Molecular Characterization of the Soybean Alcohol ... · Adh gene system reported in maize has also been observed inrice (30). Soybean(Glycine max)is less tolerantofflooding than

Plant Physiol. (1992) 100, 489-4950032-0889/92/1 00/0489/07/$01 .00/0

Received for publication December 12, 1991Accepted March 16, 1992

Molecular Characterization of the Soybean AlcoholDehydrogenase Gene Family Amplified in Vitro by the

Polymerase Chain Reaction'

Kurt D. Newman and Tara T. VanToai*

Department ofAgronomy, The Ohio State University and Soil Drainage Research Unit, U.S. Department ofAgriculture-Agricultural Research Service, 590 Woody Hayes Drive, Columbus, Ohio 43210

ABSTRACT

Soybean (Glycine max) alcohol dehydrogenase (ADH) cDNAswere amplified in vitro from total RNA by the polymerase chainreaction (PCR). The amplification strategy involved first strandcDNA synthesis from anaerobic cotyledon total RNA using an 18-thymidine primer. The second strand cDNA primer was a conservedsequence near the 5' end of known plant ADH transcripts. ThePCR products were ligated into a plasmid vector and unique cloneswere isolated on the basis of size and restriction pattern. Sequenceanalysis revealed three distinct classes of soybean ADH cDNAs, allof which showed high homology to Adh genes from maize andpeas. RNA blot hybridization analyses showed differential expres-sion patterns for these genes. One gene, expressed constitutivelyin all seedling organs, was inducible by anaerobiosis, one gene wasexpressed only in anaerobic organs, and the third gene was ex-pressed predominantly in anaerobic roots.

ADH2 (EC 1.1.1.1) in many plant species is inducible byanaerobiosis or flooding stress (6, 12, 14, 15, 18, 27). Theplant ADH enzyme is a protein dimer of about 80 kD (8, 30).In maize, the products of two unlinked genes, Adhl andAdh2, dimerize randomly to form three electrophoreticallydistinct isozymes: ADH1-ADH1 homodimer, ADH1-ADH2heterodimer, and ADH2-ADH2 homodimer (9). The two-Adh gene system reported in maize has also been observedin rice (30). Soybean (Glycine max) is less tolerant of floodingthan either maize or rice (22). The number of soybean Adhgenes has not been determined. A single ADH isozyme bandwas reported from soybean roots (1, 15, 22). Gorman andKiang (11) screened 113 soybean genotypes for variants inADH zymograms and reported that seeds of 86% of thegenotypes tested had seven-band zymograms, 10% of thegenotypes had five-band zymograms, and 4% had four-bandzymograms. From the genetic analysis of the mode of inher-itance of the observed variants, the authors proposed a four-or possibly five-gene model for soybean Adh. A four-band

1 Contribution of the Soil Drainage Research Unit, U.S. Depart-ment of Agriculture-ARS and the Ohio Agricultural Research andDevelopment Center, Wooster, OH. Ohio Agricultural Research andDevelopment Center Journal Article No. 158-9 1.

'Abbreviations: ADH, alcohol dehydrogenase enzyme; Adh, al-cohol dehydrogenase gene; PCR, polymerase chain reaction.

zymogram was also reported for ADH in soybean embryonictissues (28).We previously demonstrated that the isozyme pattern of

soybean ADH is organ specific (21). A single ADH isozymewas resolved by native PAGE of protein extracts from aerobicand anaerobic soybean leaves, hypocotyls, and roots. Thecotyledons of aerobic soybeans contained three ADH iso-zymes: two were distinct, one was faintly visible. Followinganaerobiosis, all three cotyledonous isozymes increased inactivity. Therefore, soybean ADH, like maize and rice, ap-pears to be encoded by at least two genes. These genes areexpressed in a tissue-specific manner with one gene expressedin all seedling organs, whereas the other gene appears to beexpressed only in the cotyledons.To determine the structure and organization of soybean

Adh genes, we have used PCR to amplify the Adh gene familyin vitro from total RNA of anaerobic cotyledons. We reporthere the sequences of the unique ADH cDNA clones andtheir patterns of expression. One Adh gene, expressed consti-tutively in all seedling organs, was inducible by anaerobiosis,one gene was expressed only in anaerobic organs, and a thirdgene was expressed predominantly in anaerobic roots.

MATERIALS AND METHODS

Reagents

All chemical reagents, except when stated, were fromSigma3.

RNA Extractions

Total RNA was isolated from aerobic and anaerobic roots,hypocotyls, cotyledons, and leaves of 10-d-old soybean (cvKeller) seedlings (21) by the guanidinium extraction method(2) with the addition of a CsCl (Bethesda Research Labora-tories) gradient purification step (10).

