CN108841932B - Molecular marking method for predicting and identifying fat mass of chicken abdomen and application - Google Patents
Molecular marking method for predicting and identifying fat mass of chicken abdomen and application Download PDFInfo
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Abstract
The invention discloses a molecular marking method for predicting and identifying the abdominal fat mass of a chicken and application thereof, belonging to the technical field of animal molecular genetics. The method provided by the invention comprises the steps of designing primers according to the upstream and downstream 190bp sequences of SNP site c.1880C > T of chicken protein precursor processing enzyme 1 gene, carrying out PCR amplification on chicken genome DNA according to the obtained primers, carrying out enzyme digestion on an amplification product by using restriction endonuclease, carrying out electrophoretic separation on the enzyme digestion product, and finally analyzing gene polymorphism by using PCR-RFLP technology to obtain the chicken abdominal fat marker genotype. The method provided by the invention has the characteristics of simple operation, low cost and high accuracy, can be used for automatic detection, is an effective molecular marker breeding means, and can greatly accelerate the breeding process of chickens.
Description
Technical Field
The invention relates to a molecular marking method for predicting and identifying the abdominal fat mass of a chicken and application thereof, belonging to the technical field of animal molecular genetics.
Background
The broiler chicken industry has good development prospect in the world, especially in developing Chinese furniture such as China. Over the past half century or more, significant progress has been made in breeding depending on phenotypic value selection, with significant improvements in growth rate and meat yield of broiler chickens. With the rapid development of the broiler industry in China, the physiological discomfort and related diseases of broilers are increased, such as excessive body fat accumulation, ascites syndrome, sudden death, leg diseases, reduction of body immunity and the like, and the economic loss of broilers producers caused by the problems is obvious. Excessive deposition of body fat (particularly abdominal fat) of broiler chickens can not only cause waste in the production process of broiler chickens and improve production cost, but also over-fatness of broiler breeders can seriously affect the laying rate, the fertility rate and the hatchability, and can induce the occurrence of fatty liver syndrome, over-fatness of broiler breeders can affect the semen collection amount, reduce the semen quality, and simultaneously, diseases caused by excessive obesity can increase the death and culling rate in the laying period and reduce the economic benefit. In addition, the influence of the consumers who attach importance to the diet health is received, and the low-fat poultry meat products are mainly consumed in China at present.
In recent years, with the development of molecular genetics, genetic markers are gradually applied to marker-assisted selective breeding of livestock and poultry, and the technology can effectively promote the improvement of the accuracy of genetic breeding. Molecular marker assisted selection is a method for directly selecting genomic regions affecting important economic traits. Many quantitative traits of livestock and poultry are not only controlled by micro-effective polygenes, but also influenced by single or multiple major genes. The direct selection of the genotype can be realized by selecting the molecular marker linked with the quantitative character site, thereby greatly improving the breeding efficiency of the poultry, accelerating the breeding process and providing a theoretical basis for solving the problem of excessive abdominal fat accumulation of the broiler chickens.
Protein Convertase (PC) is a family of Ca2+ -dependent serine proteases whose main function is to cleave the precursors of biologically inactive proteins or peptides into active functional molecules. Among them, Proprotein Convertase Subtilisin 1(Proprotein Convertase Subtilisin/Kexin Type 1, PCSK1) is mainly expressed in nervous and endocrine tissues, and the protein encoded by the gene can activate insulin, glucagon and proopiomelanocortin which control appetite in human body and make people produce satiety.
Results of studies on mammals have shown that the PCSK1 gene is mainly expressed in neurological and endocrine tissues. In humans, PC1/3 protein deficiency is a rare monogenic obesity disease, mainly manifested by childhood obesity, small intestine absorption dysfunction and various endocrine disorders. The results of locating the QTL affecting human obesity indicate that the gene affecting human obesity is located on chromosome 5q, and the chromosome region contains a plurality of genes including PCSK 1. In recent years, genome-wide association analysis is carried out for human obesity, and some obesity-related risk sites are found, including the PCSK1 gene. The polymorphism analysis result of the PCSK1 gene shows that the polymorphism of two sites on the gene is obviously related to human obesity. Studies in mice have shown that PCSK1 knock-out mice develop severe dysplasia due to the disruption of many neuroendocrine hormone maturation processes caused by PCSK1 deletion. The detection result of GHRH secretion level of PCSK 1-deleted mice shows that GHRH precursor of PCSK 1-deleted mice is accumulated in vivo in a large amount, and the mature GHRH level is very low. Inhibition of PCSK1 gene expression in mice results in defects in proinsulin processing, and reduction of PCSK1 gene expression results in increased proinsulin in the pancreas and circulation, with almost complete disappearance of mature insulin. So far, no report related to the study of the functions of the chicken PCSK1 gene is found, and no report is provided for selecting the abdominal fat content of the chicken by PCSK1 gene molecular marker, and no report is provided for screening genotypes by designing primers according to the upstream and downstream 190bp sequences of the SNP site c.1880C > T.
