2008, 35 (2) : 181-188 Acta Horticulturae Sinica 1, 1, 1, 1, 2, 13 ( 1,, 271018; 115214) 2, :, 5, NAD ( cymdh) cymdh cdna, M al2cymdh ( GenBank DQ221207), 996 bp (ORF) Mal2cyMDH cymdh, cymdh, cymdh MDH 37 kd, cymdh : ; ; ; ; ; : S 66111 : A : 05132353X (2008) 0220181208 Gene C lon ing, Expression and Enzym e Activ ity A ssay of a Cytosolic M a la te D ehydrogena se from Apple Fru its YAO Yu2xin 1, HAO Yu2jin 1, L IM ing 1, PANG M ing2li 1, L IU Zhi 2, and ZHA I Heng 13 ( 1 College of Horticultural Science and Engineering, Shandong A gricultural U niversity, S tate Key Laboratory of C rop B iology, Taiπan, Shandong 271018, China; 2 L iaoning Research Institute of Pom ology, X iongyue, L iaoning 115214, China) Abstract: M alate dehydrogenase (MDH) ubiquitously exists in anim als, p lants and m icrooganism s, and catalyzes the interconversion from oxaloacetate to malate. Cytosolic NAD 2dependentMDH gene ( cymdh) en2 codes a key enzyme crucial for malic acid synthesis in the cytosol, which has not been extensively charac2 terized in p lants. In this study, a full2length cdna of cymdh was isolated from app le fruits with RT2PCR as well as 3 and 5 rap id amp lification of cdna ends, and designated asm a l2cymdh ( GenB ank accession No. DQ221207). It contained a 996 bp ORF and sequence analysis showed a high sim ilarity to other p lant cymdh s. Phylogenetic analysis indicated that almost all the cymdh s could be clustered into the sam e group and it was likely to rep resent the original MDH. An about 37 kd fused p rotein was obtained by the recom2 binant p rokaryotic exp ression and its enzyme activity assay showed that it mainly catalyzed oxaloacetate to m a2 late. It was also discovered that the enzyme activity of cymdh exhibited remarkably difference between the high2 and low2acid app le germp lasm. Key words: app le; cytosolic malate dehydrogenase; gene; cloning; p rokaryotic exp ression; enzym e ac2 tivity (MDH),, : 2007-06 - 20; : 2007-11 - 22 3 Author for correspondence ( E2mail: zhaih@ sdau1edu1cn; Tel: 053828241335)
182 35, NAD NADH 32 37 kd 42 43 kd, 5 : NADP NAD NAD NAD NAD ( Yu &Ma, 2004), NAD ( cymdh) cymdh ( Kromer & Heldt, 1991; Hanning & Heldt, 1993; Yu & Ma, 2004), (Basil et al., 2002) (Natalie & Lee, 2003),, ( PEPC) (Chollet et al., 1996) (Christelle et al., 2002), (M alus dom estica Borkh. ), cymdh,, cym2 DH 1 111 ( ) 1, 30 60 90 120 150 d,,, RNA 112 4,,, : (50 m 57 cm) ; 10 kv; 3 s; ( ) 200 nm; 20 ; 100 mmol L - 1 K 2 HPO 4 + 015 mmol L - 1 CTAB; ph 710 113 cd NA (, 2005) RNA 2 g RNA M 2MLV RTase cdna Synthesis Kit ( Takara, China) cdna cymdh, M a l2cymdh, 5 2GTT CGY GTY CTY GTY ACY GG23 ( forward) 5 2CTN ACY GNC CRT NAY GAN TG23 ( reverse), ( Takara, China) 114 3 5 Rap id Amp lification Kit of cdna Ends http: www1ncbi1nlm1nih1gov Clustal W, DNASTAR 115 SD S2PAGE 5 2CG GGA TCC ATG GCG AAA GAA CCA GTT C23 ( forward, ) 5 2AGT CGA AGT GTC CGA ATA GAA T23 ( reverse) M al2cymdh, pmd182t B am H Sal pet230a ( + ) pet30a2cymdh, BL21 015 mmol L - 1 IPTG, SDS2PAGE 116 1 000 ml, 30 ml ( 50 mmol L - 1 Tris2 HCl, ph 815, 500 mmol L - 1 NaCl, 10%, 1% Triton X2100, 20 mmol L - 1 1 mmol L - 1 PMSF) 80 L 10 g ml - 1, 30 15 m in
