Molecular Characterization and Gene Expression Profiling ... - CUSAT

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Chapter 2 in the shrimp body, by the presence of haemocytes, could combine antimicrobial and chitin-binding properties that may be important in interactions between immune function and developmental function through the synthesis of exoskeleton in shrimp. These possible multifunctional properties of antimicrobial peptides represent an important new area to be investigated. The study of such AMP functions and expression in shrimp is particularly important for fundamental understanding of shrimp immunity and for further establishment of disease control in shrimp aquaculture (Bachere, 2003). Any progress in this field would contribute to a better understanding of the penaeid shrimp physiology and of their capacity to respond to pathological injuries (Bachere et al., 2000b). Moreover, from their unusual features both at the structural level and in terms of biological activity, penaeidins could represent, along with other AMPs, a new generation of therapeutic agents and find potential applications in aquaculture. The recent discovery of AMPs in crustaceans provides new clues for fundamental understanding of crustacean immunity. The study of such AMP functions and expression in shrimp is particularly important for fundamental understanding of shrimp immunity and for further establishment of disease control in shrimp aquaculture (Bachere, 2003). Moreover, from their unusual features both at the structural level and in terms of biological activity, AMPs represent a new generation of therapeutic agents and find potential applications in aquaculture. Moreover, AMP represents a new generation of therapeutic agents with potential applications in aquaculture. Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 141
Table 2.1. Primers used for the study Target gene Sequence (5’-3’) ALF-1 F- gcacgagggagcttcatatt R- gagcaaagggcctatgagtta ALF-2 F- caagggtgggaggctgtgg R- tgagctgagccactggttgg Crustin-1 F- cgcacagccgagagaaacactatcaagat Crustin-2 Crustin-3 Penaeidin-3 Penaeidin-5 R- ggcctatccctcagaacccagcacg F- tgttcccacgacttcaagtgtgc R- caaagattcaactaaataaacag F- tccctggaggtcaattgagtg R- agtcgaacatgcaggcctatcc F- aggatatcatccagttcctg R- acctacatcctttccacaag F- acctgaccctcacctgcagaggcc R- ttcgttgtcttctccatcaacc β-actin F- cttgtggttgacaatggctccg R- tggtgaaggagtagccacgctc ELF F- atggttgtcaactttgcccc R- ttgacctccttgatcacacc Product Size (bp) Annealing Temp. ( 0 C) 264 bp 62 1.5 300 bp 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 1.5 520 bp 60 1.5 Chapter 2 MgCl2 Conc. (mM) 440 bp 60 1.5 Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 142
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Molecular Characterization and Gene
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Declaration I hereby do declare tha
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Acknowledgements This work was carr
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My Seniors, Dr. Annies Joseph, Dr.
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Contents Title Page Nos. 1 General
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in response to WSSV challenge 252-2
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α-2M Alpha-2-Macroglobulin ALF Ant
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ppA Prophenoloxidase-Activating Enz
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1.1 Introduction Chapter 1 Aquacult
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Chapter 1 the giant tiger shrimp (P
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Common names: Chapter 1 Giant tiger
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1.3.1. Diseases Chapter 1 Diseases
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Lymphoidal parvo-like virus (LPV) B
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Chapter 1 and protective occlusion
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Chapter 1 small brown masses in the
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1.4.6. Natural epidemics Chapter 1
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Chapter 1 can differentiate WSSV-in
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1.5.1.1. Probiotics Chapter 1 Alter
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Chapter 1 tried in P. monodon, resu
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Chapter 1 The first and essential i
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Chapter 1 1998). The innate immunit
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1.6.1 Haemocytes Chapter 1 The haem
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Chapter 1 In crustaceans, the sheet
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Chapter 1 Theopold et al., 2002). C
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Chapter 1 kill/remove the invading
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Chapter 1 forming complexes with a
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1.7 Antimicrobial Peptides (AMPs) 1
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Chapter 1 the response is very fast
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Chapter 1 Hirsch, 1947). While they
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Chapter 1 AMPs to various degrees u
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1.7.5 Biological activity Chapter 1
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Chapter 1 including the microbes as
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Tachyplesin I Chapter 1 Crustacea P
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Anionic and cationic peptides that
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Anionic and Cationic AMPs with cyst
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Chapter 1 Fig. 1.7. Structural clas
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Chapter 1 Group III: Linear peptide
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Chapter 1 through regular processes
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Chapter 1 ultimately signal to tran
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Chapter 1 divalent polyanionic cati
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Chapter 1 type of transmembrane por
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Chapter 1 other intracellular targe
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Chapter 1 Apidaecin, a short, proli
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Chapter 1 Hultmark, 1987; Gennaro a
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Chapter 1 quite opposite characteri
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Chapter 1 depletion of mitochondria
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Chapter 1 conformations adopted by
