海藻表面附着弧菌多样性分析及方法研究
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摘要
对四种大型海藻:江篱(Gracilaria textorii)、孔石莼(Ulva pertusa)、海带苗(Laminaria japonica)和多管藻(Polysiphonia urceolata)表面附着弧菌的多样性进行了分析,同时对多样性分析中采用的不同方法进行了比较。
利用TCBS培养基从四种海藻表面总共分离到12株细菌:G1、G2分离自江篱,U3分离自孔石莼,L4、L5、L6分离自海带苗,P7、P8、P9、P10、P11、P12分离自多管藻。菌株G1,G2属于盐单胞菌(Halomonas),其余10株细菌都属于弧菌(Vibrio)。
对12株细菌的酶学活性、抗生素抗性进行了研究。发现除G1、G2外,其余菌株都可产生淀粉酶和明胶酶。菌株G1、G2和U3对氨苄青霉素、链霉素和四环素有很强抗性。
采用多种分子生物学方法(16S rDNA、gyrB、RFLP、DGGE )分析了上述12株菌的系统发育关系,对不同方法获得的结果进行了比较。
12株菌的16S rDNA系统发生树可分为4个明显分支。G1、G2与细菌H. meridiana构成一个分支。U3与V. lentus构成一个分支。L4、L5、L6、P9、P10、P11、P12与V. tasmaniesis形成一个分支。P7、P8与V. splendidus形成一个分支。
以gyrB基因序列为基础构建的系统发生树将G1、G2以外的10株弧菌分成4个较大分支,L4、P9属于同一分支,L6、P8、P10、P11和P12属于一个大的分支, U3、L5各自构成一个分支。菌株G1、G2没有得到gyrB基因序列扩增带。表明试验设计的引物是弧菌特异性引物。相对16S rDNA,不同菌株间gyrB基因序列差异性更大。
RFLP分析结果显示,12株细菌HinfI酶切后得到6种带型,SmaI酶切后得到4种带型。结合16S rDNA、gyrB基因的分析结果可知,RFLP可以在种的水平上有效进行细菌多样性分析。
12株细菌的DGGE分析结果显示,G1、G2与其它菌株电泳图谱有明显不同。菌株U3独自构成一种带型,L5、L6构成一种带型。表明DGGE技术在属的水平上分析细菌多样性效果较好,在种的水平上分析细菌多样性有一定的局限性。
The diversity of Vibrio associated with surfaces of 4 seaweeds: Gracilaria textorii、Ulva pertusa、Laminaria japonica and Polysiphonia urceolata was studied using different analysis methods.
Totally 12 strains were obtained from the surfaces of 4 seaweeds with TCBS media. Strains G1, G2 were from G. textorii, U3 were from U. pertusa, L4, L5, L6 were from L. japonica, P7, P8, P9, P10, P11, P12 were from P. urceolata. They belong to Halomonas (G1, G2) and Vibrio genus (other strains).
Enzymatic bioactivity, antibiotic resistance of the 12 strains were studied. The result showed most bacteria isolated can produce starch enzyme and glutin enzyme. Strains G1, G2, U3 are resistant to ampicillin, streptomycin and tetracyclinec.
Molecular biology methods were used to study the diversity of 12 strains. The 12 strains obviously formed 4 clusters in phylogeny tree established based on 16S rRNA gene. Each of the strains isolated from the surfaces of G. textorii、U. pertusa、L. japonica formed 1 cluster. Therefore, these strains are specific to seaweeds.
Four clusters were formed in phylogeny tree established based on gyrB gene. Strains L4, P9 formed 1 cluster. L6, P8, P10, P11, P12 formed 1 cluster. U3 formed 1 cluster. L5 formed 1 cluster. Strains G1 and G2 can not amplify gyrB gene with gyrB primers, which means that G1 and G2 were far from other strains and the primers designed are specific to Vibrio. Sequences analysis based on gyrB gene gave better resolution than 16S rDNA.
16S rDNA restriction fragment length polymorphisms of 12 strains were analyzed with 2 restriction endonucleases. Digestion of 16S rDNA fragment with HinfI enzyme gave rise to 6 restriction patterns for 12 isolates and 4 with SmaI enzyme. Comparing with 16S rDNA and gyrB analysis, RFLP is suitable for differentiation of strains on species level.