2 : 183 15 000 g 15 m in, 30 ml N i2nta 30 L SDS2PAGE B radford (B radford, 1976) 50 mmol L - 1 MOPS, ph 719, 015 mmol L - 1 OAA, 012 mmol L - 1 NADH, 3 ml 50 mmol L - 1 MOPS, ph 719, 50 mmol L - 1 L2malate, 110 mmol L - 1 NAD +, 3 ml, 30, 1 m in 340 nm, 6,,, (OD m in - 1 mg - 1 Pr) NADH mol L - 1 m in - 1 mg - 1 Pr, NADH 3 117 cymd H 0 4-80 5 g, 1% ( / ) PVPP,, 5 ml ( 100 mmol L - 1 MOPS, ph 810, 1 mol L - 1 NaCl, 250 mmol L - 1 sucrose, 100 mmol L - 1 bicine, 2 mmol L - 1 EDTA2NaOH, ph 810, 012 mmol L - 1 MgSO 4, 0175 mg ml - 1 BSA 1% PVPP), 1 h 4, 2 800 g 20 m in, 100 000 g 1 h (Bechmann J30 i ultracentrifuge), 12 h,, 116 2 211 M a l2cym D H - 80 PCR, 705 bp, 3 5 RACE cdna, 1 246 bp, ATG TAG, 8 bp 5 239 bp 3, 332, (p I) 712, 35 593 Da Mal2cyMDH cymdh 90%,, cymdh 96% 50%, Mal2cyMDH IW GNH NAD TGAAGQ I, cymdh ( 1) Mal2cyMDH N 9 ( 2), cymdh, 212 ( 3) 1 (mmdh) ( gmdh ) NAD ( cnmdh s) 2, NADP ( chmdh s) NAD ( cnmdh s) ; ( cymdh s) cymdh, 54% 72%,, MDH MDH,, cymdh 61% 51%, cymdh mmdh gmdh 1813% 14%, MDH, MDH ( 3)
184 35
2 : 185 3 cymd H F ig. 3 Phylogenetic tree of cymd H 213 M a l2cym D H pet30a2cymdh, BL21, IPTG,, 37 16,, 16 ( 4, A) 37, 16, ( 4, B) 16 015 mmol L - 1 IPTG 24 h, 37 kd,,, N i2nta, 300 mmol L - 1 ( 4, C), MDH (01105 mol L - 1 m in - 1 mg - 1 Pr), MDH (133124 mol L - 1 m in - 1 mg - 1 Pr) (01679 mol L - 1 m in - 1 mg - 1 Pr)
186 35 4 pet30a2cymd H BL21 ( A) ( B) N i2nta ( C) SD S2PAGE A: 1 4 37 30 22 16 4 h; 5, 6 16 12 24 h; 7 pet30a ( + ) B: 1 2 16 12 24 h; 3 37 12 h C: 1 4 50 100 200 300 mmol L - 1 F ig. 4 SD S2PAGE of pet30a2cymd H expression products ( A), soluble prote in in the superna tan t ( B) and recom b inan t prote in s pur if ied by N i2nta column ( C) A: 1-4 show inducement at 37, 30, 22, 16 for 4 h; 5, 6 show inducement at 16 for 12 and 24 h; 7 was pet30a ( + ). B: 1 and 2 show inducement at 16 for 12 and 24 h; 3 show inducement at 37 for 12 h. C: 1-4 were eluted by 50, 100, 200 and 300 mmol L - 1, respectively. The target fused p rotein was arrowed. 214 cymd H,,, 30 d,,, ( 5), cymdh ;, 60 d, cymdh, ( 6), 5 F ig. 5 Change of ma lic ac id con ten t dur ing fru it developm en t 6 cymd H F ig. 6 Com par ison of cymd H activ ity between the h igh2ac id and low2ac id fru its
2 : 187 cymdh 3, 30 90 d 150 d, ( 7) F ig. 7 cymd H 7 Com parison of cymd H activ ity between the h igh2ac id and low2ac id fru its 3, M al2cymdh, cymdh, cymdh MDH MDH,, MDH, MDH, MDH (McA lister2henn, 1988; Gray et al., 2001), cymdh MDH, MDH, MDH,,, (Callahan et al., 1993),, (V isser & Verhaegh, 1978; Maliepaard et al., 1998;, 2006) ( Yoshida, 1970) (Boubals et al., 1971) (Cameron & Soost, 1977) ( Stevens, 1972),, cymdh cym2 DH, MDH, MDH, H is MDH,, cymdh, cymdh ; 90 d, cymdh,,, cymdh cymdh MDH,, cymdh,
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