INTRODUCTION

Antrodia cinnamomea is a resupinate to effused-

reflexed basidiomycete with porous hymenium (Chang and

Chou, 2004). The basidiomes of Antrodia cinnamomea

(Chinese name, chang-chih) are well-known in Taiwan

as a highly-prized folk medicine. This medicine has

been used to treat drug intoxication, diarrhea, abdominal

pain, hypertension, itchy skin, and liver cancer (Tsai

and Liaw, 1982). Antrodia cinnamomea grows in the

inner cavity of a decayed tree trunk of Cinnamomum

kanehirai Hay (Lauraceae), and its large-scale cultivation

by artificial means has been unsuccessful so far. Also,

due to overexploitation, C. kanehirai, an endemic species

of evergreen tree, is becoming rare. Hence, it is being

conserved by the Taiwan government. The basidiomes of

A. cinnamomea are becoming a scarce and high priced folk

medicine in Taiwan, due to the species¡¦ host specificity and

rarity in nature and to the failure of artificial cultivation

(Chang and Chou, 2004).

Investigation of the differential mechanism of A.

cinnamomea would be beneficial for the development

of a new technique for the regulation of porous-

hymenium basidomatal formation. However, no molecular

characterization of the fruiting bodies from A. cinnamomea

has been reported so far. Expressed sequence tag (EST)

Botanical Studies (2007) 48: 387-396.

*

Corresponding author: E-mail: ttchang@tfri.gov.tw.

analysis has been successfully applied to the study of

gene expression in animals, plants, and fungi subjected

to various stresses or in different stages of development,

as is evident from the increased number of reports on

ESTs (Adams et al., 1991; Cooke et al., 1996; Yamamoto

and Sasaki, 1997; Lee et al., 2002). In addition to the

identification of pathogenicity of fungi, fundamental

aspects of fungal development have also been examined

using the EST-guided approach (Lee et al., 2002; Li et

al., 2004). In edible mushrooms, the process of fruiting

body formation is very important from a scientific and

commercial point of view, and many scientists have

therefore studied specifically expressed genes in the

fruiting bodies of Lentinula edodes, Schizophyllum

commune, and other basidiomycetes (Wessels, 1992;

Kondoh and Shishido, 1995; Fernandez Espinor and

Labarere, 1997). The EST-based approach investigated in

the present study may lead to a fundamental understanding

of basidiomatal formation in A. cinnamomea.

In the present study, two complementary DNA (cDNA)

libraries from liquid-cultured mycelia (AM) and natural

basidiomes (AT) were established in order to characterize

the gene expression during porous-hymenium basidiomatal

formation in A. cinnamomea. The potential functions of

the EST clones were determined by comparing them with

the sequences of other fungi and by obtaining specific

genes for A. cinnamomea. The results obtained in the

study may lead to the identification of genes and analysis

Basidiomatal formation in Antrodia cinnamomea

from

the perspective of gene expression

Fang-Hua CHU

1

and Tun-Tschu CHANG

2,

*

1

School of Forestry and Resource Conservation, National Taiwan University, Taipei, Taiwan

2

Division of Forest Protection, Taiwan Forestry Research Institute, Taipei, Taiwan

(Received September 8, 2006; Accepted May 30, 2007)

ABSTRACT.

In order to understand the phenomenon of morphological differentiation in medicinal fungus

Antrodia cinnamomea, two complementary DNA (cDNA) libraries were constructed from the liquid-cultured

mycelia (AM) and natural basidiomes (AT) produced from the infested wood. Using single-passed sequencing

of cDNA clones, 821 and 993 high-quality expressed sequence tags (ESTs) were generated from the liquid-

cultured mycelial and wild basidiomatal cDNA libraries, respectively. The results from BLASTX search

revealed that only 32.5% to 33.7% of ESTs showed significant similarity to protein sequences in public

databases ( E values . 10

-10

). The cDNAs encoding genes related to metabolism were found to be most

abundant, followed by genes involved in protein fate and protein synthesis in each category. Genes related to

the "Cell fate, cell cycle, and DNA processing" category showed the greatest difference between the liquid-

cultured mycelial and wild basidiomatal cDNA libraries, followed by genes involved in metabolism. The

results from this study have provided valuable sequence information, which may lead to improved production

of basidiomes of A. cinnamomea and regulation of metabolites in the future.

