Application of calcineurin gene dsRNA or calcineurin inhibitor in cotton bollworm Bt protein resistance treatment
By inhibiting the calcitonase gene of cotton bollworm or using calcitonase inhibitors, the problem of resistant to Bt transgenic crops is solved, the insecticidal effect of Bt protein is improved, and effective prevention and control of anti-coercive cotton bollworms is achieved.
Patent Information
- Application Number
- CN202510569059.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-08-22
AI Technical Summary
Bollworms are resistant to Bt transgenic crops and Bt preparations, and the prior art is difficult to effectively control.
By inhibiting the expression of calcineurin phosphatase genes or using calcineurin inhibitors such as dsRNA or prikofu (FK506), the activity of calcineurin in calcineurin is reduced and the insecticidal activity of Bt protein is enhanced.
It significantly improves the insecticidal activity of Bt protein against cotton bollworms, restores its sensitivity, and effectively curates the resistance of cotton bollworms.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biological control of agricultural pests, in particular to the application of calcineurin gene dsRNA or calcineurin inhibitor in the management of Bt protein resistance in cotton bollworm. Background Art
[0002] Bacillus thuringiensis ( Bacillus thuringiensis Bacillus thuringiensis (Bt) is a Gram-positive bacterium that produces an insecticidal protein (Bt protein) with specific insecticidal activity against a wide range of insects, including those in the Lepidoptera, Diptera, Coleoptera, and Hymenoptera orders. Due to Bt's insecticidal specificity and safety for the environment, humans, livestock, and non-target insects, the Bt insecticidal gene is the preferred choice for use in insect-resistant transgenic crops. Due to long-term use of insecticides or the continuous selective pressure of transgenic crop-expressed proteins, pests inevitably develop resistance to the insecticide or the insecticidal protein.
[0003] Currently, there are reports of resistance to transgenic crops in the fields of cotton bollworm, fall armyworm, and pink bollworm in several countries around the world. In my country, cotton bollworm has also developed early resistance to Cry1Ac expressed in transgenic cotton. To effectively delay and control resistance in cotton bollworm, in addition to the traditional high-dose / refuge strategy, the development of new Bt genes, other insecticidal genes, and transgenic crops based on RNAi technology are gaining increasing attention. Transgenic crops based on RNAi technology express specific dsRNA of a target gene in the plant. This disrupts or inhibits important genes in the pest's growth, development, detoxification, and metabolism, affecting the target pest's normal physiological activities and ultimately killing it. Furthermore, in practical applications, in addition to using transgenic plants to express dsRNA for pest control, the development of nanocarrier-dsRNA sprays is also possible. The effective combination of nanomaterials and dsRNA can significantly enhance RNAi efficiency. Therefore, identifying target genes for the effective control of resistant cotton bollworm is of great significance. Summary of the Invention
[0004] The purpose of the present invention is to provide application of calcineurin gene dsRNA or calcineurin inhibitor in the management of Bt protein resistance in cotton bollworm.
[0005] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides the use of calcineurin gene dsRNA or calcineurin inhibitors in the management of Bt protein resistance in cotton bollworm, wherein the use comprises: using dsRNA to interfere with the calcineurin gene of cotton bollworm, or feeding the cotton bollworm with calcineurin inhibitors to improve the insecticidal activity of Bt protein against resistant cotton bollworm.
[0006] In a second aspect, the present invention provides a method for improving the insecticidal activity of Bt protein against resistant cotton bollworms, the method comprising: using dsRNA to interfere with the calcineurin gene (GenBank: KR185962.1) of cotton bollworms, or feeding cotton bollworms with calcineurin inhibitors to improve the insecticidal activity of Bt protein against resistant cotton bollworms.
[0007] In the present invention, the Bt protein includes but is not limited to Cry1Ac (GenBank: AY730621.1).
[0008] Preferably, the target sequence of the dsRNA is as shown in SEQ ID NO: 1.
[0009] Furthermore, the dsRNA is injected into cotton bollworms.
