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GEM gene of Macrobrachium nipponense and encoding protein and application thereof

A 5GEM-R2, gene technology, applied in the green shrimp GEM gene and its encoded protein and application fields, achieving significant economic value and application prospects

Active Publication Date: 2019-01-15
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

At present, there is no report on the in situ hybridization of the Rev3 gene

Method used

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  • GEM gene of Macrobrachium nipponense and encoding protein and application thereof
  • GEM gene of Macrobrachium nipponense and encoding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Obtaining the full-length cDNA of the freshwater shrimp GEM gene

[0028] Total RNA extraction: Select 10 healthy and mature male freshwater shrimp androgenic glands, quickly put them into a pre-cooled mortar filled with liquid nitrogen, and grind them quickly. Total RNA was extracted using RNAiso Plus extraction reagent from Takara Company combined with the traditional phenolic extraction method, 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 and 2.0, and the concentration of the RNA was determined.

[0029]The middle fragment sequence of GEM gene cDNA was obtained from the transcriptome of the androgenic gland of freshwater shrimp. According to the sequence of the middle fragment of GEM gene cDNA, design specific forward primers 3GEM-F1 (SEQ ID NO:3), 3GEM-F2 (SEQ ID NO:4); and re...

Embodiment 2

[0030] Embodiment 2: Green shrimp GEM gene tissue expression

[0031] Based on the nucleotide sequence of SEQ ID NO:1, within the open reading frame, NCBI online primer design software ( http: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) to design and prepare primers (SEQ ID NO:7-SEQ ID NO:8) for fluorescent quantitative PCR of the green shrimp GEM gene. Tissues such as testis, androgenic gland, ovary, hepatopancreas, intestine, and heart (N≥3) were obtained from healthy and mature freshwater shrimp, and the tissues were preserved in RNA preservation solution (Takara). The total RNA of each tissue was extracted as described in Example 1, and the total RNA was reversed into cDNA using the iScriptTM cDNA Synthesis Kit perfect Real Time (Bio-Rad) kit, and the Bio-Rad iCycler iQ5 fluorescent quantitative PCR analysis system was used to detect the presence of the GEM gene in Relative expression levels in each tissue of freshwater shrimp. Such as figure 1 As shown, the expression...

Embodiment 3

[0032] Embodiment 3: In situ hybridization research of freshwater shrimp GEM gene

[0033] Based on the nucleotide sequence of SEQ ID NO:1, the in situ hybridization sense probe sequence and antisense probe sequence of the green shrimp GEM gene were designed and prepared within the open reading frame using Primer5 primer design software, and commissioned by Shanghai Sangong Bioengineering Co., Ltd. (Sangon) synthesized sense in situ hybridization probes and antisense in situ hybridization probes (SEQ ID NO: 9-SEQ ID NO: 10) labeled with a Digoxigenin signal. The testis, androgenic gland and hepatopancreas were obtained from healthy and mature male freshwater shrimp, and preserved in 4% paraformaldehyde solution for fixation. Frozen sections of each tissue were prepared. The Zytofast PLUS CISH Implementation kit (Zyto Vision GmBH, Germany) was used to perform histological localization of each tissue, and the localization results were observed under a microscope. Such as fig...

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Abstract

A GEM gene of Macrobrachium nipponense and an encoding protein and application thereof are disclosed. That full-length cDNA sequence of the GEM gene is shown in SEQ ID NO: 1. A protein encoded by theGEM gene of Macrobrachium nipponense has an amino acid sequence shown as SEQ ID NO: 2. As compared with that prior art, the gene has the following advantage: (1) the important role of the GEM gene ofthe green shrimp in the male differentiation and development process of the green shrimp provides a theoretical basis and a train of thought for the research on the male differentiation and the development mechanism of the green shrimp; 2) that Macrobrachium nipponense GEM gene of the invention can regulate the immune response mechanism of the Macrobrachium nipponense; 3) that Macrobrachium nipponense GEM gene of the invention can be use in the selection and breeding of an all-male population of Macrobrachium nipponense, and has remarkable economic value and application prospect in the procesof culturing Macrobrachium nipponense.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the development regulation of green shrimp, in particular to the green shrimp GEM gene and its coded protein 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. There is a significant difference in the growth of male and female during the culture of freshwater...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435
CPCC07K14/43509
Inventor 金舒博傅洪拓乔慧张文宜孙盛明蒋速飞熊贻伟龚永生吴滟
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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