【作者】 林欣大；

【导师】 程家安； Xinhua Lin；

【作者基本信息】 浙江大学 ， 农业昆虫与害虫防治， 2003， 博士

【摘要】 通过运用Vestigial-gal4，UAS-FLP系统把重组酶专一性地传递到翅中的新遗传筛选体系来进行遗传筛选，由于在F1即进行筛选，故称F1遗传筛选。根据翅的表型筛选出突变体以后，对这些突变体果蝇的生殖系克隆胚胎的表皮形态进行分析，从而进一步确认该突变的性质。从该F1遗传筛选中分离出了很多已知基因的突变体等位基因，其中包括了那些与Wingless(Wg)信号传导途径和Hedgehog(Hh)信号传导途径相关的突变体等位基因。同时，也分离出了一些未知基因的突变体等位基因。本文对其中与Wg信号传导途径和Hh信号传导途径相关的三类突变体等位基因(每一类都有三到四个突变体等位基因)进行了深入的研究。并通过综合分析翅的形态，生殖系克隆胚胎的表皮形态，以及缺失作图，直接测序和搜索基因组数据库，鉴定出了三个基因，这三个基因分别对应于该三类突变体等位基因。同时，对这三个基因在果蝇Wg信号传导途径和Hh信号传导途径中的作用也进行了研究。 1、分离到的第一类突变体等位基因的有B158，B140和B173。试验表明具该类突变体等位基因的果蝇呈现类似于wg突变的翅表型，生殖系克隆胚胎也呈现表皮混合的典型wg突变表型。这些突变或是无义突变(B158和B140)或是破坏原初转录本剪接的突变(B173)。sll基因编码一个高尔基体PAPS转运子。Kyte-Doolittle疏水性分析结果表明，与其它转运子相似，Sll蛋白是一个疏水性很强的蛋白(尤其在它的C端)。 通过对sll生殖系克隆胚胎进行的抗Engrailed(En)抗体染色和wg反义探针的原位杂交结果表明，与野生型果蝇的胚胎相比较，En蛋白和wg转录物条带出现了明显的缺失。胚胎分析结果表明sll基因的突变会导致wg转录和engrailed基因的表达受到抑制。进一步结合运用FLP-FRT系统和minute~-，用缺损Myc蛋白的表达来标记纯合的sll突变基因克隆，用minute~-来相对的扩大含sll突变基因的克隆，对翅成虫盘所作的克隆分析表明，在含sll突变基因的克隆中，细胞外的Wg蛋白分布明显减少，这种细胞外的Wg蛋白减少表明在翅成虫盘中Wg信号传导受到了明显抑制。 2、分离到的第二类突变体等位基因的有15-108，A164，B104和L5。具该类突变体等位基因的果蝇呈现出与Wg和Hh信号传导途径相关的翅和表皮形态。这一组突变体等位基因编码一种木糖转移酶-Oxt，这种转移酶能够将UDP-木糖中的木糖转移到核。0蛋白的丝氨酸（Serine）残基上。该组的三个突变体等位基因的突变都是无义突变，它们都破坏了。Xt基因的编码区。通过把。Xt基因加上GFP标记，发现该基因主要在细胞质中表达。将OX亡蛋白的保守结构域和哺乳动物的同源基因的保守结构域进行比较，发现这些保守区域在所比较的脊椎动物和无脊椎动物中都十分保守。 通过对 oxt生殖系克隆胚胎进行的抗 En抗体染色和 wg反义探针a杂交结果表明，与野生型果蝇的胚胎相比，En蛋白和wg转录物条带都出现了明显的缺失。胚胎分析结果表明则t基因的突变会导致wg的转录和engrailed基因的表达受抑。进一步结合运用FLP－FRT系统，用缺损GFP蛋白的表达来标记纯合的OXt突变基因克隆，针对眼睛成虫盘的克隆分析结果表明，在OXt突变克隆中，Hh下游的以蛋白的表达区域完全消失。Ci蛋白的表达区域完全消失表明Hh信号传导受到了明显抑制。 3、分离到的第三类突变体等位基因包括F66，F15亿8，F126和F107。具该类突变体等位基因的果蝇呈现出类似于把突变的表型，生殖系克隆胚胎也呈现表皮混合表型。这组的突变体等位基因编码一个Wg信号传导途径组成成分干gg由 其特点是在 N端含有一个核定位信号mLS），而在 C端则含有一个 PHO结构域（一种C4HC3锌指结构）。测序结果表明其中H个突变体等位基因F66，F15－108和F126均是无义突变。无义突变会导致不完整的蛋白产物的产生。F107有一个突变位点在PHD域内，该突变把PHD结构域（C4HC3）中的最后一个半脱氨酸（Cysteine）突变成络氨酸（Tyrosine）。 Wg信号传导途径是果蝇和脊椎动物中的一个关键性的信号传导途径。已经鉴定出来许多参与 Wg／Wnt信号传导通路的基因。Wg下游的 Wg信号传导是由核 TCFIEFI 通过 Amadillo（Ann）／P－catenin介导的。PygoPusoygo）是 W洲nt信号传导通路的成员。通过本研究的细胞定位实验发现PygO专一性的表达在细胞核中，这与其含有一个核定位信号（NLS）是一致的。运用反义探针做原位杂交观察到了pygo基因在果蝇胚胎中的表达特性。结果表明，虽然pygo普遍表达于整个胚胎中，并且在果蝇胚胎发生全过程都有，但n000在前囊胚层…亿七lastoderm冲的表达水平相对较高，这说明胚胎发生过程中来自母方的贡献较高。在幼虫组织（包括翅成虫盘，眼成虫盘和腿成虫盘）的发育过程中，PygO的表达水平总的来说比较低。然而，比较翅成虫盘，眼成虫盘和腿成虫盘的PygO表达水平，则在翅成虫盘和腿成虫盘PygO的表达水平相对较高。 6 本研究加深了对Wg、信号传导更多还原

