众所周知，果蝇之所以成为极好的遗传学模型是因为它较短的生命周期及较强的繁殖能力，能够在短时间内培养繁殖出大量特定种系的子代。目前，果蝇已成为生命科学与人类疾病研究的重要模型。所有的胚胎，不论是果蝇还是人，其发育过程和表型的正确性必须依赖细胞内信号在正确的时间、正确的地点和以正确的量传导。Snake是一种与果蝇背腹极向发育相关的蛋白酶。此蛋白酶是丝氨酸蛋白酶瀑布的一个中间点，它的缺失会导致胚胎失去腹部结构即背方化。尽管Snake在1986年就已被克隆，其基本结构及其与功能关系也在上世纪90年代被阐明，但到目前为止，关于它在胚胎内如何活化以及参与背腹极向发育的机制仍无任何文献报道。本课题试图通过体内实验观察Snake在胚胎内的剪切及其剪切是否依赖位置信号调节者Pipe来探讨Snake和位置信号之间的关系；并通过明确Snake夹子区域的关键氨基酸残基，解释Snake夹子区域结构与其功能的相互关系。 第一部分 果蝇胚胎背腹极向发育中丝氨酸蛋白酶瀑布内的Snake蛋白酶的剪切研究 目的：果蝇胚胎的背腹轴通过Toll受体信号确立，而Toll受体的配体则通过一个胞外丝氨酸蛋白酶瀑布发生的一系列蛋白水解剪切而产生。这个蛋白酶瀑布包括四个成分 Nudel、Gastrulation Defective (GD)、Snake (SNK)和 Easter(EA)。根据关于Nudel、GD和EA的研究结果推测，Snake是此蛋白酶瀑布中的一个关键酶，因为它的活化或活性可能受卵子发生期产生的位置信号所调控，而此位置信号的产生依赖于磺基转移酶Pipe。尽管snake是这些基因中第一个被克隆的，它在胚胎内的活化特征还不清楚。本研究目的通过研究Snake的剪切来探讨Snake和位置信号之间的关系。 方法：采用显微注射技术建立表达myc标记的野生型Snake (SM)、催化残基丝氨酸突变的Snake(SMSA)和酶原剪切位点突变的Snake(SNKK)等三种转基因的果蝇系。蛋白免疫印迹实验检测野生型、snake缺失、gd缺矢及pipe缺矢等不同背景下的胚胎内内源性Snake和表达myc标记的Snake的转基因胚胎内Snake的剪切情况。 结果： 1.内源性Snake酶原的大小约52kDa，但未能检测到内源性Snake的剪切产物。 2.借助表达myc标记的Snake的转基因果蝇，本实验观察到一个分子量大小约57kDa的剪切产物。此产物在发育晚期的卵室中不表达，而仅出现在胚胎中。 3.57kDa剪切产物的产生依赖于上游的蛋白酶Nudel和GD，而不依赖下游的Easter蛋白酶和Pipe。 4.此剪切产物的大小与预计的不相符，进一步的研究表明此剪切产物不是直接来源于Snake酶原剪切位点，它的产生也不依赖Snake自身的催化活性。 结论：本研究首次发现Snake转基因果蝇体内的剪切产物。此剪切作用发生在胚胎发生的早期、且依赖于产生腹侧发育信号的蛋白酶瀑布中上游蛋白酶Nudel和GD。这种时间性和瀑布依赖性表明这种剪切是Snake在瀑布通路中活化的一种反映。由于此剪切产物的产生不依赖Pipe，提示瀑布中第一个局限于腹侧、受Pipe调节的反应发生在Snake下游，即很可能是Snake剪切Easter的反应受Pipe调节。 第二部分 Snake夹子区域结构与功能分析 目的：夹子区域是在非脊椎动物的丝氨酸蛋白酶中发现的一种富含半胱氨酸的模体，因外形类似纸夹而得名。据推测它可能在蛋白酶的定位、调节催化活性或蛋白酶之间的相互作用方面发挥重要作用。本研究探讨Snake夹子区域不同突变体对果蝇胚胎发育的影响，并揭示夹子区域结构与其对蛋白酶瀑布调控功能的相互关系。 方法： 1.定点诱变技术用于将Snake夹子区域的单个和成组极性氨基酸突变为非极性的丙氨酸。 2.通过显微注射技术将体外转录的这些突变子的信使RNA注射到snake缺失的胚胎体内，并通过观察肠腔形成期的表型发育来检测这些突变子补救snake缺失的胚胎的能力。 3.将这些突变子单独转染、或与GD或/和Easter共转染果蝇S2细胞，然后通过免疫印迹实验检测这些突变的Snake在S2细胞中的表达以及这些突变子与Easter和GD的相互作用情况。 结果： 1.显微注射后，单个的极性氨基酸向丙氨酸的突变对缺失胚胎的补救有效率与野生型相似。在双重突变或三重突变中，只有loop1(R94和D98的双重突变)突变引起Snake的补救有效率明显下降。 2.S2细胞转染体系的结果表明，所有的单突变和多重突变子存在分泌障碍。 3.S2细胞共转染实验显示，loop3的多重突变引起Snake与GD的相互作用改变。与野生型Snake相比，loop3右侧的双突变(R123和D125的双重突变)和其左侧的三突变(R115，E116和R118)不能有效地剪切GD和被GD剪切。loop1中R94和D98的双重突变子被GD剪切和剪切其它Snake分子的能力与野生型相似。 4.如果将Easter加入共转染反应体系，所有突变的和野生型Snake在剪切GD和Easter的能力方面没有明显区别。 结论：夹子区域loop1中的极性氨基酸残基可能对Snake的功能至关重要；而loop3内的氨基酸残基可能在Snake与GD相互作用中起重要作用。 关键词：果绳；背腹极向；Snake蛋白酶；剪切；夹子区域；突变分析

 It is well known that Drosophilia, the fruit fly, is an excellent organism for genetics studies because it has a very short life cycle and strong reproductive capacity and produces large numbers of offspring with specific genotype in very short period. Up to now Drosophila has been widely employed in various studies related to life science and human diseases. Patterning of the embryo whether from Drosophila or human being occurs as a function of intercellular signals delivered at the right time and place, and in the right amounts. Snake, a protease at the middle point of a serine protease cascade, is involved in the development of the dorsoventral polarity of Drosophila embryo, and in the absence of which the embryo will adopt a dorsalized fate. In vivo activation of Snake has not yet been characterized although it was cloned in 1986 and its structure has been demonstrated over ten years ago. The current study planned to figure out how Snake is regulated in vivo by investigating the activation of Snake in embryo and its relationship with the regulator of the ventral cue, Pipe. We also tried to map the key amino acid residues in Snake clip domain to reveal the functional importance of the structures. Part Ⅰ Processing of Snake within a serine protease cascade defining the dorsoventral axis of the Drosophila embryo Aims: Signaling through the receptor Toll establishes the dorsoventral axis of the Drosophila embryo after an extracellular serine protease cascade, involving the proteases Nudel, Gastrulation Defective (GD), Snake (SNK), and Easter (EA), which activates the Toll ligand precursor only on the ventral side of the embryo. Based on previous studies with Nudel, GD, and EA, we hypothesize that Snake is a key protease whose activation or activity is spatially regulated by a ventral cue deposited during oogenesis. The generation of this cue relies on Pipe sulfotransferase. Snake processing has not been characterized