CN102911942A - Application of obese receptor (OB) as goose fat character genetic marker - Google Patents
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Abstract
The invention discloses an obese receptor (OB) gene and application thereof as a goose fat character genetic marker. Genetic markers in the OB gene relevant to goose abdominal fat weight and abdominal fat rate are determined, and the polymorphism in the OB gene in a nucleic acid sample obtained from a goose is determined and causes that the coded amino acid changes and is relevant to the abdominal fat weight and the abdominal fat rate. According to the single nucleotide polymorphism of the OB gene, animals containing the OB genes in a genome DNA are subjected to fatty breeding, and protogene types reducing the abdominal fat weight and abdominal fat rate of the goose are screened, thus the OB gene has important application value and economic benefit.
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 correlated characteristics such as measuring and abdomen fat rate heavy with abdomen fat, more particularly, relate to measure the difference of the OB gene that shows those features, again further specifically, be the polymorphism of measuring in the OB gene, namely weigh the existence of the single nucleotide polymorphism (Single nucleotide polymorphism is abbreviated as SNP) of goose OB (obese) 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 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.
The peptide hormone of a kind of Leptin matter of OB (obese) genes encoding (Leptin) can suppress lipopexia and keep the adipocyte normal in size.Have at present manyly about people, mouse OB gene and on a small quantity about the research report of pig OB gene, but the research of poultry OB gene report is also rarely seen.OB (obese receptor) gene plays effect of the utmost importance at the signal transduction of Lepein, and it is relevant with the body fat deposition, therefore the OB gene can be used as the fatty deposits candidate gene.The cDNA of chicken OB gene is in the Cloning and sequencings such as Taouis in 1998, code length 492bp, and 163 amino acid of encoding contain 18 amino acid signal peptides, become chicken Leptin to be comprised of 146 amino acid.Ashwell etc. detect with Northern and find, the Leptin of chicken has expression in fatty tissue and liver.The cDNA of chicken OB is cloned in Guy Horev in 2000 etc. and Takeshi etc., with the homology average out to 60% of other Mammals OB nucleotide sequences, Dunn etc. with the OB assignment of genes gene mapping of chicken on No. 8 karyomit(e).Gu Zhiliang etc. have reported the partial sequence of the exon 9 of the OB gene of chicken have been carried out Cloning and sequencing, find that a base has sudden change, have produced polymorphism, but aminoacid sequence does not change (silent mutation).Gu Zhiliang etc. further show with the OB gene of PCR-SSCP method research different varieties chicken: Beijing Fatty Chicken AA genotype frequency and other chicken kind there are differences, and high fat is that the A gene frequency is high than low fat, and infer that Gene A may be relevant with the more abdomen fat of deposition.The results of study such as Li Zhihui show, heavy and abdomen fat rate has remarkably influenced to the SNPs genotype of chicken OB gene extron 9 on Abdominal Fat in Broilers, can be for the marker assisted selection of chicken fatty character.
At present, about DNA genetic marker, the genetic diversity chicken relative to the research of correlation function gene of goose also seldom, and not about the research report of goose OB gene.
Summary of the invention
Technical problem to be solved by this invention is: for above-mentioned the deficiencies in the prior art, provide a kind of OB gene and as the purposes of goose fat traits genetic markers; The SNP that simultaneously screening is relevant with the goose fat deposition, marker assisted selection or mark are auxiliary to infiltrate to being applied to, and the application of above-mentioned genetic marker in the goose marker assisted selection is provided.
In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: a kind of OB gene is to weigh with the abdomen fat of expression goose fat proterties and abdomen fat rate and relevant gene.There is the sudden change of an A → G at the 151bp place of above-mentioned OB gene order, causes the existence of polymorphism.
The sequence of clone's OB gene and detection 151A-151G base place's sudden change the primer is as follows:
Forward primer: 5 '-TGCCAGATCTTCCAGGATGA-3 ',
Reverse primer: 5 '-GAGATTCTGAGGTCATTGGC-3 '.
The application of above-mentioned OB gene in goose genetic marker assisted Selection.
