花鳗鲡脑cDNA文库的构建及GnRH基因克隆与表达分析

CONSTRUCTION OF BRAIN cDNA LIBRARIES AND MOLECULAR CLONING AND EXPRESSION ANALYSIS OF GNRH GENE IN MARBLED EEL (ANGUILLA MARMORATA)

以花鳗鲡脑组织为材料,提取总RNA,应用CloneminerTM文库构建试剂盒构建cDNA文库。经检测,文库的滴度为4.3×106cfu/mL,总容量为5.16×107cfu/mL,阳性克隆率为99.6%,插入片段0.43—3.2kb之间,平均插入片段大小为1532bp。以文库为模板,克隆获得花鳗鲡两种类型的促性腺激素释放激素(mGnRH和cGnRH-II)cDNA序列。序列分析表明,花鳗鲡mGnRHcDNA开放阅读框(ORF)包含276个碱基,编码91个氨基酸,其中包括22个氨基酸的蛋白质前体信号肽(1—22位氨基酸)、mGnRH十肽(23—32位氨基酸)、一个三肽裂解位点(33—35位氨基酸)和56个氨基酸的GnRH相关肽(36—91位氨基酸);花鳗鲡cGnRH-IIcDNA开放阅读框包含264个碱基,编码87个氨基酸,其中包括24个氨基酸的蛋白质前体信号肽(1—24位氨基酸)、cGnRH-II十肽(25—34位氨基酸)、一个三肽裂解位点(34—36位氨基酸)和50个氨基酸的GnRH相关肽(37—87位氨基酸)。序列同源性分析表明,花鳗鲡mGnRH、cGnRH-IIcDNA与日本鳗鲡之间的相似性率高达98%;与鲑形目、鲈形目、鲽形目鱼类的相似率为73%—78%;而与鲤形目鱼类的相似性相对较低(63%—67%)。采用RT-PCR方法分析了两种GnRH基因在花鳗鲡雌雄个体中的表达,结果表明两基因在雌雄个体的组织表达模式无明显差异;但mGnRH基因在雌雄个体内表达部位多于cGnRH-II的。

Marbled eel （Anguilla marmorata） is the secondary protected animal in China. In order to save the genetic information of this rarity and clone the function genes on growth, development and reproduction, a cDNA library was constructed by CloneminerTM kit from brain of marbled eel. The titer of the amplified cDNA library was 4. 3 × 10^6 cfu/mL,the total of recombinants was 5.16 ×10^7cfu, and the percentage of recombinant efficiency was about 99.6% , the exogenous inserts of the recombinants was from 0.43kb to 3.2kb and the average size was 1532 bp. These results showed that cDNA library had excellent quality. Two types of GnRH （mGnRH and cGnRH-Ⅱ） cDNA sequences were isolated from cDNA library by PCR. Sequence analysis showed that mGnRH cDNA contained an open reading frame （ORF） of 276 bp and encoded 91 amino acid residues,which consisting of a 22-amino acid signal peptide precursor （ 1--22 amino acid residues） , mGnRH decapeptide （23--32 amino acid residues） ,3-amino acid signal processing site （33--35 amino acid residues） ,and a 56- amino acid GnRH-associated peptide （36--91 amino acid residues）, cGnRH-Ⅱ cDNA open reading frame （ORF） con- tained 264 bases encoded 87 amino acid residues,which consisting of a 24-amino acid signal peptide precursor （ 1--24 amino acid residues） ,cGnRH-Ⅱ decapeptide （25--34 amino acid residues） ,3-amino acid signal processing site （34--36 amino acid residues） , and a 50-amino acid GnRH-associated peptide （37--87 amino acid residues）. The homology analysis showed that the percentage of mGnRH and cGnRH-Ⅱ precursor sequence identity with Japanese eel Anguilla japonica is 98% ,with fishes of Salmoniformes,Perciformes and Pleuronectiformes is 73%--78%. However,it was relative low with fishes of Cypriniformes （63%--67%）. Phylogenetic tree analysis ranked the fish GnRH as three distinct groups, mGnRH and sbGnRH group,cGnRH-Ⅱ group and sGnRH group,respectively. Expression analysis by RT-PCR showed that cGnRH-Ⅱ and mGnRH gene expression had no obvious differences between female and male marbled eel individuals, but mGnRH gene could be expressed in more tissues than cGnRH-Ⅱ, mGnRH were detected in forebrain, midbrain, hindbrain, pituitary, bypothalamus,liver, heart, spleen, kidney, intestine, gill, ovary and testis, while expression of cGnRH-Ⅱ was mainly limited to forebrain, midbrain,hindbrain,ovary and testis. The present work provided evidence of two GnRH in Marbled eel reproductive system and suggested an important role of mGnRH in reproduction.