Chilo suppressalis seedless sperm formation factor TNNI3 and application thereof in male sterility

Abstract

The invention relates to a chilo suppressalis seedless sperm formation factor TNNI3 and application thereof in male sterility. The invention finds that the TNNI3 gene specifically regulates and controls the chilo suppressalis seedless spermatogenesis process for the first time, and an RNA interference technology proves that TNNI3 can inhibit the number of chilo suppressalis seedless sperm bundles and has no obvious influence on the number of nucleated sperm bundles and sperm motility, so that the egg laying amount after mating of male insects and wild female insects can be obviously reduced, and the survival rate of the male insects and the wild female insects is improved. And a theoretical basis is provided for developing a novel prevention and treatment strategy for male reproduction.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a Chilo suppressalis anucleated sperm formation factor TNNI3 and an application thereof in male sterility. Background Art

[0002] The Chilo suppressalis (Chilo suppressalis) is a major pest that severely damages crops such as rice, corn, and wheat. Its larvae bore into crop stems, causing dead seedlings, dead and pregnant ears, white ears, and insect-damaged plants, severely impacting crop growth and yield. In rice, Chilo suppressalis can cause heart and leaf dieback, weak or insufficient grain filling in ears, leading to widespread yield losses. In corn and wheat, it can also cause stem damage, lodging, and decreased fruit set. Chilo suppressalis can spread pathogens during its feeding process, exacerbating the incidence of disease. Large-scale outbreaks not only affect farmers' incomes but also threaten food security. For a long time, control of Chilo suppressalis has relied heavily on chemical pesticides, leading to increasing pesticide resistance. Chilo suppressalis male sterility technology uses genetic, chemical, or biological means to render male Chilo suppressalis individuals incapable of reproduction. The goal is to reduce the number of offspring, control population size, and mitigate damage to crops.

[0003] Lepidoptera produce two different types of sperm: nucleated sperm and anucleated sperm. Nucleated sperm carry genetic material to fertilize the egg, while many hypotheses exist regarding the function of anucleated sperm, including aiding in the transport of nucleated sperm, providing nutrition, and reducing intersperm competition. Literature has confirmed that the development and function of anucleated sperm are regulated by the BmSxl and BmPnldc1 genes. Knocking out these genes leads to abnormal development and loss of motility in anucleated sperm, thus impairing male fertility. Double mating experiments have restored female fertilization, further confirming the crucial role of anucleated sperm in reproduction.

[0004] The TNNI3 gene encodes a troponin protein, which generally plays a key role in mammalian heart development. Recent studies have reported that silencing the troponin gene leads to genital abnormalities, ovulation disorders, and impaired male accessory gland development. However, the function of the TNNI gene in male Chilo suppressalis has not been reported. Summary of the Invention

[0005] The present invention discovered for the first time that the TNNI3 gene specifically regulates the process of anucleated spermatogenesis in the Chilo suppressalis, and confirmed through RNA interference technology that TNNI3 can inhibit the number of anucleated sperm bundles in the Chilo suppressalis, without significantly affecting the number of nucleated sperm bundles and sperm motility. Furthermore, mating male insects after silencing TNNI3 with wild-type female insects can significantly reduce the egg production of the Chilo suppressalis, providing a theoretical basis for the development of new prevention and control strategies for male reproduction.

[0006] The present invention provides a TNNI3 gene of a nucleus-less sperm formation factor of Chilo suppressalis, the nucleotide sequence of which is shown as SEQ ID NO.1.

[0007] The present invention provides a protein encoded by a TNNI3 gene, a factor for forming anucleated sperm in the striped stem borer, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] The present invention provides a dsRNA for inhibiting the formation of anucleated sperm of Chilo suppressalis, which targets and interferes with the expression of TNNI3 gene. The nucleotide sequence of the dsRNA is shown in SEQ ID NO.3.

[0009] The present invention provides a method for preparing the dsRNA, which is obtained by specific amplification using the following primers:

[0010] Upstream primer: ds TNNI3-F: 5'-CCGCAGAGGAGTTGAAGAAG-3' (SEQ ID NO. 6)

[0011] Downstream primer: ds TNNI3-R: 5′-TTCTGGAGCTTTGCGAATTT-3′ (SEQ ID NO. 7).

