Double-stranded RNA preparation for regulating and controlling molting of prawns as well as synthesis method and application of double-stranded RNA preparation

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a double-stranded RNA preparation for regulating and controlling shrimp molting as well as a synthesis method and application of the double-stranded RNA preparation. The prawn shelling is regulated and controlled by synthesizing specific double-stranded RNA, and the preparation method of the preparation comprises the following steps: firstly, cloning a target gene cDNA sequence related to the key physiological process of prawn shelling; secondly, constructing the sequence into an in-vitro transcription vector, and extracting and purifying recombinant plasmids; and finally, synthesizing the high-purity and high-stability double-stranded RNA preparation aiming at the target gene by taking the plasmid as a template and utilizing an in-vitro transcription system. In-vivo injection experiments prove that when the dsRNA preparation is injected into the litopenaeus vannamei body, the molting process of the litopenaeus vannamei can be obviously delayed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a double-stranded RNA preparation for regulating the molting of prawns as well as a synthesis method and application thereof. BACKGROUND

[0002] Prawns are important marine aquaculture economic species in China, and fresh quality directly determines market value. Live transportation is a key link connecting the breeding end and the consumption end. However, the molting behavior of prawns during transportation becomes a core bottleneck restricting the survival rate of transportation, which is difficult to avoid through conventional logistics means.

[0003] The molting of prawns is regulated by both endocrine and external environment, and is periodic and sudden. During long-distance transportation, the prawns become extremely fragile during the soft shell period after molting: the new chitinous shell is easy to break and infect diseases, and the molting consumes a large amount of energy, which, combined with problems such as insufficient dissolved oxygen and ammonia accumulation in the transportation environment, will exacerbate the stress reaction, resulting in a sharp increase in mortality. According to statistics, the mortality of prawns during the soft shell period can increase from less than 5% to more than 30%, and in extreme cases, the entire batch is scrapped, causing heavy economic losses.

[0004] With the transformation and upgrading of the aquaculture industry and the growth of market demand for fresh prawns, it is urgent to establish a high-quality live prawn supply chain. The lack of molting regulation technology is a major obstacle, therefore, developing a technology that can safely and controllably delay the molting of prawns is urgently needed to ensure the survival rate of transportation. SUMMARY

[0005] In view of the above problems, the present application aims to provide a double-stranded RNA preparation for regulating the molting of prawns as well as a synthesis method and application thereof.

[0006] The technical content of the present application is as follows: The present application provides a double-stranded RNA preparation for regulating the molting of prawns, wherein the double-stranded RNA (dsRNA) is 14-3-3zeta dsRNA, and the deoxynucleotide sequence is shown in SEQ ID NO. 1. The prawns include Litopenaeus vannamei.

[0007] The present application also provides a synthesis method of the above-mentioned double-stranded RNA preparation for regulating the molting of prawns, comprising the following steps: 1) Extracting total RNA of prawns Using TransZol Up Plus RNA Kit extraction kit to extract total RNA of tissues; 2) Synthesizing first strand of cDNA Synthesizing the first strand of cDNA according to the instructions of the reverse transcription kit; 3) Cloning of gene ORF sequence PCR amplification was performed using the cDNA first strand mixture of all tissues of Penaeus orientalis as a template to obtain the ORF sequence of the target gene. The target gene fragment was purified by gel recovery and purification, and the gel recovery product was connected and transformed to obtain recombinant bacteria. After that, positive clone bacteria were screened and sequenced. The positive bacteria with correct sequencing target sequence were cultured, and the DNA plasmid was extracted; The PCR reaction was performed using PrimeSTAR Max DNA Polymerase reagent; The sequences of the PCR amplification primers Primer-ORF-F and Primer-ORF-R are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively; 4) Synthesis of 14-3-3zeta double-stranded template PCR amplification was performed using the above extracted DNA plasmid as a template to obtain DNA template 1 and DNA template 2; The PCR amplification reaction system of the DNA template 1 is: Template 1 μL, 14-3-3zeta-T7F 1 μL, 14-3-3zeta-R 1 μL, PCR Mix 10 μL, H2O 7 μL, total 20 μL; The sequences of the PCR amplification primers 14-3-3zeta-T7F and 14-3-3zeta-R are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively; The PCR amplification reaction system of the DNA template 2 is: Template 1 μL, 14-3-3zeta-T7R 1 μL, 14-3-3zeta-F 1 μL, PCR Mix 10 μL, H2O 7 μL, total 20 μL; The sequences of the PCR amplification primers 14-3-3zeta-F and 14-3-3zeta-T7R are shown in SEQ ID NO. 6 and SEQ ID NO. 7, respectively; According to the above reaction system, T7F x R and T7R x F were each made into 10 tubes (20 μL / tube); The reaction program of the PCR amplification is: 95℃ 5 min; 95℃ 30 s, 58℃ 30 s, 72℃ 60 s, a total of 34 cycles; 72℃ 10 min. The DNA template 1 and the DNA template 2 were mixed and subjected to double-stranded transcription reaction, and then purified by double enzyme digestion system to remove impurities, thereby obtaining high-purity double-stranded RNA product 14-3-3zeta dsRNA.

