CN109371033B - \28294A-FABP gene of fatty deposition of fatty liver of goose in West province and genetic marking method - Google Patents
\28294A-FABP gene of fatty deposition of fatty liver of goose in West province and genetic marking method Download PDFInfo
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Abstract
The invention discloses a fatty acid binding protein gene A-FABP (intron 2) and a genetic marker used as the character of a < 28294 >, the fatty acid binding protein gene A-FABP is used as a gene marker of a < 28294 >, the fatty acid binding protein gene A-FABP is used for screening the < 28294 >, the A-FABP gene SEQ ID NO:1 sequence 186bp has C → T mutation, resulting in polymorphism. Then, the A-FABP gene polymorphism and the fatty liver character association analysis are carried out, and the gene sequence of the A-FABP gene of the Luzhou goose is shown as SEQ ID NO:1, a C/T base mutation exists at 186bp, the CC genotype is \28294andthe size of the liver of the goose is obviously higher than that of other genotypes. The invention provides a molecular marker for marker-assisted selection of the properties of the fatty liver of \28294Heps.
Description
Technical Field
The invention belongs to the field of poultry molecular biotechnology and breeding, and mainly relates to detection of genetic markers related to the fatty acid binding protein gene of \28294andfatty acid binding protein gene of Philips, wherein the genetic markers are used for detecting the polymorphism in the A-FABP gene, and the polymorphism is used for detecting the existence of SNP.
Technical Field
\28294ThePhilips goose is a local variety in China, has the advantages of optimal fat and liver performance, large size, fast growth and strong disease resistance, particularly obviously belongs to the genetic resource of national-grade local variety, and enters the national-grade livestock and poultry genetic resource protection directory in 2014. Goose fat liver is one of three precious products in world-grade high-grade delicious health food, and the rich unsaturated fatty acid, linoleic acid, lecithin and the like have important health care effects on softening blood vessels, reducing blood fat and preventing cardiovascular and cerebrovascular diseases. Due to the fact that \28294saidgoose lacks scientific systematic breeding, the individual difference of liver size after filling fertilizer is large, and the industrialized production efficiency, product quality and economic benefit of goose are severely restricted. The conventional breeding is slower, with the development of a biomolecule technology, the major gene or molecular marker related to the fat liver character of the goose of 28294Hps can be searched from the DNA level, and the major gene or molecular marker is applied to marker-assisted selection in breeding, so that the breeding process is improved, and a larger economic effect is obtained.
Fatty acid binding proteins (A-FABP), also known as fatty acid binding protein 4(FABP4) or A-FABP, are members of the Fatty Acid Binding Protein (FABPs) family. A-FABP belongs to membrane peripheral protein, is expressed in a plurality of tissues, particularly fat tissues related to lipid metabolism, can be reversibly combined with long-chain fatty acid, and plays an important role in the synthesis, transportation and metabolism of fatty acid. Gerbens et al, in a pure Duroc study, showed that there was a significant correlation between the A-FABP polymorphism and intramuscular fat content. Related researches also exist in China, such as Rougifen and the like, in the polymorphism analysis of chicken A-FABP genes and related researches on fat traits, SNPs detection and genotype and trait association analysis are carried out on the A-FABP genes. As a result, the differences of abdominal fat rate, thick sebum and intramuscular fat content among different genotypes of A-FABP are found to be extremely obvious. The research of the leaf-full red on the bantams shows that single-base variation on the first exon and the first intron of the A-FABP gene has a remarkable influence on fat deposition. Studies of Yangxiang and the like find that single base mutation exists on an intron 2 of the Landaise goose A-FABP gene and is obviously related to fat liver performance. From this, it can be seen that the A-FABP gene is closely related to fat deposition.
Currently, research on A-FABP gene polymorphism is mainly focused on correlation analysis with intramuscular fat content, and correlation analysis with fatty liver performance is relatively poor. The study on DNA genetic markers, genetic diversity and related functional genes of the Eremon rufii goose is still in the initial stage, and no report on the genetic markers related to the fat deposition of the Eremon rufii goose fat liver is found.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an A-FABP gene and application thereof as a genetic marker of the properties of the fatty liver of the goose of \28294HJ; simultaneously, SNP related to the properties of the haunted goose liver is screened for being applied to marker-assisted selection or marker-assisted introgression, and the application of the genetic marker in the marker-assisted selection of the haunted goose liver is provided.
