Invitrogen 1.4 pet-gr, ICE-LGR, PCR TA, pmd18-t/gr pet-21a(+) (Nde I Xho I), E. coli DH-5,,,, pet-gr GR ORF cdna, GR, : PU: 5 -GGAATTCCAT

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1 42 6 Vol.42, No OCEANOLOGIA ET LIMNOLOGIA SINICA Nov., 2011 南极衣藻 (Chlamydomonas sp. ICE-L) 谷胱 * 甘肽还原酶基因的原核表达及其条件优化 1 1 燏 ( ; ) RT-PCR GR ORF cdna, ; IPTG,, pet-gr BL21, ; ; SDS-PAGE, 52.2kDa, ; GR, 1.0mmol/L IPTG, 28 3h Chlamydomonas sp. ICE-L,,,, Q936, Q78 (glutathione reductase, GR), GR, GR,,,, ( 燏, 2006;, 2010), GR ( ) (Ding et al, 2005; 燏, 2006) Chlamydomonas sp. ICE-L (Ding et al, 2007),,,, (Chlamydomonas sp. ICE-L), Provasoli (Provasoli, 1968) 1.2 : pet-21a, : DH5 ; BL21(DE3) 1.3 dntps ExTaq rtaq Reverse Transcriptase M-MLV (Rnase H) Nde I Xho I T 4 DNA Marker TaKaRa ; - -D- (IPTG) ; PCR QIAquick TM PCR Purification Kit QIAGEN ; Trizol *, , , liuying0454@163.com : 燏, dingy@gdou.edu.cn;,,, jianjc@gdou.edu.cn : , :

2 Invitrogen 1.4 pet-gr, ICE-LGR, PCR TA, pmd18-t/gr pet-21a(+) (Nde I Xho I), E. coli DH-5,,,, pet-gr GR ORF cdna, GR, : PU: 5 -GGAATTCCATATGTCTCGCTTCGCTGC AGCGAACTAC-3 (Nde I), PL: 5 -CCGCTCGAGAAGCTTGGAAGTTCCAG TGCCCATA-3 (Xho I), ICE-LGR 5 Nde I Xho I, PCR, 40.5 l 10 PCR buffer 5 l dntp mixture (10mmol/L) 1 l PU (10 mol/l) 1 l PL (10 mol/l) 1 l cdna 1 l ExTaq 0.5 l PCR, 95 5min, 94 60s 59 60s 72 75s, 30 cycles,, 72 10min, PCR 1.0%,, UNIQ-10 DNA DNA pmd 18-T Vector, E. coli DH5, X-gal IPTG, PCR 1ml LB 37, 200r/min 5 6h, 1 l, PCR,, PET-21a(+), Nde I Xho I 50 l (H Buffer 5 l Nde I 2 l Xho I 2 l DNA 21 l ddh 2 O 20 l); PET-21a(+) 50 l (H Buffer 5 l Nde I 2 l Xho I 2 l pet21a(+) 20 l ddh 2 O 21 l) 37, h, 5 l QIAquick PCR Purification Kit, PET-GR, 10 l (T 4 1 l 10 T 4 buffer 1 l pet-21a(+) 3 l GR 5 l),, 16 ( 8 12 h) DH5 PCR,, : 10 H Buffer 1 l Nde I 1 l Xho I 1 l PET-GR 3 l, DW2 10 l 37 3h, 1.0% PET-GR BL21, LB/Amp,, Amp LB, 37, 1% 3ml Amp LB, 37 (OD 600nm = ) IPTG 1mmol/L, r/min 3h 1ml 5000r/min 3min,, 500 l PBS, : 10s, 10s, 15min 13000r/min 10min, 15 l, 15 l PBS, 15 l 2 SDS, 5min, 10 l SDS-PAGE pet-gr BL2(DE3) BL21 (DE3) 1.5.2, IPTG 1mmol/L, , 100r/min ;, 28, 1mmol/L IPTG, 2h 3h 4h 5h 6h ; IPTG, mmol/L SDS-PAGE ICE-L GR ORF ICE-LGR ORF PCR 1, 1411bp, ICE-LGR, 2.2 pmd18-t/gr pet-21a (+)

3 6 : (Chlamydomonas sp. ICE-L) 819,, TA pet21a Nde I/Xho I, ( 2A B), pmd18-t GR 2.3 PCR 1 ICE-L GR ORF PCR PCR Fig.1 PCR amplification of ICE-L GR gene s ORF, 1. TAKARA DL2000; 2. GR PCR 3 pet-gr PCR,,, pet-gr, Nde I/Xho I 2 (Nde I Xho I) Fig.2 The results of double enzymatic digestion of plasmid with Nde I and Xho I A1. TAKARA DL10000; A2. pet-21 ; B1. TAKARA DL10000; B2 B4. pmd18-t/gr, ( 4) 2.4 pet-gr BL21 (DE3), IPTG, 52.2kDa ( 5),, 4 pet21-gr, Fig.4 Enzymatic digestion analysis of recombinant plasmid pet21-gr 2.5 GR 1. ; 2. 1kb DNA ladder ( GR kb) SDS-PAGE 6,,, 28, 28, SDS-PAGE ( 6 ), 2h, 3h, 4h, 3h, 3h IPTG,, IPTG,, 1.0mmol/L IPTG ( 6 ), 1.0mmol/L IPTG IPTG, GR : 1.0mmol/L IPTG, 28 3h 3 pet21-gr PCR Fig.3 Identification of recombinant plasmid pet21-gr by colony PCR 1. TAKARA DL2000; 2 9. PCR 5 GR SDS-PAGE Fig.5 SDS-PAGE analysis of GR expressed in E. coli 1 2. pet21a ; 3 4. pet-gr ; M.

