siRNA-IR sequence for sex reversal of male macrobrachium rosenbergii and application of siRNA-IR sequence

A technology of Macrobrachium rosenbergii and male, which is applied in the field of siRNA-IR sequence for male Macrobrachium rosenbergii sex transition, can solve the problems of expensive synthetic kits, high injection dosage, and high cost, and achieve the reduction of larval immune stress response and injection dosage. Small, the effect of improving the survival rate

Active Publication Date: 2020-01-21
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method can interfere with and inhibit gene expression, the injection dose is relatively high, the required sequence is relatively long, and the synthesis kit is relatively expensive and the cost is high.
For the application to production practice, this method still has some cost factors

Method used

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  • siRNA-IR sequence for sex reversal of male macrobrachium rosenbergii and application of siRNA-IR sequence
  • siRNA-IR sequence for sex reversal of male macrobrachium rosenbergii and application of siRNA-IR sequence
  • siRNA-IR sequence for sex reversal of male macrobrachium rosenbergii and application of siRNA-IR sequence

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 can interfere and suppress the siRNA screening of IR gene

[0022] According to the sequence of Macrobrachium rosenbergii IR gene (accession number: FJ409645), an interference sequence called siRNA-IR was designed and synthesized; the device was designed using online siRNA design software (https: / / www.invivogen.com / sirnawizard / design.php) Select the siRNA-IR sequence (see Table 1). Exclude fragments containing SNP sites and non-open reading frames from the alternative siRNA-IR, and remove sequences with too high and too low GC content, and finally perform Blast analysis and discard non-specific binding sequences. After obtaining the ideal siRNA-IR sequence, four oligos were designed according to the requirements of the TaKaRa siRNA in vitro transcription synthesis kit, named oligo 1, oligo 2, oligo 3 and oligo 4 (see Table 1). These four oligo sequences were entrusted to Wuhan Qingke Biotechnology Co., Ltd. to synthesize them.

[0023] Table 1 Sequences o...

Embodiment 2

[0026] Example 2 Preparation of siRNA-IR double-stranded Oligo DNA

[0027] Dissolve the synthesized single-stranded Oligo DNA with sterilized distilled water to prepare a 100 pmol / μl DNA solution, and prepare the Oligo DNA annealing reaction solution according to the components in Table 2 and Table 3. The prepared Oligo DNA annealing reaction solution was placed in a PCR amplification instrument, treated at 95°C for 2 minutes, cooled to 25°C over 45 minutes, and then kept at 25°C for 10 minutes. This step enables a stack of single-stranded oligo DNA to be annealed to form double-stranded oligo DNA. Eventually two sets of Oligo DNA will be obtained and named OligoA and Oligo B.

[0028] Table 2 Annealing reaction solution of Oligo A

[0029] Reagent dose 10X Annealing Buffer 2μl 100 pmol / μl Oligo 1 2μl 100 pmol / μl Oligo 2 2μl RNase free dH 2 o

14μl

[0030] Table 3 Annealing reaction solution of Oligo B

[0031] Rea...

Embodiment 3

[0032] Example 3 siRNA-IR in vitro transcription

[0033] The Oligo A and Oligo B formed by the annealing reaction were used to prepare the RNA in vitro transcription reaction solution according to the components in Table 4, mixed evenly and then centrifuged slightly so that the transcription reaction solution was collected at the bottom of the EP tube. The prepared transcription reaction solution was placed in a PCR amplification instrument and reacted at 42°C for 4 hours. After 4 hours, the transcription reaction solution was taken out, and 2 μl of RNasefree DNase I (5 U / μl) and 1 μl of RNase T1 (5 U / μl) were added to the original reaction tube. After mixing slightly, put it into a PCR amplification instrument and react at 37°C for 2 hours. After in vitro transcription, siRNA-IR will be purified. Add 23 μl of water-saturated acidic phenol / chloroform / isopropanol (25:24:1) to the in vitro transcription reaction solution, mix it upside down and centrifuge at 10,000 rpm at 25°...

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Abstract

The invention discloses a siRNA-IR sequence for sex reversal of male macrobrachium rosenbergii and application of the siRNA-IR sequence. Expression of an IR gene is inhibited by a siRNA interference technology to promote sex reversal of the male macrobrachium rosenbergii to obtain neo-females. IR gene fragments of macrobrachium rosenbergii are screened and obtained from macrobrachium rosenbergii transcriptome, and the siRNA is designed and synthesized according to a macrobrachium rosenbergii IR gene sequence, named siRNA-IR. The siRNA-IR is transformed into macrobrachium rosenbergii by a microscopic injection method before a sex differentiation stage to interfere and inhibit expression of the IR gene in the macrobrachium rosenbergii and promote sex interversal of the macrobrachium rosenbergii. Compared with a dsRNA interference technology, the siRNA technology has smaller injection dosage, and can reduce the cost, effectively reduce immunological stress reactions of post-larvae of themacrobrachium rosenbergii and increase the survival rate of neo-females.

Description

technical field [0001] The invention relates to the field of sex transformation of Macrobrachium rosenbergii, in particular to an siRNA-IR sequence for sex transformation of Macrobrachium rosenbergii and an application thereof. Background technique [0002] Macrobrachium rosenbergii (Macrobrachium rosenbergii) belongs to the phylum Arthropoda, Crustacea, Order Decapod, Gibrachidae, Macrobrachium genus, and is a large freshwater prawn. Macrobrachium rosenbergii belongs to tropical and subtropical shrimps. It is distributed in freshwater and brackish waters of the Indian Ocean and Pacific Ocean. It is mainly distributed in Asia, especially in Southeast Asian countries, such as India, Thailand, Myanmar, Indonesia, and Malaysia. Macrobrachium rosenbergii is one of the most important freshwater prawn species in the world. [0003] The androgenic gland (AG) is an endocrine organ unique to male crustaceans, which was first discovered in the blue crab (Callinectessapidus) [1] . F...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A01K67/033
CPCA01K67/0338A01K2207/05A01K2227/70C12N15/1138C12N2310/14
Inventor 王卫民陈健安
Owner HUAZHONG AGRI UNIV
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