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Screening for Genes Participating in the Formation of Prismatic and Nacreous Layers of the Japanese Pearl Oyster Pinctada fucata by RNA Interference Knockdown

  • Daisuke Funabara
  • Fumito Ohmori
  • Shigeharu Kinoshita
  • Kiyohito Nagai
  • Kaoru Maeyama
  • Kikuhiko Okamoto
  • Satoshi Kanoh
  • Shuichi Asakawa
  • Shugo Watabe
Open Access
Conference paper

Abstract

Many genes have been identified to participate in the shell formation so far. Nevertheless, the whole picture of the molecular mechanisms underlying the shell formation has remained unknown. In our previous study, we analyzed comprehensively genes expressed in the shell-producing tissues and identified 14 genes to be involved in the shell formation by the RNA interference (RNAi) method. In the present study, we performed further screening to find additional novel genes involved in the formation of the nacreous and prismatic layers. We here selected 80 genes from the EST data as candidates to function in the shell formation, conducted knockdown experiments by the RNAi method, and observed surface appearances on the nacreous and prismatic layers. We newly identified 64 genes that could participate in the shell formation. Taken together with our previous study, 78 genes were supposed to function in the shell formation. These findings indicate that the combination of transcriptome and knockdown analyses is a powerful tool to screen novel genes involved in the shell formation.

Keywords

EST Knockdown Nacreous layer Pearl oyster Prismatic layer RNAi Shell 

41.1 Introduction

Many genes have been identified to participate in the shell formation so far. In classical ways, proteins were purified from shells after decalcification and their properties were analyzed. Nacrein, for instance, was purified from shells of the Japanese pearl oyster Pinctada fucata and characterized in detail (Miyamoto et al. 1996). Suzuki et al. (2009) employed the RNA interference (RNAi) method to elucidate possible functions of Pif discovered as an aragonite-binding protein in the shell of P. fucata. Knockdown of the Pif gene by the RNAi method induced an abnormal crystal structure of aragonite. This finding confirmed that Pif is really involved in the nacreous layer formation and proved that the RNAi method is useful to study genes involved in shell formation. We obtained the EST data of nacreous and prismatic layer-producing tissues of P. fucata, which contained 29,682 genes, and found novel 29,550 genes (Kinoshita et al. 2011). Genes involved in the shell formation must be contained in these genes. Thus, we compared gene expression patterns among mantle pallium, edge, and pearl sac tissues using the EST data to find genes expressed in a tissue-specific manner. We selected five genes specifically expressed in the mantle pallium, three highly expressed in the mantle pallium and pearl sac, and six specifically expressed in the mantle edge as candidates to function in shell formation. Knockdown experiments for these candidate genes induced abnormal appearances on the inner surface of the shells in the oysters (Funabara et al. 2014). These findings demonstrated that a combination of transcriptome analyses and RNAi knockdown is a powerful tool to screen genes involved in the shell formation. In the present study, we conducted further screening for genes involved in the shell formation of P. fucata using the above method.

41.2 Materials and Methods

We selected 195 genes having more than 200 reads from the EST data (Kinoshita et al. 2011) of the shell-forming tissues, along with 9 genes expressed similarly to those known to be involved in the shell formation from genes having less than 200 reads in the EST data. We conducted cDNA cloning of the selected genes with primers designed using the nucleotide sequences of respective genes. dsRNAs of the selected genes were synthesized using the cDNA clones as templates with a ScriptMAX™ Thermo T7 Transcription Kit (Toyobo, Osaka, Japan). About 40 μg of dsRNA/100 μl H2O were injected into adductor muscles of 2-year-old pearl oysters (n = 3), followed by rearing them in artificial seawater at 23 °C for 8 days with feeding plankton once a day. The green fluorescence protein (GFP) and Pif genes were used as negative and positive references, respectively, to verify the RNAi experiments. Surface appearances of the prismatic and nacreous layers on the shells of the knockdown oysters were observed with a scanning electron microscope (SEM), S-4000 (Hitachi, Tokyo, Japan).

