Molecular marker method for two mutation sites of chicken MMP13 gene 5' control region and application of molecular marking method in chicken breeding

A P-MMP13, P-MMP13-F technology, applied in the field of molecular genetics, can solve the problems of affecting transcription initiation, affecting gene expression, etc.

Active Publication Date: 2016-03-23
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...

Method used

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  • Molecular marker method for two mutation sites of chicken MMP13 gene 5' control region and application of molecular marking method in chicken breeding
  • Molecular marker method for two mutation sites of chicken MMP13 gene 5' control region and application of molecular marking method in chicken breeding
  • Molecular marker method for two mutation sites of chicken MMP13 gene 5' control region and application of molecular marking method in chicken breeding

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Chicken MMP13 5' Regulatory Region Sequence Alignment and Polymorphic Site Analysis

[0018] 1. Test material

[0019] Recessive White Roc chicken (Shandong Jihua Poultry Breeding Co., Ltd.) was 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] Primer P-MMP13 was designed according to the published red jungle fowl sequence (GenBankAccessionNC_006088.3) (see Table 1 and SeqIDNo: 1 and 2 for its sequence). The sequence of the red jungle fowl is specially designed.

[0023] 2.2PCR amplification

[0024] The genomes of 36 recessive White Roc chickens were randomly selected and amplified by PCR with primer P-MMP13. The primers are shown in Table 1. The reaction system is 20 μL, including 1 μL of genomic DNA (50-100ng), 2 μL of 10×Ex-buffer, 1.6 μL of dNTPs (2.5mMeach, TaKaRa), 0.4 μL of upstream and downstream primers (10 μM), 0.1 μL of Ex-TaqD...

Embodiment 2

[0031] Example 2 Association Analysis of Chicken MMP135' Regulatory Region Polymorphism and Age at First Lay and Egg Production

[0032] 1 test material

[0033] Randomly selected 37 Jining hundred-day chickens (Jining Datang Hundred-day Chicken Breeding Farm), 53 Wenchang chickens (Hainan Province Wenchang Chicken Breeding Company), 46 Wenshang Reed Chickens (Wenshang County, Jining City, Shandong Province), 45 Hailan brown chickens (Shandong Taian Hailan Brown Breeding Co., Ltd.) and 510 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 listed in Table 1. The reaction system was 40 μL, including 2 μL of genomic DNA (50-100 ng), 4 μL of 10×Ex-buffer, 3...

Embodiment 3

[0081] Example 3 Effect of chicken MMP13 5' regulatory region polymorphic site on gene expression

[0082] 1 Test material

[0083] Random sampling of healthy Hailan brown chickens (3-5) in the peak egg production period of the farm in Linxi Village, Tai'an City

[0084] 2 test method

[0085] 2.1 Construction of polymorphic site mutant luciferase expression vector in MMP135′ regulatory region

[0086] 1) In the recessive Bailock population, two individuals with wild-type and mutant types at the -1356, -1128, -1094 and -1079 sites of the MMP135' regulatory region were selected, and their DNA was used as a template to obtain wt-MMP13 and mut -MMP13 sequence, the length of the amplified fragment is 1863bp, and the primer sequences are shown in Table 12 (SeqIDNo.4 and 5).

[0087] Table 12 constructs luciferase vector amplification primer sequence

[0088]

[0089] 2) PCR amplification

[0090] The high-fidelity enzyme PrimeSTARGXL was used for the amplification reaction,...

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Abstract

The invention relates to the field of molecular genetics, in particular to a molecular marker method for two mutation sites of a chicken MMP13 gene 5' control region and application of the molecular marker method in chicken breeding. It is found by the inventor that six mutation sites exist in the chicken MMP13 5' control region, namely, -1719 (T>C), -1661 (C>A), -1356 (G>A), -1128 (A>G), -1094 (C>A) and -1079 (T>C); linkage disequilibrium analysis is performed through the SHEsis online software, and it is found through the result that the sites -1719 and -1661, the sites -1356 and -1128 and the sites -1094 and -1079 are respectively in a completely-linked state in White Recessive Rock. The method is easy, convenient and fast to implement, beneficial for breeding chicken breeds laying eggs early at a high yield and capable of providing favorable help for the 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 MMP13 gene and its application in breeding. Background technique [0002] Extracellular matrix metalloproteinases (Matrixmetalloproteinases, MMPs) and their related endogenous inhibitors together constitute the MMP system, which is involved in the regulation of extracellular matrix (Extracellular matrix, ECM) remodeling, such as cell proliferation, granulosa cell differentiation, and the formation and formation of ovarian vascularization structures. process of degradation. MMP13, also known as collagenase 3, is able to cleave fibrillar and non-fibrillar collagen. It can catalyze the key domain of the triple helix of fibrillar collagen, resulting in changes in the stability and solubility of collagen. Balbinetal (1996) and Huangetal (1999) studies have shown that MMP13 is involved in the reg...

Claims

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Application Information

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IPC IPC(8): C12Q1/68A01K67/02
CPCA01K67/02C12Q1/6888C12Q2600/124C12Q2600/156
Inventor 姜运良袁振杰陈秋月赵纪华康丽樊新忠郭晓莉乔西波
Owner SHANDONG AGRICULTURAL UNIVERSITY
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