微生物学报

Gene name	Primer sequences (5′→3′)	Product length (bp)
astA	F: AACGGTCACGAAACTGAGCA R: GCTGCTGTGCTATTGAAGGC	264
astB	F: GTAAAATGCACTCGGCGGTC R: ACTGATGGATGCTGGCTACG	231
astD	F: CCAAGCTATCGGCCTCCATT R: GTGAACTGGAACCGCCAAAC	145
astE	F: ACAGGCGAAGAACCTGATCG R: CAACTGTGCTGGTGTTGGTG	233
argH	F: CACGCTCTTCATCGGTCAGT R: AGCCAAGCTACGAACCAGTC	178
argE	F: CGTTGAAATCACCACCGAGC R: TGAGGTGTTTCTAGCGCCTG	291
lysC	F: CAAGACGAGGACGCTATGGAAGAAC R: TCGGCGATAGGACCAAGAATACG	133
asd	F: CCCGAACGACAAAGACGCTACAG R: TCACCAACACTGAAGGCTGACAAG	132
ectA	F: CAGTCGCTGATGCTGTGGTTGG R: GAATTAACATCAAGCGGCGGACAAG	121
ectB	F: TGCGTGGTATTGATGTTGTCTCTGG R: CACTTCACTACTTCGCCGTCTTGG	112
ectC	F: GCTATGAAGGCGAAGGCGAAGTAG R: AACAGATGTTCGTCGTGCTGATCC	103
GAPDH	F: TCCTTCCCTAAACTCGCACCT R: ATACGATAGTAGCGTCAGCAT	284

Gene name	Primer sequences (5′→3′)	Product length (bp)
astA	F: AACGGTCACGAAACTGAGCA R: GCTGCTGTGCTATTGAAGGC	264
astB	F: GTAAAATGCACTCGGCGGTC R: ACTGATGGATGCTGGCTACG	231
astD	F: CCAAGCTATCGGCCTCCATT R: GTGAACTGGAACCGCCAAAC	145
astE	F: ACAGGCGAAGAACCTGATCG R: CAACTGTGCTGGTGTTGGTG	233
argH	F: CACGCTCTTCATCGGTCAGT R: AGCCAAGCTACGAACCAGTC	178
argE	F: CGTTGAAATCACCACCGAGC R: TGAGGTGTTTCTAGCGCCTG	291
lysC	F: CAAGACGAGGACGCTATGGAAGAAC R: TCGGCGATAGGACCAAGAATACG	133
asd	F: CCCGAACGACAAAGACGCTACAG R: TCACCAACACTGAAGGCTGACAAG	132
ectA	F: CAGTCGCTGATGCTGTGGTTGG R: GAATTAACATCAAGCGGCGGACAAG	121
ectB	F: TGCGTGGTATTGATGTTGTCTCTGG R: CACTTCACTACTTCGCCGTCTTGG	112
ectC	F: GCTATGAAGGCGAAGGCGAAGTAG R: AACAGATGTTCGTCGTGCTGATCC	103
GAPDH	F: TCCTTCCCTAAACTCGCACCT R: ATACGATAGTAGCGTCAGCAT	284

Sample	Clean reads	Q₂₀ (%)	Q₃₀ (%)	Error rate (%)	G+C (%)	Total mapped (%)	Uniquely mapped (%)	Multiple mapped (%)
C₁	14 866 686	96.38	90.52	0.03	53.42	92.84	86.13	6.71
C₂	15 372 448	96.39	90.53	0.03	53.78	93.04	86.52	6.52
C₃	13 642 626	96.08	90.00	0.03	53.68	92.53	85.51	7.02
L₁	15 587 564	96.54	90.82	0.03	53.86	93.19	86.74	6.44
L₂	14 179 496	96.43	90.59	0.03	53.28	92.57	86.06	6.51
L₃	13 819 380	96.52	90.75	0.03	53.52	93.09	86.94	6.15
M₁	15 599 214	96.18	90.04	0.03	53.09	91.97	85.22	6.75
M₂	15 368 656	96.49	90.66	0.03	53.57	93.11	87.06	6.05
M₃	15 754 456	96.31	90.35	0.03	53.63	92.94	86.45	6.48
H₁	14 431 560	96.06	89.84	0.03	53.36	92.13	85.53	6.60
H₂	14 331 822	96.04	89.84	0.03	53.61	93.37	87.00	6.37
H₃	17 921 678	96.48	90.70	0.03	53.41	93.43	87.23	6.20

Sample	Clean reads	Q₂₀ (%)	Q₃₀ (%)	Error rate (%)	G+C (%)	Total mapped (%)	Uniquely mapped (%)	Multiple mapped (%)
C₁	14 866 686	96.38	90.52	0.03	53.42	92.84	86.13	6.71
C₂	15 372 448	96.39	90.53	0.03	53.78	93.04	86.52	6.52
C₃	13 642 626	96.08	90.00	0.03	53.68	92.53	85.51	7.02
L₁	15 587 564	96.54	90.82	0.03	53.86	93.19	86.74	6.44
L₂	14 179 496	96.43	90.59	0.03	53.28	92.57	86.06	6.51
L₃	13 819 380	96.52	90.75	0.03	53.52	93.09	86.94	6.15
M₁	15 599 214	96.18	90.04	0.03	53.09	91.97	85.22	6.75
M₂	15 368 656	96.49	90.66	0.03	53.57	93.11	87.06	6.05
M₃	15 754 456	96.31	90.35	0.03	53.63	92.94	86.45	6.48
H₁	14 431 560	96.06	89.84	0.03	53.36	92.13	85.53	6.60
H₂	14 331 822	96.04	89.84	0.03	53.61	93.37	87.00	6.37
H₃	17 921 678	96.48	90.70	0.03	53.41	93.43	87.23	6.20

Regulate	Comparison group	Gene name
Up genes	M vs. C	fabA, hisI, pilW, surE, lysC, recX, hutC, iscR, lrgB, lpxB, rapZ, mnmC, ccsA, glnD, recF, nadB, ptsN, rnt, rnhB, xthA, flhC, gcvP, gmhB, yhbY, rnhA, uraH, rimP, rlmE, dinB, glxA, folK, yidD, umuD, gcvH, mnmA, hflD, rho, pta, der, asd, flgN/M, argE/H, XdhA/B/C, ectABC, fliE/F/M/N/P, hemA/B/C/D, astA/B/D/E, and 76 undefined genes
H vs. C	astA/B/D, ectABC, cysD/T/N/P, gcvH, xdhA/B/C, lysC, ahpC, pilW, and 10 undefined genes
Down genes	M vs. C	HutG, safE, recB/D, acrB/D/F, iclR, dctP/M/Q, malT, dgcA, fdhA, gltS, proB, soxZ, hlyD, cobG, sohB, ptsP, antC, eamA, ureD, dctM, eat, urtB/C, kdpA/C, phnE, aqpZ, betT, oprD, moaA, ligB, cobF, nhaC, sstT, ctaG, narH/K, catC, fixB, rluA, coxB, deoR, ribA, nirB/D, menA, ccoG, arsJ, pqqD, qhpC, asnC, tauE, plsY, exbB, ald, mocA, mtnA, smtB, mlaE, peaD, lysE/R, dapE, rluF, purU, xseA, yifB, eutC, chrA, ptsP, pstC/B, ubiB, nagE, thuA, iolC/D, glaH, speB, lptG, acrB/D/F, aarF, and 171 undefined genes
H vs. C	ubiB, hlyD, fecC/D, tenA, recB/D, tonB, bioD, folE, glaH, aarF, mdoG, and 19 undefined genes

Regulate	Comparison group	Gene name
Up genes	M vs. C	fabA, hisI, pilW, surE, lysC, recX, hutC, iscR, lrgB, lpxB, rapZ, mnmC, ccsA, glnD, recF, nadB, ptsN, rnt, rnhB, xthA, flhC, gcvP, gmhB, yhbY, rnhA, uraH, rimP, rlmE, dinB, glxA, folK, yidD, umuD, gcvH, mnmA, hflD, rho, pta, der, asd, flgN/M, argE/H, XdhA/B/C, ectABC, fliE/F/M/N/P, hemA/B/C/D, astA/B/D/E, and 76 undefined genes
H vs. C	astA/B/D, ectABC, cysD/T/N/P, gcvH, xdhA/B/C, lysC, ahpC, pilW, and 10 undefined genes
Down genes	M vs. C	HutG, safE, recB/D, acrB/D/F, iclR, dctP/M/Q, malT, dgcA, fdhA, gltS, proB, soxZ, hlyD, cobG, sohB, ptsP, antC, eamA, ureD, dctM, eat, urtB/C, kdpA/C, phnE, aqpZ, betT, oprD, moaA, ligB, cobF, nhaC, sstT, ctaG, narH/K, catC, fixB, rluA, coxB, deoR, ribA, nirB/D, menA, ccoG, arsJ, pqqD, qhpC, asnC, tauE, plsY, exbB, ald, mocA, mtnA, smtB, mlaE, peaD, lysE/R, dapE, rluF, purU, xseA, yifB, eutC, chrA, ptsP, pstC/B, ubiB, nagE, thuA, iolC/D, glaH, speB, lptG, acrB/D/F, aarF, and 171 undefined genes
H vs. C	ubiB, hlyD, fecC/D, tenA, recB/D, tonB, bioD, folE, glaH, aarF, mdoG, and 19 undefined genes