A kind of method of identifying the genetic marker of goose fat proterties, this goose has the heavy gene relevant with abdomen fat rate of abdomen fat with expression goose fat proterties, the method comprises measures the existence of polymorphism in the OB gene from the nucleic acid samples that goose obtains, this polymorphism is and the heavy polymorphism relevant with abdomen fat rate of abdomen fat of expression goose fat characteristic index that this polymorphism has the sudden change of a T → G at the 58bp place of OB gene order.
Aforesaid method comprises the clone and obtains an as claimed in claim 1 dna sequence dna of OB gene, amplification purpose fragment length 226bp, and there is a base mutation T58-G58 at the 58th base place.
The sequence of clone's OB gene and detection T58-G58 base place's sudden change the primer is:
Forward primer: 5 '-TGCCAGATCTTCCAGGATGA-3 ',
Reverse primer: 5 '-GAGATTCTGAGGTCATTGGC-3 '.
The application of aforesaid method in the goose breeding.
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 easily bring simultaneously a large amount of depositions of trunk stomach fat and subcutaneous lipids, cause the not good of the waste of fodder energy and carcass quality.For this problem, the inventor selects goose as research object, adopts molecular biological method screening goose fat trait related gene, and its step is as follows:
(1) select 150 of healthy day old chick seedling Xupu geese and 150 of Sichuan White Gooses to carry out feeding experiment.All carry out slaughter determining for the examination goose in 84 ages in days, and measurement and calculation is according to " " performance of poultry vocabulary of terms and measure statistical method " in the Genetic Resources of Domestic Animal technique for investigation handbook carries out.Measure that heavy, the complete clean thorax of carcass is heavy, abdomen fat is heavy and thick etc. with the kind of calliper sebum with electronic scales.Calculate trunk fatty character ratio index.
(2) according to the primers of OB 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 the OB gene is the goose fat deposition, then screen the SNP relevant with fatty character from goose OB gene, and determine that by the production practice checking OB gene is the fatty deposits genes involved, the SNP of screening is associated SNP.The SNP of the goose OB 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.
Description of drawings
Fig. 1 is the PCR-SSCP electrophorogram of goose OB Gene Partial sequence of the present invention.
Embodiment
In order to make purpose of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, 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.
The partial dna sequence that goose OB gene of the present invention obtains according to self-designed primer, the T/G of 58bp place polymorphic site marks expression with parenthesis ().
The sequence that goose OB gene of the present invention obtains is translated as aminoacid sequence figure, and the 20th L of amino acid position place (V) polymorphic site marks expression with parenthesis ().
Embodiment 1 goose genes involved SNP screening
Select 150 of the healthy young seedling Xupu geese of identical age in days, 150 of Sichuan White Gooses carry out feeding experiment.All carry out slaughter determining for the examination goose in 84 ages in days, and measurement and calculation is according to " " performance of poultry vocabulary of terms and measure statistical method " in the Genetic Resources of Domestic Animal technique for investigation handbook carries out.Measure that heavy, the complete clean thorax of carcass is heavy, abdomen fat is heavy and thick etc. with the kind of calliper sebum with electronic scales.Calculate trunk fatty character ratio index.
To the Xupu goose that is about to carry out slaughter determining and Sichuan White Goose on an empty stomach after 12 hours, only in the 5ml centrifuge tube, with 75% ethanol anti-freezing, 4 ℃ of refrigerations of refrigerator are for subsequent use with venous blood collection 1ml/ under Dispoable medical syringe (2.5ml) wing.
To selecting respectively 6 totally 12 samples in the high abdomen fat type in slaughter determining, identified and the low abdomen fat type sample, and extract respectively genomic dna.
OB gene order according to Hongyuan chicken among the GenBank, design with Primer 5.0 programs, cover this gene extron 9 encoding sequences, primer sequence is 5 '-CGGTATGCAGTGAACAAGGA-3 ' (SEQ ID NO:2), 5 '-CATATTCTGACCACGAGGTG-3 ' (SEQ ID NO:3), amplification purpose fragment length 424bp, synthetic by Shanghai English Wei Chuanjin company, respectively the goose genomic dna that extracts is increased with above-mentioned primer.
