海蜇(Rhopilema esculentum)Notch基因的cDNA克隆和表达

Cloning and Expression of Notch Gene in Rhopilema esculentum

关键词:  海蜇  Notch  cDNA  实时荧光定量PCR

Key words:  Rhopilema esculentum  Notch  cDNA  Real-time PCR

摘要:

基于转录组454 GS FLX测序结果，利用RACE和RT-PCR技术克隆了海蜇(Rhopilema esculentum) Notch基因的cDNA全长，并分析了其mRNA在海蜇不同发育阶段的表达差异，以探讨Notch基因对海蜇无性生殖的影响。结果显示，海蜇Notch基因的cDNA全长为6768 bp，包括90 bp的5¢非编码区、6066 bp的开放阅读框及612 bp包含AATAAA加尾信号的3¢非翻译区。SMART分析表明，海蜇Notch为分泌蛋白，其信号肽由21个氨基酸组成。成熟肽由2000个氨基酸组成，包括37个结构域、26个表皮生长因子样结构域、3个富含半胱氨酸的Notch/Lin-12(NL)结构域、 1个NOD结构域、1个NODP结构域、1个跨膜结构域和6个锚蛋白结构域ANK，并含有25个N-糖基化位点。同源分析表明，海蜇Notch与来自刺胞动物门的海葵(Nematostella vectensis)的Notch相似性为39%，与无脊椎动物和脊椎动物的氨基酸相似度分别为35%–37%和34%–38%。RT-PCR分析表明，Notch基因在海蜇无性繁殖的4个发育时期均有表达，螅状体阶段的表达量最高，横裂体阶段表达量最低，螅状体的表达量是横裂体的1.85倍。这些研究结果为进一步研究Notch信号通路在海蜇无性繁殖中的调控作用奠定了基础。

Abstract:

Notch Pathway is a very conservative signaling pathway in evolution, through the interaction between Notch receptor and the ligand DSL protein which conveys signals to regulate cell proliferation, differentiation, apoptosis, and determine the fate of cells. Rhopilema esculentum belongs to cnidaria, it is an ideal model organism to study cnidaria asexual propagation because of the mature artificial breeding and aquaculture technology. The cDNA cloning and expression analysis of Notch were first performed in the R. esculentum based on 454 GS-FLX sequencing technique by RT-PCR and RACE method. The results showed that the full-length cDNA of Notch was 6768 bp, containing 5¢- untranslated region (UTR) of 90 bp, an open reading frame (ORF) of 6066 bp and 3¢- untranslated region (UTR) of 612 bp which contains a polyadenylation signals of AATAAA. SMART analysis showed that R. esculentum Notch, as a secretory protein, included a putative signal peptide of 21 amino acid residues; its mature peptide included 2000 amino acid residues and consisted of 37 structure domains, which includes 26 EGF-like domains, 6 ANK domains, and a transmembrane domain except 3 Notch / Lin-12(NL) structure domain, a NOD and a NODP domains which only exist in Notch family. The homology and phylogenetic analysis showed that the amino acid sequence similarity between R.esculentum Notch and Cnidaria Nematostella vectensis amino acid was 39%; by contrast, compared with invertebrates and vertebrates, R. esculentum shares 35%-37% and 34%-38% respectively, in the similarity of the amino acid. Phylogenetic tree of the Notch based on neighbor-joining method showed that R.esculentum was clustered with Cnidaria N. vectensis. Quantitative real-time PCR analysis revealed that the expression of Notch transcript was detected in all four developmental stages, with the highest level in scyphistoma stage and the lowest in strobili stage. The expression level of scyphistoma stage was 1.85 -fold of that in strobili. The results indicate that Notch gene is involved in the regulation of asexual reproduction of R.esculentum.