【摘要】：肌鈣蛋白I(Troponin I,Tn I)不僅對肌肉的正常收縮行使功能至關重要,還能從多個方面影響到肌肉的品質。肌鈣蛋白I家族三個成員分別為肌鈣蛋白I1(TNNI1)、肌鈣蛋白I2(TNNI2)和肌鈣蛋白I3(TNNI3)。本試驗根據小尾寒羊和杜泊羊轉錄組測序數據,篩選到差異表達基因TNNI2和TNNI3。通過比對得到的相關基因保守序列,利用RT-PCR、3’RACE和5’RACE等技術方法克隆得到兩個基因的cDNA全長;對兩個基因的核苷酸序列和氨基酸序列進行了多種生物信息學比對分析;運用qRT-PCR在m RNA水平驗證了兩個基因在小尾寒羊和杜泊羊不同組織中的表達豐度。研究結果如下:(1)采用RACE等方法,以小尾寒羊股二頭肌為試驗材料,克隆得到TNNI2基因cDNA全長714bp,開放閱讀框為549bp,編碼182個氨基酸,3’端有88bp的非翻譯區(不包括polyA尾),5’端有77bp的非翻譯區。以小尾寒羊心肌提取mRNA為模板,克隆得到TNNI3基因cDNA全長847bp(不包括polyA尾),開放閱讀框為639bp,編碼212個氨基酸,3’端有65bp的非翻譯區,5’端有143bp的非翻譯區。提交GenBank,獲得登錄號分別為KX110054和KX110055。(2)使用多種軟件和在線數據庫分析,綿羊TNNI2蛋白和山羊同源性最高,分子量為21.4kDa,等電點為8.8,是一種親水性較好的堿性蛋白;二級結構以α-螺旋為主,占到了70%以上,兼有少量的無規則卷曲;該蛋白是一種細胞內功能蛋白,并沒有預測到信號肽位點;擁有1個O端糖基化位點和9個磷酸化位點。綿羊的TNNI3蛋白則與牛的該蛋白具有極高同源性,分子量為24.1kDa,等電點為9.8,也是一種親水性較好的堿性蛋白;其二級結構與TNNI2具有較高的相似性,α-螺旋的比例為70.75%,其次是不規則卷曲;該蛋白未預測到信號肽位點;預測該蛋白具有多達14個磷酸化位點,但是沒有任何糖基化位點。(3)小尾寒羊和杜泊羊不同組織中,兩個基因在mRNA水平存在較大的表達差異。TNNI2基因的mRNA在肋間肌、背最長肌和股二頭肌中高表達,在肺臟、胃、心臟和肝臟等組織中表達量很低,說明了TNNI2基因在表達上具有特異性。在不同的品種之間,TNNI2基因在杜泊羊肋間肌組織中的表達量極顯著高于小尾寒羊(P0.01);在杜泊羊股二頭肌和肺臟組織中表達量顯著高于小尾寒羊(P0.05);但是在背最長肌、肝臟、胃和心臟組織中的表達量差異不顯著。TNNI3基因在綿羊的心臟組織中高表達,而在股二頭肌、肝臟、肺和胃中少量表達甚至不表達,說明了TNNI3基因的表達在生物體組織中有一定的空間特異性。TNNI3基因在綿羊心臟中的表達量極顯著高于其他組織(P0.01),說明TNNI3基因具有心臟表達特異性。在不同的品種間,TNNI3基因在杜泊羊心臟組織中的表達量極顯著高于小尾寒羊(P0.01),而在股二頭肌、肝臟、肺和胃中兩個品種羊的表達差異不顯著。(4)TNNI2蛋白在小尾寒羊和杜泊羊背最長肌、血管、肋間肌、股二頭肌、心肌、肝臟和肺臟六種組織中的表達存在一定差異,在肌肉組織中高表達,在其他組織中不表達或者很低表達。TNNI3蛋白特異在心肌中表達。綜上所述,本研究通過對肌鈣蛋白I家族的兩個基因TNNI2和TNNI3的cDNA全長克隆,從不同的角度對兩種蛋白的結構、功能和組織表達特異性進行了分析,為小尾寒羊的生產性狀改良和分子標記輔助育種提供依據。
[Abstract]:Tropin I (Tn I) is not only essential for the normal contraction function of muscle, but also affects muscle quality from several aspects. Three members of the cardiac troponin I family are troponin I1 (TNNI1), troponin I2 (TNNI2), and troponin I3 (TNNI3), respectively. The differentially expressed genes TNNI2 and TNNI3 were screened according to the sequencing data of small tail Han sheep and Dupoyang transcription group. The full-length cDNA of the two genes was cloned by RT-PCR, 3 'RACE and 5' RACE techniques. The expression abundance of two genes in different tissues of small tail Han sheep and Dupoyang sheep was verified by qRT-PCR at m RNA level. The results are as follows: (1) The total length of TNNI12 gene cDNA is 714bp, the open reading frame is 549bp, 182 amino acids, 3 'end has 88bp non-translation region (excluding polyA tail), 5 The' end has a non-translated region of 77bp. The total length of TNNI3 gene cDNA was 847bp (excluding polyA tail), the open reading frame was 639bp, 212 amino acids, 3 'end 65bp non-translation region and 143bp non-translation region at 5' end. The GenBank accession number was KX110054 and KX110055, respectively. (2) Using a variety of software and on-line database analysis, sheep TNNI2 protein and goat have the highest homology, the molecular weight is 21. 