Application of molecular marker based on TYRP1 gene in giant salamander body color breeding

A technology of molecular markers and genes, which is applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of less research on molecular genetics

Pending Publication Date: 2022-03-08
SHAANXI INST OF ZOOLOGY NORTHWEST INSTOF ENDANGERED ZOOLOGICAL SPECIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for the rare species of giant salamander, there a

Method used

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  • Application of molecular marker based on TYRP1 gene in giant salamander body color breeding
  • Application of molecular marker based on TYRP1 gene in giant salamander body color breeding
  • Application of molecular marker based on TYRP1 gene in giant salamander body color breeding

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] TYRP1 gene cloning and sequencing

[0076] 1> RNA extraction

[0077] The RNeasy Mini kit was used to extract total RNA from giant salamander skin tissue, and all precautions for RNA manipulation were strictly followed throughout the process. Unless otherwise specified, all steps were performed at room temperature.

[0078] 2> cDNA first strand synthesis

[0079] Total RNA was reverse transcribed using the RevertAid cDNA Synthesize Kit. Unless otherwise specified, the following steps are performed on ice.

[0080] (1) In an RNase-Free 0.5 μL centrifuge tube, add 2 μg (2-4 μL) of total RNA, 1 μL of oligoDT(18) primer, and add DEPC-treated water to 12 μL.

[0081] (2) Incubate at 65°C for 5 minutes, cool on ice, and centrifuge briefly.

[0082] (3) Add the following components: 2 μL of 5× buffer, 1 μL of RNase inhibitor, 1 μL of 10 mM dNTP mix, and 1 μL of reverse transcriptase.

[0083] Instantaneous centrifugation, PCR reaction, the reaction program: 42 ° C, 60 mi...

Embodiment 2

[0147] Real-time quantitative fluorescent PCR analysis of TYRP1 gene expression in different tissues of giant salamander

[0148] 1>Total RNA extraction and reverse transcription

[0149] The Tizol one-step method was used to extract the total RNA from giant salamander muscle, skin, heart, liver, spleen, lung, stomach, pancreas, kidney, gonad (ovary / testis), and intestinal tissue, and reverse transcribed it into cDNA. The specific experimental steps were implemented in the same way. example 1.

[0150] 2>Primer design and synthesis

[0151] Using the CDS region sequence of the Chinese giant salamander TYRP1 gene sequenced, real-time fluorescent quantitative primers were designed using the online software PrimerQuest (http: / / sg.idtdna.com / Primerquest / Home / IndexPrimer). The relevant information is shown in Table 1. Primers were synthesized by Nanjing GenScript Biotechnology Company.

[0152] 3>Reaction system and conditions

[0153] The reaction system is: add 1 μL cDNA to e...

Embodiment 3

[0157] Real-time quantitative fluorescent PCR analysis of the expression of TYRP1 gene in the tail skin tissue of giant salamander with different body colors

[0158] 1>Total RNA extraction and reverse transcription

[0159] Tizol one-step method was used to extract total RNA from the tail skin tissues of giant salamanders with body colors YL, Y(B), B(Y), B(SY) and GY, and reverse transcribed it into cDNA. The specific experimental steps were the same as in Example 1.

[0160] 2>Primer design and synthesis

[0161] With embodiment 1.

[0162] 3>Reaction system and conditions

[0163] With embodiment 1.

[0164] 4> Test data analysis

[0165] With embodiment 1.

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Abstract

The invention discloses application of a molecular marker based on a TYRP1 gene in giant salamander body color breeding, the nucleotide sequence of a giant salamander TYRP1 gene coding region is cloned for the first time, the full length is 1584bp, 527 amino acid residues are coded, and a tyrosinase (177-411 amino acid) functional structure domain is included; detecting the expression profile difference of the TYRP1 gene in tissues with different body colors and skin tissues with different body colors of the wild giant salamander by adopting a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) technology; complete sequences of CDS regions of TYRP1 genes with different body colors are compared to find that TYRP1 genes of yellow body color individuals have two nucleotide insertion sites, so that a decoding frame is changed, and an amino acid sequence is interrupted. The invention provides an effective molecular marker for directional breeding of giant salamander body color, provides a method for researching a molecular genetic basis and a cell metabolism mechanism formed by body color diversity, is beneficial to breeding of new giant salamander strains, and solves the problem of germplasm degeneration of giant salamanders.

Description

technical field [0001] The invention belongs to the technical field of molecular breeding, and in particular relates to the application of a molecular marker based on the TYRP1 gene in the body color selection and breeding of giant salamanders. Background technique [0002] The rich and diverse body color characteristics of species have always been a hot issue in the study of biological evolution and epigenetics. Studies suggest that animal body color is usually caused by the different pigment cells contained in the skin surface and their different number distributions. Unlike mammals, which contain only one type of melanocyte, body color in amphibians is associated with three types of pigment cells in their dermis. For the unique body color types of amphibians, previous studies mainly elaborated and analyzed their body color formation patterns from the aspects of physiology and behavior, but there were few reports on the underlying molecular genetic characteristics of thei...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11C12N15/53C12N9/02
CPCC12Q1/6888C12N9/0071C12Y114/18001C12Q2600/124C12Q2600/156C12Q2600/158
Inventor 邓捷姜维张红星王启军赵虎孔飞张晗马红英
Owner SHAANXI INST OF ZOOLOGY NORTHWEST INSTOF ENDANGERED ZOOLOGICAL SPECIES
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