【作者】 胡晓丽；

【导师】 包振民；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2005， 博士

【摘要】 本研究采用mRNA差异显示技术分析了高温胁迫和鳗弧菌感染对栉孔扇贝基因表达的影响。克隆了栉孔扇贝色氨酸双加氧酶（TDO）,HSP70和肌球蛋白基因,并进行了表达分析。 采用3条锚定引物和8条随机引物组成24对引物组合,对高温胁迫前后栉孔扇贝基因表达变化进行了mRNA差异显示分析。在2599条扩增条带中,有59条差异条带,其中,9条在胁迫样本中特异扩增,20条只在对照样本中扩增,13条在胁迫样本中高水平扩增,17条在胁迫样本中低水平扩增。三种锚定引物H-T₁₁A,H-T₁₁C,H-T₁₁G的扩增条带总数分别为867条,865条和867条,而差异条带数占相应锚定引物总扩增条带数的比例分别为1.61%,1.84%和3.34%,特异条带数占相应锚定引物总扩增条带数的比例分别是:0.58%,0.81%和1.96%,H-T₁₁G均明显高于H-T₁₁A和H-T₁₁C。 同样利用mRNA差异显示技术,采用24对引物组合,分析了鳗弧菌应激前后栉孔扇贝基因表达的变化。共扩增得到2047条扩增条带,其中差异条带为32条。6条条带在胁迫样本中特异扩增,2条只在对照样本中扩增,9条在胁迫样本中高水平扩增,15条在胁迫样本中低水平扩增。三种锚定引物H-T₁₁A,H-T₁₁C,H-T₁₁G的扩增条带总数分别为755条,613条和679条,而差异条带数占相应锚定引物总扩增条带数的比例分别为1.10%,1.79%和1.91%。特异条带数占相应锚定引物总扩增条带数的比例分别是:0.26%,0.33%和0.59%,H-T₁₁G均高于H-T₁₁A和H-T₁₁C。 克隆了在鳗弧菌应激条件下,表达量减少的TDO基因片断,并利用5’RACE技术得到了基因全长。栉孔扇贝TDO基因全长1292 bp,编码383个氨基酸,分子量为44.8KD,等电点为6.35。编码的氨基酸序列与果蝇、人、斑马鱼、小鼠和线虫TDO的相似性分别为61%,57%,56%,55%和54%,并含有可能与TDO酶活性有关的保守性组氨酸。在大肠杆菌中表达了TDO-GST融合蛋白,并用融合蛋白制备了多克隆抗体。RT-PCR及Western blotting结果表明,栉孔扇贝TDO在鳃、消化腺、外套膜、闭壳肌、肾和性腺中都有表达。免疫组化分析显示,TDO更多还原

【Abstract】 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.更多还原