Application of the lola gene as a target in the control of fall armyworm

Abstract

This invention relates to the application of the lola gene as a target in the control of fall armyworm, belonging to the field of biotechnology for pest control. It involves the application of the lola gene as a target in pest control or the preparation of biological products for pest control. Using the fall armyworm as the research object, this invention uses the lola gene as a molecular target, combines CRISPR / Cas9 gene editing technology, and develops a highly efficient sgRNA target site for its knockout. By utilizing the effect of Lola in inhibiting the production of female eggs in fall armyworms, its reproductive capacity is reduced, thereby achieving the purpose of control. This method is convenient to operate, effective, and sensitive, enabling long-term effective control of pest populations. It also significantly reduces the negative environmental impact of chemical pesticide use and avoids the risk of pests developing resistance due to long-term exposure to chemical pesticides, providing an innovative solution for sustainable agriculture.

Description

Technical Field

[0001] This invention belongs to the field of pest and disease control in biotechnology, and in particular relates to the application of the lola gene as a target in the control of fall armyworm. Background Technology

[0002] The fall armyworm, also known as the common armyworm, is native to tropical and subtropical America and is a highly polyphagous invasive pest. It has a very wide host range, with corn, cotton, sorghum, and rice being its primary targets for larvae. It is estimated that annual crop losses due to its damage to corn, rice, sorghum, and sugarcane in Africa amount to approximately US$13 billion. The main reason for this crop damage is its high reproductive capacity. The fall armyworm reproduces in its adult stage; female moths can mate and lay eggs multiple times, and under suitable conditions, each adult female can produce more than 1,500 eggs in her lifetime.

[0003] Insects' high reproductive capacity provides crucial support for their generational reproduction and population maintenance. The ovary, as the core organ of the reproductive system, plays a vital role in insect population dynamics, ecological evolution, and species evolution. Oogenesis is a delicate and complex process that marks the transition from the proliferation and differentiation of reproductive stem cells to the birth of a mature egg. Each stage of oogenesis in an insect's life cycle is accompanied by significant changes in morphology and physiological state. Transcriptional regulation of key reproductive genes, nutritional metabolism, and hormone levels—internal and external factors—intertwine to form a complex regulatory network that synergistically regulates key physiological activities such as ovarian development and oogenesis in female insects. Errors at any step can lead to a decline in female fertility or even infertility.

[0004] Longitudinal slacking (lola) has been shown to be one of the most complex genes in Drosophila, spanning 60 kb and consisting of 32 exons arranged on the same DNA strand. Through alternating cis- and trans-splicing and multiple promoter activities, it produces at least 80 different mRNA isoforms, encoding up to 20 protein isoforms. As an important transcription factor, Lola plays a crucial role in the development of the midgut, gonads, and neurons in insects. During embryogenesis, Lola regulates axonal growth and guidance, acts as an activator of neuronal genes, and is essential for maintaining the differentiation state of neurons in the brain. Furthermore, the deletion of the lola gene leads to defects in gonadal development in Drosophila.

[0005] Currently, pest control mainly relies on traditional chemical pesticides. However, the extensive use of chemical pesticides not only leads to increased pesticide resistance in pests but also seriously affects the environment and the ecological safety of non-target organisms. Therefore, developing new, safe, non-toxic, and environmentally friendly alternatives is one of the key issues in pest control. Currently, no application of the lola gene has been found in lepidopteran insects, especially in the reproduction of noctuid moths. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing control technologies and, taking the fall armyworm as an example, to provide a lola gene that regulates the ovarian development of the pest (fall armyworm) and its application in controlling the pest (fall armyworm).

[0007] This invention utilizes CRISPR / Cas9 gene editing technology to knock out the lola gene in the fall armyworm. For the first time, it has been demonstrated that the loss of Lola protein function significantly affects oogenesis in the fall armyworm, reducing egg production and consequently leading to a significant decrease in offspring numbers. This gene-level intervention alters the reproductive capacity of the fall armyworm, providing an environmentally friendly and sustainable pest management strategy compared to traditional chemical control methods. It helps reduce the use of chemical pesticides, lessens the burden on ecosystems, and prevents the development of pesticide resistance in pests. Furthermore, the precision and reproducibility of gene editing technology provide a new and more effective means of pest control. This method is easy to implement, has broad application prospects, and can bring long-term environmental and economic benefits to agricultural production.

