CN105063037B - Hu sheep prolactin gene SNP locus and application thereof - Google Patents
Hu sheep prolactin gene SNP locus and application thereof Download PDFInfo
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- CN105063037B CN105063037B CN201510524900.0A CN201510524900A CN105063037B CN 105063037 B CN105063037 B CN 105063037B CN 201510524900 A CN201510524900 A CN 201510524900A CN 105063037 B CN105063037 B CN 105063037B
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Abstract
The invention discloses an application of Hu sheep prolactin gene SNP loci, wherein the SNP loci are PRL4244 loci, PRL1228 loci, PRL1039 loci, PRL2351 loci and PRL4591 loci. The method has obvious significance for improving the lactation yield or the lactation character of the Hu sheep by researching the SNP locus of the prolactin gene of the Hu sheep.
Description
Technical Field
The invention relates to Hu sheep prolactin gene SNP locus and application thereof, belonging to the technical field of molecular biology.
Background
The Hu sheep is originally produced in the Taihu lake basin and has the characteristics of good maternal property, strong lactation performance and early sexual maturity. And because of the property of high yield and high birth, the mutton sheep has become a good breed for the domestic mutton sheep hybridization improvement. In recent years, the research on sheep genetic breeding mainly focuses on the polymorphism research on the economic trait functional gene, and relatively few researches on the milk production performance are related. However, the sheep have the advantages of high fecundity and multiple births, relative insufficient nutrition and low lactation force of ewes, so that the lamb dysplasia, low resistance and low survival rate are caused, and the advantages of high fecundity and multiple births cannot be exerted. Therefore, the test is based on molecular genetics, researches on the high-fecundity Hu sheep lactation genes, discovers the marker genes for controlling sheep lactation amount in future, and provides technical support for sheep molecular breeding.
Studies have shown that the chemical elements in different types of sheep milk are substantially the same, but the milk yield is affected by factors such as genetics, number of fetuses, environment (v.m. russo2013), age, nutritional level, sunlight, number of young animals, physiological status, milking pattern, etc. The lactation trait of livestock is a quantitative trait and is controlled by multiple genes, and in recent years, research on the lactation genes is mainly focused on cattle. The primary function of the prolactin gene (PRL) is to promote mammary development, milk production, initiation and play an important role in the maintenance of lactation in mammalian individuals. Therefore, the PRL gene is considered as a candidate gene having an important effect on the lactation performance of livestock. The sheep Prolactin (PRL) gene has the full length of 8468bp, and contains 5 exons and 4 introns.
The lactation trait is a typical quantitative trait controlled by multiple micro-effect genes, and is a trait which is rarely studied in sheep in recent years. At present, the tissue structure, action mechanism and relation of prolactin gene with reproductive traits such as milk secretion are still little understood at home and abroad, and a great deal of exploration work needs to be carried out on the aspects so as to provide a new way for high-efficiency breeding for improving the reproductive capacity of sheep. Therefore, how to obtain the SNP locus of the Hu sheep prolactin gene to improve the milk yield of the Hu sheep or the milk secretion character of the Hu sheep is a technical problem to be solved in the field.
Disclosure of Invention
The invention aims to solve the technical problem of providing Hu sheep prolactin gene SNP locus and application thereof. In order to achieve the purpose, the invention adopts the following technical scheme:
the Hu sheep prolactin gene SNP locus application, the gene PRL4244 locus is GG type, PRL1228 locus is AC type, PRL1039 locus is AC type, PRL2351 locus is AA type or AC type or PRL4591 locus is CC type, in Hu sheep breeding application.
The invention is characterized by comprising the following steps: the application is in improving the lactation amount or improving the lactation traits of Hu sheep.
The invention has obvious significance for improving the milk yield or improving the milk secretion character of the Hu sheep by researching the SNP locus of the Hu sheep prolactin gene.
Detailed Description
The technical contents of the present invention will be described in detail with reference to specific embodiments.
