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Alternative TitleExpression characterization and functional analysis of two regulatory genes during fish embryogenesis
Thesis Advisor桂建芳
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword14kda阿朴脂蛋白 四型抗冻蛋白 形态发生运动 消化系统 脂类 卵黄合胞层
Abstract从银鲫原肠期胚胎SMART cDNA文库中筛选得到两个参与调控鱼类胚胎发育的基因,分别为14kDa阿朴脂蛋白和四型抗冻蛋白。详尽解析了各自基因的时空表达格式,并采用MO敲降的方法,研究它们在胚胎发育期间的功能。 从原肠期直到孵化期,CagApo-14的转录本定位于卵黄合胞层,在出苗后局限于消化系统,包括肝脏和消化道。CagApo-14蛋白由肝脏窦间隙内皮细胞合成。MO敲降CagApo-14将破坏鱼类消化系统的发育。CagApo-14 敲降后的鱼苗中,卵黄脂类营养物质的运输出现障碍。在鱼类发育的内营养阶段和外营养阶段,CagApo-14都参与了营养物质的吸收和运输,在消化系统器官发生的过程中发挥调控作用。 分别在斑马鱼和银鲫中克隆到两个串连重复的四型抗冻蛋白,命名为Afp4a和Afp4b。它们在斑马鱼原肠运动和卵黄吸收中发挥调节功能。DrAfp4的表达在时序上呈现接力,在空间上定位于卵黄合胞层。早期基因DrAfp4a在成体各个组织中不表达。晚期基因DrAfp4b在原肠期后的发育阶段持续存在,在成鱼中由肝脏和肠道合成。显微注射MO分别特异敲降这两个基因将导致不同的发育畸形。DrAfp4a基因敲降后的胚胎,在原肠期出现细胞运动障碍;敲降DrAfp4b不会影响原肠期,但导致晚期卵黄吸收故障。各自基因的mRNA能挽救各自基因被敲降引起的畸形,但它们相互不能交叉挽救,提示两者功能发生分歧。
Other AbstractTwo regulatory genes during fish embryogenesis, named Apo-14 and Afp4 respectively, were isolated from SMART cDNA library of gastrula-stage gibel carp embryos. The spatio-temporal expression patterns of each gene were explored in detail, and their roles during embryonic development were further investigated by morpholino knock-down approach. Apo-14 is a fish-specific apolipoprotein and its biological function remains unknown. In this study, CagApo-14 has been cloned from gibel carp (Carassius auratus gibelio) and its expression pattern has been investigated during embryogenesis and early larval development. CagApo-14 transcript and protein product are firstly expressed in yolk syncytial layer at a very high level during embryogenesis, and then restricted to the digestive system including liver and intestine in later embryos and early larvae. Immunofluorescence localization in larvae and adults indicates that CagApo-14 protein is predominantly expressed and excreted from the sinusoidal endothelial cells of liver tissue. Morpholino knockdown of CagApo-14 results in severe disruption of digestive organs including liver, intestine, pancreas, and swim bladder. And, the yolk lipid transportation and utilization have been demonstrated to be severely affected in the CagApo-14 morphants. The current study has indicated that CagApo-14 is required for digestive system organogenesis during fish embryogenesis and larval development. Antifreeze proteins have attracted a lot of attention due to their significant roles in chilling tolerance, but little is known about their functions during embryonic development. Here we identify two reduplicated homologues of polar fish type IV antifreeze proteins (Afp4) genes from gibel carp and zebrafish, and reveal their biological functions as two key regulators in zebrafish gastrulation movement and yolk absorption. They are expressed during embryogenesis in a “temporal succession” manner and spatially restricted in the yolk syncytial layer. The earlier transcribed gene, DrAfp4a, is first detected at 5hpf, peaks rapidly at 8hpf and attenuated thereafter, and absent in all the examined adult tissues. DrAfp4b is transcribed later than DrAfp4a, peaks at 30hpf, persists on during the whole process of development, and is mainly synthesized in the liver and gut in adult zebrafish. Distinct developmental defects are observed when specifically knockdown each of the two duplicated genes by corresponding MO injection. Zebrafish embryos lacking DrAfp4a function display severe defects in morphogenetic movements during gastrulation, and cell tracing experiment clarifies that DrAfp4a contributes to convergence movement. By contrast, blocking DrAfp4b expression does not perturb morphogenetic movements during gastrulation, but produces embryonic defects during later embryogenesis. As revealed by Oil Red O staining, in DrAfp4b morphants, the yolk lipid utilization and transportation are impaired. Co-injection of mRNA encoding DrAfp4a or DrAfp4b can restore the developmental defects caused by each corresponding MO, but fails to rescue the deficiency of each other, indicating that DrAfp4a and DrAfp4b have undergone functional divergence after duplication.
Document Type学位论文
Recommended Citation
GB/T 7714
夏建红. 鱼类两个胚胎调控基因的表达特征及功能分析[D]. 水生生物研究所. 中国科学院水生生物研究所,2007.
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