[0008] The first objective of this invention is to provide the application of the fall armyworm lola gene as a target in pest control.

[0009] A second aspect of this invention aims to provide an sgRNA target for the *Lola* gene of the fall armyworm.

[0010] A third aspect of the present invention is to provide a nucleic acid molecule encoding the sgRNA of the second aspect of the present invention.

[0011] A fourth aspect of this invention aims to provide the use of the lola gene or any inhibitor thereof in the control of fall armyworm and / or the preparation of products for the control of fall armyworm.

[0012] A fifth aspect of the present invention is to provide an expression cassette, vector, or mutant strain of pests comprising the nucleic acid molecule of the third aspect of the present invention.

[0013] The sixth aspect of this invention aims to provide applications of the sgRNA of the second aspect of this invention, the nucleic acid molecule of the third aspect of this invention, the expression cassette, vector, or mutant strain of the fifth aspect of this invention.

[0014] The seventh aspect of this invention aims to provide a method for controlling pests. By introducing a mixture of synthesized sgRNA and Cas9 protein into the embryos of the fall armyworm, the lola gene can be efficiently knocked out. The loss of function of the Lola protein leads to abnormal egg formation and a significant reduction in egg production, thereby effectively controlling the pest. This method has many advantages, including ease of operation, precise and effective targeting, and being environmentally friendly, showing great promise for application.

[0015] To achieve the above objectives, the present invention adopts the following technical solution:

[0016] This invention identifies the gene *lola*, which significantly affects oogenesis in the fall armyworm and consequently reduces its fertility. A technique using CRISPR / Cas9 gene editing to knock out the *lola* gene at the genomic level was developed to control the fall armyworm. The invention synthesizes the *lola* gene's sgRNA in vitro, mixes it with Cas9 protein, and injects it into fall armyworm embryos. Oogenesis and egg production are observed and recorded to comprehensively evaluate the impact of *lola* gene knockout on ovarian development in the fall armyworm. Results show that mutations in the *lola* gene in the fall armyworm cause abnormal oogenesis, leading to reduced fertility in females.

[0017] Therefore, the following contents should all be within the scope of protection of this invention:

[0018] A first aspect of the present invention provides the application of the fall armyworm *Lola* gene as a target in pest control. Preferably, the pest is the fall armyworm; more preferably, a noctuid moth; and even more preferably, a lepidopteran insect. Preferably, the nucleotide sequence of the *Lola* gene is shown in SEQ ID NO. 1.

[0019] In a second aspect, the present invention provides an sgRNA target for the fall armyworm lola gene, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0020] In a third aspect, the present invention provides a nucleic acid molecule encoding the sgRNA of the second aspect of the present invention, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0021] A fourth aspect of the invention provides the use of the lola gene or any inhibitor thereof in the control of fall armyworm and / or in the preparation of products for the control of fall armyworm, such as:

[0022] (1) Applied to biological control strategies for pests;

[0023] (2) Used for developing environmentally friendly pesticide formulations;

[0024] (3) Inhibits the development of eggs in the ovaries of pests;

[0025] (4) Prepare products that reduce the egg-laying rate of adult pests.

[0026] Preferably, the inhibitor is a substance that reduces the activity of transcription factor Iola, or a substance that degrades transcription factor Iola, or a substance that reduces the expression level of transcription factor Iola.

[0027] More preferably, the inhibitor is a CRISPR / Cas9 system targeting the lola gene, and the sgRNA nucleic acid molecule sequence of the target site in the CRISPR / Cas9 system is shown in SEQ ID NO.2.

[0028] Therefore, the present invention also protects a CRISPR / Cas9 system, said CRISPR / Cas9 system comprising any of the following:

[0029] a) sgRNA, the nucleotide sequence of which is shown in SEQ ID NO.3; the target site sequence of the sgRNA is shown in SEQ ID NO.2;

[0030] b) A CRISPR / Cas9 vector expressing the sgRNA nucleic acid molecule a)

[0031] A fifth aspect of the invention provides an expression cassette, vector, or mutant strain of pest comprising the nucleic acid molecule of the third aspect of the invention.

[0032] A sixth aspect of the present invention provides the application of the sgRNA of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the expression cassette, vector, or mutant strain of the fifth aspect of the present invention.

