应用文库构建法和生物信息学方法鉴定绵羊microRNA
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摘要
MicroRNAs (miRNAs)是一类长约22 nt的非编码单链RNA分子,广泛存在于人类及其它各种真核生物中,它通过与靶mRNA特异性的碱基互补配对,引起靶mRNA降解或者抑制其翻译,从而调节基因的转录后表达水平。研究表明,miRNA在生物体内发挥着重要作用,其主要功能是调节生物体胚胎形成、组织器官形成、细胞分化和凋亡及调控疾病发生等相关基因的表达。截止到2010年4月,已经鉴定出分布于植物、动物、单细胞藻类等130多个物种中14197个miRNA。绵羊是重要的家畜动物之一,为人类提供生活必需的肉、毛、奶等,在农业生产和人民生活中占有极其重要的地位。然而,目前已知的绵羊miRNA的数量很少,对其功能的研究则更为匮乏。所以对绵羊miRNA的研究有着非常重要的现实和理论意义。
本研究通过构建小分子RNA cDNA文库和生物信息学方法两种途径鉴定绵羊miRNA ,分析它们在不同组织中的表达情况,寻找肌肉特异miRNA,并利用在线生物信息学软件预测绵羊miRNA的靶基因,对靶基因的功能进行分析。结果小结如下:
1.通过构建绵羊背最长肌、大脑、肝脏、脾脏4个组织的小分子RNA cDNA文库,克隆得到17个绵羊miRNA。得到的miRNA中,12个克隆自背最长肌,6个克隆自大脑,其余2个克隆自肝脏和脾脏。
2.通过生物信息学方法,在绵羊表达序列标签(EST)数据库中鉴定出14个绵羊miRNA ,其中包括4对miRNA/miRNA*和1对miRNA-3p/miRNA-5p。同时鉴定出一个miRNA基因簇,由oar-miR-374b、oar-miR-374b*和oar-miR-421组成。
3.序列分析表明,鉴定出的31个绵羊miRNA属于24个miRNA家族,在绵羊中均为首次发现。其中2个miRNA (oar-NEW-1和oar-NEW-2)在其他物种中均未见报道,为新的miRNA。其余29个miRNA为其它物种中已知miRNA的绵羊同源基因。
4. Northern blot结果表明,oar-miR-1仅在心肌和背最长肌中表达,oar-miR-122仅在肝脏中表达,oar-NEW-1仅在脾脏中表达。
5. Real-time PCR结果表明,除oar-miR-425-5p没有在心肌中检测到之外,检测的其余miRNA在绵羊的6个组织中均有表达。并且,oar-miR-24在心肌和背最长肌中的表达量显著高于在其他组织中的表达量;oar-miR-24-1*在心肌中的表达量显著高于在其他组织中的表达量;oar-miR-425-3p在背最长肌中的表达量显著高于在其他组织中的表达量;oar-miR-425-5p在大脑中的表达量显著高于在其他组织中的表达量。
6.利用鉴定出的31个绵羊miRNA序列,我们在绵羊基因组中的保守3’UTR区域预测得到120个靶基因,包括230个靶位点。GO分类结果表明,这些靶基因几乎参与了全部的生物学过程,参与功能最多的是结合活性。
MicroRNAs (miRNAs), about 22nt in length, are a class of non-coding small RNA distribute extensively in human genome and other eukaryote genome. They regulate gene expression through sequence specific interactions with target mRNAs resulting in translational repression or mRNA degradation. Recent findings showed that miRNAs play an important role in embryo formation, organogenesis, cell death, cell proliferation, and cancer development, and so on. By April 2010, the number of known miRNAs has grown rapidly to 14,197 in more than 130 species. O.aries (Ovis aries) is one of the most important agricultural livestock for meat, milk, and wool production. But there is limited information about O.aries miRNAs. Thus, to research on O.aries miRNAs is very important and necessary.
In this study, we combined a computational method based on expressed sequence tag (EST) analysis with experimental identification based on small RNA cDNA library to identify O.aries miRNAs. Then, we analized their expression level in different sheep tissues to find out muscle specific miRNAs, predicted their target genes and conducted the function analysis for them. The results are as follows:
1. First, by constructing four cDNA libraries for small RNAs in the size range of 18–26nt from the tissues of longissimus muscle, brain, liver and spleen, we cloned 17 O.aries miRNAs. Among these miRNAs, 12 were isolated from longissimus muscle, 6 were cloned from brain, and 2 others were cloned from liver and spleen.
2. Second, we used a computational approach to exploit O.aries miRNAs in sheep EST database. Following a set of strict filtering criteria, we finally identified 14 O.aries miRNAs, which included 4 pairs of miRNA/miRNA*, and 1 pair of miRNA-3p/miRNA-5p. A miRNA cluster was also identified, including oar-miR-374b, oar-miR-374b* and oar-miR-421.
3. Altogether, we identified 31 oar-miRNAs belong to 24 families, all of which were first identified in sheep. 2 of them were novel miRNAs that had never been annotated in miRBase database, named oar-NEW-1 and oar-NEW-2. The remaining 29 miRNAs were sheep orthologs of known mammalian miRNAs.
4. Northern blot showed that 3 miRNAs appeared to be extremely tissue specific. oar-miR-1 could be only detected in heart muscle and longissimus muscle, oar-miR-122 could be only detected in liver, and oar-NEW-1 could be only detected in spleen.
5. We chose 4 miRNAs (the pair of oar-miR-24/oar-miR-24-1* and the pair of oar-miR-425-3p/oar- miR-425-5p) identified by computational method for expression analysis by Real-time PCR. All of these four miRNAs could be detected in total tissues, except that oar-miR-425-5p could not be detected in heart muscle. The results also showed that oar-miR-24 had a significant higher expression level in longissimus muscle and heart muscle than which in the other tissues, as same as oar-miR-24-1* in heart muscle, oar-miR-425-3p in longissimus muscle, and oar-miR-425-5p in brain.
