A barleyworm DDC gene, RNA interference sequence, primer, medicament and application thereof

Abstract

The present invention provides a DDC gene of a barleyworm, an RNA interference sequence, a primer, a medicament and an application thereof, and the nucleotide sequence of the coding region of the isolated DDC gene of the barleyworm is shown in SEQ ID NO: 1. The present invention uses the RNA interference sequence to interfere with the DDC gene of the barleyworm, so that the expression of the DDC gene of the barleyworm can be silenced, the dopamine synthesis process is blocked, the pre-pupae time in the barleyworm body is significantly reduced, the pupation rate is significantly increased, and the pupation rate of the barleyworm population is thereby improved.

Description

Technical Field

[0001] The invention belongs to the technical field of insect pupation, and specifically relates to a barleyworm DDC gene, an RNA interference sequence, a primer, a medicament and applications thereof. Background Art

[0002] The mealworm is a new type of feed protein resource with great development potential. It has the characteristics of strong stress resistance, wide feeding range, large insect biomass, and high nutritional value. The crude protein content of the mealworm can be as high as 45% or more. It is rich in amino acids and unsaturated fatty acids, chitosan, antimicrobial peptides and other bioactive substances with antibacterial and immunomodulatory effects. Using mealworms to replace conventional protein feeds in the diet can significantly improve animal growth performance, regulate the body's material metabolism, and improve the body's health level. The mealworm larvae are usually raised in groups, but the biggest feature of the mealworm is that they cannot pupate in a group state, and can only pupate smoothly in a single isolation state. Therefore, when pupating, the mature larvae need to be selected one by one and placed in a separate container (such as a pupation tray) to isolate the individuals from each other, but this process is time-consuming and labor-intensive, the breeding cost is high, and large-scale breeding is limited. Therefore, it is urgent to develop new germplasm of mealworms that can achieve group pupation.

[0003] The Chinese patent document with publication number CN 216392705 U discloses a pupation device for barleyworm larvae, which is mainly provided with a box body and a worm tube arranged horizontally and vertically on the bottom surface of the box body, the worm tube is vertically arranged, and the bottom end is contacted and positioned with the bottom surface of the box body. The method has the characteristics of space saving and simple operation of the pupation device. The Chinese patent document with publication number CN103109782 A discloses an artificial pupation method for barleyworm, using a specially selected barleyworm plastic cup as a pupation chamber for barleyworm, and each plastic cup only places one mature and healthy barleyworm larva. 20 plastic cups containing larvae are overlapped and placed on an indoor insect raising rack. The method saves space, is easy to observe, and saves labor.

[0004] Modern genetic manipulation techniques can be used to achieve precise improvement of resource insect germplasm and improve production efficiency. With the continuous improvement of insect genome information, genetic manipulation techniques based on gene interference, knockout, transgenic, gene expression regulation, etc. have greatly promoted the study of insect gene function and the creation of resource insect germplasm. At present, there is no research on target genes that regulate the pupation of mealworms. Summary of the invention

[0005] The technical problem solved by the present invention is to provide a DDC gene of a mealworm, an RNA interference sequence, a primer, a medicament and applications thereof. The RNA interference sequence is used to interfere with the DDC gene of the mealworm, so that the expression of the DDC gene of the mealworm is silenced, the dopamine synthesis process is blocked, the pre-pupae time in the mealworm is significantly reduced, the pupation rate is significantly increased, and the pupation rate of the mealworm population is thereby improved.

[0006] In order to solve the above problems, the first aspect of the present invention provides an isolated DDC gene of the barleyworm, the nucleotide sequence of the coding region of the DDC gene of the barleyworm is shown in SEQ ID NO:1.

[0007] The second aspect of the present invention provides a protein encoded by the DDC gene of the barleyworm, the amino acid sequence of the protein is shown in SEQ ID NO:2.

[0008] The third aspect of the present invention provides a primer for amplifying the coding region of the DDC gene of the barleyworm, comprising an upstream primer with a sequence as shown in SEQ ID NO: 3 and a downstream primer with a sequence as shown in SEQ ID NO: 4.

[0009] A fourth aspect of the present invention provides an RNA interference sequence of the DDC gene of the mealworm, wherein the RNA interference sequence is obtained by amplifying the coding region of the DDC gene of the mealworm or the protein encoded by the gene as a target.

