【作者】 李靓；

【导师】 薛乐勋；

【作者基本信息】 郑州大学 ， 细胞生物学， 2012， 硕士

【摘要】 背景：鞭毛／纤毛是细胞表面的突起物,在生物进化的过程中逐渐成为专门的细胞器,在细胞的运动、感知外界环境变化及信号向细胞内的传递过程中担任重要角色,纤毛的异常会导致多囊肾疾病、肥胖症、糖尿病、癌症等人类疾病。鞭毛内运输(IFT)是一种沿着微管轴丝的双向运动过程,对于绝大多数真核生物的鞭毛/纤毛的形成和维持是至关重要的,这个复杂的过程受到细胞内外各种因素的影响。驱动蛋白2是驱动蛋白家族中的一个成员,是鞭毛／纤毛内负责正向运输的关键马达蛋白。驱动蛋白2是一个异源三聚体,包括同源性较高的FLA8和FLA10亚基以及一个KAP亚基。其中FLA8和FLA10均由马达(motor)头部和螺旋一卷曲一螺旋的尾部组成,这两个亚基的尾部相互缠绕形成棒状结构,它们头部的马达则利用水解ATP产生能量沿着微管以类似行走(walking)的方式向微管正末端运动。杜氏盐藻(Dunaliella salina)是一种单细胞绿藻,有一对等长的鞭毛,培养条件简单。由于其具有1对等长的鞭毛而可以被用作研究鞭毛内运输及鞭毛相关疾病的模式生物。目的：探究驱动蛋白2亚基FLA8在鞭毛内运输中的调控机制,筛选杜氏盐藻中能与FLA8相互作用的新蛋白,扩增所捕获的蛋白FAP107的cDNA全长并对其进行原核表达,检测FAP107在杜氏盐藻鞭毛再生过程中的表达情况。方法：本实验用酵母双杂交的方法,以杜氏盐藻驱动蛋白2亚基FLA8-C端(FLA8-C)为诱饵筛选杜氏盐藻cDNA表达文库,经营养缺陷型培养及α-半乳糖苷酶活性检测,筛选与FLA8-C端相互作用的蛋白质。用5’RACE的方法扩增得到FAP107cDNA的5’端序列,与原已知序列拼接得到其cDNA全长；构建FAP107原核表达载体,用IPTG诱导对该蛋白质进行原核表达。用酵母共转化的方法对FLA8与FAP107之间的相互作用进行验证。以GAPDH作为内参,用实时荧光定量PCR的方法探索杜氏盐藻FAP107在鞭毛再生过程中的表达量变化情况,结果用2-ΔΔCT法分析。结果：酵母双杂交筛选得到的阳性克隆中所含的蛋白包括鞭毛相关蛋白107(FAP107)C端、蛋白质-蛋白质结合结构域(PDZ)、含P环的核苷三磷酸水解酶(P-loop NTPase)结构域以及微管辅助蛋白B的富含甘氨酸的细胞骨架结合蛋白结构域(CAP-Gly结构域)。扩增得到了FAP107cDNA的全长,共1207bp,其中5’UTR长133bp,3’UTR长354bp,编码区长720bp,编码239个氨基酸,该氨基酸序列与莱茵衣藻和团藻同源性为44%。该cDNA序列及氨基酸序列已提交GenBank,序列号为JN835298.1。成功的对FAP107进行了原核表达,SDS-PAGE检测HIS-FAP107融合蛋白其大小约为30kD,与预测值一致。在杜氏盐藻鞭毛再生过程中FAP107mRNA表达水平明显高于正常未处理条件下的杜氏盐藻FAP107基因的相对表达量(Fgroup=344.550,P<0.001；Ftime=63.574,P<0.001; Finteraction=54.511, P<0.001),其变化趋势为：在0-60mmin内FAP107mRNA水平逐渐降低,在60min时表达量达到最小；60-210min内FAP107mRNA水平呈不断升高的趋势,至210mmin时达到最大值；210-330min内FAP107mRNA水平又逐渐降低,并在300rnin左右降至正常水平。结论：成功用酵母双杂交的方法筛选得到与FLA8相互作用的新蛋白FAP107,该蛋白可能参与了鞭毛伸长。更多还原

