【摘要】：臭氧層變薄是地表紫外輻射(UV-B)強(qiáng)度增加的主要原因。研究表明地球UV-B輻射劑量都呈現(xiàn)逐年上升趨勢,嚴(yán)重威脅地表生物的生命活動。對無法自主移動的植物來說,高能UV-B在形態(tài)建成、生理代謝等生命活動都具有較顯著的影響。因此,研究植物如何響應(yīng)UV-B輻射有著深遠(yuǎn)的科學(xué)意義與應(yīng)用前景。植物細(xì)胞骨架一直處于動態(tài)的解聚與聚合過程中。是細(xì)胞中能夠調(diào)整自身的結(jié)構(gòu)響應(yīng)細(xì)胞的生長發(fā)育以及外界刺激的一類重要的結(jié)構(gòu)組成,在許多細(xì)胞代謝過程中起著關(guān)鍵的作用,比如細(xì)胞生長、胞質(zhì)流動、細(xì)胞器的運動以及細(xì)胞分裂。這些功能的實現(xiàn)需要一些特定的輔助蛋白來調(diào)節(jié)細(xì)胞骨架動態(tài)行為。Formin蛋白(成蛋白)被認(rèn)為是一種多結(jié)構(gòu)域的肌動蛋白結(jié)合蛋白,但近來發(fā)現(xiàn)Formin還可以與微管結(jié)合,作為一個雙功能蛋白涉及很多細(xì)胞過程的發(fā)生。與哺乳動物和酵母相比人們對植物中Formin蛋白的功能還知之甚少。擬南芥中AtFH16屬于Ⅱ類Formin,已有的研究表明,其可能參與了細(xì)胞分裂的過程。本課題組長期致力于植物響應(yīng)增強(qiáng)UV-B輻射的研究,并且前期發(fā)現(xiàn)微絲骨架的動態(tài)變化參與了UV-B輻射所導(dǎo)致的“分束分裂”現(xiàn)象的發(fā)生。綜合文獻(xiàn)分析,我們判斷微絲響應(yīng)這一過程的變化可能是依賴于Formin家族蛋白的。為了驗證這一假設(shè),(1)本研究用GFP作為融合報告基因,構(gòu)建pCAMBIA1300-AFH16-GFP融合表達(dá)載體,測序正確后轉(zhuǎn)入農(nóng)桿菌,重組質(zhì)粒通過農(nóng)桿菌介導(dǎo)法導(dǎo)入擬南芥,篩選獲得純合轉(zhuǎn)基因植株。(2)與此同時,將重組質(zhì)粒導(dǎo)入煙草表皮細(xì)胞中使其瞬時表達(dá),共聚焦檢測可見綠色熒光,進(jìn)一步證明構(gòu)建成功。(3)對野生型植株與轉(zhuǎn)基因植株做了表型對比后發(fā)現(xiàn),轉(zhuǎn)基因植株表現(xiàn)出主根伸長、葉片增大。進(jìn)一步分析原因,表明AtFH16可能參與并促進(jìn)細(xì)胞分裂。(4)最后對擬南芥中AtFH16的亞細(xì)胞定位的檢測過程中發(fā)現(xiàn),AtFH16定位在細(xì)胞質(zhì)。載體的構(gòu)建為后續(xù)研究At FH16與細(xì)胞骨架、細(xì)胞核以及探究“分束分裂”的機(jī)制提供了基礎(chǔ)的實驗材料。該載體的構(gòu)建及轉(zhuǎn)基因植物的獲得,為課題組進(jìn)一步驗證AtFH16與“分束分裂”現(xiàn)象的發(fā)生提供了重要的實驗材料。同時為課題組深入研究UV-B輻射后微絲骨架的動態(tài)調(diào)整的變化提供一定的依據(jù)。
[Abstract]:The thinning of the ozone layer is the main reason for the increase in the intensity of surface ultraviolet radiation (UV-B). The results show that the radiation dose of earth UV-B is increasing year by year, which seriously threatens the life activity of surface organisms. For plants unable to move independently, high energy UV-B has significant effects on morphogenesis, physiological metabolism and other life activities. Therefore, the study of how plants respond to UV-B radiation has profound scientific significance and application prospects. Plant cytoskeleton has been in the dynamic process of depolymerization and polymerization. It is a kind of important structural composition that can adjust its structure and respond to the growth and development of cells and external stimuli. It plays a key role in many cell metabolic processes, such as cell growth, cytoplasmic flow. Organelle movement and cell division. The realization of these functions requires specific auxiliaries to regulate cytoskeletal dynamic behavior. Formin protein is considered to be a multi-domain actin binding protein, but recently it has been found that Formin can also bind to microtubules. As a bifunctional protein, many cellular processes are involved. Little is known about the function of Formin proteins in plants compared with mammals and yeast. In Arabidopsis thaliana, AtFH16 belongs to class 鈪，

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