黄颡鱼促黄体激素受体基因克隆及在雌鱼繁殖周期中的表达

Cloning of LHR and expression analysis during the reproductive cycle in female yellow catfish(Pelteobagrus fulvidraco)

摘要:

通过简并引物扩增及Smart^TM Race 技术,首次克隆了黄颡鱼促黄体激素受体(LHR)基因cDNA 全长序列。该基因全长2 524 bp,编码701aa,含有属于糖蛋白激素受体(GpHR)家族的典型跨膜螺旋结构区域(TM helix)。9个富含亮氨酸的重复性序列(LRRs);4个潜在的N-端糖基化位点: ²⁹NFTC, ⁸²NVSR, ²⁰³NGSR, ⁵⁴⁸NLTV;24个Ser,6个Thr和5个Tyr 磷酸化位点。同时⁴⁰⁰S为潜在的PKC位点。通过RT-PCR分析组织表达,卵巢繁殖周期中卵巢和脑的表达水平并结合血浆中E₂含量的变化,发现LHR在卵巢中大量表达,其次是脑、肾脏、心脏、肝脏和肠存在少量或微量的表达;在卵巢繁殖周期中,卵巢LHR表达从Ⅲ期-Ⅴ期处于较高水平,Ⅴ期达到最高峰,随后下降到最低值;而脑的表达高峰出现在Ⅳ期,与血浆中E₂变化相一致。推测卵巢和脑中LHR基因参与卵黄的生成,调控卵母细胞的成熟和排卵。

Abstract:

The complete cDNA sequence of luteinizing hormone receptor(LHR)was cloned through degenerate primer PCR amplification and Smart^TM Race technology.The length of the cDNA of yellow catfish(Pelteobagrus fulvidraco) LHR gene is 2 524 bp and encodes the protein of 701 amino acids.This gene belongs to glycoprotein hormone receptor(GpHR)family,which contains the conserved seven transmembrane helix domains(TM helix).Meanwhile the amino acids sequence contains nine Leucine-rich repeats(LRRs).Potential N-linked glycosylation sites are ²⁹NFTC, ⁸²NVSR, ²⁰³NGSR, ⁵⁴⁸NLTV in yellow catfish LHR gene and phosphorylation site predictions identified 24 Ser,6 Thr and 5 Tyr potential phosphorylation sites.By Clustal X and MEGE 4.0 alignment revealed the highly conserved in GpHRs’ specific signature sequences: ²⁸⁷CCAF, ⁴⁷¹ERW, ⁵⁸⁴FTD and ⁶⁵⁷NPFLY.The only one protein kinase C phosphorylation site is ⁴⁰⁰S.Tissue distribution pattern showed preferential expression of LHR in ovary,and trace amounts were detected in brain,kidney,heart,liver and intestine(brain>kidney>heart>liver>intestine).Plasma 17β-Estradiol(E₂)levels in reproductive cycle were detected by radio immunoassay(RIA).The result showed that the highest peak in reproductive cycle is stage Ⅳ.The high transcript level of LHR in ovary was revealed in the reproductive phase of ovarian cycle,which was the relatively high expression from Ⅲ to Ⅴ phase and the highest level in stage Ⅴ.And we also analyzed the brain expression level in reproductive cycle,found the highest level in stage Ⅳ consistent with the level of E₂.Results demonstrated the gene of LHR in brain and ovary might also be involved in the processes which preferentially promote vitellogenesis and regulate ovulation maturation and ovulate either indirectly or directly.