Molecular Characterization and Gene Expression Profiling ... - CUSAT

More documents

Recommendations

Info

Chapter 4 Taken together, the present results show that WSSV infection modulates AMP gene expression in haemocytes of WSSV challenged P. monodon. The over expression of AMP genes, such as ALF and crustin-3 in WSSV-challenged animals, strongly suggests that these genes could be postulated as potential candidates that are involved in anti-WSSV defense in shrimps by being over expressed on WSSV infection. This implies that these peptide molecules are involved in defense mechanisms in P. monodon to WSSV invasion. This basic knowledge will provide information on AMP genes involved in bio-defence of shrimps against virus invasion. Detailed study on these molecules will allow further exploration of their particular role in the anti-WSSV response and hopefully lead to a better understanding of the disease process and survival in these animals. The information may also be of great value for a novel prophylactic strategy in shrimp culture. Table 4.1. Rearing conditions and water quality parameters Tank capacity 500 l Stocking density 15 Nos. Feeding level 4-6 % of body weight Feeding frequency Twice daily Water temperature 24-27 0 C pH 7.5-8 Salinity 15-18 %0 Ammonia 0.01-0.02 mg l -1 Nitrite 0.00-0.01 mg l -1 Nitrate Below detectable level Alkalinity 50-60 mg/l CaCO3 Dissolved oxygen 6-7 mg l -1 Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 265
Table 4.2. Primers used for the study Target Gene 18s rRNA β-actin ELF ALF Crustin-1 Crustin-2 Crustin-3 Penaeidin-3 Penaeidin-5 DNA polymerase Endonuclease Immediate Early Gene Latency Related Gene Protein Kinase Ribonucleotid e Reductase Thymidine Kinase VP 28 Primer Sequence (5' - 3') F- ttgtacgaggatcgagtgga R- atgctttcgcagtaggtcgt F- cttgtggttgacaatggctccg R-tggtgaaggagtagccacgctc F- atggttgtcaactttgcccc R- ttgacctccttgatcacacc F- caagggtgggaggctgtgg R- tgagctgagccactggttgg F- cgcacagccgagagaaacactatcaagat R- ggcctatccctcagaacccagcacg F- tgttcccacgacttcaagtgtgc R- caaagattcaactaaataaacag F- tccctggaggtcaattgagtg R- agtcgaacatgcaggcctatcc F- aggatatcatccagttcctg R- acctacatcctttccacaag F- acctgaccctcacctgcagaggcc R- ttcgttgtcttctccatcaacc F- tgggaagaaagatgcgagag R- rccctccgaacaacatctcag F- tgacgaggaggattgtaaag R- ttatggttctgtatttgagg F- gactctacaaatctctttgcca R- ctacctttgcaccaattgctag F- cttgtgggaaaagggtcctc R- tcgtcaaggcttacgtgtc F- ggagggtggggaccaacggacaaaac R- caaattgacagtagagaattttgcac F- atctgctagtccctgcacac R- aaagaggtggtgaaggcacg F- gagcagccatacgggtaaac R- gcgagcgtctaccttaatcc F- ctgctgtgattgctgtattt R- cagtgccagagtaggtgac Product Size (bp) CONTROL GENES Annealing Temp. ( 0C) 350 bp 52 2 520 bp 60 1.5 600 bp 60 1.5 AMP GENES 300 bp WSSV GENES 62 1.5 456 bp 55 3.5 299 bp 60 3.5 233 bp 60 1.5 240 bp 60 1.5 300 bp 60 1.5 586 bp 54 1.5 408 bp 50 1.5 502 bp 54 1.5 647 bp 53 1.5 512 bp 55 1.5 408 bp 53 1.5 412 bp 54 1.5 555 bp 54 1.5 Chapter 4 Final MgCl2 Conc. (mM) Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 266 Reference Bustin et al., 2002 Supungul et al., 2004 Loongyai et al., 2007 Tharantada et al., 2008 Supungul et al., 2004 Chen et al., 2004a Jimenez- Vega et al., 2004 Jiravanichp aisal et al., 2007 Chen et al., 2004b Liu et al., 2005 Liu et al., 2005 Liu et al., 2005 Liu et al., 2005 Liu et al., 2005 Liu et al., 2005 Liu et al., 2005 Liu et al., 2005
Page 1 and 2:
Molecular Characterization and Gene
Page 3 and 4:
Declaration I hereby do declare tha
Page 5 and 6:
Acknowledgements This work was carr
Page 7 and 8:
My Seniors, Dr. Annies Joseph, Dr.
