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Molecular Characterization and Gene Expression Profiling ... - CUSAT

Molecular Characterization and Gene Expression Profiling ... - CUSAT

Molecular Characterization and Gene Expression Profiling ... -

Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps THESIS SUBMITTED TO THE COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY In partial fulfillment of the degree of DOCTOR OF PHILOSOPHY IN MARINE BIOTECHNOLOGY UNDER THE FACULTY OF MARINE SCIENCES BY SWAPNA P. ANTONY DEPT. OF MARINE BIOLOGY, MICROBIOLOGY AND BIOCHEMISTRY SCHOOL OF MARINE SCIENCES COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY KOCHI-682 016, INDIA March, 2011

  • Page 2 and 3: Dedicated to My Family, My Guide &
  • Page 4 and 5: Dr. Rosamma Philip Assistant Profes
  • Page 6 and 7: and Dr. Bijoy Nandan for your help,
  • Page 8 and 9: for your unquestioning support. My
  • Page 10 and 11: 2.2.9 Cloning of the PCR product 11
  • Page 12 and 13: 6.3.2 Expression profile of AMP gen
  • Page 14 and 15: HSV Human Herpes Virus IHHN Infecti
  • Page 16 and 17: CHAPTER-1 General Introduction
  • Page 18 and 19: Chapter 1 Shrimp is one of the most
  • Page 20 and 21: Chapter 1 This species accounts for
  • Page 22 and 23: Common names: Chapter 1 Indian whit
  • Page 24 and 25: Chapter 1 mortalities by favoring c
  • Page 26 and 27: Chapter 1 Cambodia, China, India, I
  • Page 28 and 29: Chapter 1 of the shrimp cephalothor
  • Page 30 and 31: Chapter 1 (Guan et al., 2003; Withy
  • Page 32 and 33: Chapter 1 Field methods: Diagnosis
  • Page 34 and 35: Chapter 1 Dot blot hybridization: T
  • Page 36 and 37: Chapter 1 are acting as immunostimu
  • Page 38 and 39: Chapter 1 shrimp. These products ha
  • Page 40 and 41: Chapter 1 adhesion is involved in a
  • Page 42 and 43: Chapter 1 regulate clotting or mela
  • Page 44 and 45: Chapter 1 have expired or are damag
  • Page 46 and 47: 1.6.3.2 Nodulation Chapter 1 Nodula
  • Page 48 and 49: Chapter 1 appropriate (Cerenius and
  • Page 50 and 51: Chapter 1 lectins and carbohydrates
  • Page 52 and 53:

    Chapter 1 wound healing and haemost

  • Page 54 and 55:

    Chapter 1 induce innate immunity, e

  • Page 56 and 57:

    Chapter 1 quite selective for micro

  • Page 58 and 59:

    1.7.4.3 Invertebrates Chapter 1 Sin

  • Page 60 and 61:

    Chapter 1 number of excellent revie

  • Page 62 and 63:

    Chapter 1 In contrast, temporin L,

  • Page 64 and 65:

    1.7.6.1 AMPs in marine invertebrate

  • Page 66 and 67:

    Chapter 1 On the basis of the charg

  • Page 68 and 69:

    1.7.6.2 AMPs based on charge /natur

  • Page 70 and 71:

    Table 1.4.Various classes of AMPs b

  • Page 72 and 73:

    Chapter 1 and negatively charged cy

  • Page 74 and 75:

    Chapter 1 Lantibiotics contain smal

  • Page 76 and 77:

    Chapter 1 by the independent, not y

  • Page 78 and 79:

    Chapter 1 surfaces secrete AMPs fro

  • Page 80 and 81:

    1.7.10.2 Attachment Chapter 1 Once

  • Page 82 and 83:

    Chapter 1 lipid head groups (Matsuz

  • Page 84 and 85:

    Chapter 1 Fig. 1.10. The toroidal-p

  • Page 86 and 87:

    Chapter 1 but does not have any eff

  • Page 88 and 89:

    Chapter 1 indolicidin has been sugg

  • Page 90 and 91:

    Chapter 1 demonstrated to possess s

  • Page 92 and 93:

    Chapter 1 shown to cause minimal or

  • Page 94 and 95:

    Chapter 1 2004b, 2006). Interesting

  • Page 96 and 97:

    Chapter 1 interact with and permeab

  • Page 98 and 99:

    Chapter 1 structural contributions

  • Page 100 and 101:

    Chapter 1 1.7.12.3 The influence of

  • Page 102 and 103:

    Chapter 1 naturally occurring pepti

  • Page 104 and 105:

    1.7.13.2 Introduction of peptide an

  • Page 106 and 107:

    1.7.13.3 Limitations as therapeutic

  • Page 108 and 109:

    Chapter 1 which they are administer

  • Page 110 and 111:

    1.9 Objectives The present study wa

  • Page 112 and 113:

    2.1. Introduction Chapter 2 Inverte

  • Page 114 and 115:

    Chapter 2 observed in gills, heart

  • Page 116 and 117:

    Chapter 2 ALFPm1-5 (Supungul et al.

