文昌鱼被认为是现存与脊椎动物亲缘关系最近的无脊椎动物，也是脊椎动物祖先的模型，研究文昌鱼的发育相关基因对于揭示文昌鱼和脊椎动物形体结构的同源性与组织器官的发生和进化有重要的意义。 应用SMART™ cDNA文库构建技术，构建了以真核表达载体pcDNA3为基础的文昌鱼卵巢的cDNA文库。对3777个随机克隆进行了序列测定和生物信息学分析，获得了2041个基因序列。通过与GenBank中序列的同源性分析，将文库中获得的所有基因簇按照功能进行了分类。其中有几类受到我们的关注：(1)，胚胎发育中重要信号途径相关基因，包括TGF-β信号，Notch信号，视黄酸信号等； (2)与卵母细成熟相关的基因；(3)高丰度表达的基因；(4)免疫相关的基因。文昌鱼卵巢cDNA文库的成功构建以及对所得基因的进一步功能研究，将为了解文昌鱼胚胎发育中相关信号途径的组成、功能和进化机理；各组织器官发育的分子机理及与脊椎动物的同源性；免疫系统的组成与进化等方面提供更多宝贵的信息，也为进一步揭示脊椎动物的起源与进化提供新的线索。 通过对文昌鱼卵巢cDNA文库随机测序和PCR筛选，克隆得到五个荧光蛋白类似物基因。进一步应用生物信息学软件对荧光蛋白类似物基因进行了序列分析，结果表明它们是新的荧光蛋白类似物基因，这在文昌鱼中乃首次报道。进一步构建了GFPs的原核表达载体并进行诱导表达发现，低温下GFP2蛋白具有红色而其他的GFP不发光也没有颜色。通过Ni²⁺螯和层析、SP阳离子交换层析及G50分子筛等纯化步骤获得了高纯度的GFP2成熟重组蛋白，对重组GFP2蛋白的荧光波谱进行了分析，发现它只是一个红色荧光样的色素蛋白。 测定并分析了一个孕酮的细胞膜受体(mPR)基因，应用生物信息学软件分析其蛋白质序列，结果表明它含有典型的G蛋白受体的7个跨膜结构域；Northern杂交结果显示它只在生殖组织卵巢中表达，因此我们推断它在卵母细胞的成熟过程中起着重要的调节作用。 测定并分析4个与文昌鱼免疫反应相关的基因：IκBL、Ikaros、Bam32/DAPP1和TAPP基因。通过初步研究它们的基因组拷贝数、组织表达的情况和系统进化的过程，我们发现这几个基因都与文昌鱼的免疫和炎症反应相关。由于文昌鱼处于由无脊椎动物进化到脊椎动物的过渡阶段，其免疫系统也处于由天然免疫逐渐向适应性免疫进化的过程中，很多基因的功能都处在变化当中。那么我们推测在动物的演化过程中，基因倍增或基因组复制之后，多余基因从自然选择的压力下释放出来，结构和功能发生改变，并较快地积累了适应性(adaptive)的突变，从而赋予新的功能；而原先在胚胎发育过程中起着重要作用的基因，在动物发育的后期，基因重新被募集到新的组织而行使新的功能。我们的实验结果还暗示了文昌鱼的鳃和消化道很可能是原始的淋巴样组织。 关键词：文昌鱼卵巢；cDNA文库；表达序列标签(EST)；蛋白类似物基因GFP、mPR、IκBL、Ikaros、Bam32/DAPP1、TAPP；Northern杂交、Southern杂交、原位杂交

Amphioxus or lancelet is the common name of more than 20 species of marine invertebrates, which comprise the chordate subphylum of cephalochordates. Amphioxus has been popular with embryologists for over a century as a model for comparing and understanding the processes that lead to the formation of chordate structures because its body organisation seems intermediate in complexity between the tadpole larvae of tunicates and that of the jawless vertebrates, the hagfish and lamprey. Based on 18S rDNA data showed that cephalochordates are the closest chordate phylum to vertebrates. Due to its phylogenetic position amphioxus is an organism central for genome evolution and embryological studies. In this study, we have constructed a cDNA library of ovary of Chinese amphioxus, Branchiostoma belcheri tsingtauense. A total of 3,777 randomly picked expressed sequcnced tags (ESTs) were analyzed and categorized into 2041 independent genes. Some proteins were closely related to oocyte maturation and axis formation, and some showed similarities to the proteins that participated in host immune response. The most interesting findings were five high expressing fluorescent protein genes. These results suggest that the transcripts in ovary play an important role during early embryonic development and in immune response. Among these identified novel genes, five full-length cDNA fluorescent protein genes were obtained. It's the first time that fluorescent protein genes reported in amphioxus. GFPs were then expressed in the prokaryote expression system, and they were showed that the recombinant GFP proteins were nonfluorescence except GFP2. After the study of expression and purification conditions for pTRX-GFP2 in Escherichia coli, recombinant GFP2 protein with the high purity was obtained through immobilized metal-chelating affinity chromatography, SP-Sepharose fast flow and Sephadex G-50 gel filter purification steps. The excitation spectrum and pH sensitivity for recombinant GFP2 were further investigated. An expressed sequence tag with significant similarity of mammalian membrane progestin receptors (mPRs) was first time found in our cDNA library prepared from ovary of adult amphioxus, Branchiostoma belcheri tsingtauense. Characterization of the corresponding cDNA clone revealed the presence of an open reading frame encoding a 358aa long peptide. The putative protein, tentatively designed mPR appear to have seven transmembrane domains. The mPR's mRNA is only detected in the ovary tissues on northern blot. These characteristics suggest this protein mediates a "nonclassical" action of progestins to induce oocyte maturation in amphioxus. Activation of NF-κB is regulated by its inhibitor, IκB. In the present work, we have identified the complete cDNA sequence of a member of IκB family genes from our library. The amphioxus IκBL contains two ankyrin repeats and two conserved serine residues, which bear a marked similarity to those of IκB family proteins. In addition, southern blotting analyses suggest that two copies of the IκBL gene are present in this species. The expression pattern and the phylogenetic analysis of the amphioxus IκBL gene reinforces the notion that the NF-κB/IκB pathway is not only involved in the regulation of the immune and inflammatory response but also having a role during early embryogenesis. The family of zinc finger transcription factors, Ikaros, is involved in the development of lymphocytes. We have cloned a gene with similarity to members of Ikaros family in Chinese amphioxus. The predicted 276-amino-acid protein contains four zinc finger motifs but their spatial relationships have a slight difference in comparison with those of vertebrates. The results of our study about this protein suggested the possibility of isolating amphioxus genes phylogenetically related to immune system components in vertebrates despite their strong divergence. We reported here the characterization of two signal transduction proteins, Bam32 and TAPP in amphioxus. Bam32 and TAPP share several characteristics, including small size (32 to 47 kDa), lack of enzymatic domains, high conservation between humans and mice, and the presence of pleckstrin homology (PH) domains near their C termini where contain the 3-phosphoinositide-binding motif. Unlike Bam32, the N-terminal region of TAPP contains a second PH domain instead of SH2 domain. TAPP transcript in amphioxus is expressed in a variety of tissues except in notochord, but Bam32 is expressed in ovary, gill and gut. These results suggested that ovary, gill and gut tissues may be a source of lymphoid cells in amphioxus. Key words: Amphioxus, ovary; EST; fluorescent protein gene GFP; mPR; IκBL; Ikaros; Bam32; TAPP; southern blot; in situ hybridization; northern blot.