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银鲫尾芽期和心跳开始期胚胎特异表达基因的呈现
Alternative TitleScreen of Stage-specific Expression Genes in Embryos at Tail-bud Stage and Heartbeat Beginning Stage in Silver Crucian Carp
石耀华
Subtype博士
Thesis Advisor桂建芳
2002
Degree Grantor中国科学院水生生物研究所
Place of Conferral中国科学院水生生物研究所
Degree Discipline遗传学
Keyword银鲫 尾芽期 心跳开始期 胚胎发育阶段特异表达基因 抑制差减杂交
Abstract构建并筛选了银鲫(Carassiusauratusgibelio)胚胎发育尾芽期(TBS)和心跳开始期(HBS)的抑制差减杂交文库.RT-PCR检测已经得到全长cDNA的基因,研究它们的时空表达特性,初步筛选出9个差异表达基因.差异表达的五个已知基因是:一,真核翻译起始因子3亚单位2基因;二,真核翻译延伸因子1-α基因;三,FAG-2基因;四,M3-CK基因;五,SE(spl)基因.在肝脏和脾中检测不到SE(spl)的转录产物.可能的未知基因有四个:一,SC32004基因;二,SC41065-1和SC41065-2基因;三,SC42004基因;四,SC42001基因.在成熟卵细胞检测不到转录产物,胚胎发育过程中最早可在原肠期检测到该基因极其微弱的信号,神经胚期信号已经很明显,至心跳开始期和出苗前期检测到的转录产物水平最高,出苗后可能略有下降.在被检测的几种组织中均检测到转录产物,肾脏最强,依次为心脏、脾、脑、卵巢以及精巢和肝脏,其中,肝脏和性腺检测到的转录产物极少.
Other AbstractIn this dissertation, suppression subtractive hybridization (SSH) libraries were constructed from tail-bud stage (TBS) embryos and heartbeat beginning stage (FIBS) embryos in silver crucian carp (Carassius auratus gibelio). By using pre-screening, we got 1373 TBS and 1809 HBS PCR-positive clones respectively. All these clones were submitted to dot blot. 169 TBS and 272 FIBS dot blot positive clones were sequenced. 21 of 45 dot blot positive clones were identified to be differential expressed gene fragments by virtual northern hybridization. By using the technique of RACE, we have cloned complete cDNA sequences of HBS differential expressed genes of FAG-2, M3-CK, alpha actin and myosin light chains 2 SMART cDNA libraries were also constructed from TBS embryos and HBS embryos of silver crucian carp (Carasshis auratus gibelio), 1086 TBS and 676 HBS PCR positive clones were larger than 500bp. 186 TBS and 181 HBS clones of them were sequenced, so that lots of genes involving in transcriptional regulation and embryonic development were acquired. Among them, complete cDNA sequences of 23 genes are obtained. Temporal and spatial expression characters of partial genes were analyzed by RT-PCR. Results showed that at least 9 of them were temporal and spatial differential expressed. There are five differentially expressed known genes. The first one is Eukaryotic initiation factor 3, subunit 2. Its mRNA is detectable in all stages of embryonic development, however, before gastrula, detectable mRNA is very weak. Since then, detectable mRNA increases little by little until muscular contraction stage. In adult fish, the mRNA is abundant in all detected tissues but testis, in which detectable mRNA is quite weak. The second one is elongation factor 1 alpha. Its expression character is similar to Eukaryotic initiation factor 3, subunit 2. The third one is FAG-2, which is homologous gene of XAG-2. mRNA is detectable weakly at TBS and strengthen step by step until larvae. Although its mRNA could also be detected weakly in brain, spleen, heart and testis, the strongest signal was detected in kidney. In ovary and liver, no signal was detected. The fourth gene is creatine kinase M3-CK, which mRNA could be detected in every embryonic development stages. Nevertheless, detectable mRNA obviously increased since muscular contraction stage. Moreover, mRNA could only be detectable in heart. The fifth known gene is enhancer of split homology [E(spl)]. mRNA of E(spl) could be detected very weakly from eggs to blastula embryos. Gradually increasing mRNA could be detected since gastrula. Besides, at the time transformed into larvae, detectable mRNA level seemed to decrease. Brain is the space where SE(spl) exists most abundantly. As for testis and kidney, little signal was also detected respectively. In liver and spleen, no signal could be detected. There are also four possible new genes expressed differentially. The first one is SC32004, whose mRNA could be detected since neurula and increased gradually until muscular contraction stage. In adult, mRNA could mainly be detected in testis, and weakly signal could only be detected in spleen. The second one is SC41065. In the RT-PCR detection of SC41065, we got two bands belonging to 41065-1 and 41065-2 respectively. They may be similar to each other in structure and function. SC41065-1 mRNA was firstly detected at TBS and arrived the highest level at HBS. Then, the signal decreased obviously. In adult fish, its mRNA could not be detected in oocyte, liver and spleen while a decreasing trend of detectable mRNA level was drawn among testis, brain, heart and kidney. mRNA of SC41065-2 could be detected at every stage during embryonic development, and obviously increased at neurula. In HBS, detectable mRNA arrivals the highest mount. Besides, its mRNA could be observed in all detected tissues but hardly in liver. The third one is SC42004, whose expression model is similar to SC32004 during embryonic development. In adult fish, detectable mRNA is at differential level in brain and testis. The last one is SC42001. During embryonic development, detectable mRNA was initiated at gastrula and reached the crest at HBS. In the stage when embryos turn into larvae, the signal decreased little. Although mRNA was observed in every tissue, the level was different among them. In kidney, its mRNA is the most abundant, followed by heart, spleen, brain and oocyte, and in testis and liver it was hardly observed.
Pages139
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12656
Collection学位论文
Recommended Citation
GB/T 7714
石耀华. 银鲫尾芽期和心跳开始期胚胎特异表达基因的呈现[D]. 中国科学院水生生物研究所. 中国科学院水生生物研究所,2002.
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