CN110878362A - Detection method for correlation between PRL gene 5' regulatory locus point and chicken testicular character and application - Google Patents
Detection method for correlation between PRL gene 5' regulatory locus point and chicken testicular character and application Download PDFInfo
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Abstract
Description
技术领域technical field
本发明涉及生物基因领域和动物学领域，尤其涉及一种PRL基因5’调控区24bpindel位点与鸡种睾丸性状相关性的检测方法及应用。The invention relates to the field of biological genes and the field of zoology, in particular to a detection method and application of the correlation between a 24bpindel site in the 5' regulatory region of a PRL gene and chicken testis traits.
背景技术Background technique
催乳素(prolactin，PRL)是垂体前叶嗜酸细胞分泌的多肽类激素，是生长激素基因家族的重要成员，对机体生长发育、内分泌与代谢、繁殖与免疫等过程具有重要的调节作用(Bole-feysot等，1998；Gorrin等，2002；Jiang等，2005；Liang等，2006)。禽类PRL多态性及其多态性与母禽繁殖性能的相关性已有大量研究，特别是PRL基因5’调控区24bp indel位点(I58724210D)多态性与母禽繁殖性能如就巢行为等的相关性已得到广泛关注(Jiang等，2005；Liang等，2006；Xu等，2011)。但该位点与地方鸡种睾丸性状的相关性还未见有报道。Prolactin (PRL) is a polypeptide hormone secreted by eosinophils in the anterior pituitary gland and an important member of the growth hormone gene family. -feysot et al., 1998; Gorrin et al., 2002; Jiang et al., 2005; Liang et al., 2006). Avian PRL polymorphisms and their correlations with the reproductive performance of female birds have been extensively studied, especially the 24bp indel site (I58724210D) in the 5' regulatory region of the PRL gene and the reproductive performance of female birds such as nesting behavior. The correlation of et al. has received extensive attention (Jiang et al., 2005; Liang et al., 2006; Xu et al., 2011). However, the correlation between this locus and testis traits of local chicken breeds has not been reported yet.
睾丸是雄性动物重要的生殖器官，具有产生精子和分泌雄激素的功能。在优质鸡育种中，睾丸性状不仅仅是蛋鸡的重要经济性状，更是肉鸡选育的一个重要经济性状(顾敏清，2008；Orlu等，2009；Sarabia等，2013)。种公鸡睾丸大小和重量与精子、精液的数量和质量有直接联系，对鸡群受精率的高低和维持很重要，而且繁殖后期种公鸡的繁殖机能逐渐减退，精液品质大幅下降，从而缩短种公鸡的使用寿命。同时鸡睾丸作为优质鸡消费副产品有着重要的医药及经济价值，我国广东、福建、台湾等地消费者有着食用鸡鹅等禽类睾丸的习惯，目前市场售价可达100元/kg，一只公鸡的睾丸按20g计算，附加值达2元，如果能提高公鸡睾丸的重量则意味着可以提高公鸡的附加值。申请者在早期研究中发现地方鸡种睾丸性状在群体中的变异系数高达80％以上(周敏等，2019)。而评价睾丸生长发育的指标如左右两侧的睾丸重、睾丸指数、睾丸总重是屠宰性状，如直接选择这些指标，在现场育种中必须进行大量的屠宰后通过同胞值进行选择，该方法成本高，工作繁琐。随着分子遗传标记辅助选择在育种中的应用，候选基因法是家鸡进行睾丸性状分子选育行之有效、易于操作的方法。本专利以PRL基因5’调控区24bp indel位点为候选标记检测该位点在地方鸡种宁都黄公鸡中的多态性并分析其与睾丸性状的关系，以便为地方鸡种品种选育及标记辅助选择提供依据。The testis is an important reproductive organ of male animals, which has the functions of producing sperm and secreting androgens. In high-quality chicken breeding, testis trait is not only an important economic trait of laying hens, but also an important economic trait of broiler breeding (Gu Minqing, 2008; Orlu et al., 2009; Sarabia et al., 2013). The size and weight of the testicles of the rooster are directly related to the quantity and quality of sperm and semen, which are very important to the level and maintenance of the fertilization rate of the flock, and the reproductive function of the rooster gradually declines in the post-breeding period, and the quality of the semen drops significantly, thus shortening the rooster. service life. At the same time, chicken testicles have important medical and economic value as a by-product of high-quality chicken consumption. Consumers in Guangdong, Fujian, Taiwan and other places in my country have the habit of eating chicken and goose testicles. At present, the market price can reach 100 yuan/kg. The testicles of 20g are calculated, and the added value is 2 yuan. If the weight of the testicles of the rooster can be increased, it means that the added value of the rooster can