1 materials and methods
1.1 phenotypic data Collection and blood sample Collection on test animals
41 Hu sheep in lactation period with basically consistent gestation times (1-2), age, body condition, expected delivery period, nutritional status and the like are randomly selected in the Yiling breeder sheep farm outside the east region of Wulu wood Qi Mi. The experimental animals are mainly fed with leguminous and gramineous grasses, and concentrated and supplemented with concentrated feed for one time. The flocks are kept in pens to feed and drink water freely. Blood samples were taken intravenously, anticoagulated with heparin sodium, and stored at-20 ℃.
Collection of milk and determination of milk composition: at the 4 th day after production (the lambs eat the colostrums as much as possible), after the lambs and the ewes are separated, milk is squeezed out in an artificial lactation mode, and the lactation amount of the ewes is measured at 7 am, 13 am for 30 min and 20 pm respectively on the next day. Measuring the date, respectively taking 25ml of the collected milk samples in the morning, the middle and the evening, uniformly mixing, freezing and storing at the temperature of-20 ℃, and sending to a laboratory.
The samples were tested using the FOSS 5000 series tester, imported from Denmark, where Integrated Milk testing Fossmatic 5000 was used to determine Milk composition and Integrated Milk testing MilkoScan FT4000 was used to determine somatic cell count. The measurement indices include a milk protein ratio (%), a milk fat ratio (%), a lactose ratio (%), a total solid, and the like.
1.2 extraction of genomic DNA
Extracting Hu sheep genome DNA by phenol-chloroform method, sterilizing, dissolving in TE, diluting, and storing at-20 deg.C. The DNA purity was checked with a spectrophotometer and then diluted to 50 ng/. mu.l for use.
1.3 PCR primers and reagents
Primers (table 1) were designed using primer design software primer5.0 based on the sheep prolactin gene sequence published in GenBank (GenBank accession No. NW _004080183.1), and synthesized from shanghai; protease K, taq was obtained from Takara, phenol, chloroform, and ethanol from chemical reagent of Jinan.
PCR reaction system and reaction conditions
And (3) PCR reaction system: 10 × buffer (containing Mg) 2+ ) mu.L, 10pmol each of the upstream and downstream primers 2.5. mu.L, Taq DNA polymerase 3U, dNTPs (2.5. mu.l/L) 5. mu.L,1 μ L of DNA, and ultrapure water was added to 50 μ L.
The primers used were amplified by gradient PCR and the optimal annealing temperature for each primer pair was determined. Reaction conditions for PCR: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 52-62 ℃ for 45s, and extension at 72 ℃ for 1-2min for 35 cycles in total; extension at 72 ℃ for 5 min. Storing at 4 ℃. The PCR amplified product was detected by 2% agarose gel electrophoresis.
1.5 statistical analysis
And comparing the sequence and the peak image of the PRL gene obtained by sequencing by adopting BioEdit, Mega6.0 and Chromas to detect the SNP locus. Statistical data are calculated by SPSS13.0 statistical software, and the genetic effect of the PRL genotype on the milk production character is analyzed by a fixed model. And (3) carrying out significance test on the detected character difference of different genotypes of the Hu sheep.
TABLE 1 PRL Whole Gene primers
1.6 PCR results of PRL Gene
The annealing temperature of the primers is screened by adopting gradient PCR, PCR products are selected and detected by 1.5 percent agarose gel electrophoresis, the result shows that the bands are clear, no primer dimer exists, the specificity is good, and the PCR products are sent to Shanghai to carry out sequencing.
1.7SNP site screening
PRL gene sequence data returned from Shanghai workers were collated using Bioedit and aligned using MEGA 6.0. Peaks were looked at using Chromas to find 23 SNP sites.
After the correlation analysis of the single SNP locus genotype of the PRL gene and the lactation character of the Hu sheep in the lactation period, the following results are found: only PRL4244 site is associated with lactation yield, the other 4 sites are associated with lactation trait. The results are shown in tables 2 to 6.
