Molecular Characterization and Gene Expression Profiling ... - CUSAT

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2.3.1.5.2. Amino acid composition of Crustin-3 Chapter 2 The transcript had 73 amino acid residues with conserved cysteine residues characteristic of the WAP domain. The C-terminal segment included a high proportion of cysteine-rich region that participate in the formation of disulphide bonds. The partial cDNA fragment contained the 12 conserved cysteine residues. The partial cDNA fragment lacked the signal peptide region but possessed the cysteine rich region characteristic to WAP domain. The other characteristic features of the WAP domain also confirm the 3’ end of the crustin-like AMP. 2.3.1.5.3. Analysis of WAP domain structure of Crustin-3 As predicted by the ScanProsite program, a whey-acidic protein (WAP) domain signature exists in the C-terminal. According to the previous reports on the crustin-like proteins, the four-disulfide core domain has proved to play important roles in the biological function of crustins (Zhang et al., 2007). The position of the conserved cysteines for such category of ‘four-disulfide core’ domain and the location of the signature pattern is Cys 24 -Cys54, Cys31-Cys58, Cys 41 -Cys 53 , and Cys 47 -Cys 64 . In addition, searching against the Prosite database, analysis of Crustin-1 revealed the existence of WAP-type ‘four-disulfide core’ domain signature, C1-(Xn)-C2- (Xn)-C3-(X5)-C4-(X5)-C5-C6-(X3-5)-C7-(X3-4)-C8 (Bartlett et al., 2002) except for the presence of 5 residues between C7 & C8 (C7 X5 C8). 3 residues were found between C6 & C7 (C6 X3 C6). Several other consensus sequences also appears in the 4DSC domain: (1) the consensus KXGXCP containing C1; (2) a conserved aspartate (D) residue between C3 and C4; (3) KCC with C5 and C6; (4) CXP with C8 (Bartlett et al., 2002). 2.3.1.5.4. Sequence alignment of Crustin-3 BLAST analysis was performed at the nucleotide and amino acid level with other crustins in the GenBank. BLAST analysis showed that the crustin- like AMP shared maximum similarity with other crustins of P. monodon, F. Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 129
Chapter 2 chinensis and F. indicus (Table 2.10 and 2.11). Sequence alignments at the nucleotide and amino acid levels showed the presence of highly conserved sequences within the crustin molecule (Fig 2.41 and 2.42) 2.3.1.5.5. Phylogenetic analysis of Crustin-3 The bootstrap distance tree calculated for the resulting crustin sequences of shrimps could be grouped into four major groups (Fig.2.43). Group I consisted of crustins of Litopenaeus, Fenneropenaeus and Farfantepenaeus sp. Group II consisted of crustin isoforms of M. japonicus, Group III of P. monodon, and Group IV of F. chinensis. Phylogenetic analysis of crustins of all decapods could be broadly divided into two groups. Group I consisting of shrimp crustins and Group II consisting of crustins of crabs, crayfishes and lobsters (Fig.2.44). 2.3.1.6. Penaeidin-3 (GU732819) 2.3.1.6.1. Nucleotide and deduced amino acid sequences of Penaeidin-3 A partial mRNA transcript of 138 bp, encoding 45 amino acids, belonging to the penaeidin family of AMPs was obtained from the mRNA of P. monodon haemocyte by RT-PCR (Fig. 2.45). The sequence was submitted to GenBank under the accession number GU732819. Four cysteine residues could be found in the partial sequence that form the part of the six cysteine residues organized in two doublets. Penaeidin signature could be detected within the sequence using Motif Scan tool (Fig.2.46). 2.3.1.6.2. Sequence alignment of Penaeidin-3 BLAST analysis was performed at the nucleotide and amino acid level with other penaeidin-3 in the GenBank. BLAST analysis showed that the penaeidin-like AMP shared maximum similarity with penaeidin-3 of P. monodon (Table 2.12 and 2.13). Multiple alignment performed for the sequence showed the presence of highly conserved sequences both at the nucleotide and amino acid level (Fig.2.47 and 2.48). Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 130
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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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Page 103 and 104: Lactoferricin Cow Mastitis control
Page 105 and 106: 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
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Page 119 and 120: Chapter 2 and the region might be l
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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: Chapter 2 sequence also showed high
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 187 and 188: Chapter 2 LITOPENAEUS SP. FENNEROPE
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Chapter 2 LITOPENAEUS SP. FENNEROPE
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Chapter 2 Fig.2.51 Signal peptide a
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Chapter 2 FENNEROPENAEUS SP. FARFAN
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CHAPTER-3 Molecular Characterizatio
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Chapter 3 certainly help us to unra
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Chapter 3 3.3.1.1. Anti-lipopolysac
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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 rich peptide derived fro
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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 Otta, S.K., Karunasagar,
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References simplex virus has heptad
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References infected shrimp. Thesis
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References Litopenaeus vannamei cha
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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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