绵羊BMP15基因B2突变可视化检测技术的建立

doi:10.16431/j.cnki.1671-7236.2021.03.004

摘要/Abstract

摘要： 骨形态发生蛋白15（bone morphogenetic protein 15，BMP15）基因突变对绵羊排卵率、产仔数以及繁殖力有很大影响，本研究旨在建立快速可视化检测BMP15基因B2突变的技术。将改良的扩增阻滞突变系统（amplification refractory mutation system，ARMS）与核酸染料SYBR Green Ⅰ结合，针对BMP15基因B2突变设计ARMS特异性引物，使引物下游3'端的最后一个碱基为A，并在下游引物3'端的第3位碱基处设计额外的错配，以提高引物的特异性；经ARMS PCR扩增后，向产物中加入SYBR Green Ⅰ，通过肉眼观察PCR管内的颜色变化来检测BMP15基因的B2突变。经测序发现所采集的样本均为野生型，因此，利用重叠延伸PCR构建BMP15基因B2突变型模板，测序结果显示BMP15基因B2突变型模板构建成功。ARMS PCR结果显示，ARMS特异性引物能够只扩增突变型模板，而不会扩增野生型模板，且扩增效果良好。ARMS PCR扩增后加入SYBR Green Ⅰ发现已知野生型模板与阴性对照一致，呈SYBR Green Ⅰ原始颜色橙黄色，而已知突变型模板呈亮绿色，且色差明显，肉眼可辨。用建立的可视化检测BMP15基因B2突变的技术检测50个小尾寒羊基因组DNA （随机向其中20个样本中加入构建的BMP15基因B2突变型模板），将可视化检测的突变型样本编号与混合样本时记录的编号对比，结果表明该技术能够准确检测绵羊BMP15基因的B2突变，且准确率高达100%。将构建的BMP15基因B2突变型的模板进行梯度稀释，对本试验可视化检测体系的灵敏度进行检测，发现ARMS可视化检测技术的灵敏度达到0.006 ng/μL。因此，本研究建立的技术能够可视化地检测绵羊BMP15基因B2突变，且准确率高，有望为高繁殖力绵羊的选育提供技术支持。

关键词: 绵羊; BMP15基因; B2突变; 可视化检测; ARMS; SYBR Green Ⅰ

Abstract: Bone morphogenetic protein 15 (bone morphogenetic protein 15,BMP15) gene mutation had a great impact on sheep ovulation rate,litter size and fecundity.Therefore,this study was aimed to establish a rapid visual detection technology for B2 mutation of BMP15 gene.Combine the improved amplification refractory mutation system (ARMS) with the nucleic acid dye SYBR Green Ⅰ,and designed ARMS-specific primers for the B2 mutation of BMP15 gene,made the last base at the 3'end of the primer as A,meanwhile designed additional mismatches at the third base of the 3'end of the downstream primer to improve primer specificity.After ARMS PCR,added SYBR Green Ⅰ to the product,and detected the B2 mutation of BMP15 gene by visually observing the color change of the PCR tube.It was found through sequencing that the collected samples were all wild-type.Therefore,the B2 mutation of BMP15 gene template was constructed by overlap extension PCR.The sequencing results showed the B2 mutation of BMP15 gene template was successfully constructed.ARMS PCR results showed that ARMS-specific primers could only amplify mutant templates,could not amplify wild-type templates,and the amplification effect was good.After ARMS PCR,SYBR Green Ⅰ was added and it was found that the known wild-type template was consistent with the negative control and showed the original color of SYBR Green Ⅰ orange-yellow,while the known mutant template was bright green with obvious color changes.Using the established visual detection technology to detect 50 Small-tailed Han sheep genomic DNA (20 samples were added with the constructed B2 mutant template of BMP15 gene),and compare the number of the mutant sample detected visually with the number recorded when the sample was mixed.The results showed that this technology could accurately detect the B2 mutation of BMP15 gene,and the accuracy was as high as 100%.The constructed BMP15 gene B2 mutant template was serially diluted to detect the sensitivity of the visual detection system in this experiment.It was found that the sensitivity of the ARMS visual detection technology reached 0.006 ng/μL.Therefore,the technology established in this study could visually detect the B2 mutation of BMP15 gene in sheep with high accuracy,and it was expected to provide technical support for the breeding of high-fertility sheep.

