1. 毕业设计（论文）的内容和要求

1． 设计的内容 以g. lozoyensis为研究菌株，通过pcr方法克隆其cdna中负责合成纽莫康定b0六肽环的非核糖体肽合成酶（nrps）中第6位腺苷化结构域（a结构域）基因片段，导入e.coli bl21（de3）中进行诱导表达；对诱导条件进行优化（诱导剂的浓度和诱导时间），在较优的诱导产酶条件下考察镍柱纯化蛋白的条件，最后考察该酶作用的最适ph和温度、ph稳定性和热稳定性以及对不同底物的km值。

2． 要求 1) 温习各种查阅文献的方法，在此基础上阅读大量与课题相关资料（包括适量的外文文献），及时了解国内外有关非核糖体合成酶的研究进展，为以后的实验打下基础。

2) 遵守实验章程，规范实验操作，端正实验态度，并熟练使用各种常规实验仪器。

2. 参考文献

[1] Denning, D.W., Echinocandin antifungal drugs [J]. Lancet, 2003. 362(9390): 1142-51. [2] Schwartz, R.E., et al., L-671,329, a new antifungal agent. I. Fermentation and isolation [J]. J Antibiot (Tokyo), 1989. 42(2): 163-7. [3] Bills, G.F., et al., Reclassification of a pneumocandin-producing anamorph, Glarea lozoyensis gen. et sp. nov. [J], previously identified as Zalerion arboricola. Mycological Research, 1999. 103: 179-192. [4] 姚琳, 郭东林. PKS-NRPS数据库的研究进展[J]. 中国农学通报, 2009:280-3. [3] Hoffmeister D, Keller NP. Natural products of filamentous fungi: enzymes, genes, and their regulation [J]. Nat Prod Rep, 2007;24:393-416. [5] Lorenz N, Haarmann T, Pazoutova S, Jung M, Tudzynski P. The ergot alkaloid gene cluster: Functional analyses and evolutionary aspects [J]. Phytochemistry, 2009, 70:1822-32. [6] Brakhage AA, Thon M, Sprote P, Scharf DH, Al-Abdallah Q, Wolke SM, et al. Aspects on evolution of fungal beta-lactam biosynthesis gene clusters and recruitment of trans-acting factors [J]. Phytochemistry, 2009, 70:1801-11. [7] Brakhage AA, Schroeckh V. Fungal secondary metabolites - Strategies to activate silent gene clusters l[J]. Fungal Genet Bio, 2011, 48:15-22. [8] Richardt A, Kemme T, Wagner S, et al. Ebony,a novel nonribosomal peptide synthetase for bet a-alanine conjugation with biogenic amines in Drosophila [J]. J Biol Chem, 2003, 278:41160-41166. [9] Grunewald J,Marahiel MA. Chemoe nzymatic and templete directed synthesis of bioactive macrocyclic pepttide.Microbiol [J]. Mol Biol Rev,2006,7(1):121-146 [10] Mannanov RN, Sattarova RK. Chemistry of Nature Compounds, 2001, 37(2): 117~123. [11] 王世媛.非核糖体肽合成酶（NRPSs）作用机理与应用的研究进展[J]. 微生物学报, 2007,47（4）：734~737. [12] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding [J]. Analytical Biochemistry. 1976, 72: 248-254. [13] Suzuki T, Sato M, Yoshida T, et al. Rat liver mitochondrial and cytosolic fumarases with identical amino acid sequences are encoded from a single gene [J]. The Journal of Biological Chemistry, 1984, 264(5): 2581~2586. [14] Tomoko G,Shoji W, Hachiro O. Purification and Characterization of Fumarase from Corynebacterium glutamicum[J]. Biosci.Biotechnol.Biochem, 2006, 70 (5):1102~1109. [15] Yuji, U N Yumoto, M Tokushige. Ohya-Nishiguchi. Purification and characterization of two types of fumarases from Escherichia coli [J]. J Biochem, 1991, 109:728~733. [16] Kinsella, S. Doonan. Nucleotide sequence of a cDNA coding for mitochondrial fumarase from human liver [J]. Biosci Rep, 1986, 6: 921~929. [17] Kobayashi, K, T. Yamanishi. Physicochemical, catalytic, and immunochemical properties of fumarases crystallized separately from mitochondrial and cytosolic fractions of rat liver [J]. J Biochem, 1981, 89: 1923~1931. [18] Miles, J R Guest. Complete nucleotide sequence of the fumarase gene (citG) of Bacillus subtilis 168 [J]. Nucleic Acids Res, 1985, 13:131~140. [19] Weaver TM, Lees M, Zaitsev V, et al. Crystal structures of native and recombinant yeast fumarase [J]. Journal of Molecular Biology, 1998, 280: 431~442. [20] 王克夷. 蛋白质导论[M]. 北京: 科学出版社, 2007. [21] J.萨姆布鲁克, D.W.拉塞尔（美国）. 分子克隆实验指南（第三版）[M]. 黄培堂译. 北京: 科学出版社, 2002.

3. 毕业设计（论文）进程安排

2016.2-2016.3 查阅相关文献资料，掌握各种实验仪器设备的使用，准备开题报告的撰写 2016.3-2016.4 腺苷化功能域重组质粒的构建 2016.4-2016.5 表达蛋白的纯化 2016.5-2016.6 酶学性质的研究，进行中期检查报告，整理实验数据，撰写毕业论文