be increased. In an early study, the applicant found that the coefficient of variation of testicular traits in local chicken breeds in the population was as high as 80% or more (Zhou Min et al., 2019). The indicators for evaluating the growth and development of testis, such as the testis weight on the left and right sides, the testis index, and the total testis weight are slaughter traits. If these indicators are directly selected, a large number of slaughtering must be carried out in field breeding. High and tedious work. With the application of molecular genetic marker-assisted selection in breeding, candidate gene method is an effective and easy-to-operate method for molecular breeding of testis traits in chickens. In this patent, the 24bp indel site in the 5' regulatory region of the PRL gene is used as a candidate marker to detect the polymorphism of this site in the local chicken breed Ningdu yellow rooster and analyze its relationship with testis traits, so as to select and breed local chicken breeds and Mark-assisted selection provides the basis.
发明内容SUMMARY OF THE INVENTION
为了克服上述缺陷，本发明提供一种利用检测鸡种PRL基因5’调控区24bp indel位点的多态性与睾丸形状相关性的关系，并利用这种关系和方法作为鸡种品种选育和标记的的手段。In order to overcome the above-mentioned defects, the present invention provides a method for detecting the relationship between the polymorphism of the 24bp indel site in the 5' regulatory region of the chicken PRL gene and the correlation between the testis shape, and using this relationship and method as a chicken breed selection and method. means of marking.
为了实现上述发明目的，本发明采用的技术方案为：一种PRL基因5’调控区24bpindel位点与鸡种睾丸性状相关性的检测方法，其特征在于，包括以下步骤：In order to achieve the above-mentioned purpose of the invention, the technical scheme adopted in the present invention is: a method for detecting the correlation between the 24bpindel site in the 5' regulatory region of the PRL gene and the testis traits of chickens, which is characterized in that comprising the following steps:
(1)提取待检测鸡种的基因组DNA；(1) extracting the genomic DNA of the chicken species to be detected;
(2)采用PCR反应扩增扩增PRL基因5’调控区24bp indel位点上下游130bp或154bp片段；(2) PCR reaction amplification is used to amplify the upstream and downstream 130bp or 154bp fragments of the 24bp indel site in the 5' regulatory region of the PRL gene;
(3)将得到的PCR产物直接电泳分型；(3) direct electrophoresis typing of the obtained PCR product;
(4)采用SAS 9.0GLM程序进行统计分析PRL基因5’调控区24bp indel位点基因型与睾丸性状的相关性。(4) SAS 9.0GLM program was used to statistically analyze the correlation between the genotype of the 24bp indel site in the 5' regulatory region of the PRL gene and testis traits.
步骤(1)，在翅下静脉采血1～2mL，用2％EDTA抗凝，-20℃保存备用。所采血样用常规的酚/氯仿抽提法提取基因组DNA，并稀释成100ng/μL备用。In step (1), 1-2 mL of blood was collected from the subwinged vein, anticoagulated with 2% EDTA, and stored at -20°C for later use. The collected blood samples were extracted with genomic DNA by conventional phenol/chloroform extraction method, and diluted to 100ng/μL for use.
步骤(2)从NCBI上下载鸡PRL基因组序列(GenBank登录号：NC_006089)，应用Genetool软件设计上下游引物，引物序列：F:5’-tttaatattggtgggtgaagagaca-3’，R:5’-atgccactgatcctcgaaaactc-3’，扩增PRL基因5’调控区24bp indel(I59724210D)位点上下游130bp或154bp片段。引物由湖南擎科生物有限公司合成。Step (2) Download the chicken PRL genome sequence (GenBank accession number: NC_006089) from NCBI, and use Genetool software to design upstream and downstream primers, primer sequences: F: 5'-tttaatattggtgggtgaagagaca-3', R: 5'-atgccactgatcctcgaaaactc-3' , amplify the 130bp or 154bp fragments upstream and downstream of the 24bp indel (I59724210D) site in the 5' regulatory region of the PRL gene. Primers were synthesized by Hunan Qingke Biological Co., Ltd.
