红鳍笛鲷黑视蛋白基因克隆表达与进化分析

发布时间：2018-02-23 22:10

  本文关键词： 红鳍笛鲷 黑视蛋白 基因 表达 进化　出处：《广东海洋大学》2016年硕士论文　论文类型：学位论文

【摘要】：黑视蛋白作为非视觉系统中由opn4编码的一种维生素A类感光受体,是外围生物钟光传导通路上游关键作用因子,参与了生物体多种生化生理反应。为了揭示红鳍笛鲷炓视蛋白基因的结构、组织分布、早期发育各阶段表达规律及与其他笛鲷鱼类的进化关系,本研究采用RT-PCR获取红鳍笛鲷opn4基因cDNA全长,利用常规PCR结合DNA测序方法,基于opn4基因分析红鳍笛鲷与其他笛鲷的进化关系,并利用qRT-PCR分别测定opn4在红鳍笛鲷成体各组织和早期发育各阶段的表达。结果如下:1.首次成功克隆了红鳍笛鲷的opn4基因cDNA全长,该序列片段长度为4595bp,由121bp的5’非翻译区、2794bp的3’非翻译区及1680bp开放阅读框组成,编码559个氨基酸,与其他硬骨鱼类炓视蛋白氨基酸序列同源性大于83%,含有7个跨膜结构和4个高度保守的G蛋白结构功能域。2.运用PCR克隆后测序的方式,获取8种笛鲷相应的opn4基因mRNA序列(927bp)和657bp基因序列(含内含子554bp),分别进行氨基酸序列比对和序列差异分析,结果发现8种笛鲷无论外显子(927bp)之间和还是内含子(554bp)之间的序列差异性都较小,其中在8种笛鲷927bp核酸序列中均未见插入,缺失或终止密码子,对应编码的氨基酸序列中则存在14个多态位点,红鳍笛鲷和千年笛鲷均没有显示含有特有氨基酸残基,其他几种笛鲷则分别含有两个及以上的特有氨基酸残基;而在8种笛鲷554bp内含子序列中存在部分碱基的插入和缺失。3.使用RT-qPCR技术,分别测定了opn4基因在红鳍笛鲷11个组织(视网膜、脑、心、肝、脾、胃、肠、肌肉、皮肤、鳃和性腺)中的表达情况。发现opn4基因仅在红鳍笛鲷肝脏、视网膜、性腺和皮肤中表达,且在视网膜中的表达量远高于其他组织,opn4在皮肤中的高表达很可能与鱼类体色随光环境变化有关。由于目前尚未见报道该基因在鱼类肝脏和性腺中有表达,经过数次重复验证,证实在红鳍笛鲷这两部位确实检测到opn4存在少量表达,但对于其所发挥的具体作用有待进一步的探讨。4.利用RT-qPCR技术,分别检测了opn4在红鳍笛鲷早期发育10个阶段(多细胞、下包1/2、胚孔封闭、视囊、晶体出现、心脏跳动、孵化期、1天仔鱼、3天仔鱼、10天仔鱼)中的表达情况。在红鳍笛鲷早期发育各时期均可检测到opn4的表达,而在下包1/2期、胚孔封闭、晶体形成和孵化后一天的仔鱼这四个时期的表达显著,说明了opn4基因在红鳍笛鲷早期发育时期就已出现波动变化的表达,这可能与调节胚胎发育环境光协同以及满足各种生理行为的需求相关。
[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.
【学位授予单位】：广东海洋大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4

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