鄱阳湖泥鳅黑色素相关基因及其蛋白分子特征表达分析以及胚胎发育和苗种培育研究

发布时间：2018-06-09 07:04

本文选题：鄱阳湖泥鳅 + 苗种培育　；参考：《南昌大学》2016年硕士论文

【摘要】：鄱阳湖泥鳅(Misgurnus anguillicaudatus)具有较为丰富的遗传多样性,体型和体色均有明显的差异性,为本课题组的良种选育提供了极好的材料。本研究以鄱阳湖泥鳅为材料,主要开展了两个方面的工作:一是,首次克隆获得鄱阳湖泥鳅黑色素聚集激素(melanin-concentrating hormone,MCH)和黑色素刺激激素(α-melanophore-stimulating hormone,α-MSH)及它们的受体黑色素聚集激素受体1(melanin-concentrating hormone receptor 1,Mchr1)和黑色素刺激激素受体1(α-melanophore-stimulating hormone receptor 1,Mc1r)基因,初步探究了四个基因与鄱阳湖泥鳅黑斑性状形成的关系;二是,研究了不同发育温度、水体透明度和初始投喂时间对泥鳅仔鱼成活率的影响,探讨适宜条件以提高水花苗的健康程度和成活率。获得的主要研究成果如下:1、利用RACE-PCR分别克隆出泥鳅前原黑色素聚集激素1(prepro-melanin-concentrating hormone,Pmch1)基因、阿片黑素促皮质激素原(proopiomelanocortin,Pomc)基因、黑色素聚集激素受体1(Mchr1)基因和黑色素刺激激素受体1(Mc1r)基因的cDNA全长序列,分别命名为MaPmch1、MaPomc、MaMchr1、MaMc1r。运用生物信息学软件对4个基因及其蛋白质的理化性质和结构特征进行了预测分析,并构建了系统进化树。(1)MaPmch1基因cDNA全长591bp,编码含124个氨基酸的蛋白质。泥鳅PMCH1(MaPMCH1)C末端经水解可产生17个氨基酸的环形神经肽MCH1。MaPMCH1是亲水性蛋白,二级结构以无规则卷曲为主。MCH1超二级结构具有一个反平行β-折叠片层,通过二硫键连接形成环形神经肽。同源比对发现泥鳅MCH1(MaMCH1)氨基酸序列与其它硬骨鱼类完全一致。系统进化树显示泥鳅PMCH1与鲤科鱼类PMCH1聚为一支。在哺乳动物、鸟类和鱼类中,MCH存在高度保守的序列RCM*GRVYRPCW(*代表随机氨基酸)。(2)MaPomc基因cDNA全长为1133 bp,编码含221个氨基酸的蛋白质。泥鳅POMC(MaPOMC)的107-119处水解产生13个氨基酸的α-MSH。同源分析结果说明泥鳅α-MSH(Maα-MSH)氨基酸序列与其他物种完全一致,具有HFRW结构,是一个重要的功能域。系统进化树显示MaPOMC与鲤科鱼类在同一进化方向上,且独立于鲤科鱼类之外。(3)MaMchr1基因和MaMc1r基因cDNA全长分别为1364 bp和1529 bp,编码产生包含328个氨基酸的泥鳅MCHR1(MaMCHR1)和316个氨基酸的泥鳅MC1R(MaMC1R)。预测两者的二级结构均以α-螺旋为主,三级结构均存在7个跨膜结构域,并具有DRY(天冬-精-酪)结构,是典型的G蛋白偶联受体。MaMCHR1和MaMC1R与鲤科鱼类都具有较高的同源性(90%左右),系统进化树同样表明两者在进化方向上与传统分类地位相吻合。2、利用qPCR检测不同花斑泥鳅背部皮肤、腹部皮肤和全脑组织的4个基因的表达量。结果发现MaPmch1基因在不同花斑泥鳅大脑中的表达情况无明显规律。MaPomc基因在小花斑泥鳅大脑中的表达量高于大、无花斑泥鳅,推测高表达的α-MSH能促进小花斑泥鳅在黑斑处分泌更多的黑色素,从而形成较多的黑斑。MaMchr1基因的表达主要集中在大脑,且在不同花斑泥鳅脑中表达差异不显著;MaMc1r基因的表达集中在大脑和背部皮肤,大、小花斑泥鳅脑和背部皮肤表达量相当,无花斑泥鳅大脑中的表达量高于背部皮肤。推测MaMchr1基因参与黑色素沉着较少,而MaMc1r基因背部皮肤表达高有助于黑斑形成。无花斑泥鳅MaPmch1/MaPomc值高于大、小花斑泥鳅说明,在泥鳅体内通过调节MaPmch1和MaPomc基因表达量的比例来实现泥鳅不同花斑的黑色素沉着模式。3、本文详细描述了泥鳅胚胎发育的24个时期,当水温为24℃或27℃时,胚胎发育分别历时31 h 20 min和24 h。泥鳅胚胎发育的有效积温为307.65时度,生物学零点温度为14.18℃。泥鳅孵化水温对孵化率影响不明显,但对仔鱼出膜72 h后的成活率影响较显著,24℃和27℃的成活率分别为72.42%和65.84%。在一定温度范围内,水温越高,胚胎发育时间越短,仔鱼成活率越低。当水体的透明度为5 cm时,孵出72 h后的仔鱼成活率为71.83%,显著低于15 cm和25 cm的成活率。显微观察发现水中悬浮颗粒会粘附在外鳃上,从而使其活力和呼吸受到影响。大规模繁育泥鳅时,根据仔鱼成活情况,最佳的初始投喂时间为孵出后20-30 h内(24℃),即鱼苗肠道贯通后约10 h内。结合显微技术,本研究解决了泥鳅初孵仔鱼生长过程中的关键问题,对提高泥鳅苗种的成活率和科学地指导泥鳅繁育工作具有重要意义。
[Abstract]:The Poyang Lake loach (Misgurnus anguillicaudatus) has abundant genetic diversity and distinct body color and body color, which provides excellent material for the selection of good species in this study group. In this study, Poyang Lake loach was used as the material to carry out two aspects: first, to clone the melanin of the loach of Poyang Lake. Melanin-concentrating hormone (MCH) and melanin stimulating hormone (alpha -melanophore-stimulating hormone, alpha -MSH) and their receptor melanin aggregating hormone receptor 1 (melanin-concentrating hormone receptor 1, Mchr1) and melanin stimulating hormone receptor 1 (alpha -melanophore-stimulating hormone 1), The relationship between the four genes and the black spot characteristics of Poyang Lake loach was preliminarily explored. Two, the effects of different development temperatures, water transparency and initial feeding time on the survival rate of loach larvae were studied, and the suitable conditions were discussed to improve the health and survival rate of the water flower seedlings. The main results obtained were as follows: 1, using RACE-PCR The cDNA full length of the proto melanin aggregation hormone 1 (prepro-melanin-concentrating hormone, Pmch1) gene, the proopiomelanocortin (Pomc) gene, the melanin aggregation hormone receptor 1 (Mchr1) gene and the melanin stimulating hormone receptor 1 (Mc1r) gene were cloned to be named MaPmch1, MaPomc, MaMchr1, respectively. MaMc1r. uses bioinformatics software to predict the physical and chemical properties and structural characteristics of 4 genes and their proteins, and constructs a phylogenetic tree. (1) the full length 591bp of the MaPmch1 gene cDNA encodes a protein containing 124 amino acids. The circular neuropeptide MCH1.MaPMCH of the PMCH1 (MaPMCH1) C terminal of the loach is hydrolyzed to produce 17 amino acids The 1 is the hydrophilic protein, and the two grade structure has an anti parallel beta fold layer with an anti parallel beta layer with the irregular curling.MCH1 super second