Title Page
Contents
Abstract 10
1. Introduction 12
2. Materials and methods 22
2.1. Brood fish rearing and induce breeding 22
2.2. Artificial insemination and hatching 24
2.3. Embryonic and larval samples collection 25
2.4. Experimental fish rearing and samples collection 25
2.5. Isolation of IL-1β, CTSB and CTSD cDNA 26
2.5.1. RNA extraction and isolation 26
2.5.2. cDNA synthesis 27
2.5.3. RT-PCR 27
2.5.4. Purification of PCR product 28
2.6. Cloning of IL-1β, CTSB and CTSD gene 28
2.6.1. Transformation into E. coli 28
2.6.2. Purification of recombinant plasmid 29
2.7. Synthesis of IL-1β, CTSB and CTSD genomic DNA 31
2.8. Gel purification of genomic DNA 32
2.9. Distribution of IL-1β, CTSB and CTSD transcripts in embryonic development stages and tissues 34
3. Results 37
3.1. cDNA nucleotide and amino acid sequence analysis 37
3.1.1. IL-1β gene 37
3.1.2. CTSB gene 41
3.1.3. CTSD gene 45
3.2. Expression of IL-1β, CTSB and CTSD transcripts in embryonic developmental stages and early larvae stages 51
3.3. Tissue-specific expression of IL-1β, CTSB and CTSD transcripts 52
4. Discussion 54
5. Conclusion 60
7. References 63
Table 1. Primers used to amplify IL-1β, CTSB and CTSD cDNA and for... 32
Table 2. Development stages of Siberian sturgeon embryos and early larvae 35
Figure 1. Embryonic developmental stages (Stage 2, Stage 3, Stage 6, Stage 7,... 36
Figure 2. cDNA nucleotide sequence of Siberian sturgeon IL-1β gene.... 37
Figure 3. The deduced amino acid sequences of Siberian sturgeon IL-1β... 38
Figure 4. Multiple alignment of Siberian sturgeon IL-1β amino acid... 39
Figure 5. cDNA nucleotide sequence of Siberian sturgeon CTSB gene.... 41
Figure 6. The deduced amino acid sequences of Siberian sturgeon CTSB... 42
Figure 7. Multiple alignment of the Siberian sturgeon CTSB amino acid... 44
Figure 8. cDNA nucleotide sequence of Siberian sturgeon CTSD gene. Start... 46
Figure 9. The deduced amino acid sequences of Siberian sturgeon CTSD... 47
Figure 10. Multiple alignment of Siberian sturgeon CTSD amino acid... 49
Figure 11. RT-PCR analysis of Siberian sturgeon IL-1β, CTSB and CTSD... 51
Figure 12. RT-PCR analysis of Siberian sturgeon IL-1β, CTSB and CTSD... 52