멍게의 Erf 유전자 cloning 및 발현 양상
Expression pattern and cloning of Erf gene in ascidian, Halocynthia roretzi
- 발행기관 강릉대학교 대학원
- 지도교수 김길중
- 발행년도 2009
- 학위수여년월 2009. 2
- 학위명 석사
- 학과 및 전공 행양생명공학과
- 원문페이지 iv, 31 p.
- 본문언어 한국어
초록/요약
In ascidian embryos, a fibroblast growth factor (FGF) signaling induces specification of notochord, mesenchyme, and brian. Although a conserved FGF/Ras/MAPK/Ets pathway is known to be involved in this signaling, the detailed mechanisms of regulation of FGF signaling pathway are not well understood. The Ets family of transcription factors is characterized by a conserved DNA-binding domain and involved in many developmental processes. Most of the known Ets family proteins have been shown to activate transcription. However, several Ets proteins like Tel, Net and Erf subfamilies can act as transcriptional repressors. In the present study, We have isolated Hr-Erf, an ascidian ortholog of vertebrate Erf, to elucidate interactions of transcription factors involved in the FGF signaling pathway in ascidian embryos. The Hr-Erf cDNA encompassed 2154 nucleotides including 25 adenyl residues at the 3' end and encoded a predicted polypeptide of 580 amino acids. The polypeptide had the Ets DNA-binding domain in its N-terminal region. The overall degree of amino acid identity between the Hr-Erf and vertebrate Erf genes was approximately 30%, however the Ets domain was highly conserved (80%). We determined the expression pattern of Hr-Erf gene in the various adult tissues and embryos using quantitative real-time PCR (qRT-PCR). In adult animals, Hr-Erf mRNA was predominantly detected in the muscle, and at lower levels in ganglion, gills, gonad, hepatopancreas, and stomach. During embryogenesis, Hr-Erf mRNA was detected from eggs to early developmental stage embryos, whereas the transcript levels were decreased from neurula stage. To further examine the spatial expression of Hr-Erf mRNA, we carried out whole-mount in situ hybridization at various stages. Similar to the qRT-PCR results, a significant amount of maternal transcripts of Hr-Erf was presented in the fertilized eggs and early embryos. Maternal mRNA of Hr-Erf was gradually lost from the neurula stage. Zygotic expression of Hr-Erf started in most blastomeres at the 32-cell stage. At gastrula stage, Hr-Erf was specifically expressed in the precursor cells of brain and mesenchyme. These results suggest that zygotic Hr- Erf products are involved in specification of brain and mesenchyme cells. It remains to determine fundamental roles of the Hr-Erf gene during ascidian embryogenesis.
more목차
I. 서론 = 1
II. 재료 및 방법 = 5
1. Animals and embryos = 5
2. Isolation of RNA and synthesis of cDNA = 5
3. Polymerase chain reaction (PCR) amplification for full-length Erf cDNA = 6
4. Sequencing and phylogenetic analysis = 6
5. Quantitative Real-Time PCR (qRT-PCR) analysis = 7
6. Whole-mount in situ hybridization = 7
III. 결과 = 9
1. Cloning of Erf ortholog in Halocynthia roretzi = 9
2. Expression of Hr-Erf in the adult tissues = 10
3. Expression pattern of Hr-Erf in various embryos = 10
Ⅳ. 고찰 = 11
Ⅴ. 참고문헌 = 25