Disclosure of Invention
In order to solve the technical problems, the invention provides a molecular marking method for predicting and identifying the abdominal fat mass of a chicken, which adopts the following technical scheme:
the invention aims to provide a molecular marking method for predicting and identifying the fat mass of chicken belly, which is characterized in that primers are designed according to the sequence of 190bp upstream and downstream of SNP site c.1880C > T of PCSK1 gene, PCR amplification of chicken genome DNA is carried out according to the obtained primers, then restriction endonuclease is used for carrying out enzyme digestion on an amplification product, the enzyme digestion product is subjected to electrophoretic separation, and finally restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) is used for analyzing gene polymorphism to obtain the chicken belly fat marking genotype;
the nucleotide sequence of the primer is shown in SEQ ID NO.1-SEQ ID NO. 2.
The abdominal fat amount of the chicken refers to the abdominal fat weight and abdominal fat rate of the chicken.
The method comprises the following steps:
1) designing a primer according to the upstream and downstream 190bp sequences of the SNP site c.1880C > T of the PCSK1 gene of the chicken to obtain an amplification primer;
2) performing PCR amplification on chicken genome DNA by using the amplification primer obtained in the step 1) to obtain an amplification product;
3) carrying out enzyme digestion on the amplification product obtained in the step 2) by using restriction enzyme to obtain an enzyme digestion product;
4) carrying out electrophoretic separation on the enzyme digestion product obtained in the step 3) by using agarose gel to obtain a separation product;
5) carrying out genotype analysis on the separated product obtained in the step 4) by utilizing a PCR-RFLP technology to obtain the chicken abdominal fat marker genotype.
The nucleotide sequence of the primer in the step 1) is shown as SEQ ID NO.1-SEQ ID NO. 2.
Step 3), the restriction enzyme is restriction enzyme BsrGI-HF;
step 4) the agarose gel, the concentration of the agarose gel is 3%.
And 5) carrying out genotype analysis, wherein the analyzed part is a CDS region of the chicken PCSK1 gene.
Preferably, the analyzed site is missense mutation of 627 amino acid of a chicken PCSK1 gene sequence shown in SEQ ID NO.3 from alanine (Ala) to valine (Val).
Step 5) marking the genotype of the chicken abdominal fat, wherein when the C.1880C > T of the PCSK1 gene locus of the chicken is T basic group, the size of an agarose gel electrophoresis strip of a restriction enzyme digestion product is 347bp, and the restriction enzyme digestion product is named as TT genotype; when the c.1880C > T of the chicken PCSK1 gene locus is C basic group, the size of an agarose gel electrophoresis strip of a restriction enzyme digestion product is 383bp, and the restriction enzyme digestion product is named as CC genotype; the site heterozygous individual restriction enzyme product agarose gel electrophoresis band is two, the size is 347bp and 383bp respectively, and the individual restriction enzyme product agarose gel electrophoresis band is named as CT genotype.
The method is used for genetic breeding of chicken.
The invention has the beneficial effects that:
the invention proves that the distribution frequency of alleles of two genotypes of c.1880C > T mutation sites between two lines of high and low fat bidirectional selection lines is very obviously different (P <0.01), and the two sites are highly linked; in the random population of AA broiler chickens, the influence of c.1880C > T mutation sites on abdominal fat mass traits of the broiler chickens reaches an extremely significant level (P <0.01), the functions of the sites are not reported, and the characteristics of various genotypes of the gene mutation of the sites are not reported in documents.
The invention has simple operation, low cost and high precision, and can carry out automatic detection. The molecular marking method is used for selecting the abdominal fat mass of the chicken, not only provides a more effective, simple and easy molecular marking method for marking auxiliary selection in chicken breeding work, but also provides an effective molecular marking breeding means for improving the abdominal fat character of the chicken, and can accelerate the breeding process of the chicken.
