Method for improving exogenous insecticidal protein content and insecticidal activity of insect-resistant transgenic cotton

Abstract

The invention belongs to the field of agricultural pest control, and particularly relates to a method for improving exogenous insecticidal protein content and insecticidal activity of insect-resistant transgenic cotton. The method is realized through the following steps: when the cotton is in a trefoil stage, keeping the cadmium concentration of soil at 1-5 mg / kg; when the cotton is in the five-leaf stage, the cadmium concentration of the soil is kept to be 1-4 mg / kg. According to the method provided by the invention, the exogenous insecticidal protein content and the insecticidal effect of the insect-resistant transgenic cotton can be remarkably improved, and the insecticidal activity on cotton bollworms is enhanced; according to the method provided by the invention, the transgenic insect-resistant cotton is planted on the cadmium-polluted land with a certain concentration to treat cadmium pollution, meanwhile, the pest control effect can be improved, and the use of insecticides is reduced. Therefore, the invention provides an effective reference for planting the insect-resistant transgenic cotton on the cadmium-polluted land to improve the pest control effect, and has important practical value and significance.

Description

Technical Field

[0001] The 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 Art

[0002] Since 1996, the transgenic cotton planted in my country has been insect-resistant varieties (transgenic Cry1Ab / Cry1Ac Gene), the planting area of ​​genetically modified cotton has reached 3.2 million hectares. The large-scale promotion and planting of insect-resistant genetically modified cotton has effectively controlled the damage of cotton bollworms, reduced the amount of chemical pesticides and the cost of cotton farmers, and significantly increased economic, social and ecological benefits. Large-scale planting of cotton will inevitably be affected by heavy metals.

[0003] cotton bollworm ( Helicoverpa armigera Hübner belongs to the family Noctuidae of the order Lepidoptera. It is a worldwide migratory pest widely distributed in the cotton-producing areas of the Yellow River Basin and the Yangtze River Basin in my country. The cotton bollworm feeds on more than 130 to 180 species of crops from 35 to 49 families. The life cycle of the cotton bollworm is divided into four stages: egg, larva, pupa and adult. The newly hatched larvae concentrate on the growing points or the tender tips of fruit branches to feed on young leaves and young buds. The fifth to sixth instar larvae enter the gluttonous feeding stage, causing huge economic losses to the cotton industry.

[0004] For the target pest cotton bollworm, the content of exogenous insecticidal proteins (Cry1Ab / Cry1Ac) produced by insect-resistant transgenic cotton is crucial to its control effect. Therefore, by increasing the insecticidal protein content in cotton leaves, the insecticidal effect on cotton bollworm is enhanced, 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 exogenous insecticidal protein content and insecticidal activity of insect-resistant transgenic cotton. The present invention increases the exogenous insecticidal protein content in the leaves of insect-resistant transgenic cotton and increases the insecticidal activity against cotton bollworm by planting cotton in cadmium-containing soil.

[0006] In order to achieve the above object, the present invention proposes the following technical solutions: The invention provides a method for increasing the exogenous insecticidal protein content and insecticidal activity of insect-resistant transgenic cotton. When the cotton is in the three-leaf stage, the soil cadmium concentration is maintained at 1-5 mg / kg; when the cotton is in the five-leaf stage, the soil cadmium concentration is maintained at 1-4 mg / kg.

[0007] Preferably, when cotton is at the three-leaf stage and the five-leaf stage, the cadmium concentration in the soil is maintained at 3-4 mg / kg.

[0008] Preferably, the insecticidal protein is an exogenous insecticidal protein.

[0009] Preferably, the insecticidal protein is Cry1Ab, Cry1Ac, or Cry2Aa.

[0010] Preferably, the insect is cotton bollworm.

[0011] During the protein content test, the leaves selected were the first true leaves at the three-leaf stage and the five-leaf stage; The cotton bollworm described in the present invention is a second-instar larva.

[0012] The beneficial effects of the present invention are: (1) The method provided by the present invention can increase the content and insecticidal effect of exogenous insecticidal proteins in insect-resistant transgenic cotton and increase the insecticidal activity against cotton bollworms; (2) The method provided by the present invention can improve the control effect on pests and reduce the use of pesticides while controlling cadmium pollution by planting transgenic insect-resistant cotton on land with a certain concentration of cadmium pollution. It provides an effective reference for improving the control effect of insect-resistant transgenic cotton on cadmium-contaminated land against cotton bollworm, and has important practical value and significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is the corrected mortality rate (%) of the insect-resistant transgenic cotton to cotton bollworm under different cadmium concentrations described in Examples 1 and 2. DETAILED DESCRIPTION

[0014] The present invention is further described below in conjunction with embodiments.

[0015] In the following examples, cotton N15-5 (transferred Cry2A Genetically modified insect-resistant cotton) and Jimian 14 (control parent conventional cotton) were obtained from Genesis Seed Co., Ltd. The cadmium content in soil was measured by inductively coupled plasma mass spectrometry (ICP-MS), and the insecticidal protein content in cotton leaves was measured by enzyme-linked immunosorbent assay (ELISA) kit.

