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

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1 2008, 35 (2) : Acta Horticulturae Sinica 1, 1, 1, 1, 2, 13 ( 1,, ; ) 2, :, 5, NAD ( cymdh) cymdh cdna, M al2cymdh ( GenBank DQ221207), 996 bp (ORF) Mal2cyMDH cymdh, cymdh, cymdh MDH 37 kd, cymdh : ; ; ; ; ; : S : A : X (2008) 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 , China; 2 L iaoning Research Institute of Pom ology, X iongyue, L iaoning , 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),, : ; : Author for correspondence ( E2mail: zhaih@ sdau1edu1cn; Tel: )

2 182 35, NAD NADH kd 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, d,,, RNA 112 4,,, : (50 m 57 cm) ; 10 kv; 3 s; ( ) 200 nm; 20 ; 100 mmol L - 1 K 2 HPO mmol L - 1 CTAB; ph 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) 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, BL mmol L - 1 IPTG, SDS2PAGE ml, 30 ml ( 50 mmol L - 1 Tris2 HCl, ph 815, 500 mmol L - 1 NaCl, 10%, 1% Triton X2100, 20 mmol L mmol L - 1 PMSF) 80 L 10 g ml - 1, m in

3 2 : 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 cymd H 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, g 20 m in, g 1 h (Bechmann J30 i ultracentrifuge), 12 h,, M a l2cym D H - 80 PCR, 705 bp, 3 5 RACE cdna, bp, ATG TAG, 8 bp bp 3, 332, (p I) 712, 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)

4 184 35

5 2 : 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) 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 ( 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.

6 pet30a2cymd H BL21 ( A) ( B) N i2nta ( C) SD S2PAGE A: h; 5, h; 7 pet30a ( + ) B: h; h C: 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 2 : 187 cymdh 3, 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,

8 References Basil Hanss, EdgarLeal2Pinto, Avelino Teixeira, Robert Christian, Jeffery Shabanowitz, Donald Hunt, Paul Klotman Cytosolic malate de2 hydrogenase confers selectivity of the nucleic acid2conducting channel. PNAS, 99: Boubals D, Bourzeix M, Guitraud J Le Gora Chirine vari t de vigne iranienne faible teneur en acides organiques. 21: Ann Am lior Plantes, B radford M A rapid and sensitive method for the quantitation of m icrogram quantities of protein utilizing the princip le of p rotein2dye binding. Anal B iochem, 72: Callahan A M, Morgens P H, Cohen R A development. J Am Soc Hortic Sci, 118: Isolation and initial characterization of cdna s for messenger RNA s regulated during peach fruit Cameron J W, Soost R K Acidity and total soluble solids in Citrus hybrids and advanced crosses involving acidless orange and acidless pum2 melo. J Am Soc Hort Sci, 120: Chollet R, V idal J, OπLeary M H Phosphoenolpyruvate carboxylase: A ubiquitous, highly regulated enzyme in plants. Annu Rev Plant Physiol PlantMol B iol, 47: Christelle Etiennea, AnnickMoinga, Elisabeth D irlewangera, Philippe Raymond, ReneMonet and Christophe Rothan terization of six peach cdna s encoding key proteins in organic acid metabolism and solute accumulation: acidity. Physiol Plant, 114: Isolation and charac2 Involvement in regulating peach fruit GrayM W, Burger G, Lang B F The origin and early evolution of mitochondria. Genome B iol, 2 (6) : Hanning I, Heldt H W On the function of m itochondrial metabolism during hotosynthesis in sp inach (Spinacia oleracea L. ) leaves. Plant Physiol, 103: Kromer S, Heldt H W Resp iration of pea leaf mitochondria and redox transfer between the m itochondrial and extram itochondrial compart2 ment. B iochim B iophys Acta, 1057: Maliepaard C, A lston F H, A rkel G V, B rown L M, Chevreau E, Dunemann F, Evans K M, Gardiner S, Guilford P, Heusden A W, Janse J, Laurens F, Lynn J R, ManganarisA G, Periam N, R ikkerink E, Roche P, Ryder C, Sansavini S, Schm idt H, Tartarini S, King G J A ligning male and female linkage maps of app le (M alus pum ila M ill. ) using multi2allelic markers. Theor App l Genet, 97: McA lister2henn L Evolutionary relationships among the malate dehydrogenases. Trends B iochem Sci, 13: Natalie Gibson, Lee McA lister2henn The Journal of B iological Chem istry, 278: Physical and genetic Interactions of cytosolic malate dehydrogenase with other gluconeogenic enzymes. StevensM A Citrate and malate concentration in tomato fruits: Genetic control and maturational effects. J Am Soc Hortic Sci, 97: V isser T, Verhaegh J J Inheritance and selection of some fruit characters of app le. I. Inheritance of low and high acidity. Euphytica, 27: Yao Yu2xin, Zhai Heng, Zhao L ing2ling, Yi Kai, L iu Zhi, Song Ye. culturae Sinica, 33 (2) : ( in Chinese) Analysis of apple fruit acid / low2acid trait by SSR marker. Acta Horti2,,,,, / SSR., 33 (2) : Yao Yu2xin, Zhao L ing2ling, Hao Yu2jin, Zhai Heng Effective extraction of total RNA in apple flesh with imp roved hot borate method. Journal of Fruit Science, 22 (6) : ( in Chinese),,, RNA., 22 (6) : Yoshida M Genetical studies on the fruit quality of peach varieties. Bull Hortic Res Stn Jpn Series A, 9: Yu D ing, Ma Q ing2hu in wheat. B iochim ie, 86: Characterization of a cytosolic malate dehydrogenase cdna which encodes an isozyme toward oxaloacetate reduction

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