[0008] The reaction system of the double-stranded transcription is: NTP Mix 8 μL, 10×Transcription Buffer 2 μL, T7 Enzyme Mix 2 μL, DNA template 1 0.93 μL, DNA template 2 2.5 μL and RNase-free H2O 4.57 μL; The reaction condition is 4h of reaction at 37℃.

[0009] The double-enzyme digestion system is: Transcription Product 20 μL, DNase I 1 μL, RNase T1 (10 U / μL) 2 μL, RNase-free H2O 17 μL, total 40 μL; Preferably, the RNase T1 is diluted to 10 U / μL by using RNase T1 Dilution Buffer in advance; The reaction condition is 30min of incubation at 37℃.

[0010] The application further provides the application of the double-stranded RNA preparation in regulating the molting of the prawn; The double-stranded RNA preparation is 14-3-3zeta dsRNA. The application mode is to inject the 14-3-3zeta dsRNA into the prawn in vivo.

[0011] The application has the following beneficial effects: The double-stranded RNA preparation for regulating the molting of the prawn in the application regulates the molting of the prawn by synthesizing specific double-stranded RNA (dsRNA), and the preparation method comprises the following steps: firstly, cloning a target gene cDNA sequence related to the key physiological process of the molting of the prawn; secondly, constructing the sequence into an in-vitro transcription vector, extracting and purifying a recombinant plasmid; and finally, using the plasmid as a template, synthesizing the double-stranded RNA preparation with high purity and stability for the target gene by using an in-vitro transcription system. It is proved by in-vivo injection experiments that the molting process of the prawn can be significantly delayed by injecting the dsRNA preparation into the Litopenaeus vannamei in vivo.

[0012] The present application provides a kind of biological agent with clear principle and accurate effect, which can actively intervene in the molting cycle of prawns, and ensure that the target prawn group is in the non-molting period of hard shell during operation by applying the preparation before long-distance transportation, greatly enhance its stress resistance, reduce transportation loss to a minimum, ensure the quality and survival rate of live prawns reaching the terminal market, and improve economic value, which provides an innovative technical solution to solve the problem of large transportation loss caused by molting in prawn industry, and has broad industrial application prospects.Compared with traditional environmental stress or chemical induction method, the preparation has the advantages of strong targeting and programmable control. DETAILED DESCRIPTION

[0013] The present application will be further described in detail through specific implementation examples, and it should be understood that these examples are only used to illustrate the present application and not to limit the protection scope of the present application, and various equivalent modifications of the present application made by those skilled in the art after reading the present application fall within the scope defined by the appended claims.

[0014] Unless otherwise specified, all raw materials and reagents of the present application are conventional market raw materials and reagents.

[0015] The following experiments use Litopenaeus vannamei (5-7 g each) from Litopenaeus vannamei breeding farm in Donghai Island, Zhanjiang City, Guangdong Province, China, and the health status of Litopenaeus vannamei (5% of the total number) is analyzed at the beginning of the experiment, and according to the published standard operating procedures, 5% of the total number of Litopenaeus vannamei are randomly selected to check the health of the prawns.

