齐口裂腹鱼（Schizothorax prenanti）FSHβ亚基与LHβ亚基cDNA全序列克隆及生物信息学分析
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摘要
促性腺激素(GTH)是由脊椎动物脑垂体前叶细胞合成和分泌的一类糖蛋白激素,主要生理作用是刺激生殖细胞的生长、发育、成熟,正常的排精及排卵。鱼类的促性腺激素GTHⅠ和GTHⅡ分别对应着哺乳动物当中的促卵泡素(FSH)和黄体生成素(LH)。促性腺激素是由α和β两个亚基组成,以非共价键相连接形成二聚体结构,其中α亚基氨基酸组成相同,而p亚基的氨基酸组成则明显不同,具有特异的生物学作用。因此研究FSHβ亚基和LHβ亚基的结构和功能将有助于了解畜禽、鱼类生殖生理状况,为进一步提高动物繁殖效率、挖掘动物繁殖潜能等具有重要的意义。
本研究选用齐口裂腹鱼(Schizothorax prenanti)为实验材料,从齐口裂腹鱼脑垂体中提取总RNA。设计针对齐口裂腹鱼FSHβ亚基、LHβ亚基基因的特异性引物,通过cDNA末端快速扩增技术(RACE)克隆得到齐口裂腹鱼的cDNA全序列,并对序列进行相关分析。
结果显示：(1)齐口裂腹鱼FSHβ亚基cDNA全序列长度为624bp,编码区(CDS)序列长度为393bp,编码130个氨基酸；LHβ亚基cDNA全序列长度为640bp,编码区(CDS)序列长度为441bp,编码146个氨基酸。生物信息学分析显示,齐口裂腹鱼FSHβ亚基是一个具有1个跨膜螺旋和3个主要疏水区域的带负电荷的蛋白,分子量为14.51kDa,pI为5.28。其氨基酸序列具有12个磷酸化位点；LHp亚基同样也是一个具有1个跨膜螺旋和4个主要疏水区域的带负电荷的蛋白,分子量为16.34kDa,pI为4.98。其氨基酸序列具有11个磷酸化位点。(2)齐口裂腹鱼与其它脊椎动物,包括哺乳类、鸟类、爬行类、两栖类、鱼类等不同物种的两个基因编码氨基酸同源性比较分析表明：FSHp亚基与鲤形目中鲤鱼的同源性最高,达到96.9%,与两栖类中日本蟾蜍的同源性最低为37.6%；LHp亚基同源性与鲤鱼最高,达到93.1%,与人类最低为43.2%。核苷酸同源性比较显示：齐口裂腹鱼FSHβ亚基与鲤鱼的同源性最高达到96.2%,与两栖类的日本蟾蜍最低为46.9%；LHβ亚基同样与鲤鱼的同源性最高为95.8%,与鸟类的日本鹌鹑最低为50.5%。这一结果初步可以确定克隆所得到的两段序列为齐口裂腹鱼的FSHβ亚基和LHβ亚基。(3)利用MAGA4.0根据22种脊椎动物的氨基酸序列构建NJ系统树和MP系统树表明：齐口裂腹鱼属于鲤形目,与鲤鱼、草鱼、青鱼形成聚类,与传统分类学保持一致,通过相似性比对、遗传距离及系统分支程度显示FSHp亚基进化速率相对保守,LHβ亚基在鸟类、爬行类、哺乳类中进化比其他脊椎动物快。可以揭示两种糖蛋白基因的进化伴随物种进化而显示其功能。
Gonadotrophins (GTH) is a kind of glycoprotein-hormone generated and secreted by vertebrate pituitary, whose function is to stimulate the development, differentiation and maturation of germ cells and maintain spermiation and ovulation. GTH I and II of Fish play the similar roles as follicle-stimulating hormone (FSH) and luteinizing hormone (LH) of mammal respectively. GTH consists of a andβsubunits and those two subunits combine to form dipolymer by noncovalent bond. The composition of a amino is the same while (3 amino is different, which functions specifically. Therefore, the investigation of the function ofβ-subunits of FSH and LH will assist in the reproduction of vertebrate and improve the reproductive performance of farm animals.