Up genes	Function	Down genes	Function
ectA/B/C, lysC	Ectoine synthesis pathway	recB/D	Exodeoxyribonuclease Ⅴ subunit
astA/B/D	Arg and Pro metabolism	RS14435/14800/06820	DUF932/3833/1365 domain-containing protein
RS04790	Peroxiredoxin	RS06850	FAD-dependent oxidoreductase
xdhA/B/C	Xanthine dehydrogenase	RS06856/06825	NAD dependent epimerase/dehydratase family
PilW	Type Ⅳ pilus biogenesis/stability protein	aarF, ubiB	AarF/ABC1/UbiB kinase family protein
RS07420	VRR-NUC domain-containing protein	RS06830	SDR family NAD(P)-dependent oxidoreductase
RS04260	Cold-shock protein	RS01840	Efflux RND transporter periplasmic adaptor subunit
gcvH	Glycine cleavage system protein	RS10485	Glycosyltransferase
RS02725	DNA starvation/stationary phase protection protein	RS01690	PAS domain-containing methyl-accepting chemotaxis protein
RS06920/06925/11825	tRNA-Tyr/Gly/Leu	RS06855	TIGR01777 family oxidoreductase

Up genes	Function	Down genes	Function
ectA/B/C, lysC	Ectoine synthesis pathway	recB/D	Exodeoxyribonuclease Ⅴ subunit
astA/B/D	Arg and Pro metabolism	RS14435/14800/06820	DUF932/3833/1365 domain-containing protein
RS04790	Peroxiredoxin	RS06850	FAD-dependent oxidoreductase
xdhA/B/C	Xanthine dehydrogenase	RS06856/06825	NAD dependent epimerase/dehydratase family
PilW	Type Ⅳ pilus biogenesis/stability protein	aarF, ubiB	AarF/ABC1/UbiB kinase family protein
RS07420	VRR-NUC domain-containing protein	RS06830	SDR family NAD(P)-dependent oxidoreductase
RS04260	Cold-shock protein	RS01840	Efflux RND transporter periplasmic adaptor subunit
gcvH	Glycine cleavage system protein	RS10485	Glycosyltransferase
RS02725	DNA starvation/stationary phase protection protein	RS01690	PAS domain-containing methyl-accepting chemotaxis protein
RS06920/06925/11825	tRNA-Tyr/Gly/Leu	RS06855	TIGR01777 family oxidoreductase

Gene	Description of genes or factors	FPKM values of RNA-seq in different groups	Regulate
astA	Arginine N-succinyltransferase	239.65	222.21	330.96	287.26	Up
astB	N-succinylarginine dihydrolase	154.50	162.55	230.63	199.76	Up
astD	Succinylglutamate-semialdehyde dehydrogenase	303.23	298.12	350.58	418.82	Up
astE	Succinylglutamate desuccinylase	414.34	532.87	612.34	458.88	Up
argH	Argininosuccinate lyase	406.67	446.99	531.84	501.08	Up
argE	Acetylornithine deacetylase	371.84	428.38	483.91	428.41	Up
lysC	Aspartate kinase	669.51	792.61	971.45	964.37	Up
asd	Aspartate-semialdehyde dehydrogenase	182.14	172.84	216.33	203.34	Up
ectA	Diaminobutyrate acetyltransferase	604.69	764.54	940.39	925.42	Up
ectB	Diaminobutyrate transaminase	1 864.03	2 047.36	2 243.04	2 227.50	Up
ectC	Ectoine synthase	3 074.22	3 127.61	3 130.82	3 286.80	Up

Gene	Description of genes or factors	FPKM values of RNA-seq in different groups	Regulate
astA	Arginine N-succinyltransferase	239.65	222.21	330.96	287.26	Up
astB	N-succinylarginine dihydrolase	154.50	162.55	230.63	199.76	Up
astD	Succinylglutamate-semialdehyde dehydrogenase	303.23	298.12	350.58	418.82	Up
astE	Succinylglutamate desuccinylase	414.34	532.87	612.34	458.88	Up
argH	Argininosuccinate lyase	406.67	446.99	531.84	501.08	Up
argE	Acetylornithine deacetylase	371.84	428.38	483.91	428.41	Up
lysC	Aspartate kinase	669.51	792.61	971.45	964.37	Up
asd	Aspartate-semialdehyde dehydrogenase	182.14	172.84	216.33	203.34	Up
ectA	Diaminobutyrate acetyltransferase	604.69	764.54	940.39	925.42	Up
ectB	Diaminobutyrate transaminase	1 864.03	2 047.36	2 243.04	2 227.50	Up
ectC	Ectoine synthase	3 074.22	3 127.61	3 130.82	3 286.80	Up

转录组学分析Fe₃O₄纳米颗粒胁迫下盐单胞菌XH26与四氢嘧啶代谢相关的差异表达基因

PDF下载

张培霞 ¹ , 陶宇杰 ¹ , 乔丽娟 ¹ , 王嵘 ¹ , 韩睿 ² , 朱德锐 ¹ , 沈国平 ¹^,^*

微生物学报 | 研究报告 2025,65(1): 239-255

收起

微生物学报 | 研究报告 2025, 65(1): 239-255

转录组学分析Fe₃O₄纳米颗粒胁迫下盐单胞菌XH26与四氢嘧啶代谢相关的差异表达基因

全屏

张培霞¹, 陶宇杰¹, 乔丽娟¹, 王嵘¹, 韩睿², 朱德锐¹, 沈国平¹^,^*

作者信息

1 青海大学医学院, 基础医学研究中心, 青海西宁 810016

2 青海大学农林科学院, 蔬菜遗传与生理重点实验室, 青海西宁 810016

Transcriptomics reveals differentially expressed genes related to ectoine metabolism in Halomonas campaniensis XH26 under Fe₃O₄ nanoparticle stress

Peixia ZHANG¹, Yujie TAO¹, Lijuan QIAO¹, Rong WANG¹, Rui HAN², Derui ZHU¹, Guoping SHEN¹^,^*

Affiliations

1 Department of Basic Medical Sciences, Medical College, Qinghai University, Xining 810016, Qinghai, China

2 Key Laboratory of Vegetable Genetics and Physiology, Academy of Agriculture and Forestry Science, Qinghai University, Xining 810016, Qinghai, China

出版时间: 2025-01-04 doi: 10.13343/j.cnki.wsxb.20240525

文章导航

摘要

收起

【目的】探究Fe₃O₄纳米颗粒(Fe₃O₄ nanoparticles, Fe₃O₄ NPs)与坎帕尼亚盐单胞菌(Halomonas campaniensis) XH26共培养后的差异表达基因(differentially expressed genes, DEGs)，明确Fe₃O₄ NPs促进XH26胞内四氢嘧啶积聚量增长的分子机制。【方法】设置空白组(C, 0 g/L)、低浓度组(L, 0.01 g/L)、中浓度组(M, 0.10 g/L)和高浓度组(H, 0.50 g/L)的Fe₃O₄ NPs与菌株XH26共培养。采用Illumina HiSeq 300PE进行转录组测序，探究菌株不同浓度组的DEGs，并采用RT-qPCR验证关键的DEGs。【结果】与空白C组相比，M组四氢嘧啶的积累量提高了55.67% (708.87 mg/L)，M组和H组的亚铁离子和抗氧化能力显著升高，而H组的羟基自由基含量高于M组。转录组学分析显示，M组中与菌株XH26胞内代谢相关的DEGs富集于精/脯氨酸代谢(13个)、氮代谢通路(11个)、硫代谢通路(10个)，主要功能与四氢嘧啶合成通路(11个)、电子传递途径(7个)及抗氧化酶系(5个)相关。RT-qPCR验证了四氢嘧啶合成代谢关键基因lysC、asd和基因簇ectABC，精氨酸(arginine, Arg)代谢通路基因astA/B/D/E以及尿素循环基因argE/H的表达趋势，与RNA-seq测序结果相一致。【结论】四氢嘧啶是细菌细胞和生物大分子重要的稳定保护剂。Fe₃O₄ NPs胁迫下菌株XH26胞内的活性氧(reactive oxygen species, ROS)增多，且影响胞内的氨基酸和氮代谢过程。菌株XH26通过提升抗氧化能力，增加胞内的四氢嘧啶积聚量，以应对Fe₃O₄ NPs的胁迫作用。

关键词

Fe₃O₄纳米颗粒 / 坎帕尼亚盐单胞菌 / 转录组学 / 四氢嘧啶 / 氧化应激

Abstract

收起

[Objective] To studyhydroxyl radical the differentially expressed genes (DEGs) in Halomonas campaniensis XH26 after co-culture with Fe₃O₄ nanoparticles (NPs), and clarify the molecular mechanism of Fe₃O₄ NPs in increasing the ectoine accumulation in strain XH26. [Methods] Strain XH26 was co-cultured with low-, medium-, and high-concentration (0.01, 0.10, and 0.50 g/L respectively in L, M, and H groups) Fe₃O₄ NPs, and the strain cultured without Fe₃O₄ NPs (0 g/L) was taken as the control group (C). Transcriptome sequencing was performed by Illumina HiSeq 300PE. The DEGs between different groups were mined, and key genes were screened for RT-qPCR verification. [Results] Compared with group C, group M showed an increase of 55.67% (708.87 mg/L) in ectoine accumulation, and groups M and H showed increased ferrous ions and antioxidant capacity. The hydroxyl radical content in group H was higher than that in group M. The transcriptomics analysis showed that the DEGs between groups M and C were enriched in arginine/proline metabolism (13), nitrogen metabolism (11), and sulfur metabolism (10) pathways. They were mainly related to the ectoine synthesis pathways (11), electron transport pathways (7), and antioxidant enzyme systems (5). RT-qPCR was employed to verify the expression of lysC, asd, and ectABC involved in ectoine synthesis, astA/B/D/E in arginine metabolic pathway, and argE/H in urea cycle, which showed the results consistent with the results of RNA-seq. [Conclusion] Ectoine is an important stable protective agent for bacterial cells and biomacromolecules. Strain XH26 exposed to the stress of Fe₃O₄ NPs showed increased intracellular reactive oxygen species and altered amino acid/nitrogen metabolism processes. Strain XH26 increased the accumulation of ectoine to cope with the stress of Fe₃O₄ NPs by improving the antioxidant capacity.

