CN103031307B - 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 measured with the genetic marker of abdomen fat weight, abdominal fat correlated characteristic, more particularly, measure the polymorphism in PPAR α gene, namely with abdominal fat relevant goose peroxysome heavy to abdomen fat rises in value the existence of single nucleotide polymorphism (Single nucleotide polymorphism, is abbreviated as SNP) of thing activated receptor (PPAR) gene.
Background technology
The development of modern poultry genetic breeding Theory and applications, make butcher's beast particularly the speed of growth of broiler chicken, meat duck and feed efficiency increase substantially, production efficiency and factorial praluction are by adapting.But also easily bring 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 nuclear hormone receptor superfamily, and it can regulate and control the goal gene of many participation intraor extracellular lipid metabolism, also participates in Adipocyte Differentiation.PPAR has three kinds of hypotypes: α, β, γ.The target gene of PPAR α is one group of gene participating in lipolysis, can regulate and control hepatic peroxisomes oxidation; PPAR γ participates in the differentiation of adipocyte and the regulation and control of intraor extracellular lipid metabolism related objective gene, affect lipid acid to store in fatty tissue, steatogenesis is worked, the target gene of major part PPAR γ participates in lipogenesis approach directly, comprises lipoprotein lipase (LPL), fatty acid binding protein (FABP), acetyl acyl-CoA synthetase and fatty acid transport protein (FATP).CDNA long 593bp, CDS (product of mRNA reverse transcription product PCR) of chicken PPAR α gene are 1407bp.Meng He etc. are studied the possibility of PPAR gene as chicken fatty character candidate gene, result shows: scan by 7 pairs of primer pair AA broiler chicken PPAR α gene full coding region PCR-SSCP methods the single base mutation rear discovery primer 4 amplified fragments having a C-T, thus AA, AB, BB tri-genotype appear in chicken group.BB genotypic abdomen fat is heavy and abdominal fat is significantly higher, thus infers that PPAR α gene may be affect chicken lipometabolic major gene 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; PPAR α tri-kinds of genotype and abdomen fat, abdominal fat compole significant correlation, wherein BB type abdomen fat and abdominal fat the highest, AA type is minimum.There is not these type of between three kinds of genotype of PPAR γ to be correlated with.Express by Northern hybrid method known PPAR γ gene a large amount in the fatty tissue and kidney of chicken.Infer PPAR α and metabolism of fat, deposit closely related.The reports such as Xie Xianglin, there is significant correlation in PPAR Polymorphism and fatty character, the research such as Li Chunyu shows, still there is remarkable relation with abdomen fat weight, abdominal fat etc. when silent mutation in the PPAR γ gene SNP s of chicken, infers that PPAR γ gene can be used as the major gene of chicken body fat proterties.
But, the current DNA genetic marker about goose, genetic diversity chicken relative to the research of correlation function gene 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 the purposes as goose fat traits genetic markers thereof; The SNP that screening is relevant to goose fat proterties simultaneously, to being applied to marker assisted selection or the auxiliary infiltration of mark, and provides the application of above-mentioned genetic marker in goose marker assisted selection.
The embodiment of the present invention is achieved in that a kind of PPAR α gene, and described gene is and the gene that abdomen fat is heavy, abdominal fat is relevant representing goose fat characteristic index.
Further, there is a base mutation C175-G175 at the 175th base place of described gene PPAR α sequence.
Further, the sequence of cloning 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 amino acid height abdomen fat is L-glutamic acid (E), low abdomen fat be glutamine (Q).
Another object of the embodiment of the present invention is a kind of method providing PPAR α gene as the relevant genetic marker of goose fat deposition, and this comprises the steps:
(1) according to the mRNA sequence of chicken PPAR α gene in GenBank, design primer with Primer 5.0, cover this Gene Partial encoding sequence, goose genomic dna of reentrying;
(2) with above-mentioned goose genomic dna for template, pcr amplification, product purification, clone, obtain the nucleotide sequence as shown in sequence table SEQ ID NO.1;
(3) identify whether the 175th base place of this gene order exists polymorphism.
Further, described method choice goose is as research object, and adopt molecular biological method to screen goose fat trait related gene, its step is as follows:
(1) the young seedling XuPu breeder geese 200 of the health of identical age in days is selected only to carry out feeding experiment;
(2) according to the primers of PPAR α gene, sample is increased, checks order;
(3) SNP that screening is relevant to abdominal fat height.
First the method that the present invention adopts PCR to be combined with DNA sequencing filters out PPAR α gene is the genes involved that goose fat deposits, then the SNP relevant to proterties is screened from goose PPAR α gene, and determine that PPAR α gene is fatty deposits genes involved by production practice checking, 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, thus cultivates the less goose of abdomen fat, has earth shaking using value and economic benefit.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, 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, be not intended to limit the present invention.
