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对根瘤菌分类及确定根瘤菌新种的研究者来说,用多少方法才能符合要求,到现在还没绝对的标准,但根瘤菌分类分委员会委员Vineusa于2007-1-14提出的建议值得大家借鉴。大家如果按照这些方法做的话,相信发表会更顺利些。
下面是我翻译的:
定一个根瘤菌新种时至少需要多少菌株?
至少三个明显不同的菌株,这些不同指的是IGS-PCR-RFLPs, rep-PCR or AFLP genomic fingerprints, sequence data等,菌株应属于不同的地理来源或不同的宿主来源。总之,只有一个菌株来定根瘤菌新种绝对不行。
多少个分子标记方法要采用?
种内要用高分辩率的方法,如rep-PCR,AFLP等进行区分,并用至少3个编码蛋白序列的基因进行比较分析。recA 和rpoB是必须分析的两个基因。再加上至少一个共生基因,如nifH, nodA和nodC等。16S rDNA序列是必须要的,并且是几乎全长的序列。
(博主补充:关于rpoB基因,我查了一下文献,它是RNA聚合酶beta亚单位的基因,基因的长度为4150bp左右,测起来相当费事,需要设计多对引物才能将其完全测定出来。rpoB已用于一些细菌的鉴定研究,在根瘤菌上用得还不是很多,值得探索。随着基因序列测定的快速化,相信测定多个基因来比较细菌之间的关系的日子不会太远了。)
表型测定要做多少?
做尽量多的交叉结瘤实验。 菌株的生长温度范围、pH,对盐的耐受度,对C、N的利用,对抗生素的抗性,脂肪酸分析。
DNA杂交?
能做最好,将新单元的几株菌之间做杂交,看它们之间的相似性。做与最相关的参比菌株的杂交。我们更希望通过多位点分析结合表型特征来定一个新种。
Tips and suggestions for the practising rhizobial taxonomist
Submitted by vinuesa on Sun, 2007-01-14 06:57.
General recommendations for the description of symbiotic rhizobia:
From: http://edzna.ccg.unam.mx/rhizobial-taxonomy/node/12
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Felis, G. E. & Dellaglio, F. (2007). On species descriptions based on a single strain: proposal to introduce the status species proponenda (sp. pr.). Int J Syst Evol Microbiol 57, 2185-2187.
Fenchel, T. & Finlay, B. J. (2006). The diversity of microbes: resurgence of the phenotype. Philos Trans R Soc Lond B Biol Sci 361, 1965-1973.
Figueras, M. J., Alperi, A., Guarro, J. & Martinez-Murcia, A. J. (2006). Genotyping of isolates included in the description of a novel species should be mandatory. Int J Syst Evol Microbiol 56, 1183-1184.
Gevers, D., Cohan, F. M., Lawrence, J. G. & other authors (2005). Opinion: Re-evaluating prokaryotic species. Nat Rev Microbiol 3, 733-739.
Gevers, D., Dawyndt, P., Vandamme, P., Willems, A., Vancanneyt, M., Swings, J. & De Vos, P. (2006). Stepping stones towards a new prokaryotic taxonomy. Philos Trans R Soc Lond B Biol Sci 361, 1911-1916.
Goris, J., Konstantinidis, K. T., Klappenbach, J. A., Coenye, T., Vandamme, P. & Tiedje, J. M. (2007). DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57, 81-91.
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Konstantinidis, K. T., Ramette, A. & Tiedje, J. M. (2006). Toward a More Robust Assessment of Intraspecies Diversity, Using Fewer Genetic Markers. Appl Environ Microbiol 72, 7286-7293.
Martens, M., Delaere, M., Coopman, R., De Vos, P., Gillis, M. & Willems, A. (2007). Multilocus sequence analysis of Ensifer and related taxa. Int J Syst Evol Microbiol 57, 489-503.
Stackebrandt, E., Frederiksen, W., Garrity, G. M. & other authors (2002). Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 1043-1047.
Staley, J. T. (2006). The bacterial species dilemma and the genomic-phylogenetic species concept. Philos Trans R Soc Lond B Biol Sci 361, 1899-1909.
Tettelin, H., Masignani, V., Cieslewicz, M. J. & other authors (2005). Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: Implications for the microbial "pan-genome". Proceedings of the
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