亚洲璃眼蜱不同发育阶段及其组织中microRNA-10表达分析

doi:10.3864/j.issn.0578-1752.2014.04.021

摘要/Abstract

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，饱血自然脱落的成蜱是饥饿成蜱的约0.087倍。吸血初期miR-10的表达量上调，随着吸血时间的延长其表达量逐渐下调；在唾液腺、气管、卵巢、表皮、中肠中miR-10的表达情况有明显不同，其中唾液腺的表达量最高，是表皮的13.2倍；卵巢的表达量是表皮的2.98倍，气管的表达量是表皮的6.46倍。【结论】从亚洲璃眼蜱中克隆miR-10序列，序列分析显示此序列在节肢动物中相对保守。miR-10在亚洲璃眼蜱不同发育阶段及不同组织的表达明显不同，表明其表达具有选择性；依据miR-10表达的差异性及已报道miR涉及的生物学功能，推测miR-10在亚洲璃眼蜱的细胞增殖、发育及吸血方面有潜在的重要作用。本研究为寄生虫miRNAs功能的研究提供一定的依据，并有可能为防控寄生虫疾病开辟新的途径。

关键词: 亚洲璃眼蜱 , miR-10 , 克隆 , 表达分析 , qPCR

Abstract: 【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, zebra fish, 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 experiment 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 TM 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 compared 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 seed region. The expression of miR-10 that was 48.3-fold compared with the eggs and was the highest in unfed-nymph. However, the expression of miR-10 in unfed-adult and unfed-larvae was only 7.78-fold and 2.78-fold, respectively. The expression of miR-10 in the 3rd day feeding adult was 4.28-fold compared with the unfed-adult. But the expression of miR-10 in the 5th day feeding adult and fed-adult was only 0.31-fold and 0.087-fold, respectively. The relative expression levels of miR-10 increased in the early stage, but gradually decreased with the blood feeding. The relative expression levels of miR-10 among salivary gland, trachea, ovary, epidermis, midgut were remarkably different. The highest expression of miR-10 was salivary gland which was 13.2-fold compared with the epidermis that showed the lowest expression. The expression of miR-10 in ovary and trachea was 2.98-fold and 6.46-fold compared with the epidermis, respectively.【Conclusion】In the present study, the sequence of miR-10 was obtained. The analysis of miR-10 sequence suggested that it was highly conserved between arthropod. The expression of miR-10 in different developmental stages and various tissues of H. asiaticum were obviously different, which suggested that miR-10 was selectively expressed in H. asiaticum. It was deduced that according to the difference of expression and the reported function of miR-10, miR-10 may play potential roles in cell proliferation, development and blood-sucking. These results provided evidence for further study of microRNAs in parasites. MiRNA expression may be a new way for the therapeutic control of parasitic diseases in the future.

Key words: Hyalomma asiaticum , miR-10 , clone , expression analysis , qPCR亚洲璃眼蜱 , miR-10 , 克隆 , 表达分析 , qPCR

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. 亚洲璃眼蜱不同发育阶段及其组织中microRNA-10表达分析[J]. 中国农业科学, 2014, 47(4): 806-813.

YUAN Xiao-Song-1, 2 , LUO Jin-1, TIAN Zhan-Cheng-1, XIE Jun-Ren-1, WANG Fang-Fang-1, TIAN Mei-Yuan-1, ZHANG Yi-Fang-2, LIU Guang-Yuan-1. Analysis of the Relative Expression of microRNA-10 in Different Developmental Stages and Various Tissues of Hyalomma asiaticum[J]. Scientia Agricultura Sinica, 2014, 47(4): 806-813.

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