Ion transport peptide Cs-ITP-L gene of coccinella septempunctata and inhibitor and application of ion transport peptide Cs-ITP-L gene

Abstract

The invention relates to the technical field of bioengineering, in particular to a coccinella septempunctata ion transport peptide Cs-ITP-L gene as well as an inhibitor and application thereof. The invention provides a nucleotide sequence of a coccinella septempunctata ion transport peptide Cs-ITP-L gene as shown in SEQ ID NO. 1 and an amino acid sequence as shown in SEQ ID NO. 2; the invention also provides an application of the ion transport peptide Cs-ITP-L gene inhibitor in the induction of biological diapause. After dsRNA expression inhibition, the Cs-ITP-L gene of the ion transport peptide can induce diapause of coccinella septempunctata, the dsRNA of the Cs-ITP-L gene can be used as a related preparation and product for inducing diapause of coccinella septempunctata, development of the natural enemy industry is promoted, and the Cs-ITP-L gene has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and specifically relates to the Coccinella septempunctata ion transport peptide Cs-ITP-L gene, its inhibitor, and applications thereof. Background Art

[0002] The Coccinella septempunctata is widely used in biological control. It is a predatory insect that can prey on aphids, thrips, whiteflies, mites, spiders, soft scale insects, and the eggs and larvae of Lepidoptera insects, etc., and is widely used in biological control of pests. However, due to the short lifespan of the Coccinella septempunctata, the shelf life of its products is short, which limits its potential in large-scale production and application. Diapause is a state in which insects enter a growth and development arrest state in response to changes in the external environment. By regulating this characteristic, the lifespan and adaptability of the Coccinella septempunctata can be effectively extended, thereby improving its application potential in biological control. Therefore, carrying out research on the diapause regulation technology of the Coccinella septempunctata is crucial for promoting the application of biological control of the Coccinella septempunctata and the development of the natural enemy industry.

[0003] RNA interference (RNAi) is a mechanism that can precisely regulate gene expression. This technology specifically degrades target mRNA molecules through double-stranded RNA (dsRNA), thereby inhibiting the activity of specific genes. Since this technology can specifically inhibit the expression of specific genes, it has been widely used in the fields of exploring gene functions and gene therapy. RNAi can efficiently screen target genes required for biological control, laying a solid foundation for the development and application of economic insect products such as pest biological control and natural enemy insects. This technology uses the insect's own gene fragments to inhibit the expression of key genes in the growth and development of insects through RNAi, and then regulates the development progress of economic insects to meet the needs of production applications. The RNAi technology has broad development prospects in the field of agricultural plant protection and control. The key problem for achieving effective control based on RNAi is to screen specific target genes of target insects. However, the technical difficulty lies in choosing which gene to use as a target for regulation. Summary of the Invention

[0004] The present invention aims to provide a Coccinella septempunctata ion transport peptide Cs-ITP-L gene, the protein encoded thereby, and a Cs-ITP-L gene inhibitor, and the Cs-ITP-L gene inhibitor can be used to induce the diapause state of organisms.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention first provides a Cs-ITP-L gene of Coccinella septempunctata ion transport peptide, which comprises any one of the following nucleotide sequences:

[0007] (1) The nucleotide sequence shown in SEQ ID NO.1;

[0008] (2) A coding nucleotide sequence of a protein with the same function obtained by substitution, deletion or insertion of one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.1.

[0009]

[0010] Furthermore, the present invention provides a protein encoded by the above-mentioned Coccinella septempunctata ion transport peptide Cs-ITP-L gene, which has the amino acid sequence shown in SEQ ID NO.2, or an amino acid sequence having at least 95% homology with the amino acid sequence shown in SEQ ID NO.2. This shows that those skilled in the art can substitute, delete and / or add one or several amino acids to obtain a mutant sequence of the protein without affecting its activity based on the amino acid sequence disclosed in the present invention. Therefore, the Coccinella septempunctata ion transport peptide Cs-ITP-L gene of the present invention also includes a protein derived from the Coccinella septempunctata ion transport peptide Cs-TP gene, which has the same activity as the Coccinella septempunctata ion transport peptide Cs-ITP-L gene and is obtained by substituting, replacing and / or adding one or several amino acids to the amino acid sequence shown in SEQ ID NO.2.