Page 9 and 10:
Contents Title Page Nos. 1 General
Page 11 and 12:
in response to WSSV challenge 252-2
Page 13 and 14:
α-2M Alpha-2-Macroglobulin ALF Ant
Page 15 and 16:
ppA Prophenoloxidase-Activating Enz
Page 17 and 18:
1.1 Introduction Chapter 1 Aquacult
Page 19 and 20:
Chapter 1 the giant tiger shrimp (P
Page 21 and 22:
Common names: Chapter 1 Giant tiger
Page 23 and 24:
1.3.1. Diseases Chapter 1 Diseases
Page 25 and 26:
Lymphoidal parvo-like virus (LPV) B
Page 27 and 28:
Chapter 1 and protective occlusion
Page 29 and 30:
Chapter 1 small brown masses in the
Page 31 and 32:
1.4.6. Natural epidemics Chapter 1
Page 33 and 34:
Chapter 1 can differentiate WSSV-in
Page 35 and 36:
1.5.1.1. Probiotics Chapter 1 Alter
Page 37 and 38:
Chapter 1 tried in P. monodon, resu
Page 39 and 40:
Chapter 1 The first and essential i
Page 41 and 42:
Chapter 1 1998). The innate immunit
Page 43 and 44:
1.6.1 Haemocytes Chapter 1 The haem
Page 45 and 46:
Chapter 1 In crustaceans, the sheet
Page 47 and 48:
Chapter 1 Theopold et al., 2002). C
Page 49 and 50:
Chapter 1 kill/remove the invading
Page 51 and 52:
Chapter 1 forming complexes with a
Page 53 and 54:
1.7 Antimicrobial Peptides (AMPs) 1
Page 55 and 56:
Chapter 1 the response is very fast
Page 57 and 58:
Chapter 1 Hirsch, 1947). While they
Page 59 and 60:
Chapter 1 AMPs to various degrees u
Page 61 and 62:
1.7.5 Biological activity Chapter 1
Page 63 and 64:
Chapter 1 including the microbes as
Page 65 and 66:
Tachyplesin I Chapter 1 Crustacea P
Page 67 and 68:
Anionic and cationic peptides that
Page 69 and 70:
Anionic and Cationic AMPs with cyst
Page 71 and 72:
Chapter 1 Fig. 1.7. Structural clas
Page 73 and 74:
Chapter 1 Group III: Linear peptide
Page 75 and 76:
Chapter 1 through regular processes
Page 77 and 78:
Chapter 1 ultimately signal to tran
Page 79 and 80:
Chapter 1 divalent polyanionic cati
Page 81 and 82:
Chapter 1 type of transmembrane por
Page 83 and 84:
Chapter 1 other intracellular targe
Page 85 and 86:
Chapter 1 Apidaecin, a short, proli
Page 87 and 88:
Chapter 1 Hultmark, 1987; Gennaro a
Page 89 and 90:
Chapter 1 quite opposite characteri
Page 91 and 92:
Chapter 1 depletion of mitochondria
Page 93 and 94:
Chapter 1 conformations adopted by
Page 95 and 96:
Chapter 1 receptor may be a potenti
Page 97 and 98:
Chapter 1 exposure to their targets
Page 99 and 100:
Chapter 1 generating a rich spectru
Page 101 and 102:
Chapter 1 by enkelytin (Goumon et a
Page 103 and 104:
Lactoferricin Cow Mastitis control
Page 105 and 106:
Chapter 1 trial, in which peptide T
Page 107 and 108:
Chapter 1 a too limited spectrum to
Page 109 and 110:
Chapter 1 number of research groups
Page 111 and 112:
CHAPTER-2 Molecular Characterizatio
Page 113 and 114:
Chapter 2 interaction with negative
Page 115 and 116:
Chapter 2 inhibit the endotoxin or
Page 117 and 118:
Chapter 2 structure be modified to
Page 119 and 120:
Chapter 2 and the region might be l
Page 121 and 122:
Chapter 2 been made of the spectra
Page 123 and 124:
Chapter 2 their discovery and initi
Page 125 and 126:
Chapter 2 terminal pyroglutamic aci
Page 127 and 128:
Chapter 2 One can assume that the p
Page 129 and 130:
Chapter 2 by degranulation into the
Page 131 and 132:
2.2. Materials and Methods 2.2.1. E
Page 133 and 134:
Chapter 2 washed by adding 1 ml 75
Page 135 and 136:
Chapter 2 entry of recombinant DNA
Page 137 and 138:
Chapter 2 and the deduced amino aci
Page 139 and 140:
Chapter 2 Fenneropenaeus, Litopenae
Page 141 and 142:
2.3.1.3. Crustin-1 (GQ334395) Chapt
Page 143 and 144:
2.3.1.3.5. Phylogenetic analysis of
Page 145 and 146:
Chapter 2 sequence also showed high
Page 147 and 148:
Chapter 2 chinensis and F. indicus
Page 149 and 150:
Chapter 2 monodon and Group III of
Page 151 and 152:
Chapter 2 Tiger shrimp (P. monodon)
Page 153 and 154:
Chapter 2 ALF-1 was a partial seque
Page 155 and 156:
Chapter 2 As mentioned before, the
Page 157 and 158:
Chapter 2 Munoz et al., 2004). The
Page 159 and 160:
Table 2.1. Primers used for the stu
Page 161 and 162:
Table 2.4. BLASTn analysis of ALF-2
Page 163 and 164:
Table 2.8. BLASTn analysis of Crust
Page 165 and 166:
Table 2.12. BLASTn analysis of Pena
Page 167 and 168:
Fig.2.5. Agarose gel electrophoreto
Page 169 and 170:
Chapter 2 Fig.2.9. Multiple alignme
Page 171 and 172:
Chapter 2 Fig. 2.12 A bootstrapped
Page 173 and 174:
Chapter 2 Fig. 2.15. Multiple align
Page 175 and 176:
Chapter 2 Fig.2.18. A bootstrapped
Page 177 and 178:
Chapter 2 Fig.2.20. Signal peptide
Page 179 and 180:
Chapter 2 Fig.2.24 Multiple alignme
Page 181 and 182:
Chapter 2 LITOPENAEUS SP. FARFANTEP
Page 183 and 184:
Chapter 2 Fig. 2.28. Nucleotide and
Page 185 and 186:
Page 187 and 188:
Chapter 2 LITOPENAEUS SP. FENNEROPE
Page 189 and 190:
Page 191 and 192:
Page 193 and 194:
Chapter 2 Fig.2.43 A bootstrapped n
Page 195 and 196:
Page 197 and 198:
Chapter 2 LITOPENAEUS SP. FENNEROPE
Page 199 and 200:
Chapter 2 Fig.2.51 Signal peptide a
Page 201 and 202:
Chapter 2 FENNEROPENAEUS SP. FARFAN
Page 203 and 204:
CHAPTER-3 Molecular Characterizatio
Page 205 and 206:
Chapter 3 certainly help us to unra
Page 207 and 208:
Chapter 3 3.3.1.1. Anti-lipopolysac
Page 209 and 210:
Chapter 3 was predicted out to be 1
Page 211 and 212:
Chapter 3 random coiled structure t
Page 213 and 214:
3.3.1.3.5. Phylogenetic analysis of
Page 215 and 216:
Chapter 3 case has been reported in
Page 217 and 218:
Chapter 3 Multiple alignments were
Page 219 and 220:
Chapter 3 negative bacteria. In shr
Page 221 and 222:
Chapter 3 indicated a random coiled
Page 223 and 224:
Chapter 3 horseshoe crab big defens
Page 225 and 226:
Table 3.4. BLASTn analysis of ALF-2
Page 227 and 228:
Table.3.8. BLASTn analysis of Fi-pe
Page 229 and 230:
Chapter 3 Fig.3.2. Nucleotide and a
Page 231 and 232:
Chapter 3 Fig.3.5. Multiple alignme
Page 233 and 234: Chapter 3 Fig.3.8. Nucleotide and a
Page 235 and 236: Chapter 3 Fig.3.12. Multiple alignm
Page 237 and 238: Chapter 3 Fig.3.14. A bootstrapped
Page 239 and 240: Chapter 3 Fig.3.17. Signal peptide
Page 243 and 244: SHRIMPS KIND CRAB LOBSTERS CRABS Ch
Page 245 and 246: Chapter 3 Fig.3.25. Signal peptide
Page 247 and 248: Chapter 3 Fig. 3.28. Multiple align
Page 249 and 250: PENAEIDIN-2 Chapter 3 PENAEIDIN- 3
Page 251 and 252: Fig.3.31. Nucleotide and amino acid
Page 255 and 256: Chapter 3 Fig.3.37 Nucleotide and a
Page 257 and 258: 4.1. Introduction Chapter 4 The aqu
Page 259 and 260: Chapter 4 appearance and have major
Page 261 and 262: Chapter 4 the tissues has recently
Page 263 and 264: Chapter 4 with the ability of shrim
Page 265 and 266: Chapter 4 with a low dose of WSSV b
Page 267 and 268: Chapter 4 molecules they are likely
Page 269 and 270: Chapter 4 for understanding gene ex
Page 271 and 272: Chapter 4 4.3.2. Expression profile
