Freshwater shrimp molt-inhibiting hormone gene and application thereof in accelerating molting and growing of freshwater shrimps

A technology of molting inhibitory hormone and freshwater shrimp, applied in the field of biotechnology and developmental regulation

Inactive Publication Date: 2015-01-28
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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However, at present, the application in the study of im

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  • Freshwater shrimp molt-inhibiting hormone gene and application thereof in accelerating molting and growing of freshwater shrimps
  • Freshwater shrimp molt-inhibiting hormone gene and application thereof in accelerating molting and growing of freshwater shrimps

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Embodiment 1

[0021] Example 1: Obtaining the full-length cDNA of the MIH gene of freshwater shrimp

[0022] Total RNA extraction: Select two mature freshwater shrimps, remove the eye stalk tissue with sterile dissecting scissors, clip the eye stalk pigment part in RNA preservation solution (TaKaRa Bio Inc. Japan), rinse and pre-treat the freshwater shrimp eye stalk For total RNA extraction, refer to the Takara Trizol kit for specific steps.

[0023] First-strand cDNA synthesis: The first-strand cDNA was obtained after reverse transcription with reference to the Takara M-MLV reverse transcription kit with the above total RNA.

[0024] Obtaining the full-length cDNA of MIH gene in freshwater shrimp:

[0025] According to Macrobrachium rosenbergii ( Macrobrachium rosenbergii , KC990939.1) the conserved region of MIH cDNA, two pairs of intermediate primers were designed, see SEQ ID NO: 6~9,

[0026] (middle F1: 5′-CCGGCATTCCTTTGTTTACCTG-3, middle R1: 5′-ATATTCTGGCGTGTGGTCCTG-3′;

[00...

Embodiment 2

[0032] Embodiment 2: Obtaining of dsRNA of the freshwater shrimp MIH gene

[0033] According to the cloned freshwater shrimp MIH gene (Accession number: KF878973), the NCBI online sequence analysis software ( http: / / www.ncbi.nlm.nih.gov / gorf / gorf.html ) to determine the position of the open reading frame of the MIH gene as 436-795bp. According to the above results, the online dsRNA primer design software ( http: / / www.flyrnai.org / cgi-bin / RNAi_find_primers.pl ), design dsRNA primers in the MIH open reading frame of freshwater shrimp, and add the T7 promoter sequence 5'-TAATACGACTCACTATAGGG-3' in front of each primer to form dsRNA synthesis primers, and the primer sequences are the upstream primers SEQ ID NO:2 and downstream primer SEQ ID NO:3. All primers were synthesized at Bosun Biotechnology (Shanghai) Co., Ltd.

[0034] Using the above-mentioned green shrimp dsRNA primers to carry out PCR amplification on the total cDNA of green shrimp (the conditions are the same as i...

Embodiment 3

[0035] Example 3: Injection of the dsRNA of the green shrimp MIH gene on the development of the green shrimp molting

[0036] 1. Selection of Shrimp for Experiment

[0037] In order to study the silencing effect of the dsMIH gene, this study first conducted a one-time RNAi experiment to select 50 adult female shrimp with strong vitality, uniform individuals, and a body weight of about 1.50±0.35g, and divided them into two groups on average. One group was the dsRNA injection group, The other group is the DEPC water injection group (control group). Before the experiment, the snails were inflated in a 500L polyethylene plastic white bucket for 72 hours to adapt to the laboratory breeding environment, and the snails were fed once a day in the morning and evening.

[0038] The continuous RNAi experiment was designed according to the results of the one-time RNAi experiment. The shrimps used in the continuous RNAi experiment were the same batch of hatched juvenile shrimp hatched in ...

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Abstract

The invention discloses a freshwater shrimp molt-inhibiting hormone gene and application thereof in accelerating molting and growing of freshwater shrimps. The nucleic acid sequence of the freshwater shrimp molt-inhibiting hormone gene is shown by SEQ ID NO:1. A RNA interfering primer is designed by using an RNA interference technology according to an open reading frame of the sequence of the freshwater shrimp molt-inhibiting hormone gene, then PCR amplification is performed by taking freshwater shrimp total cDNA as a template to obtain a sequence shown by SEQ ID NO:4, in vitro transcription is performed according to SEQ ID NO:4 to synthesize dsRNA, and the dsRNA is injected into the hemocelom of a freshwater shrimp to accelerate the molting and growing of the freshwater shrimp. A RNAi method is used, causes small damages to the freshwater shrimps, cannot influence normal ingesting, inhabiting and mating of the freshwater shrimps, is convenient to perform long-term researches and has outstanding effects. The invention provides a new train of thought and an effective means for adjusting and controlling growth and development of the freshwater shrimps and breeding an excellent variety of the freshwater shrimps.

Description

technical field [0001] The invention relates to the fields of biotechnology and development regulation, in particular to a green shrimp molting inhibitory hormone gene and its application in accelerating the molting and growth of green shrimp. Background technique [0002] Freshwater shrimp, scientific name Japanese river prawn ( Macrobrachium nipponense ), commonly known as river shrimp, belongs to the order Decapoda, the family Palaemonidae, and the genus Macrobrachium. It is widely distributed in waters all over my country. freshwater cultured shrimp. According to the "China Fishery Yearbook" in 2012, the annual output of farmed freshwater shrimp in the country exceeds 237,000 tons, and the annual output value exceeds 15 billion yuan. Freshwater shrimp farming has played a pivotal role in my country's aquaculture. In recent years, with the continuous expansion of large-scale farming and the continuous improvement of consumer demand, the key to the current genetics and br...

Claims

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Application Information

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IPC IPC(8): C12N15/16C12N15/113A01K67/033
Inventor 江丰伟傅洪拓乔慧张文宜金舒博熊贻伟蒋速飞龚永生
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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