Key words: sheep; BMP15 gene; B2 mutant; visual detection; ARMS; SYBR Green Ⅰ

中图分类号:

S826

胡瑞瑞, 刘莉, 郭涛, 李雅心, 王小奎, 胡圣伟. 绵羊BMP15基因B2突变可视化检测技术的建立[J]. 中国畜牧兽医, 2021, 48(3): 810-817.

HU Ruirui, LIU Li, GUO Tao, LI Yaxin, WANG Xiaokui, HU Shengwei. Establishment of a Visual Detection Technique for B2 Mutation of BMP15 Gene in Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(3): 810-817.

参考文献

[1] MIAO X,LUO Q,ZHAO H,et al.Ovarian proteomic study reveals the possible molecular mechanism for hyperprolificacy of Small Tail Han sheep[J].Scientific Reports,2016,6:27606.
[2] CHEN H Y,SHEN H,JIA B,et al.Differential gene expression in ovaries of Qira Black sheep and Hetian sheep using RNA-Seq technique[J].PLoS One,2015,10(3):e0120170.
[3] DAVIS G H,MONTGOMERY G W,ALLISON A J,et al.Segregation of a major gene influencing fecundity in progeny of Booroola sheep[J].New Zealand Journal of Agricultural Research,1982,25(4):525-529.
[4] EL-HANAFY A A,EL-SAADANI M A.Fingerprinting of FecB gene in five Egyptian sheep breeds[J].Biotechnology in Animal Husbandry,2009,25(3-4):205-212.
[5] WANG W,LA Y,ZHOU X,et al.The genetic polymorphisms of TGFβ superfamily genes are associated with litter size in a Chinese indigenous sheep breed (Hu sheep)[J].Animal Reproduction Science,2018,189:19-29.
[6] GALLOWAY S M,MCNATTY K P,CAMBRIDGE L M,et al.Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner[J].Nature Genetics,2000,25(3):279-283.
[7] SHIMASAKI S,MOORE R K,OTSUKA F,et al.The bone morphogenetic protein system in mammalian reproduction[J].Endocrine Reviews,2004,25(1):72-101.
[8] HASHIMOTO O,MOORE R K,SHIMASAKI S.Posttranslational processing of mouse and human BMP-15:Potential implication in the determination of ovulation quota[J].Proceedings of the National Academy of Sciences,2005,102(15):5426-5431.
[9] NAGDY H,MAHMOUD K G M,KANDIEL M M M,et al.PCR-RFLP of bone morphogenetic protein 15(BMP15/FecX) gene as a candidate for prolificacy in sheep[J].International Journal of Veterinary Science and Medicine,2018,6(sup1):S68-S72.
[10] CHU M X,LIU Z H,JIAO C L,et al.Mutations in BMPR-ⅠB and BMP-15 genes are associated with litter size in Small Tailed Han sheep (Ovis aries)[J].Journal of Animal Science,2007,85(3):598-603.
[11] CHU M X,JIAO C L,HE Y Q,et al.Association between PCR-SSCP of bone morphogenetic protein 15 gene and prolificacy in Jining Grey goats[J].Animal Biotechnology,2007,18(4):263-274.
[12] CHU M X,LI B X,WANG J Y,et al.Association between PCR-SSCP of growth differentiation factor 9 gene and high prolificacy in Small Tail Han sheep[J].Animal Biotechnology,2004,15(2):111-120.
[13] HAYASHI K.PCR-SSCP:A simple and sensitive method for detection of mutations in the genomic DNA[J].Genome Research,1991,1(1):34-38.
[14] AHLAWAT S,SHARMA R,ROY M,et al.Association analysis of novel SNPs in BMPR1B,BMP15 and GDF9 genes with reproductive traits in Black Bengal goats[J].Small Ruminant Research,2015,132:92-98.
[15] JAVANMARD A,AZADZADEH N,ESMAILIZADEH A K.Mutations in bone morphogenetic protein 15 and growth differentiation factor 9 genes are associated with increased litter size in fat-tailed sheep breeds[J].Veterinary Research Communications,2011,35(3):157-167.
