WO2022233345A2 - 影响鸭脂肪酸组成相关基因elovl3的snp分子标记及检测方法和应用 - Google Patents

影响鸭脂肪酸组成相关基因elovl3的snp分子标记及检测方法和应用 Download PDF

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WO2022233345A2
WO2022233345A2 PCT/CN2022/111329 CN2022111329W WO2022233345A2 WO 2022233345 A2 WO2022233345 A2 WO 2022233345A2 CN 2022111329 W CN2022111329 W CN 2022111329W WO 2022233345 A2 WO2022233345 A2 WO 2022233345A2
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elovl3
gene
duck
fatty acid
upstream
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侯卓成
李晓琴
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中国农业大学
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • Y02P60/87—Re-use of by-products of food processing for fodder production

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  • the invention belongs to the field of molecular biology and poultry breeding, and particularly relates to a SNP molecular marker affecting duck fatty acid composition-related gene ELOVL3, a detection method and application.
  • Meat and poultry is an important part of China's livestock and poultry production. It is easy to raise and has the characteristics of high production performance. It is the second largest meat consumption in the world. With the demand for healthy and diversified food in modern society, the market share of poultry products has gradually increased due to the advantages of balanced nutrition, safety and health care, and unique flavor. The consumption structure has been continuously upgraded, and consumers are more interested in the quality, safety and nutritional value of poultry products.
  • Single nucleotide polymorphisms are caused by a single nucleotide variation at the genomic level and account for more than 90% of all known polymorphisms, averaging 1 in every 300 base pairs.
  • Genome-wide association analysis GWAS
  • GWAS Genome-wide association analysis
  • GS Selection
  • MAS and GS breeding can rapidly purify trait-related alleles, which not only speeds up the breeding efficiency and the progress of genetic selection, but also avoids the separation of commercial generation traits in the creation of matching lines, making breeding more efficient. Breeds of livestock and poultry with excellent traits.
  • Unsaturated fatty acid is a type of fatty acid containing double bonds in the carbon chain. Due to the different number of double bonds, it is divided into monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (ployunsaturated fatty acid). , PUFA).
  • MUFA monounsaturated fatty acid
  • PUFA polyunsaturated fatty acid
  • SFA saturated fatty acid
  • PUFA polyunsaturated fatty acids
  • LCPUFA Long chain polyunsaturated fatty acid
  • LCPUFA has important physiological functions on the human body, and has the functions of softening blood vessels, strengthening the brain and improving eyesight.
  • n-3 series PUFAs are the main components that exert these functions, and their positive effects on health are mainly by regulating and inhibiting the acid synthesis pathway of eicosanoids, thereby changing the inflammatory response and related molecular protein expression activity, regulation and normalization
  • Molecular and enzymatic activities in various signaling pathways related to pathological cell function, and n-3 fatty acids can be incorporated into membrane phospholipids to directly affect gene expression, all of these pathways are highly related, and n-3 fatty acids are biologically beneficial for health and disease The role is accomplished by a variety of coordination mechanisms.
  • PUFAs in poultry meat are higher than those in livestock meat, which can be used as an important source of human polyunsaturated fatty acid intake.
  • the poultry study found that the content of PUFAs in duck was higher than that in chicken, and in the expression data, the expression of ELOVL3 in the fatty acid extension family gene was significantly higher in duck than in chicken.
  • ELOVL3 is significantly expressed in white adipose tissue, and this gene has functions such as regulating the endogenous synthesis of saturated ultra-long-chain fatty acids and triglycerides.
  • the promoter region determines the initiation of transcription and regulates the strength of gene expression. It plays a very important role in the study of gene promoter transcription regulation.
  • the promoter structure of ELOVL3 has not been analyzed, so through the analysis of duck ELOVL3 gene promoter activity, it is clear that the Gene promoter regulatory region, while predicting candidate transcription factor binding sites in the core promoter region, combined with the results of genome-wide association analysis and selection signal analysis, to find candidate causal mutations in the regulatory region of ELOVL3, which can be verified by experiments.
  • the selection and breeding of new poultry varieties with polyunsaturated fatty acid ratio provides theoretical support and basic data, which is of great significance to improve their edible value and promote the development of related industries.
  • the present application provides a SNP molecular marker that affects duck fatty acid composition-related gene ELOVL3, the SNP molecular marker is located at -619bp upstream of the ELOVL3 gene.
  • nucleotide at the SNP molecular marker is AA, GG or GA.
  • the present application provides the application of the above SNP molecular markers in judging the expression level of ELOVL3 in ducks.
  • the present application provides the application of the above SNP molecular markers in determining the content of long-chain polyunsaturated fatty acids in duck meat.
