Cs-Tima gene of coccinella septempunctata and inhibitor and application of Cs-Tima gene

Abstract

The invention relates to the technical field of bioengineering, in particular to a coccinella septempunctata biological clock Timeless (Cs-Tims) gene as well as an inhibitor and application thereof. The invention provides a nucleotide sequence of a coccinella septempunctata biological clock Cs-Tima 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 biological clock Cs-Tims gene inhibitor in relieving biological diapause. After the biological clock Cs-Timm gene provided by the invention is subjected to dsRNA inhibition expression, the diapause of the coccinella septempunctata can be relieved, the dsRNA of the Cs-Timm gene can be used as a related preparation and product for relieving the diapause of the coccinella septempunctata, the development of the natural enemy industry is promoted, and the application prospect is wide.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a seven-spotted ladybug biological clock Cs-Tim gene and an inhibitor and application thereof. Background Art

[0002] The seven-spotted ladybird Coccinella septempunctata, a Coleoptera insect, is widely distributed throughout the world. It mainly preys on a variety of agricultural and forestry pests, such as Myzus perpersicae, Aphis glycines, Macrosiphum miscanthi, and Spodoptera frugiperda. According to statistics, it can prey on up to 38 species, making it an important predatory natural enemy insect in the farmland system. Both adults and larvae of the seven-spotted ladybird can feed on pests in large quantities, and their prey volume increases with age. It has a good control effect on the major invasive pest, the fall armyworm. Diapause is a state of growth and development stagnation that insects enter when they sense changes in the external environment. If natural enemy insects are induced to enter a diapause state, the shelf life of natural enemy products can be extended, and they can be released from diapause when they are needed. Therefore, research on the diapause regulation technology of seven-spotted ladybirds is crucial to promoting the application of seven-spotted ladybird biological control and the development of the natural enemy industry.

[0003] RNA interference (RNAi) is a mechanism that can precisely regulate gene expression. This technology uses double-stranded RNA (dsRNA, Double-stranded RNA, double-stranded ribonucleic acid) to specifically degrade target mRNA molecules, thereby inhibiting the activity of specific genes. RNAi can efficiently screen the 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. In the field of plant protection, this technology uses the insect's own gene fragments to inhibit the expression of key genes in the growth and development or biochemical metabolism of the insect through RNAi, thereby controlling pest damage or regulating the development progress of economic insects to meet the needs of production applications. However, the technical difficulty lies in choosing which gene to use as the target for regulation.

[0004] Circadian clock genes play a key role in insect diapause. They affect insects' physiological and behavioral rhythms by regulating insects' perception of environmental changes such as photoperiod and temperature. Insects use these genes to predict and adapt to seasonal changes, such as diapause to survive adverse environmental conditions. The expression pattern and regulatory mechanism of circadian clock genes are directly related to the initiation and termination of diapause, thus playing a vital role in insect survival and reproductive strategies. Summary of the invention

[0005] The object of the present invention is to provide a new circadian clock gene, namely the circadian clock Cs-Tim gene of the ladybug, and further provide the gene and its encoded protein, as well as an inhibitor of the circadian clock Cs-Tim gene, which can be used to relieve diapause of organisms.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides a circadian clock Cs-Tim gene of the ladybug, which has any one of the following nucleotide sequences:

[0008] (1) The nucleotide sequence shown in SEQ ID NO.1;

[0009] (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.

[0010]

[0011]

[0012]

[0013] Furthermore, the present invention provides a protein encoding the above-mentioned circadian clock Cs-Tim gene of the ladybug, 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. Those skilled in the art can, without affecting its activity, substitute, delete and / or add one or several amino acids to obtain a mutant sequence of the above protein according to the amino acid sequence disclosed in the present invention. Therefore, the amino acid sequence of the protein of the circadian clock Cs-Tim gene of the ladybug provided by the present invention also includes a protein derived from the circadian clock Cs-Tim gene of the ladybug, which is obtained by substituting, replacing and / or adding one or several amino acids to the amino acid sequence shown in SEQ ID NO.2 and has the same activity as the circadian clock Cs-Tim gene of the ladybug.

