Molecular Characterization and Gene Expression Profiling ... - CUSAT

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Chapter 3 sequence to be reported from F. indicus, this penaeidin was named Fi- penaeidin. 3.3.1.4.2. Amino acid composition of Fi-penaeidin The deduced amino acid sequence of Fi-penaeidin was found to be rich in amino acid residues leucine (13.1%) and serine (13.1%) followed by cysteine (9.8%) and proline (6.6%) residues as calculated by the ProtParam tool of ExPASy program. Fi-penaeidin was characterized by 10 conserved amino acid sequence in the signal peptide; a threonine and two proline residues conserved in the N-terminal domain; and the conserved cysteine array of the C-terminal structured domain. As per Destoumieux and co- workers (2000a) this overall structure of penaeidins is quite unique among the AMP families. Motif Scan performed on the deduced amino acid sequence also confirmed that the sequence belonged to penaeidin AMPs. A glycosylation motif was also detected at the 53 rd -56 th position (Fig. 3.26). 3.3.1.4.3. Sequence alignment of Fi-penaeidin BLAST analysis of the nucleotide and amino acid sequences revealed the relation of Fi-penaeidin to that of penaeidins from F. paulensis and F. subtilis (Table 3.8 and 3.9). Multiple alignments of amino acid sequences of penaeidins showed that the deduced amino acids of Fi-penaeidin shared relatively high identities with penaeidins of F. paulensis, F. chinensis and F. subtilis. It showed high similarity in the signal peptide region of all known penaeidin AMPs followed by a major gap in the ensuing region. Slight variation could be seen in the signal peptide region of L. vannamei and P. monodon. A threonine and two proline residues could be detected conserved in the N-terminal domain which is believed to be a unique feature of the penaeidin group of AMPs. Unlike majority of the penaeidins that possess six cysteine residues at the C-terminal region, only four conserved cysteine residues could be detected in the C-terminal region of Fi-penaeidin. Similar Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 196
Chapter 3 case has been reported in the penaeidins of L. vannamei (AF390145, AF390144, AF390143) and L. setiferus (AY039204). Multiple alignments were also performed for penaeidins that showed great similarity with the deduced amino acid sequence of Fi-penaeidin during BLAST analysis (Fig. 3.27 and 3.28). The results showed 100% similarity in the signal peptide region. Multiple alignments confirmed the BLAST analysis which showed that Fi-penaeidin is more similar to penaeidins of L. setiferus, F. chinensis, F. paulensis, F. subtilis and L. schmitti. 3.3.1.4.4. Phylogenetic analysis of Fi-penaeidin The phylogenetic relationship of Fi-penaeidin with that of other known penaeidins is shown in Fig 3.29 and 3.30. The tree topologies revealed the relationships of Fi-penaeidin with other shrimp penaeidins. The phylogenetic tree based on amino acid sequences suggests that all the penaeidin members possess a same ancestral origin, which has subsequently diverged at different phases of evolution. Out of all the species, penaeidin of F. indicus was found to be evolutionarily more related with penaeidins of F. subilis, F. paulensis and F. chinensis. The phylogenetic tree obtained from all known penaeidins, could be broadly classified into five major groups. Group I consisted of penaeidins belonging to subgroup 2 isolated and characterized from the Litopenaeus sp. viz. L. schmitti, L. setiferus, L. vannamei and L. stylirostris. Group II included penaeidins belonging to subgroup 3 and 5 isolated and characterized from L. stylirostris, F. chinensis and P. monodon. Interestingly penaeidins from the same species were found to form subgroups within the major group II. Group III consisted of penaeidin-4 isolated and characterized from the Litopenaeus sp. Group IV consisted of penaeidin-3 isolated and characterized from Litopenaeus sp., Fenneropenaeus sp., and Penaeus species. Group V consisted of penaeidin 2 and 5 isolated and characterized from the shrimp species L. setiferus, L. schmitti, F. subtilis, F. Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps 197
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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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Chapter 1 a too limited spectrum to
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Chapter 1 number of research groups
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CHAPTER-2 Molecular Characterizatio
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Chapter 2 interaction with negative
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Chapter 2 inhibit the endotoxin or
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Chapter 2 structure be modified to
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Chapter 2 and the region might be l
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Chapter 2 been made of the spectra
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Chapter 2 their discovery and initi
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Chapter 2 terminal pyroglutamic aci
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Chapter 2 One can assume that the p
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Chapter 2 by degranulation into the
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2.2. Materials and Methods 2.2.1. E
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Chapter 2 washed by adding 1 ml 75
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Chapter 2 entry of recombinant DNA
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Chapter 2 and the deduced amino aci
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Chapter 2 Fenneropenaeus, Litopenae
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2.3.1.3. Crustin-1 (GQ334395) Chapt
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2.3.1.3.5. Phylogenetic analysis of
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Chapter 2 sequence also showed high
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Chapter 2 chinensis and F. indicus
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Chapter 2 monodon and Group III of
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Chapter 2 Tiger shrimp (P. monodon)
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Chapter 2 ALF-1 was a partial seque
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Chapter 2 As mentioned before, the
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Chapter 2 Munoz et al., 2004). The
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Table 2.1. Primers used for the stu
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Table 2.4. BLASTn analysis of ALF-2
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Page 165 and 166: Table 2.12. BLASTn analysis of Pena
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Page 177 and 178: Chapter 2 Fig.2.20. Signal peptide
Page 179 and 180: Chapter 2 Fig.2.24 Multiple alignme
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Page 183 and 184: Chapter 2 Fig. 2.28. Nucleotide and
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Page 199 and 200: Chapter 2 Fig.2.51 Signal peptide a
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Page 211 and 212: Chapter 3 random coiled structure t
Page 213: 3.3.1.3.5. Phylogenetic analysis of
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
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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
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Page 237 and 238: Chapter 3 Fig.3.14. A bootstrapped
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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
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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 Harwig, S.S., Swiderek,
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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 Liu, C.H., Cheng, W., Ku
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References experimental system. In:
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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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