  • Page 118 and 119:

    Chapter 2 protein is released from

  • Page 120 and 121:

    Chapter 2 protein can thus, at leas

  • Page 122 and 123:

    Chapter 2 Albores et al., 2004). Ma

  • Page 124 and 125:

    Chapter 2 within cytoplasmic granul

  • Page 126 and 127:

    Chapter 2 20%, of all the sequences

  • Page 128 and 129:

    Chapter 2 assumed that the penaeidi

  • Page 130 and 131:

    Chapter 2 Fig.2.1. Schematic repres

  • Page 132 and 133:

    Chapter 2 These common sense precau

  • Page 134 and 135:

    Chapter 2 10 min. for the target ge

  • Page 136 and 137:

    Chapter 2 adding 200 µl of the lys

  • Page 138 and 139:

    Chapter 2 Peptide model of ALF-2 cr

  • Page 140 and 141:

    Chapter 2 revealed the relation of

  • Page 142 and 143:

    Chapter 2 previous reports on the c

  • Page 144 and 145:

    Chapter 2 end of the crustin-like A

  • Page 146 and 147:

    2.3.1.5.2. Amino acid composition o

  • Page 148 and 149:

    2.3.1.6.3. Phylogenetic analysis of

  • Page 150 and 151:

    Chapter 2 specific nature like phag

  • Page 152 and 153:

    Chapter 2 ALFs bind to cell walls o

  • Page 154 and 155:

    Chapter 2 SWAM2, as antibacterial p

  • Page 156 and 157:

    Chapter 2 As an important antimicro

  • Page 158 and 159:

    Chapter 2 in the shrimp body, by th

  • Page 160 and 161:

    Table.2.2. BLASTn analysis of ALF-1

  • Page 162 and 163:

    Table.2.6. BLASTn analysis of Crust

  • Page 164 and 165:

    Table 2.10. BLASTn analysis of Crus

  • Page 166 and 167:

    Table 2.14. BLASTn analysis of Pena

  • Page 168 and 169:

    Chapter 2 Fig.2.7. Signal peptide a

  • Page 170 and 171:

    Chapter 2 Fig.2.11 A bootstrapped n

  • Page 172 and 173:

    Chapter 2 Fig. 2.13 Nucleotide and

  • Page 174 and 175:

    Chapter 2 Fig.2.16. Multiple alignm

  • Page 176 and 177:

    Chapter 2 Fig. 2.19. Nucleotide and

  • Page 178 and 179:

    A B Chapter 2 Fig. 2.22. ScanProsit

  • Page 180 and 181:

    Chapter 2 Fig.2.25 Multiple alignme

  • Page 182 and 183:

    Chapter 2 Fig. 2.27. A bootstrapped

  • Page 184 and 185:

    Chapter 2 Fig.2.30 ScanProsite anal

  • Page 186 and 187:

    Chapter 2 Fig. 2.34 Multiple alignm

  • Page 188 and 189:

    Chapter 2 Fig.2.36 A bootstrapped n

  • Page 190 and 191:

    Chapter 2 Fig.2.38 ScanProsite anal

  • Page 192 and 193:

    Chapter 2 Fig.2.42 Multiple alignme

  • Page 194 and 195:

    Chapter 2 Fig.2.44 A bootstrapped n

  • Page 196 and 197:

    Chapter 2 Fig.2.47 Multiple alignme

  • Page 198 and 199:

    Chapter 2 AF387660, L. vannamei AF3

  • Page 200 and 201:

    Chapter 2 Fig.2.53 Multiple alignme

  • Page 202 and 203:

    Chapter 2 GQ334397, P. monodon FJ68

  • Page 204 and 205:

    3.1. Introduction Chapter 3 The Ind

  • Page 206 and 207:

    3.2.3. Total RNA isolation and Reve

  • Page 208 and 209:

    3.3.1.1.3. Phylogenetic analysis of

  • Page 210 and 211:

    Chapter 3 bootstrap distance tree c

  • Page 212 and 213:

    Chapter 3 et al., 2002) and Fi-crus

  • Page 214 and 215:

    Chapter 3 sequence to be reported f

  • Page 216 and 217:

    Chapter 3 chinensis, Fi-penaeidin a

  • Page 218 and 219:

    Chapter 3 on AMPs. These molecules

  • Page 220 and 221:

    Chapter 3 three α-helices crowded

  • Page 222 and 223:

    Chapter 3 presence of and α-helix

  • Page 224 and 225:

    Table 3.2. BLASTn analysis of ALF-1

  • Page 226 and 227:

    Table 3.6. BLASTn analysis of Fi-cr

  • Page 228 and 229:

    Table.3.10. BLASTn analysis of pena

  • Page 230 and 231:

    Chapter 3 Fig.3.4. Multiple alignme

  • Page 232 and 233:

    Chapter 3 Fig.3.7. A bootstrapped n

  • Page 234 and 235:

    Chapter 3 Fig.3.10 Signal peptide a

  • Page 236 and 237:

    Chapter 3 Fig.3.13. A bootstrapped

  • Page 238 and 239:

    Chapter 3 Fig.3.15. Nucleotide and

  • Page 240 and 241:

    Chapter 3 Fig.3.20 Multiple alignme

  • Page 242 and 243:

    Chapter 3 Fig.3.22. A bootstrapped

  • Page 244 and 245:

    Chapter 3 Fig.3.24. Nucleotide and

  • Page 246 and 247:

    Chapter 3 Fig.3.27 Multiple alignme

  • Page 248 and 249:

    Chapter 3 Fig.3.29. Bootstrapped ne

  • Page 250 and 251:

    Chapter 3 penaeidins (L. schmitti A

  • Page 252 and 253:

    Chapter 3 Fig.3.34. Multiple alignm

  • Page 254 and 255:

    Chapter 3 Fig.3.36 Bootstrapped nei

  • Page 256 and 257:

    CHAPTER-4 Time-Course of Antimicrob

  • Page 258 and 259:

    Chapter 4 with infectious and nonin

  • Page 260 and 261:

    Chapter 4 including Penaeus monodon

  • Page 262 and 263:

    Chapter 4 Temporal regulatory genes

  • Page 264 and 265:

    Chapter 4 initially in the first 2

  • Page 266 and 267:

    Chapter 4 in this study. It remains

  • Page 268 and 269:

    4.2. Materials and Methods 4.2.1. E

  • Page 270 and 271:

    Chapter 4 4.2). Control genes viz.

  • Page 272 and 273:

    Expression profile of penaeidin-3 g

  • Page 274 and 275:

    Chapter 4 gene transcripts were fou

  • Page 276 and 277:

    Chapter 4 tiger shrimp P. monodon c

  • Page 278 and 279:

    Chapter 4 time-course basis. Analys

  • Page 280 and 281:

    Chapter 4 haemocytes of crustaceans

  • Page 282 and 283:

    Chapter 4 shrimp cells and its comp

  • Page 284 and 285:

    Chapter 4 Taken together, the prese

  • Page 286 and 287:

    (A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C

  • Page 288 and 289:

    (A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C

  • Page 290 and 291:

    (A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C

  • Page 292 and 293:

    (A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C

  • Page 294 and 295:

    (A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C

  • Page 296 and 297:

    (A) (B) B 28 1 2 3 4 5 6 7 8 9 10 F

  • Page 298 and 299:

    (A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C

  • Page 300 and 301:

    (A) (B) Chapter 4 Fig. 4.17. Time-c

  • Page 302 and 303:

    (A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C

  • Page 304 and 305:

    5.1. Introduction Chapter 5 Penaeid

  • Page 306 and 307:

    Chapter 5 Important biotechnologica

  • Page 308 and 309:

    Chapter 5 However, very few works h

  • Page 310 and 311:

    5.2.2.3. Glucan extraction Chapter

  • Page 312 and 313:

    5.2.4. Haemolymph and other tissues

  • Page 314 and 315:

    5.3.2.1. Expression profile of ALF

  • Page 316 and 317:

    Chapter 5 for a slight down-regulat

  • Page 318 and 319:

    Chapter 5 On challenge with WSSV, a

  • Page 320 and 321:

    Chapter 5 5.3.5. Expression of WSSV

  • Page 322 and 323:

    Chapter 5 concluded that, although

  • Page 324 and 325:

    Chapter 5 quantifying an mRNA trans

  • Page 326 and 327:

    Chapter 5 to subgroups 3 and 5 pres

  • Page 328 and 329:

    Chapter 5 β-glucan as immunostimul

  • Page 330 and 331:

    Chapter 5 et al., 2007a). However,

  • Page 332 and 333:

    (A) (B) (C) C CHY CSY CHG CSG C CHY

  • Page 334 and 335:

    (A) (B) (C) C CHY CSY CHG CSG C CHY

  • Page 336 and 337:

    (A) (B) (C) C CHY CSY CHG CSG C CHY

  • Page 338 and 339:

    (A) (B) (C) C CHY CSY CHG CSG C CHY

  • Page 340 and 341:

    (A) (B) (C) C CHY CSY CHG CSG C CHY

  • Page 342 and 343:

    (A) (B) -VE CTRL +VE CTRL CHG CSG C

  • Page 344 and 345:

    (A) (B) -VE CTRL +VE CTRL CHG CSG C

  • Page 346 and 347:

    (A) (B) Chapter 5 Fig.5.15. Express

  • Page 348 and 349:

    (A) (B) -VE CTRL +VE CTRL CHG CSG C

  • Page 350 and 351:

    (A) PRE-CHALLENGE (B) POST-CHALLENG

  • Page 352 and 353:

    (C) (D) (A) PRE-CHALLENGE (B) CONTR

  • Page 354 and 355:

    (C) (D) (A) PRE-CHALLENGE (B) CONTR

  • Page 356 and 357:

    (A) (B) G M HP HR I Chapter 5 Fig.

  • Page 358 and 359:

    CHAPTER-6 Expression Profile of Ant

  • Page 360 and 361:

    Chapter 6 organisms, probiotics are

  • Page 362 and 363:

    Chapter 6 suppress the growth of pa

  • Page 364 and 365:

    Chapter 6 Six animals from each gro

  • Page 366 and 367:

    Chapter 6 produced in the control g

  • Page 368 and 369:

    Chapter 6 On WSSV challenge, a noti

  • Page 370 and 371:

    Chapter 6 ALF gene was found to be

  • Page 372 and 373:

    Chapter 6 On challenge with WSSV, a

  • Page 374 and 375:

    Chapter 6 Bacillus and Micrococcus,

  • Page 376 and 377:

    Chapter 6 gram-positive bacteria, P

  • Page 378 and 379:

    Chapter 6 probiotic bacteria applie

  • Page 380 and 381:

    Chapter 6 it is clear that ALF tran

  • Page 382 and 383:

    Chapter 6 This work proves that pro

  • Page 384 and 385:

    (A) (B) (C) Chapter 6 Fig. 6.2. Exp

  • Page 386 and 387:

    (A) (B) (C) C B M BM C B M BM Chapt

  • Page 388 and 389:

    (A) (B) (C) Fig. 6.6. Expression pr

  • Page 390 and 391:

    (A) (B) (C) Fig. 6.8. Expression pr

  • Page 392 and 393:

    (A) (B) (C) Chapter 6 Fig. 6.10. Ex

  • Page 394 and 395:

    (A) (B) -VE CTRL +VE CTRL B M BM EN

  • Page 396 and 397:

    (A) (B) -VE CTRL +VE CTRL B M BM Ch

  • Page 398 and 399:

    (A) (B) Chapter 6 Fig. 6.16. Expres

  • Page 400 and 401:

    (A) (B) -VE CTRL +VE CTRL B M BM Fi

  • Page 402 and 403:

    (C) (D) (A) PRE-CHALLENGE (B) POST-

  • Page 404 and 405:

    (C) (D) (A) PRE-CHALLENGE (B) POST-

  • Page 406 and 407:

    (A) PRE-CHALLENGE (B) POST-CHALLENG

  • Page 408 and 409:

    (A) (B) G M HP HR I Chapter 6 Fig.

  • Page 410 and 411:

    CHAPTER-7 Summary & Conclusion

  • Page 412 and 413:

    Chapter 7 their expression will pro

  • Page 414 and 415:

    � β-actin - GQ334394 � ELF - G

  • Page 416 and 417:

    Chapter 7 � Though all the three

  • Page 418 and 419:

    REFERENCES

  • Page 420 and 421:

    References of a crustin-like antimi

  • Page 422 and 423:

    References Aspan, A., Huang, T.S.,

  • Page 424 and 425:

    References Battafarano, R.J., Dahlb

  • Page 426 and 427:

    References Boman, H.G., Faye, I., G

  • Page 428 and 429:

    References Brogden, K.A., Ackermann

  • Page 430 and 431:

    References Canesi, L., Gallo, G., G

  • Page 432 and 433:

    References East, A.V., Lester, L.J.