Keywords: Antrodia cinnamomea; Expressed sequence tags; Medicinal fungus; Morphological differentiation.

mOleCUlaR BIOlOgy

388

Botanical Studies, Vol. 48, 2007

of gene expression during basidiomatal formation in A.

cinnamomea.

maTeRIalS aND meTHODS

Strains and culture conditions

Antrodia cinnamomea strain TFRIB 479 obtained from

the rotten wood of C. kanehirai at Dawu, Taitung was

identified in the study. The cultures were maintained as

reported earlier (Chang and Chou, 2004). The natural

basidiomes were obtained from the infested wood. Liquid-

cultured mycelia (AM) and natural basidiomes (AT) were

frozen in liquid nitrogen and stored at -80¢XC until use.

cDNa library construction

Total RNA for cDNA library construction was prepared

following the method described by Chang et al. (1993)

and modified by Chen et al. (2004). Poly (A)

+

RNA was

then purified with Oligotex mRNA Mini Kit (Qiagen). The

single strand cDNA was synthesized by SuperScript

TM

II

Reverse Transcrpitase (Invitrogen) while the double strand

cDNA was amplified using PCR Plus Master Mix Kit

(GeneMark) (Sized-fractionation > 100 bp). Purification

of the cDNA was performed using a DNA Clean/

Extraction Kit (GeneMark). The resulting cDNA was

cloned into the pGEM-T Easy vector system (Promega)

following the protocol for an overnight ligation. The

ligation mixture was transformed into High Efficiency

DH5£\ Competent cells (Hopegen) and then plated onto

LB-ampicillin plates containing IPTG and X-gal.

DNa sequencing

After a cDNA library was plated onto LB media plates,

the transformed white colonies were transferred into test

tubes containing 5 mL LB-amp medium. Plasmid DNA

was isolated from overnight cultures by the Plasmid

Miniprep Purification Kit (GeneMark). Sequencing was

performed with an ABI 377 automatic sequencer (Perkin

Elmer) using a T7 sequencing primer.

Sequence analysis

Nucleotide sequences of less than 100 bp were

excluded, and the remaining ones were analyzed. The

leading vector and poor quality sequences were removed

manually or via ChromasPro software (Technelysium Pty

Ltd). Individual EST was assembled into contigs using the

ContigExpress program of Vector NTI Suite 8 (InforMax,

Inc.) with parameters optimized for ESTs rather than for

genomic clones. The cDNA sequences were compared

to non-redundant (nr) protein sequence databases at the

National Center for Biotechnology Information (NCBI)

(Altschul et al., 1997). E-value results obtained from the

BLASTX were categorized into . 10

-10

, which represented

significant homology, and > 10

-10

, which implied no

hit. The former results were then grouped according to

putative function. A unique set with significant matches

was annotated on the basis of their most similar functions

following the general rules from the Functional Catalogue

by the Munich Information Center for Protein Sequences

(MIPS) (www.mips.biochem.mpg.de/proj/yeast/

catalogues/funcat/index.html) and with the aid of the Gene

Ontology Consortium (www.geneontology.org).

ReSUlTS

Sequencing and assembling of eSTs sequences

Total RNAs were isolated from liquid-cultured mycelial

(AM) and natural basidiomes (AT) of A. cinnamomea

for the construction of cDNA libraries. Initially, a total

number of 1823 clones were randomly picked and

subjected to 5¡¦ end single-pass sequencing from the

libraries. The leading vector, tailing of the sequence, and

poor-quality sequences were excluded. After excluding

fragments shorter than 150 nucleotides, 1,809 high-quality

ESTs were submitted to dbEST (GeneBank accession nos.

DV629596-DV631404). Most of the ESTs were 300-700

bp in length with an average of 538 bp. Among 817 clones

from the mycelia library, 537 clones assembled into 104

contigs while 180 ESTs were unigenes. In the basidiomatal

library, with its 992 clones, 742 clones assembled into 162

contigs while 250 ESTs remained as unigenes. As a result

of the contig analysis, 604 different genes (unigene) could

be obtained out of 1,809 analyzed cDNA clones. Among

the 604 unigenes, 512 (84.8%) expressed differently in the

mycelial and basidiomatal libraries.