[0014]

[0015] Furthermore, the present invention provides an inhibitor of the circadian clock Cs-Tim gene, and the inhibitor of the circadian clock Cs-Tim gene includes RNA, and the RNA has the function of any one or more of (1) to (5);

[0016] (1) Used to inhibit the expression of the above-mentioned circadian clock Cs-Tim gene;

[0017] (2) Reduce the level of the circadian clock Cs-Tim gene;

[0018] (3) Degradation of the biological clock Cs-Tim gene;

[0019] (4) reduce the levels of the above proteins;

[0020] (5) Degradation of the above proteins.

[0021] Furthermore, the biological clock Cs-Tim gene inhibitor includes one or more of dsRNA, miRNA, siRNA, ribozyme and shRNA that inhibit the biological clock Cs-Tim gene.

[0022] More preferably, the dsRNA comprises the sequence shown in SEQ ID NO. 3 (provided in the form of a positive strand).

[0023]

[0024] Furthermore, the dsRNA is a double-stranded RNA composed of the nucleotide sequence of the forward primer T7-dsCs-Tim-F and the nucleotide sequence of the reverse primer T7-dsCs-Tim-R shown in Table 1.

[0025] The invention provides a method for preparing a biological clock Cs-Tim gene inhibitor, comprising the following steps: taking an ORF sequence of a Cs-Tim gene as a template, amplifying with a forward primer T7-dsCs-Tim-F and a reverse primer T7-dsCs-Tim-R, and then in vitro transcribing and synthesizing dsRNA, wherein the sequence of the forward primer T7-dsCs-Tim-F is shown in SEQ ID NO.4, and the sequence of the reverse primer T7-dsCs-Tim-R is shown in SEQ ID NO.5.

[0026] The present invention provides the use of the biological clock Cs-Tim gene inhibitor in relieving biological diapause. For example, the purpose of relieving biological diapause can be achieved by introducing the biological clock Cs-Tim gene inhibitor into the organism.

[0027] The present invention provides a method for relieving biological diapause, comprising the following steps: introducing the biological clock Cs-Tim gene inhibitor into an organism. The organism may be a seven-spotted ladybug.

[0028] The seven-spotted ladybird biological clock Cs-Tim gene provided by the present invention is proved by RNA interference experiments that after the dsRNA sequence of the Cs-Tim gene is injected into the abdominal intersegmental membrane of the diapause-induced seven-spotted ladybird, the ovary is significantly developed, indicating that knocking down the Cs-Tim gene can release the diapause of the seven-spotted ladybird, and the dsRNA thereof can be used as related preparations and products for releasing the diapause of the seven-spotted ladybird, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 For the spatiotemporal expression pattern of the Cs-Tim gene in the seven-spot ladybug, where A shows the transcriptional levels of the Cs-Tim gene in different tissues under diapause conditions, and B shows the transcriptional levels of the Cs-Tim gene in different tissues under non-diapause conditions;

[0030] Figure 2 For the RNA interference efficiency of Cs-Tim;

[0031] Figure 3 For the effect of the Cs-Tim gene on the ovarian development of female insects. Among them, A shows the effect of injecting double-stranded dsGFP on the ovarian development of female insects, and B shows the effect of injecting double-stranded dsCs-Tim on the ovarian development of female insects; C shows the comparison of the widths of the ovaries of female insects 7 days after injecting dsGFP and dsCs-Tim double-strands; D shows the comparison of the lengths of the ovaries of female insects 7 days after injecting dsGFP and dsCs-Tim double-strands;

[0032] Figure 4 For the effect of the Cs-Tim gene on the reproduction of female insects. Among them, the vertical axis represents the daily egg production of female insects after injecting dsGFP and dsCs-Tim, and the horizontal axis represents the number of days. Specific implementation manner

[0033] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0034] 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 in conjunction with the drawings in the specification. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments in the present invention belong to the scope protected by the present invention. For those not specified in the embodiments regarding specific technologies or conditions, they are either conventional methods or are carried out according to the technologies or conditions described in the literature in this field, or according to the product instructions. For those reagents and instruments not specified regarding the manufacturer, they are all conventional products that can be obtained through regular channels.