[0011] MNYLSSKVSTTLLWVCMALVLFLSGSFGSPANRSSDLLSHHLAKRSFFDIQCKGVYDKSIFARLDRICEDCYNLFREPQVHSLCRRNCFTTDYFKGCLDTLQLSDELQQIQTWIKQLHGADPG(SEQ ID NO.2)

[0012] Furthermore, the present invention provides an inhibitor of the ion transport peptide Cs-ITP-L gene, which is used to inhibit the expression of the ion transport peptide Cs-ITP-L gene, or reduce the level of the ion transport peptide Cs-ITP-L gene, or degrade the ion transport peptide Cs-ITP-L gene, or dsRNA, miRNA, siRNA, ribozyme or shRNA of the ion transport peptide Cs-ITP-L gene.

[0013] Furthermore, the dsRNA includes the sequence shown in SEQ ID NO.3 (provided in the form of the sense strand).

[0014] CTTCTTCGACATTCAATGCAAAGGAGTTTATGACAAGAGCATATTCGCAAGATTAGATCGCATATGCGAAGACTGCTACAATCTCTTCAGAGAGCCCCAAGTACATTCCCTATGCAGGAGAAACTGTTTCACGACAGATTACTTCAAGGGTTGCCTTGACACACTTCAACTATCAGATGAATTGCAACAAATTCAGACATGGATAAAACAATTACACGGGG(SEQ ID NO.3)

[0015] The dsRNA is double-stranded RNA composed of the nucleotide sequence of the forward primer T7-dsCs-ITP-L-F shown in SEQ ID NO.4 and the nucleotide sequence of the reverse primer T7-dsCs-ITP-L-R shown in SEQ ID NO.5.

[0016] The present invention provides a method for preparing an inhibitor of the transport peptide Cs-ITP-L gene, comprising the following steps: using the ORF sequence of the Cs-ITP-L gene as a template, amplifying with the forward primer T7-dsCs-ITP-L-F and the reverse primer T7-dsCs-ITP-L-R and then in vitro transcribing to synthesize dsRNA. The sequence of the forward primer T7-dsCs-ITP-L-F is as shown in SEQ ID NO.4, and the sequence of the reverse primer T7-dsCs-ITP-L-R is as shown in SEQ ID NO.5.

[0017] Furthermore, the present invention provides the application of the above-mentioned inhibitor of the transport peptide Cs-ITP-L gene in inducing diapause in organisms. By introducing the inhibitor of the transport peptide Cs-ITP-L gene into organisms, the purpose of inducing diapause in organisms is achieved.

[0018] The present invention provides a method for inducing diapause in organisms, comprising the following step: introducing the above-mentioned inhibitor of the transport peptide Cs-ITP-L gene into organisms.

[0019] The organism can be Coccinella septempunctata.

[0020] For the Coccinella septempunctata transport peptide Cs-ITP-L gene of the present invention, it is proved by RNA interference experiments that after injecting the dsRNA sequence of the Cs-ITP-L gene into the intersegmental membrane of the abdomen of newly emerged Coccinella septempunctata, the ovarian development is inhibited, indicating that knocking down the Cs-ITP-L gene can induce diapause in Coccinella septempunctata, and its dsRNA can be used as a related preparation and product for inducing diapause in Coccinella septempunctata, with broad application prospects. Description of the Drawings

[0021] Figure 1 It is the spatio-temporal expression pattern of the Coccinella septempunctata Cs-ITP-L gene. Among them, A is the transcriptional level of the Cs-ITP-L gene in different tissues under diapause conditions, and B is the transcriptional level of the Cs-ITP-L gene in different tissues under non-diapause conditions.

[0022] Figure 2 It is the interference effect of Cs-ITP-L. Among them, A is the RNA interference efficiency of Cs-ITP-L, B is the expression level of the Cs-VG gene after RNA interfering with the Cs-ITP-L gene, and C is the expression level of the CsVGR gene after RNA interfering with the Cs-ITP-L gene.

[0023] Figure 3 The effects of the Cs-ITP-L gene on the ovarian development of female insects. Among them, A and B show the ovarian development conditions after injecting dsGFP and dsCs-ITP-L respectively; C shows the ovarian length 7 days after injecting dsGFP and dsCs-ITP-L respectively; D shows the ovarian width 7 days after injecting dsGFP and dsCs-ITP-L respectively; E shows the daily egg production after injecting dsGFP and dsCs-ITP-L respectively.