  • the present application provides the application of using the above SNP molecular markers in selecting ducks with high content of long-chain polyunsaturated fatty acids in duck meat.
  • the method includes detecting a nucleotide at -619 bp upstream of the ELOVL3 gene.
  • nucleotide at the upstream -619bp of ELOVL3 gene is AA genotype
  • the individual has higher carbon chain elongation ability of medium and long chain polyunsaturated fat
  • the meat has higher content of medium and long chain polyunsaturated fat.
  • the application includes using the nucleotide at the upstream -619 bp of the ELOVL3 gene to select the cultivar of the AA genotype.
  • the method used to detect the nucleotide at the upstream-619bp of the ELOVL3 gene is selected from, sequencing method, restriction fragment length polymorphism analysis, melting curve analysis, single-stranded conformational polymorphism analysis, probe amplification Blocking Mutagenesis, Chromatographic Methods.
  • the present invention provides a pair of RACE primers for amplifying duck polyunsaturated fatty acid composition-related gene ELOVL3 coding region primers and a complete cDNA sequence, the nucleotide sequence of which is:
  • CDS region amplification primer pair SEQ ID NO.1 and SEQ ID NO.2;
  • cDNARACE primer pair SEQ ID NO.3 and SEQ ID NO.4.
  • the present invention provides truncated primer pairs of different lengths for amplifying the upstream regulatory region of duck polyunsaturated fatty acid composition-related gene ELOVL3, the nucleotide sequences of which are: SEQ ID NO.5-SEQ ID NO.12.
  • the present invention provides the core promoter region of the ELOVL3 gene, which is located in the upstream region of the ELOVL3 gene at -765bp to +62bp.
  • the present application also provides a kit for carrying out the above-mentioned application or comprising the above-mentioned primers.
  • the methods for detecting polymorphisms in this application are not limited to the sequencing methods listed above, restriction fragment length polymorphism analysis, melting curve analysis, single-strand conformation polymorphism analysis, probe amplification blocking mutations, chromatography method, other known or under-study methods can also be applied to the present invention.
  • the kit of the present application may contain various reagents required for detection, including but not limited to primers, probes, buffers, enzymes, and the like.
  • ELOVL3 is a key enzyme controlling the carbon chain extension of long-chain fatty acids in animals.
  • the invention combines GWAS analysis and cell test to identify the SNP molecular marker located in the upstream region of the ELOVL3 gene that affects its expression activity, further detects the individual phenotype effect and uses it in MAS, and selects the dominant genotype that is favorable for improving the fatty acid content of duck meat Reservation of seeds can quickly improve the content of long-chain polyunsaturated fatty acids and meat quality in duck meat, which will speed up the process of breeding and improvement of high-quality ducks, and bring great impetus and objective economic benefits to the development of duck industry.
  • the present invention obtains for the first time a SNP molecular marker related to the expression activity of the polyunsaturated fatty acid composition-related gene ELOVL3, which is the A ⁇ G mutation at the upstream -619 site of the gene, and the individual with the phenotype dominant genotype AA provides a test for early selection. Data basis, thereby greatly improving the breeding efficiency of new lines, and providing a new method for marker-assisted selection of high-quality ducks.
  • Figure 1A and Figure 1B are the genome-wide association analysis (GWAS) Manhattan plot of Peking duck muscle fat content and the Manhattan plot of the association analysis of this trait on chromosome 7; the abscissa represents the duck's chromosome number; the ordinate represents the SNP locus -logP value;
  • GWAS genome-wide association analysis
  • Figure 2 is a schematic diagram of the complete cDNA structure of the duck ELOVL3 gene, including three parts: 5' UTR (5' non-coding sequence); CDs (coding sequence); 3' UTR (3' non-coding sequence);
  • Fig. 3 is the electrophoresis result of the truncated fragment in the upstream promoter region of Peking duck ELOVL3 gene;
  • Figure 4 is the measurement result of the relative activity of the double luciferase of the truncated fragment of the duck ELOVL3 promoter region, the abscissa is the relative fluorescence activity value; the ordinate is the truncated region of the promoter fragment;
  • Figure 5 shows the sequencing of the single-point mutation sequence at the -619 site (A ⁇ G) in the core promoter region of the duck ELOVL3 gene
  • Figure 6 shows the results of the relative activity assay of dual luciferase under different bases at the -619 site in the core promoter region of duck ELOVL3.
  • the abscissa is the relative fluorescence activity value; the ordinate is the different bases in the core promoter region-619 site.
  • Example 1 Amplification of the complete cDNA sequence of duck polyunsaturated fatty acid composition-related gene ELOVL3
  • the primer sequences are: 5'-GGACAGGCCAGAGGTCACT-3' (forward, SEQ ID NO.1), 5'-GAACAGGCGGCATGGTCACT-3' (reverse, SEQ ID NO.2), cDNA obtained by reverse transcription of wild duck liver RNA Sequences containing the complete protein coding regions (CDs) of the gene are amplified for templates.