Characterization of the cDNa library and the

eSTs sequences

In this study, the putative function of the cDNAs, which

is based on BLASTX¡¦s result, was assigned to those with

E-values less or equal to 10

-10

(Sterky et al., 1998). In

AM and AT libraries, only 267 (32.7%) and 325 (32.8%)

ESTs, respectively, showed similarity when these were

compared with the public sequence databases. A total

of 1809 ESTs matched with 592 distinct sequences in

the non-redundant protein database and were assigned

to 259 functional genes from both the libraries. The

developmental stage specificity and redundancy of

these 259 unigenes were analyzed to characterize the

developmental gene expression from the EST data

(Table 1). The results indicated that lanosterol 14-alpha-

demethylase, and nucleolar protein nhp2 occurred more

frequently in the basidiomatal library than in the mycelial

library. On the other hand, peroxiredoxins, ATP-binding

cassette transporter ABC1, phytase, and H/ACA snoRNP

component occurred more redundantly in the mycelial

library. The potential roles of these in basidiomatal

formation in A. cinnamomea will be explored in the future.

Cellular roles of eSTs in A. cinnamomea

To obtain an overview of how A. cinnamomea functions

at the cellular level, the transcripts identified with putative

functions were assigned nine putative cellular roles based

on those assigned them by the MIPS FunCat scheme

CHU and CHANG ¡X ESTs of

Antrodia cinnamomea

389

Table 1. Annotation of ESTs from liquid-cultured mycelia (AM) and wild basidiomes (AT) of A. cinnamomea.

Category and putative function

Related taxon

Accession No. N

M

a

N

T

b

I. Cell fate, cell cycle and DNA processing

Anaphase promoting complex subunit 10 Oryza sativa

AAU10698 1

ATP-dependent DNA helicase

Cryptococcus neoformans var. neoformans JEC21 AAW43036

1

Brefeldin a resistance protein

Schizosaccharomyces pombe

CAB44765 1 4

Bud site selection-related protein

Cryptococcus neoformans var. neoformans JEC21 AAW43110

1

Cell cycle regulatory protein

Ustilago maydis

AAN10186

1

DNA topoisomerase I

Cryptococcus neoformans var. neoformans JEC21 AAW45618

1

Fibrillarin (NOP1)

Neurospora crassa

CAC18188 1

Flap endonuclease-1

Coprinopsis cinerea

BAD14303

1

Microtubule end-binding protein EB1

Coturnix japonica

AAU12573

1

Opa-interacting protein OIP2

Cryptococcus neoformans var. neoformans

B-3501A

EAL23044 1

Prohibitin PHB1

Cryptococcus neoformans var. neoformans JEC21 AAW40684 1

RAB18

Mus musculus

BAC32402 1 1

Ras1p

Suillus bovinus

AAF65465

1

Topoisomerase I

Cryptococcus neoformans var. neoformans JEC21

AAW45618

1

Tubby-like protein 2

Arabidopsis thaliana

AAK98801

1

II. Cell rescue, defense, and virulence

Catalase

Campylobacter jejuni

CAA59444 1

Cytochrome P450, alkane-inducible

Cryptococcus neoformans var. neoformans JEC21 AAW43969 1 1

Cytochrome P450 monooxygenase

Coriolus versicolor

BAB59027 1 2

Cytochrome P450 oxidoreductase

Coriolus versicolor

BAB83588

1

Glutathione reductase

Onchocerca volvulus

CAA72516

1

Heat shock protein HSS1

Puccinia graminis f. sp. tritici

AAB93665 1

Hob1p

Cryptococcus neoformans var. neoformans JEC21 AAW40855

1

Lanosterol 14-alpha-demethylase

Phanerochaete chrysosporium

AAU01160 3 17

Methylenetetrahydrofolate reductase

Cryptococcus neoformans var. neoformans JEC21 AAW41236

5

O-methylsterigmatocystin oxidoreductase Neurospora crassa

CAE76231 1 1

Osmotic stress-related protein

Cryptococcus neoformans var. neoformans JEC21 AAW41050 1 3

Peroxiredoxin Q

Triticum aestivum

AAV66923 1

Peroxiredoxins

Phanerochaete chrysosporium

AAV53576 19 9

Protoplast regeneration and killer toxin

resistance gene

Cryptococcus neoformans var. neoformans JEC21 AAW44305

1

Snodprot-FS

Gibberella pulicaris

AAV83793 8 3

WD-repeat protein-like

Arabidopsis thaliana

BAB09052

1

III. Cellular transport, transport facilitation and transport routes

Allantoate permease

Neurospora crassa

CAE81935

1

ATPase of the ABC class

Thermoanaerobacter tengcongensis

ZP_00178674

1

ATP-binding cassette transporter ABC1 Venturia inaequalis

AAK62810 11 1

ATPase of the CDC48/PAS1/SEC18 (AAA)

