16S rDNA and ERIC （Enterobacteia Repetitive Intergenic Consensus Sequences） based on PCR method were tested for the effectiveness of the differentiation of B. thuringiensis and B. cereus. 16S rDNA-PCR primers were d...16S rDNA and ERIC （Enterobacteia Repetitive Intergenic Consensus Sequences） based on PCR method were tested for the effectiveness of the differentiation of B. thuringiensis and B. cereus. 16S rDNA-PCR primers were designed based on the sequence difference in variable regions of B. cereus 16S rDNA and B. thuringiensis 16S rDNA, 16S rDNA-PCR showed no obvious difference between B. cereus and B. thuringiensis. The only difference was that one 1600-bp amplificon could be obtained from all the three B. Cereus strains, and none amplificon from any B. thuringiensis strains. ERIC was optimized based on previous reports. The genonlic DNA was used for the template of ER1C-PCR, and the following DNA fingerprints were analyzed by the agarose gel electrophoresis. The results showed that DNA fingerprint of three B. thuringiensis strains had a unique amplicon less than 100-bp, while DNA fingerprint of three B. cereus＂ strains had none. Moreover, DNA fingerprint of B. cereus showed a 700-bp amplicon, but didn＇t have any DNA fingerprints ofB. thuringiensis genome. Therefore, ERIC-PCR technique should be able to be used for the differentiation of B. thuringiensis and B. cereus.展开更多
So far studies have focused on bacteria isolation aimed at establishing causes of fish diseases and medication methods.Now,more and more attention has been given to the composition of the microflora,its variations in ...So far studies have focused on bacteria isolation aimed at establishing causes of fish diseases and medication methods.Now,more and more attention has been given to the composition of the microflora,its variations in time,and effect on whiteleg shrimp(Litopenaeus vannamei).This is why the problem of bacterial flora in water ought to be studied.The major role of ammonifying bacteria is to break down nitrogenous organic matter to ammoniac nitrogen.The research of ammonifers in the pond water of Litopenaeus vannamei is increasing with the demand for environment friendly aquaculture.Four bacterial strains(No.zjs01,zjs02,zjs03 and zjs04),isolated from the pond water of Litopenaeus vannamei in Jinshan district of Shanghai,were cultured in ammonifying bacteria rich medium and identified by two methods.One is the sequence analysis of 16S rDNA,the other is bacteria identification system.First,sequence analysis of the 16S rDNA was done.Genomic DNA of four strains was isolated respectively,then their full length of the 16S rDNA were amplified by PCR respectively,using universal primers to the 16S rDNA.After purification by gel extraction,the PCR products were cloned and subsequently sequenced by Shanghai Invitrogen Biotechnology Company(SIBC).The phylogenetic trees were constructed,based on the result of online alignment.At the same time,the four sequences of 16S rDNA were submitted to NCBI(http://www.ncbi.nlm.nih.gov) in order to obtain accession number of strains of zjs01,zjs02,zjs03 and zjs04.Then the API 2000 Bacteria Identification System(Biomerieux Company) was applied in assessment the identification result of zjs01,zjs02,zjs03 and zjs04 by molecular method.Finally the 4 strains were identified as,zjs01: Brevundimonas diminuta,zjs02 and zjs03: Alcaligenes faecalis,zjs04: Enterobacter aerogenes.And the accession number of the strains(zjs01,zjs02,zjs03 and zjs04) is DQ857897,DQ857898, DQ857895,DQ857896 respectively.The method of detecting bacterial 16S rDNA using PCR technique is specific,sensitive,rapid and accurate in detecting bacterium in culture pond.Also an interesting thing should be indicated,it is absolutely necessary that the 16S rDNA PCR products should be cloned before DNA sequencing.This paper will establish theory groundwork for application of ammonifers microbiological preparation to bioremediation of the polluted culture water.展开更多
该研究应用PCR-DGGE技术,分析了不同种植年限正常生长、发病的黄连Coptis chinensis的根际和非根际土壤的细菌种群多样性指数,并选取代表性DGGE条带进行克隆测序,构建细菌系统发育树状图。结果表明,正常土样与发病土样土壤细菌种群差异...该研究应用PCR-DGGE技术,分析了不同种植年限正常生长、发病的黄连Coptis chinensis的根际和非根际土壤的细菌种群多样性指数,并选取代表性DGGE条带进行克隆测序,构建细菌系统发育树状图。结果表明,正常土样与发病土样土壤细菌种群差异明显,且发病土样多样性指数(H)高于正常土样;代表性条带克隆测序结果表明,黄连种植土壤中未培养细菌为优势菌群,其余类群为食酸菌属、芽孢杆菌属、不动杆菌属、未培养克吕沃尔菌属以及未培养丛毛单胞菌,未发现已报道的植物病原细菌。发病土样中Clone band d的谱带亮度高于正常土样,该条带菌株在分类上属于食酸菌属。显然,黄连种植过程中,根际土壤中细菌种群发生了改变,其中食酸菌属的细菌在发病根际土壤增加,很可能与黄连病害发生有关。展开更多
