牙鲆原始生殖细胞的标记及其迁移机制的初步研究

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	牙鲆原始生殖细胞的标记及其迁移机制的初步研究
	李美洁1,2
学位类型	硕士
导师	谭训刚
	2015-05-27
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	生物工程
关键词	原始生殖细胞（pgc）、牙鲆（olive Flounder）、nanos3、3′非翻译区（3′utr）、sdf1、cxcr4、pgc迁移
摘要	牙鲆是我国北方重要的海水养殖经济鱼类，近年来种质退化，抗病力下降，寻找保存优良种质资源的方法越来越重要。牙鲆雌雄个体差异较大，获得全雌牙鲆将促进养殖业的发展，但是目前对于牙鲆性别分化机制还不清楚。原始生殖细胞（PGC）的研究可以为这些问题的解决提供新的途径，基于PGC的分离、移植等操作技术可以发展借腹生子、转基因鱼、冷冻保存PGC等技术手段。由于PGC还与性别分化相关，因而其研究也有助于对牙鲆性别分化机制的了解。标记PGC是对PGC进行分离、移植等操作技术的前提。我们获得了PGC标记基因之一：牙鲆nanos3。通过RT-PCR和原位杂交的方法分析其在牙鲆胚胎发育过程中的表达图式。结果表明，牙鲆nanos3在胚胎发育的整个过程中均有表达，属于母源遗传物质，且在PGC中特异性表达。因此nanos3作为牙鲆PGC的标记基因是可靠的。根据牙鲆胚胎发育过程中nanos3的表达图式我们模拟了PGC的迁移模式，牙鲆PGC的迁移过程与其他硬骨鱼类类似，但在体节期发生期中期体轴两侧的PGC开始向体轴聚合，并形成比较松散的结构，然后整体向胚体后部迁移，在心跳期，PGC再次分开为两个对称的紧密的聚合体。这种迁移模式与现在报道的PGC迁移模式均不同，是青鳉、鲱鱼、虾虎鱼PGC迁移模式的综合。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。获得牙鲆sdf1、cxcr4基因，通过RT-PCR和原位杂交的方法研究在胚胎发育过程中的表达。RT-PCR结果表明，sdf1、cxcr4是母源遗传物质，在原肠胚期表达量达到最高。原位杂交结果表明，sdf1、cxcr4与胚胎发育过程中与原肠胚的形成、脑的发育、眼的发育、体节的发育、侧线原基的迁移相关。将sdf1的表达同PGC在胚胎发育过程中的迁移路线进行对比分析，我们推测Sdf1在原肠胚期以及在体节发生的早期对于PGC的迁移有引导作用，在体节发生中期时对PGC轴向迁移到胚体中轴线上具有非常重要的作用，Cxcr4在原肠胚期对于PGC的迁移有作用。
其他摘要	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.
学科领域	海洋生物学
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/22721
专题	实验海洋生物学重点实验室
作者单位	1.中国科学院大学 2.中国科学院海洋研究所
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	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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