Primers

The primers diagrammed in Figure 1 were synthesized bySynthecell Corp. (Rockville, MD). First-strand cDNA was

' Use of trade names is for the benefit of the readers and doesnot imply endorsement of the product by the U.S. Department ofAgriculture.

489

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NEWMAN AND VANTOAI

synthesized using an 18-thymidine primer (primer 1). Primer2, the sense-strand primer, was a conserved sequence whoselocation in maize ADH1 is 139 to 167 bases downstreamfrom the translation start site (3). The conserved sequencesof plant ADH were found by comparing the published se-quences of maize (3, 4), barley (29), and pea (16) ADH cDNA.There is 100% amino acid homology between the variousplant ADH proteins within the conserved regions. The de-generacies in the primers were based on codon usage ofsoybean and on sequence mismatches in the third positionof the codons that did not alter the amino acid code (13, 19,26). Restriction sites were incorporated on the 5' end of theprimers to facilitate directional cloning of the PCR productsinto plasmid vectors.

Synthesis of First-Strand cDNA

Total RNA from anaerobic cotyledons (9 ,ug) was reversetranscribed in 20 ,L of a buffer containing 50 mm Tris-HCl,pH 8.3, 75 mm KCl, 3 mm MgCl2, 10 mm DTT, 100 ,g ml-BSA (BRL), 1 unit 1AL-1 RNasin (Promega, Madison, WI), 0.5mm each dNTP (BRL), 0.75 uM primer, and 400 units ofreverse transcriptase (MMLV-RT; BRL).

PCR

The composition of the first-strand cDNA reaction wasmodified for PCR by adding 5 AL of 200 mm KCl, 8 mMMgCl2, 10 mg L- gelatin (tissue culture grade), 1.5 AL ofsense-strand primer (10 $M stock), 1 unit of Taq DNA poly-merase (Perkin-Elmer Cetus), and 23.3 AL of H20. The finalcomposition of the PCR buffer was 20 mm Tris-HCl, pH 8.3,50 mM KCl, 2 mm MgCl2, 4 mm DTT, 40 ,ug mL-' BSA, 0.2mm for each dNTP, 0.3 AM for each primer, and 1 Ag mL-'gelatin. The reaction (50 AL total volume) was covered with30 ML of mineral oil and subjected to 30 cycles of PCR in aPerkin-Elmer Cetus thermal cycler. The thermal profile was1 min at 940C; 30 x (1 min at 940C; 2 min at 550C; 3 min at720C); and 5 min at 720C. Mineral oil was placed in the wells

so A a8SC TAG A= ATC Ci - AGCbeMI TACTIC -I= GA

Me I am No139 167

I

FM AMH CII TOO ACG MAT CTT TATC T GAMaimADHI.cC.. ..

C..C ..CbaIroADHZ.cC. ..C..C ....... ... ..Barlow MMeD .c C...... ..c..C ..C

..3GlobgAH3 ..C.C...C.C ..C.

A.

1 2 3 4

s-1.6 kb-1 .8 kb

r-8 .5 kb

BSP

1 2

* w

W.,

-1.6kb-1 0 kb

-0 5 kb

Figure 2. A, PCR product evaluation by restriction with EcoRI. Lane1, Undigested PCR products; lane 2, size marker; lane 3, PCRproducts digested with EcoRI; lane 4, test restriction of lambdaDNA digested with EcoRI. B, DNA blot hybridization with the maizeADH1 cDNA (3) as probe. Lane 1, PCR products digested withEcoRI; lane 2, undigested PCR products.

of the thermal block to ensure good thermal transfer betweenthe block and the tubes.

Electrophoresis

DNA was electrophoresed through a 1% agarose (ultra-pure grade, Bio-Rad), Tris-acetate gel containing ethidiumbromide (17). Total RNA (10 ug), isolated from aerobic andanaerobic organs of 10-d-old seedlings (21), was separatedby formaldehyde (Mallinckrodt) agarose gel electrophoresis(17).

PCR Primer 2

Poly A tallI

ADH cDNAConserved sequence

Figure 1. PCR primer sequences.

M pqmbjqz uLks L"I

490 Plant Physiol. Vol. 100, 1992



SOYBEAN ALCOHOL DEHYDROGENASE GENE FAMILY

12 3 4 5 6 78910 1 2 3 4 5 6 7 8 910A_^ . .' r .oi,

D

E

Figure 3. DNA blot hybridization. A, Hybridi-zation with clone 10 at 60'C. B, Hybridizationwith clone 57 at 60°C. C, Hybridization withclone 73 at 60'C. D, Hybridization with clone10 at 55°C. E, Hybridization with clone 57 at55°C. F, Hybridization with clone 73 at 550C.Lanes 1 and 2, Clone 10; lanes 3 and 4, clone38; lanes 5 and 6, clone 57; lanes 7 and 8, clone71; lanes 9 and 10, clone 73. Clones in lanes 1,3, 5, 7, and 9 were cut with Alul. Clones inlanes 2, 4, 6, 8, and 10 were cut with Hindlil.

