猪脂肪沉积相关miRNA初步筛选
发布时间：2018-09-18 09:38
【摘要】：MicroRNAs(miRNAs)是在真核生物中发现的一类内源性的具有调控功能的非编码RNA,其大小长约20~25个核苷酸。成熟的miRNAs是由较长的初级转录物经过一系列核酸酶的剪切加工而产生的,随后组装进RNA诱导的沉默复合体,通过碱基互补配对的方式识别靶mRNA,并根据互补程度的不同指导沉默复合体降解靶mRNA或者阻遏靶mRNA的翻译。最近的研究表明mi RNA参与各种各样的调节途径,包括发育、病毒防御、造血过程、器官形成、细胞增殖和凋亡、脂肪代谢等等。为了对沂蒙黑猪肝脏中调节脂肪沉积的miRNA进行初步筛选,本研究以四头沂蒙黑猪(其中母猪、阉猪各两头)肝脏组织为研究材料,分别构建了母猪和阉猪两个小RNA文库,采用Illumina/Solexa高通量测序技术对构建的两个文库分别进行测序,并对测序数据进行了质量检测,长度分布统计并筛选出了在两个文库中差异表达的miRNA进行把基因预测、基因功能注释和KEGG通路分析。通过Solexa高通量测序取得结果如下:在母猪和阉猪两个文库中分别获得5894364和6435206纯净序列读数,分别占其总读数的54.67%和60.2%。小RNA长度分布统计结果表明:两个文库中小RNA序列主要分布在21-23nt之间,22nt序列最多,在两个文库中分别占27.32%和28.78%。其次为21nt和23nt。不同片段长度的小RNA在两个文库中呈现差异分布。通过Solexa高通量我们发现共167个miRNA在不同性别猪中表达丰度高且呈现差异表达模式,生物信息学分析表明:这些差异表达的miRNA的靶基因参与了肌动蛋白细胞骨架调节、MAPK信号通路、Notch、局部黏连、内噬作用、白细胞跨内皮迁移、神经营养因子的信号转导通路等信号通路。选取ssc-mir-4332、ssc-mir-4338、ssc-miR-127、ssc-mi R-424、ssc-mi R-194、ssc-miR-545、ssc-miR-22采用qRT-PCR进行表达量检测,其结果显示选取的miRNA皆差异显著,其中ssc-mir-4332、ssc-mir-4338、ssc-mi R-127、ssc-miR-424下调,ssc-miR-194、ssc-miR-545、ssc-miR-22上调,与测序结果在两种性别猪中表达一致。GO注释结果表明,生物学过程中,有超过50%的基因被注释到了信号传导、蛋白质磷酸化、转运、代谢过程、氧化还原过程;细胞组分中主要注释到细胞核和细胞质;分子功能主要被注释到蛋白质结合条目。本研究结果初步确定了3个与脂肪沉积相关的重要候选miRNAs,ssc-miRNA-4338、ssc-mi RNA-424和ssc-miRNA-545,这些miRNA都靶向细胞色素丙酮酸脱氢酶激酶4(Pyruvate Dehydrogenase kinase 4,PDK4)。该基因与系水力、肌内脂肪含量、肌肉色值等重要肉质性状有显著相关。为我们深入研究microRNA在脂肪沉积调控中的作用机制奠定基础,为猪的分子育种提供理论基础和科学依据。
[Abstract]:MicroRNAs (miRNAs) is an endogenous non-coding RNA, with regulatory function found in eukaryotes. Its size is about 20 ~ 25 nucleotides. Mature miRNAs is produced by a series of nuclease cleavage processes of long primary transcripts, which are then assembled into the silencing complex induced by RNA. The target mRNA, was identified by base complementary pairing and the target mRNA was degraded or repressed by silencing complex according to the degree of complementarity. Recent studies have shown that mi RNA is involved in a variety of regulatory pathways, including development, viral defense, hematopoiesis, organogenesis, cell proliferation and apoptosis, fat metabolism, and so on. In order to screen the miRNA in Yimeng black pig liver regulating fat deposition, four Yimeng black pigs (including two sows and two castrated pigs) liver tissue were used to construct two small RNA libraries of sows and castrated pigs, respectively. The two libraries were sequenced by Illumina/Solexa high-throughput sequencing technique, and the quality of the sequencing data was detected. The length distribution was counted and the differentially expressed miRNA was selected to predict the genes in the two libraries. Gene function annotation and KEGG pathway analysis. The results obtained by Solexa high-throughput sequencing were as follows: 5894364 and 6435206 pure sequence readings were obtained in sows and castrated pigs, accounting for 54.67% and 60.2% of the total reading, respectively. The statistical results of the small RNA length distribution showed that the small and medium RNA sequences of the two libraries were mainly distributed between the 21-23nt and the 22NT sequences, accounting for 27.32% and 28.78% of the two libraries, respectively. 21nt and 23nt. Small RNA with different fragment lengths showed different distributions in the two libraries. Through the high throughput of Solexa we found a total of 167 miRNA expressed in different sex pigs with high abundance and different expression patterns. Bioinformatics analysis showed that these differentially expressed miRNA target genes were involved in actin cytoskeleton regulation of MAPK signaling pathway, local adhesion, internal phagocytosis, leukocyte transendothelial migration. The signal transduction pathway of neurotrophic factor. Ssc-mir-4332,ssc-mir-4338,ssc-miR-127,ssc-mi R-424 ssc-mi R-194ssc-miR-545nsc-miR-22 was detected by qRT-PCR. The results showed that the miRNA was significantly different, and ssc-mir-4332,ssc-mir-4338,ssc-mi R-127 ssc-miR-424 down-regulated the up-regulation of ssc-miR-194 ssc-miR-194ssc-miR-545ssc-miR-22, which was consistent with the sequencing results. More than 50% of the genes were annotated to signal transduction, protein phosphorylation, transport, metabolism, redox process; cell components were mainly annotated to nucleus and cytoplasm; and molecular functions were mainly annotated to protein binding items. In this study, we preliminarily identified three important candidates for miRNAs,ssc-miRNA-4338,ssc-mi RNA-424 and ssc-miRNA-545, related to fat deposition. These miRNA were targeted at cytochrome pyruvate dehydrogenase kinase 4 (Pyruvate Dehydrogenase kinase 4 / PDK4). The gene was significantly correlated with important meat traits, such as waterpower, intramuscular fat content and muscle color value. It provides a theoretical and scientific basis for the further study of the mechanism of microRNA in the regulation of fat deposition and for the molecular breeding of pigs.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828