[0010] Preferably, the RNA interference sequence is dsRNA, and the DNA sequence encoding the RNA interference sequence is shown in SEQ ID NO:5.

[0011] In a fifth aspect, the present invention provides a primer for amplifying the RNA interference sequence of the DDC gene of the barleyworm, comprising an upstream primer having a sequence as shown in SEQ ID NO: 6 and a downstream primer having a sequence as shown in SEQ ID NO: 7.

[0012] The sixth aspect of the present invention provides a drug for regulating the pupation of mealworms, comprising the RNA interference sequence of the above-mentioned mealworm DDC gene.

[0013] The seventh aspect of the present invention provides the use of the above-mentioned DDC gene of the mealworm, or the above-mentioned protein, or the above-mentioned RNA interference sequence, or the above-mentioned agent in regulating the pupation of the mealworm.

[0014] Preferably, by reducing the expression level of the DDC gene of the mealworm in the mealworm, the pre-pupae time of the mealworm is reduced and / or the pupation rate of the group is increased.

[0015] The eighth aspect of the present invention provides a method for increasing the pupation rate of a mealworm population, comprising the following steps: injecting the above-mentioned RNA interference sequence or the above-mentioned agent into the body of the mealworm, and raising the mealworm.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides an isolated DDC gene of the mealworm. The present invention has found that RNA interference is performed on the DDC gene of the mealworm, so that the dopamine synthesis process is blocked and the pupation rate of the mealworm group can be increased, thereby providing a clear target gene for creating a mealworm germplasm capable of group pupation.

[0018] The present invention provides an RNA interference sequence of a DDC gene of a barleyworm. The RNA interference sequence is used to interfere with the DDC gene of the barleyworm, so that the expression of the DDC gene of the barleyworm is silenced, and the expression amount of the DDC gene in the barleyworm body is significantly reduced, thereby hindering the dopamine synthesis process, significantly reducing the pre-pupa time in the barleyworm body, and significantly increasing the pupation rate, thereby improving the pupation rate of the barleyworm population. The RNA interference sequence is a nucleotide fragment in the decarboxylase region of the DDC gene, and the target has high specificity to avoid non-specific silencing of other non-target genes. The RNA interference sequence has appropriate length and GC content, can effectively induce RNA interference, and has strong specificity.

[0019] Compared with the existing methods, the method for improving the pupation rate of the mealworm group of the present invention has low cost and high efficiency, and can fundamentally solve the current situation of the pupation of the mealworm group. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the expression level of DDC gene interfering RNA in barleyworm;

[0021] Figure 2 is the effect of DDC gene dsRNA on the pre-pupae time of mealworm;

[0022] Figure 3 The effect of DDC gene dsRNA on the pupation rate of mealworm. DETAILED DESCRIPTION

[0023] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] A first aspect of an embodiment of the present invention provides an isolated DDC gene of the barleyworm, wherein the nucleotide sequence of the coding region of the DDC gene of the barleyworm is shown in SEQ ID NO: 1.

[0025] The sequence structure of SEQ ID NO: 1 is as follows:

[0026]

[0027] DDC is dopa decarboxylase, a type of amino acid decarboxylase, and is an enzyme that catalyzes the decarboxylation of dopa to produce dopamine. The embodiment of the present invention provides an isolated DDC gene of the mealworm. The present invention has found that after RNA interference of the DDC gene of the mealworm, the pupation rate of the mealworm population can be increased, providing a clear target gene for creating a mealworm germplasm that can pupate in groups.

[0028] A second aspect of the embodiments of the present invention provides a protein encoded by a DDC gene of a barleyworm, the amino acid sequence of the protein being shown in SEQ ID NO:2.