PCR reaction cumulative volume is 10 μ l, and composition is as follows:
Reaction conditions: 30 PCR loop parameters are after 94 ℃ of 5 minutes denaturations: 95 ℃ 30 seconds, 50 ℃ 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 approximately 16～18 hours under the normal temperature, until blue look tetrabromophenol sulfonphthalein dyestuff is till the gel lowermost end.The gel cma staining is judged genotype according to developing result.As shown in Figure 1, the sscp analysis Bearing performance is: goose OB 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 TT type, and heterozygous is defined as the GT type.
Get pcr amplification product 50 μ l, purified, the clone is by order-checking, and having proved conclusively the PCR product is the OB gene, and the length of PCR product is 226bp, and sequence chart is:
TGCCAGATCTTCCAGGATGACACCAAACCCTCATCAAGACCATTGTCACCAGATCAAT(G)TACATTCACACACGTCGGTATCGCCAAGCAGAGGGTCACTGGCTTGGACTTATTCTGGCTTCACCCCTCTGAGTTTGTCCAAGATGACAGACTCTGCAGTCTATCACAGGTCCTCACCAGCCTGCCTTCCCAAAATGTGCTGCAGATTGCCAATGACCTCAGAATCTC
And from PCR product sequencing result, shown the variant sites of goose OB gene.The result shows that the sudden change of a T → G is arranged at the 58bp place of this gene fragment order.
Above-mentioned product is carried out amino acid sequence analysis, and the result is as follows:
CQIFQDDTKPSSRPLSPDQL(V)HSHTSVSPSRGSLAWTYSGFTPLSLSKMTDSAVYHRSSPACLPKMCCRLPMTSES
Above-mentioned amino acid sequence analysis shows, a variation also occurs the Amino Acids in Proteins sequence that a base mutation of goose OB gene causes this gene to be transcribed when expressing, i.e. the 20th amino acid: the Radix Asparagi α-amino-isovaleric acid becomes leucine.Namely all high abdomen fat is G in 58 bases, and the base of low abdomen fat is T, and what cause the high abdomen fat of coded amino acid is Radix Asparagi α-amino-isovaleric acid (V), and that hang down abdomen fat is leucine (L).
Above-mentioned experimental result shows, trait related because the OB gene from the goose fat that the goose stomach fat extracts, and in this gene order, screen the SNP relevant with abdominal fat sediment, and this base mutation has caused gal4 amino acid that a variation also occurs, 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 abdomen fat rate
Select 150 of healthy day old chick seedling Xupu geese and 150 of Sichuan White Gooses to carry out feeding experiment.
Two kind geese carry out slaughter determining to the examination goose during off-test under identical feeding and management condition, only measure body weight and blood sample collection 2ml/ before killing.Butcher and adopt the neck bloodletting, machine and manually combine and pull out lint and fine, soft fur drains that rear cooling carcass is weighed again and trunk fatty character mensuration.
Trunk fatty character measurement and calculation is according to " " performance of poultry vocabulary of terms and measure statistical method " in the Genetic Resources of Domestic Animal technique for investigation handbook carries out.Its midfield fat is heavy: refer to stomach fat and muscular stomach fatty sum on every side; Abdomen fat rate (%)=abdomen fat weighs/(complete clean thorax weight+abdomen fat is heavy) * 100%.
With each 120 goose blood sample genomic dna of two kinds extracting, utilize the primer amplification among the embodiment 1, and detect its SNP according to the method for embodiment 1.
1、PCR
The PCR reaction system, as shown in table 1:
Each moiety in the table 1 PCR reaction system
| Composition | Consumption (μ l) | Final concentration |
| 10 times of damping fluids | 4.0 | 1X |
| MgCl 2 | 4.0 | 2.5mM |
| dNTPs | 0.8 | 0.2mM |
| The F primer | 0.2 | 0.2μM |
| The R primer | 0.2 | 0.2μM |
| The Taq enzyme | 0.4 | 1U |
| Dna profiling | 0.8 | 50-500ng |
| The Total volume | 40 |
The PCR loop parameter is as follows:
34 circulations: 94 ℃, 4min; 94 ℃, 30s; 59 ℃, 40s.
