A method for increasing the content and insecticidal activity of exogenous insecticidal protein in transgenic cotton

Abstract

The present application belongs to the field of agricultural pest control, and particularly relates to a method for improving the content and insecticidal activity of exogenous insecticidal protein of insect-resistant transgenic cotton. The method is realized by the following steps: maintaining the soil cadmium concentration at 1-5 mg / kg when the cotton is at the three-leaf stage; and maintaining the soil cadmium concentration at 1-4 mg / kg when the cotton is at the five-leaf stage. The method provided by the present application can significantly improve the content and insecticidal effect of exogenous insecticidal protein of insect-resistant transgenic cotton, and enhance the insecticidal activity against cotton bollworms. The method provided by the present application can improve the control effect on pests while controlling cadmium pollution when planting transgenic insect-resistant cotton on land contaminated by a certain concentration of cadmium, and reduce the use of insecticides. Therefore, the present application provides an effective reference for improving the control effect on pests when planting insect-resistant transgenic cotton on land contaminated by cadmium, and has important practical value and significance.

Description

Technical Field

[0001] This invention belongs to the field of agricultural pest control, and in particular relates to a method for increasing the content and insecticidal activity of exogenous insecticidal proteins in insect-resistant transgenic cotton. Background Technology

[0002] Since 1996, the genetically modified cotton planted in my country has been an insect-resistant variety (GMO). Cry1Ab / Cry1Ac Genetically modified cotton (GM cotton) has been planted on 3.2 million hectares. The widespread cultivation of insect-resistant GM cotton has effectively controlled bollworm damage, reduced the use of chemical pesticides and costs for cotton farmers, and significantly increased economic, social, and ecological benefits. However, large-scale cotton cultivation inevitably exposes it to the effects of heavy metals.

[0003] cotton bollworm ( Helicoverpa armigera The cotton bollworm (Noctuidae) is a cosmopolitan migratory pest belonging to the order Lepidoptera and the family Noctuidae. It is widely distributed in cotton-growing areas of the Yellow River and Yangtze River basins in my country. The cotton bollworm feeds on more than 130-180 species of crops belonging to 35-49 families. Its life cycle consists of four stages: egg, larva, pupa, and adult. Newly hatched larvae concentrate on the growing points or tender tips of fruiting branches, feeding on young leaves and buds. Fifth and sixth instar larvae enter a voracious feeding phase, causing significant economic losses to the cotton industry.

[0004] For the target pest, the cotton bollworm, the content of exogenous insecticidal proteins (Cry1Ab / Cry1Ac) produced by insect-resistant transgenic cotton is crucial to its control efficacy. Therefore, increasing the content of insecticidal proteins in cotton leaves can enhance the insecticidal effect against the cotton bollworm, providing a reference for improving the control effect of insect-resistant transgenic cotton on cadmium-contaminated land. Summary of the Invention

[0005] In view of this, the present invention provides a method for increasing the content and insecticidal activity of exogenous insecticidal proteins in insect-resistant transgenic cotton. The present invention increases the content of exogenous insecticidal proteins in the leaves of insect-resistant transgenic cotton by planting cotton in cadmium-containing soil, thereby increasing its insecticidal activity against bollworms.

[0006] To achieve the above objectives, the present invention proposes the following technical solution:

[0007] This invention provides a method for increasing the content and insecticidal activity of exogenous insecticidal proteins in insect-resistant transgenic cotton. When cotton is in the three-leaf stage, the soil cadmium concentration is maintained at 1-5 mg / kg; when cotton is in the five-leaf stage, the soil cadmium concentration is maintained at 1-4 mg / kg.

[0008] Preferably, the soil cadmium concentration is maintained at 3-4 mg / kg when cotton is in the three-leaf and five-leaf stages.

[0009] Preferably, the insecticidal protein is an exogenous insecticidal protein.

[0010] Preferably, the insecticidal protein is Cry1Ab, Cry1Ac, or Cry2Aa.

[0011] Preferably, the insect is the cotton bollworm.

[0012] During the protein content detection process, the selected leaves were the first true leaves at the three-leaf stage and the five-leaf stage;

[0013] The cotton bollworm described in this invention is a second-instar larva.

[0014] The beneficial effects of this invention are:

[0015] (1) The method provided by the present invention can improve the content and insecticidal effect of exogenous insecticidal protein in insect-resistant transgenic cotton and increase the insecticidal activity against cotton bollworm;

[0016] (2) The method provided by the present invention can improve the control efficacy against pests and reduce the use of pesticides while planting transgenic insect-resistant cotton on cadmium-contaminated land at a certain concentration. It provides an effective reference for improving the control efficacy against bollworms on cadmium-contaminated land and has important practical value and significance. Attached Figure Description

[0017] Figure 1 The corrected mortality rate (%) of cotton bollworms against insect-resistant transgenic cotton at different cadmium concentrations as described in Examples 1 and 2. Detailed Implementation

[0018] The present invention will be further described below with reference to embodiments.

[0019] The cotton tested in the following examples is N15-5 (transfer). Cry2Aa The insect-resistant cotton (gene-based) and Jimian 14 (control parent conventional cotton) were sourced from Genesis Seed Industry Co., Ltd. Soil cadmium content was measured using inductively coupled plasma mass spectrometry (ICP-MS), and cotton leaf insecticidal protein content was measured using an enzyme-linked immunosorbent assay (ELISA) kit.

[0020] Example 1

[0021] A method for increasing the content and insecticidal activity of exogenous insecticidal proteins in insect-resistant transgenic cotton includes the following steps:

[0022] (1) Add different concentrations of CdCl2 solution to cadmium-free soil (flower nutrient soil: sand = 1:1) to correct the cadmium concentration of the soil to 0, 1, 2, 3, 4 and 5 mg / kg, and incubate for three weeks to reach equilibrium before use.