in vivo although its gene was first cloned among these members. The goals of the current study are to investigate whether the processing (activation) of Snake is regulated by the ventral cue in the Drosophila embryo. Methods: Transgenic flies expressing myc-tagged Snake(SM), SM with catalytic Serine mutated to Alanine (SMSA) or with the mutation at the cleavage site (SNKK) were generated by microinjection. Immunoblot analysis was used to examine the processing product of the endogenous or myc-tagged Snake in embryos from different background such as wild type, snake minus, gd minus and pipe minus. Results: A 52kDa band corresponding to the Snake zymogen, but no cleavage forms of the endogenous Snake was observed in wild type embryos in our experiment. Using the flies expressing a myc-tagged Snake, a 57kDa band which represents processed form of Snake was detected in the embryos, but not in the ovaries. The appearance of the 57kDa product relied on the activity of upstream proteases Nudel and GD, but was independent on downstream protease Easter or on Pipe. In addition, this 57kDa product was not a result directly from cleavage site and its generation seemed not require snake's own catalytic activity. Conclusions: The present study identifies for the first time a single processed product of Snake in vivo. This processing occurs in early embryogenesis and depends on the upstream proteases, Nudel and GD, in the cascade that generates a signal for ventral development. This time- and cascade-related processing suggests that this processing is a reflection of Snake activation within the pathway. The independence of the processing on Pipe function indicates that the first ventral side-restricted reaction may occur on downstream of Snake, that is, the processing of Easter by Snake may be the first ventrally restricted reaction regulated by Pipe in the cascade. Part Ⅱ Struture-function relationship of Snake clip domain Aims: Clip domain is very common in serine proteases in invertebrate. It is a cysteine-rich structural motif and its name was derived from the observation that the domain somewhat resembles the shape of a paper clip. The clip domain has been proposed to function in localization of protease, regulation of catalytic activity and interactions between proteases. The objective of this research was to reveal the functional importance of the structures in Snake clip domain in regulating the protease cascade. Methods: Site-directed mutagenesis was used to generate individual and group charged-to-alanine mutations in clip domain of Snake. mRNAs of each mutants were injected into the snake null embryos to score their rescuing ability for the embryo in early gastrulation. Transfections of each mutants alone, or along with Easter or/and GD were performed in Drosophila S2 cells to examine the processing product of mutated Snake and their interactions with Easter and GD by Western analysis. Results: Single charged-to-alanine mutants of Snake had no obvious changes in their rescuing ability in vivo in comparison with the wild-type. In the double or triple mutants checked, only a double-mutation of R94 and D98A in loop1 caused a dramatic reduction of the rescuing ability. Results from the S2 cell system showed that both point and loop mutants at clip domain had defects in secretion. Moreover, the double mutant of R123 and D125 on the right side of loop3, and triple mutant of R115, E116 and R118 on left side could not process GD and be processed by GD in comparison to the wild type Snake. Surprisingly, although the loop1 double-mutation (R94 and D98A) showed a very poor rescuing ability, it could be normally processed by GD and was able to process other Snake molecules. However, when Easter was present in the transfection system, no obvious differences could be observed between all mutants and wild type Snake in processing of GD or Easter. Conclusions: The loop1 of the Snake clip domain probably plays an important role in the function of Snake; and the loop3 may be involved in the interactions of Snake with GD. Key words: Drosophila; dorsoventral polarity; Snake protease; processing; clip domain; mutagenesis