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Epinephelus coioides sex control gene Rspo1 and preparation method and application thereof

A technology for regulating genes of grouper and its application in sex-regulating genes of grouper, sex-regulating gene Rspo1 of grouper and its application fields, and can solve the problem of not finding fish sex-determining genes

Active Publication Date: 2015-05-27
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Compared with higher vertebrates, most lower fishes do not have sex chromosomes, and their sex determination is mainly controlled by a variety of sex-regulating genes. So far, no fish sex-determining genes have been found

Method used

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  • Epinephelus coioides sex control gene Rspo1 and preparation method and application thereof
  • Epinephelus coioides sex control gene Rspo1 and preparation method and application thereof
  • Epinephelus coioides sex control gene Rspo1 and preparation method and application thereof

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Experimental program
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Embodiment 1

[0028] 1. Preparation of Rspo1 recombinant protein

[0029] 1. Extraction of RNA from the grouper

[0030] Healthy whole grouper fish were taken, anesthetized in ice bath for about 2 min, fish were killed and sampled, ovarian tissue was separated, and total RNA of ovarian tissue of grouper was extracted by Trizol reagent method.

[0031] 2, Rspo1 gene cloning

[0032] Synthesis of first-strand cDNA:

[0033] 1) Take out 1 μL of preserved grouper RNA, add 1 μL of Oligo (dT) 16 (10 mM), mix well and place in a water bath at 70 °C for 5 min;

[0034] 2) After standing on ice for 5 minutes, add 2 μL of dNTP Mixture (10 mM), 4 μL of 5XRT Buffer, 1 μL of RNase inhibitor (10 U / μL), and RNase-free ddH to the EP tube on ice. 2 O 8 μL, ReverTra Ace 1 μL;

[0035] 3) Place the EP tube in a PCR machine, centrifuge briefly according to the program of 30°C for 10 minutes, 42°C for 60 minutes, 99°C for 5 minutes, and 4°C for 5 minutes, and reverse transcribe to obtain the first-s...

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Abstract

The invention discloses an epinephelus coioides sex control gene Rspo1 and an application thereof. The cDNA (complementary deoxyribonucleic acid) sequence of the gene Rspo1 is as shown in SEQ (sequence) ID (identity) NO: 1, the total length is 756bp, an encoding protein of the gene Rspo1 has 251 amino acids in total, and the sequence is as shown in SEQ ID NO: 2. According to the method disclosed by the invention, a reverse transcription PCR (polymerase chain reaction) technology is utilized to clone the epinephelus coioides sex control gene Rspo1 from epinephelus coioides, and experimental results show that the Rspo1 possibly participates in control of expression of other sex control genes of the epinephelus coioides, so that a certain theoretical basis and an important clue are provided for research of sex control of relevant fishes. The encoding protein of the gene Rspo1 is expected to be applied to induced spawning, forced ripening, artificial control sex transformation, large-scale industrial farming of fishes, as well as other aspects.

Description

technical field [0001] The invention belongs to the technical field of aquatic animal genetic engineering, and relates to a grouper sex regulation gene and its application, in particular to the grouper sex regulation gene Rspo1 and its application. Background technique [0002] The Rspo1-4 superfamily, as ligands of canonical Wnt signaling, has 40%-60% protein identity and structural homology among them. The Rspo protein contains an N-terminal signal peptide, two adjacent cysteine-rich regions, a highly homologous TSP-1 module, and a carbon-terminal tail containing a bidirectional nuclear localization signal. They play important roles in various developmental and carcinogenesis pathways. The structure of Rspo1 has a 5'-terminal signal peptide, which has the function of transferring secretory proteins; two furin-like fragments containing cysteine, which are the important parts of its receptor binding; and a TSP-1 (thrombin sensitive protein-1). No C-terminal nuclear locali...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/46C12N15/12A61K38/17A61P15/00
CPCC07K14/461
Inventor 刘晓春王升鹏林浩然张勇
Owner SUN YAT SEN UNIV
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