Key words

Fe₃O₄ nanoparticles / Halomonas campaniensis / transcriptomics / ectoine / oxidative stress

引用本文

张培霞, 陶宇杰, 乔丽娟, 王嵘, 韩睿, 朱德锐, 沈国平. 转录组学分析Fe₃O₄纳米颗粒胁迫下盐单胞菌XH26与四氢嘧啶代谢相关的差异表达基因. 微生物学报, 2025 , 65 (1) : 239 -255 . DOI: 10.13343/j.cnki.wsxb.20240525

Peixia ZHANG, Yujie TAO, Lijuan QIAO, Rong WANG, Rui HAN, Derui ZHU, Guoping SHEN. Transcriptomics reveals differentially expressed genes related to ectoine metabolism in Halomonas campaniensis XH26 under Fe₃O₄ nanoparticle stress[J]. Acta Microbiologica Sinica, 2025 , 65 (1) : 239 -255 . DOI: 10.13343/j.cnki.wsxb.20240525

正文

收起

金属纳米颗粒(metal nanoparticles, MNPs)与微生物菌群共培养时，其作用表现为两重性^[1]。如氧化铁纳米颗粒(iron oxide nanoparticles)具有一定的细胞毒性，但选择适宜浓度及粒径大小时它也具有良好的生物催化活性和磁性特征^[2]。Fatollahi等^[3]研究发现，Fe₂O₃ NPs (0.01 g/L)和多壁碳纳米管(0.10 g/L)均可促进伸长盐单胞菌(Halomonas elongata)胞内积聚四氢嘧啶(ectoine)，产量分别提高了72%和67%；Wang等^[4]在食物垃圾厌氧发酵时添加Fe₃O₄ NPs，并分析微生物群落结构的组成变化和挥发性脂肪酸(volatile fatty acids, VFAs)的产量，发现螺旋体菌门(Spirochaetes)和拟杆菌门(Bacteroidetes)的相对丰度提高了4.3%和1.7%，且VFAs的产量提高了160%。其次，当MNPs与细菌共培养时，细菌能感知不同的MNPs浓度刺激，胞内催化产生活性氧(reactive oxygen species, ROS)，促进转录因子调节相关基因的表达量。ROS可同时激活多种信号通路，以诱导生物体抵抗外界的环境胁迫^[5]。如Lyu等^[6]等利用极小(直径约3 nm)的氧化铁纳米颗粒(extremely small iron oxide nanoparticles, ESIONPs)刺激斑马鱼胚胎，发现氧化应激标志物基因(Cybb, NOX1, RAC2)显著上调；Lyu等^[7]分析硫还原地杆菌(Geobacter sulfurreducens)与磁铁矿(Fe₃O₄)共培养后的基因表达差异，发现菌毛蛋白基因(PilA)的表达量显著上调，而细胞色素c基因(OmcS)的表达量显著下调。

四氢嘧啶是嗜盐菌中广泛存在的相容溶质之一，可协助细菌维持渗透压的平衡，抵抗外界极端环境条件的胁迫刺激。四氢嘧啶合成的前体物质为天冬氨酸(aspartate, Asp)，由天冬氨酸激酶(aspartate kinase, LysC)将Asp转化为天冬氨酸-β-半缩醛，再经二氨基丁酸转氨酶(diaminobutyrate transaminase, EctB)、二氨基丁酸乙酰基转移酶(diaminobutyrate acetyltransferase, EctA)和四氢嘧啶合成酶(ectoine synthase, EctC)的3步催化合成四氢嘧啶^[8]。田磊等^[9]曾从小柴旦盐湖分离获得一株高效积聚四氢嘧啶的野生型坎帕尼亚盐单胞菌(Halomonas campaniensis) XH26，产量约450 mg/L。汪明香等^[10]采用45 mg/L的Fe₃O₄ NPs与菌株XH26共培养，发现菌株胞内四氢嘧啶的积聚量提高了55.08%。然而，针对Fe₃O₄ NPs如何影响菌株XH26胞内四氢嘧啶积聚量的具体机制，尤其涉及某些基因的转录激活和表达调控是否与四氢嘧啶的代谢通路存在关联，有待深入探究。因此，本研究设置不同浓度的Fe₃O₄ NPs与菌株XH26共培养，并采用转录组学技术分析四氢嘧啶合成代谢相关基因的差异表达，以此探究Fe₃O₄ NPs促进四氢嘧啶积聚量变化的分子机制，为后续四氢嘧啶的发酵生产和代谢控制提供新的思考方向。

1 材料与方法

收起

1.1 培养基和主要试剂

菌株发酵培养基(g/L, pH 7.5)^[9]：NaCl 87.50，KCl 55.88，MgSO₄·7H₂O 24.65，l-谷氨酸钠(mono sodium glutamate, MSG) 5.61，柠檬酸钠3.00，酶水解酪素7.50，无水CaCl₂ 0.20，酵母2.0。

Fe₃O₄ NPs，昂星新型碳材料常州有限公司；HPLC级四氢嘧啶标准品，Fluka公司；乙腈，赛默飞世尔科技公司；TRIzol UP强化RNA提取试剂盒，北京全式金生物技术有限公司；PrimeScript^TM RT reagent Kit with L-gDNA Eraser逆转录试剂盒和TB Green Premix Ex Taq^TM Ⅱ qPCR试剂盒，TaKaRa公司；亚铁离子含量检测试剂盒，北京索莱宝科技有限公司；总抗氧化能力检测试剂盒、超氧化物歧化酶试剂盒和羟自由基含量试剂盒，江苏艾迪生生物科技有限公司。

1.2 主要仪器

变速组织研磨器，天根生化科技(北京)有限公司；多功能酶标仪，Bio-Rad公司；透射电镜，成都里来生物科技有限公司；高效液相色谱仪，Agilent公司；微孔过滤器，天津市津腾实验设备有限公司；色谱分析柱，Merck公司；超声波细胞粉碎机，宁波新芝生物科技股份有限公司；转录组学测序平台，武汉贝纳科技有限公司。

1.3 四氢嘧啶积聚量、Fe²⁺浓度及抗氧化能力测定

设置Fe₃O₄ NPs与菌株共培养分组：空白组[control group (C), 0 g/L]、低浓度组[low group (L), 0.01 g/L]、中浓度组[medium group (M), 0.10 g/L]和高浓度组[high group (H), 0.50 g/L]。活化菌种XH26 (光密度值OD₆₀₀约为0.8，培养约12 h)，按1%比例接种于液体培养基(100 mL，n=3/组)；37 ℃、180 r/min摇床培养至生长对数期(约12 h)，按分组浓度加入Fe₃O₄ NPs，继续培养至42 h，抽提菌株胞内的四氢嘧啶和HPLC定量检测^[9]。使用磁铁吸除培养液中的Fe₃O₄ NPs，并对4组菌株的培养液进行稀释(OD₆₀₀值约1.0)，取1.0 mL稀释后的菌液离心，菌泥使用PBS洗涤2次。参考Fe²⁺含量和总抗氧化能力(total antioxidant capacity, T-AOC)检测试剂盒的方法，建立浓度与吸光度(A₅₉₃与A₅₉₀)的标准曲线，再进行胞内的Fe²⁺含量和T-AOC检测。参考羟基(OH⁻)自由基含量和超氧化物歧化酶(superoxide dismutase, SOD)检测试剂盒的方法，测定胞内羟基/自由基的吸光度(A₅₃₂)和SOD酶活性。

1.4 转录组的文库构建和质量控制

采用TRIzol UP试剂提取菌株XH26的总RNA (n=3/组)，使用1%琼脂糖凝胶电泳评估RNA的完整性。细菌型Ribo-Zero rRNA试剂盒去除rRNA，以片段化mRNA为模板逆转录合成cDNA。AMPure XP磁珠纯化修饰后的cDNA (370−420 bp)，采用USER酶降解cDNA的第二链，然后PCR扩增并再次纯化PCR产物，采用链特异性建库法建库^[11]。使用Agilent 2100和Qubit 2.0进行文库验证和定量检测，合格文库进行高通量测序，由武汉贝纳科技有限公司完成。测序片段转化为原始序列数据reads，再去除带接头(adapter)、含未确定碱基信息和低质量的reads (质量参数Q_pred≤20的碱基数 > 50%；单个碱基错误率 < 1%)，数据过滤获得clean reads。采用Bowtie 2 (v.2.3.4.3)软件进行clean reads与参考基因组比对分析(mismatch参数为2)，并根据参考菌株的基因组(H. campaniensis XH26，NCBI登录号为SAMN18316568)，使用Rockhopper (v.1.2.1)软件进行转录本预测分析。

