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Identification method taking growth hormone receptor gene as Chinese grassland red bull excellent slaughter trait molecular marker and application thereof

A technology of red bull and grassland, applied in the field of animal genetic engineering, can solve the problems of unclear genetic characteristics, influence on milk production characteristics of cattle, and no polymorphism found.

Inactive Publication Date: 2010-01-27
JILIN UNIV +1
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Problems solved by technology

[0003] In 1996, Falaki et al. [M.Falaki, Relationships of Polymorphisms for Growth Hormone and Growth Hormone Receptor Genes with Milk Production Traits for Italian Holstein-Friesian Bulls, Journal of Dairy Science Vol.79No.8: 1446-1453] reported that they were used to encode Nine RPLR-TaqI genotypes were found in part of the C-terminal DNA sequence, and the polymorphism of this site was related to the milk protein percentage of Italian Holstein cows; S.Blott et al [Sarah Blott, Molecular Dissection of a QuantitativeTrait Locus: A Phenylalanine-to-Tyrosine Substitution in the Transmembrane Domain of the Bovine Growth Hormone Receptor Is Associated with a Major Effect on Milk Yield and Composition, Genetics, Vol.163, 253-266, January 2003] reported in Holstein-Friesian Cattle Genes The substitution of F279Y amino acid in the coding region and the transmembrane region of the intron receptor affects the milk production traits of cattle; while Gao Xue et al. Issue August 2005 (2005)] In the study of local dairy cows, no polymorphisms were found in the eighth exon, ninth exon, third exon, and 5' end of the GHR gene, because It is not clear whether the limitation of sample size or the specific genetic characteristics of local cattle

Method used

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  • Identification method taking growth hormone receptor gene as Chinese grassland red bull excellent slaughter trait molecular marker and application thereof
  • Identification method taking growth hormone receptor gene as Chinese grassland red bull excellent slaughter trait molecular marker and application thereof
  • Identification method taking growth hormone receptor gene as Chinese grassland red bull excellent slaughter trait molecular marker and application thereof

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preparation example Construction

[0068] (2) Preparation of competent DH5α host bacteria

[0069] with CaCl 2 Escherichia coli DH5α (E.coli DH5α) competent host bacteria were prepared by this method, and the whole process was carried out under strict aseptic conditions. The method is as follows: Pick a single colony of newly activated E.coli DH5α from the LB (Luria-Bertani) plate medium cultured at 37°C for 16 to 20 hours, and place it in the LB liquid medium without ampicillin, at 37°C at 200 to Shake culture at 220 rpm for 12 hours, then take 300 microliters into a 50ml Erlenmeyer flask filled with LB, shake gently for 2 hours, OD 600 The value is 0.45 to 0.55. Take the E.coli DH5α bacterial solution pre-cooled in ice for 10 minutes, put it in a centrifuge tube pre-cooled on ice, and centrifuge at 4,500 rpm for 10 minutes at 4°C. Take 0.075Mol / L of CaCl pre-cooled in 25 ml of ice bath 2 - Tris-Cl suspended precipitate, placed in ice bath for 15 minutes. Centrifuge at 4500 rpm for 10 minutes at 4°C, disc...

Embodiment 1

[0092] A 23-month-old Chinese Grassland Red Cattle was randomly selected from Sanjiazi Township, Tongyu County, Jilin Province, China. Blood was collected at the time of slaughter for genomic DNA extraction and related trait data. The extracted genomic DNA was determined by a spectrophotometer, the concentration was 32.44ug / ml, and the OD260 / OD280 Ratio was 1.824. The primers, PCR reaction system and conditions designed by the present invention are used for PCR amplification. The total PCR reaction system is 25 microliters, of which 10× reaction buffer [10mmol / L Tris-HCL (PH8.0), 50mmol / L KCl, 0.1% TritonX-100] 2 microliters, 10mmol / L dNTP 0.5 microliters , 25mmol / LMgCl 2 1.5 microliters, the primer concentration is 1.0umol / L, 0.5 microliters of upstream primers and downstream primers, 0.5 microliters (2U / ul) Taq DNA polymerase, 1 microliters of extracted genomic DNA (containing 32.44ng of genomic DNA), 19.5 μl of distilled water. Cycle program: pre-denaturation at 94°C for 5...

Embodiment 2

[0096] A 21-month-old Chinese Grassland Red Cattle was randomly selected from the Animal Husbandry Branch of Jilin Academy of Agricultural Sciences, China. Blood was collected at the time of slaughter for genomic DNA extraction and related trait data. The extracted genomic DNA was measured by a spectrophotometer, the concentration was 37.25ug / ml, and the OD260 / OD280Ratio was 1.792. The primers, PCR reaction system and conditions designed by the present invention are used for PCR amplification. The total PCR reaction system is 25 microliters, of which 10× reaction buffer [10mmol / L Tris-HCL (PH8.0), 50mmol / L KCl, 0.1% TritonX-100] 2 microliters, 10mmol / L dNTP 0.5 microliters , 25mmol / LMgCl 2 1.5 microliters, the primer concentration is 1.0umol / L, 0.5 microliters each of upstream primer and downstream primer, 0.5 microliter (2U / ul) Taq DNA polymerase, 1 microliter extracted genomic DNA (containing 37.25ng genomic DNA), 19.5 μl of distilled water. Cycle program: pre-denaturatio...

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Abstract

The invention belongs to the technical field of animal genetic engineering, and particularly relates to a growth hormone receptor gene taken as a Chinese grassland red bull excellent slaughter trait molecular marker and an application thereof. The length of a nucleotide sequence of an obtained GHR genetic segment is 489bp and the nucleotide sequence is a part of an exon. A base mutation exists at a 289-position basic group to cause the generation of restrictive fragment length polymorphism. The mutant site is taken as the molecular marker and detected by a PCR-RFLP (polymerase chain reaction-restrictive fragment length polymorphism) method using restriction enzyme BstACI, and detection results are associated with the Chinese grassland red bull slaughter traits so as to provide theoretical basis for selection breeding of Chinese grassland red bull. The invention is characterized in that partial segment of GHR gene associated with the Chinese grassland red bull excellent slaughter traits is obtained, and polymorphism analysis is conducted to the segment for providing the molecular marker for marker assisted selection of the Chinese grassland red bull.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering, and relates to the application of candidate genes for excellent slaughter traits of Chinese grassland red cattle in marker-assisted selection, specifically a growth hormone receptor (GHR) gene as a molecular marker and its application in Chinese grassland red cattle for excellent slaughter traits. applications in traits. Background technique [0002] In 1987, Lueng [DAVID W.LEUNG, Growth Hormone receptor and Serum Binding Protein: Purification, Cloning and Expression, Nature 330, 537-543 (10 December 1987)] extracted and purified growth hormone receptor (GHR) from rabbit liver , and cloned GHR cDNA from rabbit liver cDNA library for the first time according to the amino acid sequence. Subsequently, people have successively cloned the GHR cDNA of more than 20 kinds of animals such as human, mouse, rat, sheep, cow, and chicken. Growth hormone (GH) exerts effects on development ...

Claims

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Application Information

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IPC IPC(8): C12P19/34C12Q1/68
Inventor 赵志辉张国梁张嘉保赵玉民姜昊秦立红曹阳张金玉胡成华吴健
Owner JILIN UNIV
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