[0029] The sequence structure of SEQ ID NO: 2 is as follows:

[0030] MEANQFKDFAKEMIDYVSGYLENIRDRRVLPSVEPGYLRPLIPDSAPQKADKWEDVMADIERVIMPGVTHWHSPRFHAYFPTANSYPAIVADILSGAIACIGFTWIASPACTELEVVML DWLGKMIGLPKEFLACSGGKGGGVIQGTASEATLVALLGAKARTIAAIKKEKPELNDADIVAKLVGYTSSQSHSSVERAGLLGGIKLRGLQPDEKNSLRGATLEAAIKEDREAGLIPFY VVATLGTTSSCTFDNLQEIGPVCNDNSVWLHVDAAYAGSAFICPEFRYLMTGIERADSFNFNPHKWLLVNFDCSTMWLKEPNWLVDAFNVDPLYLKHEQQGAAPDYRHWQIPLGRRFRA LKLWFVLRLYGIENLQTHIRKHVELAHYFESLVVSDERFEITEEVVLGLVCFRLKASNEVNEALLKRLNGRGVIHLVPSKIRDVYFLRLAICSRYTEKEDIDVSWKEVKEAADEVLKK.

[0031] A third aspect of the embodiments of the present invention provides primers for amplifying the coding region of the DDC gene of the barleyworm, comprising an upstream primer having a sequence as shown in SEQ ID NO: 3 and a downstream primer having a sequence as shown in SEQ ID NO: 4.

[0032] The sequence structure of SEQ ID NO: 3 is as follows:

[0033] DDC-F1:ATGGAAGCTAATCAGTTCA

[0034] The sequence structure of SEQ ID NO: 4 is as follows:

[0035] DDC-R1: TTACTTCTTCAAAACCTCA.

[0036] The above primers are used to amplify the coding region of the DDC gene of the mealworm, with strong specificity and high amplification efficiency.

[0037] The fourth aspect of the embodiment of the present invention provides an RNA interference sequence of the DDC gene of the barleyworm, wherein the RNA interference sequence is amplified by targeting the coding region of the DDC gene of the barleyworm or the protein encoded by it. The RNA interference sequence of the DDC gene of the barleyworm is used to interfere with the DDC gene of the barleyworm. The RNA interference sequence enters the cell and can be degraded into 21-25nt siRNA under the action of the Dicer enzyme in the cell. The antisense strand in the siRNA will participate in guiding the synthesis of the RNA-induced silencing complex (RISC). The RISC complex guides the "guide strand" of the siRNA to bind to the complementary region in the target mRNA through its endonuclease activity. After this complementary pairing, the RISC complex will cause the degradation of the target mRNA, thereby silencing the expression of the DDC gene of the barleyworm, thereby hindering the dopamine synthesis process, and then increasing the pupation rate of the barleyworm population.

[0038] Preferably, the RNA interference sequence is dsRNA, and the DNA sequence encoding the RNA interference sequence is shown in SEQ ID NO:5.

[0039] The sequence structure of SEQ ID NO: 5 is as follows:

[0040] ACGGCCAATTCGTACCCGGCCATCGTCGCCGACATCCTCAGCGGAGCGATCGCATGCATCGGCTTCACTTGGATCGCTAGCCCTGCGTGCACAGAGCTCGAGGTCGTGATGCTGGATTGGCTGGGAAAGATGATTGGACTTCCTAAGGAGTTCCTCGCCTGCTCGGGGGGCAAGGGCGGCGGCGTGATCCAGGGCACCGCTAGTGAGGCCACTTTGGTGGCGCTGCTGGG GGCCAAGGCGCGGACGATTGCCGCGATTAAGAAAGAGAAGCCGGAATTGAACGATGCCGATATCGTTGCGAAATTGGTCGGATATACTTCGAGCCAGAGCCACTCGTCGGTGGAGCGCGGGGCTGCTTGGGGGCATTAAATTGAGGGGTTTTGCAGCCCGACGAGAAGAACAGTTTGAGAGGGGCGACTTTAGAGGCAGCCATCAAGGAAGACCGTGAAGCCGGCCTT.

[0041] The coding gene of the RNA interference sequence is a nucleotide fragment of the decarboxylase region in the DDC gene. The target site has high specificity to avoid non-specific silencing of other non-target genes. The RNA interference sequence has appropriate length and GC content, can effectively induce RNA interference, and has strong specificity.

[0042] A fifth aspect of the embodiments of the present invention provides a primer for amplifying the RNA interference sequence of the DDC gene of the barleyworm, comprising an upstream primer with a sequence as shown in SEQ ID NO: 6 and a downstream primer with a sequence as shown in SEQ ID NO: 7.

