卤化天然产物生物合成基因的克隆
毕业论文关键词：卤化酶；卤化天然产物；生物合成；
Cloning of Halogenated Natural Product Biosynthetic Genes
Abstract： In this study we constructed a metagenomic library of Yunnan soil. The whole soil genome was regarded as a unit rather than culture soil microorganisms first. Metagenomic technology could transfer the gene of uncultured microbal into cultured microbal, then research on gene function and natural products expression could be carried on. The halogenated natural products could deeply affect the biological activity of many natural products. The halogenated process of natural products was usually catalyzed by halogenase. Through phylogenetic analysis of halogenase related gene, the possibility of producing compounds could be inferred. Through sequence-based screening of clones containing halogenase gene clusters, and the relative gene was compared with the known halozyme sequence on NCBI. Six halogenated clones were obtained through sequence based screening.
Key words: Halogenated natural products, Halogenase, Biosynthetic gene.
目 录
摘要3
关键词3
Abstract3
Key words3
引言（或绪论）3
1 材料与方法4
1.1 材料和试剂 4
1.2 仪器设备 5
1.3试验方法6
1.3.1卤化酶相关基因初次验证6
1.3.2卤化酶相关基因的二次验证6
1.3.3技术路线7
1.3.4含卤化酶相关基因的单克隆筛选8
2结果与分析9
2.1 单克隆初筛 9
2.2单克隆基因筛选 9
2.3含卤化酶基因单克隆序列结果分析11
3讨论 15
致谢15
参考文献16
附录 相关序列17
卤化天然产物生物合成基因的克隆引言
从1966年发现第一个卤化酶[1]以来，共有四类卤化酶被发现：卤过氧化物酶(haloperoxidase)[2]，非血红素Fe2+ɑ-酮戊二酸及O2依赖型卤化酶(non-heme Fe II ɑ-ketoglutarate- and O2-dependent halogenase)[3]，S-腺苷甲硫氨酸依赖型氯化酶(S-adenosyl-L-methionine dependent chlorinase)[4]及FADH2依赖型卤化酶(FADH2-dependent halogenase)。很多抗生素的生物合成基因簇中存在FADH2依赖型卤化酶基因，该类酶具有很强的底物特异性和区域选择性，被认为是微生物和其他生物中的主要卤化机制[5]。研究早期,普遍认为卤代过氧化物酶是催化生物卤代反应的主要酶类,但现阶段研究表明,黄素依赖型卤化酶才是参与卤代反应的主要卤化酶类,因此近代对卤化酶的研究也主要集中在后者上[6] 。
FADH2依赖性卤化酶主要参与合成含芳香族基团的化合物、FADH2依赖型卤化酶它的作用底物含醌基吡咯环、苯酚环、吲哚环、炔基等结构中的一种或者两种，根据已知的FADH2-依赖型卤化酶底物的分析，这些卤化酶主要被分为两种，一种是以色氨酸为底物的卤化酶，被称为色氨酸卤化酶，另一种是以非色氨酸为底物的卤化酶，被称作非色氨酸卤化酶，这两类FADH2-依赖型卤化酶均需要黄素还原酶、NADH、FAD、O2和卤离子作为辅助因子。