[0033] Application of any one of (a1) to (a3) ​​in any one of (1) to (4);

[0034] (a1) The sgRNA of the second aspect of the present invention;

[0035] (a2) The nucleic acid molecule of the third aspect of the present invention;

[0036] (a3) The expression cassette, vector, or mutant strain of the fifth aspect of the present invention;

[0037] (1) Applied to biological control strategies for pests;

[0038] (2) Used for developing environmentally friendly pesticide formulations;

[0039] (3) Inhibits the development of eggs in the ovaries of pests;

[0040] (4) Prepare products that reduce the egg-laying rate of adult pests.

[0041] Furthermore, the application of the above-mentioned invasive host plants of pests can also be expanded through the following: a) regulating plant resistance to pests; b) cultivating pest-resistant plants; c) improving plant breeding; preferably, the plant is corn, cotton, rice, sorghum, or sugarcane. Preferably, the pest is the fall armyworm; further preferably, it is a noctuid moth, and even further preferably, it is a lepidopteran pest.

[0042] A seventh aspect of the present invention provides a method for controlling pests. Preferably, the method involves introducing a CRISPR / Cas9 system into the pest, comprising the sgRNA and Cas9 of the second aspect of the present invention. More preferably, the introduction is performed by injection.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] This invention discloses for the first time the application of the transcription factor lola as a target in the control of fall armyworm, and develops a highly efficient sgRNA target site for its knockout. A highly effective technology for controlling fall armyworm has been developed, namely, injecting sgRNA and Cas9 into fall armyworm embryos using CRISPR / Cas9 gene editing technology. Lola's effect of inhibiting the development of female oocytes in fall armyworms reduces their reproductive capacity, thereby achieving the control objective. This method is convenient to operate, effective, and sensitive, enabling long-term effective control of pest populations. It also significantly reduces the negative environmental impacts of chemical pesticide use and avoids the risk of pests developing resistance due to long-term exposure to chemical pesticides, providing an innovative solution for sustainable agriculture. Attached Figure Description

[0045] Figure 1 Selection of target sites for CRISPR / Cas9 technology to knock out the lola gene.

[0046] Figure 2 The amino acid sequence diagram of homozygous mutants screened after CRISPR / Cas9 knockout of the lola gene.

[0047] Figure 3 The loss of Lola function affects the phenotype of oocyte development in female fall armyworms.

[0048] Figure 4 The results of oviposition statistics after knocking out the lola gene in the fall armyworm are shown in Figure A (4 hybridization schemes and their F1 generation egg masses; Figure B: oviposition analysis of the 4 hybridization groups). Different letters (such as a and b) indicate significant differences between groups. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these embodiments are commercially available.

[0050] Example 1: Obtaining the lola gene fragment from the fall armyworm and synthesizing its sgRNA target site.

[0051] 1. Obtaining the Lola gene fragment from the fall armyworm.

[0052] Based on the fall armyworm genome and transcriptome database, the nucleotide sequence of the fall armyworm lola gene (Sflola) was obtained by BLAST sequence alignment analysis, as shown in SEQ ID NO.1.

[0053] 2. Synthesis of sgRNA target sites in the *Lola* gene of *Fall Armyworm*

[0054] (1) Target site selection

[0055] Based on the obtained nucleotide sequence of the lola gene SEQ ID NO.1, and according to the principle of designing the 5'-GG-(N)18-NGG-3' target site, a suitable site was selected. The target site sequence of the sgRNA in this application is shown in SEQ ID NO.2, and the nucleic acid molecular sequence of the sgRNA is shown in SEQ ID NO.3.

[0056] (2) Primer design

[0057] Based on the obtained sgRNA nucleic acid sequence of the lola gene (SEQ ID NO.3), primer P1 for the lola gene sgRNA was designed (Table 1). Based on the lola gene sequence (SEQ ID NO.1), primer P2 for mutant screening was designed (Table 1). Based on the pMD19-T vector sequence and the lola gene sgRNA nucleic acid sequence (SEQ ID NO.3), primer P3 for colony PCR detection was designed (Table 1).

[0058] Table 1. Primers used in this example

[0059]

[0060] (3) Synthesis of sgRNA transcription template

[0061] 1) Elongation of the short strand of the lola gene sgRNA template:

[0062] The PCR amplification reaction system is as follows:

[0063]

[0064] PCR reaction program: 98℃, 3 min; 98℃, 10 s; 55℃, 15 s; 72℃, 10 s; 30 cycles; 72℃, 10 min.