[0043] The sequence structure of SEQ ID NO: 6 is as follows:

[0044] DDC-F2: TAATACGACTCACTATAGGG ACGGCCAATTCGTACCCG.

[0045] The sequence structure of SEQ ID NO: 7 is as follows:

[0046] DDC-R2: TAATACGACTCACTATAGGG AAGGCCGGCTTCACGGTA.

[0047] The above primers are used to amplify the RNA interference sequence of the DDC gene of the barleyworm, with strong specificity and high amplification efficiency.

[0048] A sixth aspect of the embodiments of the present invention provides an agent for regulating the pupation of mealworms, comprising the RNA interference sequence of the DDC gene of the mealworms mentioned above.

[0049] A seventh aspect of the embodiments of the present invention provides the use of the above-mentioned DDC gene of the mealworm, or the above-mentioned protein, or the above-mentioned RNA interference sequence, or the above-mentioned agent in regulating the pupation of the mealworm.

[0050] Preferably, by reducing the expression level of the DDC gene of the mealworm in the mealworm, the pre-pupae time of the mealworm is reduced and / or the pupation rate of the group is increased.

[0051] An eighth aspect of the embodiments of the present invention provides a method for increasing the pupation rate of a mealworm population, comprising the following steps: injecting the above-mentioned RNA interference sequence or the above-mentioned agent into the body of the mealworm, and raising the mealworm.

[0052] Example 1 Cloning and analysis of the DDC gene of mealworm

[0053] (1) Extraction of total RNA from barleyworm

[0054] Take 30mg of barley worm tissue and place it in a 1.5ml enzyme-free tube, pre-cool it with liquid nitrogen, use a grinding rod to grind it until it is ground into powder, add 1000μl RNAisoplus lysis solution to the tube, and let it stand at room temperature for 5min. Centrifuge at 12000×g at 4℃ for 5min. Transfer the supernatant to a new 1.5ml RNase-free tube and add 200μl chloroform. Vigorously shake and mix, and let it stand at room temperature for 5min. Centrifuge at 12000×g at 4℃ for 15min, transfer the supernatant to a new 1.5ml RNase-free tube, and add isopropanol of the same volume as the supernatant. Invert and mix. Let it stand at room temperature for 10min, and centrifuge at 12000×g at 4℃ for 10min. Discard the supernatant and keep the precipitate. Prepare 75% alcohol with 750μl of anhydrous ethanol and 250μl of RNase-free H2O (ultrapure water). Add it to the tube and wash the precipitate upside down. Centrifuge at 7900×g for 5 minutes at 4℃. Discard the supernatant and keep the precipitate. Open the centrifuge tube cover and dry it in a clean bench at room temperature for 4 minutes. Add appropriate amount of ultrapure water to dissolve, and store at -80℃ after it is fully dissolved.

[0055] (2) First-strand cDNA synthesis and gDNA removal

[0056] The total RNA extracted from the barley worm was reverse transcribed to synthesize cDNA. The first-strand cDNA was synthesized according to The reaction system was as follows: 1 μg of total RNA from barley worm, Anchored Oligo (dT18 ): 1μL, 2×ES Reaction Mix: 10μL, EasyScript RT Enzyme Mix: 1μL, gDNA Remover 1μL, ultrapure water to 20μL. Reaction conditions: PCR instrument 42℃ incubation for 30min, 85℃ reaction for 5sec, 4℃ storage. Take 2μL to test its concentration, determine the cDNA concentration is 500ng / μL, store at –20℃ for later use.

[0057] (3) Full-length cloning of DDC gene

[0058] According to the DNA sequence of the DDC predicted coding region provided by the transcriptome data, primer design was completed using Primer Premier 5. The upstream primer sequence is shown in SEQ ID NO: 3, and the downstream primer sequence is shown in SEQ ID NO: 4.

[0059] The sequence structure of SEQ ID NO: 3 is as follows:

[0060] DDC-F1:ATGGAAGCTAATCAGTTCA

[0061] The sequence structure of SEQ ID NO: 4 is as follows:

[0062] DDC-R1: TTACTTCTTCAAAACCTCA.