Drawings
FIG. 1 shows an analysis map of PCSK1 gene at SNP site c.1880C > T.
Detailed Description
The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.
Example 1 buffer solution preparation and primer design
1. Laboratory animals and trait determination
The broiler chicken bred by northeast agriculture university has 542 cocks in nineteenth generation and 685 cocks in twentieth generation; AA broiler random group 348 chickens. Collecting blood from high and low fat broiler chicken and AA broiler chicken in 7 weeks old via wing vein, and collecting blood with EDTA-Na2And (4) anticoagulation. Before slaughter at 7 weeks of age, the live weight was measured, and after slaughter, the abdominal fat percentage was calculated by dividing the live weight at 7 weeks of age.
2. Drugs and enzymes
Tris (hydroxymethyl) aminomethane (Tris), Sigma Chemicals Co; tris saturated phenol, central for biotechnology development in beijing dingguo; proteinase K (protease K), MMERCK Co; DL 2000, bio-inc, dalianbao; dNTP (dATP; dTTP; dCTP; dGTP), Taq enzyme, DNAmarker, Beijing Quanjin Biotechnology Ltd; restriction enzyme BsrGI-HF, NeB, Beijing; agarose (Agarose), formerly hao.
3. Main instrument
PTC-200PCR instrument (PERKIN ELMER), Biometra gradient PCR instrument, UVP multifunctional imaging system, electrophoresis tank, electrophoresis apparatus.
4. Preparation of buffer solution and common reagent
1M Tris. Cl: 121.14g Tris base is dissolved in 800ml double distilled water, the pH value is adjusted to 8.0 by hydrochloric acid, the volume is adjusted to 1000ml, and the high pressure sterilization is carried out.
TE buffer solution: 10mM Tris. Cl,1mM EDTA, pH8.0, autoclaved.
20 × SET buffer: 3M NaCl,1M Tris. Cl (pH8.0), 20mM EDTA (pH8.0), autoclaved.
50 × TAE buffer: 242g Tris base, 57.1ml glacial acetic acid, 100ml 0.5MEDTA (pH8.0), water to 1L.
1M Tris. Cl: 121.14g Tris base is dissolved in 800ml double distilled water, the pH value is adjusted to 8.0 by hydrochloric acid, the volume is adjusted to 1000ml, and the high pressure sterilization is carried out.
0.5M EDTA: dissolving 186.1g EDTA in 800ml double distilled water, adjusting pH to 8.0 with NaOH, diluting to 1000ml, and autoclaving.
Poultry blood lysate: 10mM Tris. Cl (pH8.0), 0.1M EDTA (pH8.0), 0.5% SDS.
5. Design and Synthesis of primers
A primer is designed according to a 190bp sequence of an upstream and a downstream of a chicken PCSK1 gene SNP locus c.1880C > T, and is synthesized by EnxWeijie (Shanghai) trade company Limited:
PC1-CY-F:5’-TAAATCCCACCTTCTGATAAGTTCTGTCCCT-3’
PC1-CY-R:5’-GCACAAAGAAAAGCATTAATGAGACACTACCTGT-3’；
example 2DNA extraction, product amplification and electrophoretic separation
1. Extraction of chicken DNA
The following two methods can be adopted for extracting chicken DNA samples:
the method comprises the following steps:
(1) adding 20 μ l anticoagulated blood into 500 μ l fowl lysate, adding proteinase K to final concentration of 100-.
(2) The solution was cooled to room temperature, 5M NaCl was added to a final concentration of 1.5M and mixed well for 10 min. Adding equal volume of phenol/chloroform, repeatedly inverting the centrifuge tube, and mixing for 10 min.
(3) Centrifuge at 12,000rpm for 10min at room temperature. The supernatant was taken and mixed with chloroform of equal volume for 10 min.
(4) Centrifuge at 12,000rpm for 10min at room temperature. 2 times volume of absolute ethyl alcohol of the supernatant is taken to precipitate DNA.
(5) The DNA was picked up and placed in a 1.5ml centrifuge tube and washed 1 time with 70% ethanol.
(6)7,500rpm, centrifuge at room temperature for 5min, and discard the supernatant.
(7) The DNA was dried (note that it was not too dry) and dissolved in 200. mu.l TE.
The second method comprises the following steps:
(1) mu.l of whole blood was added to a 1.5ml centrifuge tube containing 700. mu.l of 1 XSET and gently mixed.