[0016] Example 1 Synthesis of a double-stranded RNA preparation for prawn molting 1. Total RNA extraction Total RNA of the tissue was extracted using TransZol Up Plus RNA Kit extraction kit, and the specific steps were as follows: (1) Take 150 μL TransZol Up lysis solution in a 1.5 mL enzyme-free centrifuge tube, and take about 50 mg of tissue in an enzyme-free centrifuge tube and grind it with a grinder to homogenate; (emphasis: the grinding process should be carried out on ice); (2) Add 850 μL TransZol Up lysis solution to the enzyme-free centrifuge tube in step (1), shake and mix well, so that it is fully lysed; (3) Add 200 μL chloroform to the centrifuge tube in (2), quickly upside down, shake vigorously for about 30 s, and then stand at room temperature for 3 min; ​(4) centrifuge at 12,000 rpm 4°C for 15 min using low temperature centrifuge, and then transfer the supernatant to a new 1.5 mL enzyme-free centrifuge tube; (5) add equal volume (about 550 μL) of anhydrous ethanol, mix well, and then transfer the liquid in the enzyme-free centrifuge tube to the purification column, centrifuge at 12,000 rpm 4°C for 30 s, and discard the waste liquid; (6) add 500 μL of solution CB9 to the purification column, centrifuge at 12,000 rpm 4°C for 30 s, and discard the waste liquid; (7) repeat step 6; (8) add 500 μL of WB9 with anhydrous ethanol to the purification column, centrifuge at 12,000 rpm 4°C for 30 s, and discard the waste liquid; (9) repeat step 8; (10) centrifuge at 12,000 rpm 4°C for 2 min to completely remove the residual ethanol on the membrane, and then stand at room temperature for several minutes to completely dry the purification column; (11) place the purification column in a new 1.5 mL centrifuge tube, add 60 μL of RNase-free Water, and stand at room temperature for 2 min to dissolve the total RNA, and then centrifuge at 12,000 rpm 4°C for 2 min; (12) use 1.0% agarose gel electrophoresis to detect the quality of the obtained total RNA; and use NanoDrop 2000 microspectrophotometer to detect the concentration of the total RNA; (13) store the extracted total RNA at -80°C for standby use.

[0017] 2. Synthesis of cDNA first strand Synthesize the cDNA first strand according to the reverse transcription kit instructions. Add the following reaction solution: Table 1 cDNA first strand synthesis reaction solution

[0018] After mixing the reaction solution in Table 1, incubate it in a PCR gene amplifier, and the reaction conditions are: 25°C for 12 min; 42°C for 55 min; 85°C for 10 min. Store the cDNA template solution obtained by reverse transcription at low temperature, and store it at -20°C for standby use.

[0019] 3. Cloning of 14-3-3 zeta gene ORF sequence (1) PCR amplification According to the target gene sequence obtained by screening from the Litopenaeus vannamei transcriptome database, the specific primers were designed by Primer 5.0 software, and the sequences of the PCR amplified Primer-ORF-F and Primer-ORF-R are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively. The cDNA first strand mixture of all tissues of the prawn was used as a template, and the PCR reaction was carried out in a PCR gene amplifier using PrimeSTAR Max DNA Polymerase reagent, and the target gene ORF sequence was amplified, and the reaction system is shown in Table 2:

[0020] Table 2 PCR amplification reaction system

[0021] Reaction conditions: 95℃ 5 min; 95℃ 30 s, 58℃ 30 s, 72℃ 60 s, 34 cycles; 72℃ 10 min; 4℃ storage.

[0022] 3 μL of the PCR product was taken for 1.0% agarose gel electrophoresis detection to determine the target band. The reaction system was enlarged according to the above conditions, the gel was recovered and purified by using FastPure Gel DNA Extraction Mini Kit purification kit, and then the connection, transformation and sequencing were carried out, and the specific steps were as follows: ① Add an equal volume of Buffer GDP, dissolve the gel at 52℃ for 15 min until completely dissolved, and mix well for 2 times during the period to accelerate the dissolution of the gel; ② Put the adsorption column on the collection tube, transfer the mixed solution of step ① to the adsorption column, and centrifuge at 10,000 ×g for 45 s; ③ Discard the filtrate, add 300 μL Buffer GDP to the adsorption column, stand for 1 min, and centrifuge at 12,000 ×g for 45 s; ④ Discard the filtrate, add 700 μL Buffer GW containing anhydrous ethanol to the adsorption column, and centrifuge at 12,000 ×g for 45 s; ⑤ Repeat step ④; ⑥ Discard the filtrate, put the adsorption column back into the collection tube, and centrifuge at 12,000 ×g for 90 s; ⑦ Put the adsorption column into a clean 1.5 mL sterile centrifuge tube, add 25 μL Elution Buffer to the center of the column, stand for 2 min, and then centrifuge at 12,000 ×g for 1 min; ⑧ Discard the adsorption column, and use SimpliNano microspectrophotometer to qualitatively and quantitatively determine the purified product, and store it at -20℃ for standby.

