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F$"A$+$& ; "? ,$";A %'$? $A$& ,"";, G #$$? %$+ $ & 29 &, ,$" ,%* +";& * $& *%;#$?. , "$ ,+"; $ 289 "$+& ,""; (F$"$,) $"$ 9,7% «%» "? *$ "$$?% @*$, , "$ ?$" $& 13,8% &$%& ,"";, $ "; 3,5% "$; $$F$$%$ $,, ,"?? ,$"; «» "? *$ A*,, ($. 3). ; $ $& &? @ * ?'A %$. 4.2. , ,+"; *G $%& . ,*'% " % %"$ ,$%, ! A *"$ ,"";$ A ,$" $& $, $"$ " "$ , ,"*+ $ ,$& *, $&, ,"& $$%$ %%$ "& *";*. ! %& "*+?& @ "$, ,$", $, ы $"$ G $ * %. " $%$ G*? &$ $& A$%, & ,$"A$$ $A $"$ %$, – ,$" & "? *$ $,. <& ,$ ? $%"; «& %$», ,$% %G $; $"$$ A"*& &"& %& $, "$$ /,$$ (Takishita et. al., 2007). D$"; , $ @$& & $"% %$$ "?"$ +$, * $ & $,";"$ "? "$? , *A*H – "? $? ,$";& *";* *& $,& . ,$"; *";* G$"$ 10 * @& ,$ 4 °D. ";, + ,$"; *";* 5% PYNFH (ATCC) %$$* AF GA*$, ,"";; A ,$+$ $$+ , ,"*+ $ ,$& ,. $,$+ $"$ ?$" * " ,$$ #A $?, ,$"?% Excavata (A"**H ";*H *, ,$%"; #$ $ GA*$ $ .). 30 $. 3. D#$ "$+& A*,, @*$ ,& $ ,$& * $$ $A$";& (A) $ , *";% ,$"$? $A$";& & (B). <: (Berney et al., 2004). 31 Q$"A$+$ "$ ,""; 18S ,%' " A$% *H, $"$*H "* Excavata, $*H %$% $ ,$%$%. 18S ,$" " *G A$% "$F$% "$ (Edgcomb et. al., 2002). $#$ . (Takishita et. al., 2007) ,$"$ "A GA*$, '? "; @ "; , $"$ ;H A FAF$?%$. &$ A$+$H? %$ "? *$ "%$. $ «&» «%$& stramenoplies», MUST ("? %.: Massana et al., 2004), & %G ,$&$;? 20% ,""; ", ,"*+& $ ,$," (Not et al., 2009). ," "; F$"A$+ "$ $& ,$,"& A$%, ,"*+$#$& $%$ picobiliphytes (Not et al., 2007) $"$ biliphytes (Cuvelier al., 2008). F$"A$+$& ;?& 18S $ H? $ A*,, $,F$&, &? $$+? ,G A*,,$$ ? (Cuvelier al., 2008). ; $& A ,$% * A* $,F$& $ A,F$&. = + @$ A$% $$F$$ , ,"";?% 18S $ A$$$$ in situ ,$F$+$%$ %$ (FISH), $*+ $& ,$A% , $";*H'$ ,";* $& ,$ F$*, * AAF$+$ $ % $& &G$? ("$, ,$F$, ,"? $ ,$+$ ), $*+ ,$*+; ,"% (30 °D) $ &"% (5°D) % %%. Picobiliphytes $"$ biliphytes , H? "%$ *";** $ ,$%$ A$%%$. *A$, " +H'$? ,$ +% picobiliphytes ,$"$ $"$& A*,, F$"A$+$& ;?& 18S ' G* ,$A, ?"?H? 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G $ $A$& &, G'$& A$+*H, @"A$+*H $ AAF$+*H $F%$H %$A$%& $"$ ,"";?&, ,"*+& %"*"?%$ %%$. $$ $AAF$$ (F$"AAF$$) %$A$%. (4) % $,";$? $$F%$& &. <,";$ %$+$, "? $$F$$$ %$A$% $ %$+$, % "? "$ $& F*$";A $?. $%$ &$% *"$; $& ,$F$+$ $ +*$";$. !%G $*+$ A @,$$ *";$$*%& A$% , ,$& &, "$ * *"$+ ,$F$+; $ +*$";; %. (5) *";$$*%& & ,& %$A$%, *";$$$H % +$" , ' $ *H'A $*%$? $ ,, . (6) D% @"A$+$ $"$? ** ,"G, ,%';H %, ,"?H'$& $% #$% ,$; "$ %$ $$ in situ. 50 %, + @$ $"$? $%H ";# F*%"; +$, $ A%*H ,$+*H +$%;. $? %$% $$ $ F*$$$$ %$& ' $%H @"A$+, $ @%$+ +$ $ A $& $,";$? +$. L$ $? * $*% "? $%$$$ $ $$$$$, $ "? %G " &? ?";$: &$%$+ $ ,$' ,%#"$, %$$ $ $$$. "*"?? @"A$? %$A$% ?' %? % ?"