Finally, diversity of strains was studied with DGGE. The DGGE band of G1, G2 formed 1 band type, U3 formed 1 band type, L5 and L6 formed 1 band type. The result showed that DGGE is suitable for differentiation of strains on genus level.
利用TCBS培养基从四种海藻表面总共分离到12株细菌:G1、G2分离自江篱,U3分离自孔石莼,L4、L5、L6分离自海带苗,P7、P8、P9、P10、P11、P12分离自多管藻。菌株G1,G2属于盐单胞菌(Halomonas),其余10株细菌都属于弧菌(Vibrio)。
对12株细菌的酶学活性、抗生素抗性进行了研究。发现除G1、G2外,其余菌株都可产生淀粉酶和明胶酶。菌株G1、G2和U3对氨苄青霉素、链霉素和四环素有很强抗性。
采用多种分子生物学方法(16S rDNA、gyrB、RFLP、DGGE )分析了上述12株菌的系统发育关系,对不同方法获得的结果进行了比较。
12株菌的16S rDNA系统发生树可分为4个明显分支。G1、G2与细菌H. meridiana构成一个分支。U3与V. lentus构成一个分支。L4、L5、L6、P9、P10、P11、P12与V. tasmaniesis形成一个分支。P7、P8与V. splendidus形成一个分支。
以gyrB基因序列为基础构建的系统发生树将G1、G2以外的10株弧菌分成4个较大分支,L4、P9属于同一分支,L6、P8、P10、P11和P12属于一个大的分支, U3、L5各自构成一个分支。菌株G1、G2没有得到gyrB基因序列扩增带。表明试验设计的引物是弧菌特异性引物。相对16S rDNA,不同菌株间gyrB基因序列差异性更大。
RFLP分析结果显示,12株细菌HinfI酶切后得到6种带型,SmaI酶切后得到4种带型。结合16S rDNA、gyrB基因的分析结果可知,RFLP可以在种的水平上有效进行细菌多样性分析。
12株细菌的DGGE分析结果显示,G1、G2与其它菌株电泳图谱有明显不同。菌株U3独自构成一种带型,L5、L6构成一种带型。表明DGGE技术在属的水平上分析细菌多样性效果较好,在种的水平上分析细菌多样性有一定的局限性。
The diversity of Vibrio associated with surfaces of 4 seaweeds: Gracilaria textorii、Ulva pertusa、Laminaria japonica and Polysiphonia urceolata was studied using different analysis methods.
Totally 12 strains were obtained from the surfaces of 4 seaweeds with TCBS media. Strains G1, G2 were from G. textorii, U3 were from U. pertusa, L4, L5, L6 were from L. japonica, P7, P8, P9, P10, P11, P12 were from P. urceolata. They belong to Halomonas (G1, G2) and Vibrio genus (other strains).
Enzymatic bioactivity, antibiotic resistance of the 12 strains were studied. The result showed most bacteria isolated can produce starch enzyme and glutin enzyme. Strains G1, G2, U3 are resistant to ampicillin, streptomycin and tetracyclinec.
Molecular biology methods were used to study the diversity of 12 strains. The 12 strains obviously formed 4 clusters in phylogeny tree established based on 16S rRNA gene. Each of the strains isolated from the surfaces of G. textorii、U. pertusa、L. japonica formed 1 cluster. Therefore, these strains are specific to seaweeds.
Four clusters were formed in phylogeny tree established based on gyrB gene. Strains L4, P9 formed 1 cluster. L6, P8, P10, P11, P12 formed 1 cluster. U3 formed 1 cluster. L5 formed 1 cluster. Strains G1 and G2 can not amplify gyrB gene with gyrB primers, which means that G1 and G2 were far from other strains and the primers designed are specific to Vibrio. Sequences analysis based on gyrB gene gave better resolution than 16S rDNA.
16S rDNA restriction fragment length polymorphisms of 12 strains were analyzed with 2 restriction endonucleases. Digestion of 16S rDNA fragment with HinfI enzyme gave rise to 6 restriction patterns for 12 isolates and 4 with SmaI enzyme. Comparing with 16S rDNA and gyrB analysis, RFLP is suitable for differentiation of strains on species level.
Finally, diversity of strains was studied with DGGE. The DGGE band of G1, G2 formed 1 band type, U3 formed 1 band type, L5 and L6 formed 1 band type. The result showed that DGGE is suitable for differentiation of strains on genus level.
引文
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