The study of a hematopoiesis-related gene, Lozenge (Lz), from the white shrimp, Litopenaeus vannamei

• Main Authors: Chou, Tzu-Chiao, 周子巧
• Other Authors: Leu, Jiann-Horng
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/3493w4

碩士 === 國立臺灣海洋大學 === 海洋生物研究所 === 105 === Hemocytes play important roles in crustacean immunity, and therefore it is important for a crustacean to maintain its circulating hemocyte number at a constant level. Pathogen infection induces immune responses in crustaceans, leading to the reduction of the number of circulating hemocytes. To replenish the lost hemocytes, hematopoiesis (blood cell formation) is then induced to generate new hemocytes. Studies in model organisms have revealed that hematopoiesis is a complex process that requires the participation of many transcription factors, such as the human RUNX1 and Drosophila Lozenge (Lz), both of which belong to Core Binding Factor (CBF) transcription factor family and are essential for hematopoiesis regulation. Previously, we had identified and isolated the full-length cDNA of Litopenaeus vannamei Lozenge (LvLz) from hemocytes, and in this study, we aimed at unveiling the possible role of LvLz in shrimp hematopoiesis. Tissue distribution analysis by RT-PCR/Real-time PCR showed that LvLz was ubiquitously expressed and had highest expression level in hemcoytes. Silencing the LvLz expression by RNA interference resulted in the decreased expression of immune-related genes proPO and penaeidin, the increased expression of hematopoietic-related gene astakine, and the slightly decrease of total hemocyte count as well as the proportion of granular cells. After lipopolysaccharide (LPS) injection to induce hematopoiesis, in hemocytes, the expression level of LvLz was increased, peaking at 6 h post injection (hpi) and then decreased to unstimulated level at 24 hpi, while the expressions of proPOs, penaeidin and astakine were decreased to lowest level at 3 or 6 hpi and then increased to highest levels at 24 or 48 hpi. In hematopoietic tissue, after LPS injection, the expressions of LvLz and astakine reached highest level at 6 hpi and then decreased at 48 and 24 hpi, respectively, while the expressions of proPOs and penaeidin were relatively unchanged. Subcellullar localization study showed that LvLz was localized to the nucleus of Drosophila S2 cells and to the cytoplasm of Spodoptera frugiperda Sf9 cells, suggesting that LvLz cannot translocate into the nuclei by itself. To become functionally active, Drosophila Lz has to form a heterodimeric complex with Bgb. Accordingly, LvLz should need the shrimp Bgb as well and we therefore cloned the LvBgb. When LvLz was coexpressed with LvBgb in Sf9 cells, LvLz was translocated into the nucleus. In summary, LvLz had highest expression level in hemocytes, suggesting that LvLz should play important roles in hemocytes. Its expression could be induced by hematopoietic stimuli in both hemocyotes and hematopoietic tissue. LvLz could regulate the expression of several immune and hematopoietic genes in granulocytes, and it seemed that its function depends on the association with LvBgb.