CN103031307A - PPAR (Peroxisome Proliferator Activated Receptor) alpha gene and method of applying PPAR alpha gene as goose fatty deposition related genetic marker - Google Patents
PPAR (Peroxisome Proliferator Activated Receptor) alpha gene and method of applying PPAR alpha gene as goose fatty deposition related genetic marker Download PDFInfo
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Abstract
The invention discloses a PPAR (Peroxisome Proliferator Activated Receptor) alpha gene and an application of the PPAR alpha gene serving as a goose fatty trait genetic marker. Genetic markers in the PPAR alpha gene, which are related to the abdominal fat weight and abdominal fat percentage of a goose are determined, the existence of polymorphism of the PPAR alpha gene in a nucleic acid sample obtained from the goose is determined, and the polymorphism results in the change of an encoded nucleic acid and is related to the abdominal fat weight and the abdominal fat percentage. The single nucleotide polymorphism of the gene can be used for carrying out fatty trait breeding on animals, genome DNAs (deoxyribonucleic acids) of which contain the PPAR alpha gene, so that a dominant genotype capable of reducing the abdominal fat weight and abdominal fat percentage of the goose is screened out; and the PPAR alpha gene has very great application value and economic benefits.
Description
Technical field
The present invention relates to Protocols in Molecular Biology and the breeding field of goose, specifically relate to the existence of the genetic marker of mensuration and abdomen fat weight, abdomen fat rate correlated characteristic, more particularly, it is the polymorphism of measuring in the PPAR α gene, namely weigh the existence of the single nucleotide polymorphism (Single nucleotide polymorphism is abbreviated as SNP) of goose peroxysome increment thing activated receptor (PPAR) gene relevant with abdomen fat rate with abdomen fat.
Background technology
Theoretical and the development of using of modern poultry genetic breeding, make butcher's beast particularly the speed of growth and the feed efficiency of broiler chicken, meat duck increase substantially, production efficiency is produced to pursue with batch production and is adapted.But also bring easily a large amount of depositions of trunk stomach fat and subcutaneous lipids simultaneously, cause the waste of fodder energy and carcass quality not good.
PPAR (Peroxisome Proliferators activated receptors) belongs to the nuclear hormone receptor superfamily, and it can regulate and control the goal gene of the inside and outside lipid metabolism of many participation cells, also participates in Adipocyte Differentiation.PPAR has three kinds of hypotype: α, β, γ.The target gene of PPAR α is one group of gene that participates in lipolysis, can regulate and control the oxidation of liver peroxysome; PPAR γ participates in the differentiation of adipocyte and the regulation and control of the inside and outside lipid metabolism related objective gene of cell, affecting lipid acid stores in fatty tissue, steatogenesis is worked, the target gene of most of PPAR γ is participated in the lipogenesis approach directly, comprises lipoprotein lipase (LPL), fatty acid binding protein (FABP), acetyl acyl-CoA synthetase and fatty acid transport protein (FATP).The long 593bp of cDNA, the CDS of chicken PPAR α gene (product of mRNA reverse transcription product PCR) is 1407bp.Meng He etc. are studied the possibility of PPAR gene as the chicken fatty character candidate gene, the result shows: with 7 pairs of primers the single base mutation that a C-T is arranged on rear discovery primer 4 amplified fragments is scanned with the PCR-SSCP method in the full coding region of AA broiler chicken PPAR α gene, thereby AA, AB, three genotype of BB appear in the chicken group.The genotypic abdomen fat of BB is heavy and abdomen fat rate is significantly higher, thereby infers that PPAR α gene may be to affect the lipometabolic major gene of chicken or chain with major gene, can be used for the molecular marker assisted selection of chicken fatty character.The result of study of Meng He etc. shows: the frequency of different genotype in indigenous chicken, laying hen, broiler chicken of PPAR α and PPAR γ there are differences; Three kinds of genotype of PPAR α and abdomen fat, abdomen fat rate compole significant correlation, wherein BB type abdomen fat and abdomen fat rate are the highest, and the AA type is minimum.Do not exist this type of relevant between three kinds of genotype of PPAR γ.With the Northern hybrid method as can be known PPAR γ gene a large amount in the fatty tissue of chicken and kidney express.Infer PPAR α and metabolism of fat, deposit closely related.The reports such as Xie Xianglin, there are significant correlation in PPAR Polymorphism and fatty character, Li Chunyu etc. studies show that, still there is significantly relation in the PPAR γ gene SNP s of chicken with abdomen fat weight, abdomen fat rate etc. in the situation of silent mutation, infer that PPAR γ gene can be used as the major gene of chicken body fat proterties.
But, at present the relevant relative chicken with the research of correlation function gene of DNA genetic marker, genetic diversity of goose also seldom, and do not report about the research of goose PPAR α gene.