The present invention is an A-FABP gene which is a gene related to the fatty liver trait of Erythroculter australis 28294J. The gene is shown as SEQ ID NO:1 is shown.
Furthermore, the gene has 1C/T base mutation at 186bp of the sequence table SEQ ID NO.1, which causes polymorphism.
Further, the cloning comprises that the primers used by the A-FABP gene shown as the sequence table SEQ ID NO:1 are as follows:
a forward primer: 5'-CCCAAATAAGATAAGAACAC-3'
Reverse primer: 5'-ACATCCACCAGACAAGAA-3'
The invention also aims to provide a method for using the A-FABP gene as a genetic marker related to the fatty liver trait of 28294Upu goose, which comprises the following steps:
(1) according to the sequence of goose A-FABP gene in GenBank, using Primer5.0 to design Primer, covering all the sequences from exon 2 to exon 3 of said gene;
(2) PCR amplification, product purification and cloning are carried out by taking the genomic DNA of the goose of the aforementioned \28294, and the nucleotide sequence shown in the sequence table SEQ ID NO.1 is obtained;
(3) the sequence obtained above was identified to have 1 mutation site at the 186bp position.
Furthermore, the method selects \28294thePhilips goose as a research object, and adopts a molecular biological method to screen \28294thePhilips goose is fatty
The shape related gene comprises the following steps:
(1) the same batch of health \28294simultaneousbreeding of 60 goose is selected;
(2) designing a primer according to the sequence of the A-FABP gene, and amplifying and sequencing a sample;
(3) and screening SNP related to the fatty liver character.
Detecting a primer pair mutated at 186bp of an A-FABP gene sequence related to the properties of the < 28294 >, wherein the primers are as follows:
a forward primer: 5'-CCCAAATAAGATAAGAACAC-3'
Reverse primer: 5'-ACATCCACCAGACAAGAA-3'
The application of the A-FABP gene as the genetic marker related to the fatty liver character of \28294ugoose comprises the following steps:
(1) downloading a goose A-FABP gene sequence in GenBank, designing a primer by using Primer5.0, and covering the whole sequence from exon 2 to exon 3 of the gene;
(2) the PCR amplification, product purification and forward sequencing are carried out by taking the genomic DNA of the goose of \28294Hepuas a template to obtain a nucleotide sequence shown as SEQ ID NO.1 of a sequence table;
(3) and identifying whether the 186bp of the gene sequence has mutation. 50-80 healthy Japanese geese with the same age are selected for the forced-feeding test. After 28 days, slaughter was carried out, and weight of 28294u goose livers was weighed, and genomic DNAs thereof were extracted from extreme samples (large livers and small livers), respectively.
According to the sequence of goose A-FABP gene in GenBank, Prime r5.0 is used to design a primer, which covers the whole sequence from exon 2 to exon 3 of the gene, and the sequence of the primer is as follows: 5'-CCCAAATAAGATAAGAACAC-3' (Forward) and 5'-ACATCCACCAGACAAGAA-3' (reverse), and the genomic DNA of Erwinia philippinensis gene, \28294cwas amplified using the above primers.
And (3) PCR: 20 μ l reaction: 2 × Master mix 12. mu.l, upstream and downstream primers (10. mu. mol/L)) 0.8. mu.l each, DNA template 1. mu.l, and ultrapure water to the desired volume.
PCR amplification procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 50.3 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; prolonging for 10min at 72 deg.C, and storing at 4 deg.C.
Taking 5 mul of PCR product to carry out gel electrophoresis, and detecting whether the size of the product fragment accords with the size of the target fragment, wherein the length of the PCR product is 621 bp.
Taking 30 mu l of PCR amplification product, purifying the product, sending the purified product to Hunan Qin Biotechnology Limited company for sequencing, and carrying out forward sequencing for a reaction, wherein the sequencing result confirms that the PCR product is an A-FABP gene, and the sequence is shown as SEQ ID NO:1 is shown. The above experimental results show that the A-FABP gene is extracted from the breast muscle of \28294; the Philips goose, and SNPs related to liver fat deposition are screened in the gene sequence, which is probably the reason for influencing the liver size of \28294; the Philips goose.
Verification of SNPs related to fatty deposition of the fatty liver of \28294JBrachys in production practice
And selecting 60-100 days old health \28294 ℃ under the same feeding and management conditions, and carrying out the test on 18-30 of the Phi geese. After 28 days of gavage, the test \28294ugoose was slaughtered and the fat liver was weighed.