structure. The homologous alignment found that the amino acid sequence of the loach MCH1 (MaMCH1) is exactly the same as that of other hard bone fishes. The phylogenetic tree shows that the loach PMCH1 and the cyprinid fishes are gathered into PMCH1. One branch. In mammals, birds and fish, MCH has a highly conserved sequence RCM*GRVYRPCW (* representing random amino acids). (2) the cDNA full length of the MaPomc gene is 1133 BP, encoding a protein containing 221 amino acids. The results of alpha -MSH. homology of 13 amino acids from the 107-119 hydrolysate of the loach POMC (MaPOMC) show the alpha -MSH (Ma alpha -MSH) ammonia of the loach. The base acid sequence is identical with other species, with HFRW structure and is an important functional domain. Phylogenetic tree shows that MaPOMC and cyprinid fish are in the same evolutionary direction and are independent of cyprinid fishes. (3) the total cDNA length of the MaMchr1 gene and MaMc1r gene is 1364 BP and 1529 BP respectively, which encodes the loach MCHR1 containing 328 amino acids, respectively. MaMCHR1) and 316 amino acid loach MC1R (MaMC1R). It is predicted that the two structure of both of them is dominated by alpha helix, and there are 7 cross membrane domains in the three structure and DRY (asparagine fine cheese) structure. The typical G protein coupling receptor.MaMCHR1 and MaMC1R have higher homology with cyprinid fishes (90%), and the phylogenetic tree is similar to the phylogenetic tree. The expression of the 4 genes in the back skin of different blotch loach, the abdominal skin and the whole brain tissue was detected by qPCR in the evolutionary direction. The results showed that the expression of MaPmch1 gene in the brain of different speckle loach had no obvious regularity in the expression of the.MaPomc gene in the brain of the loach loach. The high expression of alpha -MSH can promote the secretion of more melanin in the black spot of the loach loach, and the expression of the.MaMchr1 gene is mainly concentrated in the brain, and there is no significant difference in the brain and the back skin of the brain and the back, large and small flowers in the MaMc1r base. The expression of the brain and the back skin of the loach was higher than that in the back skin. It was suggested that the MaMchr1 gene was less involved in melanoma, and the high expression of the back skin of the MaMc1r gene was conducive to the formation of the black spot. The MaPmch1/MaPomc value of the loach was higher than that of the loach. The small speckle loach showed that the body was regulated by MaPmc in the body of the loach. The ratio of H1 and MaPomc gene expression to the melanin pattern.3 of loach with different blotch. In this paper, the 24 stages of the embryonic development of loach were described in detail. When the water temperature was 24 or 27, the effective accumulated temperature of embryo development of 31 h 20 min and 24 h. was 307.65 time, and the zero temperature of biological zero was 14.18 degrees. The Hatching Temperature of loach was not significantly affected by hatching temperature, but the survival rate after 72 h was significantly affected. The survival rates of 24 and 27 C were 72.42% and 65.84%. in a certain temperature range, the higher the water temperature, the shorter the embryo development time and the lower the survival rate of the larvae. The survival rate of the larvae after 72 h was hatched when the transparency of the water body was 5 cm. The survival rate of 71.83% was significantly lower than that of 15 cm and 25 cm. The microscopic observation found that the suspended particles in the water adhered to the outer gills, thus causing their vitality and respiration to be affected. In the mass breeding of loach, the optimum initial feeding time was within 20-30 H (24 degrees C) after the hatching of the larvae, that is, about 10 h after the transfixion of the larvae. Microtechnology, this study has solved the key problems in the growth process of the larvae of loach, which is of great significance to improving the survival rate of the loach seedlings and scientifically guiding the breeding work of the loach.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4

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