小单孢菌属特异寡核苷酸探针的设计及杂交条件优化

Construction of a New Oligonucleotide Probe Specific for Micromonospora and Optimization of the Hybridization Conditions

小单孢菌在生态环境中占有重要地位,也是寻找新的抗生素的重要来源。为了探索小单孢菌的生态分布和在未培养情况下的生态位,在分离和鉴定一定数量的小单孢菌的情况下利用小单孢菌属的16S rDNA基因同源性,采用ClustalX软件进行序列分析,设计了小单孢菌属的16S rRNA特异寡核苷酸探针Mn1和Mn3两个序列,将这两个序列与GenBank中小单孢和非小单孢菌属的16S rRNA序列进行Blastn比对分析,先在理论上确认Mn1和Mn3对小单孢菌属是特异的。收集小单孢和非小单孢菌株19株,通过原位杂交试验的方法对设计的探针进行特异性验证,结果证明,Mn1能和试验用到的所有小单孢菌属的标准菌株杂交和非小单孢菌均不能杂交;Mn3能和所有小单孢菌属的标准菌株杂交,但也能与链霉菌CGMCC4.891(Streptomyces microflavus)杂交。初步证明Mn1可作为小单孢菌属的特异性探针。并通过杂交条件试验优化了Mn1荧光原位杂交的条件:甲酰胺浓度为30%,溶菌酶处理时间为37℃40min,HCl处理时间为60min,杂交时间为3h。

In order to explore the ecological distribution of the cultured and uncultured of the micromonospora,on the condition of isolation and identification of a certain number of micromonospora , two new probes, which have been named as Mnl and Mn3 ,were designed based on the 16S rDNA homology. Comparative analysis of 51 sequences of 16S rDNA including those of members of the family micromonospora and of other bacteria was carried out. Comparisons of Mnl and Mn3 against the GenBank database by using the NCBI BLASTN program verified that Mnl and Mn3 are specific for micromonospora theoretically. Then whole-cell hybridization was performed with 19 strains by using Mnl and Mn3 probes , which demonstrated that Mnl was specifie for mieromonospora and Mn3 can also hybridese with Streptomyces microflavus. The optimal fluorescence in situ hybridization test conditions for Mnl were the formamide concentration in hybridization buffer 30% , lysozyme processing time for 37℃ 40 min, HC1 processing time for 60 min, bybridization time 3 h.