Using 18-25 tissues of the breast muscle tissue-like genome DNA of the goose of \28294uand using primer sequences as follows: 5'-CCCAAATAAGATAAGAACAC-3' (forward), 5'-ACATCCACCAGACAAGAA-3' (reverse), amplified and then its SNPs detected.
1、PCR
The PCR reaction system is shown in the following table:
TABLE 1 PCR reaction System
The PCR cycle parameters were as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 50.3 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; prolonging for 10min at 72 deg.C, and storing at 4 deg.C.
2. Genotype frequency analysis of SNP locus of A-FABP gene
The genotype frequency and allele frequency of the mutation site were examined by chi-square method, and the results are shown in Table 2, and the site is in Hardy-Weinberg equilibrium (P > 0.05).
TABLE 2 SNP site genotype and Gene frequency
3. Correlation analysis of A-FABP gene and \28294Hpsfatty liver character
Analysis of the effect of different SNP genotypes on fatty deposition of v 28294jps fatty liver, can be seen in table 3 below: different SNP genotypes have obvious influence on the fatty liver character of \28294Hepugoose; wherein CC genotype is \28294, liver weight of goose is high, CT and TT type individuals are 102.52% and 93.95%, and the difference reaches a significant level (P < 0.05).
TABLE 3 comparison of different genotypes of \28294J.Zasche
Note: the same column with different lower case letters is very different (P < 0.05).
The invention adopts a method of combining PCR and DNA sequencing to screen the A-FABP gene as \28294g, the gene related to the fatty liver character of the goose, then screen the SNP related to the character from the \28294g, the A-FABP gene of the goose, and the screened SNP is the SNP related to the gene determined by the production practice verification that the A-FABP gene is the fatty liver deposition related gene. The invention screens out \28294saidSNP of the A-FABP gene of the ps goose can be used as the genetic marker of \28294u, the ps goose assisted selection, thus cultivating \28294uwith more liver fat deposition and bigger liver, the ps goose has great application value and economic benefit.
Drawings
FIG. 1 is a diagram showing the sequencing peaks of the mutation sites of the A-FABP gene.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1 < 28294; screening of fatty deposition-related Gene SNP in Photinia
Healthy \28294ugoose 60 of the same day age was selected for the gavage test. After 28 days, slaughter was carried out, and weight of 28294u goose livers was weighed, and genomic DNAs thereof were extracted from extreme samples (large livers and small livers), respectively.
According to the sequence of goose A-FABP gene in GenBank, Primer5.0 is used to design a primer, which covers the whole sequence from exon 2 to exon 3 of the gene, and the sequence of the primer is as follows: 5'-CCCAAATAAGATAAGAACAC-3' (forward), 5'-ACATCCACCAGACAAGAA-3' (reverse), was synthesized by the Okagaku Biotechnology Co., Ltd, Hunan. The primers were used to amplify the genomic DNA of \28294J.though goose, respectively.
And (3) PCR: 20 μ l reaction: 2 × Master mix 12. mu.l, upstream and downstream primers (10. mu. mol/L)) 0.8. mu.l each, DNA template 1. mu.l, and ultrapure water to the desired volume.
PCR amplification procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 50.3 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; prolonging for 10min at 72 deg.C, and storing at 4 deg.C.
Taking 5 mul of PCR product to carry out gel electrophoresis, and detecting whether the size of the product fragment accords with the size of the target fragment, wherein the length of the PCR product is 621 bp.
Taking 30 mu l of PCR amplification product, purifying the product, sending the purified product to Hunan Qin Biotechnology Limited company for sequencing, and carrying out forward sequencing for a reaction, wherein the sequencing result confirms that the PCR product is an A-FABP gene, and the sequence is shown as SEQ ID NO:1 is shown. FIG. 1 is a graph of mutation sites and sequencing peaks.
The above experimental results show that the A-FABP gene is extracted from the breast muscle of \28294; the Philips goose, and SNPs related to liver fat deposition are screened in the gene sequence, which is probably the reason for influencing the liver size of \28294; the Philips goose.
Example 2 validation of SNPs associated with \28294Hebeifat deposition in manufacturing practice
The 80-day-old goose under the same feeding and management conditions was selected and tested at 28294U, 21. After 28 days of gavage, all tested \28294; the goslings were slaughtered and the livers were weighed.
