Indian Journal of Biotechnology Vol 7, January 2008, pp 66-72

Cloning and characterization of the gene encoding HMG CoA reductase from black night shade (Solanum nigrum L.)

Smitha Jose, D Girija* and P S Beena Centre for Plant Biotechnology and Molecular Biology, College of Horticulture

Kerala Agricultural University, Thrissur 680 656, India

Received 5 July 2006; revised 3 May 2007; accepted 7 June 2007

The first committed step in the pathway for biosynthesis of isoprenoids in plants is catalyzed by 3-hydroxy 3- methylglutaryl CoA reductase (HMGR; EC:1.1.1.34). Here we report for the first time, the cloning of a partial genomic DNA sequence encoding HMGR from a medicinal herb, Solanum nigrum L. This plant is associated with resistance against potato cyst nematode and the late blight pathogen, Phytophthora infestans. The clone Snhmgr (GenBank Acc.No. DQ 229901) had 582 base pairs (bp) with a 462 bp open reading frame (ORF), encoding 142 amino acid polypeptide. The in-silico analysis of Snhmgr sequence revealed about 85% identity with hmgr genes in other solanaceous plants. Phylogenetic analysis indicated that Snhmgr was more identical with tomato hmgr on evolutionary basis and belonged to the solanaceous cluster, including hmgr genes from tomato, potato, tobacco and capsicum. Functional analysis of conserved domains of Snhmgr showed CoA reductase, NAD binding and substrate binding activities.

Keywords: HMGR, in-silico analysis, mevalonate pathway, polymerase chain reaction

Introduction Several plant species belonging to the family

Solanaceae possess a rich genetic base for resistance against pests and diseases. Most of them have various medicinal uses also. Solanum nigrum L., commonly known as ‘black nightshade’, well known for its nematicidal properties1. It is reported to be associated with resistance to Phytophthora infestans2. In breeding for resistance to late blight, an economically important disease affecting potatoes, it can be used as a source of durable resistance3.

Many of the compounds involved in imparting such defence related properties to the plants are synthesized by isoprenoid pathway4. Growth regulators, phytoalexins, carotenoids and terpenoids are some of the end-roducts in this pathway. They carry out various cellular functions, like photosynthesis, chemical signaling, growth and development, and defence compound production5,6. The major rate-limiting enzyme in this pathway is 3-hydroxy 3-methyl glutaryl CoA reductase (HMGR) that is encoded by hmgr gene. The knowledge about the gene can reveal various functional aspects of different isoprenoid compounds expressed in plants.

Molecular characterization of hmgr has been done in Catharanthus roseus7, Camptotheca acuminata8, cotton9, mulberry10, melon11, pepper12 and sweet potato13. Since the enzyme HMGR catalyses synthesis of defence proteins and the plant S. nigrum is reported to possess resistance against pathogens and namatodes, we made an attempt to isolate and sequence the gene encoding HMGR from this plant.

Materials and Methods DNA Isolation

Genomic DNA was isolated from tender leaf tissue of S. nigrum following modified Doyle and Doyle14 method. The DNA was resolved in 1% agarose gel using 1× Tris acetate EDTA (TAE) buffer at 5 v/cm for 1 h. Approximate DNA yield was determined using a spectrophotometer (Spectronic Genesys 5) by measuring the absorbance at 260 nm. Primer Designing

Nucleic acid sequences coding for HMG CoA reductase were retrieved from the GenBank (http://www.ncbi.nlm.nih.gov/) and aligned by ClustalW 1.8 multiple sequence alignment programme (www.ebi.ac.uk/clustal). The bases at the most conserved regions of the alignment were considered for the primer designing and a pair of degenerate primers were designed for the specific amplification of hmgr gene. hmf2 (5′ GGGAT(C/T)GGGTTTGTTCAG 3′)

________________________ *Author for correspondence: Tel: 91 487 237 0822; Fax: 91-487-237 0019 E-mail: devakigirija@gmail.com

JOSE et al: GENE ENCODING HMG CoA REDUCTASE FROM S. NIGRUM L.

was used as the forward primer and hmr2 (5' AGATATGCCGATGAC(A/G)TCCATGTC 3') was used as the reverse primer.