TABLE 2 significant analysis of Hu sheep PRL4244 site and lactation yield
TABLE 3 analysis of the correlation between single SNP site of Hu sheep and lactation trait
TABLE 3 correlation analysis of Hu sheep prolactin gene PRL1228 site and lactation trait
TABLE 4 correlation analysis of PRL1039 site of Hu sheep prolactin gene and lactation character
TABLE 5 correlation analysis of Hu sheep prolactin gene locus PRL2351 points and lactation traits
TABLE 6 correlation analysis of Hu sheep prolactin gene site PRL4591 point and lactation character
As can be seen from tables 2, 3, 4, 5 and 6, the Hu sheep has 5 total lactation yields and lactation traits related to the SNP sites, which are shown in the statistical significance of the SNP sites.PRL4244 site GG typeThe milk secretion in the eighth week is significantly higher than that in the eighth weekTT and TG types(P < 0.05); the AC type later period milk fat rate of the PRL1228 locus is remarkably higher than that of the CC type (P is less than 0.01), and the middle total solid AC type is remarkably higher than that of the CC type (P is less than 0.05);the AC type middle-stage milk fat rate of the PRL1039 locus is remarkably higher than those of the AA type and the CC type; PRL2351 site in type AC The milk fat rate in the middle stage is obviously higher than AA type and CC type (P is less than 0.05), the protein rate in the middle stage of AA type is obviously higher than AC type and CC type, and the protein rate in the middle stage of AC type is obviously higher than that in the middle stage of AC type The total solid is obviously higher than AA type and CC type; the CC type early milk fat rate of PRL4591 locus is remarkably higher than that of TC type and TT type (P <) 0.01), the middle protein rate and early total solids are also significantly higher with type CC than with type TC and TT(P＜0.01)
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. Any obvious modifications thereof, which would be obvious to one skilled in the art without departing from the true spirit of the invention, would constitute a violation of the patent rights of the present invention and would bear corresponding legal responsibility.
Claims (2)
1. The application of Hu sheep prolactin gene SNP locus in improving lactation yield in Hu sheep breeding is characterized in that the SNP locus PRL4244 locus is GG type.
2. The application of Hu sheep prolactin gene SNP locus in improving lactation character in Hu sheep breeding is characterized in that the SNP locus PRL1228 locus is AC type, PRL1039 locus is AC type, PRL2351 locus is AA type or AC type or PRL4591 locus is CC type.
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| CN110387373A (en) * | 2019-07-24 | 2019-10-29 | 上海交通大学 | Chinese ring-necked pheasant stimulating milk secretion fibroin and its encoding gene and application |
| CN110878362B (en) * | 2019-12-18 | 2023-03-28 | 南昌师范学院 | Detection method for correlation between PRL gene 5' regulatory locus point and chicken testicular character and application |
| CN113265476B (en) * | 2021-07-21 | 2021-11-09 | 中国农业大学 | Gene chip, molecular probe combination, kit and application for analyzing milk production performance of sheep |
Citations (1)
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| CN101063169A (en) * | 2007-06-04 | 2007-10-31 | 西北农林科技大学 | PCR-RFLP method for checking goat luteotropin gene mononucleotide polymorphism |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101063169A (en) * | 2007-06-04 | 2007-10-31 | 西北农林科技大学 | PCR-RFLP method for checking goat luteotropin gene mononucleotide polymorphism |
Non-Patent Citations (4)
| Title |
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| PRL和PL基因SNP与天府肉羊哺乳期产奶量及产羔数的关联性分析;黄磊;《中国优秀硕士学位论文全文数据库农业科技辑》;20110415;D050-44 * |
| 催乳素基因多态性及其与小尾寒羊高繁殖力相关性的研究;代鸥;《中国优秀硕士学位论文全文数据库农业科技辑》;20080215;D050-83 * |
| 湖羊PRL_PRLR基因与泌乳性状相关性分析;李晓林;《中国优秀硕士学位论文全文数据库农业科技辑》;20160415;D050-130 * |
| 湖羊催乳素基因多态性与泌乳性状的相关性分析;李晓林 等;《福建农业学报》;20151115;第30卷(第11期);1032-1040 * |
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