4.1.1 in vitro
, , : TL ( Cu-asFur-TL), T1 T2 ( Cu-asFur-T12). asRNA, , overlap (!!!!!!!!!!). pSK9 , PetJ ( Cu) MidCat-r2 PpetJ/5asFur (.2). 5 , asRNA fur ( asFur). asFur 5asFur/PpetJ-c ( 5- Cu) 3asFur-T12(Bgl) (. 2) Cu-asFur-T12 5asFur/PpetJ-c 3asFur-TL(Bgl) (. 2) Cu-asFur-TL. MidCat-r2 5- EcoRI, 3asFur-TL(Bgl) 3asFur-T12(Bgl) 5- BglII. .1. - pCu (852 ..), asFur-T12 (225 ..) asFur-TL (173 ..).
.1 pfu- pPetJ-asFur-T
1-2 pPetJ pSK9 MidCatR2 5asFur/pPetJ-c
3-4 asFur-12 pPetJ/5asFur 3asFurT12(BglII)
5-6 asFur-TL pPetJ/5asFur 3asFurTL (BglII)
. pCu-asFur-TL (.2) (965 ..) pCu asFur-TL, MidCat-r2 3asFur-TL(Bgl). , pCu-asFur-T12 (.2) (1017 ..) pCu asFur-T12, MidCat-r2 3asFur-T12. (.3)
.2
.3 pfu--overlap pPetJ-asFur-T12 ( 1 2) pPetJ-asFur-TL ( 3 4).
???????????. CloneJet (Thermo Scientific), pJet (.4).
E. coli . , pJet-asFur-TL (. 5. ) pJet-asFur-T12 ( 5. ).
.5. pJet-asFur-TL(, ) pJet-asFur-T12 (,). , -.
EcoRI XhoI. , 3 (pPetJ-asFur-T12) 7 (pPetJ-asFur-TL) (. 6).
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6
pSK9, Synechocystis sp. pPetJ-asFur-T12 pPetJ-asFur-TL EcoRI BglII, . pSK9, . Synechocystis sp. (. 7 ).
.7 pSK9-pCu- asFur-T12 () pSK9-pCu- asFur-TL (). p6803L p6803R EcoRI BglII .
Up-PpetJ-s, PpetJ-sr1, PpetJ-sr2, Plat_6803-r, MidCat-r2, asFur-s , asFur , , , 3asFur-T12(Bgl) BglII.
Synechocystis sp. , bla.
.
, . fur (sll0567) Synechocystis 6803 (Kunert et al., 2003) Anabaena sp. PCC 7120 (Gonzalez et al., 2010) , . , fur . . fur Sp-, fur petJ (P petJ), (Zhang et al., 1992; Tous et al., 2001; Peter et al., 2009). , fur P petJ fur . - fur - fur P petJ .
4.1. Synechocystis 6803 fur / fur
fur
( Fur-f Fur-r; . 2) Synechocystis 6803, fur. - 1,33 ... pGEM-T E. coli , pGMFUR (. 2); Eco RI 1,33 ... (. 3). fur Nde I; 1,28 ... ( ), fur bla (. 2). fur () Van 91I Cla I I Sp-. pGMFUR-Sp, aadA , fur (. 2). Sp- pGMFUR-Sp Sph I, 2,43 ... (. 3).
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. 2. pGMFUR () pGMFUR-Sp (). pGMFUR-Sp pGMFUR, fur Van 91I Cla I Sp-, d.
. 3. pGMFUR () pGMFUR-Sp (), E. coli (1, 2, 3), . : Eco RI () Sph I ().
. ...
Synechocystis 6803 pGMFUR-Sp, . , Δfur.
fur
fur P petJ pVZExCu (. 4). p6803 Synechocystis 6803 (Los et al., 2003). , Bgl II ( Nde I ), , P petJ Cm, cat. pVZExCu Nde I Cla I ( ) Nde I Bgl II (. 4).
C Pfu - Fur-Nde-f Fur-Bgl-r (. 2), Synechocystis 6803 , 500 .., fur. - Nde I Bgl II, , pVZExCu, CIAP (Fermentas). pCuFur, fur P petJ (. 4). pCuFur Fur-Nde-f Fur-Bgl-r fur , ( CyanoBase), , ATG - TAG Nde I Bgl II, .
. 4. pVZExCu pCuFur, fur Nde I Bgl II . p6803 Synechocystis 6803 (Los et al., 2003). P petJ Cm, cat. - , fur p6803.
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Δfur pCuFur Cm, fur P petJ ( p6803; Los et al., 2003). , Δfur/CuFur, , Cm (5 /) Sp ( 5 50 /). fur .
CuFur, , fur P petJ. , Cm (20 /), fur . Anabaena sp. PCC 7120. furA ( fur Synechocystis 6803), P petE, (Gonzalez et al., 2010). furA , , , , , D1 II ( psbA), ( mreBCD) ( schT). , , psbA, mreBCD schT furA.