洼地绵羊防御素sBD-1基因克隆、序列分析及SYBR Green Ⅰ实时荧光定量检测方法的建立与应用  

Cloning and sequence analysis of Wadi sheep defensin sBD-1 gene and establishment and application of SYBR green Ⅰ real-time fluorescence quantitative PCR method

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作  者:王金良[1] 郭显坡 沈志强[1,2,3] 李敏 任艳玲[1,3] 

机构地区:[1]山东省滨州畜牧兽医研究院,山东滨州256600 [2]山东绿都生物科技有限公司,山东滨州256600 [3]山东省洼地绵羊繁育技术研究推广中心,山东滨州256600

出  处:《中国兽医学报》2011年第5期720-724,729,共6页Chinese Journal of Veterinary Science

基  金:国家"863"计划资助项目(2008AA10Z142);山东省自然科学基金资助项目(Y2007D12);山东省良种工程资助项目(2006GG22)

摘  要:根据GenBank上登录的绵羊防御素基因序列,经多重比较后,设计1对引物,从洼地绵羊舌上皮组织中扩增到防御素sBD-1基因,克隆到pMD18-T载体中进行测序。结果表明,扩增基因全长215 bp,编码64个氨基酸。基因进化树分析表明,与蒙古绵羊sBD-1基因有较近的亲缘关系,核苷酸同源性为98.5%;而与黄牛的亲缘关系最远,核苷酸同源性84.6%。氨基酸序列分析表明,序列内无信号肽区域,具有3个潜在的抗原表位。以pMD18-T/sBD-1质粒为模板建立了sBD-1基因SYBR GreenⅠ荧光定量PCR检测方法,核酸电泳、扩增动力学曲线、溶解曲线及重复性试验表明,检测方法具有良好的稳定性和特异性,得到的回归方程(R2=0.998)表明PCR产物量的对数值与起始模板量之间存在良好的线性关系,从舌、盲肠及输卵管等组织中可以进行有效的检测,检测灵敏度为83.9 copies/μL。该方法为进一步研究防御素sBD-1基因在洼地绵羊抗逆性过程中的作用奠定了基础。According to the published gene sequences of defensin gene of sheep on GenBank,one pair of primers were designed and defensin sBD-1 gene was amplified by RT-PCR from tongue epithelial tissue of Wadi sheep.PCR product was cloned into the pMD18-T vector and sequenced.The results showed that gene amplication of full-length was 215 bp,encoding 64 amino acids.Phylogenetic tree analysis showed that Wadi sheep and Menggu sheep had close phylogenetic relationship,nucleotide homology was 98.5%;kinship with the Bos taurus as far as the nucleotide homology of 84.6%.Amino acid sequence analysis showed no signal peptide amino acid sequence in the region,with three potential antigenic epitopes.sBD-1 gene SYBR Green Ⅰ fluorescence quantitative PCR method was set up using pMD18-T/sBD-1 plasmid as a template.Nucleic acid electrophoresis,amplification kinetics,dissolution curve and repeatability tests showed that the methods had good stability and specificity,the obtained regression equation(R2=0.998) showed that the logarithm of the amount of PCR product and the amount of starting template existed a good linear relationship,the gene could be effectively detect from the tongue,cecum,and fallopian tubes and other tissue,with detection sensitivity of 83.9 copies/μL.This study laid the foundation for further study on the function of defensin sBD-1 gene on resistance in low-lying land sheep.

关 键 词:洼地绵羊 sBD-1基因 序列分析 荧光定量 

分 类 号:S852.4[农业科学—基础兽医学]

 

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