F

PCR Product Purification

The PCR products were electrophoresed through a 1%agarose gel as described above. The DNA fragments were

excised from the gel and purified using either GeneClean (Bio101, Inc.) or electroelution (17) followed by Elutip (Schleicher& Schuell) chromatography.

Ligation and Transformation

PCR products (80 ng) were treated with the Klenow frag-ment of DNA polymerase I and dNTP to make blunt ends.Following treatment, the Klenow enzyme was denatured byheating at 700C for 10 min. Restriction buffer (20 mm Tris-HCl, pH 8, 100 mm KCl, 7 mm MgCl2, 100 tig ml-' BSA, and2 mm 2-mercaptoethanol) and 15 units of BamHI restrictionenzyme were added and the reaction was incubated at 370Cfor 1 h. The DNA was ethanol precipitated. Bluescript SK+(Stratagene) was cut with EcoRV and then with BamHI,separated by electrophoresis, isolated from the gel, and pu-rified with GeneClean (Bio 101, Inc.). Vector (30 ng) andPCR products (22.5 ng) were ligated in 12 ttL of 50 mm Tris,pH 7.6, 10 mm MgCl2, 1 mmATP, 100 nguL-' BSA, and 17mM DTT with 20 units of DNA ligase (New England Biolabs)and 2.5 units of RNA ligase (New England Biolabs). Thereaction was incubated at 140C for 3 h before 40 units ofDNA ligase, 5 units of RNA ligase, and additional bufferwere added to expand the volume to 90 gL. Incubation at140C continued overnight. The reaction was extractedtwice with phenol (DNA grade, American Bioanalyti-cal):chloroform:isoamyl alcohol (25:24:1) and twice with

chloroform:isoamyl alcohol (49:1) to remove the BSA. TheDNA was purified on a spin column containing Sephadex G-50 in 5 mM NH4OAc. The eluted DNA was dried undervacuum and resuspended in 10 ,L of Tris-EDTA (17). Trans-formation of XL1-Blue (Stratagene) cells was done using theprotocol supplied by Stratagene.

Isolation of Unique Clones

Over 270 transformed bacterial colonies were screened forcDNA insert size and restriction pattern using seven differentrestriction enzymes (data not shown). Conventional plasmidminipreps and PCR minipreps (20) were used for insertanalysis. Five putatively different clones were obtained basedon the screening criteria.

DNA Hybridization Analysis

Inserts from the five clones were excised from Bluescript,cut with AluI and HindIII, separated by electrophoresis, andtransferred onto nylon membranes (Zeta Probe, Bio-Rad) forcross-hybridization analysis. Undigested clones were radio-labeled by random priming (Stratagene) to a specific activityof at least 2 X 109 cpm ug-1 DNA and used as probes. Thehybridization buffer contained 5 X SSC (20 X SSC is 3 M

NaCl, 0.3 M trisodium citrate), 50% formamide (BRL), fishDNA (100 ,g ml-'), and 1% SDS. The nylon membrane was

prehybridized at the temperature indicated in the figurecaptions for 3 h prior to the addition of denatured probe (2x 106 cpm ml-' of hybridization buffer). Hybridization con-

A

B

C

491



NEWMAN AND VANTOAI

1 50tGCgAAGGGc CAGACtCCaT TgTTTCCtcG cATtTTTGGc CAtGAAGCttaGCcAAGGGa CAGACaCCtT TaTTTCCacG gATaTTTGGt CAcGAAGCagaGCcAAGGGa CAGACaCCtT TaTTTCCaaG gATaTTTGGt CAcGAAGCagaGCtAAGGGt CAGACaCCtT TaTTTCCacG tATaTTTGGt CAcGAAGCag

51 100cgGGgATTGT GGAgAGcGTa GGcGAGGGTG TGACtcATCT gAAaCCaGGtgaGGaATTGT GGAaAGtGTt GGtGAGGGTG TGACggATCT cAAgCCcGGtgaGGaATTGT GGAaAGtGTt GGtGAGGGTG TGACggATCT cAAgCCcGGggaGGaATTGT GGAaAGtGTt GGtGAGGGTG TGACagATCT tAAgCCcGGg

101 150GAcCATGccC TcCCtGTgTT cACaGGaGAg TGtggaGAtT GcgccCATTGGAtCATGtgC TtCCaGTcTT cACtGGgGAg TGcaagGAgT GtgatCATTGGAtCATGtgC TtCCaGTcTT tACcGGgGAg TGcaagGAaT GtcatCATTGGAtCATGttC TgCCgGTaTT tACgGGgGAa TGcaagGAaT GtcaaCATTG