[0035] The sequencing primers for the Cs-Tim gene, the primers for making dsRNA of the diapause hormone gene, and the RT-qPCR primers for the diapause hormone gene in the embodiments are shown in Table 1. The primers and sequencing were both synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0036] Table 1 Primer sequences and their functions

[0037]

[0038] The following is introduced by specific embodiments.

[0039] Example 1 Obtaining the ORF sequence of the ladybug circadian clock (Cs-Tim) gene

[0040] According to the annotation file of the ladybug genome library and performing Blast alignment on the NCBI website, the sequence of the Cs-Tim gene was screened. The forward primer Cs-Tim-ORF-F (5′-CAGCCGAACATACTCAAGACG-3′) and reverse primer Cs-Tim-ORF-R (5′-ATCGAAAGTTGCGTCCAGTG-3′) of the Cs-Tim gene were designed using Primer premier 5.0 software. Fragment cloning was performed using 2×TransFast Taq PCR SuperMix (AS102-01, TransGen), and ligation transformation was carried out using pEASY-T&B Cloning Kit (CTB501-01, TransGen) and DH5α (DLC101, Tsingke) competent cells. After culturing at 37°C for 8 h in an Amp-resistant petri dish, monoclonal strains were picked for sequencing. According to the sequencing results, the nucleotide sequence encoding the Cs-Tim gene was screened, verified, and obtained. The nucleotide sequence of the Cs-Tim gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by the Cs-Tim gene is shown as SEQ ID NO.2.

[0041] Example 2 Tissue-specific expression analysis of the ladybug circadian clock Cs-Tim gene

[0042] RNA of different tissues (including head, thorax, fat body, midgut, and ovary) of female ladybugs reared under diapause conditions (18±1°C, 10L:14D, relative humidity 70%) and non-diapause conditions (24±1°C, 16L:8D, relative humidity 70%) after eclosion was extracted using TransGen's TransZol up reagent. There were 3 biological replicates for each treatment, and 5 female insects were used for each replicate. cDNA was reverse transcribed using a first-strand synthesis kit (at311-03, TransGen). Using the reverse-transcribed cDNA as a template, the expression level of the circadian clock Cs-Tim gene in different developmental stages and different tissues of ladybugs was determined by quantitative real-time fluorescence PCR (Quantitative Real time RT-PCR). The real-time fluorescence quantitative PCR experiment was operated on a Lightcycler 96 (Roche, CH) instrument, and the fluorescence signal was detected using Toroivd's 5G qPCR Premix. The primers used in this example are shown in Table 1. The internal reference gene is Actin, and the target gene is Cs-Tim.

[0043] The reaction system includes: 5G qPCR Premix 10 μL, DD-H2O 7.4 μL, Forward primer (10 μM) 0.8 μL, Reverse primer (10 μM) 0.8 μL, Template DNA Dilutions 1 μL.

[0044] The reaction parameters include: 95°C for 5 min for hot start; 95°C for 3 s, 58°C for 5 s, with 40 cycles for annealing.

[0045] The RT-qPCR data was analyzed by the -ΔΔCt 2−ΔΔCt method. Using the Actin gene of Coccinella septempunctata as the internal reference gene, and the lowest expression site of the Cs-Tim gene as the reference for processing, the gene expression was normalized and plotted using GraphPad Prism 8.0 software.

