东海洋山港沿岸土壤、海水样本中Ⅰ型PKS基因的初步筛选鉴定与功能分析

Screening, Identifying and Function Analysis of Polyketide Synthase I Domains from Soil and Seawater of Yangshan Harbor

为考察土壤和海水中Ⅰ型聚酮合酶(polyketide synthase,PKS)基因的多样性和差异,本研究自行设计了一套针对PKS基因中酮缩合酶(ketosynthase,KS)片段的简并引物,使用PCR方法直接克隆东海洋山港沿岸土壤和海水DNA样本中的KS片段,去除重复序列后,共获得了23条不同的KS片段(长度为630bp^690bp),提交GenBank皆获登录号,其中19条来自土壤(DQ640993、DQ640997、DQ641926、DQ641927、DQ673137~DQ673151),4条来自海水(DQ673151,EF554859~EF554861),由核苷酸序列推断出的氨基酸序列保守,与GenBank中已知KS基因片段的相似度在45%到85%之间,种系发生分析表明,其中14条KS片段(来源于海水的KS片段皆在其中)应来源于典型的KS群,而剩余9条则来源于杂合的PKS/NRPS(Non-ribosomal peptide synthetase,非核糖体多肽合成酶)群。另外,几条KS基因特征明显,可用于进一步的研究。

To directly access type I polyketide synthases （PKS） gene diversity from soil and seawater, a set of degenerate oligonucleotide primers were designed to amplify the ketosynthase fragments （KSs） belong to the PKS Ⅰ genes from the soil and the seawater DNA samples obtained from the Yangshan Harbor （Shanghai, China）. All the unique 23 fragments （ranged from 630 bp to 690 bp after cutting off the primer and vector sequences） obtained were belonging to the conserved KS domains in PKS Ⅰ genes. Among them 19 fragments were amplified from soil （DQ640993, DQ640997, DQ641926, DQ641927, and DQ673137-DQ673151） and 4 from the seawater （DQ673151 and EF554859-EF554861）. The predicated amino acid sequences showed 45% to 85% identifies to the known KSs genes in the GenBank. Phylogenetic analysis showed that 14 of them belonged to the normal KS domains that catalyzed the condensation of the acyl groups and the other 9 KS fragments belonged to the hybrid PKS/NRPS groups. Several KS fragments were endowed with some distinct characters that could be used in the following studies.