步骤(3)，PCR反应体系为(10μL)：2×PCR mix 5μL，上下游引物各0.2μL，DNA模板0.6μL，ddH2O 4μL。PCR反应程序为：95℃预变性3min；95℃变性20s，54℃退火20s，72℃延伸20s，35个循环；后延伸72℃5min。反应结束后PRL基因取所有扩增产物用3.0％琼脂糖凝胶电泳判断基因型。In step (3), the PCR reaction system is (10 μL): 5 μL of 2×PCR mix, 0.2 μL of upstream and downstream primers, 0.6 μL of DNA template, and 4 μL of ddH 2 O. The PCR reaction program was: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 20 s, annealing at 54 °C for 20 s, extension at 72 °C for 20 s, 35 cycles; post-extension at 72 °C for 5 min. After the reaction, all the amplified products of the PRL gene were taken and 3.0% agarose gel electrophoresis was used to determine the genotype.
步骤(4)中采用SAS 9.0GLM程序进行统计分析，构建模型如下：Yij＝μ+Gi+eij。其中Yij为性状表型值，μ为该性状的总体均值，Gi为基因型效应值，eij为随机残差效应。In step (4), SAS 9.0GLM program is used for statistical analysis, and the model is constructed as follows: Yij=μ+Gi+eij. where Yij is the phenotypic value of the trait, μ is the overall mean value of the trait, Gi is the genotype effect value, and eij is the random residual effect.
PRL基因5’调控区24bp indel位点基因型在检测鸡种睾丸性状相关性中的应用。Application of the genotype of the 24bp indel site in the 5' regulatory region of the PRL gene in the detection of the correlation of testicular traits in chicken breeds.
所述应用具体为通过鸡种PRL基因5’调控区24bp indel位点为候选标记，分析其基因型与睾丸性状的关系，从而对鸡种品种进行选育。The application is specifically that the 24bp indel site in the 5' regulatory region of the PRL gene of the chicken breed is used as a candidate marker, and the relationship between its genotype and the testis traits is analyzed, so as to select and breed the chicken breed.
相对于现有技术，本发明是采用一种检测鸡种PRL基因5’调控区24bpindel位点的多态性与睾丸性状相关性的关系，并利用这种关系和方法作为鸡种品种选育和标记的的手段。这种选育方法相较于现在的屠宰选育，不仅成本低、操作简便，并且结果准确。本方法简单易行，可重复性强，在普通实验室即可进行。Compared with the prior art, the present invention adopts a method for detecting the relationship between the polymorphism of the 24bpindel site in the 5' regulatory region of the chicken breed PRL gene and the correlation between the testis traits, and utilizes this relation and method as a chicken breed selection and breeding method. means of marking. Compared with the current slaughter breeding method, this breeding method not only has low cost, simple operation, but also accurate results. The method is simple, easy to implement, and has strong repeatability, which can be carried out in ordinary laboratories.
附图说明Description of drawings
图1PRL基因5’调控区24bp indel位点的酶切结果；其中：M是DS2000 DNA Marker；5：DI型；1-4、6-9：DD型；10：II型。Fig. 1 The result of enzyme cleavage of the 24bp indel site in the 5' regulatory region of PRL gene; wherein: M is DS2000 DNA Marker; 5: DI type; 1-4, 6-9: DD type; 10: II type.
具体实施方式Detailed ways
为了阐述本发明的技术方案和技术目的，下面具体实施方式对本发明做进一步介绍。In order to illustrate the technical solution and technical purpose of the present invention, the following specific embodiments will further introduce the present invention.