本文编号：2247508
[Abstract]:MicroRNAs (miRNAs) is an endogenous non-coding RNA, with regulatory function found in eukaryotes. Its size is about 20 ~ 25 nucleotides. Mature miRNAs is produced by a series of nuclease cleavage processes of long primary transcripts, which are then assembled into the silencing complex induced by RNA. The target mRNA, was identified by base complementary pairing and the target mRNA was degraded or repressed by silencing complex according to the degree of complementarity. Recent studies have shown that mi RNA is involved in a variety of regulatory pathways, including development, viral defense, hematopoiesis, organogenesis, cell proliferation and apoptosis, fat metabolism, and so on. In order to screen the miRNA in Yimeng black pig liver regulating fat deposition, four Yimeng black pigs (including two sows and two castrated pigs) liver tissue were used to construct two small RNA libraries of sows and castrated pigs, respectively. The two libraries were sequenced by Illumina/Solexa high-throughput sequencing technique, and the quality of the sequencing data was detected. The length distribution was counted and the differentially expressed miRNA was selected to predict the genes in the two libraries. Gene function annotation and KEGG pathway analysis. The results obtained by Solexa high-throughput sequencing were as follows: 5894364 and 6435206 pure sequence readings were obtained in sows and castrated pigs, accounting for 54.67% and 60.2% of the total reading, respectively. The statistical results of the small RNA length distribution showed that the small and medium RNA sequences of the two libraries were mainly distributed between the 21-23nt and the 22NT sequences, accounting for 27.32% and 28.78% of the two libraries, respectively. 21nt and 23nt. Small RNA with different fragment lengths showed different distributions in the two libraries. Through the high throughput of Solexa we found a total of 167 miRNA expressed in different sex pigs with high abundance and different expression patterns. Bioinformatics analysis showed that these differentially expressed miRNA target genes were involved in actin cytoskeleton regulation of MAPK signaling pathway, local adhesion, internal phagocytosis, leukocyte transendothelial migration. The signal transduction pathway of neurotrophic factor. Ssc-mir-4332,ssc-mir-4338,ssc-miR-127,ssc-mi R-424 ssc-mi R-194ssc-miR-545nsc-miR-22 was detected by qRT-PCR. The results showed that the miRNA was significantly different, and ssc-mir-4332,ssc-mir-4338,ssc-mi R-127 ssc-miR-424 down-regulated the up-regulation of ssc-miR-194 ssc-miR-194ssc-miR-545ssc-miR-22, which was consistent with the sequencing results. More than 50% of the genes were annotated to signal transduction, protein phosphorylation, transport, metabolism, redox process; cell components were mainly annotated to nucleus and cytoplasm; and molecular functions were mainly annotated to protein binding items. In this study, we preliminarily identified three important candidates for miRNAs,ssc-miRNA-4338,ssc-mi RNA-424 and ssc-miRNA-545, related to fat deposition. These miRNA were targeted at cytochrome pyruvate dehydrogenase kinase 4 (Pyruvate Dehydrogenase kinase 4 / PDK4). The gene was significantly correlated with important meat traits, such as waterpower, intramuscular fat content and muscle color value. It provides a theoretical and scientific basis for the further study of the mechanism of microRNA in the regulation of fat deposition and for the molecular breeding of pigs.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828
【参考文献】
相关期刊论文 前1条
1 郭亚宁;高雪;杨公社;许尚忠;;肉质性状相关基因的研究进展[J];家畜生态学报;2006年03期
，本文编号：2247508
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