Page 273 and 274: Expression profile of endonuclease
Page 275 and 276: Chapter 4 bacterial and fungal infe
Page 277 and 278: Chapter 4 measuring TSV and YHV loa
Page 279 and 280: Chapter 4 Of particular interest he
Page 281 and 282: Chapter 4 large distribution and ab
Page 283: Chapter 4 WSSV genes viz. endonucle
Page 287 and 288: (A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
Page 289 and 290: (A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
Page 291 and 292: (A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
Page 293 and 294: (A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
Page 295 and 296: (A) (B) B 28 1 2 3 4 5 6 7 8 9 10 F
Page 297 and 298: (A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
Page 299 and 300: (A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
Page 301 and 302: (A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
Page 303 and 304: CHAPTER-5 Expression Profile of Ant
Page 305 and 306: Chapter 5 1994). The emergence of b
Page 307 and 308: Chapter 5 (Bovill et al., 2001). In
Page 309 and 310: 5.2.2. Test diets Chapter 5 Four ex
Page 311 and 312: 5.2.2.4. Preparation of Glucan inco
Page 313 and 314: Chapter 5 genes (latency related ge
Page 315 and 316: Chapter 5 On WSSV challenge, crusti
Page 317 and 318: Chapter 5 5.3.4. Expression profile
Page 319 and 320: 5.3.4.5. Expression profile of pena
Page 321 and 322: Chapter 5 must firstly rest on a so
Page 323 and 324: Chapter 5 For the present study two
Page 325 and 326: Chapter 5 ameobocytes of two marine
Page 327 and 328: Chapter 5 variation in expression o
Page 329 and 330: Chapter 5 It has been previously sh
Page 331 and 332: Chapter 5 manifested in terms of gr
Page 333 and 334: (A) (B) (C) C CHY CSY CHG CSG C CHY
Page 335 and 336:
(A) (B) (C) C CHY CSY CHG CSG C CHY
Page 337 and 338:
Page 339 and 340:
Page 341 and 342:
(A) (B) -VE CTRL +VE CTRL CHG CSG C
Page 343 and 344:
Page 345 and 346:
Page 347 and 348:
Page 349 and 350:
(A) PRE-CHALLENGE (B) POST-CHALLENG
Page 351 and 352:
(A) PRE-CHALLENGE (B) (C) (D) CONTR
Page 353 and 354:
(C) (D) (A) PRE-CHALLENGE (B) CONTR
Page 355 and 356:
(A) (B) G M HP HR I Chapter 5 Fig.
Page 357 and 358:
Chapter 5 Fig. 5.26. Post challenge
Page 359 and 360:
6.1. Introduction Chapter 6 Prophyl
Page 361 and 362:
Chapter 6 production of inhibitory
Page 363 and 364:
6.2. Materials and methods 6.2.1. E
Page 365 and 366:
Chapter 6 tissue cDNA was performed
Page 367 and 368:
Chapter 6 gene was supported by Bac
Page 369 and 370:
Chapter 6 treated group of shrimps.
Page 371 and 372:
Chapter 6 the gene was found to be
Page 373 and 374:
Chapter 6 found to support minimum
Page 375 and 376:
Chapter 6 is induced upon bacterial
Page 377 and 378:
Chapter 6 all the target genes, exc
Page 379 and 380:
Chapter 6 Detailed tissue-wise anal
Page 381 and 382:
Chapter 6 Gills and intestine was f
Page 383 and 384:
(A) (B) Fig. 6.1. Experimental diet
Page 385 and 386:
(A) (B) (C) Fig. 6.3. Expression pr
Page 387 and 388:
(A) (B) (C) C B M BM C B M BM Fig.
Page 389 and 390:
(A) (B) (C) Fig. 6.7. Expression pr
Page 391 and 392:
(A) (B) (C) Chapter 6 Fig. 6.9. Exp
Page 393 and 394:
(A) (B) -VE CTRL +VE CTRL B M BM Ch
Page 395 and 396:
Page 397 and 398:
Page 399 and 400:
Page 401 and 402:
Page 403 and 404:
Page 405 and 406:
(A) (C) (D) PRE-CHALLENGE POST-CHAL
Page 407 and 408:
(A) (B) G M HP HR I Chapter 6 Fig.