  • PCR 20 ⁇ L reaction system: 10 ⁇ L of 2 ⁇ PCR SuperMix, 0.5 ⁇ L of upstream and downstream primers (10 ⁇ mol/L), 1 ⁇ L of DNA template, and 8 ⁇ L of ultrapure water.
  • PCR amplification procedure pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30s, annealing at 60°C for 30s, extension at 72°C for 1 min, a total of 35 cycles; extension at 72°C for 10min, and finally storage at 4°C.
  • GPS1 (SEQ ID NO. 3): 5'-GATTACGCCAAGCTTGCCGCCGCCAGGCACCATCTCCTTGTA-3';
  • GPS2 (SEQ ID NO. 4): 5'-GATTACGCCAAGCTTCGCGTCTTCCATGGGATTCAAGCAGTCGG-3'.
  • the 5' and 3' RACE reactions were performed according to the operating instructions of the 5'/3' RACE kit (RACE 5'/3' Kit, Takara bio, USA).
  • the target fragment was purified using a purification kit (NucleoSpin Ge and PCR Clean-Up Kit, Takara bio, USA).
  • the 5'UTR region contains 143bp; the CDs region is 810bp; the 3'UTR region is 771bp, as shown in Figure 2.
  • the above primer synthesis and sequencing were completed by Beijing Qingke Biotechnology Co., Ltd.
  • ELOVL3-2095-F1 (SEQ ID NO. 5): ggcctaactggccggtaGGTACCAGTGAAGAGCTCCACGCACT;
  • ELOVL3-1856-F2 (SEQ ID NO. 6): ggcctaactggccggtaGGTACCTGGGAAACAAGTCATGTCCA;
  • ELOVL3-1518-F3 (SEQ ID NO. 7): ggcctaactggccggtaGGTACCCAGGGCAATTCCTCTAGCAT;
  • ELOVL3-1249-F4 (SEQ ID NO. 8): ggcctaactggccggtaGGTACCCCCTGGGACAGCTCACAGTA;
  • ELOVL3-765-F5 (SEQ ID NO. 9): ggcctaactggccggtaGGTACCCACATCCTTCCATGCCACAT;
  • ELOVL3-572-F6 (SEQ ID NO. 10): ggcctaactggccggtaGGTACCAAAGATGATGCCCAAATTGC;
  • ELOVL3-389-F7 (SEQ ID NO. 11): ggcctaactggccggtaGGTACCGATAACTGGGTGCAGGTTCC;
  • ELOVL3-+62-R (SEQ ID NO. 12): cgaggccagatcttgatatcCTCGAGCTCCGCTCCACCTCATACTC.
  • PCR 20 ⁇ L reaction system: 10 ⁇ L of 2 ⁇ PCR SuperMix, 0.5 ⁇ L of upstream and downstream primers (10 ⁇ mol/L), 1 ⁇ L of DNA template, and 8 ⁇ L of ultrapure water.
  • PCR amplification procedure pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30s, annealing at 60°C for 30s, extension at 72°C for 1 min, a total of 35 cycles; extension at 72°C for 10min, and finally storage at 4°C.
  • the target fragment was purified using a purification kit (TaKaRa MiniBEST AgaroseGel DNA Extraction Kit Ver.4.0, Takara bio, USA).
  • Each fragment was constructed into the linearized pGL4.10 reporter vector using homologous recombination reagents (Seamless Cloning and Assembly Kit, Transgen bio, China).
  • the chicken immortalized cell line ICP was cultured at 37°C, 5% CO 2 concentration and 95% air humidity.
  • the medium was DMEM/F12 containing 10% fetal bovine serum and 1% double antibody, and the cells were in good condition.
  • the counts were plated in 24-well plates, and the plating density was able to grow to 80% in 24h.
  • transfection was carried out when the adherent coverage of cells reached about 80%, and a total of 50 ⁇ L of expression vector (0.475 ⁇ g), pRL-TK (0.025 ⁇ g), liposome (1.0 ⁇ L) and Opti-MEM were mixed in each well. After incubation for 10 min, the cells were transfected into ICP1 cells and replaced with fresh medium after 6 h.
  • Embodiment 3 Verification of SNP mutation effect related to the above-mentioned duck fatty acid composition-related gene ELOVL3 core promoter activity
  • the original expression plasmid was used as the template, and the amplification primers with single base mutation were introduced from the online website (https://crm.vazyme.com/cetool/singlepoint.html) for reverse amplification.