151 200cAAgTCAGAg GAgAGcAACA TGTGTGAgCT aCTcAGGATc AACACcGAtacAAaTCAGAa GAaAGtAACA TGTGTGAcCT cCTaAGGATt AACACcGAcccAAaTCAGAa GAaAGtAACA TGTGTGAtCT tCTaAGGATt AACACtGAcctAAaTCAGAa GAaAGtAACA TGTGTGAtCT tCTtAGGATt AACACaGAcc

201 250GtgGtGTcAT GaTccATGAt ggCcAatCaA GgTTcTCtAa gAAtGGACAAGtgGaGTgAT GcTgaATGAt ggCaAggCaA GgTTtTCaAt cAAtGGACAAGgtGaGTgAT GcTgaATGAc agCaAgaCaA GaTTtTCaAt tAAgGGACAAGatGaGTaAT GcTgaATGAc aaCaAgaCcA GaTTtTCaAt tAAtGGACAA

251 300CCcaTacACC AtTTCtTgGG aACCTCtACa TTCAGtGAaT ACACTGTtGTCCaaTatACC AcTTTgTcGG aACCTCcACt TTCAGtGAgT ACACTGTaGTCCtaTttACC AcTTTgTtGG tACCTCcACt TTCAGtGAgT ACACTGTaGTCCtgTttACC AcTT.gTcGG cACCTCcACg TTCAGcGAgT ACACTGTgGT

301 350cCATGctGGa tGTGTTGCaA AgaTCAAcCC TGcTGCtCCa CTTGACAAAGcCATGttGGt tGTGTTGCcA AgaTCAAcCC TGcTGCcCCa CTTGACAAAGtCATGtcGGt aGTGTTGCcA AagTCAAcCC TGcTGCcCCa CTTGACAAAGtCATGtcGGt aGTGTTGCcA AagTCAAtCC TGaTGCaCCg CTTGACAAAG

351 400TTTGTgTTCT cAGtTGTGGA ATtTgcACaG GTtTTGGtGC TACcgTaAATTTTGTgTTCT gAGcTGTGGA ATcTcaACaG GTcTTGGtGC TACatTgAATTTTGTgTTCT gAGcTGTGGA ATcTcaACaG GTcTTGGgGC TACtaTcAATTTTGTaTTCT gAGcTGTGGA ATaTcaACtG GTcTTGGgGC TACtaTcAAT

401 450GTaGCAAAAC CAAAacctGG tTCcTCtGTt GCcaTaTTTG GAcTtGGAGcGTtGCAAAAC CAAAcaaaGG tTCaTCaGTg GCtgTaTTTG GAtTgGGAGaGTtGCAAAAC CAAAcaaaGG tTCtTCaGTg GCtgTtTTTG GAtTgGGAGaGTtGCAAAAC CAAAcaaaGG cTCtTCaGTg GCtgTcTTTG GAtTgGGAGa

451 500tGTtGccgTT GCgGCTGCtG AaGGtGCaAG agTTtCaGGT GCtTCaAGAAcGTgGagcTT GCtGCTGCcG AgGGaGCtAG acTTgCtGGT GCtTCcAGAAcGTgGggcTT GCtGCTGCcG AgGGgGCtAG gaTTgCtGGT GCaTCcAGAAcGTgGgtcTT GCtGCTGCcG AgGGgGCtAG gaTTgCtGGT GCaTCcAGAA

501 550TcATTGGaGT TGAtTTagtT TCtgcccGAT TTgaagaAGC TAAGAAGTTTTaATTGGgGT TGAcTToaaT TCtaagaGAT TTactgaAGC TAAGAAGTTTTtATTGGgGT TGAcTTgaaT TCtaggaGAT TTactgaAGC TAAGAAGTTTTtATTGGgGT TGAcTTgaaT TCcaggaGAT TTactttAGC TAAGAAGTTT

551 600GGgGTtAatG AgTTtGTgAA cccAAAgGAt cAtGAtAAaC CtGTgCAAcAGGaGTtAccG AgTTtGTgAA tggAAAaGAt tAtGAcAAgC CaGTtCAAgAGGaGTcAccG AaTTtGTgAA tggAAAaGAc tAtGAcAAgC CaGTaCAAgAGGaGTaAccG AaTTcGTaAA tggAAAaGAg tAcGAcAAgC CaGTaCAAgA