Herein, we extracted total RNA from Schizothorax prenanti and designed the primer of (3-subunits of FSH and LH genes, and obtained the cDNA of that via rapid-amplification of cDNA ends (RACE) method.
The result is described as below:(1) The total length of cDNA of P-subunit of FSH of Schizothorax prenanti is 624bp, and length of coding region is 393bp which codes 130 aminos whereas the total length of cDNA ofβ-subunit of LH is 640bp, the length of coding region is 441bp, which codes 146 aminos. Bioinformationally, the P-subunit of FSH is a negative charged protein equipped with 1 transbilayer helix and 3 hydrophobic regions, the molecular weight is 14.51kDa, the isoelectric point is 5.28, 12 phosphorylation sites are located on it. Theβ-subunit of LH is a negative charged protein equipped with 1 transbilayer helix and 4 hydrophobic regions, the molecular weight is 16.34kDa, the isoelectric point is 4.98,11 phosphorylation sites are located on it.(2)The homology analysis of the two coding genes of Schizothorax prenanti and other species including Mammals, Birds, Reptiles, Amphibian, Fishes showed:β-subunit of FSH has the highest homology as Cyprinus carpio (96.9%) and lowest homology as Bufo japonicus (37.6%), while theβ-subunit of LH has the most homology as cyprinus carpio (93.1%) and has the lowest homology as Homo (43.2%). The comparison of nucleotide homology showed that:β-subunit of FSH of Schizothorax prenanti has the highest homology as Cyprinus carpio (96.2%) and lowest homology as Japanese Quail (46.9%), while itsβ-subunit of LH has the highest homology as Cyprinus carpio (95.8%) and lowest homology as Japanese Quail (50.5%). The result can confirm that those two fragments obtained by clone are theβ-subunits of FSH and LH of Schizothorax prenanti, respectively. (3) The NJ and MP phylogenetic tree based on 22 kinds of vertebrate obtained by MAGA4.0 showed: Schizothorax prenanti belongs to Cypriniformes, which is in the cluster of cyprinus carpio, Ctenopharyngodon idellus and Mylopharyngodon piceus. This agrees with the conventional category. The result from similarity-comparison, genetic distance analysis and systematic arborization showed that the evolutionary rate ofβ-subunit of FSH is conservative whileβ-subunit of LH in amniota is faster that other vertebrates. This can reveal that the evolution of the two glycoproteins accords with the species evolution to function.
本研究选用齐口裂腹鱼(Schizothorax prenanti)为实验材料,从齐口裂腹鱼脑垂体中提取总RNA。设计针对齐口裂腹鱼FSHβ亚基、LHβ亚基基因的特异性引物,通过cDNA末端快速扩增技术(RACE)克隆得到齐口裂腹鱼的cDNA全序列,并对序列进行相关分析。
结果显示：(1)齐口裂腹鱼FSHβ亚基cDNA全序列长度为624bp,编码区(CDS)序列长度为393bp,编码130个氨基酸；LHβ亚基cDNA全序列长度为640bp,编码区(CDS)序列长度为441bp,编码146个氨基酸。生物信息学分析显示,齐口裂腹鱼FSHβ亚基是一个具有1个跨膜螺旋和3个主要疏水区域的带负电荷的蛋白,分子量为14.51kDa,pI为5.28。其氨基酸序列具有12个磷酸化位点；LHp亚基同样也是一个具有1个跨膜螺旋和4个主要疏水区域的带负电荷的蛋白,分子量为16.34kDa,pI为4.98。其氨基酸序列具有11个磷酸化位点。(2)齐口裂腹鱼与其它脊椎动物,包括哺乳类、鸟类、爬行类、两栖类、鱼类等不同物种的两个基因编码氨基酸同源性比较分析表明：FSHp亚基与鲤形目中鲤鱼的同源性最高,达到96.9%,与两栖类中日本蟾蜍的同源性最低为37.6%；LHp亚基同源性与鲤鱼最高,达到93.1%,与人类最低为43.2%。核苷酸同源性比较显示：齐口裂腹鱼FSHβ亚基与鲤鱼的同源性最高达到96.2%,与两栖类的日本蟾蜍最低为46.9%；LHβ亚基同样与鲤鱼的同源性最高为95.8%,与鸟类的日本鹌鹑最低为50.5%。这一结果初步可以确定克隆所得到的两段序列为齐口裂腹鱼的FSHβ亚基和LHβ亚基。(3)利用MAGA4.0根据22种脊椎动物的氨基酸序列构建NJ系统树和MP系统树表明：齐口裂腹鱼属于鲤形目,与鲤鱼、草鱼、青鱼形成聚类,与传统分类学保持一致,通过相似性比对、遗传距离及系统分支程度显示FSHp亚基进化速率相对保守,LHβ亚基在鸟类、爬行类、哺乳类中进化比其他脊椎动物快。可以揭示两种糖蛋白基因的进化伴随物种进化而显示其功能。
