铜胁迫下的西瓜食酸菌转录组分析

doi:10.6048/j.issn.1001-4330.2023.02.016

摘要/Abstract

摘要：

【目的】 分析西瓜食酸菌(Acidovorax citrulli)在铜胁迫下转录水平的响应特征,研究对其抗铜系统和抗铜机制,为进一步解析西瓜食酸菌的铜耐受分子机制积累数据。【方法】 以西瓜食酸菌亚群I的抗铜菌株FC440铜胁迫和无胁迫培养下的菌体为材料,提取RNA并利用RNA-Seq技术获取细菌对铜胁迫的应答数据,分析数据质量、差异表达基因的类型、KEGG富集与Pathway注释、GO富集分析等转录组水平特征,并进行qPCR验证。【结果】 共获得6个转录组文库,总数据量23.6 Gb;该细菌共有4 344个基因表达,其中有466个基因差异表达(上调266个,下调200个);差异表达的基因显著富集于ABC转运系统的跨膜转运、双组分系统通路的信号转导以及氨基酸代谢等约9大通路;基因表达趋势与转录组测序数据的趋势一致。【结论】 铜胁迫下谷氨酸、谷胱甘肽等氨基酸代谢在西瓜食酸菌抗铜胁迫中发挥重要作用;双组分系统参与西瓜食酸菌对铜离子胁迫的响应且受到负调控;该菌中可能存在包含ABC转运体的跨膜转运、氧化还原反应、胞内束缚沉淀等多种系统互作的抗铜机制。

关键词: 西瓜食酸菌; 铜胁迫; 转录组; 差异表达基因

Abstract:

【Objective】 The analysis of Acidovorax citrulli (Ac) to copper stress on transcriptome level response characteristics is helpful to promote the study of copper resistance system and mechanism The results of this study have accumulated basic data for further analysis of the molecular mechanism of copper tolerance of Ac. 【Method】 The copper-resistant FC440 strain of subgroup I of Ac was cultured under copper stress and without copper stress.Then FC440 were collected for RNA-seq to analyze data quality, types of differentially expressed genes (DEGs), KEGG enrichment and Pathway annotation, GO enrichment analysis and other transcriptome features, and verify qPCR. 【Results】 The results showed that 6 FC440 transcriptomic libraries were obtained and total data size was 23.6 Gb; Gene expression analysis showed that the bacteria had 4,344 gene expressions and 466 DEGs were identified (266 up-regulated, 200 down-regulated) ; DEGs were significantly enriched in about 9 major pathways such as transmembrane transport of the ABC transport system, signal transduction of the two-component system pathway, and amino acid metabolism; qPCR analysis results showed that the gene expression trend was consistent with the trend of transcriptome sequencing data. 【Conclusion】 The amino acid metabolism of glutamate and glutathione under copper stress plays an important role in the resistance to copper stress of Ac; the two-component systems participate in the response of Ac to copper ion stress and is negatively affected.There may be anti-copper mechanisms in this bacteria including transmembrane transport of ABC transporters, redox reactions, and intracellular binding precipitation.

Key words: Acidovorax citrulli; copper stress; transcriptome; differential expressed genes

中图分类号:

S651
S652.1

宋金迪, 刘君, 孙玉芳, 优丽图孜·乃比, 陈宝强, 颉兵兵. 铜胁迫下的西瓜食酸菌转录组分析[J]. 新疆农业科学, 2023, 60(2): 389-398.

SONG Jindi, LIU Jun, SUN Yufang, Youlituzi Naibi, CHEN Baoqiang, XIE Bingbing. Transcriptome Analysis of Acidovorax citrulli against Copper Stress[J]. Xinjiang Agricultural Sciences, 2023, 60(2): 389-398.