[0063] Using the cDNA of Phantas sphaeroides as a template, the full-length coding region of the DDC gene was amplified using the 2×TransStart FastPfu PCR SuperMix kit. The PCR reaction system was: cDNA template: 2μL; 2×Phanta Flash Master Mix: 25μL; upstream primer DDC-F1: 2μL; downstream primer DDC-R1: 2μL; ultrapure water: 19μL. The PCR amplification conditions were: 98℃ pre-denaturation for 30sec, 98℃ denaturation for 10sec, 55℃ annealing for 5sec, 72℃ extension for 5sec, and 72℃ extension for 1min after 35 cycles.

[0064] The integrity of the detection fragment was detected by 1.2% (w / v) agarose gel electrophoresis, and the target gene target band was recovered using the gel recovery kit QIAquick Gel Extraction Kit and dissolved in 20 μL ultrapure water. 4 μL of PCR recovery product and 1 μL of pEASY-T1 Simple Cloning Vector were added to the centrifuge tube and gently mixed, reacted at room temperature for 5 minutes, and the constructed vector was transferred into Escherichia coli DH5α, added to 500 μL of liquid LB medium without antibiotics for expansion and culture, centrifuged to collect the bacteria, smeared on a solid culture medium plate containing ampicillin antibiotics, and inverted in a 37°C incubator for overnight culture. White spots were selected, expanded and cultured in liquid culture medium containing ampicillin and PCR verification of bacterial liquid was performed, and positive samples were sent to Shanghai Shenggong Company for sequencing verification. The DDC gene coding region of the barleyworm was obtained, and its nucleotide sequence is shown in SEQ ID NO: 1.

[0065] The sequence structure of SEQ ID NO: 1 is as follows:

[0066]

[0067] Example 2 Synthesis of dsRNA

[0068] (1) Preparation of dsRNA

[0069] The nucleotide fragment of the decarboxylase region in DDC was amplified as the interference target site, and a dsRNA primer containing a T7 promoter was designed. The upstream primer was shown in SEQ ID NO: 6, and the downstream primer was shown in SEQ ID NO: 7.

[0070] The sequence structure of SEQ ID NO: 6 is as follows:

[0071] DDC-F2: TAATACGACTCACTATAGGG ACGGCCAATTCGTACCCG.

[0072] The sequence structure of SEQ ID NO: 7 is as follows:

[0073] DDC-R2: TAATACGACTCACTATAGGG AAGGCCGGCTTCACGGTA.

[0074] Using the DDC gene of the barleyworm as a template, the nucleotide fragment of the decarboxylase region of the DDC gene of the barleyworm was amplified. The PCR amplification system was as follows: cDNA template: 2μL; 2×Phanta Flash Master Mix: 25μL; upstream primer DDC-F2: 2μL; downstream primer DDC-R2: 2μL; ultrapure water: 19μL. The PCR amplification conditions were: 98℃ pre-denaturation for 30sec, 98℃ denaturation for 10sec, 60℃ annealing for 5sec, 72℃ extension for 5sec, and 72℃ extension for 1min after 35 cycles.

[0075] PCR product recovery, gene cloning, and sequence verification were consistent with the steps related to the full-length cloning of the DDC gene described above. The purified target gene PCR product was recovered and used as a template to synthesize dsRNA according to the instruction manual of Transcript Aid T7 HighYield Transcription Kit from Thermo Scientific. The reaction system was as follows: 5×TranscriptAidReaction Buffer: 4μL, ATP / CTP / GTP / UTP: 2μL each, TranscriptAid Enzyme Mix: 2μL, PCR template: 1μL, ultrapure water: 5μL. The above reaction solution was incubated at 37°C overnight to synthesize dsRNA.

[0076] (2) dsRNA purification

[0077] After dsRNA synthesis, 2 μL DNase I was added to the reaction solution and incubated at 37°C for 15 min; 2 μL EDTA was added and incubated at 65°C for 10 min; the reaction solution was transferred to a 1.5 mL centrifuge tube, and 115 μL ultrapure water and 15 μL 3M sodium acetate solution were added respectively; 150 μL phenol chloroform (phenol: chloroform = 1:1) was added and mixed, and then 300 μL chloroform was added, shaken and mixed, and centrifuged at 12,000 rpm and 4°C for 10 min; the supernatant was taken, 350 μL anhydrous ethanol was added, mixed thoroughly, and placed at -20°C overnight; centrifuged at 12,000 rpm and 4°C for 10 min; the supernatant was removed, 500 μL 75% pre-cooled ethanol was added to wash the precipitate, and then centrifuged at 7500 rpm and 4°C for 5 min; the supernatant was removed, the precipitate was dried at room temperature, ultrapure water was added to dissolve the precipitate, and NanoDrop RNA concentration and purity were tested by One, and the integrity of the product was tested by 1.2% (w / v) agarose gel electrophoresis.

