Molecular marker method of two mutation sites in the 5′ regulatory region of chicken pthlh gene and its application in chicken breeding

A technology of regulatory regions and sites, applied in the field of molecular genetics, can solve the problems of affecting transcription initiation and gene expression, etc.

Active Publication Date: 2019-09-27
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, transcription is regulated by the sequence of the 5' regulatory region, and its base mutation usually affects the initiation of transcription, thereby affecting gene expression

Method used

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  • Molecular marker method of two mutation sites in the 5′ regulatory region of chicken pthlh gene and its application in chicken breeding
  • Molecular marker method of two mutation sites in the 5′ regulatory region of chicken pthlh gene and its application in chicken breeding
  • Molecular marker method of two mutation sites in the 5′ regulatory region of chicken pthlh gene and its application in chicken breeding

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Chicken PTHLH 5'regulatory region sequence alignment and polymorphic site analysis

[0020] 1. Test materials

[0021] 48 recessive Bailuoke chickens (Shandong Jihua Poultry Breeding Co., Ltd.) were randomly sampled, blood was collected from the wing vein, and the genome was extracted and stored at -20°C.

[0022] 2. Test method

[0023] 2.1 Primer design

[0024] The primer P-PTHLH was designed according to the published red original chicken sequence (GenBank Accession NC_006088.4) (see Table 1 and Seq ID No:1 / Seq ID No:2 for the sequence details). This primer is for studying chicken PTHLH 5'regulation The mutation of the region is specially designed based on the sequence of the red rock fowl registered in the database.

[0025] 2.2 PCR amplification

[0026] The genomes of 48 recessive Bailuoke chickens were randomly selected and amplified by PCR with primer P-PTHLH. The primers are shown in Table 1. The reaction system is 20μL, including 1μL of genomic DNA (50-100ng)...

Embodiment 2

[0033] Example 2 Association analysis of chicken PTHLH5' regulatory region polymorphism with age at the start of laying and egg production

[0034] 1 Test materials

[0035] Randomly select 50 Jining Bairi Chickens (Jining Datang Bairi Chicken Conservation Farm), 50 Xinyang Brown Chickens (Shanghai Poultry Breeding Co., Ltd.), 50 Wenshang Reed Chickens (Wenshang County, Jining City, Shandong Province), 45 Hailan brown chickens (Shandong Tai'an Hailan Brown Breeding Company) and 550 recessive Bailuoke chickens with egg laying records (Shandong Jihua Poultry Breeding Co., Ltd.). All of the above were random sampling, blood was collected from the wing vein, and the genome was extracted and stored at -20℃.

[0036] 2 Test method

[0037] 2.1 PCR amplification

[0038] Use the genome as a template to perform PCR amplification. The primers are shown in Table 1. The reaction system is 40μL, including 2μL genomic DNA (50-100ng), 20μL 2×PrimeSTARGCBuffer, 3.2μL dNTPs (2.5mM each, TaKaRa), ups...

Embodiment 3

[0067] Example 3 Effects of polymorphic sites in the 5'regulatory region of chicken PTHLH on gene expression

[0068] 1 Test materials

[0069] Randomly sample healthy Hailan brown chickens (3-5 chickens) in the peak egg production period from the Linxi Village Farm in Tai'an City,

[0070] 2 Test method

[0071] 2.1 Construction of a mutant luciferase expression vector at a polymorphic site in the 5′ regulatory region of PTHLH

[0072] 1) In the recessive Bailock population, select PTHLH 5'regulatory regions at positions-1827 and -165 respectively for wild-type and mutant individuals, and use their DNA as a template to obtain the wt-PTHLH and mut-PTHLH sequences. The length of the amplified fragment is 2144bp, and the primer sequence is the same as Seq ID No. 1 and 2.

[0073] 2) PCR amplification

[0074] The amplification reaction adopts the high-fidelity enzyme PrimeSTAR, and the reaction system (20ul) includes: 2×PrimeSTARGCBuffer 10ul, 1.6μL dNTPs (2.5mM each, TaKaRa), plus 0.5μL (...

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Abstract

The invention relates to a molecular marking method for two mutation sites in a chicken PTHLH gene 5' regulatory region and application thereof in chicken breeding. Two mutation sites (namely the -1827 (A>G) site and the -165(C>T) site) exist in the chicken PTHLH gene 5' regulatory region, SHEsis online software is used for linkage disequilibrium analysis, and the result shows that the two sites in recessive white plymouth rock are in a complete linkage state. The two sites are related with the first-egg age and egg laying quantity at the 32th week, the first-egg age of AC / GT gene type is remarkably earlier than that of the GT / GT type and the AC / AC type, and meanwhile the egg laying quantity at the 32th week is remarkably larger than that of the other two types. The method is simple, fast and beneficial for breeding chicken variety with the early first-egg age and the large egg laying quantity and provides favorable help for marker-assisted breeding work.

Description

Technical field [0001] The invention relates to the field of molecular genetics, in particular to a molecular marking method for two mutation sites in the 5'regulatory region of chicken PTHLH gene and its application in breeding. Background technique [0002] Parathyroid hormone-like hormone (PTHLH), also called parathyroid hormone-related hormone (PTHrP), was first discovered in the 1980s and is considered to be a body fluid that causes malignant tumors. The main cause of hypercalcemia (Moseley et al, 1987). PTHLH is widely distributed in various fetal and adult tissues such as bone, pancreas, breast, kidney, stomach, uterus, etc. It acts through paracrine, autocrine, cellular endocrine and other forms, and its biological function involves calcium transport (Weir et al, 1996 ; Miao et al, 2004; Martin et al, 2005), bone formation (Cuthbertson et al, 1999; Zhao et al, 2002; Pinheiro et al, 2010; Klopocki et al, 2010), smooth muscle contraction and maintenance of pregnancy (Thied...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888A01K67/02
Inventor 姜运良郭晓莉张彤彤栾雅童李培钊辛倩康丽
Owner SHANDONG AGRICULTURAL UNIVERSITY
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