本文選題：鐵氧還蛋白 + 原核表達(dá)��； 參考：《蘭州理工大學(xué)》2017年碩士論文

【摘要】：嗜酸氧化亞鐵硫桿菌是一種應(yīng)用性很強(qiáng)的工業(yè)微生物菌種,鑒于其較好的重金屬抗性,可用于貴金屬的浸提、環(huán)境污染的治理等領(lǐng)域。鐵氧還蛋白作為一種廣泛存在并參與嗜酸氧化亞鐵硫桿菌新陳代謝過(guò)程諸如鐵硫簇組裝等重要環(huán)節(jié)的蛋白質(zhì),研究其結(jié)構(gòu)和功能對(duì)了解嗜酸氧化亞鐵硫桿菌的代謝過(guò)程、電子傳遞及重金屬抗性具有重要意義。本研究的內(nèi)容和結(jié)果如下:(1)從甘肅省蘭州市、白銀市和隴南市3個(gè)典型礦區(qū)采集的樣品中分離出四株嗜酸氧化亞鐵硫桿菌,一株氧化亞鐵鉤端螺旋菌。選取生長(zhǎng)周期短、菌體富集量大的菌株BYSW1進(jìn)行后續(xù)的研究。首先選取溫度、pH值、接種量、能源種類(lèi)利用單因素實(shí)驗(yàn)對(duì)其生長(zhǎng)條件進(jìn)行優(yōu)化。在pH為2,于30℃下接種10%的菌液,BYSW1生長(zhǎng)最好,葡萄糖和蛋白胨會(huì)明顯抑制其生長(zhǎng)。(2)在NCBI數(shù)據(jù)庫(kù)中找出鐵氧還蛋白基因序列(fdⅠ、fdⅡ),在BYSW1中擴(kuò)增出目的片段,連接到pET-32(a),轉(zhuǎn)化大腸桿菌(Escherichia coli,E.coli)DH5α。自BYSW1擴(kuò)增出的鐵氧還蛋白基因序列(fdⅠ、fdⅡ)與數(shù)據(jù)庫(kù)匯總的序列一致性分別為83.92%和85.59%,編碼的蛋白與數(shù)據(jù)庫(kù)中的序列一致性分別為89.47%和85.59%。(3)重組的質(zhì)粒轉(zhuǎn)化表達(dá)宿主E.coli BL21(DE3)。選取溫度、時(shí)間、IPTG濃度進(jìn)行單因素實(shí)驗(yàn),優(yōu)化重組蛋白的表達(dá)條件,并用蛋白質(zhì)印記法進(jìn)行驗(yàn)證。用鎳離子親和層析柱對(duì)重組蛋白進(jìn)行純化。FdⅠ和FdⅡ分別在25℃下加入0.5 mM IPTG誘導(dǎo)6 h、25℃下加入0.3 mM IPTG誘導(dǎo)6 h表達(dá)效果最佳。FdⅠ和FdⅡ分別可用100 mM咪唑和50 mM咪唑自親和柱中洗脫下來(lái)。(4)挑取五種重金屬(Cd2+、Cu2+、Mn2+、Ni2+、Zn2+),對(duì)其重金屬抗性進(jìn)行研究;對(duì)重金屬(Cu2+和Cd2+)馴化前后的嗜酸氧化亞鐵硫桿菌的菌體形態(tài)和基因組變化、重組蛋白表達(dá)差異、宿主菌體的生長(zhǎng)情況進(jìn)行了研究。結(jié)果表明:BYSW1具有較強(qiáng)的重金屬抗性,分別在0.04 mol/L Cd2+、0.04 mol/L Cu2+、0.15 mol/L Mn2+、0.08 mol/L Ni2+和0.08 mol/L Zn2+存在時(shí)生長(zhǎng)良好,但是高濃度(大于0.04 mol/L)的Cu2+和Cd2+會(huì)明顯抑制BYSW1的生長(zhǎng)。Cu2+和Cd2+脅迫后,BYSW1的菌體細(xì)胞明顯變小,且表面粗糙,但基因組仍然能保持完整。不同濃度、不同種類(lèi)的重金屬脅迫前后,鐵氧還蛋白的表達(dá)存在顯著差異,且Cd2+對(duì)嗜酸氧化亞鐵硫桿菌生長(zhǎng)的影響比Cu2+顯著;不同濃度、不同種類(lèi)的重金屬對(duì)于重組蛋白的表達(dá)宿主菌體生長(zhǎng)影響不同,0.25 m M的Cu2+和Cd2+能明顯抑制宿主菌體的生長(zhǎng)。
[Abstract]:Thiobacillus ferrooxidans acidophilus is a kind of industrial microorganism with strong application. In view of its good resistance to heavy metals, it can be used in the extraction of precious metals and the treatment of environmental pollution. Ferredoxin is a widely existed protein involved in metabolic processes of Thiobacillus ferrooxidans, such as the assembly of iron-sulfur clusters. The structure and function of ferredoxin are studied to understand the metabolic process of Thiobacillus ferrooxidans. Electron transport and heavy metal resistance are important. The contents and results of this study are as follows: (1) four strains of Thiobacillus ferrooxidans and one strain of Heptospira ferrooxidans were isolated from three typical mining areas in Lanzhou, Baiyin and Longnan of Gansu Province. The strain BYSW1 with short growth cycle and high concentration of bacteria was selected for further study. The temperature pH value, inoculation amount and energy type were selected to optimize the growth conditions by single factor experiment. The growth of BYSW1 was best when inoculated at 30 鈩，

本文編號(hào)：1863260