中国科学院水生生物研究所机构知识库
Advanced  
IHB OpenIR  > 中科院水生所知识产出(2009年前)  > 学位论文
题名: 鱼类异种核质互作过程中差异表达基因的研究
作者: 刘静
答辩日期: 2007-06-10
导师: 朱作言 ; 孙永华
专业: 水生生物学
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 斑马鱼 ; 稀有鮈鲫 ; 杂交胚胎 ; cDNA-AFLP ; 荧光定量
其他题名: Research on differentially-expressed genes
摘要: 自核移植工作开创以来,克隆胚中供体核的再程序化以及核质互作的问题就一直是发育生物学关注的焦点之一。人们认为自己可以通过克隆手段“复制”一个在遗传物质上与成体动物完全一致的个体,然而受体细胞质在遗传(线粒体基因组)和性状(早期图式形成)上或多或少地影响着克隆个体。虽然在不同物种中都有克隆成功的报道,但克隆的低成功率和成功克隆个体的早衰、发育缺陷等问题尚没有得到有效解决。影响动物克隆成功的关键因素——移植核的再程序化问题始终困扰和吸引着克隆领域的研究者,并成为动物克隆研究领域的热点和难点。另一方面,异种间的克隆能够突破不同物种间的生殖隔离障碍,为拯救濒危动物提供了一条全新的途径,同时也为核质关系研究提供了新的模型。我国学者童第周等在鱼类的克隆,特别是鱼类异种间克隆的开创性研究和卓越贡献奠定了我国在动物克隆研究领域的国际地位。 本实验室从上个世纪末开始致力于鱼类核移植工作的研究。受试的鱼类从经济淡水鱼鲤鱼,鲫鱼等逐渐转移到模式鱼斑马鱼,稀有鮈鲫。相对于同种克隆,种间克隆的成功率要降低许多,而影响种间克隆成功率的因素除了“供体细胞核的再程序化”外,更重要的是“异种间细胞核质的互作”。同种克隆是研究前一命题的有力途径,而种间克隆一直被认为是研究后一命题的有效手段。显然,以种间克隆进行核质关系的研究实际上是将这两个命题置于同一研究体系,这样并不利于对这两个命题特别是后一命题的独立分析。在本研究中,我们利用两种实验鱼— — 斑马鱼和稀有鮈鲫,通过对此两种鱼进行正反向有性杂交得到具有相同细胞核遗传物质而不同卵质的有性杂交胚胎,由于在这两种胚胎中,并不存在细胞核的再程序化事件,因而提供了研究种间核质互作的良好模型。我们利用此材料开展了关于异种间细胞质和细胞核间的相互作用及杂交胚胎中基因表达差异的研究。另一方面,鉴于在此前的研究中,本实验室所获得的由转基因鲤鱼细胞核和金鱼去核卵配合的属间克隆鱼的体节发育进程和脊椎骨数目均与受体金鱼一致,而迥异于供体鲤鱼,本研究开展了体节特异荧光蛋白基因(GFP)转基因鱼的研究。 主要内容包括: 1.利用cDNA-AFLP技术,分别比较分析了斑马鱼稀有鮈鲫正反杂交胚胎50%外包,90%外包时期部分基因的差异表达。将两个时期的正反杂交胚胎提取RNA合成cDNA建立4个cDNA池,分析64对引物组合获得13000条带,所有TDFs经过斑点杂交筛选,荧光定量分析,共鉴定25条带具有差别表达,均为量的差异,其中在ZR(Zebrafish♀×Rare minnow♂,ZR)中上调表达的有12条,RZ(Rare minnow♀×Zebrafish♂,RZ)中上调表达的13条。将差异条带进行克隆测序,BLAST分析结果表明除了未知的13条片段外已知的序列功能涉及比较广泛,其中包括线粒体蛋白,RNA假尿苷的合成,线粒体16S核糖体基因,糖基转移酶,III类组织相容性抗原,可溶性的神经因子,Huntington疾病基因等。通过cDNA-AFLP所筛选出的差异片段,对其进行荧光定量分析,胚胎原位杂交分析。结果显示片段的差异大多集中在5-10倍的范围。对于未知功能的基因片段合成反义RNA进行胚胎原位杂交。杂交结果显示,所有基因片段均为泛组织表达。 2. 利用cDNA-AFLP技术筛选斑马鱼稀有鮈鲫相互杂交胚胎50%外包,90%外包时期部分基因的差异表达。从中选择两个克隆cDNA,以进行深入的功能研究。获得两个基因ZR6和RZ10的编码区,将其体外转录为成熟mRNA,进行显微注射过表达分析。ZR6的过量表达导致胚胎发育呈现一定的表型而RZ10的过量表达没有产生变化。ZR6过表达的表型类似于BMP拮抗因子Chordin的过表达表型,即胚胎发育的背部化(dorsalization),选择若干背腹模式相关的标记基因进行原位杂交分析,揭示ZR6过表达所致的表型为背部化。由ZR6 cDNA序列所推定的蛋白序列和结构分析提示其为一个锌指蛋白,并行使转录抑制的功能。将ZR6基因的DNA结合域分别与vp16转录激活功能域和engrailed转录抑制功能域连接,构建融合基因vp16-ZR6和eng-ZR6,体外合成mRNA显微注射进行功能验证,结果显示转录抑制型eng-ZR6过表达产生类似于野生型ZR6过表达的表型,而转录激活型vp16-ZR6过表达导致类似于ZR6反义morpholino注射后的表型。进一步的研究表明,ZR6过表达所导致的背部化表型可以被bmp2b的过表达所拯救甚至反转为腹部化表型。总之,本研究发现了一个新的锌指蛋白ZR6,其行使转录抑制因子的功能,并直接或间接地参与BMP通路并调控斑马鱼背腹轴的形成。 3. 通过基因组信息学分析,快速分离克隆斑马鱼Lfng基因的上游调控序列,并将5’方向长度不同的3个上游调控序列分别与绿色荧光蛋白基因(green fluorescent protein,gfp)连接,构建表达载体pLfngEGFPa、 pLfngEGFPb、pLfngEGFPc。对斑马鱼胚胎的转基因发现,包含有GC-box, CAAT-box和TATA-box的0.2kb 的上游片段已经能够驱动GFP的组织特异性表达,但长度为0.4kb和1.2kb的上游片段所具有的启动活性更高。这表明斑马鱼Lfng启动子的关键调控序列在其5’端非编码区上溯到200bp以内,而200bp-400bp之间包含有增强调控序列。筛选获得半合子的LfngGFP转基因斑马鱼,并观察到组织特异性绿色荧光蛋白的表达。进一步研究发现,斑马鱼胚胎自1-胞期开始已能检测到Lfng的mRNA信号,Lfng-GFP转基因雌性个体的后代自1-胞期始便能观察到GFP基因的表达。这表明斑马鱼Lfng为斑马鱼卵细胞中携带的母性因子之一,提示鱼类的体 节发育受到母性因子的控制。 4. 通过基因组信息学分析,快速分离克隆斑马鱼her6基因的上游调控序列,并将5’方向长度不同的3个上游调控序列分别与gfp连接,构建表达载体pher6EGFPa、 pher6EGFPb 、pher6EGFPc。对斑马鱼胚胎的转基因发现,包含有CAAT-box,CCAAT- box,SPS sequence和TATA-box的0.5kb 的上游片段是能够正常驱动GFP的组织特异性表达的最短片段,而长度为0.2kb只包括部分SPS sequence和TATA-box的上游片段只能表达出极微弱的绿色荧光。转基因胚胎自囊胚期到尾芽期基本看不出明显的组织特异性绿色荧光蛋白的表达。从尾芽期开始,非常明显的分割图式已可以观察到,特异的GFP表达在后面发育时期依然可以持续观察到。它可以在头部包括眼睛,脑区等,每个体节的后半部分,脊索,肌肉,前体节中胚层(pre-somiticmesoderm, PSM)都可以很明显的观察到。
