【摘要】： Polo-like kinase(PLK)家族是一類高度保守的絲氨酸／蘇氨酸蛋白激酶，在細(xì)胞周期調(diào)控中發(fā)揮至關(guān)重要的作用。目前已在哺乳類細(xì)胞中鑒定出4個(gè)PLK，分別是：Polo-like kinase 1(PLK1)，Polo-like kinase 2／SNK(serum-inducible kinase)，Polo-like kinase 3／FNK／PRK(fibroblast-growth-factor-inducible kina或proliferation related kinase)及Polo-like kinase 4／SAK(SNK akin kinase)。PLK1及其同源蛋白在細(xì)胞周期的不同時(shí)相中，都有十分重要的作用：如在G2晚期／M初期參與激活Cyclin B／CDK1復(fù)合體，使細(xì)胞進(jìn)入M期，協(xié)助中心體的功能成熟以及雙極紡錘體的形成，活化細(xì)胞分裂后期促進(jìn)復(fù)合體(anaphase-promoting complex，APC)，促進(jìn)染色體正常分離，分配；決定細(xì)胞能否按期退出M期及調(diào)節(jié)而進(jìn)入胞質(zhì)分裂等事件。PLK2在G1期中表達(dá)最高，可能參與調(diào)控細(xì)胞進(jìn)入S期。PLK3主要在S期和G2期表達(dá)，可能參與DNA損傷檢查點(diǎn)信號(hào)通路和細(xì)胞進(jìn)入S期。而PLK4則主要與有絲分裂的退出有關(guān)。同時(shí)，PLK家族與腫瘤的轉(zhuǎn)移及預(yù)后密切相關(guān)，，是一個(gè)很有前景的抗癌藥物靶點(diǎn)，其小分子化合物抑制劑已經(jīng)進(jìn)入臨床Ⅰ期實(shí)驗(yàn)。人們?cè)谘芯恳陨纤膫�(gè)PLK家族成員的同時(shí)，也一直在關(guān)注該激酶家族中是否還存在其它新成員，及其在細(xì)胞周期調(diào)控中的作用。然而自1997年以來(lái)，再也沒(méi)有新的PLK基因被克隆的報(bào)道，在最新的人類蛋白激酶圖譜中也沒(méi)有新的PLK記錄。 我們綜合應(yīng)用生物信息學(xué)和RT-PCR等方法，在小鼠中克隆了一種未知基因，其N端具有一個(gè)與PLK家族催化結(jié)構(gòu)域高度同源的激酶結(jié)構(gòu)域(Kinase domain)，C端則有PLK家族特征性Polo-box結(jié)構(gòu)域(polo-box domain，PBD)，序列同源性分析顯示該基因所編碼的蛋白與PLK1的相似性為53％，同源性為33％，這比PLK4與PLK1的相似性(32％)和同源性(16％)還高。因而，我們初步將其歸為PLK激酶家族的新成員，并命名為Mouse Polo-like kinase 5(mPLK5，mammalian PLK5)。我們通過(guò)生物信息學(xué)分析，RT-PCR、原位雜交、細(xì)胞免疫組織化學(xué)實(shí)驗(yàn)以及細(xì)胞轉(zhuǎn)基因?qū)嶒?yàn)等分子細(xì)胞生物學(xué)方法和現(xiàn)代成像技術(shù)，對(duì)mPLK5的一些基本生化特征，組織表達(dá)、分布，亞細(xì)胞定位以及功能進(jìn)行了初步的研究。我們期望通過(guò)對(duì)這個(gè)新基因的深入研究，進(jìn)一步明確PLK家族在細(xì)胞周期調(diào)控中所發(fā)揮的作用，并為細(xì)胞周期調(diào)控異常的疾病(如腫瘤)提供新的治療靶點(diǎn)。 結(jié)果：1、生物信息學(xué)初步分析提示：小鼠PLK5 cDNA為2089 bp，定位于第10號(hào)染色體上。它含有兩個(gè)保守的結(jié)構(gòu)域：其N端具有一個(gè)與PLK家族催化結(jié)構(gòu)域高度同源的激酶結(jié)構(gòu)域——絲氨酸／蘇氨酸激酶結(jié)構(gòu)域，C端則有PLK家族特征性Polo-box結(jié)構(gòu)域。序列同源性分析顯示該基因所編碼的蛋白與PLK1的相似性為53％，同源性為33％。2、RT-PCR結(jié)果顯示：mPLK5的分布具有組織特異性，主要在小鼠的大腦、小腦、腦干、眼睛、肝臟、腎臟和睪丸等組織表達(dá)，而小鼠的心臟、大腸、脾臟、肌肉等組織中未見(jiàn)表達(dá)。3、原位雜交結(jié)果表明：mPLK5在小鼠腦部各區(qū)域廣泛分布，其中大腦皮層、海馬等區(qū)域表達(dá)豐度較高。4、mPLK5在小鼠發(fā)育過(guò)程中腦部分布的區(qū)域略有改變。在胎鼠時(shí)期，mPLK5主要在腦部的腦干、小腦皮層和延髓表達(dá)，而在大腦皮層和海馬等區(qū)域未見(jiàn)表達(dá)；新生鼠、幼鼠、成年鼠和老年鼠在大腦皮層、海馬、小腦皮層、腦干和延髓均有穩(wěn)定表達(dá)。5、細(xì)胞免疫組織化學(xué)研究提示，外源性表達(dá)的mPLK5(pGFP-mPLK5)主要定位于細(xì)胞核，mPLK5的Polo-box結(jié)構(gòu)域的關(guān)鍵保守氨基酸位的點(diǎn)突變(pGFP-mPLK5 W417F)不改變mPLK5的亞細(xì)胞定位。7、初步細(xì)胞轉(zhuǎn)基因?qū)嶒?yàn)發(fā)現(xiàn)與轉(zhuǎn)染pGFP-C1空載體相比，過(guò)表達(dá)pGFP-mPLK5的細(xì)胞增殖受到明顯的抑制。 結(jié)論：我們首次成功克隆了哺乳類PLK家族新成員mPLK5，其與PLK家族中的其他成員在組織分布和可能的作用等方面具有顯著的差別，如mPLK5分布具有組織特異性，主要在神經(jīng)系統(tǒng)、腎臟和睪丸等組織表達(dá)；初步細(xì)胞轉(zhuǎn)基因?qū)嶒?yàn)提示mPLK5與其它PLKs促進(jìn)細(xì)胞增殖作用不同，可能具有抑制細(xì)胞增殖 的作用。
[Abstract]:Polo - like kinase ( PLK ) family is a kind of highly conserved serine / threonine protein kinase , which plays a crucial role in cell cycle regulation . Four PLK have been identified in mammalian cells : Polo - like kinase 1 ( PLK1 ) , Polo - like kinase 2 / SNK ( serum - inducible kinase ) , Polo - like kinase 3 / FNK / keratectomy ( fibroblast - like kinase ) and Polo - like kinase 4 / SAK ( SNK akin kinase ) . PLK1 and its homologous proteins play an important role in the cell cycle , such as the activation of the Cyclin B / CDK1 complex at the early stage of G2 / M , the cell entering M phase , the function maturation of the central body and the formation of the bipolar spindle , activating the complex ( APC ) at the later stage of the cell division , promoting the normal separation and distribution of the chromosomes ;
PLK3 has the highest expression in G1 phase and may be involved in the regulation of cell entry S phase . PLK3 is mainly involved in S phase and G2 phase . PLK4 may be involved in DNA damage checkpoint signaling pathway and cell entry S phase . PLK4 is a promising anticancer drug target , and its role in cell cycle regulation has been studied . However , no new PLK gene has been cloned since 1997 , and no new PLK record has been reported in the latest human protein kinase map .