Summary of the invention
For above-mentioned the deficiencies in the prior art, provide a kind of PPAR α gene and as the purposes of goose fat traits genetic markers; The screening SNP relevant with the goose fat proterties assists infiltration in the hope of being applied to marker assisted selection or mark, and the application of above-mentioned genetic marker in the goose marker assisted selection is provided simultaneously.
The embodiment of the invention is achieved in that a kind of PPAR α gene, the gene that described gene is with the abdomen fat that represents the goose fat characteristic index is heavy, abdomen fat rate is relevant.
Further, there is a base mutation C175-G175 at the 175th base place of described gene PPAR α sequence.
Further, the sequence of clone PPAR α gene the primer is as follows:
Forward primer: 5 '-CATTGGTGTTCGCAGCTGTT-3 ',
Reverse primer: 5 '-CAGAGCTCTCCTCACCGATG-3 '.
Further, sudden change causes coded amino acid to change, and that the high abdomen fat of amino acid be L-glutamic acid (E), and that hang down abdomen fat is glutamine (Q).
It is a kind of with the method for PPAR α gene as the relevant genetic marker of goose fat deposition that another purpose of the embodiment of the invention is to provide, and this comprises the steps:
(1) according to the mRNA sequence of chicken PPAR α gene among the GenBank, with Primer 5.0 design primers, covers this Gene Partial encoding sequence, the goose genomic dna of reentrying;
(2) take above-mentioned goose genomic dna as template, pcr amplification, product purification, the clone obtains the nucleotide sequence shown in sequence table SEQ ID NO.1;
Whether the 175th base place that (3) identifies this gene order exists polymorphism.
Further, described method selects goose as research object, adopts molecular biological method screening goose fat trait related gene, and its step is as follows:
(1) select 200 of the healthy young seedling Xupu geese of identical age in days to carry out feeding experiment;
(2) according to the primers of PPAR α gene, sample is increased, checks order;
(3) screening and abdomen fat rate relevant SNP just.
The method that the present invention adopts PCR to be combined with dna sequencing at first filters out the genes involved that PPAR α gene is the goose fat deposition, then screen the SNP relevant with proterties from goose PPAR α gene, and determine that by the production practice checking PPAR α gene is the fatty deposits genes involved, the SNP of screening is associated SNP.The SNP of the goose PPAR α gene of the present invention's screening can be used as the genetic marker of goose assisted Selection, thereby cultivates abdomen fat goose still less, has earth shaking using value and economic benefit.
Embodiment
In order to make purpose of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, is not intended to limit the present invention.
Embodiment 1 goose genes involved SNP screening
Selecting the healthy young seedling Xupu goose of identical age in days supporting is that 200 of commodity geese carry out feeding experiment.Those geese are carried out high abdomen fat type and the evaluation of low abdomen fat type, and from high abdomen fat type and low abdomen fat type sample, extract respectively its genomic dna.
MRNA sequence according to chicken PPAR α gene among the GenBank, with Primer 5.0 design primers, cover this Gene Partial encoding sequence, primer sequence is: 5 '-CATTGGTGTTCGCAGCTGTT-3 ' (forward), 5 '-CAGAGCTCTCCTCACCGATG-3 ' (oppositely), synthetic by Shanghai English Wei Chuanjin company.Respectively the goose genomic dna that extracts is increased with above-mentioned primer.
PCR:10 μ l reaction system: 10 * buffer (contains Mg
2+) 1 μ l, each 0.15 μ l of upper and lower primer (20 μ mol/L), dNTPs (10 μ mol/L) 0.2 μ l, Taq enzyme 1U, DNA (100ng) 0.7 μ l, ultrapure water is mended to volume required.
The pcr amplification program: 30 PCR loop parameters are after 94 ℃ of 5 minutes denaturations: 95 ℃ 30 seconds, 57 ℃ 30 seconds, 72 ℃ 30 seconds, last 72 ℃ 7 minutes.
Get amplified production 5 μ l, add denaturing agent (95% first ammonium nitrate) 10 μ l, 95 ℃ of sex change 10 minutes, ice bath is after 5 minutes again, point sample is electrophoresis (acrylamide/N, N '-methylene-bisacrylamide are 29: 1) on 15% polyacrylamide gel, and voltage remains on 80～100V, electrophoresis is about 16～18 hours under the normal temperature, until blue look tetrabromophenol sulfonphthalein dyestuff is till the gel lowermost end.The gel cma staining, the sscp analysis Bearing performance is: goose PPAR α Gene Partial dna fragmentation has three kinds of different type of strip, illustrates that there is SNP in this dna fragmentation.Three kinds of different type of strip are defined as respectively GG type and CC type, and heterozygous is defined as the GC type.