英文摘要: Since the art of nuclear transplantation was described, the nuclear reprogramming of donor nucleus and the interaction between nucleus and cytoplasm have been studied continuously. People have considered that they themselves can copy an adult animal, and the copied animal is genetically identical to its nuclear provider. However, the cytoplasm provided by the recipient species may affect the cloned animals genetically (in the view of mitochondrial genome) or/and morphologically (in the view of early pattern formation controlled by maternal factors). Although successful cloning has been obtained in various species, there still exist many problems and questions, such as the low efficiency of cloning, the premature senility of the cloned individual and some bugs in development. In recent years, to study nuclear reprogramming which will probably provide an answer for the mentioned questions has attracted many researchers in animal cloning studies and has been emerging as a hotspot in this field. On the other hand, cross-species cloning, which can go beyond the barrier of reproductive separation, provides a new approach for rescuing the endangered animals, and a new model to study nucleo-cytoplasmic interaction. Tung T C, the late professor of fish embryologist, has laid a foundation on cloning studies in China, by his pioneering study and prominent contribution in fish cloning. The low efficiency is also the major problem in fish cloning, and fish cross-species cloning is an ideal system to study nucleo-cytoplasmic interaction during development. Our research group has been devoted in research on fish nuclear transplantation from the late 20th century. Common carp and goldfish as experimental materials have been replaced by model fish such as zebrafish (Danio rerio) and rare minnow (Gobiocypris rarus). Compared withintra-species cloning, the efficiency of cross-species cloning is rather lower. In the cross-species cloned embryos, there are two major events occurring inside, the nuclear reprogramming of donor nuclei and the cross-talk between nuclei and cytoplasm from different species. Intra-species nuclear transfer provides us a powerful tool to study nuclear reprogramming, while cross-species nuclear transfer has been considered as a useful approach to answer the latter question. However, by using cross-species nuclear transfer, we will meet the effects resulted from these two events, which does not facilitate our extensive understanding of the latter question. In our present study, we used zebrafish and rare minnow to obtain cross-bred embryos of dual-direction (zebrafish♀×rare minnow♂ and rare minnow♀×zebrafish♂), which have the same nucleus and different cytoplasm. Since there is no nuclear reprogramming in these embryos, the crossbred embryos could be considered as an excellent model to study the nucleo-cytoplasmic interaction and the differential expression profiles between two kinds of embryos. On the other hand, since our previous studies showed that the cross-genus cloned fish derived from transgenic common carp nuclei and goldfish enuclueated eggs were consistent