[0024] Figure 4 The effects of the Cs-ITP-L gene on the fat accumulation of female insects. Among them, A and B are schematic diagrams of abdominal fat accumulation after injecting dsGFP and dsCs-ITP-L in sequence respectively, C shows the TAG content in female insects after injecting dsGFP and dsCs-ITP-L, and D shows the total lipid content in female insects after injecting dsGFP and dsCs-ITP-L. Specific implementation manners

[0025] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings of the specification. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. For those not specified in the embodiments regarding specific techniques or conditions, they are conventional methods or are carried out according to the techniques or conditions described in the literature in this field, or according to the product specifications. Reagents, instruments, etc. not specified for the manufacturer can be obtained as conventional products through regular channels.

[0026] In the embodiments, the sequencing primers for the diapause induction gene, the primers for preparing dsRNA of the diapause induction gene, and the RT-qPCR primers for the diapause induction Cs-ITP-L gene are shown in Table 1. The primers and sequencing were synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0027] Table 1 Primer sequences and their functions

[0028]

[0029] The following is introduced through specific examples.

[0030] Example 1 Obtaining the ORF sequence of the ion transport peptide Cs-ITP-L gene of Coccinella septempunctata

[0031] Using the genomic data of Coccinella septempunctata, search the database-annotated files by keywords, align the unigene sequences on the NCBI website, and screen to obtain the sequence of the Cs-ITP-L gene. Then, use Primer premier 5.0 software to design the forward primer Cs-ITP-L-ORF-F and reverse primer Cs-ITP-L-ORF-R for the Cs-ITP-L gene. Use 2×TransFast Taq PCR SuperMix (AS102-01, TransGen) for fragment cloning, ligate the target fragment to the pEASY-T5 Zero cloning vector, and transfer it into Trans-T1 cells for overnight culture. The next day, after detecting by colony PCR, select the positive clone bacterial solution with the size of the target band for sequencing. Align the sequencing results with the sequence of the Cs-ITP-L gene obtained by screening to verify and obtain the nucleotide sequence encoding this gene. The nucleotide sequence of the Cs-ITP-L gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the Cs-ITP-L gene is shown in SEQ ID NO.2.

[0032] Example 2 Tissue-specific expression analysis of the ion transport peptide Cs-ITP-L gene in Coccinella septempunctata

[0033] Newly emerged Coccinella septempunctata were reared under non-diapause conditions and diapause conditions respectively. The rearing times were 5 days, 10 days, 15 days and 20 days after eclosion. Different tissues (head, thorax, fat body, midgut and ovary) of Coccinella septempunctata under different rearing conditions and different rearing days were taken, and RNA was extracted using TRIzol reagent from Takara.

[0034] Non-diapause conditions (i.e., normal development conditions) include: rearing temperature 24±1°C, photoperiod 16L:8D, relative humidity 70%.

[0035] Diapause conditions include: rearing temperature 18±1°C, photoperiod 10L:14D, relative humidity 70%.

[0036] There were 3 biological replicates for each treatment, and 5 female insects for each replicate. Use the ABM Script GII ReverseTranscriptase kit (purchased from Beijing ABTech Science & Technology Development Co., Ltd., product number: RK26500) first-strand synthesis kit to reverse transcribe to obtain cDNA. Using the reverse transcribed cDNA as a template, use quantitative real-time fluorescence PCR (Quantitative Real time RT-PCR) to determine the expression level of the ion transport peptide Cs-ITP-L gene in different developmental stages and different tissues of Coccinella septempunctata. The real-time fluorescence quantitative PCR experiment was operated on a Lightcycler 96 (Roche, CH) instrument, and The 5G qPCR Premix detects fluorescence signals.

[0037] The real-time fluorescence quantitative PCR reaction system includes: mixing 1 μL of cDNA, 0.8 μL of forward primer (concentration 10 μM), 0.8 μL of reverse primer (concentration 10 μM), and 10 μL of 5G qPCR Premix, and finally, making up to 20 μL with RNase-free water.

[0038] The reaction parameters include: 95 °C for 5 min, hot start; 98 °C for 3 s, 58 °C for 5 s, for 40 cycles.

[0039] RT-qPCR data was analyzed by the 2 -ΔΔCt method, ΔΔCt = (Ct 目标基因 − Ct 持家基因 ) 实验组 − (Ct 目标基因 − Ct 持家基因 ) 对照组 , with the housekeeping gene being Actin, normalized by the expression level of the Cs-ITP-L gene in the head, and the gene expression was standardized and graphed using GraphPad Prism 9.0 software.