Gonadotrophins (GTH) is a kind of glycoprotein-hormone generated and secreted by vertebrate pituitary, whose function is to stimulate the development, differentiation and maturation of germ cells and maintain spermiation and ovulation. GTH I and II of Fish play the similar roles as follicle-stimulating hormone (FSH) and luteinizing hormone (LH) of mammal respectively. GTH consists of a andβsubunits and those two subunits combine to form dipolymer by noncovalent bond. The composition of a amino is the same while (3 amino is different, which functions specifically. Therefore, the investigation of the function ofβ-subunits of FSH and LH will assist in the reproduction of vertebrate and improve the reproductive performance of farm animals.
Herein, we extracted total RNA from Schizothorax prenanti and designed the primer of (3-subunits of FSH and LH genes, and obtained the cDNA of that via rapid-amplification of cDNA ends (RACE) method.
The result is described as below:(1) The total length of cDNA of P-subunit of FSH of Schizothorax prenanti is 624bp, and length of coding region is 393bp which codes 130 aminos whereas the total length of cDNA ofβ-subunit of LH is 640bp, the length of coding region is 441bp, which codes 146 aminos. Bioinformationally, the P-subunit of FSH is a negative charged protein equipped with 1 transbilayer helix and 3 hydrophobic regions, the molecular weight is 14.51kDa, the isoelectric point is 5.28, 12 phosphorylation sites are located on it. Theβ-subunit of LH is a negative charged protein equipped with 1 transbilayer helix and 4 hydrophobic regions, the molecular weight is 16.34kDa, the isoelectric point is 4.98,11 phosphorylation sites are located on it.(2)The homology analysis of the two coding genes of Schizothorax prenanti and other species including Mammals, Birds, Reptiles, Amphibian, Fishes showed:β-subunit of FSH has the highest homology as Cyprinus carpio (96.9%) and lowest homology as Bufo japonicus (37.6%), while theβ-subunit of LH has the most homology as cyprinus carpio (93.1%) and has the lowest homology as Homo (43.2%). The comparison of nucleotide homology showed that:β-subunit of FSH of Schizothorax prenanti has the highest homology as Cyprinus carpio (96.2%) and lowest homology as Japanese Quail (46.9%), while itsβ-subunit of LH has the highest homology as Cyprinus carpio (95.8%) and lowest homology as Japanese Quail (50.5%). The result can confirm that those two fragments obtained by clone are theβ-subunits of FSH and LH of Schizothorax prenanti, respectively. (3) The NJ and MP phylogenetic tree based on 22 kinds of vertebrate obtained by MAGA4.0 showed: Schizothorax prenanti belongs to Cypriniformes, which is in the cluster of cyprinus carpio, Ctenopharyngodon idellus and Mylopharyngodon piceus. This agrees with the conventional category. The result from similarity-comparison, genetic distance analysis and systematic arborization showed that the evolutionary rate ofβ-subunit of FSH is conservative whileβ-subunit of LH in amniota is faster that other vertebrates. This can reveal that the evolution of the two glycoproteins accords with the species evolution to function.
引文
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