DGGE法与常规培养法对稻田蓝细菌多态性分析结果比较

Analysis on the species diversity of cyanobacteria in paddy field by comparison between normal culture and DGGE method

研究运用蓝细菌和硅藻16SrDNA特异引物,将晚季水稻生长后期稻田土壤中提取的总DNA进行PCR扩增后,以DGGE技术对PCR产物进行分析结果表明,14条DGGE带经克隆测序,经NCBI基因库比对得晚季水稻生长后期存在10种蓝细菌,包括4种Leptolyngbya、1种Chamaesiphon、1种Nostoc、1种Oscillatoria、2种Syne-chococcus和1种Chroococcidiopsis。同层不同位置土壤中蓝细菌种群亦有所不同,但每个取样点都有一些特有的蓝细菌种类。用常规方法对同一稻田土壤样品进行分离培养,根据蓝细菌鉴定图谱观察到类似Lyngbya、Oscillatori-a、Chroococcidiopsis及Nostoc的蓝细菌,但显微镜下无法准确分类。比较结果表明采用DGGE法比常规培养法能更准确进行蓝细菌多态性鉴定。

Segments of 16S rDNA genes from cyanobacteria were retrieved from paddy field samples by DNA extraction and polymerase chain reaction(PCR),and subsequently analyzed by denaturing gradient gel electrophoresis(DGGE). The cyanobacterial community was analyzed in the rice paddy field by nested PCR using universal 16S rDNA primers and subsequently primers targeting cyanobacterial specific regions in the 16S rDNA genes. The amplified products were analyzed by denaturing gradient gel electrophoresis (DGGE). 14 dominating bands were excised from the gels and further sequenced and determined by BLAST search against the GenBank database.10 bands were identified as cyanobacteria,representing 6 different cyanobacerial genera.4 of the bands were identified as belonging to the genus Leptolyngbya,1 to the genus Nostoc,2 to the genus Synechococcus,and 1 to each of the genera Oscillatoria,Chroococcidiopsis and Chamaesiphon,respectively.The cyanobacterial diversity varied both in the upper and the deeper soil fractions and different sampling points.Lyngbya,Oscillatoria,Chroococcidiopsis and Nostoc were identified under microscope by normal culture method,but can not be identified exactly,indicated that DGGE method can reflect the complex and dynamic of cyanobacteria in the community.