[0012] The present invention provides an expression cassette, a recombinant vector or a recombinant cell containing the dsRNA.

[0013] The present invention provides the use of the TNNI3 gene, protein, dsRNA, expression cassette, recombinant vector or recombinant cell in any of the following:

[0014] A1) Control of Chilo suppressalis;

[0015] A2) preparing a product for controlling the Chilo suppressalis;

[0016] A3) inhibiting the formation of anucleated sperm bundles of the Chilo suppressalis and / or reducing the egg-laying amount of the Chilo suppressalis;

[0017] A4) preparing a product for inhibiting the formation of anucleated sperm bundles of the Chilo suppressalis and / or reducing the egg production of the Chilo suppressalis.

[0018] The present invention provides a method for preventing and controlling the striped stem borer, which interferes with or inhibits the expression of the TNNI3 gene or the activity of its protein, reduces the number of anucleated sperm bundles of the striped stem borer, and further reduces the egg laying amount, thereby achieving the effect of preventing and controlling the striped stem borer.

[0019] Furthermore, the dsRNA is used to interfere with TNNI3 gene expression.

[0020] The present invention provides a method for inhibiting the formation of anucleated sperm bundles of the striped stem borer and / or reducing the egg-laying amount of the striped stem borer, interfering with or inhibiting the expression of the TNNI3 gene or the activity of its protein, reducing the number of anucleated sperm bundles of the striped stem borer, and further reducing the egg-laying amount, thereby achieving the effect of preventing and controlling the striped stem borer.

[0021] Furthermore, the dsRNA is used to interfere with TNNI3 gene expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 Representative images of testicular development and sperm bundles of the TNNI3 silenced gene. Note: A: white arrow: testis; yellow arrow: sperm bundle with nucleus; red arrow: sperm bundle without nucleus;

[0024] Figure 2 Figure 3 shows the changes in the number of sperm bundles after silencing the TNNI3 gene, where B is the interference efficiency; C is the testis area; and D is the number of sperm bundles. (Data in the figure are expressed as mean ± standard error, with n≥15 biological replicates for each treatment. The differences among treatments were analyzed using the independent sample t-test (*p<0.05, ns, not significant).

[0025] Figure 3 The number of live and dead sperm is measured for sperm motility, where A is a schematic diagram of sperm motility assay; B is the number of sperm bundles with nuclei; C is the number of sperm bundles without nuclei; and D is sperm motility (data in the figure are expressed as mean ± standard error, with n≥15 biological replicates in each treatment. Significant differences among treatments were analyzed using the independent sample t-test (*p<0.05, ns, not significant).

[0026] Figure 4 The number of eggs laid by TNNI3-silenced males mating with wild-type females. (*p<0.05, differences between treatments were analyzed using an independent samples t-test.) DETAILED DESCRIPTION

[0027] The following examples are only used to more clearly illustrate the technical scheme of the present invention, and are therefore only used as examples, and cannot limit the scope of protection of the present invention with this. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise stated, the reagents and materials used in the following examples are commercially available.

[0028] Example 1 Cloning and Analysis of the Chilo suppressalis TNNI3 Gene

[0029] (1) A Chilo suppressalis sample was placed in a 1.5 ml enzyme-free centrifuge tube, TRIZOL was added and the tube was grinded in a grinder to extract total RNA, and the total RNA extracted was synthesized into a cDNA template using a reverse transcription kit from YEASEN Biotechnology Co., Ltd.

[0030] The troponin I 3 (TNNI3) gene nucleic acid sequence (SEQ ID NO. 1) was obtained by transcriptome sequencing and cloning primers were designed using NCBI Primer-BLAST:

[0031] Upstream primer: TNNI3-F: 5'-GGCTGAACCATCTGTAGGGC-3' (SEQ ID NO. 4)

[0032] Downstream primer: TNNI3-R: 5'-ACAGCTGGCGTGAGATACAA-3' (SEQ ID NO. 5)

[0033] PCR amplification was performed using the above primers and 2× PhantaMax MasterMix (Dye Plus) enzyme from Nanjing Novozymes Biotechnology Co., Ltd., following the enzyme's recommended system and protocol. The amplification system was as follows: 2× PhantaMax Master Mix: 5.0 μL; upstream primer: 0.5 μL; downstream primer: 0.5 μL; H2O: 3 μL; cDNA: 1.0 μL.