601 650GGtaATTGCT GaAATGACca aTGGaGGtGT gGAtCGggcT gtTGAaTGtAGGgaATTGCT GaAATGACtg gTGGgGGaGT tGAcCGaagT gtTGAgTGcAGGggATTGCT GcAATGACgg gTGGgGGaGT tGAcCGtagT .TGAgTGcAGGggATTGCT GcAATGACgg gTGGgGGaGT aGAcCGaagT .TGAgTGcA

651 700CtGGcAGcAT CcAaGCcATG gTCTCaGCAT TCGAATGcGT cCAcGATGGtCtGGaAGtAT CaAtGCtATG aTCTCtGCAT TCGAATGtGT gCAtGATGGaCtGGaAGtAT CaAtGCtATG aTCTCgGCAT TCGAATGtGT gCAtGATGGaCcGGtAGtAT CaAgGCtATG aTCTCgGCAT TCGAATGtGT tCAtGATGGa

701 750TGGGGTcTTG CTGTACTTGT TGGtGTGCCt AgtAAAGATG ATGCaTTCAATGGGGTgTTG CTGTACTTGT TGGtGTGCCa AatAAAGATG ATGCtTTCAATGGGGTgTTG CTGTACTTGT TGGaGTGCCa AacAAAGATG ATGCtTTCAATGGGGTgTTG CTGTACTTGT TGGaGTGCCa AacAAAGATG ATGCcTTCAA

751 800AACTgcTCCt aTtAATtTCc TGAAcGAgAg gACtCTtAAG GGcACcTTtTAACTcaTCCa aTtAATgTCt TGAAcGAaAa gACtCTcAAG GGaACtTTcTAACTcaTCCa gTgAATgTCt TGAAcGAaAa aACtCTaAAG GGtACtTTcTAACTcaTCCa gTgAATgTCt TGAAtGAaAa aACgCTaAAG GGtACtTTcT

Plant Physiol. Vol. 100, 1992

tinued overnight. The most stringent wash was at the hybrid-ization temperature in 2 x SSC and 1% SDS for 30 min.

DNA Sequence Analysis

The clones were sequenced by the dideoxy method withSequenase (U.S. Biochemical) and [35S]dATP according to thedouble-stranded sequencing protocol supplied by the manu-facturer. Sequencing gels were 6% polyacrylamide, 0.3%bisacrylamide, and 7.7 M urea in Tris-borate. Sequences were

analyzed using the programs of Devereux et al. (5).

RNA Hybridization Analysis

Total RNA (10 ,ug) was separated by formaldehyde agarosegel electrophoresis (17) and blotted onto a nylon membrane(Zeta Probe, Bio-Rad). The clones 10, 57, 73, and the soybeanactin cDNA (26) were radiolabeled and used as the probes inthe hybridization analysis by the same procedures as de-scribed in the DNA hybridization analysis above. The hy-bridization temperature was 600C for the ADH clones and500C for the actin clone.

SOYADH1 0SOYADH57

SOYADH73PEAADH1

SOYADH10SOYADH57SOYADH7 3PEAADH1

SOYADH1 0

SOYADH57SOYADH73

PEAADH1

SOYADH1 0

SOYADH57SOYADH7 3

PEAADH1

SOYADH1 0

SOYADH57SOYADH73PEAADH1

SOYADH1 0


SOYADHI 0

SOYADH57SOYADH7 3

PEAADH1

SOYADH1 0


SOYADH57SOYADH73

801 850atGGcAACTa caAaCCaCGc aCcGAtCTTC CatctGTtGT cGAGAaGTActtGGcAACTc tgAaCCaCGt tCtGAcCTTC CatcaGTgGT gGAGAtGTAttcGGaAACTc tgAaCCcCGt tCtGAcCTTC CatcaGTgGT gGAGAtGTAttcGGaAACTc tgAgCCcCGt tCtGAcCTTC CcaaaGTqGT tGAGAtGTAt

851 900ATGAgtggGG AgCTaGAagT GGAcAAattc ATcacTCACa caGttccAttATGAacaaGG AaCTtGAacT GGAgAAt ... ... taTCACc atGaagtAccATGAacaaGG AaCTtGAacT GGAgAAt ... ... taTCACc atGaagtAccATGAagaaGG AaCTgGAgcT GGAgAAt ... .. ataTCACc atGaagtAcc

90 1 950cTCagaggTc AacaaagctT TTGAtTtaAT GcTgAAgGGa cAGtcCaTtatTCgagaaTc Aca agcT TTGAaTacAT GcTtAAaGGg gAGagCcTgctTCgagaaTc Aca .agcT TTGAaTacAT GcTtAAaGGg gAGagCcTgctTCgagaaTt Aca agcT TTGAaTacAT GtTtAAaGGg gAGagCcTcc

