【摘要】：MicroRNAs(miRNAs)是在真核生物中發(fā)現的一類內源性的具有調控功能的非編碼RNA,其大小長約20~25個核苷酸。成熟的miRNAs是由較長的初級轉錄物經過一系列核酸酶的剪切加工而產生的,隨后組裝進RNA誘導的沉默復合體,通過堿基互補配對的方式識別靶mRNA,并根據互補程度的不同指導沉默復合體降解靶mRNA或者阻遏靶mRNA的翻譯。最近的研究表明mi RNA參與各種各樣的調節(jié)途徑,包括發(fā)育、病毒防御、造血過程、器官形成、細胞增殖和凋亡、脂肪代謝等等。為了對沂蒙黑豬肝臟中調節(jié)脂肪沉積的miRNA進行初步篩選,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,分別構建了母豬和閹豬兩個小RNA文庫,采用Illumina/Solexa高通量測序技術對構建的兩個文庫分別進行測序,并對測序數據進行了質量檢測,長度分布統計并篩選出了在兩個文庫中差異表達的miRNA進行把基因預測、基因功能注釋和KEGG通路分析。通過Solexa高通量測序取得結果如下:在母豬和閹豬兩個文庫中分別獲得5894364和6435206純凈序列讀數,分別占其總讀數的54.67%和60.2%。小RNA長度分布統計結果表明: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在兩個文庫中呈現差異分布。通過Solexa高通量我們發(fā)現共167個miRNA在不同性別豬中表達豐度高且呈現差異表達模式,生物信息學分析表明:這些差異表達的miRNA的靶基因參與了肌動蛋白細胞骨架調節(jié)、MAPK信號通路、Notch、局部黏連、內噬作用、白細胞跨內皮遷移、神經營養(yǎng)因子的信號轉導通路等信號通路。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進行表達量檢測,其結果顯示選取的miRNA皆差異顯著,其中ssc-mir-4332、ssc-mir-4338、ssc-mi R-127、ssc-miR-424下調,ssc-miR-194、ssc-miR-545、ssc-miR-22上調,與測序結果在兩種性別豬中表達一致。GO注釋結果表明,生物學過程中,有超過50%的基因被注釋到了信號傳導、蛋白質磷酸化、轉運、代謝過程、氧化還原過程;細胞組分中主要注釋到細胞核和細胞質;分子功能主要被注釋到蛋白質結合條目。本研究結果初步確定了3個與脂肪沉積相關的重要候選miRNAs,ssc-miRNA-4338、ssc-mi RNA-424和ssc-miRNA-545,這些miRNA都靶向細胞色素丙酮酸脫氫酶激酶4(Pyruvate Dehydrogenase kinase 4,PDK4)。該基因與系水力、肌內脂肪含量、肌肉色值等重要肉質性狀有顯著相關。為我們深入研究microRNA在脂肪沉積調控中的作用機制奠定基礎,為豬的分子育種提供理論基礎和科學依據。
[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.
【學位授予單位】：山東農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S828

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1 郭亞寧;高雪;楊公社;許尚忠;;肉質性狀相關基因的研究進展[J];家畜生態(tài)學報;2006年03期

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本文編號：2247508