Chilo suppressalis tnni3 and application thereof in male sterility

Abstract

The application relates to a Chilo suppressalis non-nuclear sperm formation factor TNNI3 and application thereof in male sterility. It is found for the first time that the TNNI3 gene specifically regulates the non-nuclear sperm formation process of Chilo suppressalis, and it is proved by RNA interference technology that TNNI3 can inhibit the number of non-nuclear sperm bundles of Chilo suppressalis, has no significant influence on the number of nucleated sperm bundles and sperm activity, and can significantly reduce the egg production after male insects are mated with wild type female insects, thereby providing a theoretical basis for developing a new type of prevention and control strategy for male reproduction.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the nucleus-forming factor TNNI3 of the rice stem borer and its application in male infertility. Background Technology

[0002] The rice stem borer (Chilo suppressalis) is a major pest that seriously damages crops such as rice, corn, and wheat. Its larvae bore into crop stems, leading to withered seedlings, withered panicles, whiteheads, and damaged plants, severely impacting crop growth and yield. In rice, the rice stem borer can cause the death of the central leaf, unfilled panicles, or insufficient grain filling, resulting in large-scale yield reductions. In corn and wheat, it can cause stem damage, lodging, and reduced grain filling rates. During its borer feeding process, the rice stem borer may spread pathogens, exacerbating disease outbreaks. Large-scale outbreaks not only affect farmers' income but also threaten food security. For a long time, the control of the rice stem borer has heavily relied on chemical pesticides, leading to increasing pesticide resistance. Male sterilization technology for the rice stem borer is a method that uses genetic, chemical, or biological means to render male individuals infertile, aiming to reduce the number of offspring, control population size, and reduce damage to crops.

[0003] Lepidoptera produce two different types of sperm: nucleated sperm and anucleated sperm. Nucleated sperm carry genetic material and combine with an egg to form a zygote. There are many hypotheses regarding the function of anucleated sperm: they help transport nucleated sperm; they provide nutrients; and they reduce competition among sperm, etc. Literature has confirmed that the development and function of anucleated sperm are regulated by the genes BmSxl and BmPnldc1. Knockout of these genes leads to abnormal development and loss of motility in anucleated sperm, thus affecting male fertility. Double mating experiments have shown that female fertilization ability can be restored, further confirming the important role of anucleated sperm in the reproductive process.

[0004] The TNNI3 gene encodes troponin, which plays a crucial role in the development of the mammalian heart. Recent studies have reported that silencing the troponin gene leads to abnormalities in the external genitalia, causing ovulation disorders and affecting the development of male accessory glands. However, the function of the TNNI gene in male rice stem borers has not yet been reported. Summary of the Invention

[0005] This invention is the first to discover that the TNNI3 gene specifically regulates the process of nucleated spermatogenesis in the rice stem borer. Furthermore, RNA interference technology confirmed that TNNI3 can inhibit the number of nucleated sperm bundles in the rice stem borer, without significantly affecting the number of nucleated sperm bundles or sperm motility. Further, mating males with silenced TNNI3 with wild-type females significantly reduced the oviposition rate of the rice stem borer, providing a theoretical basis for developing novel control strategies based on male reproduction.

[0006] This invention provides a gene for the nucleus-free spermatogenesis factor TNNI3 from the rice stem borer, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] This invention provides a protein encoded by the TNNI3 gene of rice stem borer, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] This invention provides a dsRNA that inhibits the formation of nucleated sperm in rice stem borer by targeting and interfering with the expression of the TNNI3 gene, and its nucleotide sequence is shown in SEQ ID NO.3.

[0009] This 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, recombinant vector or recombinant cell containing the dsRNA.

[0013] This 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 rice stem borer;

[0015] A2) Prepare products for controlling rice stem borers;

[0016] A3) Inhibit the formation of nucleusless sperm bundles in the rice stem borer and / or reduce the number of eggs laid by the rice stem borer;

[0017] A4) Prepare products that inhibit the formation of nucleusless sperm bundles in the rice stem borer and / or reduce the egg production of the rice stem borer.

[0018] This invention provides a method for controlling the rice stem borer by interfering with or inhibiting the expression of the TNNI3 gene or its protein activity, reducing the number of nucleated sperm bundles in the rice stem borer, thereby reducing the number of eggs laid, and thus achieving the effect of controlling the rice stem borer.

