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Molecular marking method for two mutation sites in chicken PTHLH gene 5' regulatory region and application thereof in chicken breeding

A P-PTHLH, P-PTHLH-F technology, applied in the field of molecular genetics, can solve problems affecting transcription initiation and gene expression

Active Publication Date: 2016-10-26
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 marking method for two mutation sites in chicken PTHLH gene 5' regulatory region and application thereof in chicken breeding
  • Molecular marking method for two mutation sites in chicken PTHLH gene 5' regulatory region and application thereof in chicken breeding
  • Molecular marking method for two mutation sites in chicken PTHLH gene 5' regulatory region and application thereof in chicken breeding

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Sequence Alignment and Polymorphic Site Analysis of Chicken PTHLH 5' Regulatory Region

[0018] 1. Test material

[0019] 48 recessive White Roc 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.

[0020] 2. Test method

[0021] 2.1 Primer design

[0022] The primer P-PTHLH (see Table 1 and Seq ID No:1 / Seq ID No:2) was designed according to the published red jungle fowl sequence (GenBank Accession NC_006088.4). This primer is for the study of chicken PTHLH 5′ regulation The mutations in the region are specially designed according to the sequence of the red jungle fowl registered in the database.

[0023] 2.2PCR amplification

[0024] The genomes of 48 recessive White Roc chickens were randomly selected and amplified by PCR with primer P-PTHLH. See Table 1 for primers, 20 μL of reaction system, including 1 μL of genomic DNA (50-100ng), 10 μL o...

Embodiment 2

[0031] Example 2 Association analysis of chicken PTHLH5'regulatory region polymorphism and age at first lay and egg production

[0032] 1 test material

[0033] Randomly select 50 Jining hundred-day chickens (Jining Datang Hundred-day Chicken Breeding 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 Taian Hailan Brown Breeding Co., Ltd.) and 550 recessive White Rock chickens with egg production records (Shandong Jihua Poultry Breeding Co., Ltd.). All of the above were randomly sampled, blood was collected from the wing vein, and the genome was extracted and stored at -20°C.

[0034] 2 test method

[0035] 2.1PCR amplification

[0036] Using the genome as a template, PCR amplification was performed. The primers are shown in Table 1. The reaction system is 40 μL, including 2 μL of genomic DNA (50-100ng), 20 μL of 2×PrimeSTARGCBuffer, 3.2 μL o...

Embodiment 3

[0065] Example 3 Effect of chicken PTHLH 5' regulatory region polymorphic site on gene expression

[0066] 1 test material

[0067] Random sampling of healthy Hailan brown chickens (3-5) during the peak laying period of the farm in Linxi Village, Tai'an City,

[0068] 2 test method

[0069] 2.1 Construction of luciferase expression vector with polymorphic site mutation in PTHLH 5′ regulatory region

[0070] 1) In the recessive Bailock population, two individuals whose PTHLH 5′ regulatory region -1827 and -165 sites were wild type and mutant type were selected, and their DNA was used as a template to obtain wt-PTHLH and mut-PTHLH sequences, and amplified The length of the augmented fragment is 2144bp, and the primer sequences are the same as Seq ID No.1 and No.2.

[0071] 2) PCR amplification

[0072] The amplification reaction uses the high-fidelity enzyme PrimeSTAR, and the reaction system (20ul) includes: 2×PrimeSTARGCBuffer 10ul, 1.6μL dNTPs (2.5mM each, TaKaRa), 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 marker 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 known as parathyroid hormone-related hormone (PTHrP), was first discovered in the 1980s and is considered to be a humoral substance associated with malignant tumors. Major 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., and acts through paracrine, autocrine, endocrine, etc., 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 (Thiede et al,...

Claims

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Application Information

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IPC IPC(8): C12Q1/68A01K67/02
CPCA01K67/02C12Q1/6888C12Q2600/124C12Q2600/156
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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