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Chapter 1 receptor may be a potenti
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Chapter 1 exposure to their targets
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Chapter 1 generating a rich spectru
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Chapter 1 by enkelytin (Goumon et a
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Lactoferricin Cow Mastitis control
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Chapter 1 trial, in which peptide T
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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: Chapter 2 Munoz et al., 2004). The
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 187 and 188: Chapter 2 LITOPENAEUS SP. FENNEROPE
Page 193 and 194: Chapter 2 Fig.2.43 A bootstrapped n
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
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Chapter 3 was predicted out to be 1
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Chapter 3 random coiled structure t
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3.3.1.3.5. Phylogenetic analysis of
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Chapter 3 case has been reported in
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Chapter 3 Multiple alignments were
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Chapter 3 negative bacteria. In shr
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Chapter 3 indicated a random coiled
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Chapter 3 horseshoe crab big defens
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Table 3.4. BLASTn analysis of ALF-2
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Table.3.8. BLASTn analysis of Fi-pe
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Chapter 3 Fig.3.2. Nucleotide and a
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Chapter 3 Fig.3.5. Multiple alignme
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Chapter 3 Fig.3.8. Nucleotide and a
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Chapter 3 Fig.3.12. Multiple alignm
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Chapter 3 Fig.3.14. A bootstrapped
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Chapter 3 Fig.3.17. Signal peptide
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SHRIMPS KIND CRAB LOBSTERS CRABS Ch
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Chapter 3 Fig.3.25. Signal peptide
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Chapter 3 Fig. 3.28. Multiple align
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PENAEIDIN-2 Chapter 3 PENAEIDIN- 3
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Fig.3.31. Nucleotide and amino acid
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Chapter 3 Fig.3.37 Nucleotide and a
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4.1. Introduction Chapter 4 The aqu
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Chapter 4 appearance and have major
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Chapter 4 the tissues has recently
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Chapter 4 with the ability of shrim
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Chapter 4 with a low dose of WSSV b
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Chapter 4 molecules they are likely
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Chapter 4 for understanding gene ex
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Chapter 4 4.3.2. Expression profile
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Expression profile of endonuclease
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Chapter 4 bacterial and fungal infe
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Chapter 4 measuring TSV and YHV loa
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Chapter 4 Of particular interest he
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Chapter 4 large distribution and ab
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Chapter 4 WSSV genes viz. endonucle
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Table 4.2. Primers used for the stu
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(A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
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(A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
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(A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
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(A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
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(A) (B) B 28 1 2 3 4 5 6 7 8 9 10 F
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(A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
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(A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
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(A) (B) B 28 1 2 3 4 5 6 7 8 9 10 C
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CHAPTER-5 Expression Profile of Ant
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Chapter 5 1994). The emergence of b
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Chapter 5 (Bovill et al., 2001). In
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5.2.2. Test diets Chapter 5 Four ex
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5.2.2.4. Preparation of Glucan inco
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Chapter 5 genes (latency related ge
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Chapter 5 On WSSV challenge, crusti
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Chapter 5 5.3.4. Expression profile
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5.3.4.5. Expression profile of pena
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Chapter 5 must firstly rest on a so
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Chapter 5 For the present study two
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Chapter 5 ameobocytes of two marine
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Chapter 5 variation in expression o
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Chapter 5 It has been previously sh
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Chapter 5 manifested in terms of gr
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(A) (B) (C) C CHY CSY CHG CSG C CHY
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(A) (B) -VE CTRL +VE CTRL CHG CSG C
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(A) PRE-CHALLENGE (B) POST-CHALLENG
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(A) PRE-CHALLENGE (B) (C) (D) CONTR
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(C) (D) (A) PRE-CHALLENGE (B) CONTR
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(A) (B) G M HP HR I Chapter 5 Fig.