family

Saccharomyces cerevisiae

NP_013501

1

ATP synthase gamma chain

Cryptococcus neoformans var. neoformans JEC21 AAW43492

2

390

Botanical Studies, Vol. 48, 2007

Category and putative function

Related taxon

Accession No. N

M

a

N

T

b

Carnitine/acyl carnitine carrier

Cryptococcus neoformans var. neoformans JEC21 AAW41054 15 12

ER to Golgi transport-related protein

Cryptococcus neoformans var. neoformans

JEC211

AAW44532 3 3

GTPase

Cryptococcus neoformans var. neoformans JEC21 AAW46779 1

Intracellular transport-related protein

Cryptococcus neoformans var. neoformans JEC21 AAW41812

1

Late endosome to vacuole transport-related

protein

Cryptococcus neoformans var. neoformans JEC21 AAW42196 2

Membrane zinc transporter

Aspergillus fumigatus

AAT11930

1

Nucleoporin

Ustilago maydis

CAG26761 1

Phosphate transporter

Pholiota nameko

BAB43910

2

Protein-vacuolar targeting-related protein Cryptococcus neoformans var. neoformans JEC21 AAW40999 1

P-type cation-transporting ATPase

Blastocladiella emersonii

CAA04499

1

Sugar transporter

Neurospora crassa

CAB88582

1

Synaptobrevin (v-SNARE) homolog Bos1 Schizosaccharomyces pombe

CAB77004 1 1

Transmembrane transporter Liz1p

Cryptococcus neoformans var. neoformans JEC21 AAW43513

1

Tricarboxylate transport protein

Schizosaccharomyces pombe

CAB10116 2

Triose phosphate translocator

Cryptococcus neoformans var. neoformans

AAR82906

1

Vacuolar ATP synthase

Cryptococcus neoformans var. neoformans JEC21 AAW45529 1

V-ATPase subunit A

Fundulus heteroclitus

BAB62103

1

Vesicle-mediated transport-related protein Cryptococcus neoformans var. neoformans JEC21 AAW47173 1

Urea transporter

Cryptococcus neoformans var. neoformans JEC21 AAW43008

1

IV. Cellular communication/signal transduction

Calcium ion binding protein

Cryptococcus neoformans var. neoformans JEC21 AAW44613 1 1

Guanine nucleotide-binding protein alpha-4

subunit

Ustilago maydis 521

EAK86634

1

Sensory histidine kinase

Mesorhizobium loti

NP_103743 2 3

Serine kinase (hPAK65)

Homo sapiens

AAA75468

1

Rho GTPase activator

Cryptococcus neoformans var. neoformans JEC21 AAW44732

1

V. Metabolism

Acetamidase

Schizosaccharomyces pombe

AL023592

1

Acid sphingomyelinase

Neurospora crassa

CAD70921

1

Acyl-CoA transferases/carnitine dehydratase Ralstonia metallidurans CH34

ZP_00273947 1

Agmatinase precursor

Schizosaccharomyces pombe

NP_593990 1

Aldo-keto reductase

Cryptococcus neoformans var. neoformans

B-3501A

AAW46629 2 3

Allantoicase

Cryptococcus neoformans var. neoformans JEC21 AAW43136

2

Amidophosphoribosyltransferase

Cryptococcus neoformans var. neoformans JEC21 AAW42423

1

Aryl-alcohol dehydrogenase

Cryptococcus neoformans var. neoformans JEC21 AAW46369 1 1

Beta-1,3-mannanase

Paecilomyces lilacinus

BAD06516 1

Chorismate synthase

Schizosaccharomyces pombe

T41268

3

Cycle propane fatty acid synthase

Coprinopsis cinerea

AAL73238

1

Dihydrokaempferol 4-reductase

Cryptococcus neoformans var. neoformans JEC21 AAW43891

4

Table 1. (Continued.)

