海水养殖蟹体内与外部环境中菌群结构的PCR-DGGE比较——以三疣梭子蟹和锯缘青蟹为例

Comparison of the bacterial community structure in the crab seawater cultured and the outside environment by PCR-DGGE fingerprint technique:Portunus trituberculatus and Scylla serrata

利用PCR-DGGE指纹图谱技术和UPMGA聚类分析,对三疣梭子蟹Portunus trituberculatus、锯缘青蟹Scylla serrata的鳃组织及养殖环境中的菌群结构进行了对比分析。结果表明:三疣梭子蟹、锯缘青蟹鳃组织及养殖环境水样在指纹图谱上分别显示出21,21,23和21条信号强度不同的条带,条带的数量反映出三疣梭子蟹养殖环境中的菌群最为丰富,条带信号的强度,反映出不同样品中存在各自的优势菌种;可将4个样品中的菌群划分为2个群落,其中三疣梭子蟹和锯缘青蟹鳃组织中的菌群聚为一类,其相似度为75%,2种蟹养殖环境中的菌群聚为一类,相似度为69%。首次尝试利用16S rDNA的PCR-DGGE指纹图谱技术分析了2种健康蟹体内和各自养殖环境中微生物结构的差异性,从而寻找蟹体内的益生菌,将有助于建立一种不依赖于分离培养的原位研究蟹体内菌群组成的分子诊断技术,为今后研究锯缘青蟹、三疣梭子蟹与养殖水体微生物及相关蟹病害的关系提供基础科学依据。

At present 16S rDNA PCR-DGGE fingerprinting has become one of the popular techniques in studying the diversity of environmental microorganism.Due to the current tendency and focus on the research of the microbial role in aquiculture system,this paper introduce for the first time the PCR-DGGE fingerprint technique in the study of ecology of the microorganism from the crab gill and the aquaculture environment,and the composition and diversity of bacterial flora in the gill and culture water of healthy Portunus trituberculatus and Scylla serrata are analyzed.Direct and effective extraction of total DNA of environmental samples is crucial in the study.The method used to extract total DNA from marine fish gill and shrimp intestine microorganisms is used for reference and modified to extract the total DNA of bacterial flora of the crab gill.And the method of enzymolysis combined with hydroxybenzene/chloroform is used to extract the total DNA of the attached bacteria on the crab gill conveniently and efficiently.The electrophoretogram shows that the length of DNA segments is longer than 9.4 kb,the bands bright and clear with good integrality.Similar method is used to extract total DNA of culture water samples,and quality DNA obtained.With the bacterial universal primer of 16S rDNA V3 region： 341F and 518R（5＇ end of upstream primer has a GC-Clamp） and touchdown PCR（anneal temperature： 65~55℃,-0.5℃/cycle） method,specificity bands are amplified from the samples.The PCR products are about 250 bp long and separated in 10 mg/mL polyacrylamide gels with a denaturant gradient from 30 to 60 mg/mL.The DGGE characteristics are analyzed to reveal some information of bacteria diversity in the gill and culture water samples.Four types of samples are separated clearly in DGGE fingerprint profiles.The result shows that abundant and various bacteria are present in the gills and culture water of Portunus trituberculatus and Scylla serrata.Difference of bacterial flora of different gills and culture environment is also observed in the DGGE figure.Some bacterial species exist in four types of samples.Some are specific in certain samples.The difference of the brightness of the bands in the DGGE profiles shows different predominant bacterial community in the crab gills and culture water.The difference in life habitation of the crab and culture environment may result in the difference.Similarity coefficient and the result of clustering analysis show that the samples from the gill and from the culture water are grouped into a cluster of their own,which suggests that bacterial species in different samples from the same environment are similar.The composition of attached bacterial flora in the gills of Portunus trituberculatus and Scylla serrata is close to each other,but differs a little from the environmental samples,which indicates that the bacterial community composition is influenced not only by the habitation environment,but also the hosts.The reason may be the life habit of crab and the selective adsorption effect of the gills.DGGE figures also show that the bands from the gills also appear in the samples of the culture environment.This suggests that the bacterial flora in the gills could be found its original source in the surrounding environment.This paper is for the first time to aim to set up a method to reveal the bacterial flora in the gills of crab based on 16S rDNA PCR-DGGE fingerprinting which is of great value for establishing a kind of diagnostic technology without isolation and culture.The reveal of the composition of special bacterial flora in the crab gills is helpful to exploit microbial preparation.Besides,the comparison of the composition of the bacteria flora from healthy crab gill with that from diseased crab gill can result in finding out the pathogenic bacteria.This paper also proves that PCR-DGGE technique is convenient and effective to analyze bacterial flora within culture system of aquatic animals.