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TYR gene molecular marker related to giant salamander body color character and application of TYR gene molecular marker

A molecular marker and gene technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve problems such as less research on molecular genetics

Pending Publication Date: 2022-04-12
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 are few molecular genetic studies related to TYR gene

Method used

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  • TYR gene molecular marker related to giant salamander body color character and application of TYR gene molecular marker
  • TYR gene molecular marker related to giant salamander body color character and application of TYR gene molecular marker
  • TYR gene molecular marker related to giant salamander body color character and application of TYR gene molecular marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] TYR gene cloning and sequencing

[0051] 1> RNA extraction and reverse transcription

[0052] Take the cell or tissue sample to be studied, according to the instructions of Invitrogen’s Trizol reagent, add 1ml Trizol to each 60mm cell culture dish to lyse the cells for 5min, add 0.2mL phenol chloroform to shake vigorously for 15s, place on ice for 3-5min, 4°C , Centrifuge at 12000rpm for 15min. Carefully pipette the supernatant into a new 1.5mL EP tube, add 0.5mL isopropanol, mix well, place on ice for 10min, centrifuge at 12000rpm at 4°C for 10min, discard the supernatant, add 1mL of 75% ethanol to wash the precipitate, 4°C , Centrifuge at 12000rpm for 5min, put the ethanol on ice and add 20μL DEPC water to dissolve the precipitate after the ethanol has evaporated completely, which is the total RNA. For its quantitative determination, select the one with a ratio of A260 / 280 between 1.8-2.0 and a higher concentration for the experiment. Take 2 μg of RNA for reverse t...

Embodiment 2

[0106] Analysis of TYR Gene Expression in Different Tissues of Giant Salamander by Real-time Quantitative Fluorescent PCR

[0107] 1>Total RNA extraction and reverse transcription

[0108] 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.

[0109] 2>Primer design and synthesis

[0110] Using the CDS sequence of the Chinese giant salamander TYR gene sequence obtained by sequencing, 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.

[0111] 3>Reaction system and conditions

[0112] The reaction system is: add ...

Embodiment 3

[0116] Real-time quantitative fluorescent PCR analysis of the expression of TYR gene in the tail skin tissue of giant salamander with different body colors

[0117] 1>Total RNA extraction and reverse transcription

[0118] 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.

[0119] 2>Primer design and synthesis

[0120] With embodiment 1.

[0121] 3>Reaction system and conditions

[0122] With embodiment 1.

[0123] 4> Test data analysis

[0124] With embodiment 1.

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Abstract

The invention discloses a giant salamander body color character related TYR gene molecular marker and application thereof, the giant salamander TYR gene coding region nucleotide sequence is cloned for the first time, the full length is 1605bp, 534 amino acid residues are coded, and the giant salamander TYR gene molecular marker comprises functional structural domains such as a tyrosinase structural domain (177-408 amino acid) and an EGFLam structural domain (234-521 amino acid); detecting the expression difference of the TYR 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 TYR genes with different body colors are compared, and it is found that three nucleotide insertion sites exist in the yellow body color individual TYR gene, so that a decoding frame is changed, and an amino acid sequence is interrupted. A foundation is laid for revealing a genetic mechanism and a cell metabolism mechanism formed by body color diversity of rare species, and a scientific test method for reference is provided for research on genes related to economic characters of non-model organisms.

Description

technical field [0001] The invention belongs to the technical field of molecular breeding, and in particular relates to a TYR gene molecular marker related to the body color trait of giant salamander and its application. Background technique [0002] Animal body color is a class of phenotypic characteristics with rich diversity, especially in amphibians, the variable body color plays an important role in individual mimicry, avoiding predation, mate selection and temperature regulation, etc., and has become a field of evolution and genetics research hotspots. Since the middle and late 20th century, researchers have used the research methods of modern ecology, genetics and molecular biology to study the coloring patterns of human skin color, the color and hair of model organisms such as mice and zebrafish, and the underlying molecular genetic mechanisms. A large number of genes associated with body color traits and their genotypes were identified. These research results prov...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6851C12N9/02C12N15/53C12N15/11
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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