CHU and CHANG ¡X ESTs of

Antrodia cinnamomea

391

Category and putative function

Related taxon

Accession No. N

M

a

N

T

b

Dolichyl-phosphate beta-glucosyltransferase Schizosaccharomyces pombe

NP_596707 2 1

Exo-1,3-beta-glucanase

Agaricus bisporus

CAA63536

1

Endo-1,4-£]-xylanase A

Phanerochaete chrysosporium

AAG44993 1

Flavin-containing monooxygenase

Aspergillus fumigatus

CAE47890

2

Glucosidase 1

Caenorhabditis elegans

N P _ 5 0 2 0 5 3

1

Glutamate synthase (NADH)

Cryptococcus neoformans var. neoformans JEC21 AAW46054

1

Glycine dehydrogenase-like protein

Pleurotus djamor

AAS46734

2

Homoserine O-acetyltransferase

Saccharomyces pastorianus

Q06736 1

Hydrolases

Pseudomonas syringae pv. syringae B728a

ZP_00126253

1

3-hydroxyanthranilate 3,4-dioxygenase

Cryptococcus neoformans var. neoformans JEC21 AAW41998 1 1

3-hydroxyisobutyrate dehydrogenase

Novosphingobium aromaticivorans DSM 12444 ZP_00305232

1

Hydroxymethylglutaryl-CoA lyase

Pseudomonas mevalonii

AAA25896 1

IMP dehydrogenase

Cryptococcus neoformans var. neoformans JEC21 AAW40949 1

Laminarinase

Phanerochaete chrysosporium

BAC67687 1

Lysosomal alpha-N-acetyl glucosaminidase Gallus gallus

XP_418147

1

Mannosyl-oligosaccharide glucosidase

Schizosaccharomyces pombe

NP_594106

1

Nicotinate-nucleotide diphosphorylase Cryptococcus neoformans var. neoformans JEC21 AAW42323

1

Oxidoreductase

Agrobacterium tumefaciens str. C58

AAL43649 9 9

Phenol 2-monooxygenase

Neurospora crassa

CAF06102 1

Phospho-2-dehydro-3-deoxyheptonate

aldolase

Cryptococcus neoformans var. neoformans JEC21 AAW46670 1 1

Phosphatidylinositol phosphate phosphatase Schizosaccharomyces pombe

NP_596431

1

Phosphoglycerate mutase GPM2

Mycobacterium tuberculosis H37Rv

CAE55568 1 3

Phosphoribosyl-5-amino-1-phosphoribosyl-

4-imidazolecarboxiamide isomerase

Saccharomyces cerevisiae

NP_012244 1 2

Phosphotyrosyl phosphatase activator

Oryza sativa

BAD37240

2

Phytase

Trametes pubescens

CAC48234 46 4

Polygalacturonase

Cryptococcus neoformans var. neoformans JEC21 AAW42245

1

Polyketide biosynthesis associated protein Agrobacterium tumefaciens str. C58

NP_532458 2

Prenyltransferase

Cryptococcus neoformans var. neoformans JEC21 AAW42863

1

Riboflavin aldehyde-forming enzyme

Lentinula edodes

BAD11818

1

Short-chain alcohol dehydrogenases

Ralstonia metallidurans CH34

ZP_00271907 1 3

Urate oxidase

Aspergillus flavus

CAA43896 1 1

UTP-glucose-1-phosphate

uridylyltransferase

Cryptococcus neoformans var. neoformans JEC21 AAW42292

1

Zinc-binding dehydrogenase

Cryptococcus neoformans var. neoformans JEC21 AAW42578 1

VI. Protein fate and synthesis

Alanine-tRNA ligase

Cryptococcus neoformans var. neoformans JEC21 AAW44283 1

Arginyl tRNA synthetase

Candida albicans SC5314

EAK99505

1

Aspartyl-tRNA synthetase

Saccharomyces cerevisiae

NP_015221

1

Aspartic proteinase precursor

Botryotinia fuckeliana

AAG43236 6 6

ATP-dependent protease proteolytic subunit

ClpP

Arabidopsis thaliana

BAC43126 1

Table 1. (Continued.)

