本文選題：淇河鯽 + MSTN��； 參考：《河南師范大學》2016年碩士論文

【摘要】：淇河鯽(Carassius auratus in Qihe River)屬于銀鯽的一種,天然雌核發(fā)育,脊背寬厚是區(qū)別于其它銀鯽魚最主要的特征。目前在淇河鯽育種、疾病免疫、飼料營養(yǎng)等方面已開展較多研究,但是淇河鯽生長性狀相關(guān)的基礎(chǔ)研究還相對薄弱,其脊背寬厚的生長機制尚不清楚。肌肉生長抑制素基因(myostatin,MSTN)是一種負調(diào)控因子,它最主要功能是調(diào)節(jié)動物的骨骼肌肉生長發(fā)育。microRNAs(miRNAs)是一類非編碼單鏈小RNAs,近年來它的作用和功能越來越多地被發(fā)掘。miRNAs技術(shù)的發(fā)展為探索淇河鯽脊背寬厚這一問題開辟了一條新的途徑。提取淇河鯽總RNA,克隆,得到淇河鯽MSTN3’UTR序列。根據(jù)MSTN 3’UTR序列通過TargetScans等軟件預測出相關(guān)miRNAs,再利用RNAhybrid軟件進行驗證,最終選取miR-181a和miR-181b作為候選mi RNAs。以肌肉總DNA為模板,獲得pri-miR-181a和pri-miR-181b全長。構(gòu)建真核表達載體和miRNA慢病毒載體,通過雙熒光檢測和熒光原位雜交技術(shù)分別從分子水平和組織細胞定位方面確定MSTN和miR-181a、miR-181b之間的相互關(guān)系。采用qRT-PCR分析miR-181a、miR-181b和MSTN在淇河鯽腦、肌肉和心臟等8個組織的表達情況以及miR-181a、miR-181b和MSTN在胚胎發(fā)育及其在前期生長不同階段的表達情況。由此進一步揭示淇河鯽肌肉生長相關(guān)基因間的多層次網(wǎng)絡(luò)調(diào)控模式,探索淇河鯽肌肉生長機制。1、淇河鯽MSTN基因3’UTR的克隆提取總RNA,克隆得到MSTN基因3’UTR,拼接后獲得3’UTR長度是865bp。在NCBI中,經(jīng)過Blast比對發(fā)現(xiàn),得到片段與鯉形目魚類相似度較高,尤其與南亞野鯪(Labeo rohita)(91%,KR052242.1)和鯉魚(Cyprinus carpio)(92%,LN590705.1),淇河鯽(Carassius auratus in Qihe river)(88%,KC851952.1)同源性最高。2、淇河鯽MSTN基因相關(guān)miRNAs的預測、驗證及克隆根據(jù)MSTN基因3’UTR序列結(jié)果,利用RegRNA、Pictar、TargetScan、miRWalk、miRDB等軟件,,預測相關(guān)miRNAs,進一步用RNAhybrid軟件驗證,選取可能與MSTN基因相關(guān)的miR-181a和miR-181b作為候選miRNAs。從淇河鯽肌肉中提取總DNA,克隆pri-miR-181a和pri-miR-181b。3、真核載體的構(gòu)建及雙熒光檢測本實驗中把MSTN 3’UTR序列和加突變位點的3’UTR序列雙酶切,連接至pmirGLO載體;pri-miR-181a/b雙酶切,連接至Lenti H1載體,分別構(gòu)建真核表達載體和miRNAs慢病毒載體。構(gòu)建的兩個載體共轉(zhuǎn)染至293T細胞,雙熒光檢測發(fā)現(xiàn),miR-181a/b對MSTN基因均可能有一定的抑制作用,此外還發(fā)現(xiàn)miR-181a/b與MSTN還可能存在其他的結(jié)合位點共同作用調(diào)節(jié)MSTN基因的表達。4、MSTN和miR-181a在肌肉組織中的表達定位取淇河鯽背部和腹部肌肉,制成石蠟切片,合成探針,檢測MSTN基因和mi R-181a在肌肉組織中的表達位置。結(jié)果表明,MSTN在肌纖維中表達量較高;mi R-181a在肌漿膜中表達量較高,并且在結(jié)締組織中也有表達。此外還發(fā)現(xiàn)miR-181a在背部肌肉的表達量低于腹部,在腹部肌纖維中MSTN表達量顯著高于背部,因此推測在背部肌肉中miR-181a對MSTN的抑制作用可能沒有腹部明顯。5、淇河鯽MSTN基因及相關(guān)miR-181a/b特異性表達qRT-PCR結(jié)果顯示,淇河鯽MSTN基因在不同組織表達量存在顯著性差異,其中心肌和腦中最高,肌肉其次,腎臟最低;胚胎發(fā)育及前期生長不同階段,MSTN基因在原腸胚以前,出生20d表達量顯著高于其他時期。miR-181a在肌肉和鰓的表達豐度顯著高于其他組織,在原腸胚以前和孵化10d后也具有較高的含量。miR-181b在腦、肌肉和腸有較高的表達量,原腸胚以前和孵化10d后表達量顯著高于其他時期。
[Abstract]:The Carassius auratus (Carassius auratus in Qihe River) is one of the silver crucian carp (Carassius auratus). The natural female nucleus is developed, and the back width is the main feature different from other silver carp. At present, much research has been carried out on the breeding, disease immunity and feed nutrition of the Carassius auratus in Qihe River, but the basic research on the long traits of the Carassius auratus in Qi He is still relatively weak and its backbone is wide. Myostatin (MSTN) is a negative regulatory factor. The most important function of the gene is to regulate the growth and development of skeletal muscle in animals..microRNAs (miRNAs) is a kind of non coding single strand small RNAs. In recent years, its function and function have been more and more exploited to explore the development of.MiRNAs technology to explore Qi. The general RNA of Carassius carassius carp opened a new way. The total RNA of Qihe crucian carp was extracted, and the MSTN3 'UTR sequence of Qihe crucian carp was cloned. According to the MSTN 3' UTR sequence, the related miRNAs was predicted by TargetScans software and then the miR-181a and miR-181b were selected as the candidate mi RNAs.. The whole length of pri-miR-181a and pri-miR-181b was obtained. The eukaryotic expression vector and the miRNA lentivirus carrier were constructed. The relationship between MSTN and miR-181a and miR-181b was determined by double fluorescence detection and fluorescence in situ hybridization. QRT-PCR analysis miR-181a, miR-181b and MSTN in Qihe crucian carp were