[0016] Example 1 A method for increasing the content and insecticidal activity of exogenous insecticidal protein in insect-resistant transgenic cotton, comprising the following steps: (1) Add different concentrations of CdCl2 solution to cadmium-free soil (flower nutrient soil: sandy soil = 1:1) to correct the cadmium concentration of the soil to 0, 1, 2, 3, 4 and 5 mg / kg, incubate for three weeks and then set aside; (2) The soil treated with different concentrations of cadmium was mixed and sampled, and the cadmium content was determined by ICP-MS after pretreatment. The cadmium content of the soil under different treatments is shown in Table 1; Table 1 Cadmium content in soil under cadmium treatment (3) Soils with different cadmium concentrations were placed in small flower pots (10 cm in diameter and 10 cm in height), and five cotton seeds were sown in each pot (of which Hebei cotton 14 was only planted in the soil treated with 0 mg / kg cadmium). After germination, two cotton seeds were left in each pot to continue growing until the three-leaf stage; (4) The insecticidal protein content of the first true leaf of cotton was measured, and each treatment was repeated three times. The insecticidal protein content was determined by ELISA. The measurement results are shown in Table 2. When the cadmium concentration in the soil was 4 mg / kg, the insecticidal protein content of cotton leaves was significantly higher than that in the cadmium-free treatment.

[0017] Table 2 Insecticidal protein content of insect-resistant transgenic cotton at three-leaf stage under different cadmium concentrations Note: NC: negative control, the same below.

[0018] (5) Each cadmium-treated cotton leaf was placed in a disposable culture dish, and five second-instar cotton bollworm larvae were inoculated into each dish, with 20 cotton bollworms per replicate and three replicates for each treatment. The culture dish was placed in an artificial climate incubator (25±1℃, 65%±5%RH, 14 L:10 D) and raised for 5 days. The mortality of cotton bollworms feeding on Hebei cotton 14 was used as a control.

[0019] (6) The bioassay for cotton bollworm resistance was carried out in accordance with the agricultural industry standard “Methods for Identifying Cotton Resistance to Cotton Bollworm” (NY / T2162-2012). The results are shown in Figure 1 After feeding on cotton leaves at the three-leaf stage under the 3 mg / kg cadmium treatment, the corrected mortality of cotton bollworm was significantly higher than that under the cadmium-free treatment.

[0020] It can be seen from the above examples that when cotton grows to the three-leaf stage, the insecticidal protein content in the first true leaf increases by 14.41% to 17.14% at a cadmium concentration of 4 mg / kg. The insecticidal effect of second-instar cotton bollworm larvae on three-leaf cotton leaves treated with 4 mg / kg cadmium increases by 10.45% to 16.42%.

[0021] Example 2 A method for increasing the content and insecticidal activity of exogenous insecticidal protein in insect-resistant transgenic cotton, comprising the following steps: (1) The soil cadmium treatment method and content determination were the same as in Example 1(1); (2) Soils with different cadmium concentrations were placed in small flower pots (10 cm in diameter and 10 cm in height), and five cotton seeds were sown in each pot (of which Hebei cotton 14 was only planted in the soil treated with 0 mg / kg cadmium). After germination, two cotton seeds were left in each pot to continue growing until the five-leaf stage; (3) The insecticidal protein content of the first true leaf of cotton was measured, and each treatment was repeated three times. The insecticidal protein content was determined by ELISA. The measurement results are shown in Table 3. When the cadmium concentration in the soil was 3 mg / kg and 4 mg / kg, the insecticidal protein content of cotton leaves was significantly higher than that of the cadmium-free treatment; Table 3 Insecticidal protein content of insect-resistant transgenic cotton at five-leaf stage under different cadmium concentrations (4) Each cadmium-treated cotton leaf was placed in a disposable culture dish, and five second-instar cotton bollworms were inoculated into each dish, with 20 cotton bollworms per replicate and three replicates for each treatment. The culture dish was placed in an artificial climate incubator (25±1℃, 65%±5%RH, 14 L:10 D) and raised for 5 days. The mortality of cotton bollworms feeding on Hebei cotton 14 was used as a control.

[0022] (5) Bioassay for cotton bollworm resistance Agricultural industry standard “Method for identifying cotton resistance to cotton bollworm” (NY / T2162-2012), the results are shown in Figure 1 After feeding on cotton leaves at the five-leaf stage under 3 mg / kg and 4 mg / kg cadmium treatments, the corrected mortality of cotton bollworm was significantly higher than that under the cadmium-free treatment.

[0023] It can be seen from the above examples that at the five-leaf stage of cotton, at a cadmium concentration of 3 mg / kg, the insecticidal protein content in the first true leaf increased by 30.82%~66.26%; the insecticidal effect of inoculating second-instar cotton bollworm larvae with 3 mg / kg cadmium on the five-leaf leaves increased by 22.03%~28.81%.

[0024] At the five-leaf stage of cotton, the insecticidal protein content in the first true leaf increased by 8.26% to 58.66% at a cadmium concentration of 4 mg / kg. The insecticidal effect of inoculating the second-instar cotton bollworm larvae on the leaves at the five-leaf stage with cadmium treatment of 4 mg / kg increased by 10.17% to 16.95%.

[0025] The examples described above are only descriptions of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the design concept of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the scope of protection determined by the claims of the present invention.

Claims

1. A method for increasing the content and insecticidal activity of exogenous insecticidal protein in insect-resistant transgenic cotton, characterized in that: When cotton is in the three-leaf stage, the soil cadmium concentration should be maintained at 1-5 mg / kg; when cotton is in the five-leaf stage, the soil cadmium concentration should be maintained at 1-4 mg / kg.

2. The method according to claim 1, characterized in that When cotton is in the three-leaf and five-leaf stages, the soil cadmium concentration should be maintained at 3-4 mg / kg.

3. The method according to claim 1 or 2, characterized in that: The insecticidal protein is an exogenous insecticidal protein.

4. The method according to claim 3, characterized in that The insecticidal proteins are Cry1Ab, Cry1Ac, and Cry2Aa.

5. The method according to any one of claims 1 to 4, characterized in that: The insect is cotton bollworm.

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