[0023] (2) Connection The recycled adhesive products were linked according to the pMD19-T Vector instruction manual. The linking system is as follows: Table 3 Connection System

[0024] After gently mixing the reagents and gel recovery products from Table 3 and briefly centrifuging, the mixture was incubated overnight at 4°C to complete the ligation.

[0025] (3) Transformation ① Transform the mixture of the above-obtained ligation products into E. coli competent cells. Refer to the DH5αcompetent cells instruction manual for specific operating steps as follows: ② Remove DH5α competent cells and thaw them on ice for 3 min; ③ Add all ligation products to DH5α competent cells, mix gently with a pipette tip, and place on ice for 25 min; ④ Heat shock in a 42℃ water bath for 85 seconds, then cool on ice for 4 minutes; ⑤ Add 900 μL of LB liquid medium and incubate at 37℃ in a constant temperature shaker at 180 rpm for 1 h; ⑥ Take 200 μL of the bacterial culture from step ⑤ and spread it evenly on an LA plate (with AMP+ added). ⑦ Place the plate face up in a 37℃ constant temperature incubator and incubate for 15 min, then invert it and incubate overnight.

[0026] (4) Screening and sequencing of positive clones The identification and sequencing of positive clones on LA plates are performed as follows: ① Use a sterile inoculation loop to pick a single colony from an LA plate and inoculate it into a centrifuge tube containing 800 μL of LA liquid medium (with added AMP+); ② Incubate at 37℃ in a constant temperature shaker at 200 rpm for 3-4 h; ③ Using bacterial culture as a template, bacterial PCR was performed using universal primers M13(-47) and M13(-48). The reaction system is shown in Table 4. Table 4 Reaction System

[0027] Reaction conditions: 95℃ for 5 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 60 s, for a total of 34 cycles; 72℃ for 10 min; store at 4℃.

[0028] The bacterial culture PCR products were detected on a 1.0% agarose gel. After identifying the target band, the bacterial culture was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The deoxynucleotide sequence of 14-3-3zeta dsRNA is shown in SEQ ID NO.1.

[0029] (5) Plasmid extraction After the positive bacteria with the correct target sequence were sequenced, 50 μL was inoculated into 5 mL of LA liquid medium for expansion culture. Plasmid extraction was then performed using the FastPure® Plasmid mini kit plasmid extraction kit. The specific steps are as follows: ① Take 5 mL of overnight culture (17 h), add it to a centrifuge tube, centrifuge at 13,000 rpm for 4 min, discard the culture medium, and air dry the residual liquid; ② Add 250 μL of Buffer P1 containing RNase A to the centrifuge tube containing bacterial precipitate, and vortex to mix. ③ Add 250 μL of Buffer P2 to the centrifuge tube in step (2), gently invert and mix 8 times to fully lyse the bacteria; ④ Add 350 μL of Buffer P3 to the centrifuge tube in step (3), immediately and gently invert it 8 times to completely neutralize Buffer P2, and centrifuge at 13,000 rpm for 12 min; ⑤ Place the adsorption column in the collection tube. Carefully transfer the supernatant from step (4) into the adsorption column using a pipette, and centrifuge at 13,000 rpm for 45 s. Discard the filtrate and return the adsorption column to the collection tube; ⑥ Add 600 μL of Buffer PW2 diluted with anhydrous ethanol to the adsorption column and centrifuge at 13,000 rpm for 45 s. Discard the filtrate and return the adsorption column to the collection tube; ⑦ Repeat step ⑥; ⑧ Return the adsorption column to the collection tube. Perform empty centrifugation at 13,000 rpm for 2 min; ⑨ Place the adsorption column in a new sterile 1.5 mL centrifuge tube. Add 60 μL of Elution Buffer to the center of the membrane on the adsorption column, let stand at room temperature for 3 min, and then centrifuge at 13,000 rpm for 2 min to elute the plasmid. ⑩ Discard the adsorption column and use a SimpliNano micro spectrophotometer to perform qualitative and quantitative determination of the DNA plasmid product. Store the DNA plasmid product at -20 °C for later use.