Page 409 and 410:
Chapter 6 Fig. 6.27. Post challenge
Page 411 and 412:
7.1. Summary Chapter 7 Tropical shr
Page 413 and 414:
Salient findings Chapter 7 � From
Page 415 and 416:
Chapter 7 � Tissue-wise expressio
Page 417 and 418:
Chapter 7 application in shrimp cul
Page 419 and 420:
References Aboudy, Y., Mendelson, E
Page 421 and 422:
References Anggraeni, M.S., Owens,
Page 423 and 424:
References Bals, R., Wang, X., Zasl
Page 425 and 426:
References derived from the N-termi
Page 427 and 428:
References Breukink, E., de Kruijff
Page 429 and 430:
References Burgents, J.E., Burnett,
Page 431 and 432:
References 1,3-β-D-glucan-binding
Page 433 and 434:
References Chen, J.Y., Chuang, H.,
Page 435 and 436:
References Cole, A.M., Hong, T., Bo
Page 437 and 438:
References Dathe, M., Wieprecht, T.
Page 439 and 440:
References Diamond, G., Zasloff, M.
Page 441 and 442:
References Faber, C., Stallmann, H.
Page 443 and 444:
References Flint, S.J., Enquist, L.
Page 445 and 446:
References Gennaro, R., Zanetti, M.
Page 447 and 448:
References Gudmundsson, G.H., Lidho
Page 449 and 450:
References Harwig, S.S., Swiderek,
Page 451 and 452:
References Hirakura, Y., Kobayashi,
Page 453 and 454:
References Huang, C.C., Song, Y.L.
Page 455 and 456:
References Itami, T., Takahashi, Y.
Page 457 and 458:
References Jiravanichpaisal, P. Whi
Page 459 and 460:
References Kang, J.H., Shin, S.Y.,
Page 461 and 462:
References Kono, T., Savan, R., Sak
Page 463 and 464:
References rich peptide derived fro
Page 465 and 466:
References Li, L., Wang, J.X., Zhao
Page 467 and 468:
References Liu, C.H., Cheng, W., Ku
Page 469 and 470:
References Lorenzon, S., de Guarrin
Page 471 and 472:
References Marone, M., Mozzetti, S.
Page 473 and 474:
References Medvinsky, A., Dzierzak,
Page 475 and 476:
References Moriarty, D.J.W. 1996. M
Page 477 and 478:
References antimicrobial peptide fr
Page 479 and 480:
References Otta, S.K., Karunasagar,
Page 481 and 482:
References simplex virus has heptad
Page 483 and 484:
References infected shrimp. Thesis
Page 485 and 486:
References Litopenaeus vannamei cha
Page 487 and 488:
References Roth, B.L., Poot, M., Yu
Page 489 and 490:
References Sathish, S., Musthaq, S.
Page 491 and 492:
References a proline-arginine-rich
Page 493 and 494:
References Smith, V.J., Fernandes,
Page 495 and 496:
References Song, Y.L., Cheng, W., W
Page 497 and 498:
References experimental system. In:
Page 499 and 500:
References the black tiger shrimp P
Page 501 and 502:
References Tapay, L.M., Cesar, E.,
Page 503 and 504:
References Touhy, K.M., Probert, H.
Page 505 and 506:
References van Hulten, M.C., Reijns
Page 507 and 508:
References Vives, R.R., Imberty, A.
Page 509 and 510:
References Wang, Y.C., Chang, P.S.,
Page 511 and 512:
References Williams, M.J., Rodrigue
Page 513 and 514:
References Wu, W., Zong, R., Xu, J.
Page 515 and 516:
References Yodmuang, S., Tirasophon
Page 517 and 518:
References Zhan, W.B., Wang, Y.H. 1
Page 519 and 520:
Penaeus monodon anti-lipopolysaccha
Page 521 and 522:
Penaeus monodon crustin-like antimi
Page 523 and 524:
Penaeus monodon crustin-like antimi
Page 525 and 526:
Penaeus monodon penaeidin-like anti
Page 527 and 528:
Penaeus monodon elongation factor m
Page 529 and 530:
Fenneropenaeus indicus anti-lipopol
Page 531 and 532:
Fenneropenaeus indicus penaeidin-li
Page 533 and 534:
Fenneropenaeus indicus beta-actin m
Page 535 and 536:
PUBLICATIONS
Page 537 and 538:
Short sequence report Molecular cha
Page 539 and 540:
Fig. 2. Multiple alignment of nucle
Page 541 and 542:
220 The phylogenetic relationships
Page 543 and 544:
Table 1 Rearing conditions and wate
Page 545 and 546:
5 ′ cttgtggttgacaatggctccg3
Page 547 and 548:
Expression of WSSV genes in the hae
Page 549 and 550:
Fig. 4. Expression profile of crust
Page 551 and 552:
S.P. Antony et al. / Immunobiology
show all

Molecular Characterization and Gene Expression Profiling ... - CUSAT

Create successful ePaper yourself

Delete template?

Save as template?