1.5 表达差异基因的筛选和功能富集分析

根据定量饱和曲线确定基因的表达水平，利用软件RSeQC (v.3.0)进行总比对reads的重采样。使用Htseq (v.0.6.1) Union模型计算基因的表达量值fragments per kilobase million (FPKM)阈值> 1.0，以相对错误率(percent relative error)评估FPKM的准确性(Pearson系数R²≥0.92)。使用Bioconductor软件包DESeq 2 (v.1.20.0)进行差异表达基因(differentially expressed genes, DEGs)的基因本体论(gene ontology, GO)数据库(http://www.geneontology.org)，分析方法为负二项分布模型，以log₂ fold change > 0且P_adj < 0.05为阈值，统计差异基因的表达显著性(上调或下调)^[12]，并使用软件GO-Term Finder (v.0.86)制作GO富集图。参考京都基因和基因组百科全书数据库(Kyoto encyclopedia of genes and genomes, KEGG, http://www.kegg.jp)，使用软件KOBAS (v.3.0)进行基因组的背景关联和pathway的显著性分析(P_adj < 0.05)，确定DEGs参与的主要代谢途径^[13]。

1.6 RT-qPCR验证关键DEGs

采用TRIzol UP试剂盒提取RNA，检测纯度与浓度(满足OD₂₆₀/OD₂₈₀=1.8−2.2；有效浓度≥5 nmol/L)，然后使用逆转录试剂盒和qPCR试剂盒进行定量检测。逆转录体系(20 µL)：反应液Ⅰ 10 µL (5×gDNA eraser buffer 2.0 µL，gDNA eraser 1.0 µL，total RNA和RNase free H₂O共计7.0 µL)充分混匀后室温放置30 min，再加入反应液Ⅱ (PrimeScript RT enzyme mix Ⅰ 1.0 µL, RT primer mix 1.0 µL, 5×Primerscript buffer 4.0 µL, RNase free H₂O 4.0 µL)。定量PCR体系(20 µL)：cDNA 2.0 µL，TB Green™ Premix Ex Taq^TM Ⅱ 10.0 µL，正、反向引物(10 µmol/L)各0.8 µL，无菌水6.4 µL。RT-qPCR反应条件：95 ℃预变性3 min；95 ℃变性10 s，58 ℃退火20 s，72 ℃延伸30 s，45个循环，循环延伸阶段采集数据。四氢嘧啶合成的关键基因asd、lysC、基因簇ectABC及内参基因(GADPH)的引物设计参考文献[14]；基因astA/B/D/E和argH/E的引物设计采用NCBI Primer-BLAST程序进行(https://www.ncbi.nlm.nih.gov/)，引物合成均由生工生物工程(上海)股份有限公司完成(表1)，基因的相对表达水平采用2^−ΔΔC_t公式计算。

2 结果与分析

收起

2.1 Fe₃O₄ NPs胁迫下菌株XH26的四氢嘧啶积聚量与Fe²⁺含量分析

菌株XH26培养至对数期(12 h)时，添加不同浓度的Fe₃O₄ NPs再共培养至42 h，检测菌株胞内的四氢嘧啶积聚量、Fe²⁺含量及生长曲线(图1)。结果显示，随着Fe₃O₄ NPs浓度增加，菌株XH26胞内四氢嘧啶积聚量呈先增后减的趋势。当Fe₃O₄ NPs浓度为0−0.10 mol/L时，菌株胞内的四氢嘧啶积聚量呈上升态势，0.10 mol/L时菌株胞内的四氢嘧啶积聚量达到最大值708.87 mg/L，对比空白C组(455.36 mg/L)提高了55.67%；随着Fe₃O₄ NPs浓度继续增高，菌株胞内的四氢嘧啶积聚量呈现下降趋势(H组，604.78 mg/L，图1A)。检测胞内Fe²⁺浓度，并建立浓度(x)与吸光值A₅₉₃ (y)的标准曲线y=0.013 6x−0.017 3 (R²=0.99)。结果显示M组和H组胞内的亚铁离子含量显著升高，对比空白C组(1.81 µmol/L)分别提高了41.00%和49.00% (图1B)。酶标仪检测菌株的生长量(0−48 h)，发现菌株的生长趋势相似，但在36 h后各实验组的OD₆₀₀值存在一定差异(图1C)。透射电镜(200 nm)分析空白组(图1D)与M组(图1E)的菌株形态，发现M组的细菌结构完整未被破坏，细胞膜周围吸附大量的Fe₃O₄ NPs。

2.2 Fe₃O₄ NPs胁迫下菌株XH26胞内抗氧化水平分析

检测菌株XH26胞内的羟基自由基含量、SOD酶活性和总抗氧化能力(图2)，并建立总抗氧化能力(x)与吸光值A₅₉₀ (y)的标准曲线y=0.097 2x+0.004 2 (R²=0.99)。结果显示L、M、H实验组的羟基自由基含量对比空白C组(0.19)分别提高了13.65%、33.33%和44.15% (图2A)；M组和H组的SOD酶活性对比空白C组(9.74 U/mL)分别提高了17.17%和16.63%，对比L组(10.34 U/mL)分别提高了10.41%和9.91% (图2B)；总抗氧化能力对比空白C组(0.140 µmol/mL)分别提高了17.34%和16.34%，对比L组(0.145 µmol/mL)分别提高了13.56%和12.64% (图2C)。

2.3 转录组学数据处理与质控分析

原始数据经过滤处理，获得有效的clean reads数目 > 13 642 626条/组，G+C含量为53.86%−53.86%，Q₃₀ > 89.84%，误差为0.03% (表2)。采用Bowtie 2 (v.2.3.4.3)软件进行参考基因组比对和定位分析，结果显示样本的总匹配率 > 91%，单一匹配率 > 85%，多匹配率为6.05%−7.02%，表明测序结果可靠，满足后期的分析要求。

2.4 Fe₃O₄ NPs作用下基因表达水平分析

分析不同Fe₃O₄ NPs浓度组(C, L, M, H) DEGs的表达水平(lg FPKM+1)，并进行层次聚类分析(hierarchical clustering)。结果显示空白C组和L组的样本聚类相似，M组和H组的样本聚类相似；M组、H组与空白C组对比分析发现，M组、H组的基因转录发生逆转差异变化，即空白C组的低表达聚类区在M组、H组中转变高表达聚类区，或反之(图3A)。组间DEGs (log₂ fold change > 0且P < 0.05)和共有DEGs比较分析发现，L vs. C比较组存在上调DEGs 71个，下调DEGs 135个；M vs. C比较组存在上调DEGs 377个，下调DEGs 483个；H vs. C比较组中存在上调DEGs 207个，下调DEGs 266个；L vs. M比较组存在上调DEGs 227个，下调DEGs 259个(图3B)。共性DEGs分析显示，3个比较组(L vs. C, M vs. C, H vs. C)所特有的DEGs数目分别为56、481和127个，共有DEGs为93个(图3C)。

进一步筛选实验组与空白组的显著DEGs (log₂ fold change > 0且P_adj < 0.05)，结果显示显著DEGs主要集中在比较组M vs. C和H vs. C，包括上调基因169个，下调基因190个(表3)。分析M vs. C和H vs. C比较组所共有的36个显著DEGs发现(表4)，表达量显著上调的基因包括与四氢嘧啶合成直接相关的基因lysC和基因簇ectABC、精氨酸(arginine, Arg)代谢通路基因astA/B/D、过氧化物还蛋白基因RS04790等。表达量显著下调的基因包括辅酶Q (CoQ)合成相关基因aarF/ubiB及氧化还原酶相关基因RS06850、RS06855、RS06830等。

2.5 差异基因的GO和KEGG富集分析

利用基因功能分类的GO数据库，整体评估6个比较组(L vs. C, M vs. C, H vs. C, L vs. M, M vs. H, L vs. H)显著DEGs的表达水平(P_adj < 0.05，图4)。结果显示，M vs. C和H vs. C比较组的DEGs富集度显著高于L vs. C比较组(图4A)；高丰度DEGs主要集中于分子功能(molecular function, MF)、细胞组分(cellularcomponent, CC)和生物过程(biological process, BP)，且3个比较组的DEGs分布基本相似。重点分析M vs. C比较组的DEGs，结果显示MF类别主要富集于离子和金属离子转运蛋白活性；BP类别主要富集于细胞定位和离子转运；CC类别主要富集于细胞膜成分。不同Fe₃O₄ NPs实验组的组间对比发现，L vs. M比较组的DEGs富集度显著高于M vs. H和L vs. H比较组(图4B)，且DEGs富集情况与M vs. C比较组相似。

基于DEGs的KEGG代谢通路富集分析，绘制各比较组的气泡图(前20个，图5)。结果显示，空白C组与不同Fe₃O₄ NPs浓度实验组相比较(图5A、5C、5E)，M vs. C比较组存在显著差异(图5C)，与菌株XH26胞内代谢相关的DEGs主要富集于精/脯氨酸代谢(arginine/proline, Arg/Pro, 13个)和氮代谢(11个)。在L vs. M比较组中(图5B)，DEGs主要富集于Arg/Pro共代谢(10个)及硫代谢途径(10个)；在M vs. H比较组(图5D)，DEGs主要富集于Arg/Pro共代谢(5个)；在L vs. H比较组(图5F)，DEGs主要富集于硫代谢(10个)。