[0040] The experimental results are as Figure 1 shown. It can be seen that the Cs-ITP-L gene of the ion transport peptide in Coccinella septempunctata is highly expressed in the fat body and midgut under both diapause and non-diapause conditions, with the lowest expression in the ovary under diapause conditions and relatively low expression in the head and ovary under non-diapause conditions. Under diapause conditions, the expression level of the Cs-ITP-L gene in the midgut tissue gradually increases after the emergence of female adults; under non-diapause conditions, the expression level of the Cs-ITP-L gene in the midgut tissue first increases, then decreases, and then increases again.

[0041] Example 3 Obtaining dsRNA of the Cs-ITP-L gene of Coccinella septempunctata

[0042] Using the ORF sequence of the Cs-ITP-L gene of Coccinella septempunctata obtained in Example 1 (the nucleotide sequence of the Cs-ITP-L gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the Cs-ITP-L gene is shown in SEQ ID NO.2) as a template, dsRNA primers were designed using Primer premier6. software.

[0043] Amplify the DNA of the Cs-ITP-L gene of Coccinella septempunctata. The forward primer is T7-dsCs-ITP-L-F, and the reverse primer is T7-dsCs-ITP-L-R. The nucleotide sequence of the template is as shown in SEQ ID NO.1. Use 2×TransFast TaqPCR SuperMix (AS102-01, TransGen) for fragment cloning. The reaction system includes: 2 μL of template (cDNA), 2 μL of forward primer (10 μM), 2 μL of reverse primer (10 μM); 25 μL of 2x TransFast Taq PCR SuperMix (containing dye), and RNase-free water is added to make up the total volume to 50 μL. The reaction conditions include: pre-denaturation at 94 °C for 3 minutes; then enter the cycle reaction stage, including denaturation (94 °C, 10 seconds), annealing (55-60 °C, 20 seconds), and extension (72 °C, 30 seconds), and this cycle is repeated 35 times; finally, final extension is carried out at 72 °C for 5 minutes.

[0044] Amplify the DNA of green fluorescent protein GFP (the sequence is as shown in SEQ ID NO.14). The forward primer is T7-dsGFP-F, and the reverse primer is T7-dsGFP-R. The template is the cDNA of the strain containing GFP stored in the laboratory where the inventors are located (which can be obtained by the public and is only used for non-commercial purposes to repeat the examples described in the present invention). The reaction system and reaction procedure for amplifying the DNA of green fluorescent protein GFP refer to the reaction system and reaction procedure for amplifying the DNA of the Cs-ITP-L gene described above.

[0045] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG(SEQ ID NO.14)

[0046] After purifying the amplified PCR products, dsRNA was synthesized by in vitro transcription using the T7 RiboMAX TM Express RNAi System (Promega) kit. The DNA of the Cs-ITP-L gene was transcribed in vitro to synthesize dsCS-ITP-L (the sequence is shown in SEQ ID NO.3), and the DNA of GFP was transcribed in vitro to synthesize dsGFP.

[0047] The in vitro transcription reaction system includes: 8 μL of 10x Transcription buffer, 0.8 μL each of ATP, CTP, UTP, and GTP (each 100 mM), 1 μL of RNA Inhibitor, 0.8 μL of T7 RNA Polymerase, 1.5 μL of template, and made up to 50 μL with DEPC water.

[0048] The in vitro transcription conditions include: reacting at a constant temperature of 37°C for 4 h in a PCR instrument.

[0049] After measuring the concentration of the synthesized dsRNA using NanoDrop 2000 (Invitrogen), it was diluted with DEPC water to a final concentration of 2 μg / μL and stored in a -80°C refrigerator for later use.

[0050] Example 4: Effect of interfering with the Cs-ITP-L gene on the ovarian development of Coccinella septempunctata

[0051] Real-time fluorescence quantitative PCR was used to detect the interference efficiency of Cs-ITP-L and ovarian development. The experimental method includes the following steps:

[0052] Female Coccinella septempunctata 48 h after eclosion were selected for microinjection. 2 μg of dsCs-ITP-L was injected into the abdominal intersegmental membrane of Coccinella septempunctata, and female insects injected with an equal amount of dsGFP were used as the control group. 15 Coccinella septempunctata were taken in each group. The injected Coccinella septempunctata were reared under non-diapause conditions (photoperiod 16L:8D; humidity 70%; temperature 25°C). After 72 h of interference, total RNA of Coccinella septempunctata was extracted using TRIzol reagent, and the extracted RNA was reverse transcribed into cDNA using the ABScript GII Reverse Transcriptase (purchased from Beijing Aibote Technology Development Co., Ltd., product number RK26500) kit.