72℃，7min。
2, the correlation analysis of OB 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 table 2 as seen: heavy and abdomen fat rate has remarkably influenced to different SNP genotype on the abdomen fat of Xupu goose; Wherein TT type abdomen fat is heavy extremely significantly is lower than GG type and GT type (p＜0.01), and TT type abdomen fat rate significantly is lower than GG type and GT type (p＜0.05); The abdomen fat of GG type and GT type heavily reaches abdomen fat rate difference not significantly (P＞0.05); The thick difference of sebum between the different genotype is remarkable (P＞0.05) not.
Table 2 SNP genotype is on the impact of Xupu goose fat proterties
| Genotype | Quantity | Abdomen fat heavy (g) | Sebum thick (mm) | Abdomen fat rate (%) |
| GG | 34 | 29.51±9.05 A | 2.33±0.37 | 1.38±0.41 a |
| TT | 45 | 18.12±6.63 B | 2.12±0.24 | 0.95±0.33 b |
| GT | 41 | 27.21±4.68 A | 2.37±0.31 | 1.33±0.31 a |
Annotate: same row shoulder motes letter is all difference not remarkable (p＞0.05) mutually; Different lowercases are significant difference (p＜0.05); Different capitalizations are difference extremely significantly (p＜0.01).
2.2 different genotype is on the impact of Sichuan White Goose fatty character
Analyze different genotype to the impact of Sichuan White Goose fatty character, the results are shown in Table 3.From table 3 as seen: heavy and abdomen fat rate has remarkably influenced to different genotype equally on the abdomen fat of Sichuan White Goose; Wherein TT type abdomen fat weight and abdomen fat rate all significantly are lower than GG type and GT type (p＜0.05), and the difference of GG type and GT type is remarkable (P＞0.05) not; The thick difference of sebum between the different genotype is remarkable (P＞0.05) not.
Table 3 SNP genotype is on the impact of Sichuan White Goose fatty character
| Genotype | Quantity | Abdomen fat heavy (g) | Sebum thick (mm) | Abdomen fat rate (%) |
| GG | 46 | 29.34±7.24 a | 2.18±0.26 | 1.62±0.36 a |
| TT | 39 | 22.57±4.23 b | 2.08±0.20 | 1.25±0.23 b |
| GT | 35 | 30.33±5.56 a | 2.14±0.33 | 1.54±0.32 a |
Annotate: same row shoulder motes letter is all difference not remarkable (p＞0.05) mutually; Different lowercases are significant difference (p＜0.05).
2.5OB genetic effect
Calculate the effect of each proterties OB gene, the result is as shown in table 4.The contribution rate of an OB gene pairs fattiness proterties is larger, the heavy contribution rate to 12.466 of OB gene pairs Xupu goose abdomen fat wherein, be 13.797 to abdomen fat rate, show that having more than 12.46% in the phenotypic variation of Xupu these proterties of goose is associating variance from the OB gene SNP; The heavy contribution rate to 9.675 of OB gene pairs Sichuan White Goose abdomen fat is 8.456 to abdomen fat rate, shows that having more than 8.45% in the phenotypic variation of these proterties of Sichuan White Goose is associating variance from the OB gene SNP.Xupu goose abdomen fat contribution rate heavy and abdomen fat rate is higher than the contribution rate to Sichuan White Goose in two kinds.
Incidence coefficient between each proterties SNP associating variance and its phenotypic variance is 0.9732, shows that SNP associating variance can reflect the variation of phenotypic variance well.