41.3 Results

41.3.1 Selection of Candidate Genes Functioning in Shell Formation

We selected candidate genes having more than 200 reads in the EST data (Kinoshita et al. 2011) to be possibly involved in the shell formation, except for 14 genes which we analyzed in our previous study (Funabara et al. 2014) (Table 41.1). cDNAs of 71 genes out of the selected 181 genes above were successfully cloned and used for synthesizing dsRNAs as templates. We selected additionally 9 genes showing expression patterns similarly to those of known shell formation-related genes such as PFMG1, KRMP1, N19, and N16 series from those having less than 200 reads (Table 41.1). cDNAs of all the nine genes were cloned and used for the synthesis of dsRNAs. A total of 80 genes were subjected to the knockdown experiments.
Table 41.1

Gene expression patterns in shell- and pearl-forming tissues

Genea

TPM

Total reads

Gene

TPM

Total reads

Gene

TPM

Total reads

Gene

TPM

Total reads

ME

MP

PS

ME

MP

PS

ME

MP

PS

ME

MP

PS

1b

6042

6381

7207

1728

52b

1456

1386

93

226

104

1922

908

148

224

179

1456

1028

1129

308

2b

12,142

17,827

1240

24,620

53b

1922

1529

3099

595

105

1267

1063

805

263

187

1834

2282

0

317

3b

4833

4672

1351

869

54b

1485

1159

3238

549

106

1150

920

1933

365

188

772

1123

2396

406

4

2737

1410

5301

879

55b

1470

1326

2923

528

107

2329

2342

2156

589

190

903

836

805

219

5b

4062

4146

28

629

56b

1776

1529

0

250

108b

815

729

2535

391

191

466

430

1480

228

6b

3479

2449

5458

1034

57b

2538

2951

0

409

109

961

478

1628

282

193

1267

1195

1018

297

7b

3130

3644

4200

974

58c

2635

275

0

204

110

1616

1482

1286

374

194c

2751

1028

0

275

8b

1994

2892

185

399

59b

2140

2426

629

418

111b

1601

1864

1008

375

196

597

347

3349

432

9b

3261

2366

19

424

60b

2519

1937

1415

463

112b

975

1135

601

227

197

1092

1267

722

259

10b

2737

2892

111

442

61b

1529

1338

3654

612

113c

1019

2653

56

298

200c

2227

789

0

219

11b

3494

2844

65

485

62b

2373

1852

2563

595

114b

2504

1470

2082

520

209

189

72

1878

222

12b

2227

3597

315

488

63b

3712

2438

0

459

115b

1529

1302

1758

404

215

1631

1338

0

224

13b

3217

1972

130

400

64b

3217

2461

1878

630

116

2009

1517

0

265

216

932

1111

490

210

14b

2125

1816

3173

641

65b

3072

1972

0

376

118c

641

1924

102

216

218

670

789

860

205

15b

5008

3023

1739

785

66c

2853

48

0

200

121

830

657

1813

308

228

1325

1350

0

204

16b

2009

2593

5107

907

67b

2053

1625

3312

635

122

1077

1171

786

257

237

1194

1446

28

206

17b

2504

2665

4431

874

68b

2737

1470

2285

558

123

1077

753

1147

261

243

1529

1075

361

234

18

1732

1995

3007

611

69b

3523

3190

4496

995

124

728

693

1452

265

248

1441

1099

333

227

19

2533

1613

5097

860

70b

757

1051

833

230

125

1689

1147

2720

506

250

1296

1338

1332

345

20b

2038

1804

3626

683

71b

1878

1972

1480

454

127

1791

2246

2017

529

252

1252

1506

0

212

21

1470

1995

5190

829

72b

3028

143

0

220

128

1820

1852

1092

398

268

903

1016

1425

301

22b

2475

1697

3423

682

73

1354

2031

481

315

129

2038

1482

2044

485

272

1936

1386

37

253

23b

2358

2210

3830

761

74b

2795

2043

111

375

130

1034

1816

648

293

274

684

633

1471

259

24b

2198

1888

4330

777

75b

2562

1936

2868

648

132

1194

1565

259

241

292

903

789

1018

238

25b

2737

3405

0

473

76b

2082

1912

2646

589

133c

903

1804

46

218

300

611

203

1369

207

26b

2286

2306

4459

832

77b

2373

2414

65

372

134

888

930

962

243

301

1616

1290

0

219

27c

641

1744

3432

561

78

1441

1410

1721

403

136

1383

944

2054

396

323

1485

1147

83

207

28b

3669

3967

0

584

79b

2795

2497

2812

705

137

1237

2031

1600

428

336

1092

442

1221

244

29b

2417

2115

3867

761

80

2679

2139

1915

570

138

1558

1434

56

233

344

1310

1876

916

346

30b

2868

2270

5005

928

81c

364

1625

463

211

139

1893

1517

1767

448

384

859

1040

1203

