Molecular Characterization and Gene Expression Profiling ... - CUSAT

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Chapter 2 end of the crustin-like AMP. Multiple polyadenylation sites were observed in the present fragment. 2.3.1.4.3. Analysis of WAP domain structure of Crustin-2 As predicted by the ScanProsite program a partial WAP domain signature exists in the C-terminal region and one of the four disulphide core (DSC) domain was found to be located at Cys 6 -Cys 23 . Since only partial cDNA fragment and only five of the conserved cysteine regions could be retrieved from the sequence, other locations of the DSC could not be performed. Searching against the Prosite database, analysis of the crustin- like AMP revealed the existence of WAP type DSC domain signature. The expected WAP type 4DSC core domain signature is: C1-(Xn)-C2-(Xn)-C3-(X5)-C4-(X5)-C5-C6-(X3-5)-C7-(X3-4)-C8 where X is any amino acid residue and Xn is a stretch of n residues (Bartlett et al., 2002). The partial cDNA sequence of the crustin-like AMP showed the presence of C4-C8 and it followed the same pattern as expected in the WAP domain except for one extra residue between C7 and C8 (C7-(X5)-C8). Similar case was reported for the crustins isolated from F. chinensis, GenBank Accession No. DQ097703, DQ097704 (Zhang et al., 2007). The WAP domain signature of the present crustin-like AMP could be expressed as in Fig. 2.32. Several other consensus sequences also appear in the 4DSC domain. (1) A conserved aspartate (D) residue between C3 and C4 (2) KCC with C5 and C6. (3) CXXP with C8 (Bartlett et al., 2002). 2.3.1.4.4. Sequence alignment of Crustin-2 BLAST analysis was performed at the nucleotide and amino acid level with other crustins in the GenBank (Table 2.8 and 2.9). BLAST analysis showed that the crustin-like AMP shared maximum similarity with other crustins of F. paulensis (ABM633611), L. vannamei (AY488493), F. subtilis (ABO93323), F. brasiliensis (ABQ96197) and L. schmitti (ABM63362). The Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 127
Chapter 2 sequence also showed high similarities to the crustin isolated from P. monodon by Chen and co-workers (2004a) that have not been submitted to the GenBank. Multiple alignments showed the presence of highly conserved domains in both the nucleotide and amino acid sequences (Fig.2.33 and 2.34). 2.3.1.4.5. Phylogenetic analysis of Crustin-2 All known crustins were retrieved from the GenBank and phylogenetic analysis of both the nucleotide and amino acid sequences of the crustin-like AMP were performed. Crustins of shrimps (Fig. 2.35) and that of all groups of crustaceans (Fig. 2.36) were subjected to phylogenetic analysis. NJ trees were constructed both at nucleotide and amino acid level using MEGA 3.1 software. The phylogenetic relationship of the crustin-like AMP and other AMPs with WAP domain that showed maximum similarity with that of crustin-like AMP (FJ535568) and also with that of all the known crustins deposited in GenBank are shown in Fig. 2.35 and 2.36 respectively. The tree topologies revealed the relationships of crustin-like AMP with other invertebrate crustin / crustin-like peptides. 2.3.1.5. Crustin-3 (GQ334396) 2.3.1.5.1. Nucleotide and deduced amino acid sequences of Crustin-3 A partial mRNA transcript of 239 bp, encoding 73 amino acids, belonging to the crustin family of AMPs was obtained from the mRNA of P. monodon haemocyte by RT-PCR (Fig. 2.37). The sequence was submitted to GenBank under the accession number GQ334396. Cysteine rich region and the WAP domain signature with the characteristic 4DSC could be detected using ScanProsite tool (Fig.2.38). Motif Scan also showed the presence of cysteine-rich regions and WAP domain structure in the crustin-2 sequence (Fig. 2.39). The predicted secondary structure of crustin-3, created using SWISS-MODEL, consisted of a coiled structure with a helical segment (Fig. 2.40). Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 128
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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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Page 99 and 100: Chapter 1 generating a rich spectru
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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
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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
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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: 2.3.1.3.5. Phylogenetic analysis of
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 187 and 188: Chapter 2 LITOPENAEUS SP. FENNEROPE
Page 189 and 190: Chapter 2 Fig. 2.37. Nucleotide and
Page 193 and 194: Chapter 2 Fig.2.43 A bootstrapped n
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Chapter 2 Fig. 2.45. Nucleotide and
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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 Gennaro, R., Zanetti, M.
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References Gudmundsson, G.H., Lidho
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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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