The extracted 21 breast muscle tissue-like genomic DNA of \28294Hepsgoose was amplified using the primers in example 1, and the SNP thereof was detected according to the method of example 1.
1、PCR
The PCR reaction system is shown in the following table:
TABLE 1 PCR reaction System
The PCR cycle parameters were as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 50.3 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; prolonging for 10min at 72 deg.C, and storing at 4 deg.C.
2. Genotype frequency analysis of SNP locus of A-FABP gene
The genotype frequency and allele frequency of the mutation site were examined by chi-square method, and the results are shown in Table 2, and the site is in Hardy-Weinberg equilibrium (P > 0.05).
TABLE 2 SNP site genotype and Gene frequency
3. Correlation analysis of A-FABP gene and \28294Hpsfatty liver character
Analysis of the effect of different SNP genotypes on fatty deposition of v 28294jps fatty liver, can be seen in table 3 below: different SNP genotypes have obvious influence on the fatty liver character of \28294Hepugoose; wherein CC genotype is \28294, liver weight of goose is high, CT and TT type individuals are 102.52% and 93.95%, and the difference reaches a significant level (P < 0.05).
TABLE 3 comparison of different genotypes of \28294J.Zasche
Note: the same column with different lower case letters is very different (P < 0.05).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> Hunan agriculture university
<120> 28294musella fatty deposit A-FABP gene and genetic marking method
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 527
<212> DNA
<213> goose (goose)
<220>
<221> mutation
<222> (186)
<223> y ═ c or t
<400> 1
tgtgggattt gctaccagga aaatggctgg tgtggccaag cccaatgtaa ctatcagcat 60
aaatggtgat gtgataacca tcaaatcaga aagtaccttc aaaaatacag agatctcttt 120
caagttgggt gaagagtttg atgagaccac agcagatgac agaaaaacaa aggtgagtct 180
aaaaayggca ttttccaaat acatctctca aaggggatgt cagagcagga ggacagagtc 240
atggttttga catacgctgt tgaaatgatg ccttcagagg taagcataca aagaatcctt 300
tttgctttag tactgcttgg acatggaaac agtcttctct tcagtccata gcaccctgat 360
aaaaagggga aacttacctt ttttctttaa cagaatgtca taactttaga aaatggctca 420
ctgaaacagg tgcagaagtg ggatggcaaa gagactatca taaagagaaa agtggtggat 480
gggaacctgg tcgtggtgag tttgtttttg ttgtctaatc acagaat 527
Claims (1)
1. A method for using A-FABP gene shown as sequence SEQ ID NO.1 as a genetic marker related to the properties of 28294Hebei goose fat liver is characterized by comprising the following steps:
(1) downloading a goose A-FABP gene sequence in GenBank, designing a primer by using Primer5.0, and covering the whole sequence from exon 2 to exon 3 of the gene;
(2) the PCR amplification, product purification and forward sequencing are carried out by taking the genomic DNA of the goose of \28294Hepuas a template to obtain a nucleotide sequence shown as SEQ ID NO.1 of a sequence table;
(3) and identifying whether C-T mutation exists at 186bp position of the obtained sequence of the gene, wherein the CC genotype is \28294whilethe liver weight of the goose is higher than that of CT and TT individuals.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103031307A (en) * | 2012-08-27 | 2013-04-10 | 湖南农业大学 | PPAR (Peroxisome Proliferator Activated Receptor) alpha gene and method of applying PPAR alpha gene as goose fatty deposition related genetic marker |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103031307A (en) * | 2012-08-27 | 2013-04-10 | 湖南农业大学 | PPAR (Peroxisome Proliferator Activated Receptor) alpha gene and method of applying PPAR alpha gene as goose fatty deposition related genetic marker |
Non-Patent Citations (3)
| Title |
|---|
| A novel SNP of liver-type fatty acid-binding protein gene in duck and its associations with the intramuscular fat;He Jun等;《Mol Biol Rep》;20110515(第39期);1073-1077 * |
| Anser anser adipocyte fatty acid binding protein gene，exons 2 and 3 and partial cds;AF442493.1;《Genbank》;20160726;全文 * |
| 影响鹅肥肝形成相关基因分子标记的研究进展;陈继发等;《中国家禽》;20150625;39-42 * |
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