Food Technology & Nutrition / Spring 2013 / Vol. 10 / No S rdna. jftn.srbiau.ac.ir. . GenBank.

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1 6 2 / / 392 / 6S rdna c b * a b 39/5/6 : a 390//20 : c :... : TD0 TD4 T22 T20. 6S rdna DNA MRS 6S rdna... %99 NCBI :. GenBank :. tafvizi@piau.ac.ir 6 S rrna : *

2 .(Woses et al., 987) 6S rrna. (V9 V ) Collins et al., 99; Stackebrandt et al., ).(995; Vandamme et al., 996 6S rdna PCR Langendijk ) et al., 995; Wang et al., 996; Welling et al., 997; Lick, 2003; Caufield et al. 2007; 2.(Dimtonova et al., 2008; (T-T26).. (TD-TD6) Caco-2.. TD0 TD4 T22 T20 ) Tajabadi ) (.(Ebrahimi et al., 20a,b T20 TD0 TD4 T22 6S rdna. Alignment.... (LAB). G+C... 23S rrna 6S rrna 23S rrna 6S rrna. Woses et al., ) 6S rrna.(987 23S rrna ATPase β Tu. 6S rrna ATPase rrna 6S rrna.(schleifer et al., 995). rrna Alignment 62 2 / / 392 /

3 63 2 / / 392 / DNA - DNA Araújo et al., ) Araújo DNA 24.( ml. rpm EDTA ) TEN 3 (Tris Hcl 00Mm NaCl 50mM 00mM rpm mg/ml TEN / ph 5/ rpm 5 DNA 24:25: : : DNA. DNA. 50. DNA. TE. 0/7 6S rdna 6S rdna 66V, 630R 2.(Ehrmann et al., 2003) 66V, 5-AGAGTTTGATYMTGGCTCAG-3; 630R, 5-CAKAAAGGAGGTGATCC-3. Degenerate -.. 6S rdna 6S rrna -. V-V9 - TD0 TD4 T22 T20 Tajabadi Ebrahimi et al., 20 ) (a, b. MRS (Man ROGOSA and SHARP) Degenerate

4 6S rdna.. Clustal W bootstrap Neighbor - joining Replication 000.(Tamura et al., 2007) MEGA4 Escherchia coli (NC ). outgroup - 6S rrna Blastn (%99). Align - Reference Sequences Lactobacillus casei strain Shirota Lactobacillus casei strain YIT 0209 Lactobacillus casei strain YIT 080 Lactobacillus casei Lactobacillus paracasei Lactobacillus paracasei strain SM20 Accession Numbers AB 533. AB AB D DQ AJ T22 T20 TD0 TD4 (Tajabadi Ebrahimi et al., 20 a, b) ) 2. 6S rdna (500bp. DNA PCR Buffer PCR x 2.5 ( 0 mm Tris Hcl PH8.8, 250 mm kcl) 0.4 µm 200 µm dntp Taq DNA Polymerase 25 Corbet, ) DNA.. Palmcycler Gp-00 (Australia 2 94 DNA DNA 94 : PCR 0.5 mm ) TBE 0.5 x %/5 (EDTA, ph 8.0, 44.5 mm Tris/Borate Rima Sight DNA Stain. uv PCR.(Sambrook et al., 200) (Gene Ruler, fermentas) 00bp.(2 ) (500 bp ) PCR 630R 66V PCR. Bioneer - Chimeric Artifact Vector NTI 6S rdna 6S rdna. Blastn NCBI BLAST Search tool 64 2 / / 392 /

5 65 2 / / 392 / Lactobacillus casei strain T22 (JQ4273) Lactobacillus casei strain TD4 (JQ42732) strain TD0 (JQ42734). GenBank.. ( ). Isolates T20 T20 T20 T22 T22 T22 TD4 TD4 TD4 TD0 TD0 TD0 >%97 Schloss ) 6S rrna.(and Handelsman, 2005 NCBI Blastn (%99) ) 2.( Blastn.. Lactobacillus casei Lactobacillus casei Blastn -2 Sequence lenghts 52bp 52bp 52bp 522bp 522bp 522bp Most closely related type strain Lactobacillus casei strain Shirota Lactobacillus casei strain YIT 0209 Lactobacillus casei strain YIT 080 Lactobacillus casei strain Shirota Lactobacillus casei strain YIT 0209 Lactobacillus casei strain YIT 080 Lactobacillus casei strain Shirota Lactobacillus casei strain YIT 0209 Lactobacillus casei strain YIT 080 Lactobacillus casei strain Shirota Lactobacillus casei strain YIT 0209 Lactobacillus casei strain YIT 080 strain T20 (JQ42730) Accession numbers AB 533. AB AB AB 533. AB AB AB 533. AB AB AB 533. AB AB similarity 6S rrna (500 bp ) PCR -2 /5

6 %99 (%99) bootstrap Lactobacillus casei (D8657.) TD0 TD4 T20.. (A ) TD0 TD4 6S rdna bootstrap bootstrap %88 T20 (%99) bootstrap %99 B A. T20. %99 DNA TD0 TD4 T22. TD0 TD4.. ( ) Kullen. 500 ( V 2 V ) 6S rrna. V 3. 6S rrna wang et al., ) Wang Sisto.(2006.(Sisto et al., 2009) 6S rrna.(389 ) DNA.. (DNA/DNA) genera (DNA/RNA)... rrna. rrna.. rrna..(woese et al., 987). 6S rdna 3. GenBank B T / / 392 /