4kDa, the isoelectric point is 8. 8, it is a hydrophilic relatively good basic protein; The protein is an intracellular functional protein that is not predicted to the signal peptide site; has 1 O-terminal glycosylation site and 9 phosphorylation sites. The TNNI3 protein of sheep has extremely high homology with the protein of bovine, the molecular weight is 24. 1kDa, the isoelectric point is 9. 8, and also is a hydrophilic relatively good basic protein; The protein does not predict the signal peptide site; it is predicted that the protein has up to 14 phosphorylation sites, but there is no glycosylation site. (3) In the different tissues of the small tail Han sheep and the Dupoyang sheep, the mRNA levels of the two genes were significantly different. The mRNA of TNNI2 gene is highly expressed in the intercostal muscles, the longest muscle and quadriceps, and the expression of TNNI2 gene is very low in lung, stomach, heart and liver. The expression of TNNI2 gene in the intercostal muscle of Dupoyang sheep was significantly higher than that of small tail Han sheep (P0.01). There was no significant difference in expression between stomach and heart tissue. TNNI3 gene is highly expressed in sheep's heart tissue, but expression of TNNI3 gene in the liver, lung and stomach is not even expressed, indicating that the expression of TNNI3 gene has certain spatial specificity in organism tissue. The expression of TNNI3 gene in sheep heart was significantly higher than that of other tissues (P0.01). The expression of TNNI3 gene in the heart of Dupoyang sheep was significantly higher than that of small tail Han sheep (P0.01), but there was no significant difference in the expression of two breeds of sheep in the liver, lung and stomach. (4) The expression of TNNI2 protein in the six tissues of the longest muscle, blood vessel, intercostal muscle, quadriceps femoris, myocardium, liver and lung of the small tail Han sheep and the Dupoyang sheep has a certain difference, which is highly expressed in muscle tissue and is not expressed or expressed very low in other tissues. TNNI3 protein is specifically expressed in the myocardium, and the structure, function and tissue expression specificity of the two proteins are analyzed from different angles through the full-length cloning of the two genes TNNI2 and TNNI3 of the troponin I family. It provides the basis for the improvement of production traits and molecular marker assisted breeding for small-tailed Han sheep.
【學位授予單位】：山東農業大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S826;Q78

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本文編號：2305526