应用DGGE分析三疣梭子蟹养殖塘底泥细菌的多样性

Bacteria diversity in Portunus trituperculatus pond sediment estimated by denaturing gradient gel electrophoresis

将传统的菌落菌体形态分类方法与分子生物学方法相结合研究了三疣梭子蟹养殖塘底泥中一个细菌类群的多样性。首先采用平板稀释法对三疣梭子蟹养殖塘底泥中的细菌进行分离与纯化,根据各菌株的菌落形态特征初步将118株分离物分为8个类群,其中最大的一个类群的菌落基本特征为圆形、边缘整齐、表面凸起、菌落有光泽,对该类群的细菌多样性分析表明,通过芽孢染色可将产芽孢菌和不产芽孢菌区分开,不产芽孢菌根据其菌体形态又分为3个亚群,其中卵圆形细菌是最大的一个亚群,共有16株。应用PCR-DGGE方法对该类群进行菌株的遗传多样性分析。结果表明,卵圆形类群16株细菌中就有11株不同系统发育型的细菌。最后,将这11株细菌进行了16S rDNA部分序列(约750bp)的测定,并做系统发育学分析,结果显示,这些细菌的系统发育学地位相距很远,分属于γ-Proteobacteria、α-Proteobacteria和Firmicutes等类群。试验结果表明,以菌落特征的差异来挑选不同的菌株作为分析细菌多样性的第一个筛选步骤,会大大低估可培养异养细菌的多样性。

Bacteria diversity was investigated in culture pond sediment of Portunus trituperculatus by means of the characteristics of morphology of colony, cell and 16S rDNA. A total of 118 strains of bacteria were isolated and purified from culture pond sediment of Portunus trituperculatus by serially plate dilution, and primarily clustered into 8 groups based on colony characteristics. Further research was focused on the biggest group of them, whose colonies characterized by circular shape, flat margin, convex surface, and glistening. Among this group, 31 strains were non-spore-forming bacteria, and divided into 3 sub-groups, i.e. sphererod, middle rod and long rod group. Sphere-rod sub-group was the biggest one, including 16 isolates. The genetic diversity of sphere-rod sub-group was analyzed using PCR-DGGE （denaturing gradient gel electrophoresis） molecular fingerprinting techniques. Eleven different phylogenetic types were found within 16 isolates. 16S rRNA gene partial sequences （about 750 bp from 5end） of 11 representative isolates were determined and phylogenetically analyzed. The results indicated that the sphere-rod cultivable bacteria were clustered into three clusters in two phyla： phylum Firmicutes and phylum Proteobacteria including class γ- Proteobacteria and α-Proteobacteria. The conclusion is that using the differences of colony characteristics as the first step of pick-up of isolates will greatly underestimate the diversity of cultivable heterotrophic bacteria.