【Abstract】 Background:Flagella/cilia are protruding objects from cell surface. In the process of biological evolution, they have become specialized cell devices. Flagella/cilia play important roles in cell motility, perception of environmental changes, and transmission of the signals to cell body. Ciliary abnormalities will lead to polycystic kidney disease, obesity, diabetes, cancer, and other human diseases. Formation and maintenance of flagella/cilia are crucial for vast majority of eukaryotic cells.Intraflagellar transport (IFT) is a two-way dynamic process along the axonemal microtubules, this complex process is influenced by various factors inside and outside the cells. Kinesin2is one of the kinesin superfamily members, which present in the flagellum and responsible for the forward transport of IFT. Kinesin2is a heterologous trimer, which include two homology subunit FLA8/FLA10, and a non-dynamic subunit (KAP subunit). The C-terminal of FLA8and FLA10subunit wind around each other to form a rod-like structure, their N-terminals are motor heads, which can hydrolysis ATP to generate energy. The two heads move along the microtubule to the plus end in the form of ’walking’.Dunaliella salina is a unicellular green alga with two equal length falgella. Because of its simple culture it could be used as a model organism in the study of intraflagellar transport and flagellum/cilia-related diseases.Aim:To get insight into the regulatory mechanism of the kinesin2motor protein FLA8in intraflagellar transport, we isolated proteins that interact with C-terminal region of FLA8which is the motor subunit of kinesin2from Dunaliella salina, amplified full length of FAP107cDNA and express it in E.coli BL21(DE3), and detected transcriptional level of FAP107during flagella regeneration.Methods:Yeast two-hybrid method was performed to screen the Dunaliella salina cDNA expression library using kinesin2subunit FLA8-C as bait, positive colonies were picked out by auxotroph culture and assayed for α-galactosidase activity according the manufacturer’s instructions. The cDNA fragments of prey proteins from positive colonies were detected by colony PCR and sequencing.5’RACE method was used to amplify the5’end of FAP107cDNA, pET28a(+)-FAP107prokaryotic expression vector was constructed and transformed into E.coli BL21(DE3), IPTG was added to induce the expression of the fusion protein. The interaction of FAP107and FLA8was determined by yeast cotransformation. The transcription level of FAP gene of Dunaliella salina cells during flagella regeneration was analysed by the comparative CT (2-△△CT) method using GAPDH as control.Results:Sequence analysis of positive colonies by yeast two-hybrid screening revealed that they were fragments of flagella-associated protein107(FAP107), P-loop containing Nucleoside Triphosphate Hydrolases domain(P-loop NTPase), protein-protein binding domain (PDZ domain), and cytoskeleton-associated protein Gly-rich domain(CAP-Gly domain). A cDNA sequence of1207bp was obtained as full cDNA sequence of FAP107which encoded a protein predicted to be composed of239amino acids. The deduced amino acid sequence shared homology with Chlamydomonas reinhardtii and Volvox carteri f. nagariensis(44%). The cDNA sequence and amino acid sequence has been submitted to GenBank:JN835298.1. FAP107was expressed successfully in E.coli BL21(DE3), SDS-PAGE proved that the molecular weight of HIS-FAP107was about30kD, which was identical with the predicted30.3kD. Yeast cotransformation showed that the colonies containing FAP107and FLA8were positive by auxotroph culture and assay for a-galactosidase activity. Additionally, the results of real-time quantitative PCR showed that the transcription level of FAP in Dunaliella salina was higer than the untreated group (Fgroup=344.550, P<0.001; Ftome=63.574, P<0.001; Finteraction=54.511, P<0.001) during flagella regeneration. It showed that the expression level reduced at the first60min, then increased during60-210min, declined to a normal level at300min.Conclusion:A new protein FAP107has been demonstrated to be able to interact with FLA8by yeast two-hybrid and yeast cotransformation, and it might be involved in flagellar assembly.更多还原