951 Translation stop codons 1000GgTGtATcAT CgccATGcaa GAgTqagatg gatAtgaaaa gtccatgactGtTGcATaAT CgccATGact GAaTaaaatt tatAtaaaca actactttgaGtTGcATaAT CgccATGact GAaTaoaatt tatAtaaaca actactttgaGsTGcATaAT CaaaATGgcg GAaTaqagct ctaAaactgt aatgtaatag

1001 1050gtgtTctggg acagactact cttatgtgat ttatgaataa aAagagagaagtatTggaaa taaaataaat taaaatgaaa tatacttgag cAtgtatgtagtatTggaaa taaaataaat taaaatgaaa tatactgatt tAtgtatatatcccTaccaa gggactgtgg tcatacgcta gatatgcatt tAtatatttc

1051 1100gagagaataa aaaatttatt gataaaaTcc ccaattaggc tattaggattgttgtggtca ctgtaacaaa acattctTga gttttgcttt aaatttcataattgtggacg cgggaacaaa acattttTtg atttaatcct ccattacgcattagttgatc ccttaagtga tgatgtgTtt gtgtagtttt ccagtaagga

1101 1150atgattgtat tgttgcacat tgagtgttgg gaatgacagg catttagcccgggaagttat tgtatctctt gtaaatctca tgcgtttgta ccaaataaaaggtaggaatt atgattgttt gtttgcacat tgcgtgttta gcaagtcaagggtgggaagc atggctggga gattccaata tgcattagac ttagattttg

1151 1200tttgttactt aaaaaaaaaa aAAAAAAAAA aAAaaAaagA attccaagcttctc atgtatgctg tAtttttAtA cAAgtAtgtA tgcatAAtAcggcacttagc cctttgtttg tAtttttAtA cAAgtAtgaA taaaaAAAAAgggtcgctgc

1201 1250tccTatTGTA AAAAAAAAAA AAAAAAAAAG AATTCgaaTtc

Figure 4. Nucleic acid sequence of soybean cDNA clones aligned with the published pea ADH sequence. Identical bases are shown in

uppercase, nonidentical bases in lowercase. Dots indicate gaps inserted into the sequence to maximize the homology during the alignment.Underlined regions are discussed in the text.

492

SOYADH10SOYADH57SOYADH73PEAADH1



SOYADH1 0SOYADH57SOYADH73PEAADH1



SOYADH1 0SOYADH57SOYADH7 3PEAADH1


SOYADH1 0SOYADH57SOYADH7 3PEAADH 1











1 50. . . . . ...................... .....

. . . . . . . . . . . . . . .. . . . . .

................. ........... . . .. .. . .. .. .. .. . .

MSnTvGqiIK CrAAVAWEAG KPLvIEEVEV APPQAgEVR1 KILFTSLCHTM.aTaGkvIK CkAAVAWEAG KPLsIEEVEV APPQAmEVRV KILFTSLCHT

51.......AKGQ

......AKGQ

.AKGQ

DVYFWEAKGQDVYFWEAKGQ

101gdCaHCKSEEkeCdHCKSEEgeCpHCKSEEgeCpHCKSEEkeCaHCKSAE

151SEYTVvHaGcSEYTVvHvGcSEYTVvHaGsSEYTVvHaGcSEYTVmHvGc

TPlFPRIFGH EAsGIvESVGTPlFPRIFGH EAgGIvESVGTPlFPRIFGH EAsGIvESVGTPlFPRIFGH EAgGIvESVGTPvFPRIFGH EAgGIiESVG

100EGVThlkPGD HaLPVFTGECEGVTdlkPGD HvLPVFTGECEGVThlkPGD HaLPVFTGECEGVThlkPGD HaLPVFTGECEGVTdvaPGD HvLPVFTGEC

150SNMCeLLRIN TDsGVMihDg qsRFSknGqP ihHFlGTSTFSNMCdLLRIN TDrGVMlhDg kaRFSinGqP iyHFvGTSTFSNMCdLLRIN TDrCVMihDs qtRFSikGqP iyHFvGTSTFSNMCdLLRIN TDrGVMlnDn ksRFSikGqP vhHFvGTSTFSNMCdLLRIN TDrGVMiaDg ksRFSinGkP iyHFvGTSTF

VAKiNPaAPL DKVCvLSCGIVAKiNPaAPL DKVCvLSCGIVAKvNPaAPL DKVCvLSCGIVAKiNPdAPL DKVCiLSCGIVAKiNPqAPL DKVCvLSCGI

200cTGfGAtvNV AKPkpGSsVAsTGlGAtlNV AKPnkGSsVAcTGlGAtiNV AKPkpGSsVAcTGlGAtiNV AKPkpGSsVAsTGlGAsiNV AKPpkGStVA