1材料与方法1 Materials and methods
1.1试验材料、指标测定及样品采集1.1 Test materials, index determination and sample collection
试验鸡群由江西南师科技有限公司提供，饲养时间为2018年4月-2018年8月，鸡苗数为700羽，饲养模式为种公鸡笼养模式，按照肉种鸡常规免疫程序进行统一免疫，其它均按照常规方法进行饲养管理。在出生第一天戴上翅号，第5周戴上脚号。饲养至16周龄进行屠宰。指标测定有活体质量、左侧睾丸质量、右侧睾丸质量、睾丸总质量与睾丸指数。其中睾丸指数为(睾丸质量/活体质量)×100.去除死亡、逃逸、以及明显错误与重复数据，最后得到的试验公鸡499只。指标测定参照NY/T 823—2004《家禽生产性能名词术语和度量统计方法》规定的方法。在翅下静脉采血1～2mL，用2％EDTA抗凝，-20℃保存备用。所采血样用常规的酚/氯仿抽提法提取基因组DNA，并稀释成100ng/μL备用。The test flocks were provided by Jiangxi Nanshi Technology Co., Ltd. The breeding time was from April 2018 to August 2018. The number of chicks was 700. The breeding mode was the breeding mode of rooster cages, which was unified according to the routine immunization program of broiler breeders. Immunization and other feeding and management were carried out according to conventional methods. Wear the wing number on the first day of birth and the foot number on the fifth week. Raised to 16 weeks of age for slaughter. The indicators were measured as living body mass, left testis mass, right testis mass, total testis mass and testis index. The testis index is (testis mass/living body mass) × 100. After removing the data of death, escape, and obvious errors and duplicates, 499 test roosters were finally obtained. The index determination refers to the method specified in NY/T 823-2004 "Poultry Production Performance Terminology and Measurement Statistical Methods". 1-2 mL of blood was collected from the subwinged vein, anticoagulated with 2% EDTA, and stored at -20°C for later use. The collected blood samples were extracted with genomic DNA by conventional phenol/chloroform extraction method, and diluted to 100ng/μL for use.
1.2引物设计与合成1.2 Primer design and synthesis
从NCBI上下载鸡PRL基因组序列(GenBank登录号：NC_006089)，应用Genetool软件设计上下游引物，引物序列：F:5’-tttaatattggtgggtgaagagaca-3’，R:5’-atgccactgatcctcgaaaactc-3’，扩增PRL基因5’调控区24bp indel(I59724210D)位点上下游130bp或154bp片段。引物由湖南擎科生物有限公司合成。Download chicken PRL genome sequence (GenBank accession number: NC_006089) from NCBI, use Genetool software to design upstream and downstream primers, primer sequences: F: 5'-tttaatattggtgggtgaagagaca-3', R: 5'-atgccactgatcctcgaaaactc-3', amplify PRL A 130bp or 154bp fragment upstream and downstream of the 24bp indel (I59724210D) site in the 5' regulatory region of the gene. Primers were synthesized by Hunan Qingke Biological Co., Ltd.
PCR反应体系为(10μL)：2×PCR mix 5μL，上下游引物各0.2μL，DNA模板0.6μL，ddH2O 4μL。PCR反应程序为：95℃预变性3min；95℃变性20s，54℃退火20s，72℃延伸20s，35个循环；后延伸72℃5min。反应结束后PRL基因取所有扩增产物用3.0％琼脂糖凝胶电泳判断基因型。The PCR reaction system was (10 μL): 5 μL of 2×PCR mix, 0.2 μL of upstream and downstream primers, 0.6 μL of DNA template, and 4 μL of ddH 2 O. The PCR reaction program was: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 20 s, annealing at 54 °C for 20 s, extension at 72 °C for 20 s, 35 cycles; post-extension at 72 °C for 5 min. After the reaction, all the amplified products of the PRL gene were taken and 3.0% agarose gel electrophoresis was used to determine the genotype.
1.3统计分析1.3 Statistical analysis
由于所观察群体拥有相同的遗传背景，均为16w公鸡，且在饲养标准相同条件下饲养，所以标记与睾丸性状间的关联分析采用SAS 9.0GLM程序进行统计分析，构建模型如下：Yij＝μ+Gi+eij。其中Yij为性状表型值，μ为该性状的总体均值，Gi为基因型效应值，eij为随机残差效应。Since the observed populations have the same genetic background, all 16w roosters are raised under the same feeding standards, the association analysis between markers and testicular traits is performed by SAS 9.0GLM program for statistical analysis, and the model is constructed as follows: Y ij = μ +G i +e ij . where Y ij is the phenotypic value of the trait, μ is the overall mean value of the trait, G i is the genotype effect value, and e ij is the random residual effect.
