Key gene Sim of bHLH family and application of key gene Sim in prevention and control of spodoptera frugiperda

Abstract

The invention belongs to the field of agricultural pest control, and particularly relates to a bHLH family key gene Sim and application thereof in grassland spodoptera frugiperda control. The invention provides a bHLH family protein Sim which is a protein composed of amino acids as shown in SEQ ID NO.2, and a gene Sim for coding the bHLH family protein, and a coding region of the gene Sim is a DNA (deoxyribonucleic acid) molecule with a sequence as shown in SEQ ID NO.1. By reducing the expression of the Sim gene, spodoptera frugiperda larvae can grow slowly and die, the egg laying amount of adults is remarkably reduced, even the adults do not lay eggs, so that the population number of the adults is effectively controlled, and the harm caused by pest outbreak is reduced. A novel environment-friendly high-specificity RNA pesticide is developed based on the gene, and a new solution is provided for pest control.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant genetic engineering, and particularly relates to a key gene Sim of bHLH family and application thereof in Spodoptera frugiperda control. BACKGROUND

[0002] Spodoptera frugiperda Spodoptera frugiperda Spodoptera frugiperda (J.E.Smith) belongs to the family of noctuidae of lepidoptera, and is originally from the tropical and subtropical regions of America, and is listed as a major migratory pest by the Food and Agriculture Organization of the United Nations. The strong migration ability makes it rapidly expand and harm in a global range, and seriously threatens the crop production and food safety in the world. Spodoptera frugiperda is a polyphagous pest, and the larvae can feed on more than 76 families and 350 species of plants. At present, two kinds of host plant strains have been identified, and the "maize type" mainly feeds on corn, cotton and sorghum, and the "rice type" is mainly related to rice and various pasture. Through molecular identification, the Spodoptera frugiperda invading China in recent years is of the maize type, and likes to feed on plants of the family of poaceae. With the gradual adjustment of the agricultural structure in China, the planting amount of corn has been significantly increased, and the suddenness, gluttony and polyphagy of Spodoptera frugiperda have seriously threatened the safe production of crops such as corn in China, and brought great difficulties in prevention and control. In order to cope with the threat of Spodoptera frugiperda, China has adopted various strategies such as chemical control and biological control. Among them, chemical control is the main means to cope with the outbreak of Spodoptera frugiperda at present, and can effectively control its harm in a short period of time. However, long-term dependence on chemical control is easy to make the pests develop resistance, and pollute the environment. Therefore, it is urgent to develop green and efficient control methods based on high specificity targets.

[0003] Sim gene was first identified in Drosophila melanogaster, and belongs to the family of basic helix-loop-helix (bHLH) transcription factors, which has a DNA binding domain and a protein dimerization domain. Sim gene is a key regulatory factor for the differentiation of central nervous system (CNS) ventral midline in Drosophila. It is expressed in mesodermal cells, which constitute the ventral midline and migrate from the surface of the embryo (ectoderm), and then differentiate into midline glial cells, neurons or neuroblasts. In Drosophila Sim deletion mutant embryos, the pattern of CNS axons is deformed; this phenotype is called collapsed axon tracts, and in these embryos, midline neurons and glial cells are absent. In addition to regulating the development of nervous system, some studies have also revealed that Sim plays a crucial role in regulating the left-right asymmetry of embryonic intestine, guiding axons in larval brain, the association of basal lamina-retina neurons in visual lobe development, the development of insect olfactory system, and the development of anal pad and genital disc. In addition, as one of the downstream targets of Drosophila NR5A nuclear receptor Ftz-f1, Sim is up-regulated in 10B stage follicle cells, and finally promotes the maturation of Drosophila follicle and ovulation.

[0004] It is evident that Sim, as a developmental regulatory protein, plays multiple important roles in the development of fruit fly larvae and the reproduction of adults. However, its role in the growth regulation of fall armyworm and its application in the control of fall armyworm remain unclear. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing methods for controlling fall armyworm, and to provide a method for controlling fall armyworm by utilizing the role of the bHLH key gene Sim and its encoded protein in the growth regulation of fall armyworm.

[0006] In a first aspect, the present invention provides a bHLH family protein Sim, wherein the protein Sim is any one of the following (1)-(2): (1) A protein composed of the amino acids shown in SEQ ID NO.2; (2) A protein derived from (1) with the same function by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid shown in SEQ ID NO.2.

