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
目的：对中药白首乌根部的内生细菌进行分离、纯化、鉴定及比对,最终确定其种属并构建系统发育树。方法：运用涂布法和平板划线法从中药白首乌的块根中分离、纯化内生细菌,对分离得到的纯菌株进行形态学观察、生理生化鉴定,并结合菌株的...目的：对中药白首乌根部的内生细菌进行分离、纯化、鉴定及比对,最终确定其种属并构建系统发育树。方法：运用涂布法和平板划线法从中药白首乌的块根中分离、纯化内生细菌,对分离得到的纯菌株进行形态学观察、生理生化鉴定,并结合菌株的16S r RNA基因序列鉴定其种属,再将测序结果与Gen Bank中的已知序列进行BLAST比对,查找相似性最高的菌种进行同源性分析,最终通过软件MEGA 7.0构建系统发育树确定菌株的系统分类学地位。结果：首次从中药白首乌的根部分离得到12株内生细菌,其中9株属于芽孢杆菌属（Bacillus）,相似性为99%～100%;2株属于假单胞菌属（Pseudomonas）,相似性为100%;1株属于沙雷氏菌属（Serratia）,相似性为99%,确定优势菌属为芽孢菌属。结论：白首乌根中存在多株内生细菌,其很可能是白首乌内生菌资源的重要组成部分,本研究为白首乌内生菌的资源开发奠定了基础。展开更多
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.
文摘目的：对中药白首乌根部的内生细菌进行分离、纯化、鉴定及比对,最终确定其种属并构建系统发育树。方法：运用涂布法和平板划线法从中药白首乌的块根中分离、纯化内生细菌,对分离得到的纯菌株进行形态学观察、生理生化鉴定,并结合菌株的16S r RNA基因序列鉴定其种属,再将测序结果与Gen Bank中的已知序列进行BLAST比对,查找相似性最高的菌种进行同源性分析,最终通过软件MEGA 7.0构建系统发育树确定菌株的系统分类学地位。结果：首次从中药白首乌的根部分离得到12株内生细菌,其中9株属于芽孢杆菌属（Bacillus）,相似性为99%～100%;2株属于假单胞菌属（Pseudomonas）,相似性为100%;1株属于沙雷氏菌属（Serratia）,相似性为99%,确定优势菌属为芽孢菌属。结论：白首乌根中存在多株内生细菌,其很可能是白首乌内生菌资源的重要组成部分,本研究为白首乌内生菌的资源开发奠定了基础。