201iFGLGaVavA AAEGARvsGA SRIIGVDlvs arFeeAkKFGvFGLGdVelA AAEGARlaGA SRIIGVDlns krFteAkKFGiFGLGaVglA AAEGARisGA SRIIGVDlvs srFeeAkKFGiFGLGaVglA AAEGARisGA SRIIGVDlvs srFelAkKFGvFGLGaVglA AAEGARiaGA SRIIGVDlnp srFeeArKFG

251dKPVQqViAe MTnGGVDRaV ECTGsIqAMv sAFECVHDGWdKPVQeViAe MTgGGVDRsV ECTGsInAMi sAFECVHDGWdKPVQeViAa MTnGGVDRaV ECTGsIqAMi sAFECVHDGWdKPVQqViAe MTnGGVDRaV ECTGsIqAMi sAFECVHDGWnKPVQeVlAe MTnGGVDRsV ECTGnInAMi qAFECVHDGW

301KDdaFKTaPi NfLNErTLKG TFyGNykPRt DLPsVVEkYMKDdaFKThPi NvLNEkTLKG TFfGNsePRs DLPsVVEmYMKDdaFKThPv NfLNErTLKG TFyGNykPRt DLPsVVEkYMKDdaFKThPm NfLNErTLKG TFyGNykPRt DLPnVVEkYMKDaeFKThPm NfLNErTLKG TFfGNykPRt DLPnVVElYM

351 381tHtVPFsevn kaFdlMlKGq siRCIIRMqe*hHeVP. .sri tsFeyMlKGe slRCIIRMt.*tHtVPFsein kaFdlMlKGq siRCIIRMqe*tHtVPFsein kaFdyMlKGe siRCIIKMee*tHsVPFaein kaFdlMaKGe giRCIIRMen*

250vnEFVNpKdhvtEFVNgKdyvnEFVNpKdhvnEFVNpKehctEFVNpKdh

300GlAVLVGVPsGvAVLVGVPnGvAVLVGVPnGvAVLVGVPsGvAVLVGVPh

350sgELEvdkif InkELEleny.sgELEvdkf IkgELElekf IkkELEvekf I

Hybridization of the maize ADH1 cDNA (3) probe to thePCR products showed that the 1.3-, the 1.1-, and the 0.6-,and 0.7-kb fragments were all related to ADH cDNA (Fig.2B). The higher mol wt products that hybridized to the maizeADH1 probe may be incompletely processed mRNA contain-ing introns or may be artifacts generated by the PCR process.

DNA Blot Hybridizations

DNA blot hybridization analysis revealed three classes ofclones, based on the ability of individual clones to cross-hybridize. Clones 10, 38, and 71 could not be distinguishedby hybridization criteria (Fig. 3A) and appear to representthe same DNA clone. At high stringency, clones 57 and 73hybridized only to themselves (Fig. 3, B and C). Cross-hybridization of these clones occurred at a slightly lowerstringency. According to the hybridization results, clone 73was more closely related to clones 10, 38, and 71 (Fig. 3, Dand F) than to clone 57 (Fig. 3E).

DNA Sequencing

The DNA sequencing data confirmed that clones 10, 38,and 71 represented the same sequence, whereas clones 57and 73 represented separate ADH sequences (Fig. 4). Thesequences of the three unique clones, 10, 57, and 73, weretranslated to give the putative peptides encoded by theseamplified clones. When these peptide sequences were alignedwith pea or maize ADH sequences (Fig. 5), we observedsignificant sequence homology between all three clones andthe previously characterized Adh genes. Thus, the three am-

Figure 5. Alignment of soybean ADH peptide sequences with thoseof pea and maize. Identical residues are shown in uppercase,nonidentical residues in lowercase. Dots indicate gaps inserted intothe sequence to maximize the homology during the alignment.

RESULTS

Amplification of Soybean ADH cDNA from Total RNA

The PCR reaction yielded two major cDNA products. Themost abundant product was 1.3 kb in size, and a secondaryfragment of approximately 1.1 kb was also amplified (Fig.2A, lane 1). Both of these fragments hybridized to a maizeADH1 cDNA (3) probe (Fig. 2B, lane 2). Soybean ADHmRNA is 1.6 kb in size (21). Because the sense-strand primerin the PCR reaction was located 139 to 167 bases downstreamfrom the translation start site of the maize ADH transcripts,the 1.3- and 1.1-kb PCR fragments were of reasonable sizeto be ADH cDNA.

Digestion with EcoRI revealed that the 1.3-kb PCR productwas actually a mixture of at least two different fragments(Fig. 2A, lane 3). The digestion produced two fragments ofabout 0.6 and 0.7 kb in size on the agarose gel, whereas a

significant amount of uncut DNA remained. Because thelambda DNA was cut to completion in the test digest, theuncut fragment probably represents soybean ADH cDNAwithout EcoRI recognition sequences. The 1.1-kb PCR prod-uct was not digested by EcoRI.