  • the primer sequences are:
  • the chicken immortalized cell line ICP was cultured at 37°C, 5% CO 2 concentration and 95% air humidity.
  • the medium was DMEM/F12 containing 10% fetal bovine serum and 1% double antibody, and the cells were in good condition.
  • the counts were plated in 24-well plates, and the plating density was able to grow to 80% in 24h.
  • transfection was carried out when the adherent coverage of cells reached about 80%, and a total of 50 ⁇ L of expression vector (0.475 ⁇ g), pRL-TK (0.025 ⁇ g), liposome (1.0 ⁇ L) and Opti-MEM were mixed in each well. After incubation for 10 min, the cells were transfected into ICP1 cells and replaced with fresh medium after 6 h.

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Abstract

本申请提供了影响鸭脂肪酸组成相关基因ELOVL3的SNP分子标记及其在选育肉鸭、鉴定肉质等方面的应用，本申请中的SNP分子标记位于ELOVL3基因上游-619bp处，基因型为AA、GG或GA。本发明结合GWAS分析及细胞试验鉴别到了位于ELOVL3基因上游区域影响其表达活性的SNP，进一步检测确定AA型个体具有比GG型个体更高的多不饱和脂肪酸碳链延长能力，选择对提高鸭肉脂肪酸含量有利的优势基因型进行留种，可迅速提高鸭肉长链多不饱和脂肪酸含量以及肉品质，将加快优质鸭育种改良的进程，为鸭的产业发展带来极大的推动作用和客观的经济效益。

Description

影响鸭脂肪酸组成相关基因ELOVL3的SNP分子标记及检测方法和应用
本申请要求于2022年04月01日提交中国专利局、申请号为202210336713.X、发明名称为“影响鸭脂肪酸组成相关基因ELOVL3的SNP”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于分子生物学领域和家禽选育领域，具体涉及影响鸭脂肪酸组成相关基因ELOVL3的SNP分子标记及检测方法和应用。
背景技术
肉禽是中国畜禽生产的重要组成部分，具有易饲养，生产性能高的特点，是世界第二大消费肉类。随着现代社会对健康和多样化食品的需求，家禽产品因其营养均衡、安全保健和风味独特等优势，市场占有率逐步上升，在生活水平提高、生活方式变迁等因素的推动下，家禽产品消费结构不断升级，消费者对家禽产品质量安全和营养价值更加青睐。
单核苷酸多态性(SNP)由基因组水平上由单个核苷酸变异引起，占所有已知多态性的90％以上，平均每300个碱基对中就有1个。全基因组关联分析(GWAS)基于SNP间的连锁不平衡来鉴别影响表型和基因之间关系，可以有效挖掘与主选性状相关的分子标记，并应用于分子标记辅助选择(MAS)、全基因组选择(GS)来实现目标性状的早期选择。相比传统育种方法，MAS和GS选育使性状相关等位基因快速纯和，不但加快了选育效率和遗传选择进展，也可在配套系创制中避免商品代性状分离，更高效地选育出性状优良的畜禽品种。
不饱和脂肪酸(unsaturated fatty acid)是碳链中含有双键的一类脂肪酸，因含有双键的数量不同，分为单不饱和脂肪酸(monounsaturated fatty acid，MUFA)和多不饱和脂肪酸(ployunsaturated fatty acid，PUFA)。饱和脂肪酸(SFA)含量高不易被身体吸收，研究发现，SFA长期摄入量过多会导致高血脂症，形成动脉粥样硬化，增加心脑血管疾病的发生风险。相反，临床研究证明，多不饱和脂肪酸(PUFA)有利于人体健康，因其能调节人体脂质代谢，减少血液中胆固醇和甘油三酯含量，从而降低血液黏稠度并改善血液微循环，对心脑血管疾病的治疗和预防有重要价值；同 时还被证明，其能增强人体免疫防御功能以及排除过量SFA形成的多余脂肪，具有减肥的功效。长链多不饱和脂肪酸(long chainployunsaturated fatty acid，LCPUFA)是指链长为18-22个碳原子的PUFA，LCPUFA广泛存在于生物体内，其中最受关注且功能研究比较清楚的3个成员分别是AA、EPA和DHA。LCPUFA对人体具有重要的生理功能，具有软化血管、健脑益智、改善视力等功效。其中，n-3系列PUFAs是发挥这些功能的主要成分，其对健康的积极影响主要通过调节和抑制类二十烷酸的酸合成途径，进而改变炎症反应和相关分子蛋白表达活性、调节与正常及病理细胞功能有关的各种信号通路中分子及酶活性，且n-3脂肪酸可并入膜磷脂直接影响基因的表达，所有这些通路均高度相关，n-3脂肪酸对健康和疾病的生物有益作用是靠多种协调机制完成的。
