半滑舌鳎aqp1aa、lipocalin基因的克隆及表达分析
发布时间：2018-10-09 20:40
【摘要】：半滑舌鳎(Cynoglossus semilaevis)是我国北方沿海重要的咸水养殖经济鱼类,由于个体大小存在巨大的雌雄差异,雄性个体生长缓慢,雌性个体生长速度较快,因此培育全雌性苗种对于半滑舌鳎的育种具有重要的经济意义。半滑舌鳎的染色体类型为ZW型,并且在自然的养殖环境中存在性反转现象。由于半滑舌鳎的性别决定机制和性别决定基因尚不清楚,因此研究半滑舌鳎的性别相关基因对于半滑舌鳎全雌性优良品种的选育具有至关重要的作用。本文对半滑舌鳎的两个性别相关基因进行研究,得出以下结果:AQP(Aquaporin)家族是一组跨膜水通道蛋白,其在动物体内的主要功能是允许水分子和小分子的有机物通过细胞膜[1]。aqp1aa是第一个被发现的跨膜水通道蛋白,其在生物体内的功能包括细胞迁移,脂肪代谢、机体免疫、神经信号转导等[1,2,3,4,5]。此外有研究发现该基因还参与了雄性的精子发生。本研究对半滑舌鳎aqp1aa基因的全长进行了克隆,并进行了相关的生物信息学分析,探讨了半滑舌鳎aqp1aa基因物种间的进化关系以及在硬骨鱼类中的保守性。半滑舌鳎aqp1aa基因全长1411bp,ORF区786 bp,共编码261个氨基酸。生物信息学分析发现:半滑舌鳎aqp1aa基因包含有6个跨膜α螺旋组成的疏水性通道和一个高度保守的天冬氨酸-脯氨酸-丙氨酸基序,这是水孔蛋白超家族的特异性标签。通过序列比对并对其进行相应的进化分析发现:aqp1aa基因在硬骨鱼类中是高度保守的,尤其是α螺旋和家族标签保守性最高。qPCR结果显示:该基因在半滑舌鳎各组织中的表达水平存在差异性,在精巢中表达最高,其次是脑,其他组织表达较低,在不同发育时期中的表达结果显示:在105日龄时在精巢中的表达明显,105dph到2yph表达量一直增高,在不同表型中的表达结果显示:在雄鱼和伪雄鱼的精巢中aqp1aa基因的表达量高,在雌鱼z卵巢中的表达量次之,在dmrt1敲除的雄鱼和三倍体雄鱼中的表达量最低。通过原位杂交发现:aqp1aa杂交信号主要出现在精子细胞和精子中,在不同时期的性腺的原位杂交结果与荧光定量的结果相一致,因此我们预测aqp1aa基因在半滑舌鳎精子发生过程中具有重要的功能。脂质运载蛋白(lipocalin)超家族是一类小分子的胞外分泌蛋白,这类蛋白在序列上存在很大的差异,但是在晶体结构上高度保守,具有八个氢键组成的β反向平行,脂质运载蛋白主要是通过结合小分子来行使功能:它可以通过结合一系列的疏水性小分子或细胞表面受体形成可溶性的大分子复合物来完成其功能。本研究克隆得到了半滑舌鳎lipocalin基因的cDNA全长序列并进行了生物信息学分析,探讨了该基因的转录因子结合位点及其在物种间的进化关系,该基因mRNA在半滑舌鳎不同组织、不同性别及不同发育时期的相对表达结果表明:lipocalin基因在半滑舌鳎卵巢中表达量最高,在免疫组织中表达量较高,在精巢中表达量较低,在105日龄的卵巢中表达量显著升高。原位杂交结果显示:lipocalin基因在卵巢的Ⅲ、Ⅳ卵母细胞重出现明显的杂交信号。以上结果表明:lipocalin基因在卵子的发育过程中具有重要的作用,这为半滑舌鳎性别决定机制的探究提供了理论补充。
[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，
本文编号：2260711
[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，
本文编号：2260711
本文链接：https://www.wllwen.com/kejilunwen/jiyingongcheng/2260711.html
最近更新
教材专著
