發(fā)布時(shí)間：2019-06-14 07:39

【摘要】：肌肉生長(zhǎng)抑制素(myostatin,Mstn)屬于轉(zhuǎn)化生長(zhǎng)因子-beta(TGF-β)超家族中的一個(gè)重要成員,對(duì)動(dòng)物肌肉的生長(zhǎng)和發(fā)育具有重要的負(fù)調(diào)控作用。隨著近年凡納濱對(duì)蝦養(yǎng)殖病害的頻發(fā),墨吉明對(duì)蝦(Fenneropenaeus merguiensis)作為對(duì)蝦養(yǎng)殖業(yè)的重要對(duì)蝦養(yǎng)殖品種逐漸成為人們的研究熱點(diǎn)。本實(shí)驗(yàn)克隆獲得了墨吉明對(duì)蝦FmMstn基因組DNA的全長(zhǎng)序列和兩個(gè)高度相似的FmMstn(短型和長(zhǎng)型)cDNA序列,原因是第1個(gè)外顯子和第一個(gè)內(nèi)含子交界處有兩個(gè)可選擇的GT剪接位點(diǎn)。FmMstn基因結(jié)構(gòu)與軟體動(dòng)物、魚類和哺乳類動(dòng)物的Mstn基因類似,含有3個(gè)外顯子和2個(gè)內(nèi)含子。在FmMstn基因上游的2160bp堿基啟動(dòng)子(promoter)序列,預(yù)測(cè)的轉(zhuǎn)錄因子結(jié)合位點(diǎn)包括CAAT box,TATA box,E-boxes和Mstn基因的標(biāo)記性轉(zhuǎn)錄因子即肌增強(qiáng)因子(myocyte enhancing factor element,MEF)。短FmMstn開放閱讀框(ORF)含1260bp堿基數(shù),編碼420氨基酸。長(zhǎng)FmMstn cDNA比短FmMstn cDNA多出(GNNYTSQS)8個(gè)氨基酸序列,其他序列同源性一致。FmMstn基因與凡納濱對(duì)蝦(Litopenaeus vannamei)和斑節(jié)對(duì)蝦(Penaeus monodon)的Mstn有較高的同源性(95%)。多重序列比對(duì)分析顯示,FmMstn蛋白質(zhì)結(jié)構(gòu)與其他動(dòng)物的Mstn結(jié)構(gòu)類似,具有兩個(gè)保守的切割位點(diǎn)(RXXR),高度保守的9個(gè)半胱氨酸殘基和位于成熟肽區(qū)高保守TGF功能區(qū)。Real-time PCR結(jié)果顯示,FmMstn在對(duì)蝦心臟、肌肉、眼柄和胸節(jié)神經(jīng)組織有高表達(dá)外,在其他組織微量表達(dá);FmMstn在對(duì)蝦蛻皮后期有較高的表達(dá)水平,隨后蛻皮間期FmMstn的表達(dá)量呈現(xiàn)下降的趨勢(shì),而在蛻皮前期時(shí)呈上調(diào)趨勢(shì)直到蛻皮;同一生長(zhǎng)周期的對(duì)蝦,較小個(gè)體的FmMstn表達(dá)水平較高。通過(guò)RNA干擾技術(shù)(RNAi),沉默F(xiàn)mMstn基因的表達(dá),可顯著延長(zhǎng)對(duì)蝦的蛻皮周期,甚至使對(duì)蝦蛻皮失敗。通過(guò)DNA測(cè)序結(jié)果的直接比對(duì)分析,結(jié)果顯示FmMstn基因具有高度的多態(tài)性,且本研究識(shí)別了幾個(gè)潛在的單核酸多肽(SNPs)位點(diǎn)。綜上所述,本研究的結(jié)果顯示墨吉明對(duì)蝦FmMstn與對(duì)蝦個(gè)體的蛻皮活動(dòng)和肌肉生長(zhǎng)有一定的相關(guān)性,而單核酸多態(tài)性(SNP)的存在說(shuō)明FmMstn基因可作為墨吉明對(duì)蝦遺傳選育研究的候選基因。
[Abstract]:Myostatin (myostatin,Mstn), an important member of transforming growth factor-beta (TGF- 尾) superfamily, plays an important negative role in muscle growth and development in animals. With the frequent occurrence of diseases in Penaeus vannamei culture in recent years, (Fenneropenaeus merguiensis), as an important shrimp culture variety in shrimp culture industry, has gradually become a hot research topic. In this experiment, the full-length sequence of FmMstn genomic DNA and two highly similar FmMstn (short and long) cDNA sequences were obtained because there were two optional GT splicing sites at the junction of the first exon and the first intron. The structure of FmMstn gene was similar to that of molluscs, fish and mammals with three exons and two introns. In the (promoter) sequence of 2160bp base promoter upstream of FmMstn gene, the predicted transcription factor binding sites include the marker transcription factor of CAAT box,TATA box,E-boxes and Mstn gene, muscle enhancer (myocyte enhancing factor element,MEF). Short FmMstn open reading frame (ORF) contains 1260bp base number and encodes 420 amino acids. The (GNNYTSQS) 8 amino acid sequences of long FmMstn cDNA were more than those of short FmMstn cDNA, and the homology of other sequences was the same. FmMstn gene had high homology with (Litopenaeus vannamei) and (Penaeus monodon) Mstn of Penaeus vannamei (95%). Multiple sequence alignment analysis showed that the structure of FmMstn protein was similar to that of other animals, with two conserved (RXXR), highly conserved TGF residues and highly conserved TGF functional areas in mature peptide regions. Real-time PCR results showed that FmMstn was highly expressed in the heart, muscle, eye handle and thoracic ganglia nerve tissue of Penaeus chinensis, and was slightly expressed in other tissues. The expression level of FmMstn was higher in the late molting stage, and then decreased in the intermolting period, but up-regulated until molting in the early molting stage, and the FmMstn expression level in smaller individuals was higher in the same growth cycle. Silencing the expression of FmMstn gene by RNA interference technique can significantly prolong the molting cycle of Penaeus chinensis and even make the molting failure of Penaeus chinensis. Through the direct comparison and analysis of DNA sequencing results, the results showed that the FmMstn gene had a high degree of polymorphism, and several potential single nucleic acid polypeptide (SNPs) sites were identified in this study. To sum up, the results of this study showed that FmMstn of Penaeus Mojiming was related to molting activity and muscle growth of Penaeus vulgaris, and the existence of single nucleic acid polymorphism (SNP) suggested that FmMstn gene could be used as a candidate gene for genetic breeding of Penaeus Mojiming.
【學(xué)位授予單位】：廣東海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S917.4

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