鸭骨形态发生蛋白SYBR GreenⅠ荧光定量RT-PCR检测方法的建立  

A SYBR Green Ⅰ-based qRT-PCR Assay for Detecting Duck BMPs

作　　者：刘鸿威 林昶 王劭[2,3] 肖世峰 程晓霞[2,3] 郑敏[2,3] 陈少莺[2,3] 曾显成[1] 陈仕龙[2,3] LIU Hongwei;LIN Chang;WANG Shao;XIAO Shifeng;CHENG Xiaoxia;ZHENG Min;CHEN Shaoying;ZENG Xiancheng;CHEN Shilong(College of Animal Sciences(College of Bee Science),Fujian Agricultural and Forestry University,Fuzhou,Fujian 350002,China;Institute of Animal Husbandry and Veterinary Medicine,Fujian Academy of Agriculture Sciences,Fuzhou,Fujian 350013,China;Putian Branch,Fujian Academy of Agricultural Sciences,Fuzhou,Fujian 351100,China)

机构地区：[1]福建农林大学动物科学学院(蜂学学院),福建福州350002 [2]福建省农业科学院畜牧兽医研究所,福建福州350013 [3]福建省农业科学院莆田分院,福建莆田351100

出　　处：《福建农业学报》2022年第12期1509-1518,共10页Fujian Journal of Agricultural Sciences

基　　金：福建省农业科学院科技创新团队建设项目(CXTD2021034);福建省农业高质量发展超越“5511”协同创新工程项目(XTCXGC2021018、 XTCXGC2021012);中央引导地方科技发展专项(2022L3019)。

摘　　要：【目的】旨在建立一种检测鸭骨形态发生蛋白(BMPs)mRNA转录水平的SYBR GreenⅠ实时荧光定量RTPCR检测方法。【方法】根据GenBank中BMP1、BMP2、BMP3、BMP4、BMP5、BMP6、BMP7、BMP8a、BMP10和BMP15的核苷酸序列设计并合成特异性引物进行PCR扩增,产物回收后克隆至pMD-18-T载体,构建阳性重组质粒作为标准品,优化SYBR GreenⅠ荧光定量PCR反应条件,建立其标准曲线,对建立的方法进行特异性、敏感性和重复性试验。【结果】建立的BMP1、BMP2、BMP3、BMP4、BMP5、BMP6、BMP7、BMP8a、BMP10和BMP15基因实时荧光定量检测方法特异性强,无引物二聚体及非特异性产物,熔解曲线为单峰,相关系数R2均大于0.998,组间和组内变异系数均小于1%。应用该方法检测BMPs基因在半番鸭组织中的表达水平,结果显示,BMP1、 BMP2、 BMP5和BMP7基因在心脏中表达量较高,分别为5.5×10^(4)、 1.1×10^(5)、 5.1×10^(5)和7.7×10^(5)拷贝·μL^(-1);BMP6和BMP10基因在肝脏中表达量较高,分别为7.5×10^(4)和7.6×10^(3)拷贝·μL^(-1);而BMP3、 BMP4、BMP8a和BMP15分别在法氏囊、胸腺、喙和脾脏中表达量最高,分别为1.5×10^(5)、2.1×10^(4)、1.8×10^(3)和3.3×10^(3)拷贝·μL^(-1)。【结论】本研究建立的实时荧光定量PCR方法特异性强、重复性好,为检测鸭BMPs表达水平提供了技术手段。【Objective】 An assay for detecting mRNA transcription of bone morphogenetic proteins(BMPs) in ducks using the SYBR Green Ⅰ-based qRT-PCR was developed. 【Methods】 Specific primers were designed and synthesized according to the nucleotide sequences of duck BMPs in GenBank. The PCR amplified products were cloned into pMD-18-T vector, and the recombinant plasmids DNA used to establish standard curves. Specificity, sensitivity, and repeatability of the methodology were examined. 【Result】 The newly developed qRT-PCR assay showed singe specific melting peaks of BMP1, BMP2,BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP10, and BMP15 separately with correlation coefficients(R2) higher than0.998. The coefficients of variation within and between groups were less than 1%. The mRNA expressions of the BMPs were detected in different tissues of hybrid Muscovy ducks. The expressions of BMP1, BMP2, BMP5, and BMP7 in the heart were significantly higher than those in other tissues at 5.5×10^(4), 1.1×10^(5), 5.1×10^(5), and 7.7×10^(5) copies·μL^(-1), respectively. BMP6and BMP10 were highly expressed in the liver at 7.5×10^(4) and 7.6×10^(3) copies·μL^(-1), respectively, while BMP3 in Bursa of Fabricius, BMP4 in the thymus, BMP8a in the beak, and BMP15 in the spleen at 1.5×10^(5), 2.1×10^(4), 1.8×10^(3), and 3.3×10^(3)copies·μL^(-1), respectively. 【Conclusion】 The newly developed qRT-PCR assay for the determination of mRNA expression of BMPs in ducks was specific, sensitive, repeatable, and applicable.

关 键 词：鸭 骨形态发生蛋白(BMPs) 荧光定量PCR SYBR GreenⅠ 

分 类 号：S834[农业科学—畜牧学]