  • Page 434 and 435:

    References Chiou, T.T., Lu, J.K., W

  • Page 436 and 437:

    References Cuthbertson, B.J., Bulle

  • Page 438 and 439:

    References antimicrobial peptides f

  • Page 440 and 441:

    References Elrod-Erickson, M., Mish

  • Page 442 and 443:

    References Fehlbaum, P., Bulet, P.,

  • Page 444 and 445:

    References Gallo, R.L., Murakami, M

  • Page 446 and 447:

    References terminal bisphosphorylat

  • Page 448 and 449:

    References Hallock, K.J., Lee, D.K.

  • Page 450 and 451:

    References He, K., Ludtke, S.J., Wo

  • Page 452 and 453:

    References Hose, J.E., Martin, G.G.

  • Page 454 and 455:

    References Imjongjirak, C., Amparyu

  • Page 456 and 457:

    References Jenssen, H. 2005. Anti h

  • Page 458 and 459:

    References Jones, D.E., Bevins, C.L

  • Page 460 and 461:

    References Khoo, L., Robinette, D.W

  • Page 462 and 463:

    References Ladokhin, A.S. 1999. Ana

  • Page 464 and 465:

    References Lehrer, R.I., Lichtenste

  • Page 466 and 467:

    References Lightner, D.V., Redman,

  • Page 468 and 469:

    References syndrome baculovirus WSB

  • Page 470 and 471:

    References Lustig, F., Hoebeke, J.,

  • Page 472 and 473:

    References Antimicrobial Peptides M

  • Page 474 and 475:

    References Mitta, G., Hubert, F., D

  • Page 476 and 477:

    References Murakami, M., Lopez-Garc

  • Page 478 and 479:

    References Nile, C.J., Townes, C.L.

  • Page 480 and 481:

    References Papagianni, M. 2003. Rib

  • Page 482 and 483:

    References Pillay, T.V.R. 1998. Aqu

  • Page 484 and 485:

    References from Kuruma Shrimp Marsu

  • Page 486 and 487:

    References Robertson, P.A.W., Calde

  • Page 488 and 489:

    References In: Singh, I.S.B., Pai,

  • Page 490 and 491:

    References Scott, M.G., Hancock, R.

  • Page 492 and 493:

    References environmental studies, C

  • Page 494 and 495:

    References Soderhall, K., Smith, V.

  • Page 496 and 497:

    References Sritunyalucksana, K., So

  • Page 498 and 499:

    References Sugitha, H., Matsuo, N.,

  • Page 500 and 501:

    References Tamamura, H., Ishihara,

  • Page 502 and 503:

    References of prevalence of white s

  • Page 504 and 505:

    References Tzou, P., de Gregorio, E

  • Page 506 and 507:

    References Vargas-Albores, F., Yepi

  • Page 508 and 509:

    References Wang, C.H., Lo, C.F., Le

  • Page 510 and 511:

    References Wei, X., Decker, J.M., L

  • Page 512 and 513:

    References various tissues of black

  • Page 514 and 515:

    References Yang, D., Biragyn, A., K

  • Page 516 and 517:

    References Yu, Z.M., Li, C.W., Guan

  • Page 518 and 519:

    GENBANK ACCESSIONS

  • Page 520 and 521:

    Penaeus monodon anti-lipopolysachar

  • Page 522 and 523:

    Penaeus monodon crustin-like antimi

  • Page 524 and 525:

    Penaeus monodon penaeidin-like mRNA

  • Page 526 and 527:

    Penaeus monodon beta-actin mRNA, pa

  • Page 528 and 529:

    Fenneropenaeus indicus anti-lipopol

  • Page 530 and 531:

    Fenneropenaeus indicus crustin-like

  • Page 532 and 533:

    Fenneropenaeus indicus penaeidin-li

  • Page 534 and 535:

    Fenneropenaeus indicus elongation f

  • Page 536 and 537:

    International: List of Publications

  • Page 538 and 539:

    atg cta aag ttt gta gta tta tcc gtt

  • Page 540 and 541:

    amino acid sequences of crustins al

  • Page 542 and 543:

    Immunobiology 216 (2011) 184-194 Co

  • Page 544 and 545:

    186 S.P. Antony et al. / Immunobiol

  • Page 546 and 547:

    188 S.P. Antony et al. / Immunobiol

  • Page 548 and 549:

    190 S.P. Antony et al. / Immunobiol

  • Page 550 and 551:

    192 S.P. Antony et al. / Immunobiol

  • Page 552:

    194 S.P. Antony et al. / Immunobiol

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