Follistatin 基因在牙鲆胚胎中的表达及其在肌肉发育中作用的研究

IOCAS-IR > 海洋环流与波动重点实验室

	Follistatin 基因在牙鲆胚胎中的表达及其在肌肉发育中作用的研究
其他题名	Study of Expression of Follistatin in Flounder (Paralichthys olivaceus) Embryos and the Function on Development of Muscle
	刘庆华
学位类型	博士
	2006-06-09
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	牙鲆 Paralichthys Olivaceus Follistatin 胚胎发育肌肉原位杂交显微注射
摘要	卵泡抑素(Follistatin)是1987年由Robertson和Ueno分别从牛和猪的卵泡液中分离出的一种富含半胱氨酸的糖基化单链多肽。FS对不同类型的细胞有广泛的调节作用，具有多方面的生物学作用。本研究克隆到牙鲆Follistatin基因，并利用RT-PCR和原位杂交对其在胚胎及成体中的表达进行了分析，并对其启动子进行了组织特异性分析。 1． Follistatin基因组全长4.3 kb左右，其中启动子区长约1 kb。与cDNA序列的比较显示该基因含有5个外显子和4个内含子，内含子和外显子的交界处严格遵守GT…AG规则。Follistatin基因编码了一个含有323个氨基酸的蛋白质前体，该蛋白质前体含有信号肽区域、N-端结构域、Follistatin结构域Ⅰ、Follistatin结构域Ⅱ、Follistatin结构域Ⅲ和C-端结构域。蛋白比较分析表明Follistatin与其他物种的Follistatin的同源性较高，其结构域保守性更高。三级预测结果显示：在空间构型上，牙鲆Follistatin基因与斑马鱼Follistatin基因完全一致。在序列上高度保守的半胱氨酸在空间上两两相对，它们可能对维持Follistatin蛋白的空间结构起着重要的作用 2. RT-PCR方法和原位杂交方法研究Follistatin基因在牙鲆胚胎中的表达情况结果显示：Follistatin基因在受精后26 hrs开始在中胚层细胞处表达，其后在受精后28、30、32、34、36、38、40、42 hrs等时期Follistatin基因持续表达，并在体节腹部部位、头部，背部体节等部位表达。以MyoD为对照而进行的双色原位杂交结果显示：Follistatin与MyoD在胚胎的体节处表达有重叠区域，对双色原位杂交样品进行的冰冻切片实验结果显示：两个基因的表达区域交叉，互相混合，表明Follistatin基因也在肌肉前体细胞中表达，但并不在所有的前体细胞中表达，Follistatin可能在肌肉发育早期起一定的作用。成体组织中，Follitatin基因在体肾、肠、心脏、头肾、脾中有表达，而在肌肉中没有表达。 3．启动子序列分析结果表明：牙鲆启动子存在AP-1、C/EBP、SP1、USF、E47、MyoD等转录因子的潜在结合位点。显微注射结果显示：该基因启动子能够使绿色荧光蛋白表达在斑马鱼的胚胎肌肉组织中。
其他摘要	Follistatin (FS) was first isolated from ovarian follicular fluid of cow and pig by Robertson and Ueno in 1987 respectively. It was a cysteine-rich single chain peptide. Follistatin has broad function on different type of cells. In this study, genomic sequence of Flounder Follistatin gene was identified from genome library of flounder (Paralichthys olivaceus) by Genomic Walking. The expression of the gene in embryo and adult tissues was analyzed by RT-PCR and in situ hybridization. The tissue-specific expression of the promoter was analyzed. 1. The genomic sequence of flounder Follistatin was 4.3 kb long. The promoter was about 1.0 kb long. Compared with cDNA sequence revealed that there were five exons and four introns and the boundary of exon and intron abided by the role of GT…AG strictly. Follistatin gene encoded a 323-amino-acid preprotein. The preprotein contained signal domain, N-terminal domain, Follistatin domain Ⅰ, Follistatin domain Ⅱ, Follistatin domain Ⅲ, and C-terminal domain. Comparison of deduced amino acid sequences of flounder Follistatin with other vertebrate showed that Follistatin was highly conserved, the identity of domain was higher. Forecasting of Follistatin protein structure of flounder revealed that the model of flounder Follistatin was the same as that of zebrafish. The function of highly conserved cysteines might be maintaining the structure of Follistatin protein. 2. Analysis of expression of Follistatin during flounder embryo development revealed that Follistatin began express in mesoderm cells at 26 hours after fertilization and continues express at 28, 30, 32, 34, 36, 38, 40and 42 hours after fertilization in ventral of somite , head, dorsal of somite cells. Double in situ hybridization revealed that the region where Follistatin and MyoD expressed partly overlapped at somite and cryosection of double in situ hybridization samples showed that the expression of the two genes intercrossed and partly overlapped. But Follistatin did not express in the entire region that gives rise to muscle. The analysis of expression of Follistatin on adult tissues showed that Follistatin could express in body kidney, intestine, heart, head kidney, spleen and could not express in muscle. 3. Analysis of flounder Follistatin promoter showed that there were combine sites of AP-1, SP1, USF, E47 and MyoD. Results of microinjection showed that the promoter of Follistatin could express green fluorescent protein in zebrafish embryo muscles.
页数	97
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1453
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	刘庆华. Follistatin 基因在牙鲆胚胎中的表达及其在肌肉发育中作用的研究[D]. 海洋研究所. 中国科学院海洋研究所,2006.

条目包含的文件
文件名称/大小	文献类型	版本类型	开放类型	使用许可
10001_20031800680151（1387KB）			限制开放	--	浏览