[0053] Real-time fluorescence quantitative PCR was performed using a LightCycler 96 instrument and SYBR Premix Ex Taq II dye.

[0054] The real-time fluorescence quantitative PCR reaction system includes: 1 μL of cDNA template, 0.8 μL of forward primer (10 μM), 0.8 μL of reverse primer (10 μM), 10 μL of 5G qPCR Premix, and made up to a total volume of 20 μL with RNase free water.

[0055] The reaction conditions for real-time fluorescence quantitative PCR are as follows: pre-denaturation at 95°C for 5 min; 40 cycles are performed, and each cycle includes: denaturation at 98°C for 3 s, annealing at 55 - 60°C for 5 s, and extension at 72°C for 30 s; final extension at 72°C for 5 min.

[0056] RT-qPCR data were analyzed using the 2 -ΔΔCt -ΔΔCt method, where ΔΔCt = (Ct 目标基因 - Ct 内参基因 ) 实验组 - (Ct 目标基因 - Ct 内参基因 ) 对照组 . Among them, Actin was used as the internal reference gene, and the expression of the gene was standardized and plotted using GraphPad Prism 8.0 software.

[0057] The experimental results showed that 3 days after injecting dsCs-ITP-L double-stranded, the expression level of the ITP-L gene decreased by 90% ( Figure 2 A), and the significant decrease in Cs-ITP-L expression significantly inhibited the expression of downstream genes and CsVG and CsVGR genes ( Figure 2 B, Figure 2 C). This indicates that RNA interference of the Cs-ITP-L gene is effective.

[0058] Example 5 Effect of injecting dsCs-ITP-L on female fecundity

[0059] Female adults 48 h after initial eclosion were injected with dsCs-ITP-L (treatment group) and dsCsGFP (control group) respectively, with 30 female insects in each group. Each female insect was injected with 2 μg of dsRNA. The female insects injected with dsRNA were reared under non-diapause conditions (temperature 24 ± 1°C, photoperiod 16L:8D, relative humidity 70%). On the 5th day after injection, ovarian tissues were dissected in PBS (P1020, Solarbio) and photographed using a super-depth-of-field microscope to measure the length and width.

[0060] The results showed that compared with the control group, dsCs-ITP-L treatment inhibited the ovarian development of female insects ( Figure 3 A, Figure 3 B), and the length and width of the ovaries were significantly reduced ( Figure 3 C, Figure 3 D).

[0061] Female Coccinella septempunctata at 48 h after eclosion were selected for microinjection, and 2 μg of dsCs-ITP-L was injected into the intersegmental membrane of the abdomen of Coccinella septempunctata (treatment group). Female insects injected with an equal amount of dsGFP were used as the control. 15 Coccinella septempunctata were selected for each group. The female insects injected with dsRNA were reared under non-diapause conditions (temperature 24 ± 1°C, photoperiod 16L:8D, relative humidity 70%). The pre-oviposition period and fecundity were counted.

[0062] The experimental results are as Figure 3 shown in Figure E. The control group of Coccinella septempunctata started laying eggs on the 4th day after double-stranded injection, and the average pre-oviposition period was about 5 days; while the treatment group started laying eggs around 8 days. From the 1st day to the 7th day, the control group showed a gradually increasing fecundity; the fecundity of the treatment group also increased during the same period, but on the 7th day, compared with the control group, the fecundity of the treatment group was significantly lower than that of the control group, indicating that interfering with the Cs-ITP-L gene inhibited the fecundity of Coccinella septempunctata, and the fecundity of female insects injected with dsCs-ITP-L was significantly lower than that of female insects in the dsGFP group.

[0063] Example 6

[0064] Effect of injecting dsCs-ITP-L on fat accumulation. The experimental method includes the following steps: Female Coccinella septempunctata at 48 h after eclosion were selected for microinjection, and 2 μg of dsCs-ITP-L was injected into the intersegmental membrane of the abdomen of Coccinella septempunctata (treatment group). Female insects injected with an equal amount of dsGFP were used as the control. 15 Coccinella septempunctata were selected for each group. The female insects injected with dsRNA were reared under non-diapause conditions (temperature 24 ± 1°C, photoperiod 16L:8D, relative humidity 70%) for 5 days.

