Penaeus monodon female parent gonad maturation promoting small interfering RNA composition, kit and using method
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A technology of penaeus monodon and small interference, applied in biochemical equipment and methods, DNA/RNA fragments, drug combinations, etc., can solve problems such as applications that have not yet been reported, achieve lasting effect of promoting ripening, save economic costs, and be simple to operate. Effect
Active Publication Date: 2020-09-04
SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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In view of this, chitosan is often used as a sustained-release drug carrier in recent years, and the application of chitosan-nucleic acid polycomplexes in production has broad prospects, but the application in the gonad maturation of female broodstock of Penaeus monodon is currently Not yet reported
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Embodiment 1
[0030] Embodiment 1 preparation of the kit for promoting maturation of the female broodstock of Penaeus monodon
[0031] The kit for promoting maturation of the female broodstock of Penaeus monodon includes Reagent A, Reagent B and Reagent C.
[0032] 1.1 Preparation of Reagent A
[0033] Search the Gene Bank of the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) for P. monodon CHH (GenBank accession no.AF233295.1), MIH (GenBank accession no.GQ221086) and VIH (GenBank accession no.DQ643389) the nucleotide sequences of three hormone genes that inhibit gonad development, using BLOCK-iT TMThe RNAi Designer online program (https: / / rnaidesigner.thermofisher.com / rnaiexpress / sort.do) designed the siRNAs of the above three genes, including CHH-siRNA, and its positive-sense strand nucleotide sequence is shown in SEQ ID NO.1 The nucleotide sequence of the antisense strand is shown in SEQ ID NO.2; the nucleotide sequence of the sense strand of MIH-siRNA is shown in SEQ ID NO.3, and the nu...
Embodiment 2
[0062] Embodiment 2 Penaeus monodon female broodstock gonad maturation test
[0063] 2.1 Pretreatment of female broodstock to be promoted
[0064] Select healthy and vigorous female broodstock of Penaeus monodon as the object of maturation promotion. The size of the parent is generally >20cm in body length, >180g in weight, with normal body color, no damage to the shrimp body, clear markings, complete appendages, and no specific pathogens in the test. The broodstock prawns to be cooked are placed in the cultivation pond, and kept in the dark at about 29°C for 3 days, during which squid and clamworms are fed.
[0065] 2.2 Configuration of the working solution for the maturation-stimulating reagent of the female broodstock of Penaeus monodon
[0066] 2.2.1 Take reagents A, A1, A2 and A3 (each 70 μg) stored at -20°C and centrifuge at 4000 rpm for 30 s at room temperature, add 1 ml of reagent B, mix by inverting to obtain mixed solutions AB, A1B, A2B and A3B.
[0067] 2.2.2 Add ...
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Abstract
The invention discloses a penaeus monodon female parent gonad maturation promoting small interfering RNA composition. The composition comprises small interfering RNA aiming at three hormone genes of penaeus monodon CHH, MIH and VIH for inhibiting gonad development, wherein the three small interfering RNAs are specifically CHH-siRNA, MIH-siRNA and VIH-siRNA, wherein the nucleotide sequence of a positive-sense strand of the CHH-siRNA is shown as SEQ ID NO. 1, and the nucleotide sequence of an antisense strand of the CHH-siRNA is shown as SEQ ID NO. 2; a positive-sense strand nucleotide sequenceof the MIH-siRNA is shown as SEQ ID NO. 3, and an antisense strand nucleotide sequence of the MIH-siRNA is shown as SEQ ID NO. 4; and the nucleotide sequence of the positive-sense strand of the VIH-siRNA is shown as SEQ ID NO. 5, and the nucleotide sequence of the antisense strand of the VIH-siRNA is shown as SEQ ID NO. 6. The invention also discloses a penaeus monodon female parent gonad maturation promoting test kit containing the small interfering RNA composition and a use method thereof. The small interfering RNA composition can remarkably reduce the content of hormone for inhibiting gonaddevelopment maturity of female parent shrimps, promote the generation of egg yolk protein and accelerate the gonad development process.
Description
technical field [0001] The invention relates to a small interfering RNA composition and applications thereof, in particular to a small interfering RNA composition for promoting maturation of the female broodstock of Penaeus monodon, and also to a kit for promoting maturation of the female broodstock of Penaeus monodon and the use of the kit method. Background technique [0002] Crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), mandibular organ-inhibiting hormone (MOIH), yolk Vitellogenesis-inhibiting hormone (VIH) and other hormones that inhibit the development of gonads, it is difficult for female broodstock gonads to mature in large quantities under natural conditions, which affects the production of shrimp seedlings, so when commercially cultivating shrimp seedlings The gonads of female broodstock are usually ripened using the eye-stalk cutting technique. This technology can effectively promote the gonad development of female broodstock to a certain...
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