[0010] Preferably, the calcineurin inhibitor is Prograf (FK506).
[0011] Furthermore, the calcineurin inhibitor is added to the feed of cotton bollworm.
[0012] In a third aspect, the present invention provides a preparation for improving the insecticidal activity of Bt protein against resistant cotton bollworms, wherein the active ingredient of the preparation is dsRNA of the cotton bollworm calcineurin gene or a calcineurin inhibitor.
[0013] In the present invention, the cotton bollworm calcineurin gene is CaN (calcineurin), and its GenBank accession number is KR185962.1.
[0014] In a fourth aspect, the present invention provides the use of the calcineurin gene as a target gene in the management of Bt protein resistance in cotton bollworm.
[0015] Furthermore, the application includes: improving the insecticidal activity of Bt protein against resistant cotton bollworms by inhibiting the calcineurin gene of cotton bollworms.
[0016] Furthermore, the substance that inhibits the calcineurin gene of cotton bollworm is a substance that can inhibit the expression or activity of the calcineurin gene or protein of cotton bollworm at the transcription or translation level.
[0017] The substance can be selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, antibodies, etc.; preferably dsRNA, and more preferably the target sequence of the dsRNA is as shown in SEQ ID NO: 1.
[0018] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention utilizes dsRNA to interfere with the calcineurin gene of cotton bollworms, or by adding a calcineurin inhibitor, significantly reducing phosphatase activity in the midgut of cotton bollworms, thereby significantly enhancing the activity of Bt insecticidal proteins against cotton bollworms. The method provided by the present invention can be used to enhance the effectiveness of Bt in controlling cotton bollworms and manage resistance in Cry1Ac-resistant cotton bollworms, restoring their sensitivity to Cry1Ac. The present invention enables the control of cotton bollworms or pest resistance management, providing a safe and effective means of control and prevention, with promising application prospects in field control of cotton bollworms and in green control of cotton bollworms. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 This represents the increase in calcineurin activity in the susceptible strain of cotton bollworm induced by Cry1Ac in Example 1 of the present invention. Data in the figure are mean ± standard error. Different lowercase letters indicate significant differences between treatments (Turkey test, P < 0.05, SPSS Statistics 21). Buffer is the control. Cry1Ac-24 h: Calcineurin activity in cotton bollworms 24 hours after feeding on Cry1Ac. Cry1Ac-48 h: Calcineurin activity in cotton bollworms 48 hours after feeding on Cry1Ac.
[0020] Figure 2 The expression of calcineurin in the midgut of resistant and susceptible cotton bollworm strains in Example 1 of the present invention is shown. A: Expression level of calcineurin gene in the midgut of resistant and susceptible cotton bollworm strains; B: calcineurin activity in the midgut of resistant and susceptible cotton bollworm strains. Data in the figure are mean ± standard error. Asterisks indicate significant differences between treatments (t-test, express P <0.05, express P <0.01, SPSS Statistics 21). 96s: susceptible strain of cotton bollworm, BtR: resistant strain of cotton bollworm.
[0021] Figure 3 The mortality of resistant cotton bollworms treated with Cry1Ac after the calcineurin gene was disrupted in Example 2 of the present invention. The data in the figure are mean ± standard error. Different lowercase letters indicate significant differences between treatments (Turkey test, P<0.05, SPSS Statistics 21). A: Calcineurin silencing efficiency of resistant cotton bollworm strains; Buffer and ds-EGFP serve as controls; ds-CaN: Changes in calcineurin gene expression levels after calcineurin interference. B: Changes in sensitivity of resistant cotton bollworm strains to Cry1Ac after calcineurin interference; Buffer and ds-EGFP serve as controls; ds-CaN: Changes in sensitivity of cotton bollworm strains to Cry1Ac after calcineurin interference.
