本文選題：鄱陽湖泥鰍 + 苗種培育��； 參考：《南昌大學(xué)》2016年碩士論文

【摘要】：鄱陽湖泥鰍(Misgurnus anguillicaudatus)具有較為豐富的遺傳多樣性,體型和體色均有明顯的差異性,為本課題組的良種選育提供了極好的材料。本研究以鄱陽湖泥鰍為材料,主要開展了兩個(gè)方面的工作:一是,首次克隆獲得鄱陽湖泥鰍黑色素聚集激素(melanin-concentrating hormone,MCH)和黑色素刺激激素(α-melanophore-stimulating hormone,α-MSH)及它們的受體黑色素聚集激素受體1(melanin-concentrating hormone receptor 1,Mchr1)和黑色素刺激激素受體1(α-melanophore-stimulating hormone receptor 1,Mc1r)基因,初步探究了四個(gè)基因與鄱陽湖泥鰍黑斑性狀形成的關(guān)系;二是,研究了不同發(fā)育溫度、水體透明度和初始投喂時(shí)間對(duì)泥鰍仔魚成活率的影響,探討適宜條件以提高水花苗的健康程度和成活率。獲得的主要研究成果如下:1、利用RACE-PCR分別克隆出泥鰍前原黑色素聚集激素1(prepro-melanin-concentrating hormone,Pmch1)基因、阿片黑素促皮質(zhì)激素原(proopiomelanocortin,Pomc)基因、黑色素聚集激素受體1(Mchr1)基因和黑色素刺激激素受體1(Mc1r)基因的cDNA全長(zhǎng)序列,分別命名為MaPmch1、MaPomc、MaMchr1、MaMc1r。運(yùn)用生物信息學(xué)軟件對(duì)4個(gè)基因及其蛋白質(zhì)的理化性質(zhì)和結(jié)構(gòu)特征進(jìn)行了預(yù)測(cè)分析,并構(gòu)建了系統(tǒng)進(jìn)化樹。(1)MaPmch1基因cDNA全長(zhǎng)591bp,編碼含124個(gè)氨基酸的蛋白質(zhì)。泥鰍PMCH1(MaPMCH1)C末端經(jīng)水解可產(chǎn)生17個(gè)氨基酸的環(huán)形神經(jīng)肽MCH1。MaPMCH1是親水性蛋白,二級(jí)結(jié)構(gòu)以無規(guī)則卷曲為主。MCH1超二級(jí)結(jié)構(gòu)具有一個(gè)反平行β-折疊片層,通過二硫鍵連接形成環(huán)形神經(jīng)肽。同源比對(duì)發(fā)現(xiàn)泥鰍MCH1(MaMCH1)氨基酸序列與其它硬骨魚類完全一致。系統(tǒng)進(jìn)化樹顯示泥鰍PMCH1與鯉科魚類PMCH1聚為一支。在哺乳動(dòng)物、鳥類和魚類中,MCH存在高度保守的序列RCM*GRVYRPCW(*代表隨機(jī)氨基酸)。(2)MaPomc基因cDNA全長(zhǎng)為1133 bp,編碼含221個(gè)氨基酸的蛋白質(zhì)。泥鰍POMC(MaPOMC)的107-119處水解產(chǎn)生13個(gè)氨基酸的α-MSH。同源分析結(jié)果說明泥鰍α-MSH(Maα-MSH)氨基酸序列與其他物種完全一致,具有HFRW結(jié)構(gòu),是一個(gè)重要的功能域。系統(tǒng)進(jìn)化樹顯示MaPOMC與鯉科魚類在同一進(jìn)化方向上,且獨(dú)立于鯉科魚類之外。(3)MaMchr1基因和MaMc1r基因cDNA全長(zhǎng)分別為1364 bp和1529 bp,編碼產(chǎn)生包含328個(gè)氨基酸的泥鰍MCHR1(MaMCHR1)和316個(gè)氨基酸的泥鰍MC1R(MaMC1R)。預(yù)測(cè)兩者的二級(jí)結(jié)構(gòu)均以α-螺旋為主,三級(jí)結(jié)構(gòu)均存在7個(gè)跨膜結(jié)構(gòu)域,并具有DRY(天冬-精-酪)結(jié)構(gòu),是典型的G蛋白偶聯(lián)受體。MaMCHR1和MaMC1R與鯉科魚類都具有較高的同源性(90%左右),系統(tǒng)進(jìn)化樹同樣表明兩者在進(jìn)化方向上與傳統(tǒng)分類地位相吻合。2、利用qPCR檢測(cè)不同花斑泥鰍背部皮膚、腹部皮膚和全腦組織的4個(gè)基因的表達(dá)量。結(jié)果發(fā)現(xiàn)MaPmch1基因在不同花斑泥鰍大腦中的表達(dá)情況無明顯規(guī)律。MaPomc基因在小花斑泥鰍大腦中的表達(dá)量高于大、無花斑泥鰍,推測(cè)高表達(dá)的α-MSH能促進(jìn)小花斑泥鰍在黑斑處分泌更多的黑色素,從而形成較多的黑斑。MaMchr1基因的表達(dá)主要集中在大腦,且在不同花斑泥鰍腦中表達(dá)差異不顯著;MaMc1r基因的表達(dá)集中在大腦和背部皮膚,大、小花斑泥鰍腦和背部皮膚表達(dá)量相當(dāng),無花斑泥鰍大腦中的表達(dá)量高于背部皮膚。推測(cè)MaMchr1基因參與黑色素沉著較少,而MaMc1r基因背部皮膚表達(dá)高有助于黑斑形成。無花斑泥鰍MaPmch1/MaPomc值高于大、小花斑泥鰍說明,在泥鰍體內(nèi)通過調(diào)節(jié)MaPmch1和MaPomc基因表達(dá)量的比例來實(shí)現(xiàn)泥鰍不同花斑的黑色素沉著模式。3、本文詳細(xì)描述了泥鰍胚胎發(fā)育的24個(gè)時(shí)期,當(dāng)水溫為24℃或27℃時(shí),胚胎發(fā)育分別歷時(shí)31 h 20 min和24 h。泥鰍胚胎發(fā)育的有效積溫為307.65時(shí)度,生物學(xué)零點(diǎn)溫度為14.18℃。泥鰍孵化水溫對(duì)孵化率影響不明顯,但對(duì)仔魚出膜72 h后的成活率影響較顯著,24℃和27℃的成活率分別為72.42%和65.84%。在一定溫度范圍內(nèi),水溫越高,胚胎發(fā)育時(shí)間越短,仔魚成活率越低。當(dāng)水體的透明度為5 cm時(shí),孵出72 h后的仔魚成活率為71.83%,顯著低于15 cm和25 cm的成活率。顯微觀察發(fā)現(xiàn)水中懸浮顆粒會(huì)粘附在外鰓上,從而使其活力和呼吸受到影響。大規(guī)模繁育泥鰍時(shí),根據(jù)仔魚成活情況,最佳的初始投喂時(shí)間為孵出后20-30 h內(nèi)(24℃),即魚苗腸道貫通后約10 h內(nèi)。結(jié)合顯微技術(shù),本研究解決了泥鰍初孵仔魚生長(zhǎng)過程中的關(guān)鍵問題,對(duì)提高泥鰍苗種的成活率和科學(xué)地指導(dǎo)泥鰍繁育工作具有重要意義。
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S917.4

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