Siniperca chuatsi male specific anti-mullerian hormone AMHY gene and application thereof

A technology of Mullerian hormone and mandarin fish, which can be applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of increasing the difficulty of interpreting the molecular mechanism of sex determination in fish, and achieve a short, high-efficiency, sex identification method. The effect of fast and accurate identification

Active Publication Date: 2020-08-28
广东梁氏水产种业有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although high-throughput sequencing methods have been used to find sex-specific molecular markers in a variety of fish, the sex-determining genes of most of them are still unknown, which increases the difficulty of interpreting the molecular mechanism of fish sex determination

Method used

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  • Siniperca chuatsi male specific anti-mullerian hormone AMHY gene and application thereof
  • Siniperca chuatsi male specific anti-mullerian hormone AMHY gene and application thereof
  • Siniperca chuatsi male specific anti-mullerian hormone AMHY gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0041] Firstly, the genomes of the male and female Siniperca sinensis were sequenced, and then the genome sequences of the male and female were analyzed for bioinformatics to enrich the male-specific molecular sequences ( figure 1 ). Then, annotation analysis was performed on the obtained male-specific sequence, and the genomic sequence of AMHY was obtained as shown in SEQ ID NO:1.

[0042] The 5'UTR and 3'UTR sequences of the AMHY gene were determined by comparative analysis with the genome sequences of autosomal AMH and other related fish AMH. The upstream and downstream primers AMHY-orf-F / R were designed according to the sequence of UTR, see SEQ ID NO:2 and SEQ ID NO:3.

[0043] Extract the total RNA of the testis of Siniperca sinensis, and then use Oligo dT primers to reverse-transcribe the synthetic testis cDNA template, and then use the template as the upstream and downstream primers with the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3, by PCR Amplified to obtain ...

Embodiment 2

[0072] The present embodiment provides a method for sex identification of mandarin fish with cocked mouth based on the AMHY gene of mandarin fish, comprising the following steps:

[0073] (1) Design the primer AMHY-F / R according to the homologous difference segment of the above-mentioned AMHY and AMH genome sequences, see SEQ ID NO: 6 and SEQ ID NO: 7 for details, the length of the amplification product corresponding to AMHY is 427bp, and the length of the corresponding AMH gene The product length is 803bp.

[0074] (2) DNA sample preparation of mandarin fish with cocked mouth:

[0075] Obtained visually mature mandarin fish from Sanshui Baijin Aquatic Seedling Co., Ltd., Foshan City, Guangdong Province, and obtained 48 male and female mandarin fishes by dissection. Tail fins were cut off separately, and DNA samples were prepared by using the conventional column centrifugation method (Universal Column Genome Extraction Kit, Beijing Kangwei Century Biotechnology Co., Ltd.) and...

Embodiment 3

[0084] This embodiment provides the application of using the above-mentioned cocked mandarin fish AMHY gene to carry out the all-female breeding of cocked mandarin fish, including the following steps:

[0085] (1) Construct the pcDNA4.0-AMHY recombinant protein expression plasmid, and embed the plasmid with a new type of liposome.

[0086] (2) When the mandarin carp fry are raised to 10 days old, mix the eel feed with the embedded plasmid in step (1) and then feed the bait fish. Feed the mandarin fish and feed them twice a day. Continuous feeding for more than two months, natural light is used during the breeding period, the cultivation water temperature is 26-28°C, and the feeding is nearly full.

[0087] (3) After the feeding, use the method in Example 2 to screen the mandarin fish fed with androgen, and obtain the genotype as XX male fish, that is, pseudo-male fish. Refer to Example 2 for the specific process.

[0088] (4) Continue raising the XX pseudo-male identified by...

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Abstract

The invention discloses a siniperca chuatsi male specific anti-mullerian hormone AMHY genome sequence and a construction method thereof, and a siniperca chuatsi male specific anti-mullerian hormone AMHY gene and an encoded protein thereof. The AMHY genome sequence is as shown in SEQ ID NO: 1, the sequence of the AMHY gene is as shown in SEQ ID NO: 4, and the sequence of the encoded protein is shown as SEQ ID NO: 5. The invention also discloses male molecular marker primers based on the siniperca chuatsi AMHY gene, a method for sex determination of siniperca chuatsi, and application of the AMHYgene, the encoded protein and the male molecular marker primers in all-female breeding of the siniperca chuatsi. The sequences of the male molecular marker primers are shown as SEQ ID NO: 6 and SEQ ID NO: 7, and the male molecular marker primers can realize rapid and accurate sex identification of the siniperca chuatsi. The sex identification method is short and high in efficiency.

Description

technical field [0001] The invention belongs to the technical field of Siniperca sinensis, in particular to the male-specific anti-Müllerian hormone AMHY gene of Siniperca sinensis and its application. Background technique [0002] Sex-specific molecular markers are an important prerequisite for the realization of fish parthenogenesis and the elucidation of its sex-determining molecular mechanism. In recent years, with the rapid development of high-throughput sequencing technology, more and more sex-specific molecular markers of fish have been identified, such as large yellow croaker (Larimichthyscrocea), black snakehead (Channaargus), silver carp (Hypophthalmichthysnobilis ) and bighead carp ( Hypophthalmichthys molitrix) and so on. However, although high-throughput sequencing methods have been used to find sex-specific molecular markers in a variety of fish, the sex-determining genes of most of them are still unknown, which increases the difficulty of interpreting the mol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/46C12N15/10C12Q1/6879C12N15/11
CPCC07K14/461C12N15/1096C12Q1/6879C12Q2531/113Y02A40/81
Inventor 张勇韩崇朱巧莹李水生韩林强李桂峰
Owner 广东梁氏水产种业有限公司
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