2.6 RT-qPCR验证四氢嘧啶合成的关键DEGs

Fe₃O₄ NPs胁迫作用下，菌株XH26胞内的四氢嘧啶积聚量均有所增长，筛选Fe₃O₄ NPs实验组(L, M, H)与四氢嘧啶合成直接相关的共有DEGs (11个，表5)，包括四氢嘧啶合成通路的关键基因lysC、asd和基因簇ectABC；Arg代谢通路的基因astA/B/D/E；尿素循环的基因argE/H。RT-qPCR验证11个关键的DEGs，结果显示(图6)，(1) 四氢嘧啶的合成通路涉及5个关键基因，在3个比较组中的基因簇ectABC均显著上调表达，且高于RNA-seq结果；基因lysC表达验证结果略低于RNA-seq测序；基因asd的表达验证结果与RNA-seq测序相一致。(2) Arg代谢通路涉及4个相关基因，基因astB/D/E在3个比较组中均上调表达；基因astA在M vs. C和H vs. C比较组中上调表达，但在L vs. C比较组中的表达量较低。(3) 尿素循环涉及2个相关基因，基因argE在3个比较组中的表达量均上调，且在H vs. C组中高于RNA-seq测序结果；基因argH在L vs. C、M vs. C比较组中的表达量显著上调，而在H vs. C比较组中表达量较低。总体而言，关键DEGs在M vs. C比较组中的上调趋势显著高于L vs. C和H vs. C比较组，RT-qPCR验证的表达趋势与RNA-seq测序结果相一致。

3 讨论与结论

收起

3.1 Fe₃O₄ NPs胁迫刺激促进菌株XH26合成四氢嘧啶

MNPs刺激细胞产生氧化应激是毒理学的主要假设之一，可通过检测细胞产生的氧化应激能力进行验证^[15]。适宜浓度Fe₃O₄ NPs的细菌毒性远低于其他的MNPs，但仍可促进ROS的产生^[16-17]。细菌胞内的ROS稳态和氧化还原平衡，主要由酶/非酶系统调节完成^[18]，如烷基氢过氧化物还原酶/硫醇特异性抗氧化剂家族蛋白(AhpC/TSA)、硫氧还蛋白(thioredoxin) 等^[19]。本研究M和H实验组中，AhpC/TSA家族过氧化物还蛋白基因RS04790显著上调，FAD依赖性氧化还原酶基因RS06850、NAD(P)⁺依赖性氧化还原酶基因RS06830、TIGR01777家族氧化还原酶(TIGR01777 family oxidoreductase)基因RS06855的表达量显著下调。此外，M实验组硫氧还蛋白RS07995的表达量显著上调。分析菌株XH26的抗氧化能力，发现M和H实验组菌株胞内的羟基自由基含量、总抗氧化能力和SOD酶活性显著升高，且H实验组羟基自由基的含量远高于M组，可能导致细菌的增殖和能量代谢能力下降。

细菌胞内的ROS水平升高可以激活多种蛋白激酶，通过信号转导调节细胞的代谢活动，以产生应激适应^[20]。同时，ROS升高可能破坏细胞的氧化还原平衡，损害蛋白质、DNA和RNA等生物大分子^[21]。四氢嘧啶属于高度亲水性的相容溶质，具有良好的生物稳定和细胞保护作用，能够维持细胞膜、酶或功能蛋白质的空间结构稳定，缓解ROS的细胞损伤，以此抵抗外界环境的胁迫^[22]。Fe₃O₄ NPs胁迫下，M组与四氢嘧啶代谢直接相关基因的表达量显著上调(lysC、asd和基因簇ectABC)，如图7红色通路所示，这些基因的表达量上调，可以直接促进Asp代谢合成四氢嘧啶。Hernández等^[23]曾证实细菌Arg的代谢通路与三羧酸循环(tricarboxylic acid cycle, TAC)、尿素循环(urea cycle)以及谷氨酸(l-glutamate, Glu)代谢密切相关。本研究中，基因argE/H的上调表达，有效促进中间产物精氨酸代琥珀酸(l-arginosuccinate)转化为Arg和延胡索酸(fumarate)；同时，基因astA/B/D/E的上调表达，促进Arg转化为Glu和α-酮戊二酸(图7)。由此表明，在Fe₃O₄ NPs胁迫下，菌株XH26胞内的ROS水平升高，可能通过激活多种蛋白激酶来实现代谢调节，最终促进菌株合成四氢嘧啶，以此缓解ROS引起的细胞和生物大分子损伤作用。

3.2 Fe₃O₄ NPs-Fe²⁺作为酶蛋白EctC的辅因子促进四氢嘧啶合成

在生物体酸性物质或溶酶体的作用下，Fe₃O₄ NPs可以释放铁离子至细胞，参与胞内的酶促代谢和金属酶类的调节作用^[24-25]。Zhong等^[26]分析膨胀颗粒污泥床(expanded granular sludge bed, EGSB)中Fe₃O₄ NPs的Fe²⁺释放量，发现在添加Fe₃O₄ NPs 20 d后，Fe²⁺浓度从0.71 mg/L迅速增至5.10 mg/L。酶蛋白EctC (14.70 kDa)隶属于Cupin超家族成员，辅助因子为Fe²⁺，负责催化底物(N-γ-乙酰基-l-2, 4-二氨基丁酸)的羰基与α-氨基分子内环化，脱水缩合成四氢嘧啶(图8)^[27]。Widderich等^[28]分析阿拉斯加鞘氨醇盒菌(Sphingopyxis alaskensis) EctC酶活性与金属辅因子的关系，发现Fe²⁺可能通过结合EctC的3个氨基酸残基(Glu⁵⁷, Tyr⁸⁵, His⁹³)，促进酶与底物的结合，从而提高酶的催化活性。本研究M和H实验组中，菌株胞内的Fe²⁺浓度分别提高了41.00%和49.00%，且基因ectC的表达量均显著上调。因此，我们推测Fe₃O₄ NPs与菌株XH26共培养时，可能通过释放Fe²⁺激活酶EctC，促进XH26胞内积聚四氢嘧啶，具体的反应机制需进行体外Fe₃O₄ NPs与EctC酶活性实验验证。

3.3 Fe₃O₄ NPs可能影响电子传递促进四氢嘧啶合成

细菌具有复杂的细胞外电子摄取系统(extracellular electron uptake, EEU)，可从不溶性电子供体中攫取电子^[29]。Fe₃O₄ NPs作为一种多孔水性介质，其末端的电子受体(水合铁)可作为细菌胞内的能量转换驱动器^[30]。如Vu等^[31]利用Fe₃O₄ NPs改良的微生物电化学系统(microbial electrochemical system, MES)进行厌氧发酵，发现Fe₃O₄ NPs可增强生物膜导电率或电极募集，促进电子转移，从而增加甲烷的产量(与未添加Fe₃O₄ NPs的MES系统相比提高22.1%)。Zheng等^[32]采用磁铁矿(Fe₃O₄)与金属还原地杆菌(Geobacter metallireducens)和硫还原地杆菌(G. sulfurreducens)共培养并进行转录组学分析，发现金属还原地杆菌细胞色素b6基因(RS02690)、细胞色素c相关基因(RS15860, RS02890, RS02575, RS01255, RS09110)的表达量显著上调；硫还原地杆菌细胞色素c相关基因(omcX, omcI, ppcE, ppcH)的表达量显著上调。本研究中，M实验组卟啉代谢通路基因hemA/B/C/D、细胞色素c组装蛋白基因ccsA的表达量均显著上调，辅酶Q合成相关基因aarF、ubiB的表达量显著下调。血红素是血红蛋白(氧转运)、肌红蛋白(氧储存)和细胞色素(电子传递)的辅助因子^[33]，辅酶Q和细胞色素c主要参与呼吸链的电子传递^[34]，且辅酶Q还参与呼吸过程中ROS的产生^[35]。因此，Fe₃O₄ NPs可能影响电子传递过程，从而为四氢嘧啶的合成提供能量，但具体涉及呼吸链电子转移的影响作用，尚需体外电化学实验验证。

综上所述，本文设置不同浓度Fe₃O₄ NPs与H. campaniensis XH26菌株共培养，利用转录组学分析探究了Fe₃O₄ NPs培养条件下的差异表达基因。Fe₃O₄ NPs胁迫下，菌株XH26胞内的DEGs主要与四氢嘧啶合成通路、电子传递途径及抗氧化酶系相关，表明菌株XH26通过促进四氢嘧啶合成和提升抗氧化能力，以应对胞外Fe₃O₄ NPs的胁迫作用。

基金

收起

国家自然科学基金(32260019)
青海中央引导地方科技发展资金(2024ZY015)

参考文献

收起

文献

收起

参考文献引证文献

排序方式：

[1]

DINALI R, EBRAHIMINEZHAD A, MANLEY-HARRIS M, GHASEMI Y, BERENJIAN A.Iron oxide nanoparticles in modern microbiology and biotechnology[J].Critical Reviews in Microbiology,2017,43(4):493-507.

https://doi.org/10.1080/1040841X.2016.1267708

[2]

孙剑飞, 张宇, 杨芳, 马明, 熊非, 顾宁.医药磁性氧化铁纳米材料的研究和发展[J].科学通报,2019,64(8):842-853.