276

31c

4586

1267

0

421

82

2053

2605

583

422

141

1252

1577

0

218

395

58

36

2618

290

32b

2519

3202

2877

752

83

1776

1972

851

379

143

1558

2031

65

284

399

1441

1673

333

275

33b

2446

1613

3719

705

84

1339

2210

453

326

145c

4047

167

0

292

407

1325

1517

296

250

34b

2067

2151

786

407

85

1689

2402

1295

457

147

1150

1398

361

235

411c

131

211

259

214

35b

4367

2784

6642

1251

86

1412

1840

546

310

148

1601

1601

0

244

3840

0

0

2812

304

36b

2868

2258

2655

673

87

1645

693

1878

374

150

1747

1374

2461

501

3969

0

0

1896

205

37b

4906

4756

0

735

88

1150

36

1147

206

152

670

442

3275

437

4121

0

0

1896

205

38b

1951

1685

1970

488

89

1456

2031

1610

444

154

670

609

1129

219

4600

0

0

2109

228

39b

2140

1995

2997

638

90

1005

621

1018

231

155

1063

741

814

223

5656

0

0

2017

218

40

3188

2485

0

427

91

1208

801

1110

270

157

1398

908

1591

344

7101

0

0

2054

222

41

3596

3465

2711

830

92b

1631

2342

0

308

161

1077

1804

0

225

7147

0

0

1952

211

42b

2795

2772

2396

683

93

2198

2449

1795

550

162

1063

645

1480

287

11,232

0

0

1961

212

43

1922

1458

3867

672

94

1514

1398

1425

375

164

1776

1792

1230

405

390b

1267

896

0

162

44b

1907

2629

194

372

95

2140

1756

2738

590

165

2024

1458

1304

402

493b

422

574

259

105

45b

1645

1649

3210

598

96c

437

1446

2701

443

166

2693

2019

0

354

496b

87

585

0

55

46b

2955

1900

2600

643

97

2096

1792

851

396

167

1019

1243

1489

335

1362b

335

36

204

48

47b

2636

2342

3034

705

98c

1194

2760

194

334

168

320

454

2785

361

3968b

0

550

0

4

48b

2198

2856

0

390

99

1893

2175

315

346

170

742

442

1175

215

4254b

0

0

1138

123

49

2446

2222

157

371

101

1718

2306

2211

550

171

742

382

1499

245

6605b

0

574

0

48

50b

2941

2330

3451

770

102

1310

1350

1563

372

172

495

693

1674

273

14278b

0

48

0

4

51b

2417

2103

3608

732

103

2315

2067

1832

530

176

1048

1040

749

240

16419b

0

0

28

55

TPM templates per million, ME mantle edge, MP mantle pallium, PS pearl sac

aData and gene numbers from Kinoshita et al. (2011)

bGenes subjected to RNAi experiments in the present study

cGenes analyzed in our previous study Funabara et al. (2014)

41.3.2 Observation of the Appearances on the Inner Surface of the Knockdown Oyster Shells

Knockdown of 64 out of 80 genes induced abnormal appearances on the inner surface of the shells (Table 41.2). Among them, 18 knockdown oysters had abnormal appearances on both the prismatic and nacreous layers, 45 only on the nacreous layers, and 1 only on the prismatic layers. The data combined with our previous study are shown in Fig. 41.1. Ninety-four genes, 80 in the present and 14 in our previous studies, contained 78 genes that are suggested to be involved in the shell formation processes. Only one gene changed the surface appearance on the prismatic layer.
Table 41.2

Appearances of the inner surface of shells injected with dsRNAs of the subject genes

Genea

Prismatic

Nacreous

Gene

Prismatic

Nacreous

Gene

Prismatic

Nacreous

1

n

a

39

a

a

79

a

a

2

a

a

42

n

a

92

a

a

3

a

a

44

n

a

108

n

a

5

n

a

45

n

a

111

n

a

6

a

a

46

a

a

112

n

a

7

a

a

47

n

a

114

a

n

8

a

a

48

n

a

115

n

n

9

n

a

50

n

a

390

n

n

10

a

a

51

n

a

493

n

n

11

a

a

52

n

a

496

n

n

12

n

a

53

n

a

1362

n

n

13

a

a

54

n

n

3968

n

n

14

a

a

55

n

a

4254

n

n

15

a

a

56

n

a

6605

n

a

16

n

a

57

n

a

14,278

n

n

17

n

a

59

a

a

16,419

n

n

20

n

a

60

n

a

27b

a

a

22

n

a

61

n

n

31b

a

a

23

a

a

62

n

a

58b

n

a

24

n

a

63

n

a

66b

n

a

25

n

a

64

n

n

81b

a

a

26

n

a

65

n

a

96b

a

a

28

n

a

67

n

n

98b

a

a

29

a

a

68

n

a

113b

n

a

30

n

a

69

n

a

118b

n

a

32

n

a

70

n

a

133b

n

a

33

n

a

71

n

n

145b

n

a

34

n

a

72

n

n

194b

a

a

35

n

a

74

n

a

200b

n

a

36

a

a

75

n

a

411b

n

a

37

n

a

76

n

n

   