Get pcr amplification product 50 μ l, serve the order-checking of Hai Yingwei wound Tianjin company behind the product purification, it is PPAR α gene that sequencing result has been proved conclusively the PCR product, and the length of PCR product is 464bp, and sequence is shown in the SEQ ID NO:1 in the sequence table.Listed PPAR α gene order is carried out sequence analysis among the GenBank with goose PPAR alpha gene fragment sequence and other 16 species, the result shows: goose PPAR alpha gene fragment sequence has 95% above homology with animals such as kind duck, family duck, mouse, and with the having more than 80% of the animals such as chicken, people, ox, sheep, further specifying PPAR α gene has higher conservative property during evolution.
Above-mentioned product is carried out amino acid sequence analysis, result's following (seeing SEQ ID NO:2):
HWCSQLFWGLEGSWSPGTGATLGPLVVPWRGGHTGGDIGALPSHGVVHLGVSHREAFPE(Q)TLGELRGEPSASSRPCLPCELTFVAGQVREQDVVITALRSVACLATSIQHGGHREPHLSSLPTGGRGPGKSLIARILTRNGKHSRDFSVHRAGEL
Amino acid sequence analysis shows that the Amino Acids in Proteins sequence that a base mutation of goose PPAR α gene order causes this gene to be transcribed morphs when expressing, L-glutamic acid (E) sports glutamine (Q).
Above-mentioned experimental result shows, the trait related because PPAR α gene of the goose fat that extracts from the goose stomach fat, and in this gene order, screen the SNP relevant with abdominal fat sediment, probably Here it is affects the reason of abdominal fat sediment.
The checking of SNP in production practice that embodiment 2 is relevant with abdomen fat rate
Select 200 of the healthy Xupu geese of identical age in days and identical feeding and management condition to carry out feeding experiment.All carry out slaughter determining for the examination goose in 84 ages in days, only measure body weight and blood sample collection 2ml/ before the government official, and carcass is weighed and trunk fatty character mensuration again.
With 186 goose blood sample genomic dnas that extract, and utilize primer amplification among the embodiment 1, and detect its SNP according to the method for embodiment 1.
1、PCR
The PCR reaction system, as follows:
The PCR loop parameter is as follows:
34 circulations: 94 ℃, 4min; 94 ℃, 30s; 59 ℃, 40s.
72℃，7min。
2, the correlation analysis of PPAR α genotype and goose fat proterties
2.1 different genotype is on the impact of Xupu goose fat proterties
Analyze different SNP genotype to the impact of Xupu goose fat proterties, the results are shown in Table 2.From following table 2 as seen: different SNP genotype are heavy on the abdomen fat of Xupu goose, abdomen fat rate has remarkably influenced; Wherein the heavy and abdomen fat rate of the abdomen fat of CC type all significantly is lower than GC type and GG type (p＜0.05), and the heavy and abdomen fat rate difference of the abdomen fat of GC type and GG type is not remarkable (P＞0.05) all; Liver weight between the different genotype and liver rate difference is remarkable (P＞0.05) not.
Table 1SNP genotype is on the impact of fatty character
| Genotype | Quantity | Abdomen fat heavy (g) | Liver heavy (g) | Abdomen fat rate (%) | Liver rate (%) |
| GG | 63 | 12.26±1.51 a | 52.8±3.20 | 0.59±0.11 a | 1.92±0.20 |
| CC | 81 | 10.11±1.25 b | 50.6±3.76 | 0.46±0.09 b | 1.95±0.18 |
| GC | 42 | 12.64±1.78 a | 52.3±2.96 | 0.63±0.10 a | 2.01±0.21 |
Annotate: same row shoulder motes letter is all difference not remarkable (p＞0.05) mutually; Different lowercases are significant difference (p＜0.05).
2.2PPAR the α genetic effect is analyzed
Calculate the contribution rate of each proterties of PPAR α gene pairs, the result is as shown in table 2.The contribution rate of PPAR α gene pairs fatty character is larger.Wherein goose abdomen fat heavy contribution rate to 10.16% in PPAR α gene pairs Xupu is 11.35% to abdomen fat rate, shows that having more than 10% in the phenotypic variation of these two proterties of Xupu goose is associating variance from PPAR α gene SNP.Incidence coefficient between each proterties SNP associating variance and its phenotypic variance is 0.982, shows that SNP associating variance can reflect the variation of phenotypic variance well.
The contribution rate of table 2PPAR α gene pairs fatty character
| Sample number | Abdomen fat heavy (g) | Liver heavy (g) | Abdomen fat rate (%) | Liver rate (%) |
| 186 | 10.16 | 2.534 | 11.350 | 2.267 |
In sum, the SNP genotype of the PPAR α gene of Xupu goose is heavy on the abdomen fat of goose, abdomen fat rate has significant impact, wherein the heavy and abdomen fat rate of the abdomen fat of CC type all significantly is lower than GC type and GG type (p＜0.05), and the heavy and abdomen fat rate difference of the abdomen fat of GC type and GG type is not remarkable (P＞0.05) all; Liver weight between the different genotype and liver rate difference is remarkable (P＞0.05) not.PPAR α gene can be used as the major gene of goose body fat proterties.The CC genotype is that reduction goose abdomen fat is heavy, the preponderant genotype of abdomen fat rate, and its contribution rate surpasses 10%.