SUN JF, ZHANG Y, YANG F, MA M, XIONG F, GU N.Research and development of medical magnetic nanomaterials[J].Chinese Science Bulletin,2019,64(8):842-853 (in Chinese).

[3]

FATOLLAHI P, GHASEMI M, YAZDIAN F, SADEGHI A.Ectoine production in bioreactor by Halomonas elongata DSM2581: using MWCNT and Fe-nanoparticle[J].Biotechnology Progress,2021,37(1):e3073.

https://doi.org/10.1002/btpr.3073

[4]

WANG LT, LEI ZF, YANG XJ, ZHANG C, LIU C, SHIMIZU K, ZHANG ZY, YUAN T.Fe₃O₄ enhanced efficiency of volatile fatty acids production in anaerobic fermentation of food waste at high loading[J].Bioresource Technology,2022,364:128097.

https://doi.org/10.1016/j.biortech.2022.128097

[5]

CHEN S, LIU HL, YANGZONG ZX, GARDEA-TORRESDEY JL, WHITE JC, ZHAO LJ.Seed priming with reactive oxygen species-generating nanoparticles enhanced maize tolerance to multiple abiotic stresses[J].Environmental Science & Technology,2023,57(48):19932-19941.

[6]

LYU ZJ, KOU Y, FU Y, XIE YX, YANG B, ZHU HJ, TIAN J.Comparative transcriptomics revealed neurodevelopmental impairments and ferroptosis induced by extremely small iron oxide nanoparticles[J].Frontiers in Genetics,2024,15:1402771.

https://doi.org/10.3389/fgene.2024.1402771

[7]

LIU FH, ROTARU AE, SHRESTHA PM, MALVANKAR NS, NEVIN KP, LOVLEY DR.Magnetite compensates for the lack of a pilin-associated c-type cytochrome in extracellular electron exchange[J].Environmental Microbiology,2015,17(3):648-655.

https://doi.org/10.1111/1462-2920.12485

[8]

张鑫, 舒志万, 李永臻, 邢江娃, 王嵘, 沈国平, 朱德锐.相容溶质四氢嘧啶的微生物合成研究进展[J].生物工程学报,2022,38(3):868-881.

ZHANG X, SHU ZW, LI YZ, XING JW, WANG R, SHEN GP, ZHU DR.Advances in the microbial production of the compatible solute ectoine: a review[J].Chinese Journal of Biotechnology,2022,38(3):868-881 (in Chinese).

[9]

田磊, 张芳, 沈国平, 高翔, 龙启福, 朱德锐.Ectoine高产菌株Halomonas sp. XH26的鉴定及紫外诱变选育[J].生物学杂志,2020,37(4):31-35.

TIAN L, ZHANG F, SHEN GP, GAO X, LONG QF, ZHU DR.Identification of high-yielding strain Halomonas sp. XH26 for producing ectoine and UV mutagenesis breeding[J].Journal of Microbiology,2020,37(4):31-35 (in Chinese).

[10]

汪明香, 郭敏, 高翔, 李永臻, 韩睿, 朱德锐, 沈国平.磁性纳米金属对盐单胞菌生长特性及胞内Ectoine的积聚影响[J].中国生物工程杂志,2023,43(5):45-54.

WANG MX, GUO M, GAO X, LI YZ, HAN R, ZHU DR, SHEN GP.Effects of magnetic nano-metals on growth characteristics and intracellular ectoine accumulation of Halomonas sp. XH26[J].China Biotechnology,2023,43(5):45-54 (in Chinese).

[11]

PARKHOMCHUK D, BORODINA T, AMSTISLAVSKIY V, BANARU M, HALLEN L, KROBITSCH S, LEHRACH H, SOLDATOV A.Transcriptome analysis by strand-specific sequencing of complementary DNA[J].Nucleic Acids Research,2009,37(18):e123.

https://doi.org/10.1093/nar/gkp596

[12]

PINEL GD, HORDER JL, KING JR, McINTYRE A, MONGAN NP, LÓPEZ GG, BENEST AV.Endothelial cell RNA-seq data: differential expression and functional enrichment analyses to study phenotypic switching[J].Methods in Molecular Biology,2022,2441:369-426. http://link.springer.com/protocol/10.1007/978-1-0716-2059-5_29

[13]

DAS P, BEHERA BK, CHATTERJEE S, DAS BK, MOHAPATRA T.De novo transcriptome analysis of halotolerant bacterium Staphylococcus sp. strain P-TSB-70 isolated from East coast of India: in search of salt stress tolerant genes[J].PLoS One,2020,15(2):e0228199.

https://doi.org/10.1371/journal.pone.0228199

[14]

张鑫, 王智博, 缪增强, 邢江娃, 王嵘, 李永臻, 朱德锐, 沈国平.转录组学分析盐单胞菌四氢嘧啶合成代谢相关的表达差异基因与RT-qPCR验证[J].微生物学报,2022,62(3):1083-1098.

https://doi.org/10.13343/j.cnki.wsxb.20210402

ZHANG X, WANG ZB, MIAO ZQ, XING JW, WANG R, LI YZ, ZHU DR, SHEN GP.Transcriptome analysis of differential genes associated with the ectoine synthesis pathway of Halomonas campaniensis under salt stress and expression verification by RT-qPCR[J].Acta Microbiologica Sinica,2022,62(3):1083-1098 (in Chinese).

https://doi.org/10.13343/j.cnki.wsxb.20210402

[15]

DUAN JC, KODALI VK, GAFFREY MJ, GUO J, CHU RK, CAMP DG, SMITH RD, THRALL BD, QIAN WJ.Quantitative profiling of protein S-glutathionylation reveals redox-dependent regulation of macrophage function during nanoparticle-induced oxidative stress[J].ACS Nano,2016,10(1):524-538.

https://doi.org/10.1021/acsnano.5b05524

[16]

LEWIŃSKA A, RADOŃ A, GIL K, BŁONIARZ D, CIURASZKIEWICZ A, KUBACKI J, KĄDZIOŁKA-GAWEŁ M, ŁUKOWIEC D, GĘBARA P, KROGUL-SOBCZAK A, PIOTROWSKI P, FIJAŁKOWSKA O, WYBRANIEC S, SZMATOŁA T, KOLANO-BURIAN A, WNUK M.Carbon-coated iron oxide nanoparticles promote reductive stress-mediated cytotoxic autophagy in drug-induced senescent breast cancer cells[J].ACS Applied Materials & Interfaces,2024,16(12):15457-15478.

[17]

KICHEEVA AG, SUSHKO ES, BONDARENKO LS, BAIMURATOVA RK, KYDRALIEVA KA, SCHWAMINGER SP, PRASSL R, TROPSKAYA NS, DZHARDIMALIEVA GI, SMIRNYKH DV, MARTYNOVA AA, KUDRYASHEVA NS.Cytotoxic and radical activities of metal-organic framework modified with iron oxide: biological and physico-chemical analyses[J].Chemico-Biological Interactions,2024,399:111150.

https://doi.org/10.1016/j.cbi.2024.111150

[18]

BU T, YANG JX, LIU JX, FAN XF.NaHS immersion alleviates the stress effect of chromium (Ⅲ) on alfalfa seeds by affecting active oxygen metabolism[J].Plant Signaling & Behavior,2024,19(1):2375673.

[19]

MYKYTCZUK NCS, TREVORS JT, FOOTE SJ, LEDUC LG, FERRONI GD, TWINE SM.Proteomic insights into cold adaptation of psychrotrophic and mesophilic Acidithiobacillus ferrooxidans strains[J].Antonie Van Leeuwenhoek,2011,100(2):259-277.

https://doi.org/10.1007/s10482-011-9584-z

[20]

SNIECKUTE G, RYDER L, VIND AC, WU ZZ, ARENDRUP FS, STONELEY M, CHAMOIS S, MARTINEZ-VAL A, LELEU M, DREOS R, RUSSELL A, GAY DM, GENZOR AV, CHOI BSY, BASSE AL, SASS F, DALL M, DOLLET LCM, BLASIUS M, WILLIS AE, et al.ROS-induced ribosome impairment underlies ZAKα-mediated metabolic decline in obesity and aging[J].Science,2023,382(6675):eadf3208.

https://doi.org/10.1126/science.adf3208

[21]

SCHIEBER M, CHANDEL NS.ROS function in redox signaling and oxidative stress[J].Current Biology,2014,24(10):R453-R462.

https://doi.org/10.1016/j.cub.2014.03.034

[22]

许慧娴, 陈永涛, 黄建忠, 陶勇, 柯崇榕, 杨欣伟.四氢嘧啶生物合成及其关键酶生化特性研究进展[J].生物工程学报,2024,40(6):1620-1643.

XU HX, CHEN YT, HUANG JZG, TAO Y, KE CR, YANG XW.Advances in ectoine biosynthesis and biochemical characteristics of key enzymes[J].Chinese Journal of Biotechnology,2024,40(6):1620-1643 (in Chinese).