[16] YANG D Y,FUSHIMI H,CAI S Q,et al.Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system (ARMS) analyses of medicinally used rheum species and their application for identification of Rhei rhizoma[J].Biological and Pharmaceutical Bulletin,2004,27(5):661-669.
[17] PARK M J,KIM M K,IN J G,et al.Molecular identification of Korean ginseng by amplification refractory mutation system-PCR[J].Food Research International,2006,39(5):568-574.
[18] DONOHOE G G,SALOMÄKI A,LEHTIMÄKI T,et al.Rapid identification of apolipoprotein E genotypes by multiplex amplification refractory mutation system PCR and capillary gel electrophoresis[J].Clinical chemistry,1999,45(1):143-146.
[19] 刘世佳,潘香羽,李发弟,等.绵羊BMP15基因特征、组织表达及其与产羔数性状的关联性分析[J].甘肃农业大学学报,2015,50(3):40-48. LIU S J,PAN X Y,LI F D,et al.Characteristics and expression of BMP15 gene and its association with litter size in sheep[J].Journal of Gansu Agricultural University,2015,50(3):40-48.(in Chinese)
[20] 杨艳影.苏尼特羊BMPR-ⅠB、BMP15和GDF9基因与多胎性状关联性分析及BMP15基因突变检测[D].呼和浩特:内蒙古大学,2017. YANG Y Y.Association analyses of mutations in BMPR-ⅠB,BMP15 and GDF9 genes with litter size and identification of mutation in BMP15 gene in Sonid sheep[D].Hohhot:Inner Mongolia University,2017.(in Chinese)
[21] QIAN L,DING G,ZHOU Q,et al.Molecular authentication of Dendrobium loddigesii Rolfe by amplification refractory mutation system (ARMS)[J].Planta Medica,2008,74(4):470-473.
[22] NEWTON C R,GRAHAM A,HEPTINSTALL L E,et al.Analysis of any point mutation in DNA.The amplification refractory mutation system (ARMS)[J].Nucleic Acids Research,1989,17(7):2503-2516.
[23] HAYASHI K,HASHIMOTO N,DAIGEN M,et al.Development of PCR-based SNP markers for rice blast resistance genes at the Piz locus[J].Theoretical and Applied Genetics,2004,108(7):1212-1220.
[24] WU D Y,UGOZZOLI L,PAL B K,et al.Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia[J].Proceedings of the National Academy of Sciences,1989,86(8):2757-2760.
[25] KWOK S,KELLOGG D E,MCKINNEY N,et al.Effects of primer-template mismatches on the polymerase chain reaction:Human immunodeficiency virus type 1 model studies[J].Nucleic Acids Research,1990,18(4):999-1005.
[26] DRENKARD E,RICHTER B G,ROZEN S,et al.A simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis[J].Plant Physiology,2000,124(4):1483-1492.
[27] LI M,SUN X,JIANG J,et al.Tetra-primer ARMS-PCR is an efficient SNP genotyping method:An example from SIRT2[J].Analytical Methods,2014,6(6):1835-1840.
[28] LI W,LEE S Y,BACK C G,et al.Loop-mediated isothermal amplification for the detection of Xanthomonas arboricola pv.pruni in peaches[J].The Plant Pathology Journal,2019,35(6):635.
[29] LAN C,YAO J,YANG X,et al.Specific and sensitive detection of the guava fruit anthracnose pathogen (Colletotrichum gloeosporioides) by loop-mediated isothermal amplification (LAMP) assay[J].Canadian Journal of Microbiology,2020,66(1):17-24.
[30] 李月华.LAMP与SYBR Green Ⅰ实时荧光定量PCR方法检测酸奶中葡糖杆菌的研究[D].保定:河北农业大学,2015. LI Y H.Study on LAMP and SYBR Green Ⅰ real time PCR for detection of Gluconobacter in yoghourt[D].Baoding:Hebei Agricultural University,2015.(in Chinese)