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

[0020] This invention provides a method for inhibiting the formation of nucleated sperm bundles in the rice stem borer and / or reducing the number of eggs laid by the rice stem borer. By interfering with or inhibiting the expression of the TNNI3 gene or its protein activity, the number of nucleated sperm bundles in the rice stem borer is reduced, thereby reducing the number of eggs laid, thus achieving the effect of preventing and controlling the rice stem borer.

[0021] Furthermore, the dsRNA was used to interfere with TNNI3 gene expression. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 These are representative images of testicular development and sperm bundles for the silencing gene TNNI3. Note: A: White arrow: testis; Yellow arrow: nucleated sperm bundle; Red arrow: anucleate sperm bundle;

[0024] Figure 2 The change in sperm bundle count after silencing the TNNI3 gene is shown in Figure 1. B represents interference efficiency; C represents testicular area; and D represents sperm bundle count. (Data in the figure are expressed as mean ± standard error, with n≥15 biological replicates per treatment. Independent samples t-tests were used to analyze the significance of differences between treatments (*p<0.05, ns not significant).

[0025] Figure 3 The number of live and dead sperm was measured to determine sperm motility. Figure A shows a schematic diagram of sperm motility testing; Figure B shows the number of nucleated sperm bundles; Figure C shows the number of anucleated sperm bundles; and Figure D shows sperm motility. (Data in the figures are expressed as mean ± standard error. The number of biological replicates (n ≥ 15) in each treatment was used. Independent samples t-tests were used to analyze the significance of differences between treatments (*p < 0.05, ns difference not significant).

[0026] Figure 4 The number of eggs laid after mating between a silent TNNI3 male and a wild-type female. (*p<0.05, independent samples t-test was used to analyze the significance of differences between treatments) Detailed Implementation

[0027] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0028] Example 1: Cloning and Analysis of the TNNI3 Gene in the Rice Stem Borer

[0029] (1) Take the rice stem borer sample and place it in a 1.5ml enzyme-free centrifuge tube. Add TRIZOL and grind it in a grinder to extract total RNA. Use the reverse transcription kit of YEASEN Biotechnology to synthesize cDNA template from the extracted total RNA.

[0030] The nucleic acid sequence of the troponin I 3 (TNNI3) gene (SEQ ID NO.1) was obtained using transcriptome sequencing. 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 the 2×PhantaMax MasterMix (Dye Plus) enzyme from Nanjing Novizan Biotechnology Co., Ltd., following the recommended enzyme system and procedure. The amplification system was as follows: 2×PhantaMax Master Mix: 5.0 μL; forward primer: 0.5 μL; reverse primer: 0.5 μL; H2O: 3 μL; cDNA: 1.0 μL.

[0034] The reaction procedure is as follows:

[0035] Pre-denaturation: 95℃, 5 min; denaturation: 95℃, 15 sec; annealing: 60℃, 15 sec; extension: 72℃, 55 sec; final extension: 72℃, 5 min; storage: 16℃, ∞; repeat 32 cycles from denaturation to extension.

[0036] (2) After identifying the amplified product with 1.2% agarose gel, the gel was cut and the target fragment was purified and recovered using the gel recovery kit of Sangon Biotech Co., Ltd. The recovered product was then ligated into the Blunt vector, transformed into E. coli DH5α competent cells, and single colonies were picked and sent to Changsha Qingke Biotechnology Co., Ltd. for Sanger sequencing.

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

[0038] (1) Preparation of dsRNA template

[0039] Based on the TNNI3 gene sequence obtained in Example 1, suitable dsRNA segments were predicted using SnapDrogon-dsRNA Design (https: / / www.flyrnai.org / cgi-bin / RNAi_find_primers.pl), and specific amplification primers were designed for the amplification of the dsRNA fragments of this gene. The designed specific primers are as follows:

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

[0041] Downstream primer: dsTNNI3-R:5'-TTCTGGAGCTTTGCGAATTT-3' (SEQ ID NO.7)

[0042] Using the target fragment correctly sequenced in Example 1 as a template, the dsRNA (SEQ ID NO.3) of the TNNI3 gene was synthesized using a double-stranded reagent kit from Thermo Fisher Scientific.

[0043] (2) Microinjection of dsRNA of TNNI3 gene

[0044] TNNI3 dsRNA was injected twice consecutively on days 1 and 3 after pupation. The relative expression level of the TNNI3 gene was detected on day 5 after pupation, and the number of nucleated and non-nucleated sperm bundles in the testes was statistically analyzed. Figure 1 The study showed changes in testicular development and sperm count after the silencing of the TNNI3 gene. Figure 2 Using membrane-permeable nucleic acid dyes 14. The results of staining sperm bundles in the testes were used to statistically analyze the activity of nucleated sperm. Figure 3 The results show the number of eggs laid after mating with a wild-type female following the silencing of the TNNI3 gene.