to the goldfish in the aspects of somatic development and vertebral number, instead of common carp, we were interested in fish somitogenesis and carried out the study of somite-specific transgenic zebrafish carrying green fluorescent protein (gfp) gene. 1. To determine partial differential expression of genes between crossbred embryos between zebrafish and rare minnow, cDNA-AFLP was analyzed in two developmental stages (50%-epiboly stage and 90%-epiboly stage). Expression profiles were generated from 13000 cDNA fragments in four cDNA pools with 64 primer combinations. 27 fragments were differentially expressed including 13 in ZR (Zebrafish♀×Rare minnow♂, ZR) and 14 in RZ (Rare minnow♀×Zebrafish♂, RZ). The fragments were sequenced and blasted. Results showed that the fragments were related to mitochondrial protein gene, RNA pseudouridylate synthesis, 16S ribosomal RNA, glycylpeptide N-tetradecanoyl transferase, soluble neuropilin 2b1, glycosyl transferase, class III histocompatibility, Huntington's disease and so on, besides some gene of unknown functions fragments. Anyway, the differential expression of genes in crossbred embryos mostly was related to the biological process of mitochondria and gene modification etc. Studies of real-time PCR showed most genes had the transcriptional differences of 5-10 folds between two kinds of embryos. For the unknown genes, we did whole-mount in situ hybridization in zebrafish embryos and the result showed that all the examined genes were expressed ubiquitously. 2. We cloned some differentially expressed genes between ZR and RZ embryos, and selected two TDFs (transcript-derived fragments) to clone the full-length cDNA for their functionary study in zebrafish embryos. In our experiment, we obtained the coding region sequences of ZR6 and RZ10, conducted in vitro synthesis of mRNA, and injected their mRNA into 1-cell embryos of zebrafish. The ectopic overexpression results showed that the overexpression of RZ10 had no obvious impact on the development of injected embryos, while injection of ZR6 leaded to a typical dorsalization phenotype, just mimicking the upregulation of chordin, an antagonist of BMP. Through bioinformatics analysis of the predicted amino acid sequence of ZR6, we proposed that ZR6 is a zinc finger protein and functions as a transcriptional repressor. With the help of transcriptional activator vp16 and transcriptional repressor engrailed, we constructed the fusion cDNA expression vectors, the transcriptional activation form vp16-ZR6 and the transcriptional repression form eng-ZR6. By injection of vp16-ZR6 and eng-ZR6 mRNA, we found that vp16-ZR6 injection gave a phenotype which was similar to the inhibition of ZR6 expression by morpholino injection, while the eng-ZR6 overexpression phenocopied the overexpression of orginal ZR6. Furthermore, the dorsalized phenotype resulted from ZR6 overexpression was able to be attenuated or even reversed by the injection of bmp2b mRNA. All these revealed that ZR6 is a novel zinc finger gene and this protein acts as a transcriptional repressor in the dorso-ventral patterning of zebrafish embryo. In addition, ZR6 may be involved in BMP pathway directly or indirectly. 