Spermatogenesis gene and use thereof
By identifying and utilizing the LTNAT gene of the lepidopteran pest fall armyworm, sgRNA was designed for CRISPR/Cas9 gene editing to construct a male-sterile strain. This solved the problem of insufficient spermatogenesis gene mining in the control of lepidopteran pests and achieved highly efficient pest control.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANJING AGRICULTURAL UNIVERSITY
- Filing Date
- 2026-01-07
- Publication Date
- 2026-05-29
AI Technical Summary
The lack of exploration of specific spermatogenesis genes in lepidopteran insects in existing technologies limits the application of CRISPR/Cas9 technology in the control of lepidopteran pests.
We identified and utilized the LTNAT gene of the fall armyworm, a lepidopteran pest, as a molecular target for CRISPR/Cas9 technology. By designing sgRNA and performing gene editing, we constructed a male-sterile strain to achieve genetic control of the lepidopteran pest.
A male-sterile strain of lepidopteran pests was successfully constructed. By mating with wild-type females, the population size was significantly reduced, providing an efficient and environmentally friendly pest control strategy.
Smart Images
Figure CN122104718A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, to a spermatogenesis gene and its application, and more particularly to a spermatogenesis gene that regulates male reproduction in lepidopteran pests and its application. Background Technology
[0002] The fall armyworm, belonging to the Noctuidae family of Lepidoptera, has highly voracious larvae and a high reproductive rate in adults. For a long time, chemical pesticides and the planting of genetically modified Bt maize have been the primary methods for controlling fall armyworm damage. However, the fall armyworm has developed high levels of resistance to many chemical pesticides and Bt maize. The extensive use of chemical pesticides leads to environmental pollution and pest resurgence. Therefore, it is urgent to explore new, green control strategies to manage fall armyworm infestations.
[0003] CRISPR / Cas9-based precision-guided insect sterilization is a highly efficient and environmentally friendly pest control strategy. It weakens male fertility by targeting and knocking out genes involved in male reproductive development. The sterile males are then released into the field to mate with wild-type females, preventing the females from producing offspring and thus reducing the population size. Spermatogenesis is a fundamental biological process of male sexual reproduction. However, the current lack of discovery of spermatogenesis-specific genes in lepidopteran insects severely limits the development of this technology. Summary of the Invention
[0004] The purpose of this invention is to provide a spermatogenesis gene and its application, which solves the problem that the insufficient discovery of specific spermatogenesis genes in lepidopteran insects seriously restricts the development of CRISPR / Cas9 technology-guided insect sterility technology, and can be used for the genetic control of lepidopteran pests.
[0005] To solve the above-mentioned technical problems, the present invention is implemented using the following technical solution:
[0006] In a first aspect, the present invention provides a spermatogenesis gene, wherein the spermatogenesis gene is the LTNAT gene that regulates male reproduction in lepidopteran pests, and its nucleotide sequence is shown in SEQ ID NO. 1.
[0007] Given the conservation of spermatogenesis gene pathways in lepidopteran insects, identifying genes involved in male spermatogenesis using the fall armyworm, a major pest, as a model can provide safe and efficient molecular targets for controlling lepidopteran pests using CRISPR / Cas9-based insect sterility technology. The above-mentioned scheme provides the spermatogenesis gene for male reproduction in lepidopteran pests: the LTNAT gene and its nucleotide sequence. The successful discovery of lepidopteran-specific spermatogenesis genes provides possibilities and pathways for insect sterility research using CRISPR / Cas9 technology.
[0008] In a second aspect, the present invention provides an LTNAT protein encoded by the LTNAT gene described in the first aspect, the amino acid sequence of which is shown in SEQ ID NO. 2.
[0009] In the above scheme, the LTNAT gene mutation also causes the protein to mutate, forming a blocking protein that cannot function, thus causing male infertility.
[0010] Thirdly, the present invention provides a method for constructing a male-sterile strain of lepidopteran pests, comprising the following steps:
[0011] sgRNA1 and sgRNA2 were synthesized based on the gene sequence information of the LTNAT gene described in the first aspect;
[0012] After mixing sgRNA1 and sgRNA2 with Cas9, the mixture was injected into the eggs of lepidopteran pests to obtain the G0 generation mutant.
[0013] The G0 mutant was crossed with the wild type to obtain the G1 generation heterozygous mutant;
[0014] The G1 generation carrying the same mutation was self-crossed to obtain the G2 generation homozygous mutant, among which the males were male-sterile strains of lepidopteran pests.
[0015] The above scheme provides a specific method for studying insect sterility using CRISPR / Cas9 technology. The sterile strains obtained by designing and synthesizing sgRNA from the spermatogenesis genes of lepidopteran pests can effectively control pests.
[0016] Furthermore, the sequence of sgRNA1 is shown in SEQ ID NO. 3, and the sequence of sgRNA2 is shown in SEQ ID NO. 4.
