南极海冰细菌Pseudoalteromonas sp.的Trx与TrxR基因克

发布时间：2018-04-24 14:44

  本文选题：南极海冰细菌 + 硫氧还蛋白　； 参考：《哈尔滨工业大学》2013年硕士论文

【摘要】：硫氧还蛋白系统是生物体内重要的抗氧化系统之一，主要包含有硫氧还蛋白（Trx）、硫氧还蛋白还原酶（TrxR）和NADPH，该系统通过调节酶活性、转录因子、各种胁迫应答和信号传导等，在生物抗逆系统中起着重要的作用。南极由于其独有的地理及气候特征，形成了一个干燥、酷寒、强辐射的自然环境，蕴育了丰富的微生物资源。在长期的进化和生长过程中，微生物形成了极为独特的基因资源、遗传背景及新的代谢特征。本论文以南极海冰细菌Pseudoalteromonas sp.为材料，对硫氧还蛋白系统中两个重要成员Trx与TrxR的基因进行了克隆、异源表达以及活性研究，以期进一步完善南极微生物低温适应性的分子机制，获得具有自主知识产权的功能基因，为抗逆生物遗传育种提供新的基因。 （1）在前期从海冰细菌Pseudoalteromonas sp.ANT178中克隆出Trx全长基因（PsTrx）的基础上，本论文利用生物信息学方法对PsTrx进行了分析和预测。该基因大小为327bp，编码108个氨基酸，理论分子量为11.9kDa，等电点pI=4.50，推测其关键活性位点为Cys33和Cys36，结构模型中含4个α-螺旋和5个β-折叠。将PsTrx基因转入E.coli BL21中，实现了异源表达，并对该重组蛋白进行了Ni-亲和层析纯化及活性测定。结果表明，经纯化后的Trx重组蛋白比活力为96.67U/mg，纯化倍数达22.22倍，总回收率为27.26%；其最适反应温度为25℃，最适反应pH为7.0。PsTrx在高盐（2M NaCl）环境中仍保留一定的活性。变性剂SDS对蛋白活性的抑制力最强，相对活性仅为22.94%，氧化剂H2O2对PsTrx的抑制作用最小，相对活性可高达95.36%。 （2）PsTrx基因转入E.coli BL21后，重组菌株耐盐性显著提高，在盐度为9%培养条件下，其最大OD600吸光值可以达到1.316，而野生型大肠杆菌的OD600仅为0.393。利用FQ-PCR相对定量2-△△Ct法测定不同盐度下海冰细菌PsTrx表达情况，高盐度下（5%-9%NaCl）的PsTrx表达量高于对照组（3.3%NaCl），其中9%盐度下的表达量是对照组的2.5倍，这表明PsTrx基因在菌体的耐盐机制中起着重要作用，进而可为耐盐遗传育种提供新的基因。 （3）从菌株Pseudoalteromonas sp.ANT178克隆出TrxR基因，命名为PsTrxR，该基因具有完整的基因阅读框，大小为951bp。生物信息学分析表明该基因编码316个氨基酸，PsTrxR的理论分子量为33.7kDa，等电点为4.98，推断的活性位点为Cys136和Cys139，蛋白结构内含5个α-螺旋和18个β-折叠。将PsTrxR转入E.coli BL21中成功表达，利用Ni-亲和层析对重组蛋白进行纯化，重组蛋白比活力为29.71U/mg，总纯化倍数达26.29倍，总回收率为35.75%。重组PsTrxR的最适反应温度为20℃，最适反应pH为7.0，该酶在50℃处理40min后，酶活性下降至66%。以DTNB为底物测定酶动力学参数Km和Vmax分别为1.15mM和5.17nmol/mL/min。变性剂DTT对PsTrxR酶活的抑制作用为100%，，金属离子Zn2+对酶的抑制作用小，相对剩余活性可达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 伪 -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

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