豬脂肪沉積相關(guān)miRNA初步篩選
發(fā)布時間：2018-09-18 09:38
【摘要】：MicroRNAs(miRNAs)是在真核生物中發(fā)現(xiàn)的一類內(nèi)源性的具有調(diào)控功能的非編碼RNA,其大小長約20~25個核苷酸。成熟的miRNAs是由較長的初級轉(zhuǎn)錄物經(jīng)過一系列核酸酶的剪切加工而產(chǎn)生的,隨后組裝進(jìn)RNA誘導(dǎo)的沉默復(fù)合體,通過堿基互補(bǔ)配對的方式識別靶mRNA,并根據(jù)互補(bǔ)程度的不同指導(dǎo)沉默復(fù)合體降解靶mRNA或者阻遏靶mRNA的翻譯。最近的研究表明mi RNA參與各種各樣的調(diào)節(jié)途徑,包括發(fā)育、病毒防御、造血過程、器官形成、細(xì)胞增殖和凋亡、脂肪代謝等等。為了對沂蒙黑豬肝臟中調(diào)節(jié)脂肪沉積的miRNA進(jìn)行初步篩選,本研究以四頭沂蒙黑豬(其中母豬、閹豬各兩頭)肝臟組織為研究材料,分別構(gòu)建了母豬和閹豬兩個小RNA文庫,采用Illumina/Solexa高通量測序技術(shù)對構(gòu)建的兩個文庫分別進(jìn)行測序,并對測序數(shù)據(jù)進(jìn)行了質(zhì)量檢測,長度分布統(tǒng)計并篩選出了在兩個文庫中差異表達(dá)的miRNA進(jìn)行把基因預(yù)測、基因功能注釋和KEGG通路分析。通過Solexa高通量測序取得結(jié)果如下:在母豬和閹豬兩個文庫中分別獲得5894364和6435206純凈序列讀數(shù),分別占其總讀數(shù)的54.67%和60.2%。小RNA長度分布統(tǒng)計結(jié)果表明:兩個文庫中小RNA序列主要分布在21-23nt之間,22nt序列最多,在兩個文庫中分別占27.32%和28.78%。其次為21nt和23nt。不同片段長度的小RNA在兩個文庫中呈現(xiàn)差異分布。通過Solexa高通量我們發(fā)現(xiàn)共167個miRNA在不同性別豬中表達(dá)豐度高且呈現(xiàn)差異表達(dá)模式,生物信息學(xué)分析表明:這些差異表達(dá)的miRNA的靶基因參與了肌動蛋白細(xì)胞骨架調(diào)節(jié)、MAPK信號通路、Notch、局部黏連、內(nèi)噬作用、白細(xì)胞跨內(nèi)皮遷移、神經(jīng)營養(yǎng)因子的信號轉(zhuǎn)導(dǎo)通路等信號通路。選取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進(jìn)行表達(dá)量檢測,其結(jié)果顯示選取的miRNA皆差異顯著,其中ssc-mir-4332、ssc-mir-4338、ssc-mi R-127、ssc-miR-424下調(diào),ssc-miR-194、ssc-miR-545、ssc-miR-22上調(diào),與測序結(jié)果在兩種性別豬中表達(dá)一致。GO注釋結(jié)果表明,生物學(xué)過程中,有超過50%的基因被注釋到了信號傳導(dǎo)、蛋白質(zhì)磷酸化、轉(zhuǎn)運(yùn)、代謝過程、氧化還原過程;細(xì)胞組分中主要注釋到細(xì)胞核和細(xì)胞質(zhì);分子功能主要被注釋到蛋白質(zhì)結(jié)合條目。本研究結(jié)果初步確定了3個與脂肪沉積相關(guān)的重要候選miRNAs,ssc-miRNA-4338、ssc-mi RNA-424和ssc-miRNA-545,這些miRNA都靶向細(xì)胞色素丙酮酸脫氫酶激酶4(Pyruvate Dehydrogenase kinase 4,PDK4)。該基因與系水力、肌內(nèi)脂肪含量、肌肉色值等重要肉質(zhì)性狀有顯著相關(guān)。為我們深入研究microRNA在脂肪沉積調(diào)控中的作用機(jī)制奠定基礎(chǔ),為豬的分子育種提供理論基礎(chǔ)和科學(xué)依據(jù)。
[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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S828
本文編號：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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S828
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 郭亞寧;高雪;楊公社;許尚忠;;肉質(zhì)性狀相關(guān)基因的研究進(jìn)展[J];家畜生態(tài)學(xué)報;2006年03期
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