然而，对于PUFA，人体自身大多不能合成，需要从外界摄取。由此，多不饱和脂肪酸在食品、健康辅料、化妆品和制药中的潜在市场是十分巨大的。目前，不饱和脂肪酸仅能从高等植物种子和深海鱼油中获取。但是受限于其易氧化的性质和工艺复杂的提炼过程，无法满足市场及广大人民需求。因此，长期以来，人们在不断探求可替代的生物资源。因禽肉具有普遍性，禽类养殖产量庞大，且已有研究表明，禽肉中的PUFAs高于畜肉，可作为人类多不饱和脂肪酸摄入的重要来源。禽类研究发现，鸭肉中PUFAs含量高于鸡肉，而在表达数据中，脂肪酸延伸家族基因中ELOVL3的表达鸭显著高于鸡。且ELOVL3在白色脂肪组织中具有显著表达，该基因具有调节饱和超长链脂肪酸和甘油三酯的内源性合成等作用。目前，国内外对其它家畜脂肪酸组成的研究较多，但有关禽肉的脂肪酸组成及合成机制报道还较少。启动子区域决定转录起始，调控基因表达的强弱，研究基因的启动子转录调控具有非常重要的作用，关于ELOVL3的启动子结构尚未解析，所以通过对鸭ELOVL3基因启动子活性分析，明确该基因启动子调控区，同时预测核心启动子区的候选转录因子结合位点，并结合全基因组关联分析及选择信号分析结果，找到ELOVL3调控区的候选因果突变，通过实验加以验证，可为后续高多不饱和脂肪酸比例禽类新品种的选育工作提供理论支撑和基础数据，对提高其食用价值和推动相关产业发展具有重要意义。
发明内容
为解决上述问题，一方面，本申请提供了影响鸭脂肪酸组成相关基因ELOVL3的SNP分子标记，所述SNP分子标记位于ELOVL3基因上游-619bp处。
进一步地，所述SNP分子标记处核苷酸为AA、GG或GA。
另一方面，本申请提供了上述SNP分子标记在判断鸭中ELOVL3表达水平中的应用。
另一方面，本申请提供了上述SNP分子标记在判断鸭肉长链多不饱和脂肪酸含量中的应用。
另一方面，本申请提供了使用上述SNP分子标记在选育鸭肉中长链多不饱和脂肪酸含量高的鸭中的应用。
进一步地，所述方法包括检测ELOVL3基因上游-619bp处的核苷酸。
进一步地，ELOVL3基因上游-619bp处的核苷酸为AA基因型时个体有更高的中长链多不饱和脂肪碳链延长能力，其肉中有更高的中长链多不饱和脂肪含量。
进一步地，所述应用包括使用ELOVL3基因上游-619bp处的核苷酸为AA基因型的个体选育品种。
进一步地，检测ELOVL3基因上游-619bp处的核苷酸使用的方法选自，测序法、限制性片段长度多态性分析、熔解曲线分析、单链构象异构多态性分析、探针扩增阻滞突变、色谱方法。
另一方面，本发明提供了扩增鸭多不饱和脂肪酸组成相关基因ELOVL3编码区引物及完整cDNA序列的RACE引物对，其核苷酸序列为：
CDS区域扩增引物对：SEQ ID NO.1和SEQ ID NO.2；
cDNARACE引物对：SEQ ID NO.3和SEQ ID NO.4。
另一方面，本发明提供了扩增鸭多不饱和脂肪酸组成相关基因ELOVL3上游调控区不同长度的截断引物对，其核苷酸序列为：SEQ ID NO.5-SEQ ID NO.12。
另一方面，本发明提供了ELOVL3基因的核心启动子区域，其位于ELOVL3基因上游区域为-765bp～+62bp处。
另一方面，本申请还提供了用于进行上述应用或者包含上述引物的试 剂盒。
本申请中检测多态性的方法不限于上面所列的测序法、限制性片段长度多态性分析、熔解曲线分析、单链构象异构多态性分析、探针扩增阻滞突变、色谱方法，其他已知或研究中的方法也可以应用于本发明。
根据所用方法不同，本申请的试剂盒中可以包含各种检测所需的试剂，包括但不限于引物、探针、缓冲液、酶等。
有益效果：ELOVL3是动物体内控制长链脂肪酸碳链延伸的关键酶。本发明结合GWAS分析及细胞试验鉴别到了位于ELOVL3基因上游区域影响其表达活性的SNP分子标记，进一步检测个体表型效应后将其用于MAS中，选择对提高鸭肉脂肪酸含量有利的优势基因型进行留种，可迅速提高鸭肉长链多不饱和脂肪酸含量以及肉品质，将加快优质鸭育种改良的进程，为鸭的产业发展带来极大的推动作用和客观的经济效益。本发明首次得到与多不饱和脂肪酸组成相关基因ELOVL3表达活性相关的一个SNP分子标记，为该基因上游-619位点的A<G突变，表型优势基因型AA的个体，为早期选择提供试验数据依据，从而大大地提高新品系选育效率，为优质鸭的标记辅助选择提供了新的方法。