[0065] 2) Construction of positive plasmids:

[0066] After PCR amplification products were purified by agarose gel electrophoresis, the purified products were ligated into pMD19-T vector for sequencing. The ligation reaction system is as follows:

[0067]

[0068] After incubating the ligation product overnight in a ligator at 16°C, it was added to competent cells, gently mixed by pipetting, and incubated on ice for 30 min. Then, it was heat-shocked in a 42°C water bath for 90 s, followed by cooling on ice for 10 min. 500 μL of antibiotic-free LB broth was added, and the cells were incubated at 37°C and 180 rpm for 1 h in a shaker, followed by centrifugation at 5000 rpm for 5 min at room temperature. The bacterial culture was concentrated to 100 μL, gently resuspended with a pipette tip, and then evenly spread onto ampicillin-resistant LB agar plates. The plates were incubated inverted mode overnight at 37°C. Positive clones were screened, and single-clone colony PCR and sequencing verification were performed; colonies without nucleotide mutations in the sequencing results were considered the desired positive colonies.

[0069] 3) Amplification of the sgRNA transcription template of the lola gene:

[0070] After expanding the culture of the selected positive clones, plasmids were extracted and used as DNA templates. PCR was then used to synthesize sgRNA templates for in vitro transcription. The reaction system is as follows:

[0071]

[0072] PCR reaction program: 98℃, 3 min; 98℃, 10 s; 55℃, 15 s; 72℃, 10 s; 30 cycles; 72℃, 10 min.

[0073] The above reaction system was repeated in 8 tubes. After the reaction, the PCR products were combined and 100 μL of Nuclease-free water was added to make up to 500 μL. Then, an equal volume of Tris-saturated phenol:chloroform:isoamyl alcohol (25:24:1, pH=8.0) was added for purification. After purification, the DNA concentration was determined by NanoDrop ND-2000 and verified by gel electrophoresis.

[0074] (4) In vitro transcription and purification of sgRNA

[0075] sgRNA was synthesized and purified in vitro according to the MEGAScript T7 kit instructions.

[0076] 1) Add the following reaction system:

[0077]

[0078] After mixing and centrifuging the above reaction solution, transcribe at 37°C for 8 hours.

[0079] 2) After transcription is complete, add 1 μL LTURBO DNase, mix well and incubate at 37°C for 15 min.

[0080] 3) Add 390 μL of Nuclease-free water to bring the transcription product to 400 μL, then add 400 μL of Tris-saturated phenol:chloroform:isoamyl alcohol (25:24:1, pH>8.0), mix thoroughly, and centrifuge at 12000 rpm for 10 min at 4 °C.

[0081] 4) Transfer the supernatant to a new centrifuge tube, add 1 mL of anhydrous ethanol and 50 μL of NaOAc (pH = 5.2), mix well, and precipitate at -20°C for 30 min.

[0082] 5) After precipitation, centrifuge at 4℃ and 12000rpm for 15min, and discard the supernatant.

[0083] 6) Add 1 mL of 75% ethanol to the centrifuge tube to wash the precipitate, centrifuge at 12000 rpm for 5 min at 4°C, discard the supernatant, and wash twice.

[0084] 7) Open the cap of the centrifuge tube and let it air dry on ice for 10 minutes to evaporate any residual ethanol.

[0085] 8) Add an appropriate amount of Nuclease-free water to dissolve the RNA.

[0086] 9) Measure the RNA concentration and verify it by gel electrophoresis. Store in a -80℃ freezer for later use.

[0087] 3. Microinjection of Spodoptera litura fertilized eggs

[0088] Before injection, all necessary experimental equipment, including ddH2O, glass needles, and insect rearing boxes, should be sterilized or disinfected. The sgRNA and Cas9 protein should be mixed to a final concentration of 300 ng / μL, and then centrifuged at 12,000 rpm for 5 min at 4°C.

[0089] After mating, fresh egg masses laid by the fall armyworm within 2 hours are collected. The egg masses, sterilized with formaldehyde, are brushed to remove surface hairs. While still moist, the eggs are transferred to a glass slide and arranged in straight rows with the face up using an oviposition needle. A mixture of sgRNA and Cas9 is injected using a microinjector. After injection, the eggs are placed in an incubator for incubation at 25±1℃.