[23]

HERNÁNDEZ VM, ARTEAGA A, DUNN MF.Diversity, properties and functions of bacterial arginases[J].FEMS Microbiology Reviews,2021,45(6):fuab034.

https://doi.org/10.1093/femsre/fuab034

[24]

张珺哲. 四氧化三铁纳米颗粒的代谢过程研究[D]. 北京: 中国科学院大学博士学位论文, 2019.

ZHANG JZ. Study on metabolic process of Fe₃O₄ nanoparticles[D]. Beijing: Doctoral Dissertation of University of Chinese Academy of Sciences, 2019 (in Chinese).

[25]

LUO T, GAO JL, LIN N, WANG JK.Effects of two kinds of iron nanoparticles as reactive oxygen species inducer and scavenger on the transcriptomic profiles of two human leukemia cells with different stemness[J].Nanomaterials,2020,10(10):1951.

https://doi.org/10.3390/nano10101951

[26]

ZHONG D, LI JX, MA WC, XIN HM.Magnetite nanoparticles enhanced glucose anaerobic fermentation for bio-hydrogen production using an expanded granular sludge bed (EGSB) reactor[J].International Journal of Hydrogen Energy,2020,45(18):10664-10672.

https://doi.org/10.1016/j.ijhydene.2020.01.095

[27]

CZECH L, HÖPPNER A, KOBUS S, SEUBERT A, RICLEA R, DICKSCHAT JS, HEIDER J, SMITS SHJ, BREMER E.Illuminating the catalytic core of ectoine synthase through structural and biochemical analysis[J].Scientific Reports,2019,9(1):364.

[28]

WIDDERICH N, KOBUS S, HÖPPNER A, RICLEA R, SEUBERT A, DICKSCHAT JS, HEIDER J, SMITS SHJ, BREMER E.Biochemistry and crystal structure of ectoine synthase: a metal-containing member of the cupin superfamily[J].PLoS One,2016,11(3):e0151285.

[29]

GUPTA D, GUZMAN MS, BOSE A.Extracellular electron uptake by autotrophic microbes: physiological, ecological, and evolutionary implications[J].Journal of Industrial Microbiology & Biotechnology,2020,47(9/10):863-876.

[30]

ZHANG JR, HUANG S, YIN YG, YANG LQ, LI XN, JIAO WT, SAKAMAKI T.Fe₂O₃ micron particles are critical for electron transfer and the distribution of electrochemically active bacteria in soil MFCs[J].Science of the Total Environment,2023,893:164909.

[31]

VU MT, NOORI MT, MIN B.Conductive magnetite nanoparticles trigger syntrophic methane production in single chamber microbial electrochemical systems[J].Bioresource Technology,2020,296:122265.

[32]

ZHENG SL, LIU FH, LI M, XIAO LL, WANG OM.Comparative transcriptomic insights into the mechanisms of electron transfer in Geobacter co-cultures with activated carbon and magnetite[J].Science China Life Sciences,2018,61(7):787-798.

[33]

FUJIWARA T, HARIGAE H.Biology of heme in mammalian erythroid cells and related disorders[J].BioMed Research International,2015,2015:278536.

[34]

汤明芳, 盛光遥, 李长鑫, 丁静.基于细胞色素c的胞外电子传递过程[J].微生物学报,2023,63(2):509-522.

https://doi.org/10.13343/j.cnki.wsxb.20220424

TANG MF, SHENG GY, LI CX, DING J.The process of extracellular electron transfer based on cytochrome c[J].Acta Microbiologica Sinica,2023,63(2):509-522 (in Chinese).

https://doi.org/10.13343/j.cnki.wsxb.20220424

[35]

WANG Y, LILIENFELDT N, HEKIMI S.Understanding coenzyme Q[J].Physiological Reviews,2024,104(4):1533-1610.

2025年第65卷第1期

PDF下载

106

引用本文

BibTeX

文章信息

doi: 10.13343/j.cnki.wsxb.20240525

接收时间：2024-08-26
首发时间：2026-03-21
出版时间：2025-01-04

补充材料

相关文章

文章信息

作者

出版历史

收稿日期：2024-08-26
录用日期：2024-09-30

基金

National Natural Science Foundation of China(32260019)

国家自然科学基金(32260019)

Qinghai Central Government Guide Local Science and Technology Development Fund(2024ZY015)

青海中央引导地方科技发展资金(2024ZY015)

作者信息

1 青海大学医学院, 基础医学研究中心, 青海西宁 810016

2 青海大学农林科学院, 蔬菜遗传与生理重点实验室, 青海西宁 810016

通讯作者:

参考文献

分享链接

https://castjournals.cast.org.cn/joweb/wswxb/CN/10.13343/j.cnki.wsxb.20240525

分享至

全文二维码

扫描看全文

引用本文

BibTeX

本文的引用情况

2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

关闭全屏

BibTeX
EndNote
RefWorks
TxT

Gene name	Primer sequences (5′→3′)	Product length (bp)
astA	F: AACGGTCACGAAACTGAGCA R: GCTGCTGTGCTATTGAAGGC	264
astB	F: GTAAAATGCACTCGGCGGTC R: ACTGATGGATGCTGGCTACG	231
astD	F: CCAAGCTATCGGCCTCCATT R: GTGAACTGGAACCGCCAAAC	145
astE	F: ACAGGCGAAGAACCTGATCG R: CAACTGTGCTGGTGTTGGTG	233
argH	F: CACGCTCTTCATCGGTCAGT R: AGCCAAGCTACGAACCAGTC	178
argE	F: CGTTGAAATCACCACCGAGC R: TGAGGTGTTTCTAGCGCCTG	291
lysC	F: CAAGACGAGGACGCTATGGAAGAAC R: TCGGCGATAGGACCAAGAATACG	133
asd	F: CCCGAACGACAAAGACGCTACAG R: TCACCAACACTGAAGGCTGACAAG	132
ectA	F: CAGTCGCTGATGCTGTGGTTGG R: GAATTAACATCAAGCGGCGGACAAG	121
ectB	F: TGCGTGGTATTGATGTTGTCTCTGG R: CACTTCACTACTTCGCCGTCTTGG	112
ectC	F: GCTATGAAGGCGAAGGCGAAGTAG R: AACAGATGTTCGTCGTGCTGATCC	103
GAPDH	F: TCCTTCCCTAAACTCGCACCT R: ATACGATAGTAGCGTCAGCAT	284

Gene name

Primer sequences (5′→3′)

Product length (bp)

astA

F: AACGGTCACGAAACTGAGCA
R: GCTGCTGTGCTATTGAAGGC

264

astB

F: GTAAAATGCACTCGGCGGTC
R: ACTGATGGATGCTGGCTACG

231

astD

F: CCAAGCTATCGGCCTCCATT
R: GTGAACTGGAACCGCCAAAC

145

astE

F: ACAGGCGAAGAACCTGATCG
R: CAACTGTGCTGGTGTTGGTG

233

argH

F: CACGCTCTTCATCGGTCAGT
R: AGCCAAGCTACGAACCAGTC

178

argE

F: CGTTGAAATCACCACCGAGC
R: TGAGGTGTTTCTAGCGCCTG

291

lysC

F: CAAGACGAGGACGCTATGGAAGAAC
R: TCGGCGATAGGACCAAGAATACG

133

asd

F: CCCGAACGACAAAGACGCTACAG
R: TCACCAACACTGAAGGCTGACAAG

132

ectA

F: CAGTCGCTGATGCTGTGGTTGG R:
GAATTAACATCAAGCGGCGGACAAG

121

ectB

F: TGCGTGGTATTGATGTTGTCTCTGG
R: CACTTCACTACTTCGCCGTCTTGG

112

ectC

F: GCTATGAAGGCGAAGGCGAAGTAG
R: AACAGATGTTCGTCGTGCTGATCC

103

GAPDH

F: TCCTTCCCTAAACTCGCACCT
R: ATACGATAGTAGCGTCAGCAT

284

Sample	Clean reads	Q₂₀ (%)	Q₃₀ (%)	Error rate (%)	G+C (%)	Total mapped (%)	Uniquely mapped (%)	Multiple mapped (%)
C₁	14 866 686	96.38	90.52	0.03	53.42	92.84	86.13	6.71
C₂	15 372 448	96.39	90.53	0.03	53.78	93.04	86.52	6.52
C₃	13 642 626	96.08	90.00	0.03	53.68	92.53	85.51	7.02
L₁	15 587 564	96.54	90.82	0.03	53.86	93.19	86.74	6.44
L₂	14 179 496	96.43	90.59	0.03	53.28	92.57	86.06	6.51
L₃	13 819 380	96.52	90.75	0.03	53.52	93.09	86.94	6.15
M₁	15 599 214	96.18	90.04	0.03	53.09	91.97	85.22	6.75
M₂	15 368 656	96.49	90.66	0.03	53.57	93.11	87.06	6.05
M₃	15 754 456	96.31	90.35	0.03	53.63	92.94	86.45	6.48
H₁	14 431 560	96.06	89.84	0.03	53.36	92.13	85.53	6.60
H₂	14 331 822	96.04	89.84	0.03	53.61	93.37	87.00	6.37
H₃	17 921 678	96.48	90.70	0.03	53.41	93.43	87.23	6.20

Sample

Clean reads

Q₂₀ (%)

Q₃₀ (%)

Error rate
(%)

G+C (%)

Total mapped
(%)

Uniquely mapped
(%)

Multiple mapped
(%)

