Molecular Characterization and Gene Expression Profiling ... - CUSAT

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2.2.9 Cloning of the PCR product 117-119 2.2.10 Sequencing and sequence analysis 119-120 2.3 Results 120-132 2.3.1 Molecular characterization of AMP genes in P.monodon 120-132 2.3.1.1 Anti-lipopolysaccharide factor -1 (ALF-1) (GU732817) 120-122 2.3.1.2 Anti-lipopolysaccharide factor -2 (ALF-2) (HM588914) 122-123 2.3.1.3 Crustin-1 (GQ334395) 124-126 2.3.1.4 Crustin-2 (FJ535568) 126-128 2.3.1.5 Crustin-3 (GQ334396) 128-130 2.3.1.6 Penaeidin-3 (GU732819) 130-131 2.3.1.7 Penaeidin-5 (GQ334397) 131-132 2.3.2 Control / Reference genes 132 2.4 Discussion 130-140 Tables 140-148 Figures 149-188 3 Molecular characterization and phylogenetic analysis of antimicrobial peptides in Fenneropenaeus indicus 186-237 3.1 Introduction 186-187 3.2 Materials and Methods 187-188 3.2.1 Experimental animals 187 3.2.2 Haemolymph collection 187 3.2.3 Total RNA isolation and Reverse transcription 188 3.2.4 PCR amplification 188 3.2.5 Agarose gel electrophoresis 188 3.2.6 Cloning of the PCR product 188 3.2.7 Sequencing and sequence analysis 188 3.3 Results 188-199 3.3.1 Molecular characterization of AMP genes in F. indicus 188-199 3.3.1.1 Anti-lipopolysaccharide factor (ALF-1) (GU732814) 189-190 3.3.1.2 Anti-lipopolysaccharide factor (ALF-2) (HM366921) 190-192 3.3.1.3 Fi-Crustin (GQ469987) 192-195 3.3.1.4 Fi-penaeidin (HM243617) 195-198 3.3.1.5 Penaeidin-5 (HM243616) 198-199 3.3.2 Control / Reference genes 199 3.4 Discussion 199-205 Tables 205-210 Figures 211-237 4 Time-course of antimicrobial peptide gene expression in response to White spot syndrome virus challenge 238-283 4.1 Introduction 238-248 4.1.1 Antiviral immune response 243-248 4.2 Materials and Methods 249-251 4.2.1 Experimental animals and rearing conditions 249 4.2.2 Sampling 249-250 4.2.3 WSSV challenge 250 4.2.4 Haemolymph collection 250 4.2.5 Total RNA isolation and reverse transcription 250 4.2.6 Semi-quantitative RT-PCR analysis of target gene expression 250-251 4.3 Results 251-255 4.3.1 Expression profile of control genes in the haemocytes of P. monodon in response to WSSV challenge 251 4.3.2 Expression profile of AMP genes in the haemocytes of P. monodon
in response to WSSV challenge 252-253 4.3.3 Expression profile of WSSV genes in the haemocytes of P. monodon in response to WSSV challenge 253-255 4.4 Discussion 255-265 Tables 265-266 Figures 267-283 5 Expression profile of antimicrobial peptide genes in response to the administration of immunostimulants 284-337 5.1 Introduction 284-288 5.2 Materials and Methods 288-293 5.2.1 Experimental animals and rearing conditions 288 5.2.2 Test diets 289 5.2.2.1 Yeast strains 289 5.2.2.2 Preparation of yeast incorporated experimental feed 289 5.2.2.3 Glucan extraction 290 5.2.2.4 Preparation of glucan incorporated experimental feed 291 5.2.3 Feeding experiments and WSSV challenge 291 5.2.4 Haemolymph and other tissues collection 292 5.2.5 Total RNA isolation and reverse transcription 292 5.2.6 Semi-quantitative RT-PCR analysis of target gene expression 292-293 5.2.7 Statistical analysis of the post-challenge survival data 293 5.3 Results 293-300 5.3.1 Expression profile of control genes in the haemocytes of P. monodon in response to various immunostimulants 293 5.3.2 Expression profile of AMP genes in the haemocytes of P. monodon in response to various immunostimulants 293-296 5.3.3 Expression of WSSV genes in haemocytes of P. monodon in response to the administration of various immunostimulants 296 5.3.4 Expression profile of AMP genes in various tissues of P. monodon in response to the administration of Candida haemulonii yeast 297-299 5.3.5 Expression of WSSV genes in various tissues of P. monodon in response to the administration of Candida haemulonii yeast 300 5.3.6 Post-challenge survival 300 5.4 Discussion 300-311 Tables 311 Figures 312-337 6 Expression profile of antimicrobial peptide genes in response to the administration of probiotic bacteria 338-388 6.1 Introduction 338-341 6.1.1 Role of probiotics in aquaculture 339 6.1.2 Modes of action of probiotics 339-341 6.2 Materials and methods 342-344 6.2.1 Experimental animals and rearing conditions 342 6.2.2 Test diets 342 6.2.3 Feeding experiment and WSSV challenge 342-343 6.2.4 Haemolymph and other tissues collection 343 6.2.5 Total RNA isolation and reverse transcription 343 6.2.6 Semi-quantitative RT-PCR analysis of gene expression 343-344 6.2.7 Statistical analysis 344 6.3 Results 344-353 6.3.1 Expression profile of control genes in the haemocytes of P. monodon in response to the administration of probiotic bacteria 344
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: Contents Title Page Nos. 1 General
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
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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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Table 2.8. BLASTn analysis of Crust
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Table 2.12. BLASTn analysis of Pena
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Fig.2.5. Agarose gel electrophoreto
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Chapter 2 Fig.2.9. Multiple alignme
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Chapter 2 Fig. 2.12 A bootstrapped
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Chapter 2 Fig. 2.15. Multiple align
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Chapter 2 Fig.2.18. A bootstrapped
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Chapter 2 Fig.2.20. Signal peptide
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Chapter 2 Fig.2.24 Multiple alignme
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Chapter 2 LITOPENAEUS SP. FARFANTEP
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Chapter 2 Fig. 2.28. Nucleotide and
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Chapter 2 LITOPENAEUS SP. FENNEROPE
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Chapter 2 Fig.2.43 A bootstrapped n
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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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SHRIMPS KIND CRAB LOBSTERS CRABS Ch
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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
Page 421 and 422:
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
Page 429 and 430:
References Burgents, J.E., Burnett,
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References 1,3-β-D-glucan-binding
Page 433 and 434:
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.
Page 439 and 440:
References Diamond, G., Zasloff, M.
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References Faber, C., Stallmann, H.
Page 443 and 444:
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 Hirakura, Y., Kobayashi,
Page 453 and 454:
References Huang, C.C., Song, Y.L.
Page 455 and 456:
References Itami, T., Takahashi, Y.
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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
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References Marone, M., Mozzetti, S.
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References Medvinsky, A., Dzierzak,
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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
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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
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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
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References Wu, W., Zong, R., Xu, J.
Page 515 and 516:
References Yodmuang, S., Tirasophon
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References Zhan, W.B., Wang, Y.H. 1
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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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