凡纳滨对虾海水养殖系统内细菌群落的PCR-DGGE分析

PCR-DGGE analysis of bacterial communities in marine Litopenaeus vannamei culture system

采用PCR-DGGE(PCR-denaturing gradient gel electrophoresis)技术对一个典型的凡纳滨对虾(Litopeneaus vannamei)海水养殖系统细菌群落进行分子分析。结果表明,沿岸水、蓄水池、养殖池水具有较高的细菌种类多样性,而蓄水池进水、对虾粪样、肠壁定植细菌样以及排水渠污水的细菌多样性程度低。每种环境群落的优势种明显。3个养殖池水样(Y1、Y2、Y3)、2个沿岸水样(W1、W2)、2个粪样(F1、F2)、蓄水池水样(B1、B2)及2个肠壁定植细菌样(G1、G2)各自具有高度群落相似性。BLAST结果表明,12个条带克隆序列所代表优势种很可能来源于以下几个属:柔发菌属(Flexithrix)、黏纤维菌属(Cytophaga)、Dyella属、聚球菌属(Synechococcus)、Chlorarachnion属、支原菌属(Mycoplasma)、草螺菌属(Herbaspirillum)、河氏菌属(Hahella)、Ruegeria属。本研究表明,PCR-DGGE技术可以用于海水对虾养殖系统的细菌群落结构分析。对于海水对虾养殖系统来说,一些序列所代表的主要细菌种类极有可能是很少被注意到或研究过的,具有潜在的研究价值。

The bacterial communities in a typical culture system of marine shrimp （Litopenaeus vannamei） was analyzed at molecular level. DNA was extracted from three samples from farming water, three samples from longshore water, three sample from impounding reservoir, one sewage water sample, three feces samples, and three bacterial samples on the gut wall, and PCR was carried out with primer pairs GC968f/1401r followed by agarose electrophoresis and DGGE. The band fingerprinting in DGGE gel was performed with cluster analysis by MVSP software. The main bands in DGGE gel were recovered for reamplification and following clone sequencing. The acquired sequences were performed BLAST searches against NCBI database, and the sequences including the closely related sequences were aligned with Clustal W program in BioEdit software for phylogenetic analysis. The results indicated that all the samples could be well amplified in PCR and gave rise to predicted 470 bp fragments. DGGE result showed that environmental water samples from longshore, impounding reservoir, and farming pond have much higher bacterial diversity than the samples from impounding reservoir, the feces, intestinal walls, and sewage water. The dominant bacterial species were obvious in any environment samples. The bacterial communities profile from three samples farming water had high similarity. So did two samples from longshore water, two feces samples, two samples from impounding reservoir and two samples from intestinal walls. The ＂genetic distance＂ of the samples evaluated by the cluster with MVSP revealed the difference of the bacterial communities, but the order of the samples in the cluster did not correspond with the spatial order of the samples and the similarity due to adjacent spatial order. Obviously, it could be explained by the fact that the shrimp culture system investigated was managed under high-density and intensive mode, and more easily influenced by human effort. The main microbes represented by 12 main bands in DGGE gel were closely related to Flexithrix, Cytophaga, Dyella, Syneehoeoceus, Chloraraehnion, Mycoplasma, Herbaspirillum, Hahella, and Ruegeria, most of which belong to the members of Proteobacteria and Cytophaga-Flexibacter-Bacteroides. Thus it is suggested that conventional culture methods can cause huge bias on analysis of environmental bacterial communities. The present study demonstrates that PCRDGGE can be used for molecular analysis of bacterial communities from marine shrimps culture system. As for marine shrimp culture system, it is most likely that some bacteria have not been noticed or investigated, and has potential research value.