4 GR SDS-PAGE Fig.6 SDS-PAGE analysis of induced expression of GR in recombinant bacteria at different conditions : (1. 16 ; ; ; ; ; ; M. ); : (1. 2h; 2. 3h; 3. 4h; 4. 5h; 5. 6h); : IPTG (1. 0.1mmol/L; mmol/L; mmol/L; mmol/L) (6.5kDa, 14.3kDa, 20.1kDa, 29.0kDa, 44.3kDa, 66.4kDa, 97.2kDa, 116kDa, 200kDa, 4 ) 3,,, BL21,,, pgex 4-T-1, (, 2006) 1) pet21a, Chlamydomonas sp. ICE-LGR, BL21(DE3) ( 5) pet21a, BL21 PCR, ( 3 4) pet-gr BL21 (DE3), IPTG 52.2 kda ( 5),, ICE-L GR 54.6kDa (Ding et al, 2007), GR 55kDa (Gutterer et al, 1999), 52kDa (Pigiet et al, 1977) ICE-LGR cdna, IPTG IPTG,, IPTG (Patnaik, 2001) IPTG,, (Sakamoto et al, 1996), IPTG 1.0mmol/L GR ( 6),, GR IPTG, GR,,, 3 6h, 6h,, GR 4h, 3h, 4 ICE-L GR, 1), (SGIV). :, 32 50

5 6 : (Chlamydomonas sp. ICE-L) 821 Chlamydomonas sp. ICE-L GR, GR, 燏,,, ICE-L(Chlamydomonas sp. ICE-L)., 37(2): ,,, Pseudoalteromonas sp. S UGD., 41(6): Ding Y, Miao J, Wang Q et al, Effect of Cd on GSH and GSH-related enzymes of Chlamydomonas sp. ICE-L existing in Antarctic ice. J Environ Sci, 17(4): Ding Y, Miao J, Wang Q et al, Purification and characterization of a psychrophilic glutathione reductase from Antarc- tic ice microalgae Chlamydomonas sp. Strain ICE-L. Polar Biol, 31: Gutterer J M, Ralf D, Johannes H, Purification of glutathione reductase from bovine brain, generation of an antiserum, and immuneocytochemical localization of the enzyme in neural cells. J Neurochem, 73(4): Patnaik P R, Investigation of induction effect on the steady state performance of a continuous for recombinant - galactosidase. Process Biochemistry, 36(11): Pigiet V P, Conley R R, Purification of thioredoxin, thioredoxin reductase, and glutathione reductase by affinity chromatography. J Bio Chem, 252(18): Provasoli L, Media and prospects for the cultivation of marine algae. In: Watanabe A, Hattori A ed. Cultures and Collections. Proc US-Japan Conf, Hakone, Japan, Sakamoto S, Terada L, Lee Y C et al, Efficient production of thermus protease aqualysin 1 in Escherichia coli: effects of cloned gene structure and two-stage culture. Appl Microbiol Biotechnol, 45(1 2): PROKARYOTIC EXPRESSION AND ITS CONDITIONAL OPTIMIZATION OF GLUTATHIONE REDUCTASE GENE OF ANTARCTIC CHLAMYDOMONAS SP. ICE-L LIU Ying 1, DING Yu 1, JIAN Ji-Chang 1, WU Zao-He 1, MIAO Jin-Lai 2 (1. Guangdong Provincial Key Lab of Pathogenic Biology and Epidemiology for Aquatic Economic Animals; Fisheries College, Guangdong Ocean University, Zhanjiang, ; 2. Key Lab of Marine Bioactive Substances, First Institute of Oceanography, State Oceanic Administration, Qingdao, ) Abstract Glutathione reductase (GR) is an important antioxidative enzyme in organisms. In order to construct prokaryotic expression vector and to study the recombinant gene expression in Escherichia coli, complete gene of Chlamydomonas sp. ICE-L GR ORF was cloned by the RT-PCR method followed by restriction enzymes cutting, connection. The induced time, IPTG concentration and temperature of prokaryotic expression were optimized using SDS-PAGE. The results showed that a recombinant of prokaryotic expression vector pet-gr was constructed successfully, and ICE GR protein in inclusion body can express effectively after the expression vector pet-gr was transformed to E. coli BL21. The SDS-PAGE results indicated that the molecular weight (52.2kDa) of the target protein was similar to the theoretic molecular weight. The optimal expression condition was set as 1.0mmol/L IPTG, induced temperature 28, and induced time 3 hours. These results are expected to lay a foundation for further studies on the properties and function of this gene. Key words Antarctic Chlamydomonas sp. ICE-L, Glutathione reductase (GR), Prokaryotic expression, Induced condition, Ice algae

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr PCR 龙平, 崔占虎, 李虔全, 徐建平, 张春红, 周立社 * *, 李旻辉 ( 包头医学院, 内蒙古包头 014060) DNA 30 28 DNA trnl-trnf PCR PCR 136 bp 323 bp 10% PCR PCR Astragalus membranaceus Fisch. Bge. var. mongho- licus Bge. Hsiao A. membranaceus