非无菌培养的微小卡罗藻次生代谢产物的细胞毒及其宏基因组酮基合成酶基因的筛选

Screening Cytotoxicity of Crude Extract and Existence of Ketosynthas Gene in Metagenome of Karlodinium micrum Under Nonaxenie Cluture

目的:检测微小卡罗藻(Karlodinium micrum)粗提物的细胞毒活性,筛选其KS基因。方法:非无菌条件培养微小卡罗藻,用乙酸乙酯提取其粗产物,以脐静脉内皮细胞(HUVEC)为目标细胞,测定粗提取物的细胞毒活性;运用分子生物学方法对多聚酮合酶(PKS)基因的酮基合成酶(KS)基因进行筛选,运用BLAST进行比对和系统发生树进行分析。结果:微小卡罗藻培养液粗产物具有细胞毒活性,IC50值为70.8μg/mL。KS基因筛选获得680 bp的片段,并经测序推导出其氨基酸序列,该氨基酸序列与纤维多囊菌和点型念珠蓝细菌KS域的氨基酸序列同源性分别为60%和57%。结论:首次从非无菌培养的微小卡罗藻中筛选到PKS中KS域基因片段,为从中分离聚酮类化合物提供了基因学的依据。

Objective： To screen cytotoxic activity of crude extract of Karlodinium micrum and ketosynthases（KS） domain of polyketone synthetase（PKS）. Methods： After HUVEC cells were routinely cultured, tetrazolium-based calorimetric assay for cytotoxiciy was performed to screen the K.micrum crude extract showing active. Furthermore, matagenome of K.micrum were also screened for PKS genes which could be responsible for bioactive secondary metabolites biosynthesis showing positive. Results： Crude extracts of K.micrum under nonaxenie culture showed cytotoxic effects with IC50 70.8μg/mL. KS domains approximately 680 bp in size were detected by degenerate PCR. BLAST analysis in GenBank （tBlastn） showed that the putative KS fragments gave greatest similarity （60% and 57%, respectively） to the KS fragments of Polyangium cellulosum and Nostoc punctiforme. Conculsion： This is the first report of KS domain in metagenome of K.micrum under nonaxenie cluture. The results provide evidence that K.micrum maybe produce natural products through PKS pathways.