牙鲆Lhx1基因的克隆和表达分析

Cloning and expression analysis of Lhx1 in Paralichthys olivaceus

Lhx1基因对于哺乳动物卵巢缪勒氏管的形成具有重要作用,但鱼类中的报道仅限于其在体轴和肾脏形成中的作用,尚未见到其与性别及性腺发育相关的报道。本研究克隆获得了牙鲆(Paralichthys olivaceus)Lhx1a和Lhx1b开放阅读框(ORF)序列,长度分别为1224 bp和1206 bp(GenBank注册号为:JX999940、JX999941),同源序列比较显示牙鲆Lhx1a和Lhx1b基因均具有两个LIM及一个HOX结构域,符合Lhx1进化保守性。这两个基因在牙鲆雌、雄成鱼各组织RT-PCR差异表达谱不尽相同,Lhx1a在精巢中弱表达,卵巢中不表达,在其他组织中雌、雄表达谱差异不大,都只在脑和眼组织有弱表达;而Lhx1b的表达在性腺中相对较高,且卵巢的表达量高于精巢。进一步检测牙鲆Lhx1a和Lhx1b在雌、雄性腺发育各期的表达谱,发现这两个基因表达集中于Ⅲ、Ⅳ期性腺,Lhx1a在精巢中弱表达,Lhx1b卵巢中的表达明显高于精巢,而在Ⅰ、Ⅱ、Ⅱ、Ⅴ期性腺几乎均不表达。由此推断牙鲆Lhx1a和Lhx1b均为性别相关基因,但在两性性腺发育中的作用可能各有不同。

LIM homeobox protein 1 gene （Lhxl）, was reported to play an important role in mammal mullerian duct development. But there is no report on this gene in fish so far, and only few papers studied its function during the formation of axis and kidney. In this study, the open reading frames of Lhxla and Lhxlb were cloned from olive flounder, Paralichthys olivaceus. Their lengths are 1224 bp （GenBank Accession number, JX999940） and 1206 bp （JX999941）, respectively. LHX 1 a and LHXlb proteins both have two LIM and one HOX highly conserved domains The different expression patterns of Lhxla and Lhxlb genes in male and female flounder tissues measured by RT-PCR showed that Lhxla was expressed in testis but not in ovary. There are almost no expression signals in other tissues, and only brain and eye samples from both male and female individuals showed lower expression signals. The expression of Lhxlb in gonads was totally opposite to that of Lhxla. Its expression in ovary is higher and that in testis is lower. Moreover, the expression patterns of these two genes at different gonad developmental stages were studied by RT-PCR. The results showed that there were almost no signals for these two genes at stage Ⅰ, Ⅱ and Ⅴ in gonads. But, at stage IU and IV, the expression could be detected. Lhxla has a lower expression at stageⅢ and Ⅳ of testis tissues, and Lhxlb has a higher expression in ovary and lower expression in testis. Then, both genes were sex-related genes, and their possible roles in testis and ovary development might be different.