曼氏无针乌贼(Sepiella japonica)促性腺激素释放激素(GnRH)基因的鉴定、特征及成熟期的表达研究

IDENTIFICATION,CHARACTERIZATION,AND EXPRESSION ANALYSIS OF A GNRH-LIKE GENE AT MATURE STAGE IN THE CEPHALOPOD SEPIELLA JAPONICA(SEPIIDAE)

促性腺激素释放激素(GnRH)在脊椎动物的繁殖过程中起着至关重要的作用,同时也存在于许多无脊椎动物中。利用cDNA末端快速扩增技术(RACE)克隆出曼氏无针乌贼(Sepiella japonica)促性腺激素释放激素(命名为SjGnRH,KP 982885.1)。SjGnRH基因的cDNA全长710 bp,开放阅读框(ORF)为273 bp,编码90个氨基酸,包括一条含有31个氨基酸的信号肽、12个氨基酸的GnRH多肽和44个氨基酸的GnRH相关肽(GAP);SjGnRH与剑尖枪乌贼(Uroteuthis edulis)和真蛸(Octopus vulgaris)的同源性分别为86.7%和73.3%;SjGnRH高度保守的十二肽与剑尖枪乌贼和真蛸的octGnRH相似度达100%,其信号肽的相似性分别为66.7%和54.8%,GAP区的相似性分别为95.5%和77.3%;实时荧光定量PCR(qRT-PCR)结果显示,SjGnRH基因在成熟期的表达具有特异性,在脑中表达量最高,且与其他组织差异显著(P<0.05);采用原位杂交技术检测SjGnRH mRNA在脑内的表达模式,结果表明:SjGnRH在食道上神经团、食道下神经团和视叶的不同部位均有表达,暗示SjGnRH可能参与曼氏无针乌贼的生殖调控作用。

Gonadotropin-releasing hormone(GnRH)is known for playing a crucial role in vertebrate reproduction and has been characterized in many invertebrate species.In this study,a GnRH-like gene(designated SjGnRH,KP 982885.1)was firstly identified in the common Chinese cuttlefish Sepiella japonica using rapid amplification of cDNA ends(RACE)method.The full-length sequence of SjGnRH cDNA is 710 bp and the open reading frame(ORF)contains 273 bp,encoding 90 amino acids,a putative signal peptide containing 31 amino acids,a 12 amino acid GnRH peptide and a 44 amino acid GnRH related peptide(GAP)sequence.It has 86.7%and 73.3%identity with Uroteuthis edulis and Octopus vulgaris,respectively.The highly conserved dodecapeptide of S.japonica shares 100%similarity with octGnRH of U.edulis and O.vulgaris,exhibits 66.7%and 54.8%similarity in the signal peptide region,and 95.5%and 77.3%in the GAP region,respectively.According to the results of qRT-PCR,the SjGnRH gene is tissue-specific at the mature stage,with the highest expression in brain,which is significantly different from other tissues(P<0.05).The expression pattern of SjGnRH mRNA was detected in the brain using in situ hybridization method.Therefore,SjGnRH is expressed in different regions of the supraoesophageal mass,subesophageal mass,and optic lobes,which suggests that SjGnRH might be involved in the reproduction of the cuttlefish S.japonica.