[0078] The DNA sequence encoding the dsRNA is shown in SEQ ID NO:5.

[0079] The sequence structure of SEQ ID NO: 5 is as follows:

[0080] ACGGCCAATTCGTACCCGGCCATCGTCGCCGACATCCTCAGCGGAGCGATCGCATGCATCGGCTTCACTTGGATCGCTAGCCCTGCGTGCACAGAGCTCGAGGTCGTGATGCTGGATTGGCTGGGAAAGATGATTGGACTTCCTAAGGAGTTCCTCGCCTGCTCGGGGGGCAAGGGCGGCGGCGTGATCCAGGGCACCGCTAGTGAGGCCACTTTGGTGGCGCTGCTGGG GGCCAAGGCGCGGACGATTGCCGCGATTAAGAAAGAGAAGCCGGAATTGAACGATGCCGATATCGTTGCGAAATTGGTCGGATATACTTCGAGCCAGAGCCACTCGTCGGTGGAGCGCGGGGCTGCTTGGGGGCATTAAATTGAGGGGTTTTGCAGCCCGACGAGAAGAACAGTTTGAGAGGGGCGACTTTAGAGGCAGCCATCAAGGAAGACCGTGAAGCCGGCCTT.

[0081] Example 3 Gene silencing efficiency after injection of DDC gene dsRNA and its effect on pre-pupae time and pupation rate of barleyworm

[0082] (1) Injection method

[0083] Prepare microinjection needles, set the needle puller parameters as: P = 500, Heat = 575, Pull = 145, VEL = 145, DEL = 100, cut the tip of the glass capillary under a stereo microscope to ensure that the needle tip is sharp and hard. Select mature larvae of mealworms as the subjects, inject at the base of their thoracic feet, gently pierce into the body, and inject 120nL dsRNA solution at an injection speed of 20nL / s; after the injection, let it stand for 5sec and gently pull out the needle. After the injection, use plastic tweezers to transfer the larvae to a disposable plastic cup (9.8cm×5.6cm) and place them in an artificial climate box for breeding; the control group was injected with the same dose of dsGFP.

[0084] (2) Silencing efficiency detection method

[0085] The steps of total RNA extraction and reverse transcription are as described above. The SuperReal PreMix Plus (SYBR Green) (Tiangen) fluorescence quantitative kit was used for qRT-PCR experiment, and the relative expression of genes was detected by ABI 7500 Real-Time PCR System (Applied Biosystems, Carlsbad). Actin was used as the internal reference gene, the nucleotide sequence of the Actin gene of the barleyworm is shown in SEQ ID NO: 8, and the amino acid sequence of Actin of the barleyworm is shown in SEQ ID NO: 9. Primers were designed using Beacon Designer 7, the upstream primer is shown in SEQ ID NO: 10, the downstream primer is shown in SEQ ID NO: 11, the upstream primer of the internal reference gene Actin is shown in SEQ ID NO: 12, and the downstream primer is shown in SEQ ID NO: 13.

[0086] The sequence structure of SEQ ID NO: 8 is as follows:

[0087]

[0088] The sequence structure of SEQ ID NO: 9 is as follows:

[0089] MCDDDVAALVVDNGSGMCKAGFAGDDAPRAVFPSIVGRPRHQGVMVGMGQKDSYVGDEAQSKRGILTLKYPIEHGIITNWDDMEKIWHHTFYNELRVAPEEHPILLTEAPLNPKANREKMTQIMFETFNAPAMYVAIQAVLSLYASGRTTGIVLDSGDGVSHTVPIYEGYALPHAILRLDLAGRDLTD YLMKILTERGYSFTTTAEREIVRDIKEKLCYVALDFEQEMATAAASTSLEKSYELPDGQVITIGNERFRCPEALFQPSFLGMESCGIHETVYNSIMKCDVDIRKDLYANTVLSGGTTMYPGIADRMQKEITALAPSTIKIKIIAPPERKYSVWIGGSILASLSTFQQMWISKQEYDESGPGIVHRKCF.