The heavy contribution rate to 10.16% of PPAR α gene pairs Xupu goose abdomen fat is 11.35% to abdomen fat rate, shows that having more than 10% in the phenotypic variation of these two proterties of Xupu goose is associating variance from PPAR α gene SNP.Incidence coefficient between each proterties SNP associating variance and its phenotypic variance is 0.982, shows that SNP associating variance can reflect the variation of phenotypic variance well.This further show PPAR α gene can be used as the goose fat proterties the main effect gene or with control ventral fat character key-gene chain.
The above only is preferred embodiment of the present invention, not in order to limiting the present invention, all any modifications of doing within the spirit and principles in the present invention, is equal to and replaces and improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. a PPAR α gene is characterized in that, the gene that described gene is with the abdomen fat that represents the goose fat characteristic index is heavy, abdomen fat rate is relevant.
2. PPAR α gene as claimed in claim 1, it is characterized in that: there is a base mutation C175-G175 at the 175th base place of described gene PPAR α sequence.
3. PPAR α gene as claimed in claim 1 is characterized in that, the sequence of clone PPAR α gene the primer is as follows:
Forward primer: 5 '-CATTGGTGTTCGCAGCTGTT-3 ',
Reverse primer: 5 '-CAGAGCTCTCCTCACCGATG-3 '.
4. PPAR α gene as claimed in claim 2 is characterized in that, sudden change causes coded amino acid to change, and that the high abdomen fat of amino acid be L-glutamic acid (E), and that hang down abdomen fat is glutamine (Q).
5. one kind with the method for PPAR α gene as the relevant genetic marker of goose fat deposition, it is characterized in that this comprises the steps:
(1) according to the mRNA sequence of chicken PPAR α gene among the GenBank, with Primer 5.0 design primers, covers this Gene Partial encoding sequence, the goose genomic dna of reentrying;
(2) take above-mentioned goose genomic dna as template, pcr amplification, product purification, the clone obtains the nucleotide sequence shown in sequence table SEQ ID NO.1;
Whether the 175th base place that (3) identifies this gene order exists polymorphism.
6. method as claimed in claim 5 is characterized in that, described method selects goose as research object, adopts molecular biological method screening goose fat trait related gene, and its step is as follows:
(1) select 200 of the healthy young seedling Xupu geese of identical age in days to carry out feeding experiment;
(2) according to the primers of PPAR α gene, sample is increased, checks order;
(3) screening and abdomen fat rate relevant SNP just.
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| CN109371033A (en) * | 2018-11-23 | 2019-02-22 | 湖南农业大学 | The method of Xupu geese fatty liver fat deposition A-FABP gene and genetic marker |
| CN109797223A (en) * | 2018-12-14 | 2019-05-24 | 湖南农业大学 | Obr gene and the method marked as Xupu geese fatty liver fat deposition correlated inheritance |
| CN110241235A (en) * | 2019-07-26 | 2019-09-17 | 湖南农业大学 | ADSL gene and genetic marking method as XuPu breeder geese inosine acid content |
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| CN101921770A (en) * | 2010-04-27 | 2010-12-22 | 曲湘勇 | PPAR (Peroxisome Proliferator-Activated Receptor) alpha gene and application thereof as goose fat traits genetic markers |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109371033A (en) * | 2018-11-23 | 2019-02-22 | 湖南农业大学 | The method of Xupu geese fatty liver fat deposition A-FABP gene and genetic marker |
| CN109371033B (en) * | 2018-11-23 | 2021-10-22 | 湖南农业大学 | \28294A-FABP gene of fatty deposition of fatty liver of goose in West province and genetic marking method |
| CN109797223A (en) * | 2018-12-14 | 2019-05-24 | 湖南农业大学 | Obr gene and the method marked as Xupu geese fatty liver fat deposition correlated inheritance |
| CN109797223B (en) * | 2018-12-14 | 2022-06-21 | 湖南农业大学 | OBR gene and method for using same as genetic marker related to fatty deposition of \28294ugoose fat liver |
| CN110241235A (en) * | 2019-07-26 | 2019-09-17 | 湖南农业大学 | ADSL gene and genetic marking method as XuPu breeder geese inosine acid content |
| CN110241235B (en) * | 2019-07-26 | 2022-06-21 | 湖南农业大学 | ADSL gene and genetic marking method for its content of \28294u-goose inosinic acid |
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