[0017] Furthermore, the method for preparing sgRNA1 and sgRNA2 includes the following steps:
[0018] Primers for sgRNA1 were designed and synthesized based on the LTNAT gene sequence. The primer sequences are shown in SEQ ID NO. 5 and SEQ ID NO. 6.
[0019] Primers for synthesizing sgRNA2 were designed and synthesized based on the LTNAT gene sequence. The primer sequences are shown in SEQ ID NO. 7 and SEQ ID NO. 8.
[0020] Fusion PCR was performed to synthesize in vitro transcription templates, and in vitro transcription was performed using the in vitro transcription templates.
[0021] The RNA transcribed in vitro was purified to obtain sgRNA1 and sgRNA2.
[0022] Furthermore, the concentration of the Cas9 protein is 300±50 ng / μL, and the concentration of the sgRNA is 150±50 ng / μL.
[0023] Fourthly, the present invention provides a male-sterile strain of lepidopteran pests, prepared by the method for constructing a male-sterile strain of lepidopteran pests as described in any one of the third aspects.
[0024] Furthermore, the lepidopteran pests include one of the following: fall armyworm, beet armyworm, rice stem borer, Asian corn borer, rice leaf roller, codling moth, cotton bollworm, tobacco budworm, tobacco hawk moth, and diamondback moth.
[0025] Of the above technical solutions, the preferred application is to the genetic control of fall armyworm.
[0026] Fifthly, the application of a male-sterile strain of lepidopteran pests described in the fourth aspect in the control of lepidopteran pests.
[0027] Furthermore, the method for controlling lepidopteran pests using male-sterile strains includes the following steps:
[0028] Male-sterile strains of lepidopteran pests are released into the field and mated with wild-type female lepidopteran pests to suppress their egg production and reduce their population size.
[0029] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:
[0030] This invention identifies the previously unreported spermatogenesis gene LTNAT in the fall armyworm. This gene is specifically present in lepidopteran pests and its sequence is highly conserved. This invention knocks out the LTNAT gene using CRISPR / Cas9 gene editing technology, resulting in complete male sterility. This invention provides a new gene resource for the control of lepidopteran pests using CRISPR / Cas9-based insect sterilization technology. Attached Figure Description
[0031] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0032] Figure 1 These are the LTNAT gene sequence structure diagram of Example 1 and the sgRNAs site design diagram of Example 2 of the present invention;
[0033] Figure 2This is a graph showing the detection results of the homozygous mutant of the LTNAT gene in Example 2 of the present invention;
[0034] Figure 3 This is an analysis diagram of the male infertility caused by the LTNAT gene in Example 3 of the present invention;
[0035] Figure 4 This is a diagram showing the result of the female's eggs failing to hatch after mating with a male of the LTNAT homozygous mutant in Example 3 of the present invention.
[0036] Figure 5 This is a schematic diagram of the LTNAT mutation staining results in Example 4 of the present invention;
[0037] Figure 6 This is a schematic diagram of the image processing and analysis results of the LTNAT mutation in Embodiment 4 of the present invention;
[0038] Figure 7 This is a schematic diagram of the sequence conservation detection results of LTNAT in different families of Lepidoptera in Example 5 of the present invention. Detailed Implementation
[0039] The above content is further illustrated below with specific embodiments, but it should not be construed as limiting the scope of the invention to the following embodiments. All technologies implemented based on the above content of this invention fall within the scope of this invention.
[0040] It should be understood that all experimental procedures not detailed in the experiment are routine experimental procedures well known to those skilled in the art.
[0041] Table 1: Source of materials.
[0042]
[0043] The sources of the materials in the following embodiments are shown in Table 1, and the sequences are shown in Table 2.
[0044] Example 1
[0045] This embodiment provides a method for cloning the LTNAT gene, including the following steps: extracting total RNA from the testes of 1-day-old male fall armyworms using VeZol reagent.
[0046] First-strand complementary DNA (cDNA) was synthesized using the HiScript III first-strand cDNA synthesis kit with gDNA removal agent. The open reading frame (ORF) of the LTNAT gene was predicted using the online tool NCBI-ORF Finder. Primers for gene cloning were designed based on the ORF Finder results: LTNAT-F was 5'-ATCATCAATCCTCAGCTGCC-3' and LTNAT-R was 5'-CTGAACAGCCGTCATTAAGC-3'.
[0047] PCR amplification was performed using 2×Phanta Max Master Mix. The PCR reaction system consisted of 12.5 µL 2×Phanta Max Master Mix, 1 µL forward primer F (10 µmol / L), 1 µL reverse primer R (10 µmol / L), 2 µL cDNA, and 8.5 µL ddH2O.