C₁

14 866 686

96.38

90.52

0.03

53.42

92.84

86.13

6.71

C₂

15 372 448

96.39

90.53

0.03

53.78

93.04

86.52

6.52

C₃

13 642 626

96.08

90.00

0.03

53.68

92.53

85.51

7.02

L₁

15 587 564

96.54

90.82

0.03

53.86

93.19

86.74

6.44

L₂

14 179 496

96.43

90.59

0.03

53.28

92.57

86.06

6.51

L₃

13 819 380

96.52

90.75

0.03

53.52

93.09

86.94

6.15

M₁

15 599 214

96.18

90.04

0.03

53.09

91.97

85.22

6.75

M₂

15 368 656

96.49

90.66

0.03

53.57

93.11

87.06

6.05

M₃

15 754 456

96.31

90.35

0.03

53.63

92.94

86.45

6.48

H₁

14 431 560

96.06

89.84

0.03

53.36

92.13

85.53

6.60

H₂

14 331 822

96.04

89.84

0.03

53.61

93.37

87.00

6.37

H₃

17 921 678

96.48

90.70

0.03

53.41

93.43

87.23

6.20

Regulate	Comparison group	Gene name
Up genes	M vs. C	fabA, hisI, pilW, surE, lysC, recX, hutC, iscR, lrgB, lpxB, rapZ, mnmC, ccsA, glnD, recF, nadB, ptsN, rnt, rnhB, xthA, flhC, gcvP, gmhB, yhbY, rnhA, uraH, rimP, rlmE, dinB, glxA, folK, yidD, umuD, gcvH, mnmA, hflD, rho, pta, der, asd, flgN/M, argE/H, XdhA/B/C, ectABC, fliE/F/M/N/P, hemA/B/C/D, astA/B/D/E, and 76 undefined genes
H vs. C	astA/B/D, ectABC, cysD/T/N/P, gcvH, xdhA/B/C, lysC, ahpC, pilW, and 10 undefined genes
Down genes	M vs. C	HutG, safE, recB/D, acrB/D/F, iclR, dctP/M/Q, malT, dgcA, fdhA, gltS, proB, soxZ, hlyD, cobG, sohB, ptsP, antC, eamA, ureD, dctM, eat, urtB/C, kdpA/C, phnE, aqpZ, betT, oprD, moaA, ligB, cobF, nhaC, sstT, ctaG, narH/K, catC, fixB, rluA, coxB, deoR, ribA, nirB/D, menA, ccoG, arsJ, pqqD, qhpC, asnC, tauE, plsY, exbB, ald, mocA, mtnA, smtB, mlaE, peaD, lysE/R, dapE, rluF, purU, xseA, yifB, eutC, chrA, ptsP, pstC/B, ubiB, nagE, thuA, iolC/D, glaH, speB, lptG, acrB/D/F, aarF, and 171 undefined genes
H vs. C	ubiB, hlyD, fecC/D, tenA, recB/D, tonB, bioD, folE, glaH, aarF, mdoG, and 19 undefined genes

Regulate

Comparison group

Gene name

Up genes

M vs. C

fabA, hisI, pilW, surE, lysC, recX, hutC, iscR, lrgB, lpxB, rapZ, mnmC, ccsA, glnD, recF, nadB, ptsN, rnt, rnhB, xthA, flhC, gcvP, gmhB, yhbY, rnhA, uraH, rimP, rlmE, dinB, glxA, folK, yidD, umuD, gcvH, mnmA, hflD, rho, pta, der, asd, flgN/M, argE/H, XdhA/B/C, ectABC, fliE/F/M/N/P, hemA/B/C/D, astA/B/D/E, and 76 undefined genes

H vs. C

astA/B/D, ectABC, cysD/T/N/P, gcvH, xdhA/B/C, lysC, ahpC, pilW, and 10 undefined genes

Down genes

M vs. C

HutG, safE, recB/D, acrB/D/F, iclR, dctP/M/Q, malT, dgcA, fdhA, gltS, proB, soxZ, hlyD, cobG, sohB, ptsP, antC, eamA, ureD, dctM, eat, urtB/C, kdpA/C, phnE, aqpZ, betT, oprD, moaA, ligB, cobF, nhaC, sstT, ctaG, narH/K, catC, fixB, rluA, coxB, deoR, ribA, nirB/D, menA, ccoG, arsJ, pqqD, qhpC, asnC, tauE, plsY, exbB, ald, mocA, mtnA, smtB, mlaE, peaD, lysE/R, dapE, rluF, purU, xseA, yifB, eutC, chrA, ptsP, pstC/B, ubiB, nagE, thuA, iolC/D, glaH, speB, lptG, acrB/D/F, aarF, and 171 undefined genes

H vs. C

ubiB, hlyD, fecC/D, tenA, recB/D, tonB, bioD, folE, glaH, aarF, mdoG, and 19 undefined genes

Up genes	Function	Down genes	Function
ectA/B/C, lysC	Ectoine synthesis pathway	recB/D	Exodeoxyribonuclease Ⅴ subunit
astA/B/D	Arg and Pro metabolism	RS14435/14800/06820	DUF932/3833/1365 domain-containing protein
RS04790	Peroxiredoxin	RS06850	FAD-dependent oxidoreductase
xdhA/B/C	Xanthine dehydrogenase	RS06856/06825	NAD dependent epimerase/dehydratase family
PilW	Type Ⅳ pilus biogenesis/stability protein	aarF, ubiB	AarF/ABC1/UbiB kinase family protein
RS07420	VRR-NUC domain-containing protein	RS06830	SDR family NAD(P)-dependent oxidoreductase
RS04260	Cold-shock protein	RS01840	Efflux RND transporter periplasmic adaptor subunit
gcvH	Glycine cleavage system protein	RS10485	Glycosyltransferase
RS02725	DNA starvation/stationary phase protection protein	RS01690	PAS domain-containing methyl-accepting chemotaxis protein
RS06920/06925/11825	tRNA-Tyr/Gly/Leu	RS06855	TIGR01777 family oxidoreductase

Up genes

Function

Down genes

Function

ectA/B/C, lysC

Ectoine synthesis pathway

recB/D

Exodeoxyribonuclease Ⅴ subunit

astA/B/D

Arg and Pro metabolism

RS14435/14800/06820

DUF932/3833/1365 domain-containing protein

RS04790

Peroxiredoxin

RS06850

FAD-dependent oxidoreductase

xdhA/B/C

Xanthine dehydrogenase

RS06856/06825

NAD dependent epimerase/dehydratase family

PilW

Type Ⅳ pilus biogenesis/stability protein

aarF, ubiB

AarF/ABC1/UbiB kinase family protein

RS07420

VRR-NUC domain-containing protein

RS06830

SDR family NAD(P)-dependent oxidoreductase

RS04260

Cold-shock protein

RS01840

Efflux RND transporter periplasmic adaptor subunit

gcvH

Glycine cleavage system protein

RS10485

Glycosyltransferase

RS02725

DNA starvation/stationary phase protection protein

RS01690

PAS domain-containing methyl-accepting chemotaxis protein

RS06920/06925/11825

tRNA-Tyr/Gly/Leu

RS06855

TIGR01777 family oxidoreductase

Gene	Description of genes or factors	FPKM values of RNA-seq in different groups	Regulate
astA	Arginine N-succinyltransferase	239.65	222.21	330.96	287.26	Up
astB	N-succinylarginine dihydrolase	154.50	162.55	230.63	199.76	Up
astD	Succinylglutamate-semialdehyde dehydrogenase	303.23	298.12	350.58	418.82	Up
astE	Succinylglutamate desuccinylase	414.34	532.87	612.34	458.88	Up
argH	Argininosuccinate lyase	406.67	446.99	531.84	501.08	Up
argE	Acetylornithine deacetylase	371.84	428.38	483.91	428.41	Up
lysC	Aspartate kinase	669.51	792.61	971.45	964.37	Up
asd	Aspartate-semialdehyde dehydrogenase	182.14	172.84	216.33	203.34	Up
ectA	Diaminobutyrate acetyltransferase	604.69	764.54	940.39	925.42	Up
ectB	Diaminobutyrate transaminase	1 864.03	2 047.36	2 243.04	2 227.50	Up
ectC	Ectoine synthase	3 074.22	3 127.61	3 130.82	3 286.80	Up

Gene

Description of genes or factors

FPKM values of RNA-seq in different groups

Regulate

astA

Arginine N-succinyltransferase

239.65

222.21

330.96

287.26

astB

N-succinylarginine dihydrolase

154.50

162.55

230.63

199.76

astD

Succinylglutamate-semialdehyde dehydrogenase

303.23

298.12

350.58

418.82

astE

Succinylglutamate desuccinylase

414.34

532.87

612.34

458.88

argH

Argininosuccinate lyase

406.67

446.99

531.84

501.08

argE

Acetylornithine deacetylase

371.84

428.38

483.91

428.41

lysC

Aspartate kinase

669.51

792.61

971.45

964.37

asd

Aspartate-semialdehyde dehydrogenase

182.14

172.84

216.33

203.34

ectA

Diaminobutyrate acetyltransferase

604.69

764.54

940.39

925.42

ectB

Diaminobutyrate transaminase

1 864.03

2 047.36

2 243.04

2 227.50

ectC

Ectoine synthase

3 074.22

3 127.61

3 130.82

3 286.80