(2) Proteinase K (10mg/ml) was added to a final concentration of 100-.
(3) After digestion is completed, adding equal volume of Tris saturated phenol, reversing back and forth, and mixing uniformly
(4) Centrifuge at 12,000rpm for 10min, carefully transfer the upper aqueous phase to another centrifuge tube with tip-off tip, and discard the organic phase. The third and fourth steps are repeated once.
(5) Adding the mixed solution of phenol, chloroform and isoamyl alcohol (volume ratio is 24: 23: 1) with the same volume into the water phase, and mixing for 10 min. Centrifuge at 12,000rpm for 10min and remove the aqueous phase to another centrifuge tube.
(6) Adding equal volume of chloroform and isoamyl alcohol mixture (23: 1) into the water phase, mixing by reversing back and forth for 10min at 12,000rpm, centrifuging for 10min, and removing the water phase to another centrifuge tube.
(7) 1/10 volumes of NaAc (3M, pH5.2) and 2 volumes of absolute ethanol were added to the aqueous phase, and the mixture was inverted to precipitate DNA.
(8) The DNA was picked up and placed in a 1.5ml centrifuge tube and washed 1 time with 70% ethanol.
(9) Centrifuge at 7,500rpm for 5 min. Carefully pour the tube off the ethanol, invert it on filter paper, let the ethanol run out, and dry it in air.
(10) 200. mu.l of TE was added and the DNA was dissolved overnight in a water bath at 50 ℃. After dissolution, the mixture is stored at-20 ℃ for later use.
2. Amplifying chicken DNA;
PCR reaction
(1) The chicken DNA is taken as a template for PCR amplification, and a 10ul reaction system comprises the following solutions or reagents:
(2) the above solutions were mixed and subjected to PCR reaction under the following conditions.
Denaturation at 94 deg.C for 5 min; 30 cycles of 94 ℃ for 30sec, 55 ℃ for 30sec, 72 ℃ for 30 sec; extension at 72 ℃ for 10 min.
(3) After the reaction is finished, taking the PCR reaction solution (5-10 mu l) for agarose gel electrophoresis, and detecting the PCR product.
PCR-RFLP digestion reaction and electrophoresis
The following reaction solutions were prepared in a 0.2ml EP tube and mixed well
The reaction was carried out at 37 ℃ for 3 hours, and the cleavage was checked with 3% agarose gel and genotyping was carried out.
Example 3 statistical modeling
According to the characteristics of the population, the following linear model is constructed:
Y＝μ+G+F+D(F)+BW7+e①
Y＝μ+G+S+G×S+F+D(F)+BW7+e②
y is a character observed value, mu is a population mean value, G is a genotype fixing effect, S is a gender fixing effect, F is a family random effect, D (F) is a family hen random effect, BW7 is a covariance variable, and e is a residual value. The model is used for analyzing the correlation of site polymorphism and abdominal fat and other characters in the 19 th and 20 th generation groups of high and low-fat broiler bidirectional strains of the northeast agricultural university respectively; and the model II is used for analyzing the correlation of site polymorphism and abdominal fat and other characters in the AA broiler random population. Statistical software JMP 7.0(SAS Institute, 2000) was used to test the degree of correlation between the genotype of the population and the trait and to estimate the least squares mean of the trait.
Example 4 correlation analysis of polymorphism of chicken PCSK1 gene and abdominal fat amount of broiler chicken in two-way selection line and AA broiler chicken random population
The primers (PC1-CY-F, PC1-CY-R) are utilized to perform PCR amplification on the genomic DNA of 542 cocks in the nineteenth generation, 685 cocks in the twentieth generation and 348 chickens in the AA broiler random population of the broiler high and low-fat bidirectional selection line bred by the northeast agriculture university, and then PCR-RFLP analysis is performed on sites c.1880C > T. A total of 3 genotypes were detected at each SNP site in both populations. For the site c.1880C > T, when the size of an agarose gel electrophoresis strip of the enzyme digestion product is 347bp, the enzyme digestion product is named as TT genotype; when the size of the agarose gel electrophoresis band of the enzyme digestion product is 383bp, the enzyme digestion product is named as CC genotype; the heterozygous individual restriction enzyme product agarose gel electrophoresis strip of this position is two, the size is 347bp and 383bp respectively, its name is CT genotype (as shown in figure 1).