PCR AmpW~cation of hmgr PCR amplification reaction was set-up with 1 pL

(25 ng) of genomic DNA, 2.5 pL of 10 x Taq assay buffer, 1 pL of (10 pM) hmf2, 1 pL (10 ph4) of hmr2,

i 1 pL of 10 mM cNlT mix and 2 pL of 0.3 U Taq DNA polymerase in a total reaction volume of 25 pL. The cycling conditions were set to an initial denaturation of 94°C for 2 min, 30 cycles of denaturation at 94OC for 1 rnin, annealing at 58OC for 1 rnin and extension at 72°C for 2 min, followed by final extension of 72°C for 5 rnin. 10 pL of PCR

F- product was checked in a 0.8% agarose gel. The - - relevant PCR product was eluted from the gel using perfectprepR Gel Cleanup Kit (Eppendorf AG, Germany) and ligated to pGEM-T Easy vector - (Promega Corporation, USA).

Cloning and Sequencing The ligation reaction was set-up with 100 ng of

eluted PCR products, 1 pL (50 ng) of pGEM-T Easy vector, 1 pL of T4 DNA ligase and 5 pL of 2 x rapid ligation buffer in a 10 pL total reaction volume at room temperature for 1 h and then kept at 4OC overnight. The ligation product was transformed into competent cells of Escherichia coli DH5a prepared by CaC12 treatment and plated on LB/ampicillin (50 ppm) plates layered with IPTG (6 pL) and X-gal (60 pL) (Stock: Ampicillin-5 mg/mL in water; IPTG-200 mg/rnL in water; X-gal-20 mg/mL in DMSO) and incubated overnight at 37OC. Recombinants were selected through blue-white screening on Luria agar. Presence of the insert in single white colonies was confirmed by PCR with the same primer combination, hm.-hmr2. A secondary confirmation was also done by restriction analysis of the recombinant plasmid using EcoRI digestion. The insert was sequenced at the DNA Sequencing Facility, Department of Biochemistry, Delhi university South Campus, New Delhi, using T7 primer.

In-silico Analysis of Sequence The nucleic acid and deduced protein sequences

were analyzed by various online algorithms for structural prediction, presence of exons, motifs and domains, analysis of phylogenetic Elation with other published hmgr genes, etc. The cloned and sequenced

PCR products were analyzed by online BLAST (http://www.ncbi.nlrn.nih.gov/blast/). Nucleic acid and protein sequences of other hmgr genes were obtained from NCBI (htep://www.ncbi.nlm.nih.gov/). Alignments of sequences were carried out using 'ClustalW 1.83' (www.ebi.ac.uk/clustal). Phylogenetic tree was constructed using the 'Phylogram' tool. The other nucleotide sequence analysis tools used were 'Genscan' (www.genes.rnit.edu/genscan/) and nucleic acid tools of 'Biology workbench'(http://seqtool.sdsc.edu/). Protein sequence was analyzed using 'MOTIF' (http://motif.genome.jpl), InterProscan (www.ebi.- ac.uk/InterProScan/), Kyte and Doolittle hydropathy plot analysis (http://occawlonline.pearsoned.com), domain structure prediction (www.biochem.uc1.ac.uk- /bsm/cath/) and amino acid tools of 'Biology Workbench'(http://seqtool.sdsc.edu/).

