本文關(guān)鍵詞： 紅鰭笛鯛 黑視蛋白 基因 表達 進化　出處：《廣東海洋大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：黑視蛋白作為非視覺系統(tǒng)中由opn4編碼的一種維生素A類感光受體,是外圍生物鐘光傳導(dǎo)通路上游關(guān)鍵作用因子,參與了生物體多種生化生理反應(yīng)。為了揭示紅鰭笛鯛炓視蛋白基因的結(jié)構(gòu)、組織分布、早期發(fā)育各階段表達規(guī)律及與其他笛鯛魚類的進化關(guān)系,本研究采用RT-PCR獲取紅鰭笛鯛opn4基因cDNA全長,利用常規(guī)PCR結(jié)合DNA測序方法,基于opn4基因分析紅鰭笛鯛與其他笛鯛的進化關(guān)系,并利用qRT-PCR分別測定opn4在紅鰭笛鯛成體各組織和早期發(fā)育各階段的表達。結(jié)果如下:1.首次成功克隆了紅鰭笛鯛的opn4基因cDNA全長,該序列片段長度為4595bp,由121bp的5’非翻譯區(qū)、2794bp的3’非翻譯區(qū)及1680bp開放閱讀框組成,編碼559個氨基酸,與其他硬骨魚類炓視蛋白氨基酸序列同源性大于83%,含有7個跨膜結(jié)構(gòu)和4個高度保守的G蛋白結(jié)構(gòu)功能域。2.運用PCR克隆后測序的方式,獲取8種笛鯛相應(yīng)的opn4基因mRNA序列(927bp)和657bp基因序列(含內(nèi)含子554bp),分別進行氨基酸序列比對和序列差異分析,結(jié)果發(fā)現(xiàn)8種笛鯛無論外顯子(927bp)之間和還是內(nèi)含子(554bp)之間的序列差異性都較小,其中在8種笛鯛927bp核酸序列中均未見插入,缺失或終止密碼子,對應(yīng)編碼的氨基酸序列中則存在14個多態(tài)位點,紅鰭笛鯛和千年笛鯛均沒有顯示含有特有氨基酸殘基,其他幾種笛鯛則分別含有兩個及以上的特有氨基酸殘基;而在8種笛鯛554bp內(nèi)含子序列中存在部分堿基的插入和缺失。3.使用RT-qPCR技術(shù),分別測定了opn4基因在紅鰭笛鯛11個組織(視網(wǎng)膜、腦、心、肝、脾、胃、腸、肌肉、皮膚、鰓和性腺)中的表達情況。發(fā)現(xiàn)opn4基因僅在紅鰭笛鯛肝臟、視網(wǎng)膜、性腺和皮膚中表達,且在視網(wǎng)膜中的表達量遠高于其他組織,opn4在皮膚中的高表達很可能與魚類體色隨光環(huán)境變化有關(guān)。由于目前尚未見報道該基因在魚類肝臟和性腺中有表達,經(jīng)過數(shù)次重復(fù)驗證,證實在紅鰭笛鯛這兩部位確實檢測到opn4存在少量表達,但對于其所發(fā)揮的具體作用有待進一步的探討。4.利用RT-qPCR技術(shù),分別檢測了opn4在紅鰭笛鯛早期發(fā)育10個階段(多細胞、下包1/2、胚孔封閉、視囊、晶體出現(xiàn)、心臟跳動、孵化期、1天仔魚、3天仔魚、10天仔魚)中的表達情況。在紅鰭笛鯛早期發(fā)育各時期均可檢測到opn4的表達,而在下包1/2期、胚孔封閉、晶體形成和孵化后一天的仔魚這四個時期的表達顯著,說明了opn4基因在紅鰭笛鯛早期發(fā)育時期就已出現(xiàn)波動變化的表達,這可能與調(diào)節(jié)胚胎發(fā)育環(huán)境光協(xié)同以及滿足各種生理行為的需求相關(guān)。
[Abstract]:Melanin, as a type of vitamin A photoreceptor encoded by opn4 in the non-visual system, is a key factor in the upstream of the circadian clock light transduction pathway. In order to reveal the structure, tissue distribution, expression law of early development stages and the evolutionary relationship with other snappers, we have participated in a variety of biochemical physiological responses of the organism, in order to reveal the structure, tissue distribution, expression pattern and evolution of the protein gene in red snapper. In this study, the full-length cDNA of opn4 gene was obtained by RT-PCR. The evolutionary relationship between Lutinus macrocephalus and other snappers was analyzed based on opn4 gene by using conventional PCR and DNA sequencing methods. QRT-PCR was used to determine the expression of opn4 in the adult tissues and early stages of development of Lutanus macrocephalus. The results were as follows: 1. The full length of opn4 gene cDNA was cloned successfully for the first time. The length of the fragment is 4595 BP, which consists of 121bp 5'untranslated region (121bp), 3'untranslated region (2794bp) and 1680bp open reading frame (ORAF), encoding 559 amino acids. The amino acid sequence of the protein is more than 833, and contains 7 transmembrane structures and 4 highly conserved functional domains of G protein structure. 2. The sequence was sequenced by PCR cloning. The corresponding mRNA sequence of opn4 gene (927bpp) and 657bp gene sequence (containing intron 554 BP) were obtained for amino acid sequence alignment and sequence difference analysis, respectively. The results showed that the sequence differences between exon 927bp) and intron 554bp) were small, and no insertion, deletion or termination codon was found in the nucleic acid sequence of 927bp. There were 14 polymorphic loci in the corresponding amino acid sequence. Neither Lutinus macrocephalus nor Lutonic bream had specific amino acid residues, while the others had two or more unique amino acid residues respectively. The insertion and deletion of partial bases in the 554 BP intron sequence of Lutanus macrocephalus were studied by RT-qPCR technique. The opn4 gene was detected in 11 tissues (retina, brain, heart, liver, spleen, stomach, intestine, muscle and skin). The expression of opn4 gene was found only in the liver, retina, gonad and skin of Lutanus macrocephalus. Moreover, the high expression of Pn4 in the retina is much higher than that in other tissues, which may be related to the changes of fish body color with the light environment. As there are no reports of the expression of the gene in fish liver and gonad, it has been repeatedly verified several times. It was confirmed that there was a small amount of opn4 expression in the two parts of Lutinus macrocephalus, but the specific role of opn4 in the early development of Lutanus macrocephalus was studied by using RT-qPCR technique, and 10 stages (multicellular) of opn4 were detected in the early stage of development of Lutanus macrocephalus. The expression of opn4 was detected at the early development stage of Lutanus macrocephalus, but at the 1/2 stage, the expression of opn4 was detected at the early development stage of Lutanus macrocephalus, and the expression of opn4 was detected at the early stage of development, and the expression of opn4 was detected during the early development of Lutanus macrocephalus, and the expression of opn4 was detected during the early development of Lutanus macrocephalus. The expression of opn4 gene in the four stages of embryo pore closure, crystal formation and hatching was significant, which indicated that the expression of opn4 gene had been fluctuating in the early development period of Lutinus macrocephalus. This may be related to the regulation of the environment of embryonic development and to meet the needs of various physiological behaviors.
【學(xué)位授予單位】：廣東海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S917.4

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