7 67 2 / / 392 / ( ) 6S rrna -3 Caufield, P. W., Li, Y., Dasanayake, A. & Saxena, D. (2007). Diversity of lactobacilli in the oral cavities of young women with dental caries. Caries Res, 4(), 2-2. Collins, M. D., Rodrigues, U., Ash, C., Aguirre, M., Farrow, J. A. E., Martinez- Murcia, A., Phillips, B. A., Williams, A. M. & Wallbanks, S. (99). Phylogenetic analysis of the genus Lactobacillus and related lactic acid bacteria as determined by reverse transcriptase sequencing of 6S rrna. FEMS Microbiol. Lett, 77, 5-2. Dimtonova, S. P., Bakalov, B. V., Aleksandrova-Georgieva, R. N. & Danova, S. T. (2008). Phenotypic and and molecular identification of lactobacilli isolated from vaginal secretions. J Microbiol Immunol Infect, 4, Ehrmann, M. A., Mu ller, M. R. A. & Vogel, R. F. (2003). Molecular analysis of sourdough reveals Lactobacillus mindensis sp. nov. International Journal of Systematic and Evolutionary Microbiology, 53, 7 3. Kullen, M. J., Sanozky-Dawes, R. B., Crowell, D. C. & Klaenhammer, T. R. (2000). Use of the DNA sequence of variable regions of the 6S rrna gene for rapid and accurate identification of bacteria in the Lactobacillus acidophilus complex. Journal of Applied Microbiology, 89, Langendijk, P. S., Schuts, F., Jansen, G. J., Raangs, G. C., Kamphuis, G. R., Wilkinson, M. H. F. & Welling, G. J. (995). Quantitative fluorescence in situ hybridization of GenBank. (%) bootstrsp. Outgroup E. coli (NC02947.).....(389) /3 Araújo, W. L., Angellis, D. A. & Azevedo, J. L. (2004). Direct RAPD evaluation of bacteria withoutconventional DNA extraction. Brazilian Archives of Biology and Technology, 47,

8 Bifidobacterium spp. with genus-specific 6S rrna-targeted probes and its application in fecal samples. Appl. Environ. Microbiol, 6, Lick, S. (2003). Review: Typing systems for lactobacilli, Milchwissenschaft, 58, Schleifer, K. H., Ehrmann, M., Beimfohr, C., Brockmann, E., Ludwig, W. & Amann, R. (995). Application of molecular methods for the Classification and identification of Lactic Acid Bacteria. Int. Dairy Journal, 5, Schloss, P. D. & Handelsman, J. (2005). Introducing DOTUR, a computer program fordefining operational taxonomic units and estimating species richness. Appl.Environ. Microbiol, 7, Sisto, A., De Bellis, P., Visconti, A., Morelli, L. & Lavermicocca, P. (2009). Development of a PCR assay for the strainspecific identification of probiotic strain Lactobacillus paracasei IMPC2.. Int J Food Microbiol, 36(), Stackebrandt, E. & Rainey, F. A. (995). Partial and complete 6S rdna sequences, their use in generation of 6S rdna phylogenetic trees and their implications in molecular ecological studies, p. -7. In: A. D. L. Akkeramns, J. D. van Elsas, and F. J. de Bruijn (ed.), Molecular microbial ecology manual. Kluwer Academic Publishers, Dordrecht. Tajabadi Ebrahimi, M., Bahrami, H. & Ziary, Z. (20 a). Tarkhineh source of probiotic lactic acid bacteria. The Quarterly Journal of Biological Sciences, Islamic Azad University, Zanjan. 4(2): -9. Tajabady, E. M., Ouwehand, A. C., Jafari, P., Bahrami, H. & Heidary Nasrabadi, M. (20 b). Evaluation probiotic effects of lactic acid bacteria isolated from Tarkhineh, International Scientific Conference on Probiotic and prebiotic, Slovakia, June 4-6. Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0.Mol Biol Evol, 24, Vandamme, P., Pot, B., Gillis, V., de Vos, P., Kersters, K. & Swings, J. (996). Polyphasic taxonomy, a consensus approach to bacterial systematics. Microb.Rev, 60, Wang, R. F., Cao, W. W. & Cerniglia, C. E. (996). PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples. Appl. Environ. Microbiol, 62, Wang, Y. Y., Li, H. R., Jia, S. F., Wu, Z. J. & Guo, B. H. ( 2006). Analysis of bacterial diversity of kefir grains by denaturing gradient gel electrophoresis and 6S rdna sequencing. Wei Sheng Wu Xue Bao, 46(2), Welling, G., Elfferich, W. P., Raangs, G. C., Wildeboer-Veloo, A. C. M., Jansen, G. J. & Degener, J. E. (997). 6S ribosomal RNAtargeted oligonucleotide probes for monitoring of intestinal tract bacteria. Scand. J. Gastroenterol. 32 Supplement 222, 7-9. Woese, C. R. (987). Bacterial evolution. Microbial. Rev, 5, / / 392 /