[0034] The reaction procedure is as follows:

[0035] Pre-denaturation: 95°C, 5 min; denaturation: 95°C, 15 sec; annealing: 60°C, 15 sec; extension: 72°C, 55 sec; final extension: 72°C, 5 min; storage: 16°C, ∞; 32 cycles of denaturation-extension steps were performed.

[0036] (2) The amplified product was identified using a 1.2% agarose gel, and the gel was cut and the target fragment was purified and recovered using a gel recovery kit from Sangon Biotech. The recovered product was then ligated to a Blunt vector and transformed into Escherichia coli DH5α competent cells. A single colony was picked and sent to Changsha Qingke Biotech Co., Ltd. for Sanger sequencing.

[0037] Example 2 Effect of gene silencing efficiency and spermatogenesis after microinjection of TNNI3 gene dsRNA

[0038] (1) Preparation of dsRNA template

[0039] Based on the TNNI3 gene sequence obtained in Example 1, SnapDrogon-dsRNA Design (https: / / www.flyrnai.org / cgi-bin / RNAi_find_primers.pl) was used to predict appropriate dsRNA segments and design specific amplification primers for amplification of the dsRNA fragment of the gene. The designed specific primers are as follows:

[0040] Upstream primer: ds TNNI3-F: 5′-CCGCAGAGGAGTTGAAGAAG-3′ (SEQ ID NO. 6);

[0041] Downstream primer: ds TNNI3-R: 5'-TTCTGGAGCTTTGCGAATTT-3' (SEQ ID NO. 7)

[0042] The target fragment that was sequenced correctly in Example 1 was used as a template to synthesize dsRNA of the TNNI3 gene (SEQ ID NO. 3) using a double-stranded kit from Thermo Fisher Scientific.

[0043] (2) Microinjection of TNNI3 gene dsRNA

[0044] TNNI3 dsRNA was injected twice on the 1st and 3rd day after pupation. The relative expression of TNNI3 gene was detected on the 5th day after pupation, and the number of sperm bundles with nuclei and sperm bundles without nuclei in the testis was statistically analyzed. Figure 1 The figure shows the changes in testicular development and sperm bundle number after silencing the TNNI3 gene. Figure 2 Use membrane-permeable nucleic acid dyes 14. The results of staining of sperm bundles in the testis were used to count the activity of nucleated sperm. Figure 3 The figure shows the number of eggs laid by wild-type females after mating with TNNI3-silenced females.

[0045] (3) Sperm bundle staining

[0046] On the 1st and 3rd day after pupation, TNNI3 dsRNA was injected twice. Testes were dissected on the 5th day after pupation. A 10x10 2 mm grid was drawn on a glass slide with a marker. The testes were then placed on a slide containing complete culture medium supplemented with fetal bovine serum. A hole was pierced with a dissecting needle and the testes were gently pressed with forceps to release all sperm bundles onto the slide. An equal volume of 4% paraformaldehyde was added for fixation for 10 minutes. One-fifth the volume of DAPI dye was then added and the cells were incubated in a 37°C incubator in the dark for 10 minutes. The cells were then counted under a fluorescence microscope (blue light).

[0047] (4) Sperm motility measurement

[0048] Place the testis on a glass slide with 1xPBS, poke a hole with a dissecting needle, gently press the testis with tweezers to release all the sperm bundles in the testis onto the slide, add 1.5ul of nucleic acid dye SYBR14, then place the slide in a 37℃ constant temperature incubator and incubate in the dark for 10 minutes, then add 1.5ul of PI dye and continue to incubate in a 37℃ constant temperature incubator in the dark for 10 minutes, and count under a fluorescence microscope. Live sperm are observed under green wavelengths, and dead sperm are observed under red wavelengths (such as Figure 3 A).