[0046] The experimental results showed that the circadian clock Cs-Tim gene of Coccinella septempunctata was expressed in the body of Coccinella septempunctata, and was highly expressed in the midgut under both diapause and non-diapause conditions ( Figure 1 ).

[0047] Example 3 Obtaining dsRNA of the circadian clock Cs-Tim gene of Coccinella septempunctata

[0048] Based on the ORF of the circadian clock Cs-Tim of Coccinella septempunctata obtained in Example 1 (its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2), dsRNA primers were designed using Primer premier 6 software.

[0049] Amplify the DNA of the circadian clock Cs-Tim gene of Coccinella septempunctata. The forward primer is T7-dsCs-Tim-F, the reverse primer is T7-dsCs-Tim-R, and the template is the plasmid extracted from the bacterial solution with correct sequencing in Example 1. The reaction system includes: template 2 μL, T7-dsCs-Tim-F (10 μM) 2 μL, T7-dsCs-Tim-R

[0050] (10 μM) 2 μL, 2×TransFast Taq PCR SuperMix (+dye) 25 μL, RNase free water 19 μL. The reaction program is shown in Table 2.

[0051] Table 2 PCR reaction program

[0052]

[0053] Amplify the ORF sequence DNA of GFP (the sequence is 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 inventor is located (which can be publicly obtained and used only for non-commercial purposes to repeat the examples described in the present invention). The reaction system includes: 2 μL of template, 2 μL of T7-dsGFP-F (10 μM), 2 μL of T7-dsGFP-R (10 μM), 25 μL of 2×TransFast Taq PCR SuperMix (+dye), and 19 μL of RNase free water. The reaction procedure refers to Table 2.

[0054]

[0055]

[0056] After purifying the amplified PCR product, use T7 RNA Polymerase (NEB ENGLAND Biolabs) reagent to synthesize dsRNA by in vitro transcription. The reaction system for synthesizing dsRNA is shown in Table 3.

[0057] Table 3 Reaction system for synthesizing dsRNA

[0058]

[0059] The reaction condition is to react at 37 °C for 4 hours, and add 1 μL of DNase I reagent to each tube 15 minutes before the end. Use phenol-chloroform extraction to obtain dsRNA double strands, including the following steps: Collect the product into a 1.5 mL centrifuge tube, add the lower layer reagent of water-saturated phenol with a volume half of the product volume and chloroform with a volume half of the product volume, gently mix and centrifuge at a speed of 12000×g in a high-speed centrifuge at 4 °C for 15 minutes (the following centrifugation processes all adopt this condition). Take the upper layer, add an equal volume of chloroform, gently mix, and centrifuge for 15 minutes. Take the upper layer again, add 1 / 10 volume of 3M NaAc and 2.5 times volume of absolute ethanol, gently mix, and let it stand at -20 °C for 15 minutes. Centrifuge for 30 minutes, white precipitate will form. After sucking out the supernatant, add 1 mL of 75% ethanol, mix well, use it to wash the precipitate, and centrifuge for 5 minutes. Slowly suck out the ethanol, and blow in the ultra-clean workbench for 5 minutes to remove the ethanol. Add 50 μL of RNase-Free water, gently flick the bottom of the tube to dissolve the precipitate. After measuring the concentration of the synthesized dsRNA using NanoDrop 2000 (Invitrogen), dilute it to a final concentration of 2 μg / μL with RNase-Free water (Tiangen Biotech) and store it in a -80 °C refrigerator for standby. Name the above dsRNA as dsCs-Tim (the sequence is shown in SEQ ID NO.3).

[0060] Referring to the above method, dsGFP was synthesized using the primers T7-dsGFP-F and T7-dsGFP-R, and the green fluorescent protein GFP was used as a negative control.