[0045] (3) Staining of sperm bundles

[0046] TNNI3 dsRNA was injected twice consecutively on days 1 and 3 post-pupae, and the testes were dissected on day 5 post-pupae. A 10x10 2mm grid was drawn on a glass slide with a marker. The testes were then placed on a glass slide containing complete culture medium supplemented with fetal bovine serum. A hole was made with a dissecting needle, and the testes were gently pressed with forceps to release all the sperm bundles onto the glass slide. An equal volume of 4% paraformaldehyde was added, and the slide was fixed for 10 minutes. Then, 1 / 5 volume of DAPI dye was added, and the slide was incubated in the dark at 37°C for 10 minutes. The sperm were then counted under a fluorescence microscope (blue light).

[0047] (4) Sperm motility assay

[0048] Place the testes on a glass slide containing 1xPBS, make a small hole with a dissecting needle, and gently press the testes with forceps to release all the sperm bundles onto the slide. Add 1.5 μL of SYBR14 nucleic acid dye, then incubate the slide in the dark at 37°C for 10 min. Add 1.5 μL of PI dye and incubate again in the dark at 37°C for 10 min. Count the sperm under a fluorescence microscope. Live sperm are observed under green wavelength, and dead sperm are observed under red wavelength (e.g., ...). Figure 3 A).

[0049] (4) Next, place the slide under bright field and count all sperm bundles to obtain the total sperm bundles. Anucleate sperm bundles = total sperm bundles - nucleated sperm bundles.

[0050] like Figure 1 , Figure 2 BC analysis showed that injecting TNNI3 dsRNA twice, on days 1 and 3 post-pupae, and then detecting the relative expression level of the TNNI3 gene using qRT-PCR on day 5 post-pupae, revealed a significantly lower expression level of the TNNI3 gene compared to the control. Furthermore, the area of ​​the testes was measured using ImageJ software, and SPSS statistical analysis showed no significant difference in testicular area.

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

[0052] like Figure 4As shown, dsRNA of TNNI3 and EGFP was injected on days 1, 3, and 5 after pupation. On day 1 after emergence, male adults were mated with wild-type female adults to lay eggs until the female adults died and stopped laying eggs. The number of eggs laid was then counted. Compared with the control, the number of eggs laid was significantly reduced by 58.5%.

[0053] Based on the above experiments, the TNNI3 gene specifically regulates the process of nucleus-free spermatogenesis in the rice stem borer, thereby reducing egg production, providing a theoretical basis for developing new prevention and control strategies for male reproduction.

[0054] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in 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 protection scope of the present invention.

Claims

1. The use of dsRNA targeting the TNNI3 gene of rice stem borer and / or expression cassettes, recombinant vectors, or recombinant cells containing said dsRNA in any of the following: A1) Inhibits the formation of nucleated sperm bundles in the rice stem borer, thereby reducing the number of eggs laid by the rice stem borer; A2) Prepare products that inhibit the formation of nucleusless sperm bundles in the rice stem borer, thereby reducing the egg production of the rice stem borer; The nucleotide sequence of the TNNI3 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the dsRNA targeting the TNNI3 gene of the rice stem borer is shown in SEQ ID NO.

3.

3. A method for controlling the rice stem borer, characterized in that, Inhibiting the expression of the TNNI3 gene or its protein activity reduces the number of nucleated sperm bundles in the rice stem borer, thereby reducing egg production and achieving the effect of preventing and controlling the rice stem borer; wherein the nucleotide sequence of the TNNI3 gene is shown in SEQ ID NO.

1.

4. The method according to claim 3, characterized in that, TNNI3 gene expression was suppressed using dsRNA as shown in SEQ ID NO.

3.

5. A method for inhibiting the formation of enucleated sperm bundles in the rice stem borer, thereby reducing the oviposition rate of the rice stem borer, characterized in that, Inhibiting the expression of the TNNI3 gene or its protein activity reduces the number of nucleated sperm bundles in the rice stem borer, thereby reducing the number of eggs laid; wherein the nucleotide sequence of the TNNI3 gene is shown in SEQ ID NO.

1.

6. The method according to claim 5, characterized in that, TNNI3 gene expression was suppressed using dsRNA as shown in SEQ ID NO.3.

Images