Results and Discussion Genomic DNA isolated from leaves of S. nigrum

yielded a single intact band with no RNA contamination, as revealed by electrophoresis on 1% agarose gel (Fig. 1). Approximate DNA yield was 8 pg g-' tissue as revealed by spectrophotometry. PCR reaction with primer combination hmf2-hmr2 at an annealing temperature of 58°C yielded two bands of size 1535 and 945 bp, respectively (Fig. 2). The 945 bp band was eluted and used for bacterial transformation. This amplicon is designated as 'Snhmgr'. Plasmids from true recombinants yielded the same 945 bp amplicon in PCR with the primer combination hmf2-hmr2, indicating the presence of insert (Fig. 3). Restriction analysis of the recombinant plasrnid yielded two separate bands corresponding to the vector and the released insert (Fig. 4). The insert length was 582 bp as reveled by sequencing using T7 universal primer (Fig. 5).

The sequence Snhmgr (GenBank Acc.No.: DQ 229901) had four open reading frames. Homology search using translated BLAST (blastx) revealed that the longest ORF of 426 bp (142 amino acid residues) showed more than 80% identity with HMGR in Lycopersicon esculentum, Nicotiana tabucum and S. tuberosum (Table 1). The high level of identity of the sequences with other plant hmgr genes might be due to the conserved catalytic domains in the sequence. All HMGRs show high-level homology in the C- terminal catalytic region and the membrane spanning region15. But at the N terminal, they have sequences with less conserved regions, except the hydrophobic domains16. Genetic relatedness among 15 plant genera

INDIAN J BIOTECHNOL, JANUARY 2008

I 1 1 4 M

Fig. l 4 e n o m i c DNA isolated from S. nigrum: Lanes 1 to 4, Genomic DNA; Lane M, Molecular weight marker h Fig. LCRestriction analysis of cloned insert: Lane M, Molecular DNNHindIIYEcoRI. weight marker h DNNHind IIYEcoR I; Lane 1, Non-recombinant

plasmid; Lane 2, Recombinant plasmid. The insert released after restriction is seen as the lower band

to tobacco and capsicum. Snhmgr was analysed for the presence of

recognition sequences of ten different restriction enzymes. The analysis revealed that many of the commonly used restriction enzymes, BamHI, DpnI, HpaI and MboI did not have recognition sequences within. The enzyme AluI had five sites; HinfI had

L three sites; BtgI, HaeII, HindIII and PstI had one site each, within the cloned sequence (Table 2). The exon

Fig. 2-Amplification of hmgr using hmfl-hmr2 primer analysis of the sequence using 'Genscan' tool combination: Lane 1, hmgr-gene amplified by PCR; Lane M, revealed the presence of an initial exon only. No Molecular weight marker h QNNHindmlEcoRI. terminal exon or poly-A tail could be detected. The

length of exon was found to be 400 bp (Fig. 7).

Amino acid analysis revealed composition of different amino acids in HMGR (Table 3). The molar per cent of glycine was found to be high (8.89%). The conserved glycine residues are important in the maintenance of the correct structure of U-domains17. HMGR requires a high concentration of thiol- reducing agents for its activity and needs some

1835 1 conserved cysteine residues. Snhmgr had 3.7%

835 I cystein residues that reflect their importance, not only for the appropriate conformation of the catalytic site of the enzyme, but also for its active role in the

Fig. 3-Reamplification of hmgr gene from the recombinant plasmids: Reamp13ed hmgp insm recombinant catalytic process'8. No histidine residues were plasmid; Lane 2, hmgr amplification from the plant DNA; Lane reported in the sequence. There are only two M, Molecular weight marker h DNAIHind IWEcoR I. conserved histidine residues in other plant HMGR

proteins, in which the residue present in the b2 sharing homology with S. nigrum hmgr gene as domain get protonated during the conversion of HMG revealed by BLAST was determined by constructing CoA to ~nevalonate'~. Since the cloned hmgr sequence phylogram (Fig. 6). Plants belonging to family was not corresponding to the b2 domain, no histidine Solanaceae, including S. nigrum formed a single residues were detected in the deduced amino acid major cluster with two sub-clusters, revealing more sequence. Glycosylation sites of the protein are genetic similarity with potato and tomato as compared usually associated with asparagines residues7 and it

JOSE et al: GENE ENCODING HMG CoA REDUCTASE FROM S. NIGRUM L.