392

Botanical Studies, Vol. 48, 2007

Category and putative function

Related taxon

Accession No. N

M

a

N

T

b

Calmodulin-dependent protein kinase

Cryptococcus neoformans var. neoformans JEC21 AAW45617

1

Carboxypeptidase C

Saccharomyces cerevisiae

S46008

1

Carboxypeptidase Y

Trichophyton rubrum

AAS76668

1

Chaperone regulator

Cryptococcus neoformans var. neoformans JEC21 AAW42057

2

Dipeptidyl aminopeptidases/ acylaminoacyl-

peptidases

Microbulbifer degradans 2-40

ZP_00315190 1

Elongation factor 1-gamma

Cryptococcus neoformans var. neoformans JEC21 AAW40932 3 7

Endopeptidasee

Cryptococcus neoformans var. neoformans JEC21 AAW43542 1 4

Eukaryotic translation initiation factor SUI1

family protein

Arabidopsis thaliana

NP_177291 1

FKBP-type peptidyl-prolyl cis-trans

isomerases 1

Rubrivivax gelatinosus PM1

ZP_00245218

1

Glutaminyl-trna synthetase

Schizosaccharomyces pombe

NP_596745

1

Glyceraldehyde-3-phosphate dehydrogenase

(GAPDH)

Schizophyllum commune

P32638

1

Insulin degrading enzyme

Cryptococcus neoformans var. neoformans JEC21 AAW46588 2 2

Leucine aminopeptidase

Coprinopsis cinerea

BAB87833 1

Nucleolar peptidyl-prolyl cis-trans

isomerase (PPIase)

Saccharomyces cerevisiae

NP_013637 2 3

Polyubiquitin 6

Gracilaria verrucosa

S53719 1

20S proteasome alpha-type subunit

Saccharomyces cerevisiae

NP_014604

3

Ribosomal large subunit assembly and

maintenance-related protein

Cryptococcus neoformans var. neoformans

B-3501A

AAW41360

2

Ribosomal protein

Cryptococcus neoformans var. neoformans JEC21 AAW42014

3

40S ribosomal protein S8

Schizophyllum commune

AAC69196 5 6

Ribosomal protein l15 homologue

Aspergillus fumigatus

CAE47918

1

50S ribosomal protein L22

Cryptococcus neoformans var. neoformans JEC21 AAW43715 1

60s ribosomal protein l5-b

Cryptococcus neoformans var. neoformans JEC21 AAW42426

1

60s ribosomal protein l27

Cryptococcus neoformans var. neoformans JEC21 EAK83063 1

LSU ribosomal protein L1P

Thermus thermophilus HB27

YP_005708 1

RING/C3HC4/PHD zinc finger-like protein Cucumis melo

AAO45753 1

t-complex protein 1, beta subunit

Cryptococcus neoformans var. neoformans JEC21 AAW40957 1 1

t-complex protein 1, delta subunit

Cryptococcus neoformans var. neoformans JEC21 AAW44504 6 6

t-complex protein 1, theta subunit

Cryptococcus neoformans var. neoformans JEC21 AAW42275

2

Translation initiation factor

Cryptococcus neoformans var. neoformans JEC21 AAW43820 2 1

Ubiquitin-like protein

Plasmodium yoelii

EAA18158

1

Ubiquitin-like protein 5

Drosophila melanogaster

Q9V998

1

Ubiquitin carboxyl-terminal hydrolase Schizophyllum commune

AF077976 1

VII. Transcription

Cofilin

Pichia angusta

AAK85273 1 1

DNA-directed RNA polymerase I

Schizosaccharomyces pombe

NP_594382

1

Glia maturation factor beta

Cyprinus carpio

BAA95482 1 1

H/ACA snoRNP component

Candida albicans SC5314

EAK98515 16

Hexamer-binding protein HEXBP

Leishmania major

A47156 1 1

Table 1. (Continued.)

CHU and CHANG ¡X ESTs of

Antrodia cinnamomea

393

(Figure 1). A major portion (79 ESTs) in the AM library

represented transcription, which is in the metabolism

category, corresponding to 9.6% of all ESTs. The next one

was involved in protein fate and protein synthesis. Except

for the "unknown function" category, ESTs related to

metabolism were found to be the most abundant, followed

by the category "protein fate and protein synthesis"

categories in the AT library. In these categories, 40S

ribosomal protein, S8, and the delta subunit of t-complex

protein 1 (tcp-1-delta) were found in both libraries. In the

metabolism category, there were 23 and 33 unigenes in the

AM and AT libraries, respectively (Table 1). However,

in the differential expression during the mycelial and

basidiomatal stages, the cell fate, cell cycle, and DNA

processing category was found to be the most divergent,

followed by those involved in cellular transport, transport

facilitation, and transport routes (Table 2). There were

15 kinds of ESTs in the cell fate, cell cycle, and DNA

processing category while only two unigene ESTs occurred

in both stages. The difference between the mycelial and

basidiomatal stages was 86.7%.