2结果与分析2 Results and Analysis
PRL基因5’调控区24bp indel(I59724210D)位点不同基因型与睾丸性状的相关性见表1。从表1可以看出，在所测定的6个性状中，不同基因型间的睾丸性状存在显著差异(P<0.05)，DD型个体的16w总睾丸质量、16w睾丸总质量指数、16w左侧睾丸质量、16w左侧睾丸指数极显著高于DI型个体(P<0.01)，DD型个体的16w右侧睾丸质量与16w右侧睾丸指数显著高于DI个体(P<0.05)，6个指标与II型个体差异不显著。The correlation between different genotypes and testis traits at the 24bp indel (I59724210D) site in the 5' regulatory region of the PRL gene is shown in Table 1. It can be seen from Table 1 that among the 6 traits measured, there are significant differences in testicular traits among different genotypes (P<0.05). Testicular mass and 16w left testis index were significantly higher than those of DI type individuals (P<0.01). The 16w right testis mass and 16w right testis index of DD type individuals were significantly higher than those of DI individuals (P<0.05). Six indicators There was no significant difference with type II individuals.
表1位点24bp indel(I59724210D)基因型与笼养宁都黄公鸡睾丸性状的相关性Table 1. Correlation between 24bp indel (I59724210D) genotype and testis traits in caged Ningdu yellow roosters
可根据上述获得的相关性分析结果，针对数据优异的基因型鸡种进行筛选育种。Based on the correlation analysis results obtained above, screening and breeding can be performed for genotype chicken breeds with excellent data.
以上显示和描述了本发明的基本原理和主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。The basic principles and main features of the present invention and the advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments, and the descriptions in the above-mentioned embodiments and the description are only to illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have Various changes and modifications fall within the scope of the claimed invention. The claimed scope of the present invention is defined by the appended claims and their equivalents.
Claims (5)
- The detection method of the correlation between the PRL gene 5' regulatory site and the chicken testicular character is characterized by comprising the following steps:(1) extracting the genome DNA of the chicken species to be detected;(2) amplifying 130bp or 154bp fragments on the upstream and downstream of a 24bp indel site of a PRL gene 5' regulatory region by adopting a PCR reaction;(3) carrying out direct electrophoretic typing on the obtained PCR product;(4) the SAS 9.0GLM program is adopted to carry out statistical analysis on the correlation between different genotypes of 24bp indel sites in the PRL gene 5' regulatory region and the testis traits.
- 2. The method for detecting correlation between PRL gene 5' regulatory site and chicken testicular character according to claim 1, wherein the primer sequence in step (2): 5'-tttaatattggtgggtgaagagaca-3' for F and 5'-atgccactgatcctcgaaaactc-3' for R.
- 3. The method for detecting correlation between PRL gene 5' regulatory locus and chicken testicular character according to claim 1, wherein the step (4) is performed by statistical analysis using SAS 9.0GLM program, and the model is constructed as follows: yij ═ μ + Gi + eij. Wherein YIj is a trait phenotypic value, mu is an overall mean value of the trait, Gi is a genotype effect value, and eij is a random residual effect.
- And 4, application of the 24bp indel locus genotype of the PRL gene 5' regulatory region in detecting the testis character correlation of the chicken breeds.
- 5. Use according to claim 4, characterized in that: the application specifically comprises the steps of analyzing the relationship between the genotype and the testicular character of the cock by taking the 24bp indel site of the 5' regulatory region of the PRL gene of the local chicken as a candidate marker, and breeding local chicken breeders.
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| WO1992013102A1 (en) * | 1991-01-15 | 1992-08-06 | Genmark | Polymorphic dna markers in bovidae |
| CN101063169A (en) * | 2007-06-04 | 2007-10-31 | 西北农林科技大学 | PCR-RFLP method for detecting goat prolactin gene single nucleotide polymorphism |
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