附图说明
图1A和图1B为北京鸭肌肉脂肪含量的全基因组关联分析(GWAS)曼哈顿图及该性状在7号染色体上关联分析的曼哈顿图；横坐标表示鸭的染色体编号；纵坐标表示SNP位点的-logP值；
图2为鸭ELOVL3基因完整的cDNA结构示意图，包括三个部分：5’UTR(5’非编码序列)；CDs(编码序列)；3’UTR(3’非编码序列)；
图3为北京鸭ELOVL3基因上游启动子区域截断片段电泳结果；
图4为鸭ELOVL3启动子区域截断片段双荧光酶相对活性测定结果，横坐标为相对荧光活性值；纵坐标为启动子片段截断区域；
图5为鸭ELOVL3基因核心启动子区域-619位点(A<G)单点突变序列测序情况；
图6为鸭ELOVL3核心启动子区域-619位点不同碱基情况下的双荧光酶相对活性测定结果。横坐标为相对荧光活性值；纵坐标为核心启动子区域-619位点的不同碱基情况。
具体实施方式
为了使本发明的目的、技术方案更加清楚明白，以下结合实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。若未特别指明，实施例所用技术手段为本领域技术人员所熟知的常规手段。
实施例1鸭多不饱和脂肪酸组成相关基因ELOVL3完整cDNA序列的扩增
根据Ensembl(http://asia.ensembl.org/)数据库中获取鸭7号染色体ELOVL3(ENSAPLG00020013672)的cDNA序列，分别在5’UTR和3’UTR区域设计扩增引物。
引物序列为：5’-GGACAGGCCAGAGGTCACT-3’(正向，SEQ ID NO.1)，5’-GAACAGGCGGCATGGTCACT-3’(反向，SEQ ID NO.2)，以野鸭肝脏RNA反转录获得的cDNA为模板扩增包含该基因完整蛋白编码区域(CDs)的序列。
PCR：20μL反应体系：2×PCR SuperMix 10μL，上下游引物(10μmol/L)各0.5μL，DNA模板1μL，超纯水补8μL。
PCR扩增程序：94℃预变性5min；94℃变性30s，60℃退火30s，72℃延伸1min，共35个循环；72℃后延长10min，最后4℃保存。
以扩增得到的CDs序列为模板，首先分别设计5’RACE特异性引物GPS1和3’RACE特异性引物GPS2，引物前端根据线性化pUC19载体序列分别添加15bp同源序列GATTACGCCAAGCTT，引物序列为：
GPS1(SEQ ID NO.3)：5’-GATTACGCCAAGCTTGCCGCCGCCAGGCACCATCTCCTTGTA-3’；
GPS2(SEQ ID NO.4)：5’-GATTACGCCAAGCTTCGCGTCTTCCATGGGATTCAAGCAGTCGG-3’。
按照5’/3’RACE试剂盒(RACE 5’/3’Kit,Takara bio，USA)操作指南分别进行5’和3’RACE反应。
获得目的长度片段后利用纯化试剂盒(NucleoSpin Gel and PCR Clean-Up Kit，Takara bio，USA)对目的片段进行纯化。
通过使用无缝克隆试剂盒(HD Cloning Kit，Takara bio，USA)同 源重组连接至线性化的pUC19载体上，测序获得序列信息，拼接获得ELOVL3基因的完整cDNA结构，共包含1724碱基对，其中，5’UTR区含有143bp；CDs区810bp；3’UTR区771bp，如图2。以上引物合成及测序均由北京擎科生物技术有限公司完成。
实施例2鸭多不饱和脂肪酸组成相关基因ELOVL3的核心启动子区域确认
从Ensembl(http://asia.ensembl.org/)数据库获取鸭7号染色体ELOVL3(ENSAPLG00020013672)上游2500bp序列，利用在线网站Primer3(https://bioinfo.ut.ee/primer3-0.4.0/)进行不同长度片段引物设计，并添加同源重组臂及酶切位点，扩增不同长度的缺失片段，截断引物序列分别为：
上游引物(5’-3’)：
ELOVL3-2095-F1(SEQ ID NO.5):ggcctaactggccggtaGGTACCAGTGAAGAGCTCCACGCACT；
ELOVL3-1856-F2(SEQ ID NO.6):ggcctaactggccggtaGGTACCTGGGAAACAAGTCATGTCCA；
ELOVL3-1518-F3(SEQ ID NO.7):ggcctaactggccggtaGGTACCCAGGGCAATTCCTCTAGCAT；
