放射污染区古菌分离及多样性分析

Separation and identification of archaea from radioactive pollution

摘要:

【目的】研究放射污染区古菌多样性。【方法】放射污染区采集土样，采用甘油-精氨酸培养基(GJ)、甘油-天冬氨酸培养基(C1)、海藻糖-肌酸培养基(B7)、甘露醇-丙氨酸培养基(Z5)、干酪素-甘露醇培养基(CMKA)、壳聚糖-天冬酰胺培养基(F6)、甘露醇-酸水解酪蛋白培养基(GW1)、CM培养基、HP培养基和KC培养基10种分离培养基，采用梯度稀释法对古菌进行分离，将分离获得的菌株经形态特征，16S rRNA基因片段扩增及限制性内切酶酶切，选取酶切图谱中存在差异性的条带进行测序，最终通过序列比对，聚类分析，获得不同种类的古菌资源。【结果】从该土样中共获得了256株古菌，最终筛选出71株不同类型的古菌，这71株古菌均属于广古菌门，盐杆菌纲，盐杆菌目，盐杆菌科，分布于盐陆生菌属(Haloterrigena)、纳白菌属(Natrialba)、盐球菌属(Halococcus)、盐红菌属(Halorubrum)、盐长寿菌属(Halovivax)、纳线菌属(Natrinema)、盐碱球菌属(Natronococcus)、盐二型菌属(Halobiforma)、盐惰菌属(Halopiger)、盐池栖菌属(Halostagnicola)、富盐菌属(Haloferax) 11个属，26个种，其中31株菌的16S rRNA基因序列与已有效发表菌株的序列相似性小于98%，Haloterrigena为该土样的优势菌属。对于分离效果较好的F6培养基采用了梯度营养成分的稀释，最终获得了19株古菌，这些菌株相互之间存在一定的差异性。【结论】本次分离获得了大量的古菌，表明放射污染区存在着较为丰富的古菌资源，其中蕴藏着多种新的物种类型，具有较大的研究价值。

Abstract:

[Objective] This study was to investigate the diversity of archaea from radioactive pollution environment. [Methods] Glycerin-arginine medium (GJ), Glycerin-aspartic acid medium (C1), Trehalose-creatine medium (B7), Mannitol-alanine medium (Z5), Casein-mannitol medium (CMKA), Chitosan-asparagine medium (F6), Mannitol-acid hydrolysis of casein medium (GW1), CM, HP, and KC media with different salinities were used for isolating archaea from radioactive pollution areas. Different isolates selected on the basis of morphological characteristics and Hae III digestion patterns of 16S rRNA genes. The results of 16S rRNA gene sequences were compared with sequences obtained from GenBank databases. Phylogenetic tree was constructed by the Neighbor-Joining method. [Results] Total 256 archaeal strains were obtained by different selective media, and belonged to 71 different types. They were members of Haloterrigena, Natrialba, Halococcus, Halorubrum, Halovivax, Natrinema, Natronococcus, Halobiforma, Halostagnicola, Halopiger, Natronorubrum and Haloferax genera. The similarities between detected sequences (34% of total strains) and published sequences were less than 98%. They formed distinct clades in phylogenetic tree and might represent new taxa. Haloterrigena was the dominant group. Nineteen strains were obtained by different Chitosan-asparagine medium (better isolating medium) ten-fold dilution culture. Compared with strains by Chitosan-asparagine medium, they were different groups. [Conclusion] The radioactive pollution area harbors abundant archaea, including large number of unknown bacterial groups, has a great research value.