We used bioinformatics and RT - PCR to clone an unknown gene in mice . The N - terminus has a kinase domain highly homologous to the PLK family catalytic domain , and the C - terminal has a PLK family characteristic Polo - box domain ( PBD ) . The homology of the protein encoded by the gene with PLK1 is 53 % , and the homology is 33 % , which is higher than that of PLK4 and PLK1 ( 32 % ) and homology ( 16 % ) . Therefore , we initially classified them as a new member of PLK kinase family and named Mouse Polo - like kinase 5 ( mPL5 ) . We hope to further clarify the role of PLK family in cell cycle regulation by means of bioinformatics analysis , RT - PCR , in situ hybridization , cellular immune histochemistry experiment and cell transgenic experiment and so on . We expect to further clarify the role of PLK family in cell cycle regulation and provide a new treatment target for diseases such as tumors .









Results : 1 . Preliminary analysis of biological information showed that the cDNA of PLK5 was 2089 bp , located on chromosome 10 . It contained two conserved domains : its N - terminal has a kinase domain _ serine / threonine kinase domain which is highly homologous to the catalytic domain of PLK family . The homology is 33 % . 2 . The results of RT - PCR show that the expression of the gene is higher in the brain , cerebellum , brain stem , eye , liver , kidney and testis .
The expression of pGFP - mPLK562 was mainly localized in the nucleus , and the point mutation of the key conserved amino acid position of the Polo - box domain ( pGFP - mPL5W417F ) was significantly inhibited compared with that of pGFP - C1 empty vector transfected with pGFP - C1 empty vector .









Conclusion : The new member of PLK family of mammals has been successfully cloned , which is different from other members of PLK family in the aspects of tissue distribution and possible roles , such as mPL5 distribution with tissue specificity , which is mainly expressed in nervous system , kidney and testis .
The results suggest that mPL5 can inhibit the proliferation of cells and inhibit the proliferation of cells .









and the like .
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 林莉,王升啟,管偉,楊秉呼,胡小電;Plk1基因?yàn)榘械姆戳x寡核苷酸對(duì)HepG2肝癌細(xì)胞的體內(nèi)外抗腫瘤作用[J];癌癥;2001年11期

本文編號(hào)：2132572