[0030] 4. Synthesis of 14-3-3zeta double-stranded template Using the extracted DNA plasmids as templates, PCR amplification was performed to obtain DNA template 1 and DNA template 2. The amplification reaction systems are shown in Tables 5 and 6, respectively. Table 5 PCR amplification reaction system for DNA template 1

[0031] Table 6 PCR amplification reaction system for DNA template 2

[0032] The sequences of the PCR amplification primers 14-3-3zeta-T7F and 14-3-3zeta-R for the DNA template 1 are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively; The sequences of the PCR amplification primers 14-3-3zeta-F and 14-3-3zeta-T7R for the DNA template 2 are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively; The PCR amplification reaction program was as follows: 95℃ for 5 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 60 s, for a total of 34 cycles; 72℃ for 10 min.

[0033] Based on the above reaction system, expand the system by making 10 tubes of T7F×R and 10 tubes of T7R×F (20 μL / tube). Take one tube each of the T7F×R product and the T7R×F product, and take 5 μL for electrophoresis to check for a single bright band. The band should match the length of the original PCR product. Use a purification kit to purify the PCR product to obtain DNA template 1 and DNA template 2.

[0034] The concentrations of the purified DNA are shown in Table 7. Table 7 DNA Concentration

[0035] 5. Synthesis of 14-3-3 zeta double-stranded RNA 1. Double-stranded transcription reaction DNA template 1 and DNA template 2 were mixed and subjected to double-stranded transcription to obtain the transcription product. The transcription reaction system is shown in Table 8. Table 8 Transcription reaction system

[0036] DNA template 1 and DNA template 2 should be added in a 1:1 ratio, with a recommended amount of 0.5 μg each. After mixing the components in Table 8, gently pipette to mix them thoroughly, and briefly centrifuge the reagents to the bottom of the tube. The reaction conditions are 37°C for 4 hours.

[0037] 2. Purification The above double-stranded transcripts were purified using a dual-enzyme digestion system to remove impurities and obtain high-purity double-stranded RNA product 14-3-3zeta dsRNA.

[0038] The two-enzyme digestion system is as follows: Table 9 Two-enzyme digestion system

[0039] RNase T1 was pre-diluted to 10 U / μL using RNase T1 Dilution Buffer.

[0040] After mixing the components in Table 9, gently blow them to mix them thoroughly and then briefly centrifuge them; incubate at 37°C for 30 minutes.

[0041] Take 1 μL of the obtained product and dilute it 10-fold. Take 1 μL of the diluted 14-3-3 zeta dsRNA to test the double-strand concentration. Take another 3 μL and mix it with RNA loading buffer, then inject it into the gel well for electrophoresis. The electrophoresis result should be a single band of uniform length.

[0042] Test case In vivo injection experiment The experiment was divided into 4 groups: a control group (PBS group) and 3 experimental groups (14-3-3zeta dsRNA group, 20E group and lv14-3-3zeta dsRNA+20E), with 3 replicates in each group and 10 shrimp in each replicate; the injection method was intramuscular injection in the abdomen (between the second and third abdominal segments of the shrimp); the injection concentrations were 2 ug / g BW for lv14-3-3zeta dsRNA and 1 ug / g BW for 20E.

[0043] Notes: PBS group: saline group, serving as the control group; 20E is molting hormone, which promotes molting; BW: short for shrimp body weight.

[0044] Experimental results: After 96 hours, the cumulative molting rates of the PBS group, the 14-3-3zeta dsRNA group, the 20E group, and the lv14-3-3zeta dsRNA+20E group were 16.7%, 13.3%, 43.3%, and 20.0%, respectively.

[0045] Results analysis: The 14-3-3 zeta dsRNA injection group reduced the cumulative molting rate by 3.4% compared with the PBS group (under normal conditions); under high concentrations of ecdysone (molting was easier after injection of 20E), the 14-3-3 zeta dsRNA injection group showed a more significant effect, reducing the cumulative molting rate by 23.3%.

[0046] In summary, the present invention injects 14-3-3 zeta dsRNA into shrimp, which can significantly reduce the cumulative molting rate and has broad application prospects in long-distance shrimp transportation, especially in the transportation of broodstock.