ELOVL3-1249-F4(SEQ ID NO.8):ggcctaactggccggtaGGTACCCCCTGGGACAGCTCACAGTA；
ELOVL3-765-F5(SEQ IDNO.9):ggcctaactggccggtaGGTACCCACATCCTTCCATGCCACAT；
ELOVL3-572-F6(SEQ IDNO.10):ggcctaactggccggtaGGTACCAAAGATGATGCCCAAATTGC；
ELOVL3-389-F7(SEQ IDNO.11):ggcctaactggccggtaGGTACCGATAACTGGGTGCAGGTTCC；
下游引物(5’-3’)：
ELOVL3-+62-R(SEQ IDNO.12):cgaggccagatcttgatatcCTCGAGCTCCGCTCCACCTCATACTC。
利用基因组提取试剂盒(TIANamp Genomic DNA Kit，Tiangen bio，China)提取北京鸭血液基因组作为模板，使用PCR mix(2×PCR  SuperMix，Transgen bio，China)进行PCR扩增，
PCR：20μL反应体系：2×PCR SuperMix 10μL，上下游引物(10μmol/L)各0.5μL，DNA模板1μL，超纯水补8μL。
PCR扩增程序：94℃预变性5min；94℃变性30s，60℃退火30s，72℃延伸1min，共35个循环；72℃后延长10min，最后4℃保存。
取PCR产物5μL进行凝胶电泳，检测产物片段大小符合目的片段大小，鉴定结果如图3，并将PCR产物送北京擎科生物技术有限公司测序，测序结果确认PCR产物为目标序列则片段扩增成功。
片段扩增成功后利用纯化试剂盒(TaKaRa MiniBEST AgaroseGel DNAExtraction Kit Ver.4.0，Takara bio，USA)纯化目的片段。
利用软件SnapGene 5.3.1分析pGL4.10限制性内切酶位点，选择利用Kpn I和XhoI(NEB，US)对其进行双酶切并纯化(TaKaRa MiniBEST DNAFragment Purification KitVer.4.0，Takara bio，USA)线性化载体。
利用同源重组试剂(Seamless Cloning and Assembly Kit，Transgen bio，China)将各片段构建至线性化的pGL4.10报告载体上。
鸡永生化细胞系ICP在37℃、5％CO 2浓度和95％空气湿度条件下进行培养，培养基为含10％胎牛血清和1％双抗的DMEM/F12，细胞状态良好时进行细胞计数铺于24孔板中，铺板密度为在24h能生长至80％。
铺板24h后，细胞贴壁覆盖度达80％左右时进行转染，每孔表达载体(0.475μg)、pRL-TK(0.025μg)、脂质体(1.0μL)和Opti-MEM共50μL混匀孵育10min，转染到ICP1细胞中，6h后更换新鲜培养基。
转染48h后收集细胞样品，加入100μL 1×的细胞裂解液裂解5min后，离心取20μL上清液，利用多功能酶标仪(Infinite F200，CH)测定并计算萤火虫荧光素酶与海肾荧光素酶的相对活性(Dual Luciferase Reporter Assay Kit，Vazyme bio，China)，试验进行3次系统重复，每次包含3次技术重复。测定结果如图4所示。
上述结果表明，ELOVL3基因启动子核心区域为-765bp～+62bp区域，此结果为后续探究ELOVL3基因表达活性的影响因素提供了重要基础。
实施例3与上述鸭脂肪酸组成相关基因ELOVL3核心启动子活性相关的SNP突变效应验证
获得核心启动子区域候选突变后以原始表达质粒为模板，利用在线网站(https://crm.vazyme.com/cetool/singlepoint.html)引入单碱基突变的扩增引物进行反向扩增，引物序列为：
正向(SEQ ID NO.13)：5’-ACgCAATTCTTGTAAAGAGAGACATGACTATG-3’；
反向，(SEQ ID NO.14)5’-CTTTACAAGAATTGcGTAATAATACTTTGCTGAATTCTCCTGC-3’，针对扩增产物进行DpnI消化，去除甲基化模板质粒后进行重组连接反应，最终进行产物转化、涂板及克隆鉴定(Mut II Fast Mutagenesis Kit V2，Vazyme bio，China)。克隆菌液送北京擎科生物技术有限公司测序，测序结果如图5，确认单点突变构建成功。提取相应表达质粒备用。
鸡永生化细胞系ICP在37℃、5％CO 2浓度和95％空气湿度条件下进行培养，培养基为含10％胎牛血清和1％双抗的DMEM/F12，细胞状态良好时进行细胞计数铺于24孔板中，铺板密度为在24h能生长至80％。
