Institutional Repository of Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences
|Place of Conferral||北京|
我们获得牙鲆nanos3 3′UTR，构建了驱动GFP表达的载体GFP-ypnanos3 3′UTR-pSP64，通过显微注射人工合成的GFP-ypnanos3 3′UTR RNA到斑马鱼受精卵中，研究其对于PGC的标记功能。结果表明，牙鲆nanos3 3′UTR可以最早在8体节期将斑马鱼PGC标记出来，因此牙鲆nanos3 3′UTR含有PGC特异性调控序列。对牙鲆nanos3 3′UTR序列进行分析，我们预测到3个与miR-430结合的GCAC位点（GCAC1、GCAC2、GCAC3），以及与Dnd蛋白结合的U-rich区域（U-rich region1和U-rich region2）。通过对不同长度牙鲆nanos3 3′UTR的PGC特异调控效率的分析，我们找到了68bp（38-105bp）的调控片段，并验证了GCAC1和GCAC2可能是miR430的结合位点，U-rich region1可能是Dnd蛋白的结合位点。
|Other Abstract|| The olive flounder (Paralichthys olivaceus) is an important cultured marine fish. Facing the trend of degradation of genetical characterization, it is important to preserve excellent germplasm resources. In olive flounder, female is larger than male and acquiring all-female offspring means great for aquaculture. However, sex differentiation of olive flounder is still undiscovered. Primordial germ cells (PGC) provide new ways for solving these problems. The research on PGC can improve the technology such as surrogate propagation, transgenic fish and PGC frozen preservation. Also, it also helps us to understand the sex differentiation mechanism, which is related with the PGC development.|
The labeling of PGC is the premise of its separation and transplantation. We cloned nanos3 of olive flounder, a PGC specific gene. RT-PCR and Whole-mount in situ hybridization were used to analyze its expression patterns during flounder embryogenesis. The results indicated that nanos3 was consistently expressed during embryogenesis and maternally supplied. nanos3 was specifically expressed in PGC. Therefore, it was reliable that nanos3 was PGC specific marker gene in olive flounder.
We simulated PGC migration model according nanos3 expression pattern during embryogenesis. The PGC migration model of olive flounder was almost similar with others teleosts fish. But at middle somitogenesis stage, the PGC, aligned in two elongated lines at both side of body axis during early somitogenesis stage, aggregated into a single loose cluster and migrated posteriorward. At heart-beating stage, PGC re-aligned into two tight clusters on both side of body axis. This PGC migration pattern was different from the teleost fishes reported and it was a new model that combines elements of PGC of medaka, herring and goby.
We injected chimeric RNA containing green fluorescent protein (GFP) and olive flounder nanos3 3′UTR into zebrafish fertilized eggs and PGC can be successfully visualized in vivo as early as the 8-somites stage. These results indicated that there was a PGC specific regulatory elements in nanos3 3′UTR of olive flounder. Furthermore, Dnd binding region (U-rich region) and three noncanonical miR-430 binding sites (GCAC) were predicated in nanos3 3′UTR of olive flounder. By injecting different length of olive flounder nanos3 3′UTR to zebrafish embryos, we found a 67bp (68-135bp) length functional region which belong to the PGC specific regulatory elements in olive flounder nanos3 3′UTR. By injecting different RNAs which were composed of GFP and olive flounder nanos3 3′UTR mutants into zebrafish fertilized eggs, we identified that the GCAC1 and GCAC2 in this region might be the miR430 binding site and the U-rich region1 might be the Dnd binding region.
Furthermore, we cloned sdf1 and cxcr4 and analyze their expression by RT-PCR and whole-mount in situ hybridization during embryogenesis. RT-PCR results indicated that sdf1 and cxcr4 were maternally supplied and had the highest expression level during gastrulation stage. Whole-mount in situ hybridization results indicated that Sdf1and Cxcr4 were related to the brain development, eye development, somites development and lateral line primordium migration during embryogenesis. Comparison analysis of sdf1 expression pattern and the PGC migration model showed that Sdf1 guided PGC migration during gastrulation and somitogenesis. In particular, Sdf1 play an important role in directing PGC aggregation into a single loose cluster from the two elongated lines at middle somitogenesis stage. In addition, the expression pattern indicated that Cxcr4 guided PGC migration during gastrulation.
|First Author Affilication||Institute of Oceanology, Chinese Academy of Sciences|
|李美洁. 牙鲆原始生殖细胞的标记及其迁移机制的初步研究[D]. 北京. 中国科学院大学,2015.|
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