69

accounts for 0.74% of the Snhmgr sequence.

Motif scan of deduced amino acid sequence revealed presence of HMGR family profile extending from 29th upto 135th residue. The secondary structure

prediction of the sequences showed the proportion of different structures, viz. α-helix, β-sheet and random coil (Fig. 8). Penetrating through most parts of the snhmgr, random coils formed the most abundant

Fig. 5—The cloned sequence of Snhmgr: The translation initiation codon atg is given in bold. The amino acid sequence encoding largest ORF is underlined. The cloned sequence data can be obtained from Genbank under the accession number DQ 229901.

Table 1—Deduced amino acid sequence of Snhmgr ORF and identity with other plant hmgr genes

Details of sequences sharing homology ORF & Length (bp)

Aminoacid sequence Acc. No. Plant species % identity

+ 1 426 M V P Q R P A K V A L S Q A E K P A P I I I P A L S E D D E E I I Q S V V Q G K T P S Y S L E S K L G D C L R A A S I R K E A L Q R I T G K S L E G L P L E G F D Y E S I L G Q C C E M P V G Y V Q I P V G I A G P L L L D G R E Y S V P M A T T E G C L V A S T N R G C K A

L01400 L40938 U60452 AF110383 AF542543 X63649 AF038046 AY623812 AY706757 AF303583 U72146

Solanum tuberosum Lycopersicon esculentum Nicotiana tabacum Capsicum annuum N. attenuata N. sylvestris Gossypium hirsutum Catharanthus roseus Hevea braziliensis Pisum sativum Camptotheca acuminata

93 92 88 84 85 85 82 80 80 82 81

Table 2—Restriction analysis of Snhmgr sequence Restriction enzyme Recognition sequence No. of cut(s) Position of restriction sites Fragment sizes (bp)

Alu I AG'CT 5 61, 104, 185, 304, 323 19, 43, 61, 81, 119, 259 Bam HI G'GATC_C 0 - - Btg I C'CryG_G 1 85 85, 497 Dpn I GA'TC 0 - - Hae II r_GCGC'y 1 582 0, 582 Hind III A’AGCT_T 1 302 280, 302 Hinf I G'AnT_C 3 26, 296, 404 26, 108, 178, 270 Hpa I GTT’AAC 0 - - Mbo I ‘GATC_ 0 - - Pst I C_TGCA'G 1 103 103, 479 n-any nucleotide; r-purine; y-pyrimidine

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Fig. bphylogenetic tree showing diversity among various plant hmgr genes. Snhmgr is indicated with an arrow mark.

Table 3-Amino acid composition of Snhmgr protein sequence

Uncharged

Basic

Amino acid Non-wlar

GIY Ala Val Leu Ile

Met Pro Phe Trp

polar

Ser Thr C Y ~ T Y ~ Asn Gln L Y ~

Mol %

c. - ...--.-.'-.--..-.--. Y Arg 4.44 U w4 SS w M His 0.00

7-Exon sequences present in sn%mgr gene. The length of Acidic ASP 3.70 the exon region is indicated in the scale. Glu 8.89

Fig. 8-Predicted secoridary structure of the amino acid sequence encoded by the longest open reading frame in Snhrngr.

structural elements (59.7%), while a-helices (20.89%) and P-sheets (19.4%) were intermittently distributed in the protein. Important functional domains were located using Interproscan. The structures of major domains were compared with CATH structural database and are depicted in Fig. 9. These constitute oxidoreductase domain and domain 2 of HMGR chain A. These are similar'to conserved regions in class I and I1 HMGR enzymes. Class I is predominant in eukaryotes and contains N-terminal membrane region. Class 11, found in prokaryotes, are soluble due to lack of membrane region2'. Yeast and human HMGR are divergent in their N-terminal region, but conserved in their active site. In contrast, human and bacterial HMGR differ in their active site architecture. While

the prokaryotic enzyme is a homodimer, the eukaryotic enzyme a homotetrame?'. Another domain of HMGR associated with lipid metabolism was also detected in the sequence. The analysis revealed that all the conserved domains associated with HMGR were conserved in the cloned sequence also.