Claims

1. A double-stranded RNA preparation for regulating molting in shrimp, characterized in that, The double-stranded RNA is 14-3-3 zeta dsRNA, and the deoxynucleotide sequence is shown as SEQ ID NO.

1.

2. The double stranded RNA agent of claim 1, wherein, The penaeid shrimp includes Litopenaeus vannamei.

3. A method for synthesizing a double-stranded RNA preparation for regulating the molting of shrimps according to claim 1 or 2, characterized in that, The method comprises the following steps: 1) extracting total RNA of the penaeid shrimp; 2) synthesizing the first strand of cDNA; 3) cloning of the ORF sequence of the gene PCR amplification is performed with the mixture of the first strand of cDNA of all tissues of the penaeid shrimp as a template, and a DNA plasmid is extracted; 4) synthesis of the double-stranded template of 14-3-3 zeta PCR amplification is performed with the DNA plasmid of step 3) as a template to obtain DNA template 1 and DNA template 2; 5) synthesis of 14-3-3 zeta double-stranded RNA DNA template 1 and DNA template 2 are mixed to perform a double-stranded transcription reaction, and then a double-enzyme digestion system is used for purification to remove impurities, thereby obtaining high-purity double-stranded RNA product 14-3-3 zeta dsRNA.

4. The method of synthesis of a double stranded RNA agent according to claim 3, wherein, The operation of step 3) comprises the following steps: PCR amplification is performed with the mixture of the first strand of cDNA of all tissues of the penaeid shrimp as a template to obtain the ORF sequence of the target gene, the target gene fragment is gel-recovered and purified, the gel-recovered product is connected and transformed to obtain recombinant bacteria, and then positive clone bacteria are screened and sequenced, and the positive bacteria with correct sequencing target sequence are cultured to extract a DNA plasmid; The PCR amplification is performed by using PrimeSTAR Max DNA Polymerase reagent for PCR reaction; The sequences of the primers Primer-ORF-F and Primer-ORF-R used in the PCR amplification are shown as SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

5. The method of claim 3, wherein the double stranded RNA agent is synthesized by, The PCR amplification reaction system of the DNA template 1 comprises the following components: Template 1 μL, 14-3-3 zeta-T7F 1 μL, 14-3-3 zeta-R 1 μL, PCR Mix 10 μL, H2O 7 μL, and a total of 20 μL. The sequences of the primers 14-3-3 zeta-T7F and 14-3-3 zeta-R used in the PCR amplification of the DNA template 1 are shown as SEQ ID NO. 4 and SEQ ID NO. 5, respectively.

6. The method of synthesis of a double stranded RNA agent according to claim 3, wherein, The PCR amplification reaction system of the DNA template 2 comprises the following components: Template 1 μL, 14-3-3 zeta-T7R 1 μL, 14-3-3 zeta-F 1 μL, PCR Mix 10 μL, H2O 7 μL, and a total of 20 μL. The sequences of the primers 14-3-3 zeta-F and 14-3-3 zeta-T7R used in the PCR amplification of the DNA template 2 are shown as SEQ ID NO. 6 and SEQ ID NO. 7, respectively.

7. The method of claim 3, wherein the double stranded RNA agent is synthesized by a method comprising: The reaction procedure of the PCR amplification of step 4) is as follows: 95℃ for 5 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 60 s, for a total of 34 cycles; and 72℃ for 10 min.

8. The method for synthesizing the double-stranded RNA preparation according to claim 3, characterized in that, The reaction system of the double-stranded transcription is: NTP Mix 8 μL, 10×Transcription Buffer 2 μL, T7 Enzyme Mix 2 μL, DNA template 10.93 μL, DNA template 2 2.5 μL and RNase-free H2O 4.57 μL; The reaction condition is 4h of reaction at 37℃.

9. The method of claim 3, wherein the double stranded RNA agent is synthesized by, The double-enzyme digestion system is: Transcription Product 20 μL, DNase I 1 μL, RNase T1(10U / μL) 2 μL, RNase-free H2O 17 μL, total 40 μL; The reaction condition is 30min of incubation at 37℃.

10. Use of the double-stranded RNA preparation according to claim 1 or 2 for the regulation of molting in shrimps, characterized in that, The application mode is to inject the 14-3-3zeta dsRNA into the prawn in vivo.

Images