椎体虫细胞周期调控蛋白FOR20的质粒构建开题报告

1. 研究目的与意义

锥体虫trypanosoma brucei是一种古老的单细胞的原生生物，主要寄生在人和牛的体内，造成人的嗜睡病和牛的那加那病。

锥体虫trypanosoma brucei是一种古老的单细胞的原生生物，主要寄生在人和牛的体内，造成人的嗜睡病和牛的那加那病.中心体一般被认为通过调节微管聚合及解聚的动态平衡而在细胞周期的分裂期起重要作用。

最近，研究显示中心体的异常也会导致g1期的阻塞，但是，目前对于中心体及其结合蛋白在结合期的功能并不清楚我们发现for20定位于中心体上，下调for20不仅导致细胞周期s期的异常，而且还使得有丝分裂蛋白激酶与中心体解离。

2. 研究内容和预期目标

生物信息学分析

椎体虫基因组的提取和目的片段的扩增椎

质粒的构建和检验

3. 研究的方法与步骤

实验前生物信息学分析

基因组的获取和目的基因的扩增

质粒的构建和目的基因的测序分析

4. 参考文献

Goldberg AL (2003) Protein degradation and protection against misfolded or damaged proteins. Nature. 426: 895–899Sauer RT et al. (2004) Sculpting the proteome with AAA( ) proteases and disassemblymachines. Cell. 119: 9–18Catic A. et al. (2007) Sequence and structure evolved separately in a ribosomal ubiquitinvariant. EMBO J. 26: 3474-3483.Hicke, L., and Dunn, R. (2003) Regulation of membrane protein transport by ubiquitin andubiquitin-binding proteins. Annu. Rev. Cell Dev. Biol. 19: 141-172.Muratani, M., and Tansey, W. P. (2003) How the ubiquitin-proteasome system controlstranscription. Nat. Rev. Mol. Cell. Biol. 4: 192-201.Hoege C. et al. (2002) RAD6-dependent DNA repair is linked to modification of PCNA byubiquitin and SUMO. Nature. 419: 135-141.Stelter P., and Ulrich H. D. (2003) Control of spontaneous and damage-induced mutagenesisby SUMO and ubiquitin conjugation. Nature. 425: 188-191.Gregory R. C. et al. (2003) Regulation of the Fanconi anemia pathway bymonoubiquitination. Semin. Cancer Biol. 13: 77-82.Haglund K. at al. (2003) Distinct monoubiquitin signals in receptor endocytosis.TrendsBiochem. Sci. 28: 598-603.Pickart C. M., and Fushman D. (2004) Polyubiquitin chains: polymeric protein signals. Curr.Opin. Chem. Biol. 8: 610-616.Varadan R. et al. (2002) Structural properties of polyubiquitin chains in solution. J. Mol. Biol.324: 637-647.Varadan R. et al. (2004) Solution conformation of Lys63-linked di-ubiquitin chain providesclues to functional diversity of polyubiquitin signaling. J. Biol. Chem. 279: 7055-7063.Hershko A., and Ciechanover, A. (1998) The ubiquitin system. Annu. Rev. Biochem. 67:425-479.Wu C. J. et al. (2006) Sensing of Lys 63-linked polyubiquitination by NEMO is a key eventin NF-kappaB activation. Nat. Cell Biol. 8: 398-406.Hofmann R. M., and Pickart, C. M. (2001) In vitro assembly and recognition of Lys-63polyubiquitin chains. J. Biol. Chem. 276: 27936-27943.Peng J. et al. (2003) A proteomics approach to understanding protein ubiquitination. Nat.Biotechnol. 21: 921-926.

Kim I., and Rao, H. (2006) What's Ub chain linkage got to do with it? Sci. STKE. 2006: pe18.Dahlmann B., et al. (1988) The multicatalytic proteinase: a high-Mr endopeptidase. Biochem.J. 255: 750–751.Zwickl P. et al. (1999) An archaebacterial ATPase, homologous to ATPases in the eukaryotic26S proteasome, activates protein breakdown by 20 S proteasomes. J. Biol. Chem. 274:26008–26014.

5. 计划与进度安排

1，2022年3月2号-3月30号 目标基因的扩增，酶切，插入及检验

2，2022年4月1号-4月15号 目标基因的蛋白表达及检验