[0007] Secondly, the present invention also provides a gene Sim for a bHLH family protein, wherein the nucleotide sequence of the gene Sim is any one of the following (3)-(5): (3) A DNA molecule whose coding region is the sequence shown in SEQ ID NO.1; (4) DNA molecules that hybridize with the DNA sequence defined in (4) under strict conditions and encode proteins with the same function; (5) DNA molecules that have at least 95% sequence identity with the DNA sequence defined in (3) and encode proteins with the same function.

[0008] Thirdly, the present invention also provides the application of the protein Sim described in the first aspect or the gene Sim described in the second aspect as a target, wherein the application is any one of the following: A1) Regulate the growth rate of fall armyworm larvae; A2) Regulate the reproductive capacity of fall armyworm larvae and adults; A3) Regulate the mortality rate of fall armyworm larvae.

[0009] Fourthly, the present invention also provides the use of a substance capable of reducing the activity of the protein Sim described in the first aspect or capable of reducing the expression level of the gene Sim described in the second aspect, in any of the following applications: B1) Reduce the growth rate of fall armyworm larvae; B2) Reduce the reproductive capacity of fall armyworm larvae and adults; B3) Increase the mortality rate of fall armyworm larvae; The substance capable of reducing the activity of the protein Sim as described in the first aspect or the substance capable of reducing the expression level of the gene Sim as described in the second aspect is any one of the following. C1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the gene encoding the protein; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant expression vector containing the nucleic acid molecule described in C1) or the expression cassette described in C2); C4) Recombinant microorganisms containing the recombinant expression vector described in C3).

[0010] Furthermore, the nucleic acid molecule is an sgRNA molecule that targets the protein-coding gene described in the second aspect, and the target sequence of the sgRNA molecule is shown in SEQ ID NO.6 or SEQ ID NO.7.

[0011] Furthermore, the sequence of the sgRNA molecule is shown in SEQ ID NO.8-11.

[0012] Using the above technical solution, sgRNAs are prepared according to the target sequence provided in the fourth aspect. The sgRNAs and Cas9 protein are introduced into fresh eggs of fall armyworm, which can effectively reduce the activity of protein Sim in the first aspect or reduce the expression level of gene Sim in the second aspect.

[0013] Fifthly, the present invention also provides a method for controlling fall armyworm, the method comprising introducing the substance described in the fourth aspect into the fall armyworm via a CRISPR / Cas9 system.

[0014] Furthermore, the importation is carried out into the eggs of the fall armyworm.

[0015] Furthermore, the concentration of the substance described in the fourth aspect is 500-700 ng / μl, preferably 600 ng / μl.

[0016] Using the above-mentioned technical method, sgRNAs at a concentration of 500-700 ng / μL and Cas9 at a volume ratio of 200-400 ng / μL were mixed evenly at a 1:2 volume ratio. The preferred concentration of sgRNAs was 600 ng / μL, and the preferred concentration of Cas9 was 300 ng / μL. This mixture was then introduced into fresh eggs of *S. fall armyworm*. It was found that after injection of sgRNAs containing the Sim gene, the body length growth of *S. fall armyworm* larvae was significantly inhibited, and a large number of larvae died. These results indicate that the Sim gene plays a crucial role in the growth and development of *S. fall armyworm* larvae.

[0017] Compared with existing technologies, this invention reveals for the first time the crucial role of the Sim gene in the larval development and adult reproduction of the fall armyworm. By inhibiting Sim gene expression, the development of fall armyworm larvae can be effectively delayed, and the reproductive capacity of adults can be weakened, thereby curbing its population growth. This discovery provides an innovative strategy for controlling large-scale outbreaks of the fall armyworm and also lays a solid scientific foundation for developing green and efficient control methods to combat the fall armyworm and other lepidopteran pests. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below.

[0019] Figure 1 The figure shows the reduction efficiency of Sim gene expression in the fall armyworm after injection of Sim gene-specific sgRNAs. The abbreviations in the figure are: "WT" represents the wild-type fall armyworm, "Sim" represents the wild-type fall armyworm. mut "Represents the injection group that received the Sim gene sgRNAs."

