發(fā)布時間：2018-04-24 14:44

  本文選題：南極海冰細(xì)菌 + 硫氧還蛋白��； 參考：《哈爾濱工業(yè)大學(xué)》2013年碩士論文

【摘要】：硫氧還蛋白系統(tǒng)是生物體內(nèi)重要的抗氧化系統(tǒng)之一，主要包含有硫氧還蛋白（Trx）、硫氧還蛋白還原酶（TrxR）和NADPH，該系統(tǒng)通過調(diào)節(jié)酶活性、轉(zhuǎn)錄因子、各種脅迫應(yīng)答和信號傳導(dǎo)等，在生物抗逆系統(tǒng)中起著重要的作用。南極由于其獨(dú)有的地理及氣候特征，形成了一個干燥、酷寒、強(qiáng)輻射的自然環(huán)境，蘊(yùn)育了豐富的微生物資源。在長期的進(jìn)化和生長過程中，微生物形成了極為獨(dú)特的基因資源、遺傳背景及新的代謝特征。本論文以南極海冰細(xì)菌Pseudoalteromonas sp.為材料，對硫氧還蛋白系統(tǒng)中兩個重要成員Trx與TrxR的基因進(jìn)行了克隆、異源表達(dá)以及活性研究，以期進(jìn)一步完善南極微生物低溫適應(yīng)性的分子機(jī)制，獲得具有自主知識產(chǎn)權(quán)的功能基因，為抗逆生物遺傳育種提供新的基因。 （1）在前期從海冰細(xì)菌Pseudoalteromonas sp.ANT178中克隆出Trx全長基因（PsTrx）的基礎(chǔ)上，本論文利用生物信息學(xué)方法對PsTrx進(jìn)行了分析和預(yù)測。該基因大小為327bp，編碼108個氨基酸，理論分子量為11.9kDa，等電點(diǎn)pI=4.50，推測其關(guān)鍵活性位點(diǎn)為Cys33和Cys36，結(jié)構(gòu)模型中含4個α-螺旋和5個β-折疊。將PsTrx基因轉(zhuǎn)入E.coli BL21中，實(shí)現(xiàn)了異源表達(dá)，并對該重組蛋白進(jìn)行了Ni-親和層析純化及活性測定。結(jié)果表明，經(jīng)純化后的Trx重組蛋白比活力為96.67U/mg，純化倍數(shù)達(dá)22.22倍，總回收率為27.26%；其最適反應(yīng)溫度為25℃，最適反應(yīng)pH為7.0。PsTrx在高鹽（2M NaCl）環(huán)境中仍保留一定的活性。變性劑SDS對蛋白活性的抑制力最強(qiáng)，相對活性僅為22.94%，氧化劑H2O2對PsTrx的抑制作用最小，相對活性可高達(dá)95.36%。 （2）PsTrx基因轉(zhuǎn)入E.coli BL21后，重組菌株耐鹽性顯著提高，在鹽度為9%培養(yǎng)條件下，其最大OD600吸光值可以達(dá)到1.316，而野生型大腸桿菌的OD600僅為0.393。利用FQ-PCR相對定量2-△△Ct法測定不同鹽度下海冰細(xì)菌PsTrx表達(dá)情況，高鹽度下（5%-9%NaCl）的PsTrx表達(dá)量高于對照組（3.3%NaCl），其中9%鹽度下的表達(dá)量是對照組的2.5倍，這表明PsTrx基因在菌體的耐鹽機(jī)制中起著重要作用，進(jìn)而可為耐鹽遺傳育種提供新的基因。 （3）從菌株P(guān)seudoalteromonas sp.ANT178克隆出TrxR基因，命名為PsTrxR，該基因具有完整的基因閱讀框，大小為951bp。生物信息學(xué)分析表明該基因編碼316個氨基酸，PsTrxR的理論分子量為33.7kDa，等電點(diǎn)為4.98，推斷的活性位點(diǎn)為Cys136和Cys139，蛋白結(jié)構(gòu)內(nèi)含5個α-螺旋和18個β-折疊。將PsTrxR轉(zhuǎn)入E.coli BL21中成功表達(dá)，利用Ni-親和層析對重組蛋白進(jìn)行純化，重組蛋白比活力為29.71U/mg，總純化倍數(shù)達(dá)26.29倍，總回收率為35.75%。重組PsTrxR的最適反應(yīng)溫度為20℃，最適反應(yīng)pH為7.0，該酶在50℃處理40min后，酶活性下降至66%。以DTNB為底物測定酶動力學(xué)參數(shù)Km和Vmax分別為1.15mM和5.17nmol/mL/min。變性劑DTT對PsTrxR酶活的抑制作用為100%，，金屬離子Zn2+對酶的抑制作用小，相對剩余活性可達(dá)71.38%。
[Abstract]:Thioredoxin system is one of the most important antioxidant systems in organisms, including thioredoxin (TRX), thioredoxin reductase (TRXR) and NADPH.The system regulates enzyme activity, transcription factors, stress response and signal transduction. It plays an important role in biological stress resistance system. Because of its unique geographical and climatic characteristics, Antarctica has formed a dry, cold, strong radiation natural environment, rich in microbial resources. During the long term evolution and growth, microorganisms have formed unique genetic resources, genetic background and new metabolic characteristics. In this paper, the Antarctic sea ice bacteria Pseudoalteromonas sp. The genes of Trx and TrxR, two important members of thioredoxin system, were cloned, heterologous expression and activity were studied in order to further improve the molecular mechanism of low temperature adaptation of Antarctic microorganisms. Functional genes with independent intellectual property rights were obtained, which provided new genes for genetic breeding of stress-resistant organisms. 1) based on the cloning of the full-length Trx gene PsTrx from the sea ice bacteria Pseudoalteromonas sp.ANT178, the bioinformatics method was used to analyze and predict the PsTrx. The gene is 327bp, encoding 108 amino acids, with theoretical molecular weight of 11.9 kDa and isoelectric point Pi 4.50. The key active sites are presumed to be Cys33 and Cys36. There are 4 偽 -helix and 5 尾 -folds in the structural model. The PsTrx gene was transferred into E.coli BL21, and its heterologous expression was realized. The recombinant protein was purified by Ni-affinity chromatography and its activity was determined. The results showed that the specific activity of purified Trx recombinant protein was 96.67 U / mg, the purification multiple was 22.22 times, the total recovery was 27.26%, the optimum reaction temperature was 25 鈩

本文編號：1797073