Macrobrachium nipponense FoxL2 (Forkhead box protein L2) protein as well as coding gene and application thereof

A technology of foxl2 and freshwater shrimp, applied in the fields of biotechnology and reproductive development regulation, can solve problems that have not yet been studied

Inactive Publication Date: 2015-05-06
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Studies on the FoxL2 gene are mostly found in mammals an...

Method used

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  • Macrobrachium nipponense FoxL2 (Forkhead box protein L2) protein as well as coding gene and application thereof
  • Macrobrachium nipponense FoxL2 (Forkhead box protein L2) protein as well as coding gene and application thereof
  • Macrobrachium nipponense FoxL2 (Forkhead box protein L2) protein as well as coding gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The acquisition of the full-length cDNA of the freshwater shrimp FoxL2 gene is realized by the following steps:

[0022] Step 1: Total RNA extraction:

[0023] Select the testes of 5 mature male shrimps, quickly put them into a pre-cooled mortar filled with liquid nitrogen, and grind them rapidly. The RNAiso Plus extraction reagent from Takara Company combined with the traditional phenolic extraction method was used to extract the total RNA of the testis, and the extraction method was referred to the instruction manual. The quality of the RNA was detected by agarose gel electrophoresis, and the OD260 / 280 ratio of the sample was analyzed by a spectrophotometer to be between 1.8-2.0, and the concentration of the RNA was determined.

[0024] Step 2: Cloning of the full-length cDNA sequence of the freshwater shrimp FoxL2 gene:

[0025] According to the FoxL2 gene cDNA intermediate fragment sequence (SEQ ID NO.9) obtained from the androgenic gland transcriptome library of ...

Embodiment 2

[0029] Embodiment 2: the synthesis of dsRNA of FoxL2 gene of freshwater shrimp

[0030]Based on the nucleotide sequence (SEQ ID NO.2) of the freshwater shrimp FoxL2 gene, NCBI online dsRNA primer design software (http: / / www.flyrnai.org / cgi-bin / RNAi_find_primers.pl ) designed primers (SEQ ID NO.3 and SEQ ID NO.4) for preparing double-stranded RNA, and synthesized dsRNA by in vitro transcription according to the instructions of the Transcript AidTM T7 High Yield Transcription kit (Fermentas, Inc., USA). The prepared dsRNA was dissolved in DEPC water, its concentration was measured and its purity was detected on 1.5% agarose gel. The above dsRNA solution was injected into the pericardial cavity of freshwater shrimp at a dose of 4 ug / g. The control group was injected with the same volume of normal saline. The male shrimps used in the experiment were the same batch of hatched shrimps to ensure that the testes were at the same developmental stage, and the female shrimps were all in...

Embodiment 3

[0032] Embodiment 3: the application of FoxL2 gene of freshwater shrimp in production

[0033] According to the method provided in Example 2, the crayfish FoxL2 gene dsRNA was synthesized. Select 100 healthy male shrimps, adopt the method provided in Example 2, inject FoxL2 gene dsRNA into the selected male shrimps, and raise them with 30 healthy female shrimps in the same pond. The control group consisted of another 100 male shrimps, which were reared in the same pond with another 30 healthy female shrimps. The ovaries of the female shrimps are all mature. The control group had egg-bearing female shrimp after 6 days of feeding, but the experimental group did not find egg-bearing female shrimp until the 11th day. It only took 12 days for the female shrimps in the control group to conceive all the eggs, while it took 19 days for the female shrimps in the experimental group to conceive all the eggs.

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Abstract

The invention discloses a macrobrachium nipponense FoxL2 (Forkhead box protein L2) protein as well as a coding gene and an application thereof. An FoxL2 gene is cloned from testes or ovaries of the macrobrachium nipponense by using primer groups provided by the invention, dsRNA is prepared by using the FoxL2 gene, after the dsRNA is injected into the pericardial cavity of male or female macrobrachium nipponense, the expression of the FoxL2 gene in the testes or ovaries can be silenced, the development of the testes of the macrobrachium nipponense can be delayed, but the influence on the development of the ovaries of the macrobrachium nipponense is not obvious. Thus, the FoxL2 gene of the macrobrachium nipponense can be used for promoting the development of the testes and has the function against the action of the FoxL2 gene in mammals and fishes. According to the macrobrachium nipponense FoxL2 protein as well as the coding gene and the application thereof, new thinking and effective means are provided for overcoming the sexual precocity phenomenon of the macrobrachium nipponense and breeding good varieties.

Description

technical field [0001] The invention relates to the fields of biotechnology and reproductive development regulation, in particular to the FoxL2 protein of freshwater shrimp and its coding gene and application. Background technique [0002] Freshwater shrimp, scientific name Macrobrachium nipponense, commonly known as river shrimp, belongs to Decapoda, Palaemonidae, and Macrobrachium. It is widely distributed in waters all over my country. It grows fast and has good adaptability. It is an important freshwater cultured shrimp in my country because of its high economic efficiency and high economic efficiency. According to the "China Fishery Yearbook" in 2012, the annual output of cultured freshwater shrimp in the country exceeds 230,000 tons, and the annual output value exceeds 15 billion yuan. It is one of the most promising species in the freshwater aquaculture industry. In recent years, with the sharp reduction of its natural resources and the expansion of its breeding scale...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/11A61K31/713A61P15/08
CPCA61K31/713C07K14/43509
Inventor 金舒博傅洪拓熊贻伟蒋速飞龚永生乔慧张文宜
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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