?? $% $ & ,"$ *+& $"$, ,$%& ";H $*+$? $ &$? $$? @$% & * "%. 5" #)+)%( 1. 0 .. $$"A$?. $"A$? ,$: +$. ! 3-& . – D=.: <- D.-=. *-, 2007. 2. !4* 4!%*. =$: 2-& . =. A". / = . . KA", J. , J. P"A"?. – .: $, 2005. 3. Alonso C., Pernthaler J. Roseobacter and SAR11 dominate microbial glucose uptake in coastal North Sea waters. // Environ. 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Nitrospira, " Deltaproteobacteria, G ,$"$ "$$ Gemmatimonadetes $ Actinobacteria =" %,"$F$$$ =-,* "$$"$ @"F% 1,5% A% A" 0,5× *F (20 % $-, 7,6). =" 60 *G "$ "$ $ A"? $ @"H$"$ %G$$%-$$%. "? @A ,"$ A"? ,$& @,,F %G$"$ ,$ –20qD, % $F*A$"$ 10 %$ ,$ 12000 /%$. +*H G$; $"$ $ $,";"$ "? "$A$$? $"$ "? ,A$ =,* $$H. 4. 1$+' I-5!%)+ )+'"3+=& #) E.coli "? "$A$$? $,";"$ GeneJETTM PCR Cloning Kit (Fermentas). =-,* *,"?"$ ,%';H F% -blunding. $%* *F* "?"$ 2 %" +$'A =-,* $ 1 %" F% DNA-blunding. D%; '"; ,%#$"$ $ $*$"$ ,$ %,* 70°D +$ 5 %$. &"G"$ ";* "; * $ * G $"$ $H "$A$$?, "?? 50 A pJET/blunt $ 1 %" 4 -"$A. $*H %; $*$"$ 30 %$ ,$ % %,*. %, "$ E.coli (#%% XL-1) "? F%$$ ,"*+"$, $,";*? %$* F%$$ CaCl2-$$%& " (Sambrook et al., 1989). "? F%$$ G*H ,$* %,%$ "%$ "?"$ , 3 %" "$A %$, '"; ,%#$"$, G$"$ 30 %$ $ ,$"$ «," #». "? «G$"$?» " "?"$ 800 %" SOC, *,$H $*$"$ 45 %$ ,$ 37qD. "? $$A "$ "$ "$ +#$ =$ LB, G' 20 %A/%" %,$$""$. "$$"$ #$ "$$. "? A $$A ";#A +$" "$ $,";"$ % «$,?+$?» ". "? @A $%* " & "$ ,$"$ ,$$ @,,F, G'$ 20 %" $"; , *,$H " $,?$"$ 5 %$, % %G$"$. "$ " ," $F*A$$? (15 %$, 12000 /%$) $,";"$ + %$ = ,"%$& ,%&. 61 5. +$')#;'(< +'+#- '%#)!'(7 5"#!$+)#;'")< "$ ,"*+& ,""; ,$"$ ,*% $& $? ,"";?%$, A$$%$ %G* & ,%';H (http://www.ebi.ac.uk/fasta33). "$+$ , &$%& ,A%% ** FASTA ,"?"$ "$% ,""; $%H'? & ,%';H , ,A%% CHECK CHIMERA (http://rdp8.cme.msu.edu/html/analyses.html). 5" #)+)%( 1. .. %$+ $ %$A ' "'$ ". // F. $. … . $". *. !"$: !J, 2004. 20 . 2. 5!0 .., &+ .<., =!% .. "$? +$' "? $,";$? ,"$% , $$. // "*"?? $"A$?. 2006. . 40. 1. D. 180–183. 3. 1 .>., .., " .., ?20 .(. $$ $ "$+& A"*$& HG "$ ", ?" , ,"";?% 16S . // $$"A$?. 1999. . 68. 4. D. 475-483. 4. Blackwood C.B., Oaks A., Buyer J.S. Phylum- and class-specific PCR primers for general microbial community analysis. // Appl. Environ. Microbiol. 2005. Vol. 71, No. 10. P. 6193–6198. 