[0022] Figure 4 The data in the figure are the mortality of resistant cotton bollworms treated with Bt after adding Prograf in Example 3 of the present invention. The data in the figure are mean ± standard error. Asterisks indicate significant differences between treatments (t test, express P <0.01, express P <0.001, P <0.0001, SPSS Statistics 21). Mortality of resistant cotton bollworms after feeding with Prograf (FK506) at concentrations of 10, 20, 50, 100, and 200 µM, Cry1A protoxin alone, and a mixture of Cry1Ac and FK506.
[0023] Figure 5 1 is a comparison of the interference efficiency of ds-CaN-1 and ds-CaN-2 in Example 3 of the present invention. DETAILED DESCRIPTION
[0024] The present invention provides an application for controlling Bt resistance of cotton bollworm by inhibiting calcineurin, so as to solve the problem of cotton bollworm resistance to Bt transgenic crops or Bt preparations existing in the prior art, and achieve a significant improvement in the effect of controlling cotton bollworm resistance.
[0025] The present invention adopts the following technical solutions: The present invention finds that by inhibiting the expression of the calcineurin gene of cotton bollworm or by using a cotton bollworm calcineurin inhibitor, the activity of calcineurin in the midgut of cotton bollworm can be significantly reduced, thereby promoting the insecticidal activity of Bt against resistant cotton bollworms and significantly improving the toxicity of Bt.
[0026] The present invention provides a method for controlling the resistance of cotton bollworm to Bt by inhibiting the expression of the calcineurin gene of cotton bollworm or by using Bt in conjunction with a cotton bollworm calcineurin inhibitor.
[0027] In the above application, the dsRNA of the calcineurin gene of cotton bollworm, the dsRNA targeting the calcineurin gene is a specific dsRNA fragment for the calcineurin gene, and can inhibit the expression of the calcineurin gene of cotton bollworm.
[0028] Preferably, the nucleotide sequence of the target sequence to which the dsRNA binds is shown in SEQ ID NO: 1.
[0029] The present invention provides dsRNA that can inhibit the expression of the calcineurin gene. The dsRNA with a target sequence such as SEQ ID NO: 1 can reduce the activity of calcineurin in the intestine of cotton bollworms, enhance the toxicity of Bt insecticidal proteins, improve the insecticidal activity against resistant cotton bollworms, and slow down the development of Bt resistance in cotton bollworms.
[0030] The present invention provides a preparation for managing Bt resistance, wherein the preparation is a calcineurin inhibitor of cotton bollworm, Prograf (FK506) or others.
[0031] In the above application, the insecticidal protein of Bacillus thuringiensis is preferably Cry1Ac.
[0032] In the above application, the cotton bollworm is Cry1Ac-resistant cotton bollworm.
[0033] The present invention further provides the use of dsRNA of the calcineurin gene of cotton bollworm or a calcineurin inhibitor of cotton bollworm in improving the insecticidal activity of Bacillus thuringiensis (Bt) Cry1Ac against resistant cotton bollworm.
[0034] The nucleotide sequence of the target sequence to which the dsRNA binds is shown in SEQ ID NO: 1.
[0035] Based on the nucleotide sequence of the target sequence to which the dsRNA binds, those skilled in the art can obtain the sequence of the positive strand of the dsRNA and obtain the dsRNA by a commonly used dsRNA synthesis method.
[0036] The present invention also provides a preparation for controlling Bt resistance in cotton bollworm, including but not limited to the cotton bollworm calcineurin inhibitor Prograf (FK506).
[0037] In the above method, the above preparation can be administered to the cotton bollworm by injecting insecticidal protein of Bacillus thuringiensis, injecting dsRNA of calcineurin gene of cotton bollworm, or adding inhibitor of Prograf to the feed of cotton bollworm.
[0038] The above method can be used for the control of Cry1Ac-resistant cotton bollworm.
[0039] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.
[0040] In the following examples, upstream and downstream primers were designed based on the calcineurin gene of the cotton bollworm (GenBank: KR185962.1). dsRNA was obtained by PCR using cotton bollworm cDNA as a template. The upstream primer sequence is: 5'-CATGTATGGACGCGTTTGAC-3' (SEQ ID NO: 2), and the downstream primer sequence is: 5'-GCCAGTATGGGTGAGGAGAA-3' (SEQ ID NO: 3).
