【作者】 张玉；

【导师】 晏磊；

【作者基本信息】 黑龙江八一农垦大学 ， 微生物学， 2021， 硕士

【摘要】 磁小体是具有磁性、纳米级、均一性、分散性和生物相容性良好的矿物晶体,在医学和生物工程领域有广阔的应用前景。目前发现的能生产磁小体的微生物主要是趋磁细菌(Magnetotactic bacteria,MTB),但是其生产磁小体的条件较为苛刻,需要严格控制铁和氧浓度,导致磁小体难以实现规模化生产。嗜酸性氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans,A.ferrooxidans)作为驱动环境铁硫循环的重要微生物之一,其在适宜环境条件下也能产生磁小体,并且其分离、培养和磁小体合成条件较MTB简单,有望成为实现磁小体规模化生产的潜力菌种,但目前A.ferrooxidans磁小体的合成机制尚不清楚,极大地制约了其磁小体的产业化。为全面挖掘A.ferrooxidans磁小体合成的相关基因,构建磁小体合成的基因调控网络,本论文对不同Fe(II)浓度、不同培养时间和不同静磁场强度处理下的A.ferrooxidans BYM菌体样本进行透射电镜观察和转录组测序,采用生物信息学分析方法对样本差异表达基因进行功能注释和基因表达趋势分析;利用权重基因共表达网络分析方法筛选与磁小体合成相关的关键基因;通过RT-q PCR获得基因的相对表达量,利用多元拟合和冗余分析对基因与环境条件和基因与性状的相关性进行分析;最后对基因组中的转录因子及其与差异表达基因之间的互作模式进行预测,构建磁小体合成的基因调控网络。透射电镜和胞内总铁含量分析结果显示,磁小体数量和胞内总铁含量在Fe(II)浓度为40 g/L时最大,分别达到15.67±2.08个/cell和2.82±0.22μg/mg,而磁小体粒径在Fe(II)浓度为80 g/L时最大,为44.00±4.58 nm;当培养时间为36 h时,磁小体数量和粒径最小,分别为7.00±1.00个/cell和20.00±2.00 nm,而胞内总铁含量最大,为5.93±1.03μg/mg;外加静磁场强度为3.5 m T时,磁小体数量和胞内总铁含量最大,分别是19.33±2.52个/cell和4.69±0.66μg/mg,磁小体粒径与磁场强度为15 m T时相当,均为50.00 nm左右。培养基中Fe(II)浓度、培养时间和外加静磁场强度与磁小体数量、粒径和胞内总铁含量具有显著相关性(R~2>0.683,P<0.05)。差异表达基因和基因表达趋势分析结果表明,不同处理条件主要影响细胞的离子吸收和转运、氧化还原反应、质膜结构组成、信号转导和群体感应等生物学过程;基于权重基因共表达网络分析,得到与磁小体数量和粒径呈正相关(R~2>0.35,P<0.09)和负相关(R~2<-0.42,P<0.04)的基因模块各2个,并从中筛选获得24个与磁小体合成相关的关键基因;通过NCBI数据库注释和序列比对分析,最终挑选了10个与磁小体合成相关的基因作为RT-q PCR分析的对象,包括基因chr＿2549、chr＿1390、chr＿1157、chr＿251、chr＿974、chr＿2620、chr＿1779、chr＿2224、chr＿1243和chr＿1062,这些基因的功能主要与细胞氧化还原、离子转运、亚铁螯合、膜结构和细胞趋化性有关;对挑选出的10个基因进行相对表达量的分析发现,基因的表达与不同处理条件大多具有显著相关性(P<0.01);基于RDA(冗余分析)发现,挑选出的10个基因的表达与磁小体数量和粒径具有较大相关性;预测构建的A.ferrooxidans BYM磁小体合成基因调控网络显示,构成网络的生物学过程主要有转录调控、能量产生和转化、氧化还原、无机离子转运和代谢、细胞壁/膜/被膜的生物合成等,这与磁小体的合成过程相对应。综上所述,适宜的Fe(II)浓度和磁场强度能促进A.ferrooxidans BYM磁小体的形成,并且细胞在对数生长末期或稳定期的磁小体合成能力最强;环境条件主要通过改变细胞的离子转运、氧化还原反应、膜结构等生物学过程来影响胞内磁小体的合成;预测构建的A.ferrooxidans BYM磁小体合成的基因调控网络具有与MTB磁小体合成相对应的生物学过程,说明其具有可参考性。本研究为今后针对磁小体合成相关基因对A.ferrooxidans进行遗传学操作,进而揭示磁小体合成机制提供了理论基础。更多还原