The contribution rate (%) of table 4 OB gene pairs fatty character
| Kind | Sample number | Abdomen fat is heavy | Sebum is thick | Abdomen fat rate |
| The Xupu goose | 120 | 12.466 | 7.813 | 13.797 |
| Sichuan White Goose | 120 | 9.675 | 6.265 | 8.456 |
In sum, the SNP genotype of OB gene is heavy and abdomen fat rate has extremely significantly or significant impact on the goose abdomen fat of this research, and the TT type is to reduce that goose abdomen fat is heavy, the preponderant genotype of abdomen fat rate, and its contribution rate surpasses 12% to the Xupu goose, to Sichuan White Goose above 8%.Goose abdomen fat heavy contribution rate to 12.466% in OB gene pairs Xupu is 13.797% to abdomen fat rate; The heavy contribution rate to 9.675% of OB gene pairs Sichuan White Goose abdomen fat is 8.456 to abdomen fat rate.Therefore, OB gene of the present invention can be used as the main effect gene of goose fat proterties.The SNP that the present invention screens causes amino acid to change, high abdomen fat be Radix Asparagi α-amino-isovaleric acid (V), low abdomen fat be leucine (L).The polymorphic site of this discovery can be used as goose abdomen fat rate and the heavy molecular genetic marker of abdomen fat carries out marker assisted selection and breeding.
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 (9)
1. an OB gene is characterized in that, described OB gene is to weigh the gene relevant with abdomen fat rate with the abdomen fat of expression goose fat proterties.
2. OB gene as claimed in claim 1 is characterized in that, there is the sudden change of a T → G at the 58bp place of described OB gene order, causes the existence of polymorphism.
3. OB gene as claimed in claim 1 is characterized in that, the sequence of clone's OB gene and detection 58T-58G base place's sudden change the primer is as follows:
Forward primer: 5 '-TGCCAGATCTTCCAGGATGA-3 ',
Reverse primer: 5 '-GAGATTCTGAGGTCATTGGC-3 '.
4. such as each described OB gene application in goose genetic marker assisted Selection in the claims 1 to 3.
5. method of identifying the genetic marker of goose fat proterties, this goose has the heavy gene relevant with abdomen fat rate of abdomen fat with expression goose fat proterties, it is characterized in that, the method comprises measures the existence of polymorphism in the OB gene from the nucleic acid samples that goose obtains, this polymorphism is and the heavy polymorphism relevant with abdomen fat rate of abdomen fat of expression goose fat characteristic index that this polymorphism has the sudden change of a T → G at the 58bp place of OB gene order.
6. method as claimed in claim 5 is characterized in that: the method comprises the clone and obtains an as claimed in claim 1 dna sequence dna of OB gene, amplification purpose fragment length 226bp, and there is a base mutation T58-G58 at the 58th base place; Namely all high abdomen fat is G in 58 bases, and the base of low abdomen fat is T, and what cause the high abdomen fat of coded amino acid is the Radix Asparagi α-amino-isovaleric acid, and what hang down abdomen fat is leucine.
7. method as claimed in claim 6 is characterized in that, the sequence of clone's OB gene and detection T58-G58 base place's sudden change the primer is:
Forward primer: 5 '-TGCCAGATCTTCCAGGATGA-3 ',
Reverse primer: 5 '-GAGATTCTGAGGTCATTGGC-3 '.
8. method as claimed in claim 6 is characterized in that, the described method of the inventor selects goose as research object, adopts molecular biological method screening goose fat trait related gene, and its step is as follows:
(1) select 150 of healthy day old chick seedling Xupu geese and 150 of Sichuan White Gooses to carry out feeding experiment;
(2) according to the primers of OB gene, sample is increased, checks order;
(3) screening and abdomen fat rate relevant SNP just.
9. each described method application in the goose breeding in the claim 5 to 8.
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| CN109022593A (en) * | 2018-08-28 | 2018-12-18 | 扬州大学 | The method that the assisted Selection of liver goose abdominal fat weight and carcass weight marks and utilizes molecular marker assisted selection |
| 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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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109022593A (en) * | 2018-08-28 | 2018-12-18 | 扬州大学 | The method that the assisted Selection of liver goose abdominal fat weight and carcass weight marks and utilizes molecular marker assisted selection |
| CN109022593B (en) * | 2018-08-28 | 2021-07-16 | 扬州大学 | Auxiliary selection marker for abdominal fat weight and carcass weight of liver goose and method for auxiliary selection by using molecular 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 |
| 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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