图/表 11

表1 转录组数据qPCR验证分析所用引物

Table 1 Primers used for validation qPCR of transcriptome data

基因ID Gene ID	本地数据库基因ID Local database gene ID	正向引物(5'~3') Forward primer	反向引物(5'~3') Reverse primer
NMDCN0000J7Q	GE00642	GCGTGACCGCCATCCAG	CCTTGCGGGCGATGATG
NMDCN0000J7N	GE00282	AACAACTACCAGGTGGGACT	GGTGAGCTGCACGGATT
NMDCN0000J7P	GE01538	AGCCTCTTGCCATTGCC	CACCACCTGGGTATCGC
NMDCN0000J7O	GE02901	CCCTCCGCCAGAACAAT	TCCACCGACACGATGATG
NMDCN0000J7M	GE00281	GCGCTACAACGGCTACC	GCCAGGCAGAGGAACAC
NMDCN0000J7L	GE01342	TGCCGTGACGACTGCTT	GCGTATTGTTGGCCTTGT
NMDCN0000J7K	GE04032	AACAACTACCAGGTGGGACT	GGTGAGCTGCACGGATT
NMDCN0000J7J	GE01848	CGCCTATCCGTCCAAGC	AGCACCGTGTAGCCGTC
NMDCN0000J7I	GE02981	CGCCGTCCCTCCCGTTT	GCCAGGATCGTCCCCAG
NMDCN0000J7H	GE01751	GTCAACCTCTGGGGCTACA	AACTCGTACACGAAGGTCTTG
NMDCN0000J7G	GE01955	GAACCTGGAAGTCATCATCG	CCTTGGTCTTGTTGTCGG
NMDCN0000Q79	GE04262	GGCTGGCTGGGTCATAC	ATTGTAGATTTCGCCGTTG
NMDCN0000Q7A	GE02797	CACAAGACTGTTTCTGTTCCAAG	GCACGTATGTCCGTCACCT
NMDCN0000Q7B	GE00664	CGTCCCGTATCGCCGTCA	GCGTGATGCCCGTGAGC
NMDCN0000Q7C	GE01086	GCTGCTGTCGGAAATGG	TTGTTGGTGCGGAAGTTAC
rpoB	-	GCGACAGCGTGCTCAAAGTG	GGCCTTCGTTGGTGCGTTTCT

表2 铜胁迫处理下FC440菌株转录本的碱基质量统计

Table 2 Base mass statistics of the transcript of Acidovorax citrulli FC440 under copper stress

样品名 Sample	Reads总数 Total reads	碱基总数 Bases(bp)	N (%)	Q20 (%)	Q30 (%)
CK₁	29 540 900	4 460 675 900	0.04	96.90	92.01
CK₂	31 451 022	4 749 104 322	0.04	96.80	91.82
CK₃	27 676 892	4 179 210 692	0.02	97.09	92.40
T₁	29 357 120	4 432 925 120	0.02	96.93	92.02
T₂	27 027 648	4 081 174 848	0.01	96.99	92.14
T₃	27 104 738	4 092 815 438	0.02	97.04	92.24

表3 FC440菌株的参考基因组比对统计

Table 3 Reference genome comparison of Acidovorax citrulli FC440

样品名 Sample	可用读取 Useful Reads	总映射读取 Total Mapped Reads	占比 Proportion (%)	单映射读取 Uniquely Mapped Reads	占比 Proportion (%)	多映射读取 Multiple Mapped Reads	占比 Proportion (%)
CK₁	29 395 754	29 311 584	99.71	28 350 407	96.72	961 177	3.28
CK₂	31 279 164	31 182 372	99.69	30 036 408	96.32	1 145 964	3.68
CK₃	27 553 118	27 468 703	99.69	26 767 147	97.45	701 556	2.55
T₁	29 214 302	29 096 642	99.60	28 357 073	97.46	739 569	2.54
T₂	26 898 484	26 777 059	99.55	26 086 501	97.42	690 558	2.58
T₃	26 987 212	26 892 876	99.65	26 195 932	97.41	696 944	2.59

图1 FC440菌株铜胁迫处理下的相关性检验

Fig.1 Correlation test between samples of Acidovorax citrulli FC440 under copper stress

图2 FC440菌株铜胁迫处理下的PCA变化

Fig.2 PCA analysis between samples of Acidovorax citrulli FC440 under copper stress

图3 FC440菌株铜胁迫处理下的差异表达基因聚类热图注:横向表示基因,每一列为一个样本,红色表示高表达基因,绿色表示低表达基因

Fig.3 Clustering heat map of differentially expressed genes between samples of Acidovorax citrulli FC440 under copper stress Note: Horizontal represents the gene, each column is a sample, red represents the high expressed gene, green represents the low expressed gene

图4 FC440菌株铜胁迫处理下的差异表达基因MA变化

Fig.4 MA map of differentially expressed genes in samples of Acidovorax citrulli FC440 under copper stress

图5 FC440菌株铜胁迫处理组下的差异表达基因火山图

Fig.5 Volcanic map of differentially expressed genes in samples of Acidovorax citrulli FC440 under copper stress

图6 FC440菌株铜胁迫处理下的差异表达基因GO功能富集

Fig.6 The GO function of differentially expressed genes in copper stress treated Acidovorax citrulli FC440 sample

图7 KEGG通路显著富集气泡注:纵坐标为 KEGG Pathway 条目;横坐标为Richfactor,图中圆点的大小表示注释到该通路的差异基因的多少,颜色表示该通路的显著性 FDR 值

Fig.7 The KEGG pathway significantly enriched bubble diagram Note: The ordinate is KEGG Pathway entry;The horizontal axis is Richfactor.The size of the dots in the figure indicates the number of differential genes annotated to this pathway, and the color indicates the significant FDR value of this pathway

图8 铜胁迫处理后西瓜食酸菌15个基因相对表达量

Fig.8 The relative expression levels of 15 genes in Acidovorax citrulli FC440 under copper stress

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