[0020] Figure 2 This is a comparison of the body size of 6th instar larvae injected with Sim gene-specific sgRNAs with that of wild-type control larvae.

[0021] Figure 3 The survival rates of fall armyworm larvae at various instars in the injection group and the control group are given. The abbreviations are: "1, 2, 3, 4, 5, 6" represent the instars of the larvae, and "p" represents the pupal stage.

[0022] Figure 4 The total number of eggs laid by a single female fall armyworm in the wild and injected groups after mating. Detailed Implementation

[0023] The following embodiments are further illustrations of the present invention, but not limitations thereof. In the following embodiments, unless otherwise stated, the molecular biology techniques used are conventional techniques that can be easily obtained and applied by those skilled in the art through public or commercial means.

[0024] The fall armyworm experimental materials used in the following examples were raised on artificial feed in the laboratory of Nanjing Agricultural University.

[0025] Example 1: Sequencing and analysis of the proposed knockout region of the Sim gene in the fall armyworm. The Sim gene of the fall armyworm has the nucleotide sequence shown in SEQ ID NO.1 and the encoded amino acid sequence shown in SEQ ID NO.2.

[0026] Late-stage embryos of the fall armyworm were collected, and gDNA was extracted from the mixture. Three different biological replicates were set up, and sequencing primers were designed as follows: Upstream primer sequence Sim-F: 5'-AAAAGCCACTTCAATCTG-3' (SEQ ID NO.3) Downstream primer sequence Sim-R: 5'-CCAACAAATTCTTAACCC-3' (SEQ ID NO.4).

[0027] Using fall armyworm gDNA as a template, the target knockout region of the Sim gene in fall armyworm was amplified by PCR using the primers described above. Its nucleotide sequence is shown in SEQ ID NO. 5. After amplification, the target fragment was identified, purified, and recovered by 1% agarose gel electrophoresis. The recovered product was ligated into the pCE2 TA / Blunt-Zero vector, transformed, and cultured. Positive clones were screened and sent to a biotechnology company for sequencing. Sequencing results showed that the nucleotide sequence of exon 2 of the Sim gene in this fall armyworm sequencing segment is shown in SEQ ID NO. 6, and the nucleotide sequence of exon 3 is shown in SEQ ID NO. 7.

[0028] Example 2: Injection of fresh eggs of fall armyworm Based on the target sequences of exons 2 and 3, the required sgRNAs were designed. The nucleotide sequence of sgRNA1 is shown in SEQ ID NO. 8, the nucleotide sequence of sgRNA2 is shown in SEQ ID NO. 9, the nucleotide sequence of sgRNA3 is shown in SEQ ID NO. 10, and the nucleotide sequence of sgRNA4 is shown in SEQ ID NO. 11. The target sequence of sgRNA1 and sgRNA2 is exon 2, and the target sequence of sgRNA3 and sgRNA4 is exon 3. Then, the DNA templates of the four sgRNAs were amplified by PCR using the primers in Table 2.

[0029] Table 1 sgRNA sequences

[0030] Table 2 Primer sequences

[0031] After purification of the PCR amplification products, the required sgRNAs were synthesized and purified, and stored at -80℃ for later use. Fall armyworms were reared in an artificial climate incubator at a temperature maintained at 27±1℃, relative humidity at 70%, and a photoperiod of 8 hours day / 16 hours night. The day before injection, newly emerged female and male adults were placed in a gauze-covered barrel-shaped culture box at a 1:2 ratio and placed in the incubator. One hour after the start of the second dark cycle, fresh eggs were collected and arranged on a glass slide. By microinjection, a final concentration of 600 ng / μL of sgRNAs was mixed with 300 ng / μL of Cas9 at a 1:2 volume ratio and introduced into the fresh eggs of the fall armyworms.

[0032] Example 3: Detection of Sim gene expression after injection of Sim gene sgRNAs. To detect Sim gene expression, RNA was extracted from both injected larvae and adults. Specifically, RNA from larvae was extracted from their molted bodies, while RNA from adults was extracted from their pupal shells shed during initial emergence. These RNAs were then reverse transcribed into cDNA.

[0033] The results are as follows Figure 1 As shown, the expression level of the Sim gene decreased significantly after injection.