[0023] (2) The cadmium contents of the soils treated with different concentrations of cadmium were determined by ICP-MS after pretreatment. The cadmium contents of the soils under different treatments are shown in Table 1.

[0024] Table 1. Cadmium contents of the soils treated with cadmium

[0025]

[0026] (3) The soils treated with different concentrations of cadmium were put into small flower pots (10 cm in diameter and 10 cm in height), and five cotton seeds were sown in each pot (Jimmun 14 was only planted in the soil treated with 0 mg / kg cadmium). After germination, two plants were left to grow in each pot until the three-leaf stage.

[0027] (4) The first true leaves of cotton were taken for the determination of the content of insecticidal protein, and each treatment was repeated three times. The determination method of the content of insecticidal protein was ELISA. The measurement results are shown in Table 2. When the cadmium concentration of the soil was 4 mg / kg, the content of insecticidal protein in the cotton leaves was significantly higher than that without cadmium treatment.

[0028] Table 2. Contents of insecticidal protein of the three-leaf stage insect-resistant transgenic cotton under different cadmium concentrations

[0029]

[0030] Note: NC: negative control, same below.

[0031] (5) Each cadmium-treated cotton leaf was put into a disposable culture dish, and 5 two-instar cotton bollworm larvae were introduced into each dish, and 20 cotton bollworms were used for each repetition, and each treatment was repeated three times. The culture dishes were placed in an artificial climate incubator (25±1℃, 65%±5% RH, 14L:10D), and the cotton bollworms were fed for 5 days. The mortality rate of the cotton bollworms feeding on Jimmun 14 was used as a control.

[0032] (6) The cotton bollworm resistance bioassay was carried out according to the agricultural industry standard "Cotton Resistance to Cotton Bollworm Identification Method" (NY / T2162-2012), and the results are shown in Figure 1 , the corrected mortality rate of the cotton bollworms feeding on the three-leaf stage cotton leaves treated with 3 mg / kg cadmium was significantly higher than that without cadmium treatment.

[0033] As can be seen from the above examples, when the cotton grows to the three-leaf stage, the content of insecticidal protein in the first true leaf increases by 14.41%~17.14% under the concentration of 4 mg / kg cadmium. The two-instar cotton bollworm larvae are introduced into the three-leaf stage cotton leaves treated with 4 mg / kg cadmium, and the insecticidal effect increases by 10.45%~16.42%.

[0034] Example 2

[0035] A method for increasing the content and insecticidal activity of exogenous insecticidal protein in insect-resistant transgenic cotton, comprising the following steps:

[0036] (1) The soil cadmium treatment method and content determination are the same as in Example 1(1);

[0037] (2) Different cadmium concentrations of soil were loaded into small flower pots (diameter 10 cm, height 10 cm), and five cotton seeds were sown in each pot (JiMian 14 was only planted in 0 mg / kg cadmium treated soil), and after germination, two plants were left to grow in each pot until the five-leaf stage;

[0038] (3) The first true leaf of cotton was taken for measurement of insecticidal protein content, with three replicates for each treatment. The determination method of insecticidal protein content was ELISA. The measurement results are shown in Table 3. When the cadmium concentration of the soil is 3 mg / kg and 4 mg / kg, the insecticidal protein content of the cotton leaf is significantly higher than that without cadmium treatment;

[0039] Table 3 Insecticidal protein content of insect-resistant transgenic cotton at five-leaf stage under different cadmium concentrations

[0040]

[0041] (4) Each cadmium-treated cotton leaf was placed in a disposable culture dish, 5 two-instar cotton bollworms were introduced into each dish, and 20 cotton bollworms were introduced into each replicate, with three replicates for each treatment. The culture dishes were placed in an artificial climate incubator (25±1℃, 65%±5%RH, 14L:10D) and fed for 5 days. The mortality rate of cotton bollworms feeding on JiMian 14 was used as a control.

[0042] (5) The cotton bollworm resistance bioassay was carried out according to the agricultural industry standard "Cotton Bollworm Resistance Identification Method" (NY / T2162-2012). The results are shown in Table 4. The corrected mortality rate of cotton bollworms feeding on five-leaf stage cotton leaves treated with 3 mg / kg and 4 mg / kg cadmium was significantly higher than that without cadmium treatment. Figure 1

[0043] As can be seen from the above examples, at the five-leaf stage of cotton, the insecticidal protein content of the first true leaf increases by 30.82%~66.26% under 3 mg / kg cadmium concentration; the insecticidal effect of two-instar cotton bollworm larvae introduced into five-leaf stage leaves treated with 3 mg / kg cadmium increases by 22.03%~28.81%.

[0044] At the five-leaf stage of cotton, the insecticidal protein content of the first true leaf increases by 8.26%~58.66% under 4 mg / kg cadmium concentration. The insecticidal effect of two-instar cotton bollworm larvae introduced into five-leaf stage leaves treated with 4 mg / kg cadmium increases by 10.17%~16.95%.

[0045] ​The above examples are merely used to describe the preferred modes of the present application, and are not used to limit the scope of the present application. Any modifications and improvements made by those skilled in the art to the technical solutions of the present application without departing from the design concept of the present application shall fall within the protection scope of the present application.

Claims

1. A method for increasing the content and insecticidal activity of exogenous insecticidal proteins in insect-resistant transgenic cotton, characterized in that, When cotton is in the three-leaf stage, the soil cadmium concentration should be maintained at 3-4 mg / kg; when cotton is in the five-leaf stage, the soil cadmium concentration should be maintained at 3-4 mg / kg; the insecticidal proteins are Cry1Ab, Cry1Ac, and Cry2Aa; the insect is the cotton bollworm.

Images