DISCUSSION

From a scientific and economic point of view,

differentiation of sexual fruiting bodies from mycelia

at vegetative is an interesting phenomenon in

homobasidiomycetes, especially while A. cinnamomea

possesses a porous-hymenium basidiome without stipe.

Although several mycologists have made efforts to study

gene expression during the formation of fruit bodies (Lee

et al., 2002; Sunagawa and Magae, 2005; Yamada et al.,

2006), porous-hymenium basidiomatal formation and

biosynthesis of active components at the molecular level

have not yet been explained.

High-throughout single-run partial sequencing,

generation, and analysis of ESTs have proven to be a

rapid and efficient approach to obtaining information on

mRNA expression (Adams et al., 1991). To study the

gene regulation during porous-hymenium basidiomatal

formation in A. cinnamomea, cDNA clones were

randomly sequenced at different stages of the culture of

A. cinnamomea. In total, 1,809 ESTs matched with 592

distinct sequences in the non-redundant protein database

and assigned to 259 functional genes from all libraries.

Although the number of ESTs in this study is low, one may

obtain some useful information from the genes encoded

by the A. cinnamomea genome. The cDNA libraries and

the accompanying database are valuable resources for

researchers hoping to understand the genetic control of

basidiomatal formation and secondary metabolism in A.

cinnamomea.

It is customary in genome annotation to establish a

cutoff for "statistically significant" database hits. By

correlating an observed alignment score with the expected

distribution, we can quantify statistical significance in

Category and putative function

Related taxon

Accession No. N

M

a

N

T

b

Leucine zipper protein

Oryza sativa

BAD38167 1 1

Mammalian swi/snf complex 60 kda subunit

homolog

Schizosaccharomyces pombe

T50184

2

mRNA processing-related protein

Cryptococcus neoformans var. neoformans JEC21 AAW43157

2

nhp2

Schizosaccharomyces pombe

AL158056 3 7

Nucleus protein

Cryptococcus neoformans var. neoformans JEC21 AAW40929 1 3

Reptin

Apis mellifera

XP_395860 1

Pre-mRNA splicing factor RNA helicase

PRP28

Cryptococcus neoformans var. neoformans JEC21 AAW41184 1 6

TATA box binding protein (TBP)

Homo sapiens

AB010067 2

tRNA dihydrouridine synthase

Cryptococcus neoformans var. neoformans JEC21 AAW45443 1

trp-asp repeats containing protein

Cryptococcus neoformans var. neoformans JEC21 AAW46166 1

VIII. Energy

Cytochrome-b5 reductase

Cryptococcus neoformans var. neoformans JEC21 AAW45007 1

Cytochrome c1

Cryptococcus neoformans var. neoformans JEC21 AAW44407 1 2

Glyceraldehyde 3-phosphate dehydrogenase Arabidopsis thaliana

NP_172801 2

NADP-dependent oxidoreductases

Pseudomonas syringae pv. syringae B728a

ZP_00124416 3 8

a

Redundancy number of the sequences in liquid-cultured mycelia ESTs.

b

Redundancy number of the sequences in wild-type basidiomatal ESTs.

Table 1. (Continued.)

394

Botanical Studies, Vol. 48, 2007

the form of an E value. It can be expressed in terms

of the false-positive expectation value for the BLAST

searches and is set routinely at values such as E = 0.001

or E = 10

-5

. The problem with this approach is that the

distribution of similarity scores for evolutionarily and

functionally relevant sequence alignments is very broad,

and a considerable fraction of them may fail the E-value

cutoff, resulting in undetected relationships and missed

opportunities for functional prediction (false negatives)

(Galperin and Koonin, 2001). In the present study, to

minimize the false-positive rate, it was critical to set

the E values at less or equal to 10

-10

while filtering low-

complexity sequences. BLASTX analysis revealed

that only 22.1% and 33.7% of ESTs showed significant

similarity to published protein sequences in the AM and

AT libraries, respectively. The absence of these sequences

from public databases might indicate specific roles for

these proteins in A. cinnamomea. Similarly, with E values

less than or equal to 10

-5

as the cutting point, only 39.5%

to 40.8% ESTs showed no significant similarity to any

known proteins in the existing databases.

