Effect Of Heat Stress And Vibrio Anguillarum Challenge On Gene Expression Of Scallop (Chlamys Farreri) And Cloning And Expression Of Related Genes

DDRT-PCR is used to analyze effect of heat stress and Vibrio anguillarum challenge on gene expression of scallop (Chlamys farreri). Cloning and expression analysis of scallop tryptophan 2,3-dioxygenase, HSP70 and myosinVI are carred out.Differences of gene expression between control and heat stressed scallops are analyzed by mRNA differential display with 24 primer combinations (3 anchored primers and 8 random primers). A total of 2599 bands are amplified and 59 bands are different between the two groups. The number of bands specific to heat stressed scallops, specific to control scallops, up-regulated in heat stressed scallops and down-regulated in heat stressed scallops is 9, 20, 13 and 17, respectively. The number of bands amplified with 3 anchored primers H-T₁₁A, H-T₁₁C and H-T₁₁G is 867, 865 and 867, respectively. The number of different bands account for 1.61%, 1.84% and 3.34% in the total bands amplified with anchored primers H-T₁₁A , H-TnC and H-TnG, respectively; while specific bands amplified with H-TnA , H-T₁₁C and H-T₁₁G are 0.58%, 0.81% and 1.96%, respectively. Obviously, more differential and specific bands are obtained with H-TnG than with H-T₁₁A and H-TuC.Differences of gene expression between control and Vibrio anguillarum challenged scallops (Chlamys farreri) are analyzed by mRNA differential display too, with 24 primer combinations (3 anchored primers and 8 random primers). A total of 2047 bands are amplified and 32 bands are different between the two groups. The number of bands specific to Vibrio anguillarum challenged, specific to control scallops, up-regulated in Vibrio anguillarum challenged and down-regulated Vibrio anguillarum challenged is 6, 2, 9 and 15, respectively. The number of bands amplified with 3 anchored primers H-T₁₁A, H-TnC and H-TuG is 755, 613 and 679, respectively. The number of different bands account for 1.10%, 1.79% and 1.91% inthe total bands amplified with anchored primers H-TnA , H-TnC and H-TuG, respectively. Specific bands amplified with H-TUA, H-TnC and H-TnG are 0.26%, 0.33% and 0.59%, respectively. More different and specific bands are obtained with H-TuG than with H-TuAand H-TUC.Scallop Chlamys farreri Tryptophan 2, 3-dioxygenase (TDO) gene fragment, down-regulated by Vibrio anguillarwn challenge, is isolated using mRNA differential display. Full-length TDO gene sequence is obtained by 5' RACE. Scallop TDO gene consists of 1292 nucleotides encoding an expected polypeptide of 383 amino acids with the estimated molecular weight of 44.8 kDa and isoelectric point of 6.35. The deduced amino acid sequence is homologous to TDOs from various organisms with similarities of 61%, 57%, 56%, 55% and 54% to those of Drosophila melanogaster, Homo sapiens, Danio rerio, Mus musculus and Caenorhabditis elegans, respectively. Conserved histidines that probable related to enzyme activity are found in scallop TDO. TDO-GST fusion protein is expressed in E. coli and polyclonal antibody against the fusion protein is obtained. Scallop TDO is expressed widely in mantle, gill, digestive gland, gonad, adductor muscle and kidney. Immunohistochemical analysis showed that scallop TDO is located mainly in the cytoplasm of most cell types.DNA and cDNA sequences of C. farrery HSP70 gene were obtained. The HSP70 gene consists of 4368 nucleotides encoding an expected polypeptide of 651 amino acids. Like the organization of oyster Hsc70 gene, sequence of the C. farrery HSP70 gene contains six coding exons and five introns. All the intron borders of C. farrery HSP70 start and end with the consensus GT and AG splicing signals and C. farrey HSP70 has longer intron sequence than that of oyster Hsc70. The corresponding amino acid sequences contain characteristic motifs of HSP70 family. RT-PCR analysis shows that C. farrery HSP70 is expressed in mantle, gill, digestive gland, gonad, adductor muscle and kidney. Through native PAGE and WAVE analysis, intra-individual insert-deletions and point mutations are found in C. farrery HSP70 3'UTR. Sites probable related to stress resistance are found by analyzing C. farrery HSP70 3'UTR of tree populations (Changdao wild population, Changdao one-year selective population, Changdao two-year selective population) and the heat tolerance of individuals in Changdao wild population.A 143 bp cDNA fragment encoding 47 amino acids with high similarity to myosin VI was amplified from chlamys ferrary mantle tissue by RT-PCR with degenerate primers. Longer cDNA sequence was obtained by 3' RACE and 5' RACE. It comprises 2940 bp with 5'-noncoding sequences and has an open reading frame of 2835 bp that encodes 945 amino acids. The deduced amino acid sequence is homologous to myosin VI from various organisms with similarities of 60%, 59% and 56% to those of Gallus gallus, Homo sapiens and Drosophila melanogaster, respectively. Conserved motifs of myosin classes and features specific to class VI myosin are also found. This result suggests that the encoded protein belongs to myosin VI family. RT-PCR analysis showed that scallop myosin VI is expressed in mantle, digestive gland, kidney, adductor muscle, gill and gonad. More myosin VI transcript is detected in digestive gland and kidney. Myosin VI-GST fusion protein is expressed in E. coli.