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Freshwater shrimp legumain-like gene and application of dsRNA of freshwater shrimp legumain-like gene

A green shrimp and gene technology, applied in the direction of DNA / RNA fragments, applications, genetic engineering, etc.

Active Publication Date: 2022-04-26
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are relatively few studies on the role of legumain in the normal development of the body and in normal tissues

Method used

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  • Freshwater shrimp legumain-like gene and application of dsRNA of freshwater shrimp legumain-like gene
  • Freshwater shrimp legumain-like gene and application of dsRNA of freshwater shrimp legumain-like gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Obtaining the full-length nucleotide sequence of the freshwater shrimp Legumain-like gene.

[0028] The full-length sequence of the gene Legumain-like was obtained by comparative analysis of the transcriptome at each developmental stage of the female green shrimp nest.

Embodiment 2

[0029] Example 2: Acquisition of Legumain-like gene fragments and dsRNA of freshwater shrimp

[0030] Based on the nucleotide sequence of the freshwater shrimp Legumain-like gene shown in SEQ ID NO.1, the NCBI online dsRNA primer design software (https: / / www.flyrnai.org / cgi-bin / RNAi_find_primers.pl) design RNA interference specific primers (SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7), and add the T7 promoter sequence before the primers: TAATACGACTCACTATAGGG.

[0031] Use the upstream primer SEQ ID NO.4 that contains T7 promoter and downstream primer SEQ ID NO.5 and upstream primer SEQ ID NO.6 and downstream primer SEQ ID NO.7 to obtain PCR product (sequence such as SEQ ID NO .3, shown in SEQ ID NO.8), was purified and recovered by the PCR product purification kit, and the PCR product obtained by purification and recovery was used as a template for in vitro transcription of dsRNA1 and dsRNA2, according to the manufacturer's instructions, using TranscriptAidTM T7 Hig...

Embodiment 3

[0035] Example 3: Injection of dsRNA synthesized by Legumain-like gene fragments inhibits ovarian development of female green shrimp

[0036] (1) Injection of dsRNA

[0037] According to the appearance characteristics of the ovary development of freshwater shrimp, 0.60 ± 0.12g of 180 healthy freshwater shrimp with ovarian development in stage IV (oogonia proliferation stage, white and transparent) were selected to ensure the consistency of the initial ovarian development period, and dsGFP injection was set. Control group, blank control group, dsRNA1 experimental group and dsRNA2 experimental group, 60 freshwater shrimps in each group, set 3 parallel repetitions (n=20). Before the injection, they were temporarily raised in a glass tank for three days to adapt to the breeding environment in the laboratory (water temperature 25±1°C), and fresh snails were fed once a day in the morning and evening.

[0038]Before the injection, the freshwater prawns were kept in glass tanks for t...

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Abstract

The invention discloses a freshwater shrimp Legumain-like gene and application of dsRNA of the freshwater shrimp Legumain-like gene, and belongs to the technical field of biology. The invention provides a freshwater shrimp Legumain-like gene and an application of dsRNA of the freshwater shrimp Legumain-like gene, and comprises the freshwater shrimp Legumain-like gene, a gene segment, the dsRNA of the gene segment and the application of the dsRNA in inhibition of freshwater shrimp ovary development. Firstly, a complete sequence of Legumain-like is obtained through gene cloning, the nucleotide sequence of the complete sequence is SEQ ID NO.1, and the amino acid sequence of the complete sequence is SEQ ID NO.2. An RNA interference primer is designed by taking the complete sequence as a template, and an interference gene segment SEQ ID NO.3 is obtained; and the dsRNA is synthesized by the primer. The dsRNA and the dsGFP are injected into the pericardial cavity of the female freshwater shrimp for interference, and detection finds that the development of the ovary of the female freshwater shrimp is effectively inhibited.

Description

Technical field [0001] The invention relates to the application of green shrimp legumain-like gene and its dsRNA, and belongs to the field of biotechnology. Background technique [0002] Green shrimp, whose scientific name is Macrobrachium nipponense, belongs to the family Palaemonidae and the genus Macrobrachium. Its body color is blue-gray with brown markings, hence its name. It is also commonly known as river shrimp. Naturally distributed in China, Japan, South Korea, North Korea, Laos, Malaysia, Myanmar (mainland Myanmar), Vietnam and the Russian Far East, and gradually spread to Singapore, the Philippines, Uzbekistan, southern Iraq, Moldova, Kazakhstan, Iran and other places. Green shrimp is an important economic freshwater aquaculture species in my country, but it has always been troubled by the problem of "rapid maturation". "Rapid maturation" means that female shrimp reproduce for multiple generations during the breeding season. This phenomenon can cause problems s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N9/64C12N15/113C12N15/10C12N15/89A01K67/033A01K61/59
CPCC12N9/641C12N15/1137C12N15/10C12N15/89A01K67/0338A01K61/59A01K2207/05A01K2217/058A01K2227/70Y02A40/81
Inventor 乔慧傅洪拓程丹张文宜蒋速飞熊贻伟金舒博龚永生
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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