[0041] The Cry1Ac protein (Cry1Ac protoxin) used in the following examples was purchased from Beijing Genralpest Biotechnology Co., Ltd. (www.genralpest.com); Tacrolimus (FK506) was purchased from Abcam with a trade name of CAS No: AB120223-1001, and a calcineurin (CaN) test kit was purchased from Nanjing Jiancheng Bioengineering Institute with a trade name of A068-1.
[0042] Example 1 Determination of calcineurin expression and enzyme activity in resistant and susceptible strains of cotton bollworm 1. Fourth-instar larvae of the susceptible strain of cotton bollworm were fed with 24 µg / mL Cry1Ac protein for 24 and 48 h, respectively. Midgut enzymes were extracted and the activity of calcineurin was detected.
[0043] 2. The results show that ( Figure 1 ), after feeding for 24 h and 48 h, the activity of calcineurin increased significantly.
[0044] 3. The midguts of sensitive and resistant strains of cotton bollworm were dissected respectively, RNA was extracted, reverse transcribed into cDNA template, and the expression levels of calcineurin gene in the midguts of different strains were detected.
[0045] 4. The results show that ( Figure 2 A) The expression level of calcineurin was significantly increased in the resistant lines.
[0046] 5. Dissect the midguts of sensitive and resistant strains of cotton bollworms respectively, extract midgut enzymes, and detect the activity of calcineurin in the resistant and susceptible strains of cotton bollworms.
[0047] 6. The results show that ( Figure 2 B) The calcineurin activity in the resistant strain was significantly higher than that in the sensitive strain.
[0048] Example 2 Gene Silencing Efficiency and Its Effect on Bt Virulence after Injection of Calcineurin (CaN) Gene dsRNA 1. Synthesize calcineurin (CaN) gene dsRNA (target nucleotide sequence shown in SEQ ID NO: 1). Double-stranded dsRNA for the green fluorescent protein (GFP) gene was used as a control. Calcineurin gene dsRNA (1000 ng) was microinjected into the midgut of resistant cotton bollworms (above the third pair of thoracic legs and below the first pair of abdominal legs) into early third-instar larvae.
[0049] 2. 48 hours after injection, 15 cotton bollworm larvae from each treatment were collected, their midguts were dissected, RNA was extracted and cDNA was reversed to detect the silencing efficiency of the calcineurin gene. The results are as follows: Figure 3 As shown in A, injection of dsRNA of the calcineurin gene of cotton bollworm can lead to a significant decrease in the expression level of the calcineurin gene in resistant cotton bollworm.
[0050] 3. Transfer the remaining bollworms that are in good condition to an artificial diet containing 500 µg / g of Cry1Ac protein. After 7 days, examine the larvae for mortality and perform statistical analysis on the bioassay results. Larvae that have been fed on a normal diet for 48 hours serve as a control.
[0051] 4. The results show that ( Figure 3 B) After dsRNA injection, cotton bollworms were treated with Cry1Ac. The mortality rate of resistant bollworms increased significantly, from 19.4% to 48.6%. These results indicate that interfering with the calcineurin gene enhances the bollworm's sensitivity to Cry1Ac, significantly improving resistance control in the bollworm.
[0052] Neither dsRNA nor FK506 alone resulted in mortality in cotton bollworms.
[0053] Example 3: Prograf significantly improves the insecticidal activity of Bt against resistant cotton bollworms 1. Add different concentrations of 10, 20, 50, 100, and 200 µM of Prograf (FK506) to an artificial diet containing 100 µg / g Cry1Ac protein for cotton bollworms. Mix thoroughly and distribute the mixture evenly across 24-well plates. Newly hatched resistant larvae of cotton bollworms were seeded and reared in an insectary. Cotton bollworms reared on a normal artificial diet served as a control.
[0054] 2. After 7 days, check the larvae for death and make statistical analysis on the bioassay results. Figure 4 ), after the Cry1Ac protein and Prograf (FK506) were mixed and fed to cotton bollworms, the mortality rate of resistant cotton bollworm larvae increased significantly.