No putative transmembrane regions could be detected in the amino acid sequence by the Kyte Doolittle hydropathy plot (Fig. 10). This could be due to non-representation of the N-terminal portion of the protein in the cloned sequence. In plant HMGRs, two transmembrane regions are generally present in the amino terminal, anchoring the enzyme to endoplasmic reticulum2'.

JOSE et al: GENE ENCODING HMG CoA REDUCTASE FXOM S. NIGRUM L.

Fig. 9-a. Oxido reductase domain, sequence family 3.30.70.420.1; and b. I-IMGR chain A, domain 2, sequence family 3.90.770.10.1 protein structure of various domains of Snhmgr. The alpha helix is shown in magenta (helix shaped), U-sheets in yellow (wide ribbon shaped) and the random coils in gray (line shaped).]

Fig. 10-Hydtropathy plots of the cloned Snhmgr sequence. The average hydrophobicity of each amino acid residue was calculated using the algorithm of Kyte and Doolittle over a window of 19 amino acids and was plotted as a function of window position.

The cloned and sequenced fragments can be used to design primers and probes that can aid in the isolation of complete gene by rapid amplification of cDNA ends (RACE) or by screening the genomic or cDNA library. The different patterns of expression of hmgr gene can be studied in different tissues under various environmental conditions. The hmgr genes are well known for their tissue specific- expressions and regulation by environmental factors such as light23924. Hence, mRNA expression study will be useful in

understanding the regulational aspects. Further investigations are required for the future applications of the gene in resistance breeding. This will be useful in pyramiding of different resistant genes in improving the effectiveness of protection and durability of resistance.

References 1 Scholte K, Screening of non-tuber bearing Solanaceae for

resistance to and induction of juvenile hatch of potato cyst nematodes and their potential for trap cropping, Ann Appl Biol, 136 (2000) 239-246.'

2 Polkowska-Kowalczyk L, Wielgat B & Maciejewska U, The elicitor-induced oxidative processes in leaves of Solanurn species with differential polygenic resistance to Phytophthora infestans, J Plant Physiol, 161 (2004) 913-920.

3 Zimnoch-Guzowska E, Lebecka R, Kryszczuk A, Maciejewska U, Szczerbakowa A et al, Resistance to Phytophthora infestans in somatic hybrids of Solanurn nigrum L. and diploid potato, Theor Appl Genet, 107 (2003) 43-48.

4 Bach T J, Some new aspects of isoprenoid biosynthesis in plants: A review, Lipids, 30 (1995) 191-202.

5 Arigoni D, Sagner S, Latzel C, Eisenreich W, Bacher A et al, Terpenoid biosynthesis from 1-deoxy D-xylulose in higher plants by intramolecular skeletal rearrangement, Proc Natl Acad Sci USA, 94 (1 997) 10600- 10605.

6 Newman J D & Chappell J, Isoprenoid biosynthesis in plants: Carbon partitioning within the cytoplasmic pathway, Crit Rev Biochem Mol Biol, 34 (1999) 95-106.

7 Maldonado-Mendoza I E, Burnett R J & Nessler C L,

INDIAN J BIOTECHNOL, JANUARY 2008

Nucleotide sequence of a cDNA encoding 3-hydroxy 3- methylglutaryl Co A reductase from Catharanthus roseus, Plant Physiol, 100 (1992) 1613-1614.

8 Maldonado-Mendoza I E, Vincent R M & Nessler C L, Molecular characterization of three differentially expressed members of the Camptotheca acuminata 3-hydroxy-3- methylglutaryl CoA reductase (HMGR) gene family, Plant Mol Biol, 34 (1997) 781-790.