The method is characterized in that the nineteenth and twenty-th generation chickens of a high-low-fat bidirectional selection line of broiler chickens bred by northeast agriculture university are taken as materials, and the allelic distribution condition of sites c.1880C > T between two lines is analyzed. The results show that there is a very significant difference in the distribution of alleles at position c.1880C > T between the two lines (P <0.01) (Table 1).
TABLE 1SNP site c.1880C > T allele frequency in the nineteenth and twentieth generation population
A random group of AA broilers is taken as a material, the allelic distribution situation of sites c.1880C > T between two lines is analyzed, and two individuals with extreme abdominal fat rates (15% of extreme individuals of the two individuals are selected according to the abdominal fat rates and are divided into two groups with low abdominal fat rates and high abdominal fat rates) are selected for allelic frequency difference analysis. The results show that the allele frequencies at position c.1880C > T are very significantly different between two individuals with extreme abdominal fat rates (P <0.01) (Table 2).
TABLE 2 differential analysis of allele frequencies between two individuals with extreme abdominal fat rates at SNP site c.1880C > T in random population of AA broilers
The nineteenth and twentieth generation groups of high-low-fat bidirectional selection series of the broilers and the random group of AA broilers are used as materials, the influence of the site c.1880C > T on abdominal fat mass characters of the broilers of the two groups is calculated, and the result shows that the influence of the site c.1880C > T on abdominal fat rate and abdominal fat weight reaches a very significant level (p <0.01) (Table 3)
TABLE 3 influence of c.1880C > T sites on abdominal fat mass traits of broilers of two groups (P value)
Note: p <0.01
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> northeast university of agriculture
<120> molecular marking method for indicating and identifying abdominal fat mass of chicken and application
<130> 1
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 31
<212> DNA
<213> c.1880C > T site upstream primer PC1-CY-F
<400> 1
taaatcccac cttctgataa gttctgtccc t 31
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<212> DNA
<213> c.1880C > T site downstream primer PC1-CY-R
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gcacaaagaa aagcattaat gagacactac ctgt 34
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<213> site sequence for polymorphism analysis
<400> 3
TAAATCCCAC CTTCTGATAA GTTCTGTCCC TAATGTGTCA GTCTTTTGGG GCTTACAAAT 60
CATGGTCTAA ATTTATATAT TGACAGAGTC ATTTTTTATT TTAGTGAATT CTCACTGCTT 120
TGTGATCAAA TATCCTCTAT TTATTCCTCT TACATTCTGA GAACCTATGC TATTGCTGTC 180
TTGTCCTTTA GTCCAAAAGA ATACAAAATG AAGGAAGGAT TGTAAACTGG AAATTGATTT 240
TGCATGGCAC TGATACCCAG CCTGAACATA TGAAACAACC ACGTGTATAC ACATCTTACA 300
ATGCTGTGCA AAATGACAGA AGAGGAGTGG AGAAGATGAC AGACCTTGCA GAGGTAGTGT 360
CTCATTAATG CTTTTCTTTG TGC 383
Claims (3)
1. A molecular marking method for predicting and identifying abdominal fat mass of broiler chickens is characterized by comprising the following specific steps:
1) designing a primer according to an upstream 190bp sequence and a downstream 190bp sequence of a broiler PCSK1 gene SNP locus c.1880C > T to obtain an amplification primer; the nucleotide sequence of the primer is shown as SEQ ID NO.1-SEQ ID NO. 2;
2) carrying out PCR amplification on broiler chicken genome DNA by using the amplification primer obtained in the step 1) to obtain an amplification product;
3) carrying out enzyme digestion on the amplification product obtained in the step 2) by using restriction enzyme to obtain an enzyme digestion product; the restriction enzyme is BsrGI-HF;
4) carrying out electrophoretic separation on the enzyme digestion product obtained in the step 3) by using agarose gel to obtain a separation product;
5) carrying out genotype analysis on the separated product obtained in the step 4) by utilizing a restriction fragment length polymorphism polymerase chain reaction technology to obtain a broiler abdominal fat marker genotype; the abdominal fat amount of the broiler chickens refers to the abdominal fat weight and abdominal fat rate of the broiler chickens; the broiler abdominal fat marker genotype: when the c.1880C > T of the PCSK1 gene locus of the broiler chicken is T basic group, the size of an agarose gel electrophoresis strip of a restriction enzyme digestion product is 347bp, and the restriction enzyme digestion product is named as TT genotype; when the c.1880C > T of the PCSK1 gene locus of the broiler chicken is C basic group, the size of an agarose gel electrophoresis strip of a restriction enzyme digestion product is 383bp, and the agarose gel electrophoresis strip is named as CC genotype; the site heterozygous individual restriction enzyme product agarose gel electrophoresis band is two, the size is 347bp and 383bp respectively, and the individual restriction enzyme product agarose gel electrophoresis band is named as CT genotype.