[0055] The present invention specifically optimizes the target sequence of dsRNA, screens out the highly conserved region in the target sequence of SEQ ID NO: 1, reduces the loss of gene silencing efficiency, and designs dsRNA of appropriate length and stability; further, the dsRNA is coated with a nanocarrier to enhance the dsRNA delivery efficiency.
[0056] The study showed that, first, the target sequence of SEQ ID NO:1 is consistent in the gene sequence of the resistant and susceptible strains of cotton bollworm, and has a certain degree of conservation. Second, according to the dsRNA design principles, the target sequence of SEQ ID NO:1 has a better GC content and the smallest secondary structure. Third, preliminary experiments showed that the target sequence of SEQ ID NO:1 has the highest silencing efficiency. Figure 5 As can be seen, among the designed candidate dsRNAs, ds-CaN-1 and ds-CaN-2 showed significant differences in their interference efficiency, with ds-CaN-1 having the highest interference efficiency, indicating that the target sequence SEQ ID NO: 1 had the highest silencing efficiency. The nucleotide sequence of the ds-CaN-2 target sequence is shown in SEQ ID NO: 4. The upstream primer sequence used to synthesize ds-CaN-2 is: 5'-GTATAGAATGTGTTCTTTACCTC-3' (SEQ ID NO: 5), and the downstream primer sequence is: 5'-CTACAAAGAAACAATTTACTGT-3' (SEQ ID NO: 6).
[0057] The present invention analyzes the resistance mechanism of the target gene, optimizes the design of dsRNA, and mixes it with the inhibitor of the target gene (calcineurin gene) for feeding, thereby achieving precise prevention and control of resistant cotton bollworms and having significant application value.
[0058] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.
Claims
1. Application of calcineurin gene dsRNA or calcineurin inhibitor in the management of Bt protein resistance in cotton bollworm, characterized in that: The application includes: using dsRNA to interfere with the calcineurin gene of cotton bollworm, or feeding cotton bollworm with calcineurin inhibitor to improve the insecticidal activity of Bt protein on resistant cotton bollworm.
2. A method for improving the insecticidal activity of Bt protein against resistant cotton bollworm, characterized in that: The method comprises: using dsRNA to interfere with the calcineurin gene of cotton bollworm, or feeding cotton bollworm with a calcineurin inhibitor to enhance the insecticidal activity of Bt protein against resistant cotton bollworm; The Bt protein includes Cry1Ac.
3. The method according to claim 2, characterized in that The target sequence of the dsRNA is shown in SEQ ID NO:
1.
4. The method according to claim 3, characterized in that The dsRNA was injected into cotton bollworms.
5. The method according to claim 2, characterized in that The calcineurin inhibitor is Prograf.
6. The method according to claim 5, characterized in that The calcineurin inhibitor is added to the feed of cotton bollworms.
7. A preparation for improving the insecticidal activity of Bt protein against resistant cotton bollworm, characterized in that: The active ingredient of the preparation is dsRNA of calcineurin gene of cotton bollworm or calcineurin inhibitor.
8. The preparation according to claim 7, characterized in that The target sequence of the dsRNA is shown in SEQ ID NO: 1; and / or, The calcineurin inhibitor is Prograf.
9. Application of calcineurin gene as target gene in the management of Bt protein resistance in cotton bollworm.
10. The use according to claim 9, characterized in that The application includes: improving the insecticidal activity of Bt protein against resistant cotton bollworm by inhibiting the calcineurin gene of cotton bollworm; The substance that inhibits the calcineurin gene of cotton bollworm is a substance that can inhibit the expression or activity of the calcineurin gene or protein of cotton bollworm at the transcriptional or translational level, and the substance is selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, and antibodies; preferably dsRNA, and more preferably the target sequence of the dsRNA is as shown in SEQ ID NO: 1; and / or, The cotton bollworm is Cry1Ac-resistant cotton bollworm.