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Chapter 5 Fig. 5.26. Post challenge
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6.1. Introduction Chapter 6 Prophyl
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Chapter 6 production of inhibitory
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6.2. Materials and methods 6.2.1. E
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Chapter 6 tissue cDNA was performed
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Chapter 6 gene was supported by Bac
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Chapter 6 treated group of shrimps.
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Chapter 6 the gene was found to be
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Chapter 6 found to support minimum
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Chapter 6 is induced upon bacterial
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Chapter 6 all the target genes, exc
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Chapter 6 Detailed tissue-wise anal
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Chapter 6 Gills and intestine was f
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(A) (B) Fig. 6.1. Experimental diet
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(A) (B) (C) Fig. 6.3. Expression pr
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(A) (B) (C) C B M BM C B M BM Fig.
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(A) (B) (C) Fig. 6.7. Expression pr
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(A) (B) (C) Chapter 6 Fig. 6.9. Exp
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(A) (B) -VE CTRL +VE CTRL B M BM Ch
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(A) (C) (D) PRE-CHALLENGE POST-CHAL
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(A) (B) G M HP HR I Chapter 6 Fig.
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Chapter 6 Fig. 6.27. Post challenge
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7.1. Summary Chapter 7 Tropical shr
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Salient findings Chapter 7 � From
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Chapter 7 � Tissue-wise expressio
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Chapter 7 application in shrimp cul
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References Aboudy, Y., Mendelson, E
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References Anggraeni, M.S., Owens,
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References Bals, R., Wang, X., Zasl
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References derived from the N-termi
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References Breukink, E., de Kruijff
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References Burgents, J.E., Burnett,
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References 1,3-β-D-glucan-binding
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References Chen, J.Y., Chuang, H.,
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References Cole, A.M., Hong, T., Bo
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References Dathe, M., Wieprecht, T.
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References Diamond, G., Zasloff, M.
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References Faber, C., Stallmann, H.
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References Flint, S.J., Enquist, L.
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References Gennaro, R., Zanetti, M.
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References Gudmundsson, G.H., Lidho
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References Harwig, S.S., Swiderek,
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References Huang, C.C., Song, Y.L.
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References Itami, T., Takahashi, Y.
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References Jiravanichpaisal, P. Whi
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References Kang, J.H., Shin, S.Y.,
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References Kono, T., Savan, R., Sak
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References Li, L., Wang, J.X., Zhao
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References Liu, C.H., Cheng, W., Ku
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References Lorenzon, S., de Guarrin
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References Medvinsky, A., Dzierzak,
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References Moriarty, D.J.W. 1996. M
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References Litopenaeus vannamei cha
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References Sathish, S., Musthaq, S.
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References a proline-arginine-rich
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References experimental system. In:
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References the black tiger shrimp P
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References Tapay, L.M., Cesar, E.,
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References Touhy, K.M., Probert, H.
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References van Hulten, M.C., Reijns
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Penaeus monodon anti-lipopolysaccha
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Penaeus monodon crustin-like antimi
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Penaeus monodon crustin-like antimi
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Penaeus monodon penaeidin-like anti
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Penaeus monodon elongation factor m
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Fenneropenaeus indicus anti-lipopol
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Fenneropenaeus indicus penaeidin-li
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Fenneropenaeus indicus beta-actin m
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PUBLICATIONS
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Short sequence report Molecular cha
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Fig. 2. Multiple alignment of nucle
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220 The phylogenetic relationships
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Table 1 Rearing conditions and wate
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5 ′ cttgtggttgacaatggctccg3
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Expression of WSSV genes in the hae
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Fig. 4. Expression profile of crust
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S.P. Antony et al. / Immunobiology
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