【作者】 陈琪；

【导师】 华跃进；

【作者基本信息】 浙江大学 ， 生物物理学， 2021， 博士

【摘要】 耐辐射奇球菌(Deinococcus radiodurans)作为极端微生物家族的重要成员,对外界环境胁迫有极强的抗性。大量研究结果显示,耐辐射奇球菌对于极端环境的适应能力源自于其超强的抗氧化能力,其中细胞内积累的高锰铁比率一直被认为是其抗氧化机制之一。耐辐射奇球菌作为研究氧化应激的模式生物,研究其锰离子相关调控机制,有助于深化了解耐辐射奇球菌极端的抗性机制,同时拓展细菌对锰离子的转运机制。本论文主要研究了耐辐射奇球菌锰离子转运相关蛋白的功能和应用。首先,我们通过生物信息学方法预测发现了耐辐射奇球菌中Ter C家族蛋白锰离子转运相关蛋白DR1187和DRB0131,并与大肠杆菌内锰离子外排蛋白Mnt P以及枯草芽孢杆菌Ter C家族蛋白Yce F进行比对分析。通过预测分析发现DR1187与DRB0131均具有跨膜区域。根据亲疏水性分析,跨膜区域为亲水肽键相连。两者跨膜区域数量不同,可能是由于细胞膜结构不同产生的影响。最后通过多序列比对以及同源建模结构分析发现,两个基因编码的蛋白均具有Mnt P蛋白中非常重要的两个天冬氨酸位点。相较于野生型,dr1187基因缺失突变株对锰离子的抗性下降,胞内锰离子含量升高,说明DR1187在参与了耐辐射奇球菌中的锰离子吸收转运。同时,dr1187基因缺失突变株对紫外线、过氧化氢和电离辐射的抗性增强,且突变株中ROS含量低于野生型菌株。通过点突变DR1187中的D67和D183两个位点,发现该点突变导致胞内锰离子富集,同时比野生型表现出更强的氧化抗性,表明DR1187中的D67和D183两个在参与锰离子转运和细胞氧化抗性上起到重要作用。细胞壁的结构也是影响金属离子的转运重要因素,通过构建S层蛋白缺失突变株△dr2577,发现胞内锰离子含量上升。研究发现铅和镉的胁迫下,锰离子外排基因表达上调,同时突变体△dr2577释放更多锰离子。铅和镉是水稻中积累的主要有毒重金属,能引起细胞内的氧化应激反应产生大量ROS。将水稻幼苗与耐辐射奇球菌共培养,电镜和细菌荧光标记结果显示耐辐射奇球菌定殖于水稻根外层,并且突变株△dr2577能有效地改善镉或铅处理后导致的水稻幼苗黄化,株高、根长、干生物量和叶绿素等含量显著下降的现象。同时△dr2577突变株能吸附更多的铅和镉,且能通过释放胞内锰离子和谷氨酸来提高水稻中锰离子和谷氨酸含量、抗氧化酶活性,从而降低水稻中镉和铅的积累并缓解其引起的氧化压力。本研究探讨研究了耐辐射奇球菌中的锰离子转运相关基因,发现DR1187参与了锰离子的吸收转运及对氧化应激的抗性,是一个潜在的锰离子外排蛋白。同时发现S层蛋白DR2577影响胞内锰离子的积累,其突变株△dr2577通过外排锰离子等抗氧化小分子,有效缓解了镉和铅离子对水稻胁迫产生的毒性。研究结果为阐明耐辐射奇球菌锰离子代谢和抗逆机制提供基础,并拓展了极端微生物在缓解作物重金属污染中的重要作用。更多还原

【Abstract】 As an important member of the extremist microbial family,Deinococcus radiodurans has strong resistance to environmental stress.More and more research results have shown that the adaptability of D.radiodurans to extreme environments stems from its super strong antioxidant capacity.The high ratio of manganese to iron accumulated in cells has always been considered to be one reason for its antioxidant mechanisms.D.radiodurans is a model organism for studying oxidative stress.Studying the related regulatory mechanism of manganese ions can not only explore the extreme resistance mechanism of D.radiodurans,but also expand the regulatory mechanism of bacteria on metal ions,and widely apply this characteristic.In this paper,we studied the functions and application of manganese ion transport-related proteins in D.radiodurans through studying the proteins that affect intracellular manganese ion level.First,through bioinformatics method we predicted and found the manganese ion transport-related proteins DR1187 and DRB0131 in D.radiodurans belonging to Ter C family.Then we compared them with the manganese ion efflux protein Mnt P in Escherichia coli and the protein Yce F belonging to Ter C family in Bacillus subtilis.Through predictive analysis,both DR1187 and DRB0131 have transmembrane regions.According to the hydrophilicity and hydrophobicity analysis,the transmembrane region was connected by hydrophilic peptide bonds.The difference in the number of transmembrane regions between these proteins may be because of the cells’ different membrane structures.Finally,through multi-sequence alignment and homology modeling structure analysis,it was found that the proteins encoded by the two genes both had two very important aspartic acid sites in the Mnt P protein.Compared with the wild-type,the dr1187 gene deletion mutant showed decreased resistance to manganese ions and increased intracellular manganese ion content,indicating that DR1187 was involved in the absorption and transport of manganese ions in D.radiodurans.At the same time,the dr1187 gene deletion mutant showed enhanced resistance to ultraviolet light,hydrogen peroxide,and ionizing radiation,and the ROS content in the mutant was lower than that in the wild-type strain.The point mutation at D67 and D183 in DR1187 revealed that the point mutation resulted in intracellular enrichment of manganese ions and exhibited higher oxidative resistance than wild-type,indicating that D67 and D183 in DR1187 played important roles in their participation in manganese ion transport and cellular oxidative resistance.The structure of cell wall was also an important factor affecting the transport of metal ions.The intracellular manganese ion content was found to be increased by constructing the S-layer protein deletion mutant △dr2577.The results showed that under the stress of lead and cadmium,the expression of manganese efflux gene was upregulated,and the mutant △dr2577 released more manganese ions.Lead and cadmium are the main toxic heavy metals accumulated in rice,which can cause intracellular oxidative stress and produce large amounts of ROS.The rice seedlings were co-cultured with D.radiodurans,and the results of electron microscopy and bacterial fluorescence labeling showed that D.radiodurans colonized the outer layer of rice roots,and the mutant △dr2577 could effectively improve the yellowing of rice seedlings,and the significant decreases in plant height,root length,dry biomass,and chlorophyll contents caused by cadmium or lead treatment.At the same time,the △dr2577 mutant could adsorb more lead and cadmium,and improve the content of manganese ions and glutamic acid in rice and the activity of antioxidant enzymes by releasing intracellular manganese ions and glutamic acid,thereby reducing the accumulation of cadmium and lead in rice and relieving the oxidative stress caused by them.Taking together,we investigated the manganese ion transport-related genes in D.radiodurans and found that DR1187 was involved in the absorption and transport of manganese ions as well as its resistance to oxidative stress,and it was a potential manganese ion efflux protein.At the same time,it was found that the S-layer protein DR2577 affected the accumulation of intracellular manganese ions,and its mutant△dr2577 effectively alleviated the toxicity of cadmium and lead to rice under stress by efflux of antioxidant small molecules such as manganese ions.The results provide a basis for elucidating the manganese ion metabolism and the stress resistance mechanism of D.radiodurans,and expand the important role of extremophiles in alleviating heavy metal pollution of crops.更多还原