基金Supported by Genetically Modified Organisms Breeding Major Projects (2009ZX08009-031B)State Key Laboratory for Biology of Plant Diseases and Insect Pests Open Fund (DKL2010OP13)
文摘16S rDNA and ERIC （Enterobacteia Repetitive Intergenic Consensus Sequences） based on PCR method were tested for the effectiveness of the differentiation of B. thuringiensis and B. cereus. 16S rDNA-PCR primers were designed based on the sequence difference in variable regions of B. cereus 16S rDNA and B. thuringiensis 16S rDNA, 16S rDNA-PCR showed no obvious difference between B. cereus and B. thuringiensis. The only difference was that one 1600-bp amplificon could be obtained from all the three B. Cereus strains, and none amplificon from any B. thuringiensis strains. ERIC was optimized based on previous reports. The genonlic DNA was used for the template of ER1C-PCR, and the following DNA fingerprints were analyzed by the agarose gel electrophoresis. The results showed that DNA fingerprint of three B. thuringiensis strains had a unique amplicon less than 100-bp, while DNA fingerprint of three B. cereus＂ strains had none. Moreover, DNA fingerprint of B. cereus showed a 700-bp amplicon, but didn＇t have any DNA fingerprints ofB. thuringiensis genome. Therefore, ERIC-PCR technique should be able to be used for the differentiation of B. thuringiensis and B. cereus.
文摘So far studies have focused on bacteria isolation aimed at establishing causes of fish diseases and medication methods.Now,more and more attention has been given to the composition of the microflora,its variations in time,and effect on whiteleg shrimp(Litopenaeus vannamei).This is why the problem of bacterial flora in water ought to be studied.The major role of ammonifying bacteria is to break down nitrogenous organic matter to ammoniac nitrogen.The research of ammonifers in the pond water of Litopenaeus vannamei is increasing with the demand for environment friendly aquaculture.Four bacterial strains(No.zjs01,zjs02,zjs03 and zjs04),isolated from the pond water of Litopenaeus vannamei in Jinshan district of Shanghai,were cultured in ammonifying bacteria rich medium and identified by two methods.One is the sequence analysis of 16S rDNA,the other is bacteria identification system.First,sequence analysis of the 16S rDNA was done.Genomic DNA of four strains was isolated respectively,then their full length of the 16S rDNA were amplified by PCR respectively,using universal primers to the 16S rDNA.After purification by gel extraction,the PCR products were cloned and subsequently sequenced by Shanghai Invitrogen Biotechnology Company(SIBC).The phylogenetic trees were constructed,based on the result of online alignment.At the same time,the four sequences of 16S rDNA were submitted to NCBI(http://www.ncbi.nlm.nih.gov) in order to obtain accession number of strains of zjs01,zjs02,zjs03 and zjs04.Then the API 2000 Bacteria Identification System(Biomerieux Company) was applied in assessment the identification result of zjs01,zjs02,zjs03 and zjs04 by molecular method.Finally the 4 strains were identified as,zjs01: Brevundimonas diminuta,zjs02 and zjs03: Alcaligenes faecalis,zjs04: Enterobacter aerogenes.And the accession number of the strains(zjs01,zjs02,zjs03 and zjs04) is DQ857897,DQ857898, DQ857895,DQ857896 respectively.The method of detecting bacterial 16S rDNA using PCR technique is specific,sensitive,rapid and accurate in detecting bacterium in culture pond.Also an interesting thing should be indicated,it is absolutely necessary that the 16S rDNA PCR products should be cloned before DNA sequencing.This paper will establish theory groundwork for application of ammonifers microbiological preparation to bioremediation of the polluted culture water.
文摘该研究应用PCR-DGGE技术,分析了不同种植年限正常生长、发病的黄连Coptis chinensis的根际和非根际土壤的细菌种群多样性指数,并选取代表性DGGE条带进行克隆测序,构建细菌系统发育树状图。结果表明,正常土样与发病土样土壤细菌种群差异明显,且发病土样多样性指数(H)高于正常土样;代表性条带克隆测序结果表明,黄连种植土壤中未培养细菌为优势菌群,其余类群为食酸菌属、芽孢杆菌属、不动杆菌属、未培养克吕沃尔菌属以及未培养丛毛单胞菌,未发现已报道的植物病原细菌。发病土样中Clone band d的谱带亮度高于正常土样,该条带菌株在分类上属于食酸菌属。显然,黄连种植过程中,根际土壤中细菌种群发生了改变,其中食酸菌属的细菌在发病根际土壤增加,很可能与黄连病害发生有关。
