亚洲璃眼蜱成蜱不同发育期miR-451与MIF表达谱的动态分析  

Dynamic Analysis of Expression Level of miR-451 and MIF Gene in Different Developmental Stages of Adult Hyalomma asiaticum

作　　者：罗金[1] 袁小松[1] 郝佳伟[1] 田占成[1] 谢俊仁[1] 陈泽[1] 任巧云[1] 殷宏[1,2] 罗建勋[1] 刘光远[1] 

机构地区：[1]中国农业科学院兰州兽医研究所/家畜疫病病原生物学国家重点实验室/甘肃省动物寄生虫病重点实验室,兰州730046 [2]扬州大学/江苏高校动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国农业科学》2015年第21期4366-4373,共8页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31201899;31471967);农业科技创新工程(ASTIP);农业部肉牛牦牛项目(NBCIS CARS-38)

摘　　要：[目的]micro RNA(miRNA)作为一类内源性的非编码RNA,仅有18—25 nt,其广泛存在于动植物细胞中,可诱导生物基因沉默,参与细胞生长、发育、基因转录和翻译等诸多生命活动的调控过程。以亚洲璃眼蜱为研究对象,对miR-451及其靶基因(巨噬细胞游走因子,MIF)在相互作用关系进行分析。[方法]参考miR-451成熟体序列(AAA CCG UUA CCA UUA CUG AGU UU)来自研究单元亚洲璃眼蜱高通量测序所获得的结果。根据该成熟体序列设计stem-loop及PCR引物,RT-PCR扩增获得蜱源性miR-451序列;并对不同物种来源的miR-451序列特征进行分析。基因合成方法获得抑制miR-451的ds RNA序列,用于亚洲璃眼蜱饥饿成蜱体内注射。参考美洲钝眼蜱MIF基因(登录号:AF289543.2),设计亚洲璃眼蜱的特异性实时荧光定量引物,以β-actin作为内参基因,采用SYBR Green real-time RT-PCR方法检测注射ds RNA后亚洲璃眼蜱饥饿成蜱不同发育时间点MIF基因的表达水平,以及miR-451的表达谱特征。[结果]琼脂糖凝胶电泳检测miR-451的PCR产物条带与预期大小一致,为72 nt。不同物种来源的miR-451具有较高的保守性,特别是种子序列极度保守,仅在短尾负鼠miR-451成熟体的19位点处存在A→U的突变。miR-451的RNA干扰实验证实,0-30 h miR-451表达逐渐上调,6 h达到最高值,其拷贝数为1.0×108。随后表达丰度逐渐降低。30 h其表达水平与PBS对照组相同。48-60 h miR-451再次出现一个表达上调微小变化过程。而miR-451抑制后的MIF直到30 h才有表达的上调,42 h达到峰值(拷贝量仅为9.0×103),此后其表达规模受到抑制,48 h时与PBS对照组水平一致。84 h后表达才逐渐上调,直到90 h达到峰值,并呈现正态分布趋势。[结论]利用RT-PCR获得亚洲璃眼蜱成蜱阶段miR-451序列,生物信息学方法分析了miR-451序列在不同物种间具有较高保守性。miRNA的保守性决定其靶标基因的特异性,因此,以上结果提示miR-451[Objective]The microRNAs（miRNAs） are a class of small single-stranded noncoding RNAs（18—25 nt） in both unicellular and multicellular organisms.Previous studies showed that miRNAs play pivotal roles in regulating diverse developmental processes by targeting m RNAs for translational repression,development,or transcription.To dissect the interaction between miR-451 and target gene（Macrophage Migration Inhibitory Factor,MIF）,Hyalomma asiaticum ticks were analyzed.[Method]The primers were designed for stem-loop and PCR based on the mature sequence of miR-451（AAA CCG UUA CCA UUA CUG AGU UU）from High-through sequence results of Hyalomma asiaticum.The mature sequence of miR-451 was amplified by RT-PCR and analyzed miR-451 sequences from different species.The ds RNA of miR-451 was obtained by gene synthesis and it was injected into unfed-adult H.asiaticum.Referencing the MIF gene of Amblyomma americanum in Gen Bank（Accession number：AF289543.2） for specific primers of q PCR to H.asiaticum,and the β-actin gene as an internal reference gene.After injecting ds RNA of miR-451,the MIF and miR-451 expression level of various developmental stages of unfed-adult ticks were detected by SYBR Green real-time RT-PCR.[Result]The miR-451 PCR product was consistent with the prediction that it is 72 nt by agarose gel electrophoresis.The result showed miR-451 has a high conservatism in different species by multiple sequence alignment analysis,only Monodelphis domestica has a base mutations（A→U） at 19 sites.Our results showed that the miR-451up-regulation at 0-30 h and 6h had highest expression level with a copy number of 1.0×10^8.Subsequently,the expression level gradually reduced,such that they had same level at 30 h with PBS control.The expression had an up-regulation until 48-60 h again.After the miR-451 was restrained the MIF was up-regulated at 30 h,and 42 h peaks（with copy levels of 9.0×10^3）,since the expression is restrained,and the level is a same with PBS control at 48.At 84 h the expression l

关 键 词：亚洲璃眼蜱 微小RNA(microRNA) miR-451 巨噬细胞抑制游走因子 MIF 

分 类 号：S852.746[农业科学—基础兽医学]