[0090] 4. Detection and screening of mutants

[0091] Following PCR amplification using mutant genomic DNA as a template, base sequencing was performed. The sequencing results were analyzed using SnapGene software for base sequence alignment to identify different sequence base mutations. After detection, a mutant genotype exhibiting premature termination of Lola protein translation in the fall armyworm was identified. Figure 2 The details are as follows:

[0092] (1) Extraction of mutant genomic DNA:

[0093] 1) Collect the exoskeleton of the mutant pupa, add 600 μL of digestion solution (EDTA, SDS, NaCl, proteinase K and Tris-HCl) and two grinding beads, grind thoroughly and digest overnight in a water bath at 55°C.

[0094] 2) After digestion, add 600 μL of DNA extraction buffer to the mixture, gently invert to mix, and centrifuge at 12,000 rpm for 10 min.

[0095] 3) Take off the upper aqueous phase and repeat step 2) until there is no obvious white layer at the boundary between the phenol phase and the aqueous phase.

[0096] 4) Add an equal volume of chloroform:isoamyl alcohol (24:1) to the supernatant, mix by inverting the container, centrifuge at 12000 rpm for 10 min, and collect the supernatant.

[0097] 5) Add 2 volumes of anhydrous ethanol (pre-cooled) to the supernatant, invert and mix well, precipitate at -20℃ for 20 min, centrifuge at 12000 rpm for 15 min, and discard the supernatant.

[0098] 6) Add 1 mL of 70% ethanol (pre-cooled) to wash the precipitate, centrifuge at 12000 rpm for 5 min, discard the supernatant, and repeat twice.

[0099] 7) Open the cap of the centrifuge tube and let it air dry for 10-15 minutes to allow the residual ethanol to evaporate completely until the precipitate becomes transparent. Add an appropriate amount of ddH2O to dissolve the DNA.

[0100] (2) Mutant screening:

[0101] Using mutant genomic DNA as a template, PCR amplification and base sequencing were performed. The PCR amplification reaction system is as follows:

[0102]

[0103] The PCR reaction program was as follows: 98℃, 3 min; 98℃, 10 s; 55℃, 15 s; 72℃, 10 s; 30 cycles; 72℃, 10 min.

[0104] Example 2: Phenotypic results of *Lola* gene knockout in *Fall Armyworm* (using CRISPR / Cas9 system).

[0105] 1. Abnormal development of adult eggs

[0106] from Figure 3 It can be seen that the fall armyworm (Sflola) - / - On the second day, the ovaries of homozygous mutant adults were significantly smaller than those of wild-type, with numerous immature oocytes arranged in the ovarian ducts, and a significant reduction in the number of mature oocytes in the ovarian stalk. These results indicate that knockout of the Sflola gene effectively inhibits oocyte production in the ovaries of female fall armyworms.

[0107] 2. Significantly reduced egg production

[0108] Sflola, the fall armyworm - / - Four hybridization experiments were conducted between the homozygous mutant and the wild type, namely: Sflola - / - Hybridization of homozygous mutant females and males, Sflola - / - Hybridization of homozygous mutant females and wild-type males, wild-type females and Sflola - / - Homozygous mutant males were crossed with wild-type females and males, and the number of eggs laid in each group was observed.

[0109] from Figure 4 As can be seen, compared with the control group of wild-type females and males hybridized, Sflola - / - Crossing homozygous mutant females with wild-type males resulted in significantly smaller offspring egg masses and a significantly reduced egg production; similarly, Sflola... - / - A significant reduction in egg production was also observed when homozygous mutant females were crossed with males. This result indicates that knockout of the Sflola gene can effectively suppress the number of offspring in the fall armyworm.

[0110] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Knockout lola The application of genes in pest control or the preparation of biological products for pest control is characterized by, The lola The gene sequence is shown in SEQ ID NO:1; the pest is the fall armyworm.

2. The application according to claim 1, characterized in that, The application is through lola Gene knockout inhibits the production and / or egg-laying rate of female insect eggs, thereby reducing their reproductive capacity.

3. The application according to claim 1, characterized in that, The biological product is a bio-environmentally friendly pesticide formulation.

4. A kind of inhibition lola The application of gene-expressed sgRNA in the control of fall armyworm is characterized by, The target site sequence of sgRNA via the CRISPR / Cas9 system is shown in SEQ ID NO.2; the nucleotide sequence of the sgRNA is shown in SEQ ID NO.

3.

5. A method for controlling fall armyworm, characterized in that, This includes introducing the CRISPR / Cas9 system described in claim 4 into the fall armyworm embryo to inhibit the presence of [certain substances] in the fall armyworm. lola The expression of nucleic acid molecules of genes.

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