining high-dose expression of Bt protein in transgenic insect-resistant cotton, characterized in that: When the transgenic insect-resistant cotton meets the following conditions at the same time, the transgenic insect-resistant cotton Bt protein is judged to be expressed at a high dose: 1) The average Bt protein expression in cotton tissue is greater than 450 ng / g (fresh weight); 2) The average corrected mortality rate of larvae in the bioassay results of cotton leaves against target pests was greater than 60%.

2. The method for determining high-dose expression of Bt protein in transgenic insect-resistant cotton according to claim 1, characterized in that: The cotton tissue is one or more of cotton leaves at the seedling stage, leaves at the bud stage, budlets at the bud stage, leaves at the boll stage, and bolllets at the boll stage.

3. The method for determining high-dose expression of Bt protein in transgenic insect-resistant cotton according to claim 1, characterized in that: The expression level of Bt protein in cotton tissue is determined by ELISA method.

4. The method for determining high-dose expression of Bt protein in transgenic insect-resistant cotton according to claim 1, characterized in that: The cotton leaves are collected from cotton sown at an appropriate time and managed normally during the peak period of occurrence of the second generation of target pests, the peak period of occurrence of the third generation of target pests, and the peak period of occurrence of the fourth generation of target pests.

5. The method for determining high-dose expression of Bt protein in transgenic insect-resistant cotton according to claim 1, characterized in that: The target pest is cotton bollworm.

Images