牙鲆kiss2基因的分子鉴定及表达分析

Molecular characterization and expression patterns of kiss2 gene in Japanese flounder

为克隆与牙鲆生殖相关的神经内分泌因子kiss2基因,并检测该基因在牙鲆成体不同组织及早期不同发育阶段的表达变化,本研究采用Trizol法提取牙鲆成鱼各组织及早期不同发育阶段样品总RNA;参照其他物种的kiss2 c DNA序列,设计并合成kiss2引物进行PCR扩增;运用实时荧光定量PCR检测kiss2基因在牙鲆成鱼各组织及早期不同发育阶段的表达情况。结果成功地获得长度为449 bp的牙鲆kiss2 c DNA序列,其中编码区序列为366 bp,编码121个氨基酸。在该序列中发现了高度保守的"FNYNPLTLRF"序列,此序列可以调节促性腺激素释放激素和黄体生成素的合成与释放。定量PCR结果显示,牙鲆kiss2基因在脑组织中的表达量最高,在卵巢和精巢中也均有表达;牙鲆kiss2基因在胚胎阶段表达量相对较低,而在孵化后第7 d其表达量显著增高,且在孵化后的第29 d达到高峰。说明牙鲆kiss2基因具有明显的时期和组织差异性表达,其在牙鲆脑中的丰富表达及在精巢和卵巢中的表达表明该基因可能在牙鲆神经系统与生殖中发挥重要作用。

A reproduction-related neuroendocrine gene,kiss2,was cloned and its expression changes in different adult tissues and developmental stages were examined by real-time quantitative PCR in Japanese flounder（ Paralichthys olivaceus）. A c DNA sequence of 449 bp was obtained,which included an open reading frame（ ORF） of 366 bp encoding a polypeptide of 121 amino acids. A highly conserved Kisspeptin-10 region ＂FNYNPLTLRF ＂, a functional peptide stimulating the synthesis and release of Gn RH / LH,was found. The most abundant expression of kiss2 was detected in brain.The kiss2 was also expressed in gonad including ovary and testis. During embryonic and larval development,kiss2 showed low levels of expression. After 7-d hatching,the expression of kiss2 markedly increased and reached a peak at 29 d. These results demonstrated that the kiss2 gene exhibits stage- and tissuespecific expression. Its expressions in brain and the gonad suggest that the kiss2 gene might be involved in nervous system and reproductive development of Japanese flounder.