2. The method according to claim 1, wherein the agarose gel of step 4) has a agarose gel concentration of 3%.
3. The application of the method of claim 1 or 2 in selective breeding of broiler abdominal fat mass traits, wherein the broiler abdominal fat mass traits are abdominal fat weight and abdominal fat rate of broilers.
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| CN110578008B (en) * | 2019-10-28 | 2023-03-31 | 东北农业大学 | Molecular marking method for predicting and identifying chicken abdominal fat weight |
| CN110904243B (en) * | 2019-12-12 | 2020-11-10 | 东北农业大学 | Molecular marking method for predicting and identifying fat mass of chicken abdomen and application |
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| CN113373238A (en) * | 2021-06-02 | 2021-09-10 | 华南农业大学 | Application of 61-bp indel polymorphism of chicken RIN2 gene in predicting and identifying chicken abdominal fat traits |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010072608A1 (en) * | 2008-12-22 | 2010-07-01 | F. Hoffmann-La Roche Ag | Pcsk1 single nucleotide polymorphism in type 2 diabetes |
| CN101962684A (en) * | 2010-11-04 | 2011-02-02 | 徐州师范大学 | Single nucleotide polymorphism for cattle PCSK1 gene and detection method thereof |
| WO2013188605A2 (en) * | 2012-06-15 | 2013-12-19 | Nuclea Biotechnologies, Inc. | Predictive markers for cancer and metabolic syndrome |
| CN104513858A (en) * | 2014-12-29 | 2015-04-15 | 东北农业大学 | Molecular marker method for predicting and authenticating abdominal fat amount of chicken and application of molecular marker method |
| WO2017059076A1 (en) * | 2015-09-30 | 2017-04-06 | Rhythm Pharmacueticals, Inc. | Method of treating melanocortin-4 receptor pathway-associated disorders |
| CN107108708A (en) * | 2014-12-22 | 2017-08-29 | 加州大学评议会 | For generating antigen, the composition of antibody and method and immunotherapeutical compositions and method |
-
2018
- 2018-07-13 CN CN201810768924.4A patent/CN108841932B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010072608A1 (en) * | 2008-12-22 | 2010-07-01 | F. Hoffmann-La Roche Ag | Pcsk1 single nucleotide polymorphism in type 2 diabetes |
| CN101962684A (en) * | 2010-11-04 | 2011-02-02 | 徐州师范大学 | Single nucleotide polymorphism for cattle PCSK1 gene and detection method thereof |
| WO2013188605A2 (en) * | 2012-06-15 | 2013-12-19 | Nuclea Biotechnologies, Inc. | Predictive markers for cancer and metabolic syndrome |
| CN107108708A (en) * | 2014-12-22 | 2017-08-29 | 加州大学评议会 | For generating antigen, the composition of antibody and method and immunotherapeutical compositions and method |
| CN104513858A (en) * | 2014-12-29 | 2015-04-15 | 东北农业大学 | Molecular marker method for predicting and authenticating abdominal fat amount of chicken and application of molecular marker method |
| WO2017059076A1 (en) * | 2015-09-30 | 2017-04-06 | Rhythm Pharmacueticals, Inc. | Method of treating melanocortin-4 receptor pathway-associated disorders |
Non-Patent Citations (3)
| Title |
|---|
| Identifiation of a potential functional single nucleotide polymorphism for fatness and growth traits in the 3′-untranslated region of the PCSK1 gene in chickens;K. Zhang等;《J Anim Sci》;20171130;第95卷(第11期);4776-4786 * |
| PCSK1和PCSK2单核苷酸多态性与新诊断2型糖尿病的相关性分析;郑晓雅等;《解放军医学杂志》;20141201;第39卷(第12期);961-964 * |
| Selection Signature Analysis Implicates the PC1/PCSK1 Region for Chicken Abdominal Fat Content;Hui Zhang等;《PLoS ONE》;20120711;第7卷(第7期);e40736 * |
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