[0048] The reaction procedure was as follows: 95℃ for 3 min, 95℃ for 15 s, 54℃ for 15 s, 72℃ for 2 min, and a final extension at 72℃ for 10 min, repeated 35 times. The PCR product was purified using a gel extraction kit, then cloned into the pClone007 blunt-ended vector and sequenced to obtain the nucleotide sequence of the LTNAT gene.
[0049] See the LTNAT gene structure diagram. Figure 1 The gene consists of 5 exons and encodes 242 amino acids. The nucleotide sequence of the LTNAT gene is SEQ ID NO. 1.
[0050] The amino acid sequence of the protein encoded by the LTNAT gene is shown in SEQ ID NO. 2.
[0051] Example 2
[0052] In this embodiment, two sgRNAs were synthesized based on the gene sequence information of the LTNAT gene and microinjected into the embryos of the fall armyworm to carry out CRISPR / Cas9 gene editing, including the following:
[0053] To improve gene editing efficiency, this invention designs two highly specific sgRNAs targeting exon 3 based on the gene sequence information of the LTNAT gene in the fall armyworm and the off-target prediction software CasOT. The sgRNA site design diagram is shown below. Figure 1 The primers for synthesizing sgRNA1 are shown in SEQ ID NO. 5 and SEQ ID NO. 6, and the primers for synthesizing sgRNA2 are shown in SEQ ID NO. 7 and SEQ ID NO. 8.
[0054] sgRNA was synthesized in vitro using the GeneArt™ Precision gRNA Synthesis Kit. The sequence of sgRNA1 is shown in SEQ ID NO. 3, and the sequence of sgRNA2 is shown in SEQ ID NO. 4. The Cas9 protein used was the commercial product TrueCut™ Cas9 protein v2.
[0055] Ten pairs of 2-day-old adults were placed in gauze-lined plastic boxes for mating and oviposition. Fresh egg masses were collected every 30 minutes after the onset of darkness. Individual, dispersed eggs were transferred to double-sided adhesive tape on a glass slide using a fine brush. A mixture of Cas9 protein (300 ng / μL) and sgRNA (150 ng / μL) was injected into the eggs using an InjectMan NI 2 microinjection system. The injected eggs and hatched larvae were placed in a climate chamber at 26 ± 1°C, 70% ± 5% relative humidity, with a 14:10 light / dark cycle.
[0056] PCR primers flanking two sgRNA target sites were designed to detect chimeric mutations in G0 generation individuals. The PCR primers were: LTNAT-MF: 5'-CTCGCAGTTCTATGCCTTAG-3' and LTNAT-MR: 5'-TGCAGAACATTATGCACTCG-3'. DNA was extracted from the hind legs of G0 generation adults for PCR amplification and Sanger sequencing. Overlapping chromatographic peaks in the target region were considered indicators of mutation. G0 generation mutants were crossed with wild-type fall armyworms to produce the G1 generation.
[0057] To assess whether the mutation was heritable, mixed DNA was extracted and analyzed from 20 randomly selected G1 generation larvae. PCR amplification products from the G1 individuals were cloned into the pClone007 blunt-ended vector, and positive clones were sequenced to determine whether the mutation resulted in a premature stop codon. G1 individuals carrying the same effective frameshift mutation were hybridized to obtain homozygous G2 mutant lines. The mutation type and sequencing peak diagram of the G2 homozygous mutant lines are shown in [the original text]. Figure 2 .
[0058] Example 3
[0059] This embodiment provides a method for determining the fertility of a homozygous LTNAT mutant line, including the following steps:
[0060] To assess the impact of the LTNAT gene mutation on male fertility, 2-3 day old unmated males and females were placed in 490 ml plastic cups for pairing. Mating behavior was observed every 15 minutes under red light after entering the dark period. Eggs laid by each female fall armyworm were collected for four consecutive days after mating. The total number of eggs, the number of hatched offspring, and the hatching rate were recorded. All experiments were conducted in a climate-controlled chamber with a temperature set at 26 ± 1 °C, a relative humidity of 75% ± 5%, and a photocycle of 14 hours of light and 10 hours of darkness. Adults were fed a 10% honey solution as their nutrient source. Fertility test results are as follows: Figure 3 and Figure 4 As shown, Figure 3 A graph illustrating the results of LTNAT gene-induced male infertility. Figure 4 The image shows the result of females failing to hatch after mating with males of the LTNAT homozygous mutant. The results indicate that mating between males of the LTNAT homozygous mutant and wild-type or homozygous mutant females failed to induce females to produce offspring, suggesting that the LTNAT mutant causes complete male sterility.