[0034] Example 4: Effects of the Sim gene on the growth and development of fall armyworm larvae Two hundred wild-type newly hatched larvae and two hundred newly hatched larvae injected with sgRNAs were randomly selected from each group to observe their growth rate and count the mortality rate at each instar.

[0035] The results are as follows Figure 2 and Figure 3 As shown, injection of sgRNAs from the Sim gene significantly inhibited the body length growth of fall armyworm larvae and resulted in a large number of deaths. These results indicate that the Sim gene plays a crucial role in the growth and development of fall armyworm larvae.

[0036] Example 5: Effect of the Sim gene on the reproductive capacity of the fall armyworm. After the female adults in the injection group emerged, they were paired one by one with newly emerged wild-type male adults and placed in disposable plastic cups for mating. The plastic cups were covered with gauze for egg laying. If any male died, a newly emerged male was immediately replaced. The gauze was changed regularly every day, and the total number of eggs laid by a single female was counted to assess the impact of reduced Sim gene expression on adult fertility.

[0037] The results are as follows Figure 4As shown, after the Sim gene expression was reduced, the total number of eggs laid by a single female adult fall armyworm was significantly reduced compared to the wild-type control group, and most adults even stopped laying eggs completely. Therefore, the Sim target sequence proposed in this invention can effectively reduce the reproductive capacity of fall armyworm and prevent its population from exploding, making it suitable for developing transgenic plants to achieve environmentally friendly pest control.

[0038] Unless otherwise specifically stated, the numerical values ​​set forth in these embodiments do not limit the scope of the invention. In all examples shown and described herein, any specific value should be interpreted as merely exemplary and not as a limitation, unless otherwise specified; therefore, other examples of exemplary embodiments may have different values.

Claims

1. A bHLH family protein Sim, characterized in that, The protein Sim is any one of the following (1)-(2): (1) A protein composed of the amino acids shown in SEQ ID NO.2; (2) A protein derived from (1) with the same function by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid shown in SEQ ID NO.

2.

2. A gene Sim encoding a bHLH family protein, characterized in that, The nucleotide sequence of the gene Sim is any one of the following (3)-(5): (3) A DNA molecule whose coding region is the sequence shown in SEQ ID NO.1; (4) DNA molecules that hybridize with the DNA sequence defined in (4) under strict conditions and encode proteins with the same function; (5) DNA molecules that have at least 95% sequence identity with the DNA sequence defined in (3) and encode proteins with the same function.

3. The application of the protein Sim of claim 1 or the gene Sim of claim 2 as a target, characterized in that, The application is any of the following: A1) Regulate the growth rate of fall armyworm larvae; A2) Regulate the reproductive capacity of fall armyworm larvae and adults; A3) Regulate the mortality rate of fall armyworm larvae.

4. The use of a substance capable of reducing the activity of the protein Sim as described in claim 1 or capable of reducing the expression level of the gene Sim as described in claim 2, in any of the following, characterized in that, The application is, B1) Reduce the growth rate of fall armyworm larvae; B2) Reduce the reproductive capacity of fall armyworm larvae and adults; B3) Increase the mortality rate of fall armyworm larvae; The substance capable of reducing the activity of the protein Sim as described in claim 1 or the substance capable of reducing the expression level of the gene Sim as described in claim 2 is any one of the following. C1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the gene encoding the protein; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant expression vector containing the nucleic acid molecule described in C1) or the expression cassette described in C2); C4) Recombinant microorganisms containing the recombinant expression vector described in C3).

5. The application according to claim 4, characterized in that, The nucleic acid molecule is an sgRNA molecule that targets the protein-encoding gene of claim 2, and the target sequence of the sgRNA molecule is shown in SEQ ID NO.6 or SEQ ID NO.

7.

6. The application according to claim 5, characterized in that, The sequence of the sgRNA molecule is shown in SEQ ID NO.8-11.

7. A method for controlling fall armyworm, characterized in that, The method includes introducing the substance described in any one of claims 4-6 into the fall armyworm via a CRISPR / Cas9 system.

8. The method according to claim 7, characterized in that, The importation refers to the introduction into the eggs of the fall armyworm.

9. The application according to claim 8, characterized in that, The concentration of the substance described in any one of claims 4-6 is 500-700 ng / μl.

10. The application according to claim 9, characterized in that, The concentration of the substance described in any one of claims 4-6 is 600 ng / μl.

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