四种红树植物根际土壤微生物Ⅰ型和Ⅱ型PKS基因的检测与多样性分析

Detection of type Ⅰ and Ⅱ polyketide synthase genes in microorganisms from mangrove rhizosphere soil

【目的】探究红树林土壤中聚酮合酶(Polyketide synthase,PKS)基因的多样性和新颖性。【方法】用Ⅰ型和Ⅱ型PKS基因酮基合成酶(Ketosynthase,KS)域的简并引物对海南清澜港红树林海莲、黄槿、银叶、老鼠簕4种红树根际土壤样品中DNA进行PCR扩增,之后利用PCR-限制性酶切片段多样性(PCR-RFLP)和测序分析法对Ⅰ型和Ⅱ型PKS基因的多样性进行探讨。【结果】对得到的72条Ⅰ型PKS基因的酮基合成酶(Ketosynthase,KS)域DNA序列进行PCR-RFLP分析,共得到51个可操作分类单元(Operational taxo-nomic unit,OTUs),其中37个OTUs为单克隆产生,没有明显的优势OTU。选取了26个代表不同OTU的克隆进行测序分析,这些序列与GenBank中已知序列的最大相似率均未超过85%。KS域氨基酸序列的系统发育分析显示,所得KS域来源广泛,包括蓝细菌门(Cyanobacteria)、变形杆菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和一些未可培养细菌;对55条PKSⅡ基因KS域DNA序列的PCR-RFLP分析后共得到25个OTUs,有两个明显的优势OTUs,代表的克隆子数所占比例超过10%。【结论】PCR-RFLP分析表明红树林根际土壤中存在着丰富多样的Ⅰ型和Ⅱ型PKS基因,且前者多样性更高;低的序列相似度表明所获得的PKSⅠ基因KS域序列独特;系统发育分析表明得到的PKSⅠ基因来源广泛。

[Objective] We identified polyketide synthase （PKS） genes from mangrove soil. [Methods] Degenerate PCR primers were used to amplify ketosynthase （KS） domains associ- ated with type I and II PKS genes from DNA of soil samples derived from the Qinglan Harbor （Hainan, China）. The molecular diversity and phylogeny of type I and II PKS genes were ana- lyzed by PCR-RFLP based cloning approach. [Results] A total of 72 clones and 51 OTUs were obtained, there was no dominant OTU and 37 OTUs were from single clone. Twenty-six clones of different OTUs were sequenced and the DNA sequences were translated into amino acid （AA） sequences. All identified KS domains showed the identities at AA level to their closest matches in GenBank at less than 85%. Phylogenetic analysis of these sequences indicated that the identified ketosynthase （KS） domains were clustered with those from diverse bacterial group, including Cyanobacteria, Proteobacteria, Firmicutes, Actinobacteria and some uncul- tured bacteria. A total of 55 clones containing Type II PKS gene KS domain DNA sequences were analyzed by PCR-RFLP. Only 25 OTUs were generated and there were two dominant OTUs which present more than 10% of clones in the clone library. [Conclusionl These results suggested the presence of diverse PKS I and PKS II genes in microorganisms from mangrove rhizosphere soil, and the diversity of PKS II gene is lower than the PKS I gene. Low KS do- main sequence similarities indicated they possessed unique PKS I genes. Phylogeny deduced from the sequences of KS domains in PKS I genes showed that they distributed in different bacterial phyla.