3. To characterize the promoter of zebrafish lfng and generate somite-specific transgenic zebrafish, we isolated the upstream regulatory region of zebrafish lfng by blast search at the Ensembl genome database (www.ensembl.org) and analyzed the promoter activity using green fluorescent protein (GFP) as a reporter. Promoter activity assay in zebrafish shows that the 0.2kb fragment containing GC-box, CAAT-box and TATA-box can direct tissue-specific GFP expression, while 0.4kb and 1.2kb with further upstream sequence included drive GFP expression more efficiently. We produced the lfngGFP-trasngenic founders showing somite-specific expression of GFP and consequently generated hemizygous lfngGFP-trasngenic line. The eggs from lfngGFP-transgenic female zebrafish show strong GFP expression, which is consistent to the reverse-transcription PCR (RT-PCR) detection of lfng transcripts in the fertilized eggs. This reveals that zebrafish lfng is a maternal factor existing in matured eggs, suggesting that fish somitogenesis may be influenced by maternal factors. 4. By blast search at the Ensembl genome database (www.ensembl.org) we characterized the promoter of zebrafish her6. We isolated the upstream regulatory region of zebrafish her6 and analyzed the promoter activity using green fluorescent protein (GFP) as a reporter. Promoter activity assay in zebrafish shows that the 0.2kb fragment containing partial SPS sequence and TATA-box is not sufficient to direct GFP expression, while 0.5kb and 1.2kb with further upstream sequence included drive GFP expression more efficiently. We tracked the patterns of expression of her6 in transgenic zebrafish embryos. A typical strong GFP expression is first observed in blastula but there is no obvious tissue-specific expression from blastula to the tailbud stage. From the tailbud stage, a clear segmental pattern of expression starts to appear. Specific GFP expression is observed from the later stages. GFP was observed in head including eyes, brain etc, caudal half of somites, notochord, PSM (pre-somitic mesoderm) and muscle.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12156
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

Files in This Item:
File Name/ File Size Content Type Version Access License
鱼类异种核质互作过程中差异表达基因的研究.pdf(7485KB)----暂不开放 联系获取全文
Service
Recommend this item
Sava as my favorate item
Show this item's statistics
Export Endnote File
Google Scholar
Similar articles in Google Scholar
[刘静]'s Articles
CSDL cross search
Similar articles in CSDL Cross Search
[刘静]‘s Articles
Related Copyright Policies
Null
Social Bookmarking
Add to CiteULike Add to Connotea Add to Del.icio.us Add to Digg Add to Reddit
所有评论 (0)
暂无评论
 
评注功能仅针对注册用户开放,请您登录
您对该条目有什么异议,请填写以下表单,管理员会尽快联系您。
内 容:
Email:  *
单位:
验证码:   刷新
您在IR的使用过程中有什么好的想法或者建议可以反馈给我们。
标 题:
 *
内 容:
Email:  *
验证码:   刷新

Items in IR are protected by copyright, with all rights reserved, unless otherwise indicated.

 

 

Valid XHTML 1.0!
Copyright © 2007-2016  中国科学院水生生物研究所 - Feedback
Powered by CSpace