Cloning of Carp Septin7 and Its Interaction with sperm specific protein, Msap

Septin是一種保守性相當高的細胞骨架(cytoskeleton)蛋白質，除了植物之外幾乎所有的真核生物中都含有Septin。septin基因最早在酵母菌（Saccharomyces cerevisiae）中發現，其在胞質分裂中佔有重要的地位，缺失septin的酵母菌在出芽生殖時會產生細胞形狀異常的現象。最近的研究顯示，Septin家族成員也參與許多其他的細胞內反應，如細胞極性、計畫性細胞死亡、細胞週期調控和細胞形狀形成等。在胺基酸序列方面，Septin家族主要有三個共同的結構：N端的basic domain、GTPase domain和C端的coiled-coil domain。在哺乳類，Septin的basic domain負責phosphoinositides之結合，而coiled-coil domain則和Septin家族蛋白質間的結合有關。 本實驗室在之前進行和精子發生相關基因Msap的二維電泳分析時，發現其和Septin家族蛋白質中的Septin7之間可能有結合的情形，且Septin7在鯉魚精子中主要分佈在basal body的位置，和近期的研究結果相似。於是本論文利用SMART RACE的系統選殖鯉魚睪丸中septin7的cDNA，結果顯示可能有兩種長度不同的septin7 transcripts存在於鯉魚的睪丸組織。利用特定引子進行PCR發現兩種transcripts都廣泛存在於各組織中，但彼此之間有表現量差異的情形。免疫組織化學染色可以看出在鯉魚睪丸組織中，Septin7蛋白質並沒有特殊的分佈位置，而是呈現散佈在組織中的情形。而利用Septin7重組蛋白質抗體進行各組織萃取液的西方轉印，結果也顯示Septin7在各個組織中廣泛存在，但以在睪丸的表現量為最多，而且50kDa為主要的表現形式。為了確認Msap和Septin7蛋白質之間的結合情形，利用二維電泳的方法分析pull down和免疫共沉的產物，發現Septin7和Msap之間的確有結合的關係。Septin is a highly conserved group of cytoskeleton protein that have been found in almost all eukaryotes but not in protozoa or plants. The septin genes were originally identified in Saccharomyces cerevisiae, and have an important role during cytokinesis. The budding yeast mutants that exhibited defects in septins will produce abnormal morphology during the budding process. Recent researches implicate that different Septins have diverse cellular roles including cell polarity, programmed cell death, cell cycle regulation and cell morphogenesis. The septin family of genes possess three common structures: an N-terminal basic domain, GTPase domain and a C-terminal coiled-coil domain. In mammal cells, the function of the N-terminal basic region is possibly responsible for the interaction with phosphoinositides. Some data point that the C-terminal coiled-coil domain is also responsible for intermolecular interaction upon Septin complex formation. Previous studies about the spermatogenesis associated gene, msap, of our laboratory by using two dimensional electrophoresis discovered the possible interaction between Septin7 and Msap. The studies also revealed that Septins are localized in the basal body of carp sperm, which is consistent with other research. In this study, the septin7 cDNA is cloned by SMART RACE system and two variants with different length are obtained. By using PCR, we found that both two transcripts exist in all kind of tissue, but the relative expression is different. The result of immunohistochemistry revealed that the Septin7 is ubiquitously expressed present in all testicular tissue. Western blot by Septin7 antibody showed the Septin7 is expressed as 50 kDa as a major form in all kind of tissue, althouth enriched in testis. To confirm the interaction between Septin7 and Msap, pull down and co-immunoprecipitation were used. The results favored the possibility that Septin7 interacts with Msap.中文摘要………………………………………………………………………………1 英文摘要………………………………………………………………………………2 緒論……………………………………………………………………………………3 Septin的蛋白質結構…………………………………………………………….4 酵母菌中的Septins………………………………………………………………5 線蟲中的Septins…………………………………………………………………6 果蠅中的Septins…………………………………………………………………6 哺乳類中的Septins……………………………………………...……………….7 Septin filament的形成…………………………………………………………...8 Msap……………………………………………………………………………...8 Septin7……………………………………………………………………………9 材料與方法…………………………………………………………………………..11 萃取total RNA………………………………………………………………….11 北方轉印法……………………………………………………………………..11 RT-PCR………………………………………………………………………….13 南方轉印法……………………………………………………………………..13 SDS-膠體電泳………………………………………………………………….14 蛋白質西方轉印法……………………………………………………………..15 免疫組織化學…………………………………………………………………..16 抗血清的誘導與製備…………………………………………………………..17 抗體純化………………………………………………………………………..17 蛋白質濃度測定………………………………………………………………..18 免疫共沉分析…………………………………………………………………..19 二維電泳………………………………………………………………………..19 銀染色………………………………………………………………………….20 Pull down………………………………………………………………………..21 結果…………………………………………………………………………………..22 鯉魚septin7基因的選殖……………………………………………………….22 septin7 cDNA推導的胺基酸序列之分析與比較……………………………..22 septin7基因表現的組織特異性………………………………………………..23 septin7 transcripts的分析與比較........................................................................24 Septin7重組蛋白質的誘導及純化.....................................................................24 Septin7在睪丸組織中的空間分布.....................................................................25 Septin7和Msap之間的結合關係.......................................................................26 討論..............................................................................................................................29 參考文獻......................................................................................................................33 圖表..............................................................................................................................38 圖1-鯉魚septin7-L及septin7-S兩型transcripts的核酸序列與所預測的胺基 酸序列……………………………………………………………………..39 圖2-鯉魚Septin7與其他生物Septin7胺基酸序列比對…………………….43 圖3-septin7基因的組織特異性表現…………………………………………..45 圖4-septin7基因在鲤魚各組織的表現………………………………………..46 圖5-septin7 transcripts在各組織的存在情形…………………………………47 圖6-Septin7重組蛋白質的誘導……………………………………………….48 圖7-Septin7蛋白質在各組織中的表現……………………………………….49 圖8-睪丸組織中Septin7之免疫定位………………………………………….50 圖9-Msap pull down……………………………………………………………51 圖10-Septin7 pull down………………………………………………………...52 圖11-睪丸組織萃取液的二維電泳分析………………………………………53 圖12-Septin7純化抗體二維電泳之西方轉印………………………………...54 圖13-Msap免疫共沉之二維電泳分析………………………………………...55 圖14-Septin7免疫共沉之二維電泳分析……………………………………...56 附錄............................................................................................................................5