Embodiment 1 goose genes involved SNP screens
The health of identical age in days young seedling XuPu breeder geese breed system commercial goose 200 is selected only to carry out feeding experiment.High abdomen epoxy-type and the qualification of low abdomen epoxy-type are carried out to those geese, and extract its genomic dna respectively from high abdomen epoxy-type and low abdomen epoxy-type sample.
According to the mRNA sequence of chicken PPAR α gene in GenBank, primer is designed with Primer 5.0, cover this Gene Partial encoding sequence, primer sequence is: 5 '-CATTGGTGTTCGCAGCTGTT-3 ' (forward), 5 '-CAGAGCTCTCCTCACCGATG-3 ' (oppositely), is synthesized by Ying Weichuan Tianjin, Shanghai company.Respectively the goose genomic dna extracted is increased with above-mentioned primer.
PCR:10 μ l reaction system: 10 × buffer is (containing Mg
2+) 1 μ l, upper and lower primer (20 μm of ol/L) each 0.15 μ l, dNTPs (10 μm of ol/L) 0.2 μ l, Taq enzyme 1U, DNA (100ng) 0.7 μ l, ultrapure water is mended to volume required.
Pcr amplification program: after 94 DEG C of 5 minutes denaturations, 30 PCR loop parameters are: 95 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 30 seconds, last 72 DEG C 7 minutes.
Get amplified production 5 μ l, add denaturing agent (95% first ammonium nitrate) 10 μ l, 95 DEG C of sex change 10 minutes, ice bath is after 5 minutes again, point sample is electrophoresis (acrylamide/N, N '-methylene-bisacrylamide be 29: 1) on the polyacrylamide gel of 15%, and voltage remains on 80 ~ 100V, electrophoresis about 16 ~ 18 hours under normal temperature, until blue look Bromophenol Blue dye to gel lowermost end.Gel cma staining, sscp analysis result shows as: goose PPAR α Gene Partial DNA fragmentation has three kinds of different type of strip, illustrates that this DNA fragmentation exists SNP.Three kinds of different type of strip are defined as GG type and CC type respectively, and heterozygous is defined as GC type.
Get pcr amplification product 50 μ l, serve the order-checking of Hai Yingwei Chuan Jin company after product purification, it is PPAR α gene that sequencing result confirms PCR primer, and the length of PCR primer is 464bp, and sequence is as shown in the SEQ ID NO:1 in sequence table.Listed PPAR α gene order in the GenBank of goose PPAR alpha gene fragment sequence and other 16 species is carried out sequence analysis, result shows: the animals such as goose PPAR alpha gene fragment sequence and kind duck, family duck, mouse have more than 95% homology, and with the animals such as chicken, people, ox, sheep have more than 80%, further illustrating PPAR α gene has higher conservative property during evolution.
Above-mentioned product is carried out amino acid sequence analysis, result following (see SEQ ID NO:2):
HWCSQLFWGLEGSWSPGTGATLGPLVVPWRGGHTGGDIGALPSHGVVHLGVSHREAFPE(Q)TLGELRGEPSASSRPCLPCELTFVAGQVREQDVVITALRSVACLATSIQHGGHREPHLSSLPTGGRGPGKSLIARILTRNGKHSRDFSVHRAGEL
Amino acid sequence analysis shows, the Amino Acids in Proteins sequence that a base mutation of goose PPAR α gene order causes this gene to be transcribed when expressing morphs, and L-glutamic acid (E) sports glutamine (Q).
Above-mentioned experimental result shows, the goose fat extracted from goose stomach fat is trait related because PPAR α gene, and screens the SNP relevant with abdominal fat sediment in this gene order, and probably Here it is affects the reason of abdominal fat sediment.
The checking of SNP in production practice that embodiment 2 is relevant with abdominal fat
The healthy XuPu breeder geese 200 of identical age in days and identical feeding and management condition is selected only to carry out feeding experiment.Allly carry out slaughter determining for examination goose in 84 ages in days, only measure body weight blood sample collection 2ml/ before killing, and carcass is weighed and carcass lipid property determination again.
With 186 the goose blood sample genomic dnas extracted, and utilize the primer amplification in embodiment 1, and detect its SNP according to the method for embodiment 1.
1、PCR
PCR reaction system, as follows:
PCR loop parameter is as follows:
34 circulations: 94 DEG C, 4min; 94 DEG C, 30s; 59 DEG C, 40s.
72℃，7min。
2, the correlation analysis of PPAR α genotype and goose fat proterties
2.1 different genotype are on the impact of XuPu breeder geese fatty character
Analyze different SNP genotype to the impact of XuPu breeder geese fatty character, the results are shown in Table 2.From following table 2: different SNP genotype is heavy on the abdomen fat of XuPu breeder geese, abdominal fat has remarkably influenced; Wherein the abdomen fat of CC type is heavy and abdominal fat is all remarkable in GC type and GG type (p < 0.05), the heavy and abdominal fat difference all remarkable (P > 0.05) of the abdomen fat of GC type and GG type; Liver between different genotype weighs and liver rate difference remarkable (P > 0.05).
