长松萝中一个聚酮合酶基因簇的克隆和鉴定

Isolation and charcterizaiton of a polyketide synthase gene cluster from Usnea longissima

摘要:

摘要:【目的】探索药用地衣长松萝(Usnea longissima Ach)聚酮化合物的生物合成基因簇，克隆聚酮合酶(PKS)基因并分析其功能。【方法】以长松萝地衣型真菌为材料，通过巢氏PCR获得聚酮合酶基因片段和原位杂交筛选基因组文库获得聚酮合酶基因及相邻基因簇。并对获得聚酮合酶进行分子系统进化分析和基因表达分析。【结果】获得药用地衣长松萝中的编码聚酮合酶基因UlPKS5的全长序列以及相邻修饰基因β-内酰胺酶和脱水酶。聚酮合酶UlPKS5含有酮体合成酶(KS)，酰基转移酶(AT)，产物模板(PT)以及酰基载体蛋白(ACP)结构域。分子系统进化分析显示UlPKS5属于非还原型聚酮合酶中第五组，与蒽醌类化合物生物合成相关。通过半定量RT-PCR分析表明山梨醇(10%)和蔗糖(2% 和10%)能够强烈诱导UlPKS5基因表达。【结论】聚酮合酶(UlPKS5)及相邻修饰基因β-内酰胺酶和脱水酶与长松萝中蒽醌类化合物生物合成相关。

关键词:关键词:地衣型真菌，长松萝，聚酮合酶，半定量PCR

Abstract:

Abstract:［Objective］To isolate polyketide synthase (PKS) gene from medicinal Usnea longissima lichen forming fungi，and identify the function of obtained PKS.［Methods］We used Usnea． longissima lichen forming fungi to isolate PKS gene by nested PCR using degenerate primers and screening a Fosimid genomic library．MEGA 4.0. 2 program was used for phylogenetic analysis and RT-PCR was used to detect gene expression.［Results］ We obtained a gene cluster including non-reducing PKS (UlPKS5)，putative β-lactamase and putative dehydratase from Usnea longissima lichen forming fungi．UlPKS5 contained ketosynthase (KS)，acyl transferase (AT)，product template (PT) and acyl carrier protein (ACP) domain． Phylogenetic analysis shows that UlPKS5 belonged to non-reducing PKS group V，which involved anthraquinone biosynthesis.RT-PCＲ analyses reveal that the expression of UlPKS5 was up-regulated by sucrose (2% and 10%) and sorbitol (10%).［Conclusion］PKS(UlPKS5)，putative β-lactamase and putative dehydratase were related with anthraquinone biosynthesis in U. longissima.

Key words:Keywords: lichen forming fungi，Usnea longissima，polyketide synthase，RT-PCR