38

n

a

77

n

a

   

n normal appearance, a abnormal appearance

aGene numbers from Kinoshita et al. (2011)

bData from Funabara et al. (2014)

Fig. 41.1

The numbers of individuals having normal and abnormal appearances on the inner surface of the shells of the Japanese pearl oysters Pinctada fucata subjected to the RNAi experiments as observed by SEM. “Prismatic and nacreous layers,” “nacreous layers,” “prismatic layers,” and “normal appearances” indicate individuals having abnormal appearances on “both the prismatic and nacreous layers,” “only on the nacreous layers,” “only on the prismatic layers,” and “normal appearances” on the shell inner surface, respectively. Numerals indicate the numbers of the genes

41.4 Discussion

We have obtained the data of gene expression patterns and genes possibly involved in shell formation (Tables 41.1 and 41.2). It is not easy to discuss how genes play roles in shell formation based on expression patterns in the EST and knockdown data. We have only short sequences of the respective genes in the EST data. Full-length sequences or at least open reading frame (ORF) regions of the interest genes are required to discuss their function. To determine the full-length sequences, it is reasonable that we choose genes in descending order of the numbers of their reads in the EST data. We can also search the genome database for their gene models by BLAST searching using the EST sequence data (Takeuchi et al. 2012).

Many studies on shell formation-related proteins have focused on those secreted from mantle tissues into shells. This way is incapable of analyzing regulatory pathways to form shells. We found in our previous study that some shell formation-related genes encoded proteins lacking a signal peptide, suggesting that such cytoplasmic proteins function in shell formation together with secretory ones (Funabara et al. 2014). We have not determined the full-length sequences for the newly identified 64 genes to be involved in shell formation yet. They may contain cytoplasmic proteins which function in shell formation. The combination of transcriptome and knockdown analyses would give us some useful information on the shell formation processes from genes to shells.

References

  1. Funabara D, Ohmori F, Kinoshita S, Koyama H, Mizutani S, Ota A, Osakabe Y, Nagai K, Maeyama K, Okamoto K, Kanoh S, Asakawa S, Watabe S (2014) Novel genes participating in the formation of prismatic and nacreous layers in the pearl oyster as revealed by their tissue distribution and RNA interference knockdown. PLoS One 9:e84706CrossRefGoogle Scholar
  2. Kinoshita S, Wang N, Inoue H, Maeyama K, Okamoto K, Nagai K, Kondo H, Hirono I, Asakawa S, Watabe S (2011) Deep sequencing of ESTs from nacreous and prismatic layer producing tissues and a screen for novel shell formation-related genes in the pearl oyster. PLoS One 6:e21238CrossRefGoogle Scholar
  3. Miyamoto H, Miyashita T, Okushima M, Nakano S, Morita T, Matsushiro A (1996) A carbonic anhydrase from the nacreous layer in oyster pearls. Proc Natl Acad Sci U S A 93:9657–9660CrossRefGoogle Scholar
  4. Suzuki M, Saruwatari K, Kogure T, Yamamoto Y, Nishimura T, Kato T, Nagasawa H (2009) An acidic matrix protein, Pif, is a key macromolecule for nacre formation. Science 325:1388–1390CrossRefGoogle Scholar
  5. Takeuchi T, Kawashima T, Koyanagi R, Gyoja F, Tanaka M, Ikuta T, Shoguchi E, Fujiwara M, Shinzato C, Hisata K, Fujie M, Usami T, Nagai K, Maeyama K, Okamoto K, Aoki H, Ishikawa T, Masaoka T, Fujiwara A, Endo K, Endo H, Nagasawa H, Kinoshita S, Asakawa S, Watabe S, Satoh N (2012) Draft genome of the pearl oyster Pinctada fucata: a platform for understanding bivalve biology. DNA Res 19:117–130CrossRefGoogle Scholar

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Authors and Affiliations

  • Daisuke Funabara
    • 1
  • Fumito Ohmori
    • 2
  • Shigeharu Kinoshita
    • 3
  • Kiyohito Nagai
    • 4
  • Kaoru Maeyama
    • 2
  • Kikuhiko Okamoto
    • 2
  • Satoshi Kanoh
    • 1
  • Shuichi Asakawa
    • 3
  • Shugo Watabe
    • 5
  1. 1.Graduate School of BioresourcesMie UniversityTsuJapan
  2. 2.MIKIMOTO COSMETICSMieJapan
  3. 3.Department of Aquatic Bioscience, Graduate School of Agricultural and Life SciencesThe University of TokyoTokyoJapan
  4. 4.Pearl Research Laboratory, K. MIKIMOTO & CO., LTD.Hamajima, ShimaJapan
  5. 5.School of Marine BiosciencesKitasato UniversityMinami, SagamiharaJapan

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