[0049] (4) Next, place the slide under bright field and count all sperm bundles to obtain the total number of sperm bundles. Sperm bundles without nuclei = total sperm bundles - sperm bundles with nuclei.

[0050] like Figure 1 、 Figure 2 BC showed that TNNI3 dsRNA was injected twice, on the 1st and 3rd day after pupation. qRT-PCR was used to detect the relative expression of the TNNI3 gene on the 5th day after pupation. Compared with the control, TNNI3 gene expression was significantly reduced. Furthermore, testicular area was measured using ImageJ software, and SPSS statistical analysis showed no significant difference in testicular area.

[0051] like Figure 2 、 3 Statistical analysis of sperm bundles in the testes of 15 male pupae in both the control and treatment groups revealed that silencing TNNI3 significantly reduced the total number of sperm bundles and the number of anucleated sperm bundles in the Chilo suppressalis. However, there were no significant differences in the number of nucleated sperm bundles or sperm motility. The number of anucleated sperm bundles decreased by 25.8%.

[0052] like Figure 4As shown, TNNI3 and EGFP dsRNA were injected on the 1st, 3rd and 5th day after pupation, respectively. On the 1st day after emergence, the male adults were mated with wild-type female adults to lay eggs until the female adults died and stopped laying eggs. The egg production was then counted, and compared with the control, the egg production decreased significantly, by 58.5%.

[0053] Based on the above experiments, the TNNI3 gene specifically regulates the process of anucleate spermatogenesis in the chilo suppressalis, thereby reducing egg production, providing a theoretical basis for the development of new control strategies for male reproduction.

[0054] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. The TNNI3 gene of the Chilo suppressalis anucleate sperm formation factor, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The protein encoded by the TNNI3 gene, a factor in the formation of anucleated sperm in the Chilo suppressalis, has an amino acid sequence as shown in SEQ ID NO.

2.

3. A dsRNA for inhibiting the formation of anucleated sperm in Chilo suppressalis, characterized in that: Targeted interference with the expression of TNNI3 gene, the nucleotide sequence of which is shown in SEQ ID NO.

3.

4. The method for preparing dsRNA according to claim 3, characterized in that: The following primers were used for specific amplification: Upstream primer: ds TNNI3-F: 5'-CCGCAGAGGAGTTGAAGAAG-3' Downstream primer: ds TNNI3-R: 5'-TTCTGGAGCTTTGCGAATTT-3'.

5. An expression cassette, recombinant vector or recombinant cell containing the dsRNA according to claim 3.

6. Use of the TNNI3 gene of claim 1, the protein of claim 2, the dsRNA of claim 3, the expression cassette, recombinant vector or recombinant cell of claim 5 in any of the following: A1) Control of Chilo suppressalis; A2) preparing a product for controlling the Chilo suppressalis; A3) inhibiting the formation of anucleate sperm bundles of the Chilo suppressalis and / or reducing the egg production of the Chilo suppressalis; A4) preparing a product for inhibiting the formation of anucleated sperm bundles of the Chilo suppressalis and / or reducing the egg laying amount of the Chilo suppressalis.

7. A method for preventing and controlling Chilo suppressalis, characterized in that: Interfering with or inhibiting the expression of the TNNI3 gene or its protein activity can reduce the number of anucleated sperm bundles of the Chilo suppressalis, thereby reducing the amount of egg production, thereby achieving the effect of preventing and controlling the Chilo suppressalis.

8. The method according to claim 7, characterized in that: The dsRNA according to claim 3 is used to interfere with TNNI3 gene expression.

9. A method for inhibiting the formation of anucleated sperm bundles of the Chilo suppressalis and / or reducing the egg-laying amount of the Chilo suppressalis, characterized in that: Interfering with or inhibiting the expression of the TNNI3 gene or its protein activity can reduce the number of anucleated sperm bundles of the Chilo suppressalis, thereby reducing the amount of egg production, thereby achieving the effect of preventing and controlling the Chilo suppressalis.

10. The method according to claim 9, characterized in that: The dsRNA according to claim 3 is used to interfere with TNNI3 gene expression.

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