used. The expression of 8 tissues, such as brain, muscle and heart, as well as the expression of miR-181a, miR-181b and MSTN in the development of embryo and the different stages of early growth, thus further reveal the multi-level network regulation mode between the muscle growth related genes of Qi He carp, explore the mechanism of muscle growth of Qi River Carassius auratus,.1, and the MSTN gene of Qi He crucian carp, 3 'UTR. The total RNA was extracted, and the MSTN gene was cloned to 3 'UTR, and the length of 3' UTR after splicing was 865bp. in NCBI. After Blast comparison, it was found that the similarity between the fragment and the carp shaped fish was higher, especially with the South Asian wild carp (Labeo rohita) (91%, KR052242.1) and the carp (Cyprinus carpio). (88%, KC851952.1) the highest homologous.2, the prediction, verification and cloning of the miRNAs related to the MSTN gene of Qi River Carassius auratus, verification and cloning based on the results of the MSTN gene 3 'UTR sequence, using RegRNA, Pictar, TargetScan, miRWalk, miRDB and other software to predict the related miRNAs. The total DNA, pri-miR-181a and pri-miR-181b.3 were extracted from the muscle of Qi He crucian carp, pri-miR-181a and pri-miR-181b.3 were cloned. In the construction of eukaryotic vector and double fluorescence detection, the MSTN 3 'UTR sequence and the 3' UTR sequence of the mutation site were cut into the pmirGLO carrier; pri-miR-181a/b double enzyme cut, connected to Lenti H1 vector, constructed the eukaryotic expression vector. The two vectors constructed with miRNAs were transfected into 293T cells. Double fluorescence detection found that miR-181a/b could inhibit the MSTN gene. Furthermore, it was found that miR-181a/b and MSTN may also have other binding sites to regulate the expression of the MSTN gene, and the expression of MSTN and miR-181a in the muscle tissue. Locating the muscles of the back and abdomen of the crucian carp, the paraffin section was made into a paraffin section and a probe was synthesized to detect the expression of the MSTN gene and MI R-181a in the muscle tissue. The results showed that the expression of MSTN in the muscle fibers was higher; the expression of MI R-181a in the serous membrane was higher, and the expression of MI was also expressed in the connective tissue. Moreover, miR-181a was also found in the back muscles. The expression of MSTN in the abdominal muscle was significantly higher than that in the back. Therefore, it is presumed that the inhibitory effect of miR-181a on MSTN in the back muscles may not be obvious in the abdominal.5. The qRT-PCR results of the MSTN gene and related miR-181a/b specific expression of the Qi He crucian carp found that the MSTN gene in Qi He crucian carp has a significant difference in the expression of different tissues. Among them, the highest in the heart and the brain, the second of the muscle and the lowest in the kidney; the MSTN gene was significantly higher in the expression of 20d in the muscles and gills than in other tissues before the embryo of the gastrula and at the different stages of the early growth of the embryo. The expression of the MSTN gene was significantly higher than that of the other tissues before and after the incubation of the proembryo and after the incubation of the 10d. In the brain, muscle and intestine, the expression level was higher, and the expression level of gastrula before and after hatching 10d was significantly higher than that in other periods.

【學位授予單位】：河南師范大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S917.4

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