栉孔扇贝17β-hsd4基因的克隆和表达分析

Cloning and expression analysis of 17β-hsd4 gene in Chlamys farreri

为了研究17β-hsd4基因在无脊椎动物生殖过程中的作用,实验从栉孔扇贝转录组数据库中获得一个表达序列标签,采用cDNA末端快速扩增技术克隆得到1条全长为2 417 bp的cDNA序列,其开放阅读框为2 223 bp,编码740个氨基酸,推测的氨基酸序列含有17β-HSDs特有的4个保守区和17β-HSD4的3个酶结构域,且与人等脊椎动物的序列相似性均在58%以上。半定量RT-PCR显示,该基因在栉孔扇贝精巢、卵巢、肌肉、外套膜、鳃、肝胰腺和肾脏等各组织中均有表达,其中在肝胰腺和肾脏中表达量明显高于其他组织。对不同发育时期性腺中该基因表达的qRT-PCR检测发现,其在精卵巢发育周期的表达模式不同,且在生长期和成熟期的精巢中表达量显著性高于同时期的卵巢。由此推测,栉孔扇贝多种组织中均可以合成17β-HSD4,在精巢的发育和成熟的作用明显高于卵巢。

To study the role of 17β-hsd4 in the reproductive process of invertebrates,this study got a partial expression sequence tag from the transcription group database of Chlamys farreri. Then a cDNA was cloned by using Rapid Amplification of cDNA End method, of which the full-length was 2 217 bp. It encoded a peptide of 740 amino acids which contained 4 conserved domains and 3 enzyme structural domains,and the amino acid sequence had identities of over 58% with the sequence of vertebrates. For example, Homo sapiens. Semi-quantative RT-PCR revealed the mRNA was detected widely in the testis, ovary, adductor muscle,mantle, gill, hepatopancreas and kidney, especially higher in hepatopancreas and kidney, qRT-PCR was adopted at different development stages of gonad in which the gene expressed. It showed different expression patterns between testis and ovary,and the gene expression in testis at growing stage and mature stage was obviously higher than the ovary at the same stages（ P 〈 0.05 ）. It could be concluded that 17β-HSD4 could be synthesized in many tissues of C. farreri, and the gene might be more important in regulating the development and maturity of testis than ovary.