6. Further, we predicted 120 potential target genes, included 230 target sites, for 31 oar-miRNAs in O.aries mRNAs. Gene ontology analysis showed that these target genes nearly took part in all kinds of biological process, and the molecular function which involved most is binding activity.
本研究通过构建小分子RNA cDNA文库和生物信息学方法两种途径鉴定绵羊miRNA ,分析它们在不同组织中的表达情况,寻找肌肉特异miRNA,并利用在线生物信息学软件预测绵羊miRNA的靶基因,对靶基因的功能进行分析。结果小结如下:
1.通过构建绵羊背最长肌、大脑、肝脏、脾脏4个组织的小分子RNA cDNA文库,克隆得到17个绵羊miRNA。得到的miRNA中,12个克隆自背最长肌,6个克隆自大脑,其余2个克隆自肝脏和脾脏。
2.通过生物信息学方法,在绵羊表达序列标签(EST)数据库中鉴定出14个绵羊miRNA ,其中包括4对miRNA/miRNA*和1对miRNA-3p/miRNA-5p。同时鉴定出一个miRNA基因簇,由oar-miR-374b、oar-miR-374b*和oar-miR-421组成。
3.序列分析表明,鉴定出的31个绵羊miRNA属于24个miRNA家族,在绵羊中均为首次发现。其中2个miRNA (oar-NEW-1和oar-NEW-2)在其他物种中均未见报道,为新的miRNA。其余29个miRNA为其它物种中已知miRNA的绵羊同源基因。
4. Northern blot结果表明,oar-miR-1仅在心肌和背最长肌中表达,oar-miR-122仅在肝脏中表达,oar-NEW-1仅在脾脏中表达。
5. Real-time PCR结果表明,除oar-miR-425-5p没有在心肌中检测到之外,检测的其余miRNA在绵羊的6个组织中均有表达。并且,oar-miR-24在心肌和背最长肌中的表达量显著高于在其他组织中的表达量;oar-miR-24-1*在心肌中的表达量显著高于在其他组织中的表达量;oar-miR-425-3p在背最长肌中的表达量显著高于在其他组织中的表达量;oar-miR-425-5p在大脑中的表达量显著高于在其他组织中的表达量。
6.利用鉴定出的31个绵羊miRNA序列,我们在绵羊基因组中的保守3’UTR区域预测得到120个靶基因,包括230个靶位点。GO分类结果表明,这些靶基因几乎参与了全部的生物学过程,参与功能最多的是结合活性。
MicroRNAs (miRNAs), about 22nt in length, are a class of non-coding small RNA distribute extensively in human genome and other eukaryote genome. They regulate gene expression through sequence specific interactions with target mRNAs resulting in translational repression or mRNA degradation. Recent findings showed that miRNAs play an important role in embryo formation, organogenesis, cell death, cell proliferation, and cancer development, and so on. By April 2010, the number of known miRNAs has grown rapidly to 14,197 in more than 130 species. O.aries (Ovis aries) is one of the most important agricultural livestock for meat, milk, and wool production. But there is limited information about O.aries miRNAs. Thus, to research on O.aries miRNAs is very important and necessary.
In this study, we combined a computational method based on expressed sequence tag (EST) analysis with experimental identification based on small RNA cDNA library to identify O.aries miRNAs. Then, we analized their expression level in different sheep tissues to find out muscle specific miRNAs, predicted their target genes and conducted the function analysis for them. The results are as follows:
1. First, by constructing four cDNA libraries for small RNAs in the size range of 18–26nt from the tissues of longissimus muscle, brain, liver and spleen, we cloned 17 O.aries miRNAs. Among these miRNAs, 12 were isolated from longissimus muscle, 6 were cloned from brain, and 2 others were cloned from liver and spleen.
2. Second, we used a computational approach to exploit O.aries miRNAs in sheep EST database. Following a set of strict filtering criteria, we finally identified 14 O.aries miRNAs, which included 4 pairs of miRNA/miRNA*, and 1 pair of miRNA-3p/miRNA-5p. A miRNA cluster was also identified, including oar-miR-374b, oar-miR-374b* and oar-miR-421.
3. Altogether, we identified 31 oar-miRNAs belong to 24 families, all of which were first identified in sheep. 2 of them were novel miRNAs that had never been annotated in miRBase database, named oar-NEW-1 and oar-NEW-2. The remaining 29 miRNAs were sheep orthologs of known mammalian miRNAs.
4. Northern blot showed that 3 miRNAs appeared to be extremely tissue specific. oar-miR-1 could be only detected in heart muscle and longissimus muscle, oar-miR-122 could be only detected in liver, and oar-NEW-1 could be only detected in spleen.
5. We chose 4 miRNAs (the pair of oar-miR-24/oar-miR-24-1* and the pair of oar-miR-425-3p/oar- miR-425-5p) identified by computational method for expression analysis by Real-time PCR. All of these four miRNAs could be detected in total tissues, except that oar-miR-425-5p could not be detected in heart muscle. The results also showed that oar-miR-24 had a significant higher expression level in longissimus muscle and heart muscle than which in the other tissues, as same as oar-miR-24-1* in heart muscle, oar-miR-425-3p in longissimus muscle, and oar-miR-425-5p in brain.
6. Further, we predicted 120 potential target genes, included 230 target sites, for 31 oar-miRNAs in O.aries mRNAs. Gene ontology analysis showed that these target genes nearly took part in all kinds of biological process, and the molecular function which involved most is binding activity.
引文
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