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Heat Shock Cognate 70(hsc70)and Gata Transcription Factor as the Regulators of Vitellogenesis in the Shrimp Metapenaeus Ensis

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This dissertation, "Heat Shock Cognate 70(HSC70)and Gata Transcription Factor as the Regulators of Vitellogenesis in the Shrimp Metapenaeus Ensis" by Pui-kei, Chung, 鍾沛基, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered This dissertation, "Heat Shock Cognate 70(HSC70)and Gata Transcription Factor as the Regulators of Vitellogenesis in the Shrimp Metapenaeus Ensis" by Pui-kei, Chung, 鍾沛基, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled HEAT SHOCK COGNATE 70 (HSC70) AND GATA TRANSCRIPTION FACTOR AS THE REGULATORS OF VITELLOGENESIS IN THE SHRIMP Metapenaeus ensis Submitted by Chung Pui Kei for the degree of Master of Philosophy at The University of Hong Kong in August 2005 Ovarian maturation in shrimp is characterized by the synthesis and accumulation of yolk protein precursor vitellogenin (Vg) in the oocytes. The 5' flanking region of the shrimp (Metapenaeus ensis) vitellogenin gene (MeVg2) consists of two potential binding sites for heat shock transcription factor (HSF) and one site for GATA transcription factor (GATA-TF). Deletion of the HSF and GATA response elements increased and decreased the promoter activity respectively. These suggest a negative role of HSF and a positive role of GATA-TF in the regulation of MeVg2 gene expression. In other animal models, there are reports for the interaction of HSF with heat shock cognate 70 (hsc70) to form a regulatory complex for gene transcription. To study the role of hsc70 in the regulation of vitellogenin gene expression in the shrimp M. ensis, the ORF of 1,947 bp encoding for hsc70 was cloned from the hepatopancreas and ovary. The hsc70 mRNA level was enriched in the ovary and hepatopancreas of female shrimp at early vitellogenic stage but the level dropped dramatically during ovarian maturation. In addition, Western blot analysis revealed that hsc70 was predominantly present in the nuclear fraction of various organs but not in the cytoplasm. The results from electrophoretic mobility shift assay (EMSA) showed that the ovary nuclear extract contained a factor that bound to the HSF response element. It is possible that the hsc70 interacts with HSF to form a transcription repressor and suppresses vitellogenin gene expression. For further analysis, a partial cDNA encoding the zinc finger DNA binding domain of GATA-TF was cloned by RT-PCR. Northern blot analysis revealed that GATA-TF was predominantly expressed in the hepatopancreas with increasing expression level throughout ovarian maturation. Therefore, GATA-TF was implicated to be a positive regulator of vitellogenin gene expression. An in vitro organ explant culture was developed and a RNAi technique was used to study the gene function of hsc70 and GATA-TF. Double-stranded RNA (dsRNA) for hsc70 caused the knockdown of hsc70 mRNA level and led to a 54 % and 62 % increase in MeVg2 mRNA level in the ovary and hepatopancreas respectively. On the other hand, gene knockdown of GATA-TF led to a 50 % reduction in MeVg2 expression level in the hepatopancreas. In conclusion, hsc70 and GATA-TF were characterized as the negative and positive regulators of MeVg2 gene expression respectively. They probably act on the MeVg2 promoter and regulate MeVg2 gene expression at transcription level. The result from this study is important in opening a new research area for transcription control of vitellogenesis and it may provide new insights to develop new techniques to improve shrimp aquaculture. DOI: 10.5353/th_b3483516 Subjects: Heat shock proteins Transcription factors Genetic regulation Gene expression Oogenesis Shrimps - Genetics


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This dissertation, "Heat Shock Cognate 70(HSC70)and Gata Transcription Factor as the Regulators of Vitellogenesis in the Shrimp Metapenaeus Ensis" by Pui-kei, Chung, 鍾沛基, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered This dissertation, "Heat Shock Cognate 70(HSC70)and Gata Transcription Factor as the Regulators of Vitellogenesis in the Shrimp Metapenaeus Ensis" by Pui-kei, Chung, 鍾沛基, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled HEAT SHOCK COGNATE 70 (HSC70) AND GATA TRANSCRIPTION FACTOR AS THE REGULATORS OF VITELLOGENESIS IN THE SHRIMP Metapenaeus ensis Submitted by Chung Pui Kei for the degree of Master of Philosophy at The University of Hong Kong in August 2005 Ovarian maturation in shrimp is characterized by the synthesis and accumulation of yolk protein precursor vitellogenin (Vg) in the oocytes. The 5' flanking region of the shrimp (Metapenaeus ensis) vitellogenin gene (MeVg2) consists of two potential binding sites for heat shock transcription factor (HSF) and one site for GATA transcription factor (GATA-TF). Deletion of the HSF and GATA response elements increased and decreased the promoter activity respectively. These suggest a negative role of HSF and a positive role of GATA-TF in the regulation of MeVg2 gene expression. In other animal models, there are reports for the interaction of HSF with heat shock cognate 70 (hsc70) to form a regulatory complex for gene transcription. To study the role of hsc70 in the regulation of vitellogenin gene expression in the shrimp M. ensis, the ORF of 1,947 bp encoding for hsc70 was cloned from the hepatopancreas and ovary. The hsc70 mRNA level was enriched in the ovary and hepatopancreas of female shrimp at early vitellogenic stage but the level dropped dramatically during ovarian maturation. In addition, Western blot analysis revealed that hsc70 was predominantly present in the nuclear fraction of various organs but not in the cytoplasm. The results from electrophoretic mobility shift assay (EMSA) showed that the ovary nuclear extract contained a factor that bound to the HSF response element. It is possible that the hsc70 interacts with HSF to form a transcription repressor and suppresses vitellogenin gene expression. For further analysis, a partial cDNA encoding the zinc finger DNA binding domain of GATA-TF was cloned by RT-PCR. Northern blot analysis revealed that GATA-TF was predominantly expressed in the hepatopancreas with increasing expression level throughout ovarian maturation. Therefore, GATA-TF was implicated to be a positive regulator of vitellogenin gene expression. An in vitro organ explant culture was developed and a RNAi technique was used to study the gene function of hsc70 and GATA-TF. Double-stranded RNA (dsRNA) for hsc70 caused the knockdown of hsc70 mRNA level and led to a 54 % and 62 % increase in MeVg2 mRNA level in the ovary and hepatopancreas respectively. On the other hand, gene knockdown of GATA-TF led to a 50 % reduction in MeVg2 expression level in the hepatopancreas. In conclusion, hsc70 and GATA-TF were characterized as the negative and positive regulators of MeVg2 gene expression respectively. They probably act on the MeVg2 promoter and regulate MeVg2 gene expression at transcription level. The result from this study is important in opening a new research area for transcription control of vitellogenesis and it may provide new insights to develop new techniques to improve shrimp aquaculture. DOI: 10.5353/th_b3483516 Subjects: Heat shock proteins Transcription factors Genetic regulation Gene expression Oogenesis Shrimps - Genetics

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