[0061] Example 4

[0062] (1) Detection of the interference efficiency of Cs-Tim by real-time fluorescence quantitative PCR

[0063] Female Coccinella septempunctata with consistent developmental progress (within 48 h after eclosion) in the laboratory-reared population were selected for microinjection. 2 μg of dsCs-Tim was injected into the intersegmental membrane of the abdomen of Coccinella septempunctata, and female insects injected with an equal amount of dsGFP were used as a control. 8 Coccinella septempunctata were taken in each group, and the injected Coccinella septempunctata were reared under diapause conditions (18 ± 1 °C, 10L:14D, relative humidity 70%). Fluorescent quantitative PCR (the detection method refers to Example 2) was used to detect the RNA interference effect. The results showed that 3 days after injecting the dsCs-Tim double strand, the expression level of the Cs-Tim gene in the whole insect sample decreased by 72% ( Figure 2 ).

[0064] (2) Effects of injecting dsCs-Tim on the ovarian development and fecundity of female insects

[0065] The female insects injected with the above dsCs-Tim were paired with untreated male insects at a ratio of 1:1. After pairing, they were reared under diapause conditions (18 ± 1 °C, 10L:14D, relative humidity 70%). After 7 days of injection, some female insects were dissected in PBS (P1020, Solarbio) to obtain ovarian tissues, and the length and width were measured by photographing with a super-depth-of-field microscope. Another part was continuously reared under diapause conditions (18 ± 1 °C, 10L:14D, relative humidity 70%) to count the egg production. The results showed that compared with the control, the dsCs-Tim treatment promoted the significant development of the female insect ovaries ( Figure 3 A, Figure 3 B), and the length and width of the ovaries increased significantly ( Figure 3 C, Figure 3 D). In addition, the Coccinella septempunctata treated with dsCs-Tim started to lay eggs 11 days after the double-strand injection, and the average pre-oviposition period was about 12 days. However, the Coccinella septempunctata injected in the control group never laid eggs ( Figure 4 ).

[0066] 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-Tim gene of the biological clock of Coccinella septempunctata is 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 Cs-Tim gene of the ladybug circadian clock 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. A biological clock Cs-Tim gene inhibitor, characterized in that, Comprising RNA, said RNA having the function of any one or more of (1) to (5); (1) For inhibiting the expression of the gene as claimed in claim 1; (2) Reducing the level of the gene as claimed in claim 1; (3) Degrading the gene as claimed in claim 1; (4) Reducing the level of the protein as claimed in claim 2 or 3; (5) Degrading the protein as claimed in claim 2 or 3.

5. The circadian clock Cs-Tim gene inhibitor according to claim 4, wherein The circadian clock Cs-Tim gene inhibitor comprises one or more of dsRNA, miRNA, siRNA, ribozyme and shRNA that inhibit the circadian clock Cs-Tim gene.

6. The circadian clock Cs-Tim gene inhibitor according to claim 5, wherein The dsRNA comprises the sequence shown in SEQID NO 3.

7. A preparation method of a biological clock Cs-Tim gene inhibitor, characterized in that, Comprising the following steps: using the ORF sequence of the Cs-Tim gene as claimed in claim 1 as a template, amplifying with the forward primer T7-dsCs-Tim-F and the reverse primer T7-dsCs-Tim-R and then synthesizing dsRNA by in vitro transcription. The sequence of the forward primer T7-dsCs-Tim-F is as shown in SEQ ID NO.4, and the sequence of the reverse primer T7-dsCs-Tim-R is as shown in SEQ ID NO.

5.

8. Use of the circadian clock Cs-Tim gene inhibitor as claimed in any one of claims 4-6 in relieving diapause.

9. Use of the biological clock Cs-Tim gene inhibitor according to claim 8 in relieving biological diapause, characterized in that, Introducing the circadian clock Cs-Tim gene inhibitor as claimed in any one of claims 4-6 into an organism.

10. A method for relieving biological diapause, characterized in that: Comprising the following steps: introducing the circadian clock Cs-Tim gene inhibitor as claimed in any one of claims 4-6 into an organism.

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