【Abstract】 By using a vestigial-ga14 to deliver the tissue specific flipase to the wing, a F1 genetic screen was performed. A number of mutant alleles of known or unknown genes were isolated from this Fl screen. This study focused on three groups of mutant alleles from mutant alleles of unknown genes. All these three groups of alleles related to either Wingless (Wg) signaling pathway or Hedgehog (Hh) signaling pathway (each group composed of 3-4 mutant alleles). By analyzing the wing phenotype, cuticle phenotype of germline clone embryo, deficiency kit mapping, direct sequencing and searching the genome sequence database, three genes: sll, oxt and pygo were identified (each gene corresponding to one group of mutant alleles).1) The first group of the mutant alleles included B158, B140 and B173. These alleles showed wg-like phenotype (notch et al.), the germline clone embryo showed cuticle fusion phenotype. This group of mutant alleles turned out to be a gene encoding a Golgi adenosine 3’-phosphate 5’-phosphsulfate transporter (adenosine PAPS transporter). Kyte-Doolittle hydrophilicity plots showed that Sll is a very hydrophobic protein, especially in C terminus. The nature of the mutant alleles is either nonsense mutation or the mutation which disrupted the splicing of the primary transcripts.Anti-En antibody staining and in situ hybridization on sll Germline clone (GLC) embryo using wg antisense probe showed defective En protein bands and defective wg transcripts bands. Furthermore, the wing imaginal disc clonal analysis showed that in the homozygous sll mutant clones, the extracellular Wg was dramatically reduced. The reduction of extracellular Wg indicated that the Wg signaling in the wing imaginal disc was disrupted. The reason leaded to this phenomenon is that sll encodes a PAPS transporter, so the disruption of the sll gene would generate the unsulfated Heparan Sulfate Proteoglycan (HSPG), which most likely would lose normal function.2) The second group of mutant alleles included 15-108. A164, B104 and L5. These alleles showed wing and cuticle phenotypes related to both Wg and Hh signaling pathways (notch and vein defect et al.). This group of mutant alleles turned out to be a gene encoding a xylosyltransferase, which is an enzyme transfers the xylose from UDP-xylose to the serine of the protein core. The nature of all the three mutant alleles of this group is nonsense mutation disrupting the coding region of oxt gene. By tagging the oxt gene with a GFP tag, it was found that this gene mainly expressed in the cytosol. Comparison of conserved domains (Core-2/ I-Branching and Wsc domain) of Oxt with mammalian homologues showed that both of domains are conserved throughout the invertebrate and vertebrate.Anti-En antibody staining and in situ hybridization on oxt GLC embryo using wg antisense probe showed defective En protein and defective wg transcripts bands. The embryonic analysis results indicated that the disruption of oxt gene would lead to the disruption of wg transcription and the expression of engrailed gene, as the wgtranscription is dependant on the Engrailed. Clonal analysis in this study on the eye imaginal disc showed that in the oxt mutant clones, the Ci expression was completely deleted. On the Hh signal receiving cells, the function of HSPG in HhN movement might be that it competes with Ptc for binding of HhN, following the releasing from the Disp, HhN binds to the HSPG to prevent the HhN from being captured by the Ptc, hence facilitate it’s transfer to the more distantly located cells. Also, HSPG might also be required for secreting cells: as a co-factor for secreting cells to displace the HhN from the Disp.3) The 3rd group of mutant alleles included F66, F15-28, F126 and F107. These alleles showed wing and cuticle phenotypes related to Wg signaling pathway. This group of mutant alleles turned out to be a new component of Wg singaling pathway. The sequence result showed that the nature of pygo mutant alleles F66, F15-108 and F126 were nonsense mutation, which probably led to tru更多还原