5. Bottos E.M., Vincent W.F., Greer C.W., Whyte L.G. 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Zhou J., Bruns M.A., Tiede J.M. DNA recovery from soils for diverse composition // Appl. Environ. Microbiol. 1996. V. 62. 2. P. 316–322. 89 #)$'+& !)=& >$(7 1+'-$ $ 5!'(7 +""=+=&7 #%#&'-1')*" )! <.!. 1, .. Q1, <.=. ?1, !.";#1, .K. ";2,3 1 – 1/// % /!" ) # 2 – 4012 1/// # 3 – !/12 1)!1/$2 !1// !#? ? $";; ,$F$%, ,'$ "$+& A$+$& $ %$";& ', ,*$*%& ,$ A$$ $ ,$ %$$?, ?"?? %'% F% ,& F%$$? A$&$%$+A G$% %. "A? $% %F"A$+$% $ @"A$+$% ?%, %F$ $AH +$";*H "; ,"$$ ** $ +$"A ,$G& $, ,@%* $& "* %$; *, F%$*H'*H . $? $ ?"?H? "G% $ $& , %" $*+% '% & A$%. $A$%, &?'$ ,$F$& ', $AH *'*H "; $"A$+$& ,& *A ', " $& *$*H ?";; $," " %F$ %G "?; 60– 74% (/*#$, 1996). ! *"; $$A $$? %$F" ,&$ %F$ F$F$";? F* ,A*G $& +$ $" , $%* * $ A "?H'$%$ A$%%$. $";? ,", ,H'? $?, "$? ,$,"$ *A$& A$%, ", ,#$&, " $ .. $"; $ ,$F$ $$ ,$ A $$?. L$% *"" ";#$ "$? G$$ $ ,&$ $ ,?G$$ A , $ $ $"$?. , "% * A$ 90 F$"",", ? ,$G$%$ "$?%$ $ $ ,,+$H'$ "? A $$? $ %,* $ *&. ;H, A %,* ,$G?, $? %$H, $ +$H $;? 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(96.8%) B5-06-26 1 B5-06-16 1 B5-06-18 1 B5-06-10, B506-17 B5-06-33 2 1 B5-06-8 1 B5-06-19 1 B5-06-4 1 B5-06-31 1 L#1')*"+& 1%55+ =$$ ";F-,A*,, =$$ ";F-,A*,, =$$ ";F-,A*,, =$$ -,A*,, =$$ -,A*,, =$$ A%%-,A*,, =$$ A%%-,A*,, =$$ A%%-,A*,, =$$ A%%-,A*,, EF471903 Pseudomonas sp. (92.9%) =$$ DQ321570 *";. $? (93.0%) A%%-,A*,, EF428995 Pseudomonas sp. (95.3%) =$$ AY850288 *";. $? (98.1%) A%%-,A*,, DQ295890 Crenothrix sp. (96.3%) =$$ AB240465 *";. $? (96.0%) A%%-,A*,, AJ414655 Methylobacter sp. (91.7–95.9%) =$$ EF125436 *";.$?(92.3–96.4%) A%%-,A*,, AB246770 Myxobacterium sp. (91.2%) =$$ AY622263 *";. $? (93.3%) ";-,A*,, AM230484 Flavobacterium sp. (90.7%) Bacteroidetes/ EF516915 *";. $? (92.0%) Chlorobi DQ244076 Niastella sp. (94.1%) Bacteroidetes/ DQ004770 *";. $? (97.8%) Chlorobi X99392 Opitutus sp. (94.4%) Chlamydiae/ EF516582 *";. $? (99.5%) Verrucomicrobia AY493572 Leptolyngbya sp. (93.3%) $$$ DQ513910 *";. $? 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(95.2% ) 97 L#1')*"+& 1%55+ J%%,$$ Eukaryota; Ochromonas ";F,$$ Eukaryota J%%,$$ Chlamydiae/ Verrucomicrobia Eukaryota; Acricotopus J%%,$$ ";F,$$ #*")$ 1')5$, M). 14 12 10 8 6 4 2 #; 3 + ) , + )+ ) - ) + , + ) + ) , # + ;) ) + Ch la ) m , yd + ia ) e/ Ve rr uc om Ba ic ct ro er bi oi de a te s/C hl or ob I + i ' ,+ ) Fi rm ic ut es Eu ka ry ot a 0 ,- ,+,) ,+,)+ ". 