亚洲璃眼蜱不同发育阶段及其组织中microRNA-10表达分析  

Analysis of the Relative Expression of microRNA-10 in Different Developmental Stages and Various Tissues of Hyalomma asiaticum

作　　者：袁小松[1,2] 罗金[1] 田占成[1] 谢俊仁[1] 王芳芳[1] 田美媛[1] 张以芳[2] 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]云南农业大学动物科学技术学院,昆明650201

出　　处：《中国农业科学》2014年第4期806-814,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金项目(31201899);甘肃省创新研究群体计划项目(1210RJIA006)

摘　　要：【目的】MicroRNA(miRNA)是一类长度为20—22个核苷酸(nt)且高度保守的非编码小RNA。迄今为止,在病毒、动物、植物中都有发现,如在Mareks病毒、果蝇、斑马鱼、人类以及拟南芥等多种生物中都有存在。其通过与靶基因特异性的碱基互补配对使靶基因降解或者受到抑制,在转录后水平调控靶基因的表达水平。miRNAs参与多种生物的细胞增殖、分化、代谢与死亡等多种生物学过程。为了解microRNA-10在亚洲璃眼蜱中的潜在生物学功能,试验获得亚洲璃眼蜱miR-10的前体、成熟体序列;分析亚洲璃眼蜱不同发育阶段及组织miR-10的表达水平,及亚洲璃眼蜱miR-10的生物学意义;为进一步研究miR-10与亚洲璃眼蜱生长发育间的关系奠定基础。【方法】用Trizol Reagent分别提取不同发育阶段及组织的总RNA,用SYBR?Prime Script TMmiRNA RT-PCR Kit(TaKaRa Code:RR716)制备cDNA。参考miRBase数据库中isc-miR-10(登录号:MI0012262)序列,设计特异性引物,通过PCR的方法从亚洲璃眼蜱中扩增获得miR-10前体序列,通过MEGA4软件将此前体序列与已知物种的miR-10前体序列进行比对分析;利用qPCR技术分析亚洲璃眼蜱不同发育阶段及组织miR-10的表达情况;推测miR-10在亚洲璃眼蜱中的生物学功能。【结果】获得亚洲璃眼蜱miR-10的前体序列CUACAUCUACCCUGUAGAUCCGAAUUUGUUUGCCACUAGACUACAAAUUCGGUUCUAGAGAGGCUUUGUGUGG,大小为73bp;亚洲璃眼蜱的miR-10前体序列与肩突硬蜱的相似性最高,是95.9%,且与其他节肢动物相似性在88.9%—91.7%之间;miR-10在不同物种间高度保守。miR-10的成熟体序列为UACCCUGUAGAUCCGAAUUUGU,种子序列为ACCCUGU。在亚洲璃眼蜱的不同发育阶段中,饥饿若蜱的miR-10的表达水平最高,是卵的48.3倍;饥饿成蜱是卵的7.78倍;饥饿幼蜱是卵的2.78倍。在饥饿雌性成蜱吸血过程中,吸血3d的成蜱是饥饿成蜱的约4.28倍,吸血5d的成蜱是饥饿成蜱的约0.31倍,饱血自然脱落的�[Objective] MicroRNAs （miRNAs） are a conserved class of non-coding 20-22 nt small RNAs. miRNAs have been reported in many viruses, animals and plants such as Mareks diseased virus, fruit flies, zebrafish, humans and Arabidopsis so far. MiRNAs regulate gene expression by binding to mRNA at post-transcriptional levels, leading to mRNA inhibition or degradation. MiRNAs regulate a variety of biological processes, including cell proliferation, differentiation, metabolism and apoptosis. The purpose of this expreiment is to understand the potential biological function of miR-10 in Hyalomma asiaticum. To obtain the precursor and mature of microRNA-10 （miR-10）, its relative expression and the biologic significance in different developmental stages and various tissues from Hyalomma asiaticum were analyzed, which will be helpful for further study the relationship between the function of miR-10 and development of H. asiaticum. [Method] Total RNA of the different developmental stages and various tissues were extracted using Trizol Reagent, then transcripted to cDNA using SYBR ~Prime Script Tra miRNA RT-PCR Kit（ TaKaRa Code： RR716）. To obtain the miR-10 precursor sequence from H. asiaticum, the specific primers were designed according to the miRBase database （Accession number： MI0012262）. Then its homology was comparied with the sequence from miRBas database by the MEGA4 software. The expression of miR-10 from different developmental stages and various tissues of H. asiaticum was assessed by qPCR. The biological function of miR-10 in H. asiaticum was presumed. [ Result ] A 73 bp gene was cloned by PCR, which was CUACAUCUACCCUGUAGAUCCGAAUUUGUUUGCCA CUAGACUACAAAUUCGGUUCUAGAGAGGCUUUGUGUGG. There was a relatively high genetic similarity among 16 varieties The similarity between H. asiaticum and Ixodes scapularis was 95.9%, and 88.9%-91.7% compared with other arthropods. The miR-10 sequence was highly conserved in various species. The mature sequence of miR-10 was UACCCUGUAGAUCCGAAUUUGU, in which ACCCUGU was see

关 键 词：亚洲璃眼蜱 MIR-1 0 克隆 表达分析 qPCR 

分 类 号：Q969.91[生物学—昆虫学]