【Abstract】 Magnetosome is a kind of mineral crystal with magnetism,nanoscale,homogeneity,dispersion and biocompatibility,which has a broad application prospect in medicine and bioengineering.Magnetotactic bacteria(MTB)are the main microorganisms that can produce magnetosome.However,the production conditions of magnetosome in MTB are very strict,requiring tight control of iron and oxygen concentration.It is difficult to achieve large-scale production of magnetosome.Acidithiobacillus ferrooxidans(A.ferrooxidans),as one of the important microorganisms driving iron and sulfur circulation in the environment,can also produce magnetosome under suitable environmental conditions,and its separation,culture and magnetosome synthesis conditions are simpler than MTB.Therefore,it has become a potential strain to achieve large-scale production of magnetosome.At present,the magnetosome synthesis mechanism of A.ferrooxidans is not clear.So,the industrial production of magnetosome has been greatly restricted.In order to fully explore the genes related to magnetosome synthesis and construct the magnetosome synthesis gene regulatory network in A.ferrooxidans.In this paper,transmission electron microscope and transcriptome sequencing were performed on A.ferrooxidans BYM treated with different Fe(II)concentration,growth time and static magnetic field intensity.Bioinformatics analysis was used to analyze the function of differentially expressed gene(DEGs)and gene expression trend in samples.The key genes related to magnetosome synthesis were screened by weighted gene co-expression network analysis(WGCNA).The relative expression levels of genes were obtained by RT-q PCR,and the correlation between genes and environmental conditions and genes and traits was analyzed by polynomial fitting and redundancy analysis(RDA).Finally,the transcription factors in the genome and their interaction patterns with DEGs were predicted to construct a gene regulatory network of magnetosome synthesis in A.ferrooxidans BYM.The results of transmission electron microscopy and the analysis of intracellular total iron content showed that the number of magnetosome and intracellular total iron content reached the maximum(15.67±2.08 per cell and 2.82±0.22μg/mg)at 40 g/L Fe(II).The maximum size of magnetosome was 44.00±4.58 nm at 80 g/L Fe(II).When the growth time was 36 h,the number and size of magnetosome were the minimum(7.00±1.00 per cell,20.00±2.00 nm),and the intracellular total iron content was the maximum(5.93±1.03μg/mg).When the magnetic field intensity was 3.5 m T,the number of magnetosome and the intracellular total iron content reached the maximum(19.33±2.52 per cell,4.69±0.66μg/mg),and the size of magnetosome was about 50.00 nm,which was similar to that of the magnetic field intensity was 15 m T.Fe(II)concentration,growth time and static magnetic field intensity were significantly correlated with the number and size of magnetosome and intracellular total iron content in A.ferrooxidans BYM(R~2>0.683,P<0.05).The results of DEGs and gene expression trend analysis showed that treatment conditions mainly affected the biological processes such as ion absorption and transport,redox reaction,plasma membrane structure,signal transduction and quorum sensing.Based on WGCNA,the results showed that there were 2 gene profile of positive correlation with the number and size of magnetosome(R~2>0.35,P<0.09)and 2 gene profile of negative correlation with the number and size of magnetosome(R~2<-0.42,P<0.04),and 24 key genes related to magnetosome synthesis were screened from them.Through NCBI database annotation and sequence alignment analysis,10 genes related to magnetosome synthesis were selected as the objects of RT-q PCR,including chr＿2549,chr＿1390,chr＿1157,chr＿251,chr＿974,chr＿2620,chr＿1779,chr＿2224,chr＿1243 and chr＿1062.The functions of these genes are mainly related to cell redox,ion transport,ferrous chelation,membrane structure and cell chemotaxis.The relative expression levels of 10 genes were analyzed and it was found that the gene expression were significantly correlated with conditions(P<0.01).Based on RDA,the results showed that the expression of10 genes was significantly correlated with the number and size of magnetosome.The predicted constructed magnetosome synthesis gene regulation network shows that the biological processes constituting the network mainly include transcription regulation,energy generation and transformation,redox,inorganic ion transport and metabolism,cell wall/membrane/envelope biogenesis,which correspond to the magnetosome synthesis process.In conclusion,appropriate Fe(II)concentration and magnetic field intensity can promote the formation of magnetosome,and the synthesis ability of magnetosome is the strongest at the end of logarithmic growth or stable stage.Environmental conditions mainly affect the magnetosome synthesis by changing the biological processes such as ion transport,redox reaction and membrane structure.The predicted constructed magnetosome synthesis gene regulatory network has biological process corresponding to MTB magnetosome synthesis,indicating that it has reference value.This study provides a basis for genetic manipulation of A.ferrooxidans based on magnetosome synthesis genes and revealing the magnetosome synthesis mechanism.更多还原