2. $A%% $";A $? A$,, ,"*+A $ $ & $ & %$% @$$*% %$. ,G A% QQ< 09-04-00977, $A$% ,% D 122, =A%% F*%";& $"$ «$"A$+$ * $$». 5" #)+)%( 1. .., &'% .., " .., \ .. ,$? % %"*"?-A$+A "$ "? $*+$? %$A$%, $$& %$ // $"A$? *$& . 2008. 2. D. 91 – 94. 2. 5! .. D? $%$ $, %$%$ $ GG "$;?& Butomus umbellatus L. // $ IV !.F. , % $?%. , 1995. D.26. 3. 8$ . ., 18 . . $$ "$+& $, $A &$"$' , ' +$"$ $ "$+* 98 $, G'$& *"$ // $$"A$?, 1998, . 67, 6, . 859-864. 4. 8$ . ., !$ . . D** $," $A &$"$' // D% ?$ @$% $A &$"$'. D,.: J$%$, 1993. . 141-173. 5. " . . D,F$? %$F" #$& & $ & % // $"A$? $ F$,"A$?. 1970, 4, 1. . 8-13. 6. )+ . ., 8/ . . F$$*H'? $; @,$F$ & %F$& /,A %? // $"A$? %?, 1984, 1. . 60-62. 7. 2 5. ., > . . $"; $ $#+ A*,, #$& & $?& " DD // J$$". G*. 1979. . 15, 1. . 60-63. 8. #) .5. $$ G"$ ,$ "% *$$ & ,* // $$"A$?. 1953. . 22. !,. 2. D. 171-178. 9. #$% . . $"; ' ,$F$ %F$ $A &$"$' // $"A$? *$& , 2002, 4, . 99101. 10. #$% . . F$"?H'$ %$A$% ,$F$ %F$ $A &$"$' // . . *+,$+. F. «Q$$"A$? $ $ @"A$? *G ». /"";, 26-28 %? 2003 A. /"";, 2003. . 173. 11. #$% .., .., 8/ .., " .. ,$? %"*"?-A$+$& % "? $*+$? %$+A $? %$& ', $$& %F$%$ // $"A$? *$& . 2009. 1. 102 – 110. 12. 4 . . "G$ "+$ <;% $ A"$+% &$"$'& // $"A$+? ,*$; $ + !"A$ $ &$"$'. 1984, ., «*». D. 123-124. 99 13. (4 .. $," $ $"; ,$F$ "$+& $,& D?% // $$"A$?. 1965. . 34. !,. 1. D. 133-139. 14. > . . $$ $ $ " $ ,$F$ $ D-%% " // J$$". G*., 1978, 14, 4. . 36-40. 15. > . . "; ,$F$ #$& & $ *$$ A$+A ' // J$$". G*. 1996. . 32. ¹ 2. D. 41-47. 16. Amann R.I., Ludwig W., Schleifer K.H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation // Microbiological Reviews. 1995. Vol. 59. P. 143 – 169. 17. Nocker A., Camper A.K. Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide. // Appl. Environ. Microbiol. 2006. V. 72. No. 3. P. 1997 – 2004. 18. Rudi K., Moen B., Droemtorp S.M., Holck A.L. Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples. // Appl. Environ. Microbiol. 2005. V. 71. No. 2. P. 1018 – 1024. 100 #&' %"#$< 5$*'< ,+,) $!'(7 5, '+ +-',+- 5#%*+(7 1')5$ '+ 5 ,'(7 ",8")$ 5"'1 -+ +7#< (+,+<+#;) .. HA$1, .K. ";2,3 1 – 1/// 8!!)$" !1!1, : ! # 2 – 4012 1/// # 3 – !/12 1)!1/$2 !1// =$$%$ F%$, *""$H'$%$ F%$$ "$ & @$%, ?"?H? %F%$+$ $ A$&$%$+$ &$$$ %. D$% <-&"$& – $ *,& & $% C";? – ,"G " KA $ A-$A . &" ?"?? $";#$% , ,"'$ – 5900 A, %$%"; A"*$ 17,0 % ($. 1). - +7#< $. 1. -&% ,"G$? $% <-&"$& . 101 &$*? 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