In redundant ESTs analysis, most ESTs occurred one

to four times while only few occurred several times in

the cDNA libraries. In this study, 16 unigenes occurred

more than five times in the libraries. There were 46

instances when cDNAs were identified as phytase among

these unigenes. These unigenes showed high expression

in the liquid-cultured mycelial library compared to the

basidiomatal library. BLASTX analysis showed that these

ESTs of phytases had 62~67% identity with Trametes

pubescens phytase. Moreover, cDNA was identified

as lanosterol 14-alpha-demethylase 17 times in the

basidiomatal library, but only thrice in the liquid-cultured

mycelia library. The lanosterol 14-alpha-demethylase

belongs to the CYP51 family of the cytochrome P450

superfamily, and it is notable that lanosterol 14-alpha-

demethylase is the only member of the cytochrome P450

family. Also, this is true in all biological kingdoms

(Revankar et al., 2004). In addition, the ESTs identified as

putative methylenetetrahydrofolate reductase were found

five times in the basidiomatal libraries only. This suggests

the possibility that this gene was expressed specifically at

the basidiomatal stage and may be related to basidiomatal

formation.

According to the number of unigenes in each category,

cDNAs encoding for the genes related to "metabolism"

were found to be the most abundant. Among the 45

unigenes isolated from these two libraries, 22 unigenes

(48.9%) were expressed only in the basidiomatal library.

In another study, riboflavin aldehyde-forming enzyme

specifically and abundantly was expressed in the mature

basidiomes of Lentinula edodes (Hirano et al., 2004).

Thus, the promoter region of the riboflavin aldehyde-

forming enzyme may be the emitter of the expressing

signal of the vector for gene manipulation in a mature

fruiting body. In our study, the putative riboflavin

aldehyde-forming enzyme appeared only in the wild-type

A. cinnamomea. This result indicates that the promoter of

the aldehyde-forming enzyme may be the vector regulator

when the mature basidiome is being manipulated.

Regardless of the cultural conditions or different

expression patterns during the mycelial and basidiomatal

stages, the group of ESTs tentatively assigned to the cell

fate, cell cycle, and DNA processing-related category

varied the most. Among the 27 unigenes isolated from

these two libraries, only 18 unigenes (66.7%) were

expressed in the basidiomatal library. On comparing our

results with the sequence expression of P. ostreatus in the

same category, the putative Ras 1p gene also appeared

only at the fruiting body stage (Lee et al., 2002).

Table 2. Divergency analysis of unigenes in each category from mycelia and basidiomatal stages.

Category

I

II

III

IV

V

VI

VII

VIII

Total

a

15

16

24

5

45

37

15

4

Both

b

2

7

4

2

10

9

7

2

Divergency (%)

c

86.7

56.3

83.3

60

77.8

75.7

53.3

50

(Mention all the categories in Table 1)

a

Total number of independent unigenes derived from two libraries.

b

Number of independent unigenes appearing in both stages.

c

Percentage of unigenes appearing only in mycelia or basidiomatal stage.

Figure 1. Fun cti onal cat egori es of E STs from d iffere nt

s tages of A. cinnamomea. I: Cell fate, cell cycle and DNA

processing; II: Cell rescue, defense and virulence; III: Cellular

transport, transport facilitation and transport routes; IV: Cellular

communication/signal transduction; V: Metabolism; VI: Protein

fate and protein synthesis; VII: Transcription; VIII: Energy; IX:

Unknown. AM: liquid-cultured mycelia; AT: wild basidiomes of

A. cinnamomea. The number listed on top of the bar indicates

the percentage of EST.

CHU and CHANG ¡X ESTs of

Antrodia cinnamomea

395

The EST database established in this study provides

the sequence information of genes expressed during

basidomatal formation at the whole-gene level.

Combining the analysis with metabolomics, proteomics,

microarray analysis, transformation system, and

bioinformatics, we hope to explore the genes and to

understand how basidiomatal formation can be regulated

in the future.

Acknowledgements. The authors are grateful for

expert technical assistance from Ms. Yi-Ru Lee and Ms.

Shang-Fen Chi. Financial assistance from the Council

of Agriculture Executive Yuan (94AS-5.2.1-ST-a1) is

gratefully acknowledged.

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