铺板24h后，细胞贴壁覆盖度达80％左右时进行转染，每孔表达载体(0.475μg)、pRL-TK(0.025μg)、脂质体(1.0μL)和Opti-MEM共50μL混匀孵育10min，转染到ICP1细胞中，6h后更换新鲜培养基。
转染48h后收集细胞样品，加入100μL 1×的细胞裂解液裂解5min后，离心取20μL上清液，利用多功能酶标仪(Infinite F200，CH)测定并计算萤火虫荧光素酶与海肾荧光素酶的相对活性(Dual Luciferase Reporter Assay Kit，Vazyme bio，China)，试验进行3次系统重复，每次包含3次技术重复。测定结果如图6所示。
上述试验结果表明，当ELOVL3基因上游-619位点为AA基因型时，其核心启动子区域有更高的转录活性，表明AA型个体具有比GG型个体更高的多不饱和脂肪酸碳链延长能力，为其在检测和提升鸭肉长链多不饱和脂肪酸含量的应用提供了重要依据。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其它实施例，这些实施例都属于本发明保护范围。

Claims (15)

  1. 一种影响鸭脂肪酸组成相关基因ELOVL3的SNP分子标记，其特征在于，所述SNP分子标记位于ELOVL3基因上游-619bp处。
  2. 根据权利要求1所述的SNP分子标记，其特征在于，所述SNP分子标记处核苷酸为AA、GG或GA。
  3. 扩增鸭多不饱和脂肪酸组成相关基因ELOVL3的引物对，其特征在于，包括扩增编码区的引物对和扩增完整cDNA序列的RACE引物对，其中扩增编码区的引物对的核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示；
    扩增完整cDNA序列的RACE引物对的核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示。
  4. 利用权利要求3所述引物对扩增鸭多不饱和脂肪酸组成相关基因ELOVL3的方法，其特征在于，包括以下步骤：
    以野鸭肝脏RNA反转录获得的cDNA为模板，利用所述扩增编码区的引物对进行扩增，得基因完整蛋白编码区域的序列；
    以基因完整蛋白编码区域的序列为模板，利用扩增完整cDNA序列的RACE引物对，按照5’/3’RACE试剂盒操作指南分别进行5’和3’RACE反应，对获得目的片段利用纯化试剂盒进行纯化；
    使用无缝克隆试剂盒，将纯化后的序列同源重组连接至线性化的pUC19载体上，测序获得序列信息，拼接获得ELOVL3基因的完整cDNA结构。
  5. 根据权利要求4所述的方法，其特征在于，所述扩增的程序，包括：94℃预变性5min；94℃变性30s，60℃退火30s，72℃延伸1min，共35个循环；72℃后延长10min，最后4℃保存。
  6. 根据权利要求4所述的方法，其特征在于，ELOVL3基因的完整cDNA结构，共包含1724碱基对，其中，5’UTR区含有143bp；CDs区810bp；3’UTR区771bp。
  7. 扩增鸭多不饱和脂肪酸组成相关基因ELOVL3上游调控区不同长度的截断引物对，其特征在于，包括上游引物和下游引物，其中上游引物的核苷酸序列如SEQ ID NO.5-SEQ ID NO.11所示，下游引物的核苷酸序 列如SEQ ID NO.12所示。
  8. 权利要求1或2所述的SNP分子标记或权利要求3所述引物对在判断鸭中ELOVL3表达水平中的应用。
  9. 权利要求1或2所述的SNP分子标记或权利要求3所述引物对在判断鸭肉长链多不饱和脂肪酸含量中的应用。
  10. 权利要求1或2所述的SNP分子标记或权利要求3所述引物对在选育鸭肉中长链多不饱和脂肪酸含量高的鸭中的应用。
  11. 根据权利要求8-10任一项所述的应用，其特征在于，所述应用包括检测ELOVL3基因上游-619bp处的核苷酸。
  12. 根据权利要求11所述的应用，其特征在于，ELOVL3基因上游-619bp处的核苷酸为AA基因型时个体ELOVL3表达水平高，有更高的中长链多不饱和脂肪碳链延长能力，其肉中有更高的中长链多不饱和脂肪含量。
  13. 根据权利要求10所述的应用，其特征在于，所述应用包括使用ELOVL3基因上游-619bp处的核苷酸为AA基因型的个体选育品种。
  14. 根据权利要求10所述的应用，其特征在于，检测ELOVL3基因上游-619bp处的核苷酸使用的方法选自：测序法、限制性片段长度多态性分析、熔解曲线分析、单链构象异构多态性分析、探针扩增阻滞突变和色谱方法中的一种。
  15. 执行根据权利要求8-14任一项所述应用的试剂盒。
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