Table 1SNP genotype is on the impact of fatty character
| Genotype | Quantity | Abdomen fat heavy (g) | Liver heavy (g) | Abdominal fat (%) | 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 |
Note: same row shoulder note letter is all difference not significantly (p > 0.05) mutually; Different lowercase is significant difference (p < 0.05).
2.2PPAR α gene effect analysis
Calculate the contribution rate of each proterties of PPAR α gene pairs, result is as shown in table 2.The contribution rate of PPAR α gene pairs fatty character is larger.The contribution rate that wherein PPAR α gene pairs XuPu breeder geese abdomen fat is heavy, to 10.16%, is 11.35% to abdominal fat, shows to have in the phenotypic variation of these two proterties of XuPu breeder geese more than 10% to be associating variance from PPAR α gene SNP.The incidence coefficient that each proterties SNP combines between variance and its phenotypic variance is 0.982, shows that SNP associating variance can reflect the change of phenotypic variance well.
The contribution rate of table 2PPAR α gene pairs fatty character
| Sample number | Abdomen fat heavy (g) | Liver heavy (g) | Abdominal fat (%) | Liver rate (%) |
| 186 | 10.16 | 2.534 | 11.350 | 2.267 |
In sum, the SNP genotype of the PPAR α gene of XuPu breeder geese is heavy on the abdomen fat of goose, abdominal fat has significant impact, wherein the abdomen fat of CC type is heavy and abdominal fat is all remarkable in GC type and GG type (p < 0.05), the heavy and abdominal fat difference all remarkable (P > 0.05) of the abdomen fat of GC type and GG type; Liver between different genotype weighs and liver rate difference remarkable (P > 0.05).PPAR α gene can be used as the major gene of goose body fat proterties.CC genotype is that reduction goose abdomen fat is heavy, the preponderant genotype of abdominal fat, and its contribution rate is more than 10%.
The heavy contribution rate of PPAR α gene pairs XuPu breeder geese abdomen fat, to 10.16%, is 11.35% to abdominal fat, shows to have in the phenotypic variation of these two proterties of XuPu breeder geese more than 10% to be associating variance from PPAR α gene SNP.The incidence coefficient that each proterties SNP combines between variance and its phenotypic variance is 0.982, shows that SNP associating variance can reflect the change of phenotypic variance well.This shows that PPAR α gene can be used as the main effect gene or chain with the key-gene controlling ventral fat character of goose fat proterties further.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. a molecule marker for goose PPAR α gene, is characterized in that having SNP site, and this site is positioned at Position Number based on the 175th of such as nucleotide sequence shown in SEQ ID NO.1, is 175C/G.
2. molecule marker according to claim 1, is characterized in that, nucleotides sequence is classified as SEQ ID NO.1.
3. a goose PPAR α gene mutation body, it is characterized in that, compared with being distinguished as of wild-type goose PPAR α gene, there is single base mutation 175C-175G, this Position Number is based on the such as nucleotide sequence shown in SEQ ID NO.1, this gene is heavy to the abdomen fat of expression goose fat characteristic index, abdominal fat is relevant, described single base mutation causes the amino acid coded by it to sport L-glutamic acid by glutamine, changes into high ventral fat character by low ventral fat character.
4. increase the primer pair of molecule marker described in claim 1 or 2, it is characterized in that,
Forward primer: 5 '-CATTGGTGTTCGCAGCTGTT-3 ',
Reverse primer: 5 '-CAGAGCTCTCCTCACCGATG-3 '.
5. the application of molecule marker in goose marker assisted selection of a claim 1 or 2.
6. the molecule marker of claim 1 or 2 is as a method for the relevant genetic marker of goose fat deposition, it is characterized in that, comprises the steps:
(1) according to the mRNA sequence of chicken PPAR α gene in GenBank, design primer pair with Primer 5.0, cover this Gene Partial encoding sequence, goose genomic dna of reentrying, described primer pair is the primer pair of claim 4;
(2) use the primer pair of design in step (1) and goose genomic dna to be template, pcr amplification, product purification, clone, obtain the molecule marker of claim 2;
(3) identifying the polymorphism situation at the 175th base place in described molecule marker, is low ventral fat character when its encode glutamine, is high ventral fat character during encoding glutamate, and described Position Number is based on the such as nucleotide sequence shown in SEQ ID NO.1.
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| 家禽单核苷酸多态性研究与应用新进展;卢立志;《农业生物技术学报》;20101231;第18卷(第2期);374-381 * |
| 用PCR-SSCP筛选PPAR基因SNP的研究;曲湘勇;《中国家禽业—机遇与挑战》;20071231;331-334 * |
| 鹅脂肪性状候选基因的研究;曲湘勇;《中国博士论文全文数据库》;20080715;第2008年卷(第7期);第四章、摘要 * |
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