[0065] The female Coccinella septempunctata were dissected in PBS buffer (P1020, Solarbio) to observe the fat accumulation in the abdomen of Coccinella septempunctata and obtain adipose tissue. A triglyceride (TG) assay kit (purchased from Nanjing Jiancheng Bioengineering Institute, product number A110-1-1) was used to detect the triglyceride content in the fat body.

[0066] The total lipid content was detected by chloroform-methanol extraction method. The specific operation was as follows: First, weigh the fresh weight of the treated test insects (denoted as M), and then place them in a constant temperature oven at 72°C for 48 hours until constant weight (denoted as DW). Then, add 1 mL of chloroform-methanol solution (volume ratio of chloroform to methanol is 2:1) to the dried test insect residue, homogenize and grind it with a high-speed electric grinding pestle, and then centrifuge at a centrifugal force of 2600×g for 10 minutes to remove the supernatant, and repeat centrifugation once. Finally, put the centrifuged residue into a constant temperature oven at 70°C for 48 hours until constant weight (denoted as LDW). The formula for calculating the total lipid content is: total lipid content = (DW - LDW) / M × 100%.

[0067] The experimental results are as Figure 4 shown. Compared with the injection of dsGFP, significant fat accumulation occurred after the injection of dsCs-ITP-L ( Figure 4 A, Figure 4 B), the triglyceride content increased significantly ( Figure 4 C), the total lipid content increased significantly after the injection of dsCs-ITP-L ( Figure 4 D), and the results after the injection of dsCs-ITP-L were consistent with the phenotype under diapause conditions, indicating that knocking down the Cs-ITP-L gene can induce diapause in Coccinella septempunctata.

[0068] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. The Cs-ITP-L gene of Coccinella septempunctata ion transport peptide, characterized in that, Having any one of the following nucleotide sequences: (1) The nucleotide sequence shown in SEQ ID NO.1; (2) A coding nucleotide sequence of a protein with the same function obtained by substitution, deletion or insertion of one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.

1.

2. A protein encoding the Coccinella septempunctata ion transport peptide Cs-ITP-L gene as claimed in claim 1.

3. The protein according to claim 2, wherein Having the amino acid sequence shown in SEQ ID NO.2, or having an amino acid sequence with at least 95% homology to the amino acid sequence shown in SEQ ID NO.

2.

4. An inhibitor of the ion transport peptide Cs-ITP-L gene, characterized in that, Comprising RNA, said RNA having any one or more of the functions of (1) to (3); (1) For inhibiting the expression of the gene as claimed in claim 1; (2) For reducing the level of the protein as claimed in claim 2 or 3; (3) For degrading the protein as claimed in claim 2 or 3.

5. The ion transport peptide Cs-ITP-L gene inhibitor according to claim 4, wherein The ion transport peptide Cs-ITP-L gene inhibitor comprises one or more of dsRNA, miRNA, siRNA, ribozyme, shRNA that inhibit the ion transport peptide Cs-ITP-L gene.

6. The ion transport peptide Cs-ITP-L gene inhibitor according to claim 5, characterized in that, The dsRNA comprises the sequence shown in SEQ ID NO.

3.

7. A preparation method of an inhibitor of the ion transport peptide Cs-ITP-L gene, characterized in that, Comprising the following steps: Using the ORF sequence of the Cs-ITP-L gene as a template, amplifying with the forward primer T7-dsCs-ITP-L-F and the reverse primer T7-dsCs-ITP-L-R and then synthesizing dsRNA by in vitro transcription. The sequence of the forward primer T7-dsCs-ITP-L-F is as shown in SEQ ID NO.4, and the sequence of the reverse primer T7-dsCs-ITP-L-R is as shown in SEQ ID NO.

5.

8. Use of the Cs-ITP-L gene inhibitor as claimed in any one of claims 4 - 6 in inducing diapause.

9. Use of the Cs-ITP-L gene inhibitor according to claim 8 in inducing diapause, characterized in that, Introducing the ion transport peptide Cs-ITP-L gene inhibitor as claimed in any one of claims 4 - 6 into an organism.

10. A method for inducing diapause in organisms, characterized in that, Comprising the following steps: Introducing the ion transport peptide Cs-ITP-L gene inhibitor as claimed in any one of claims 4 - 6 into an organism.

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