【摘要】：半滑舌鰨(Cynoglossus semilaevis)是我國北方沿海重要的咸水養(yǎng)殖經(jīng)濟魚類,由于個體大小存在巨大的雌雄差異,雄性個體生長緩慢,雌性個體生長速度較快,因此培育全雌性苗種對于半滑舌鰨的育種具有重要的經(jīng)濟意義。半滑舌鰨的染色體類型為ZW型,并且在自然的養(yǎng)殖環(huán)境中存在性反轉(zhuǎn)現(xiàn)象。由于半滑舌鰨的性別決定機制和性別決定基因尚不清楚,因此研究半滑舌鰨的性別相關(guān)基因?qū)τ诎牖圉嵢菩詢?yōu)良品種的選育具有至關(guān)重要的作用。本文對半滑舌鰨的兩個性別相關(guān)基因進行研究,得出以下結(jié)果:AQP(Aquaporin)家族是一組跨膜水通道蛋白,其在動物體內(nèi)的主要功能是允許水分子和小分子的有機物通過細胞膜[1]。aqp1aa是第一個被發(fā)現(xiàn)的跨膜水通道蛋白,其在生物體內(nèi)的功能包括細胞遷移,脂肪代謝、機體免疫、神經(jīng)信號轉(zhuǎn)導(dǎo)等[1,2,3,4,5]。此外有研究發(fā)現(xiàn)該基因還參與了雄性的精子發(fā)生。本研究對半滑舌鰨aqp1aa基因的全長進行了克隆,并進行了相關(guān)的生物信息學(xué)分析,探討了半滑舌鰨aqp1aa基因物種間的進化關(guān)系以及在硬骨魚類中的保守性。半滑舌鰨aqp1aa基因全長1411bp,ORF區(qū)786 bp,共編碼261個氨基酸。生物信息學(xué)分析發(fā)現(xiàn):半滑舌鰨aqp1aa基因包含有6個跨膜α螺旋組成的疏水性通道和一個高度保守的天冬氨酸-脯氨酸-丙氨酸基序,這是水孔蛋白超家族的特異性標(biāo)簽。通過序列比對并對其進行相應(yīng)的進化分析發(fā)現(xiàn):aqp1aa基因在硬骨魚類中是高度保守的,尤其是α螺旋和家族標(biāo)簽保守性最高。qPCR結(jié)果顯示:該基因在半滑舌鰨各組織中的表達水平存在差異性,在精巢中表達最高,其次是腦,其他組織表達較低,在不同發(fā)育時期中的表達結(jié)果顯示:在105日齡時在精巢中的表達明顯,105dph到2yph表達量一直增高,在不同表型中的表達結(jié)果顯示:在雄魚和偽雄魚的精巢中aqp1aa基因的表達量高,在雌魚z卵巢中的表達量次之,在dmrt1敲除的雄魚和三倍體雄魚中的表達量最低。通過原位雜交發(fā)現(xiàn):aqp1aa雜交信號主要出現(xiàn)在精子細胞和精子中,在不同時期的性腺的原位雜交結(jié)果與熒光定量的結(jié)果相一致,因此我們預(yù)測aqp1aa基因在半滑舌鰨精子發(fā)生過程中具有重要的功能。脂質(zhì)運載蛋白(lipocalin)超家族是一類小分子的胞外分泌蛋白,這類蛋白在序列上存在很大的差異,但是在晶體結(jié)構(gòu)上高度保守,具有八個氫鍵組成的β反向平行,脂質(zhì)運載蛋白主要是通過結(jié)合小分子來行使功能:它可以通過結(jié)合一系列的疏水性小分子或細胞表面受體形成可溶性的大分子復(fù)合物來完成其功能。本研究克隆得到了半滑舌鰨lipocalin基因的cDNA全長序列并進行了生物信息學(xué)分析,探討了該基因的轉(zhuǎn)錄因子結(jié)合位點及其在物種間的進化關(guān)系,該基因mRNA在半滑舌鰨不同組織、不同性別及不同發(fā)育時期的相對表達結(jié)果表明:lipocalin基因在半滑舌鰨卵巢中表達量最高,在免疫組織中表達量較高,在精巢中表達量較低,在105日齡的卵巢中表達量顯著升高。原位雜交結(jié)果顯示:lipocalin基因在卵巢的Ⅲ、Ⅳ卵母細胞重出現(xiàn)明顯的雜交信號。以上結(jié)果表明:lipocalin基因在卵子的發(fā)育過程中具有重要的作用,這為半滑舌鰨性別決定機制的探究提供了理論補充。
[Abstract]:The semi-smooth tongue is an important salt water fish in the northern coast of China. Because of the large difference of male and female, the growth of male individual is slow, and the growth rate of female individual is faster. Therefore, it is of great economic significance to breed whole female fry for semi-smooth tongue breeding. The type of the chromosome of the semi-smooth tongue is of the normal type, and there is a phenomenon of sexual reversal in the natural culture environment. Because the gender decision mechanism and the gender decision gene of the semi-smooth tongue are not clear, the study of sex-related genes in the semi-smooth tongue is of great importance to the selection and breeding of the semi-smooth tongue. The AQP (Aquaporin) family is a group of transmembrane water channel proteins, and its main function in animal body is to allow water molecules and small molecules to pass through cell membrane[1]. aqp1aa is the first transmembrane water channel protein found, which functions within the organism including cell migration, fat metabolism, body immunity, nerve signal transduction, etc.[1, 2, 3, 4, 5]. In addition, studies have found that the gene is also involved in spermatogenesis in males. In this study, the full length of aqp1aa gene was cloned and related bioinformatic analysis was carried out. The full length of aqp1aa gene is 1411bp, and the ORF region is 786 bp. It coencodes 261 amino acids. Bioinformatics analysis revealed that the semi-smooth laqp1aa gene consists of six hydrophobic pathways consisting of six transmembrane vesicles and a highly conserved aspartate-proline-alanine motif, which is a specific label for aquaporin. It was found that the aqp1aa gene was highly conserved in the hard-bone fish, especially with the highest conservativeness of helix and family label. The results of qPCR showed that the expression level of the gene in the tissues of the semi-smooth tongue was the highest, followed by the brain, and the expression of other tissues was low, and the expression of the gene in different development periods showed that the expression of the gene in the testis was obvious at the age of 105 days. The expression of aqp1aa in the sperm nest of male and pseudo-male fish was high, and the expression of aqp1aa gene in male fish and pseudo-male fish was higher. In situ hybridization, the hybridization signal of aqp1aa appears mainly in sperm cells and sperm, and in situ hybridization of gonads in different periods is consistent with the results of fluorescence quantification, so we predict that the aqp1aa gene plays an important role in spermatogenesis. Lipid-carrying proteins are extracellular secretory proteins of a class of small molecules, which differ greatly in sequence, but are highly conserved in crystal structure and have eight hydrogen bonds in opposite directions. Lipoproteins are primarily functional by binding small molecules: it can accomplish its function by combining a series of hydrophobic small molecules or cell surface receptors to form soluble macromolecular complexes. In this study, the full-length cDNA sequence of lipocalin gene was cloned and bioinformatic analysis was carried out. The transcription factor binding site of the gene and its relationship between species were discussed. The results of relative expression in different sex and different developmental stages showed that the expression of lipocalin gene in the ovary of semi-smooth tongue was the highest, the expression level was higher in the immune tissue, the expression level was lower in the testis, and the expression level of the ovary was significantly increased in the age of 105 days. The results of in situ hybridization showed that the lipocalin gene had obvious hybridization signal in the 鈪，

本文編號：2260711