[0090] The sequence structure of SEQ ID NO: 10 is as follows:

[0091] DDC-qF: GGCATTCACCCAGATTCCAC.

[0092] The sequence structure of SEQ ID NO: 11 is as follows:

[0093] DDC-qR:CAATCCAGCATCACGACCTC.

[0094] The sequence structure of SEQ ID NO: 12 is as follows:

[0095] Actin-F:GGGGATGGGGTGTCTCATAC.

[0096] The sequence structure of SEQ ID NO: 13 is as follows:

[0097] Actin-R:CTCCTGCTCGAAATCCAACG.

[0098] The single-well system of qRT-PCR reaction is as follows: cDNA template: 1μL, 2×SuperRed premix plus: 12.5μL, upstream primer: 0.75μL, downstream primer: 0.75μL, 50×RO5 Reference DyeⅡ: 0.5μL, ultrapure water: 9.5μL. qRT-PCR reaction conditions: 95℃15min; 95℃10sec; 60℃30sec, 40 cycles. The experiment was set with 3 biological replicates and 3 technical replicates. 2 -△△Ct The relative expression level of each gene among different treatments was calculated.

[0099] (3) Methods for observing pupation rate

[0100] Five mature larvae of mealworms injected with dsDDC were placed in each disposable plastic cup. The number of pre-pupae was observed every 2 days until all pupated. The pupation rate in each treatment group was calculated. Eight biological replicates were set for each treatment group. The mealworms injected with dsGFP were set as the control group.

[0101] (4) Test results and analysis

[0102] DDC gene silencing efficiency

[0103] The results are as follows Figure 1 As shown in the figure, "**" indicates P<0.01; the meanings of the English abbreviations in the figure: "dsDDC" indicates the treatment group (dsDDC) injected with DOPAdecarboxylase gene dsRNA; "control group" indicates the injection of green fluorescent protein (GFP) gene dsRNA. Compared with the control group, after the injection of DDC gene dsRNA, the expression of DDC gene in the barleyworm decreased significantly, indicating that the interference sequence of the DDC gene can significantly inhibit the expression of the DDC gene.

[0104] Effects of injection of DDC gene dsRNA on pre-pupae time and pupation rate of Euryale ferruginea

[0105] The results are as follows Figure 2 , Figure 3 As shown in the figure, "**" indicates P<0.01; the meanings of the English abbreviations in the figure: "dsDDC" indicates the treatment group injected with DOPA decarboxylase gene dsRNA; "control group" indicates the group injected with green fluorescent protein (GFP) gene dsRNA. Compared with the control group, after injection of DDC gene dsRNA, the pre-pupa time in the body of the mealworm was significantly reduced, and the pupation rate was significantly increased, indicating that the interference of the DDC gene can significantly improve the efficiency of group pupation.

[0106] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. An RNA interference dsRNA of the DDC gene of barleyworm, characterized in that The DNA sequence encoding the RNA interference dsRNA is shown in SEQ ID NO:

5.

2. A primer for amplifying RNA interference dsRNA of the DDC gene of barleyworm, characterized in that: It includes an upstream primer with a sequence as shown in SEQ ID NO: 6 and a downstream primer with a sequence as shown in SEQ ID NO:

7.

3. A medicament for regulating the pupation of barley worms, characterized in that: An RNA interference dsRNA comprising the barleyworm DDC gene as claimed in claim 1.

4. Use of the RNA interference dsRNA as claimed in claim 1 or the medicament as claimed in claim 3 in reducing the pre-pupae time of mealworms and / or increasing the pupation rate of the population.

5. The use according to claim 4, characterized in that: By reducing the expression level of the DDC gene of the mealworm in the mealworm, the pre-pupae time of the mealworm is reduced and / or the pupation rate of the group is increased.

6. A method for increasing the pupation rate of barleyworm populations, characterized in that: The following steps are involved: The RNA interference dsRNA as claimed in claim 1 or the agent as claimed in claim 3 is injected into the body of the mealworm, and the mealworm is raised.

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