[0061] Example 4
[0062] This embodiment provides a method for staining sperm from LTNAT homozygous mutant males, including the following steps:
[0063] Sperm samples were fixed for 1 hour at room temperature using 1 mL of 4% paraformaldehyde. The fixed samples were then washed three times with PBS, each time for 10 minutes. The sperm were then incubated for 1 hour in 400 µL of 1:1000 diluted TRITC-phalloidin, followed by incubation for 10 minutes with 400 µL of 1:1000 diluted DAPI. After staining, the samples were washed three times with PBST (PBS containing 0.1% Tween-20), each time for 10 minutes. 10 µL of the stained sperm suspension was dropped onto a glass slide, covered with a coverslip, and gently pressed to ensure even distribution. To count sperm counts, a five-point sampling method was used to image the samples at 20x magnification. Fluorescence images were acquired using a Zeiss LSM 980 confocal microscope equipped with Airyscan 2 technology. ImageJ software was used for image processing and analysis. LTNAT mutation staining results are shown below. Figure 5 As shown, LTNAT mutations lead to abnormal development of nucleated sperm. Furthermore, the image processing and analysis results of LTNAT mutations are as follows: Figure 6 As shown, the results indicate that the LTNAT mutation prevents nucleated and non-nucleated sperm from transferring from the copulatory sac to the seminal vesicle, suggesting impaired motility of non-nucleated sperm.
[0064] Example 5
[0065] This embodiment provides a method for detecting the sequence conservation of LTNAT in different families of Lepidoptera, including the following steps:
[0066] Based on the NCBI and InsectBase 2.0 databases, using the LTNAT amino acid sequence of *Pseudomonas aeruginosa* as a reference sequence, searches using Blastp and tBlastn revealed that the LTNAT amino acid sequence exists only in lepidopteran insects and is highly conserved among different lepidopteran species, suggesting that the function of this gene is conserved among lepidopteran pests. The sequence conservation results of LTNAT in different lepidopteran families are as follows: Figure 7 As shown.
[0067] Table 2: Nucleotide sequence listing.
[0068]
[0069] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A spermatogenesis gene, characterized in that, The spermatogenesis gene is the LTNAT gene, which regulates male reproduction in lepidopteran pests, and its nucleotide sequence is shown in SEQ ID NO.
1.
2. An LTNAT protein, characterized in that, Encoded by the LTNAT gene of claim 1, the amino acid sequence of the LTNAT protein is shown in SEQ ID NO.
2.
3. A method for constructing a male-sterile strain of a lepidopteran pest, characterized in that, Includes the following steps: According to claim 1, the gene sequence information of the LTNAT gene is used to synthesize sgRNA1 and sgRNA2; After mixing sgRNA1 and sgRNA2 with Cas9, the mixture was injected into the eggs of lepidopteran pests to obtain the G0 generation mutant. The G0 mutant was crossed with the wild type to obtain the G1 generation heterozygous mutant; The G1 generation carrying the same mutation was self-crossed to obtain the G2 generation homozygous mutant, among which the males were male-sterile strains of lepidopteran pests.
4. The method for constructing male-sterile strains of lepidopteran pests according to claim 3, characterized in that, The sequence of sgRNA1 is shown in SEQ ID NO. 3, and the sequence of sgRNA2 is shown in SEQ ID NO.
4.
5. The method for constructing male-sterile strains of lepidopteran pests according to claim 3, characterized in that, The method for preparing sgRNA1 and sgRNA2 includes the following steps: Primers for sgRNA1 were designed and synthesized based on the LTNAT gene sequence. The primer sequences are shown in SEQ ID NO. 5 and SEQ ID NO.
6. Primers for synthesizing sgRNA2 were designed and synthesized based on the LTNAT gene sequence. The primer sequences are shown in SEQ ID NO. 7 and SEQ ID NO.
8. Fusion PCR was performed to synthesize in vitro transcription templates, and in vitro transcription was performed using the in vitro transcription templates. The RNA transcribed in vitro was purified to obtain sgRNA1 and sgRNA2.
6. The method for constructing male-sterile strains of lepidopteran pests according to claim 3, characterized in that, The concentration of the Cas9 protein was 300±50 ng / μL, and the concentration of the sgRNA was 150±50 ng / μL.
7. A male-sterile strain of a lepidopteran pest, characterized in that, It is prepared by the method for constructing male-sterile strains of lepidopteran pests as described in any one of claims 3-6.
8. The male-sterile strain of lepidopteran pests according to claim 7, characterized in that, The lepidopteran pests include one of the following: fall armyworm, beet armyworm, rice stem borer, Asian corn borer, rice leaf roller, codling moth, cotton bollworm, tobacco budworm, tobacco hawk moth, and diamondback moth.
9. The application of the male-sterile strain of lepidopteran pests as described in claim 7 in the control of lepidopteran pests.
10. The application according to claim 9, characterized in that, The method for controlling lepidopteran pests using male-sterile strains includes the following steps: Male-sterile strains of lepidopteran pests are released into the field and mated with wild-type female lepidopteran pests to suppress their egg production and reduce their population size.