【摘要】：多環(huán)芳烴(Polycyclic Aromatic Hydrocarbons,簡稱PAHs)是一類致癌性很強的有機污染物。課題組前期研究證實了植物根系吸收PAHs的過程同時存在主動和被動運輸兩種轉(zhuǎn)運方式,并揭示了植物根系的主動吸收是以H+/PAHs共轉(zhuǎn)運的方式進行。關(guān)于菲脅迫下植物根系質(zhì)膜蛋白差異組學(xué)的研究迄今尚未見有報道,而質(zhì)膜蛋白質(zhì)差異組學(xué)的研究對于闡釋植物根系吸收PAHs的生物學(xué)機制至關(guān)重要。為此,本文首先以小麥為材料,探索根系質(zhì)膜蛋白含量與影響根系生長的重要環(huán)境因素(溫度、光照強度及培養(yǎng)時間)之間的關(guān)系,然后對SDS-PAGE凝膠濃度、等電聚焦時間、上樣量等雙向電泳條件進行優(yōu)化,建立了小麥根系質(zhì)膜蛋白的雙向電泳技術(shù)條件;接著以菲(PHE)為多環(huán)芳烴代表,采用建立的雙向電泳條件進行小麥根系菲脅迫下質(zhì)膜蛋白質(zhì)的差異表達分析,并對部分差異表達蛋白進行質(zhì)譜鑒定,為多環(huán)芳烴質(zhì)膜轉(zhuǎn)運蛋白的分離鑒定以及PAHs污染環(huán)境的植物修復(fù)提供科學(xué)依據(jù)與技術(shù)支持。論文的主要結(jié)論如下:1、以小麥為材料,研究溫度、光照強度和培養(yǎng)時間對根系生物量和質(zhì)膜蛋白含量的影響。結(jié)果表明,當(dāng)小麥在溫度25℃、光照強度18 000 Lux、培養(yǎng)時間為3d時根系質(zhì)膜蛋白質(zhì)含量達到最高。正交試驗結(jié)果進一步證實,25℃和18 000 Lux下培養(yǎng)3d,根系質(zhì)膜蛋白含量最高,達3.7891 μμg/(10g),這一結(jié)果為質(zhì)膜差異蛋白質(zhì)組學(xué)的研究提供了培養(yǎng)方法。2、通過對小麥根系質(zhì)膜蛋白雙向電泳條件進行優(yōu)化,確定了以450μg/gel的最佳蛋白上樣量,10%的最佳SDS-PAGE凝膠濃度,18 h的最佳等電聚焦時間的雙向電泳體系,得到了分辨率較高,重復(fù)性較好的質(zhì)膜蛋白雙向電泳圖譜,并通過正交試驗進一步驗證了這一結(jié)果。3、以菲為多環(huán)芳烴代表,以菲處理4 h后和未經(jīng)菲處理的小麥根系為試驗材料,對兩組試驗材料的質(zhì)膜蛋白質(zhì)進行雙向電泳分離,雙向電泳圖譜經(jīng)過PDQuest軟件分析,每組圖譜均能得到200余個清晰的蛋白點;通過對兩組圖譜的比較,共得到了 14個顯著差異表達的質(zhì)膜蛋白;采用MALDI-TOF/TOFMS質(zhì)譜系統(tǒng)對14個差異表達的蛋白進行一級質(zhì)譜(PMF)和二級質(zhì)譜(MS/MS)分析,并利用NCBI蛋白數(shù)據(jù)庫檢索質(zhì)譜數(shù)據(jù)。被鑒定的差異蛋白包括白蛋白、鈷氨素非依賴蛋氨酸合成酶、烯醇化酶、熱休克蛋白、β-葡萄糖苷酶、甲胎蛋白、甘油醛-3-磷酸脫氫酶,這些蛋白分別參與了協(xié)同調(diào)控、信號傳導(dǎo)、脅迫反應(yīng)、物質(zhì)運輸和能量代謝等過程。綜上試驗結(jié)果,菲脅迫對小麥根系質(zhì)膜蛋白的組成造成了很大的影響,并迫使根系質(zhì)膜啟動了多種應(yīng)對菲脅迫的應(yīng)激反應(yīng)。
[Abstract]:Polycyclic aromatic hydrocarbons (Polycyclic Aromatic Hydrocarbons,) is a kind of organic pollutants with strong carcinogenicity. The previous studies of the research group confirmed that there were both active and passive transport modes in the process of PAHs uptake by plant roots, and revealed that the active absorption of plant roots was carried out by H / PAHs co-transport. Up to now, there are no reports on the differential histology of plasma membrane proteins in plant roots under phenanthrene stress, and the study of plasma membrane proteome is very important to explain the biological mechanism of PAHs uptake by plant roots. In this paper, wheat was used as material to explore the relationship between the content of plasma membrane protein and the important environmental factors (temperature, light intensity and culture time) affecting root growth, and then the concentration of SDS-PAGE gel and isoelectric focusing time. The two-dimensional electrophoresis conditions such as sample size and other two-dimensional electrophoresis conditions were optimized, and the technical conditions of two-dimensional electrophoresis of wheat root plasma membrane proteins were established. Then, using phenanthrene (PHE) as representative of polycyclic aromatic hydrocarbons (PAHs), the differential expression of plasma membrane proteins under phenanthrene stress in wheat roots was analyzed by two-dimensional electrophoresis, and some differentially expressed proteins were identified by mass spectrometry. It provides scientific basis and technical support for the isolation and identification of polycyclic aromatic hydrocarbon membrane transporter and the phytoremediation of PAHs pollution environment. The main conclusions of this paper are as follows: 1. The effects of temperature, light intensity and culture time on root biomass and plasma membrane protein content were studied. The results showed that the protein content of root plasma membrane reached the highest when the temperature was 25 鈩，

本文編號：2478176