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A kind of shrimp masculinization gene and its application

A masculinization and shrimp technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of low efficiency, high operation skills, and lengthy methods of single male shrimp and crab groups

Active Publication Date: 2019-05-14
ZHEJIANG WANLI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current methods for producing single-male populations of shrimp and crabs using transplanted androgenic glands from shrimp and crabs are inefficient and involve lengthy protocols such as manual separation and microsurgical removal of androgenic glands from immature males, both of which Programs require high operational skills

Method used

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  • A kind of shrimp masculinization gene and its application
  • A kind of shrimp masculinization gene and its application
  • A kind of shrimp masculinization gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Identification of masculinizing genes and polypeptides

[0029] 1) By obtaining the complete sequence of the masculinization gene of Macrobrachium rosenbergii from the shrimp gene bank, the full length of the full-masculinization gene was obtained using the ExPASx proteomics server (http: / / ca.expasy.org / tools / dna.html) computer translation, and choose the most likely frame. Deduced amino acid and polypeptide sequences were further evaluated by SMART and the CBS prediction server (http: / / www.cbs.dtu.dk / services / ).

[0030] The cDNA sequence of the masculinization gene of Macrobrachium rosenbergii is 3264bp in full length (SEQ ID NO: 1), including 3' untranslated region 318bp, open reading frame 1314bp, 5' untranslated region 1632bp, deduced to generate a polypeptide sequence containing 438 amino acids ( SEQ ID NO: 2). There are two CCCH-containing zinc finger structures in the deduced open reading frame: GDDDNICRDFLRNVCRRGKRCKYRHPED and KTELTFCHDFQNGNCSRPMCRF...

Embodiment 2

[0035] Example 2 Silencing of masculinizing gene expression

[0036] Double-stranded RNA preparation

[0037] According to the cDNA sequence of the masculinization gene of Macrobrachium rosenbergii, the forward primer 5'-CGC with restriction site was designed GGATCC TCACAGCCCTCCCAATTACA-3' (nucleotide (nt) 1249-1269; SEQ ID NO:7, the underline is the BamHI restriction site) and reverse primer 5'-CCC AAGCTT ATAGGACACAAGATGACCACCAGA-3' (nucleotides (nt) 1567-1590; SEQ ID NO: 8, the underline is the HindIII restriction site), a dsRNA expression vector containing the masculinization gene of Macrobrachium rosenbergii was constructed. Using the cDNA containing the masculinization gene of M. 72°C for 10 minutes), clone the masculinization gene fragment, and obtain the sequence of the target gene sequence as SEQ ID NO: 3;

[0038] PCR amplification products were electrophoresed on a 1.2% agarose gel, visualized in ethidium bromide using UV light, excised from the gel and purified u...

Embodiment 3

[0046] Example 3 Sex regulation based on masculinization gene silencing

[0047] Breeding of False Female Parents

[0048] For the RNAi experiment of long-term silencing of masculinizing genes, male shrimp seedlings with a healthy body and complete appendages (Post Larvae, PL, PL20-PL120) (N=200) and male shrimp seedlings with a body length of 0.8-1.2cm were selected for the experiment. The RNA interference of the masculinizing gene, the microinjection of the RNA interference and the operating procedures are as described above. In order to maintain efficient and durable RNA interference efficiency, the same double-stranded RNA sample was re-injected into shrimp every other month, and the injection cycle was 3 months. Shrimp bodies after RNA interference were placed in indoor cement ponds and continued to be reared until the body length was 3-5 cm, and the male and female sexes were captured and identified. Firstly, identify the male and female sexes from the aspect of appear...

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Abstract

The invention provides a masculinization gene of shrimps, and application of the masculinization gene of the shrimps in all-male seed production of the shrimps, and in particular relates to a culture technology of unisexual all-male seeds of macrobrachium rosenbergii. The encoded amino acid sequence of the masculinization gene of the shrimps is as shown in SEQ ID No: 2; a nucleotide sequence of the masculinization gene of the shrimps is as shown in SEQ ID No: 1. According to nucleic acid and protein sequence information of the masculinization gene, biotechnologies such as an RNA interference technology and a gene expression regulation technology are comprehensively applied, so that the effective silencing of the masculinization gene of the shrimps is realized, and directional sex control is carried out on male individuals; after the masculinization gene of the shrimps is adopted, sex reversion is induced, sex-controllable female parents can be obtained by screening, the unisexual all-male seeds capable of rapidly growing are cultured, and all-masculinization monosex culture practical application is carried out.

Description

technical field [0001] The invention belongs to the technical field of sex gene screening of aquaculture animals, and in particular relates to a shrimp masculinization gene and its application. Background technique [0002] my country is a big shrimp and crab farming country in the world. Shrimp and crab farming in coastal areas has formed a huge industry, which has become an important pillar of the local economy and promoted the improvement of farmers' living standards. During the growth of shrimp and crabs, the size and growth speed of females and males of the same age are very different, showing a bimodal growth pattern. Therefore, an all-male / all-female population of shrimp and crabs produces higher marketable yields, grows faster, and requires less time to grow for higher benefits per unit area. If the advantage of this male / female characteristic is fully utilized to carry out all-female / all-male single-sex farming, the specifications of marketed commodities and aquacu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/113C12N15/63C07K14/435A01K67/033
CPCA01K67/0338A01K2227/70C07K14/43509C12N15/113C12N2310/14
Inventor 马文明李东瑞孟慧佳张梦钱国英杨劲树杨卫军
Owner ZHEJIANG WANLI UNIV
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