1 2 3 4 5 6 7 8

A

B

C

D

E

F..

F CM :

Figure 6. RNA blot hybridization. A, RNA gel. B, Hybridization withclone 10 at 60'C. C, Hybridization with clone 57 at 60°C. D,Hybridization with clone 73 at 60°C. E, The same as D, but withlonger exposure time. F, Hybridization with the soybean actin gene(pSAC 111) at 50°C. Lane 1, Control cotyledon RNA; lane 2, anaer-

obic cotyledon RNA; lane 3, control hypocotyl RNA; lane 4, anaer-obic hypocotyl RNA; lane 5, control leaf RNA; lane 6, anaerobicleaf RNA; lane 7, control root RNA; lane 8, anaerobic root RNA.

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SOYADH1 0SOYADH57SOYADH73PEAADH1ZEAADH1 f








Plant Physiol. Vol. 100, 1992

plified soybean clones clearly represent different members ofthe soybean Adh gene family.

RNA Hybridization Analysis

Hybridization analysis of RNA from aerobic and anaerobiccotyledons, hypocotyls, leaves, and roots was performed at astringency that did not allow the DNA clones to cross-hybridize. Because the melting temperature for RNA-DNAhybrids is higher than for DNA-DNA hybrids, and gene-specific probes were not used, some cross-hybridization ofthe clones could have occurred. Despite these limitations, aunique expression pattern of the ADH transcripts correspond-ing to each clone was observed (Fig. 6). ADH transcriptscorresponding to clone 10 were detected in anaerobic coty-ledons, hypocotyls, and roots (Fig. 6B). ADH transcriptscorresponding to clone 57 were detected primarily in anaer-obic roots and, to a much lesser extent, in anaerobic cotyle-dons and hypocotyls (Fig. 6C). Clone 73 was the only onethat hybridized to aerobic RNA (Fig. 6, D and E). Figure 6, Dand E, shows that the ADH transcripts corresponding to thisclone were present in both aerobic and anaerobic cotyledons,hypocotyls, leaves, and roots.

Hybridization with the soybean actin probe pSAC III (26)revealed that the actin message abundance declined duringanaerobiosis at the time when the ADH message abundanceincreased (Fig. 6F).

DISCUSSION

This study demonstrates that PCR can be used to amplifymembers of a gene family simultaneously from total RNA.Clones 10, 57, and 73 all coded for ADH, but were distinctlydifferent both in their nucleic acid sequences and in theirexpression pattems.Both maize Adhl and Adh2 genes are expressed at a low

level in aerobic seedlings and are induced simultaneously inresponse to anaerobiosis (7, 8). The anaerobic induction ofsoybean ADH reported in this study appeared to be genespecific. Although the transcripts corresponding to the threesoybean ADH clones isolated were induced by anaerobiosis,ADH transcripts corresponding to clone 73 were the onlytranscripts that accumulated in aerobic tissues. Transcriptscorresponding to clones 10 and 57 were expressed only inanaerobic tissues.The organ-specific expression of ADH has been docu-

mented in maize (23-25), rice (30), and soybean seedlings(21). Using gel electrophoresis, Xie and Wu (30) reported thatboth ADH1 and ADH2 proteins were detected in rice leaves,sheaths, nodes, and roots. However, although ADH1 is thepredominant isozyme in rice leaves, sheaths, and nodes,ADH2 is the predominant isozyme in rice roots. It remainsto be clarified whether clone 57, which detected the expres-sion of the predominantly root-specific ADH transcripts, isrelated to the rice Adh2. Comparison of amino acid sequencesindicated that clone 57 was somewhat more related to maizeAdh2 than to maize Adhl-F (data not shown). The promoterregion of this gene, once identified, will be useful in researchinvolving transgenic soybean, where it is necessary to havethe target genes expressed predominantly in anaerobic roots.

In this study, total RNA from anaerobic soybean cotyledonswas used as the template for the PCR amplification. How-ever, the expected cotyledon-specific gene was not cloned.Because soybean is an allo-tetraploid, it is likely that anotherAdh gene exists that was not amplified by PCR, or was missedduring the cloning of the PCR products. Using the soybeanADH clones isolated in this study as probes, we are currentlyin the process of isolating full length ADH cDNA clones andgenomic clones from soybean libraries.

ACKNOWLEDGMENTS

We would like to thank Dr. Karen Oishi (University of Arizona,Tucson, AZ) for the maize ADH1 cDNA clone, Dr. Thomas Sims forhis valuable input to the manuscript, and Dr. Joe Kamalay (The OhioState University) for advice and technical assistance.

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