9 Scott H C, Loguercio L L, Trolinder N L & Wilkins T A, HMG CoA reductase gene family in cotton: Unique structural features and differential expression of hmg2 potentially assoc~ated with synthesis of specific isoprenoids in developing embryos, Plant Cell Physiol, 40 (1999) 750-761.

10 Jain A K, Vincent R M & Nessler C L, Molecular characterization of a 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) gene from mulberry (Morus alba L.), Plant Mol Biol, 42 (2000)554-@.

11 Kato-Emori S, Higashi @.&?pya K, Kobayashi T & Ezura H, Cloning and characteri@tion 8f thi gene encoding 3-hydroxy- 3-methyl glutaryl-coenzyme A reductase in melon (Cucumis melo L. reticulatus), Mol Genet Genomics, 265 (2001)135- 142.

12 Ha S H, Kim J B, Hwang Y S & Lee S W, Molecular characterization of three 3-hydroxy 3-methylglutaryl coenzyme A reductase genes imluding pathogen induced hmg2 from pepper (Capsrcum annuum), Biochim Biophys Acta, 1625(2003) 253-260.

13 Kondo K, Uritani I & Oba K, Induction mechanism of 3- hydroxy 3-methyl glutaryl-CoA reductase in potato tuber and sweet potato root tissues, Biosci Biotechnol Biochem, 67 (2003) 1007-1017.

14 Doyle J J & Doyle J L, A rapid DNA isolation procedure for small quantities of fresh leaf tissues, Phytochrn Bull, 19 (1987) 11-15.

15 Denbow C J, Lang S & Cramer C, The N-terminal domain of

tomato 3-hydroxy-3-methylglutaryl CoA reductases, J Biol Chem, 271 (1996) 9710-9715.

16 Chye M L, Khush A, Tan C T & Chua N H, Characterization of cDNA and genomic clones encoding 3-hydroxy-3- methylglutaryl coenzyme A reductase from Hevea brasiliensis, Plant Mol Biol, 16 (1991) 567-577.

17 Caelles C, Ferrer A, Balcells L, Hegardt F G & Boronat A, Isolation and structural characterization of a cDNA encoding 3-hydroxy-methyl glutaryl coenzyme A reductase, Plant Mol Biol, 13 (1989) 627-638.

18 Roitelman J & Shechter I, Regulation of rat liver 3-hydroxy 3- methyl g lu tq l coenzyme A reductase, J Biol Chem, 259 (1984) 870-877.

19 Liscum L, Finer-moore R J, Stroud R M, Luskey K L & Brown M S et al, Domain structure of 3-hydroxy-3- methylglutaryl-coenzyme A reductase, a glycoprotein of the endoplasmic reticulum, J Biol Chm, 260 (1985) 522-530.

20 Goldstein J L & Brown M S, Regulation of the mevalonate pathway, Nature (Lond), 343 (1990) 425-430.

21 Basson M E, Thorsness M, Finer-Mmre J, Stroud M R & Rine J, Structural and functional conservation between yeast and human 3-hydroxy-3-methylglutql coenzyme A reductases, the rate limiting enzyme of sterol biosynthesis, Mol Cell Biol, 8 (1988) 3797-3808.

22 Chin D J, Gil G, Russell D W, Liscum L & Luskey K L, Nucleotide sequence of 3-hydroxy 3-methyl gIutaryl coenzyme A reductase, a glycoprotein of endoplasrnic reticulum, Nature (Lond), 308 (1984) 61 3-6 17.

23 Ji W, Hatzios K K & Cramer C, Expression of 3-hydroxy-3- methylglutaryl coenzyme A reductase activity in maize tissues, Physiol Plant, 84 (1992) 185-192.

24 Moore K B & Olshi K K Characterization of 3-hydroxy-3- methylglutaryl coenzyme A reductase activity during maize seed development, germination and seedling emergence, Plant Physiol, lOl(1993) 485-49 1.