鼠伤寒沙门菌sRNA SdsR靶基因的预测及验证分析

张家莉; 令狐远凤; 段世宇; 潘永; 杨琦

doi:10.19303/j.issn.1008-0384.2022.004.003

鼠伤寒沙门菌sRNA SdsR靶基因的预测及验证分析

doi: 10.19303/j.issn.1008-0384.2022.004.003

张家莉^{1, 2,},
令狐远凤^{1, 2},
段世宇^{1, 2},
潘永³,
杨琦^{1, 2, 4, ,}

1.
贵州大学动物科学学院，贵州　贵阳　550025
2.
贵州大学动物疫病研究所，贵州　贵阳　550025
3.
贵州省农业科学院畜牧兽医研究所，贵州贵阳　550025
4.
贵州省动物疫病研究室，贵州　贵阳　550025

基金项目: 国家自然科学基金项目（31760740、31602065）

详细信息

作者简介:
张家莉（1998−），女，硕士研究生，研究方向：动物微生物（ E-mail：1142153482@qq.com）

通讯作者:
杨琦（1983−），男，博士，副教授，研究方向：动物微生物（ E-mail：yangqinmg@163.com）

中图分类号: S 852.61
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出版历程
- 收稿日期: 2021-12-02
- 修回日期: 2022-03-09
- 网络出版日期: 2022-06-19
- 刊出日期: 2022-04-28

Prediction and Verification of sRNA SdsR Target Genes in Salmonella typhimurium

ZHANG Jiali^{1, 2
,},
LINGHU Yuanfeng^{1, 2},
DUAN Shiyu^{1, 2},
PAN Yong³,
YANG Qi^{1, 2, 4
, ,}

1.
College of Animal Science, Guizhou University, Guiyang, Guizhou　550025, China
2.
Institute of Animal Diseases, Guizhou University, Guiyang, Guizhou 550025, China
3.
Institute of Animal Husbandry and Veterinary Medicine, Guizhou Academy of Agricultural Science, Guiyang, Guizhou 550025, China
4.
Guizhou Animal Disease Laboratory， Guiyang, Guizhou　550025, China

摘要

摘要: 目的筛选鼠伤寒沙门菌sRNA SdsR的靶基因，为明确sRNA与靶基因的互作以及沙门菌的致病机制奠定基础。方法利用TargetRNA2软件预测sRNA SdsR的靶标，基于前期的sRNA SdsR敲除后转录组测序结果，将预测到的基因注释到GO、KEGG以及eggNOG数据库中进行分析，通过RT-qPCR对预测到的杂交能量较高的部分靶基因进行验证。结果 TargetRNA2软件预测到29个靶标，其中hemA、STM0951、mreC、STM1252、dcoC的杂交能量较高，且能与sRNA SdsR有连续的碱基匹配，分别与鼠伤寒沙门菌血红素合成、氧化还原过程、草酰乙酸脱羧酶的合成、膜的组成部分、细胞质蛋白的合成有关。RT-qPCR结果显示，相对于野生菌株3409，敲除sRNA SdsR后hemA、mreC的表达分别下调0.70和0.39倍；STM0951、STM1252、dcoC的表达分别上调0.51、0.35和1.86倍。结论 hemA、 STM0951 、mreC、STM1252 、dcoC基因很可能受sRNA SdsR的直接调控且对某些靶基因的表达有促进作用。
- 鼠伤寒沙门菌 /
- sRNA SdsR /
- TargetRNA2 /
- 靶基因 /
- 转录组测序
Abstract: Objective Target genes of Salmonella Typhimurium sRNA SdsR were investigated to further understand the interactions between the sRNA and the target genes as well as the pathogenic mechanism of S. typhimurium. Method The TargetRNA2 software was used to predict the target of sRNA SdsR in the pathogen. According to the results obtained in a previous sRNA SdsR knock-out transcriptome sequencing study, the predicted genes were annotated into GO, KEGG, and eggNOG databases for analysis. Those with high hybridization energy were further verified by RT-qPCR. Result There were 29 targets predicted by TargetRNA2. Among them, hemA, STM0951, mreC, STM1252, and dcoC showed high hybridization energy with a possibility of having a continuous base to match the sRNA SdsR. They might be associated with the heme synthesis, redox process, oxaloacetate decarboxylase synthesis, and membrane components and cytoplasmic protein synthesis in S. typhimurium. The RT-qPCR showed, after sRNA SdsR knockout, hemA to be downregulated by 0.70 times and mreC 0.39 times, while STM0951, STM1252, and dcoC upregulated by 0.51, 0.35 and 1.86 times, respectively, over the wild strain 3409. Conclusion It appeared that the genes identified in this study, including hemA, STM0951, mreC, STM1252 and dcoC, could directly be regulated by the sRNA SdsR and might affect the expressions of some target genes.
- Salmonella Typhimurium /
- sRNA SdsR /
- TargetRNA2 /
- target gene /
- transcriptome sequencing

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图 1 RT-qPCR验证结果

Figure 1. Results of RT-qPCR verification

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表 1 引物序列

Table 1. Primer sequence

引物名称 Primer name	序列（5′-3′） Sequence (5′-3′)	预扩增片段长度 Pre-amplified fragment length/bp
hemA-F	ACGAATCTGCTCCGACTGAC	200
hemA-R	GCAAACTGGCGAACGCTTAT	200
STM0951-F	ACATTGAGCGGCGAGAAAGT	116
STM0951-R	ACCCACGATGTTATCCCGAC	116
mreC-F	ACAACAGCAGCAGATAGCGT	173
mreC-R	CGATATTCGCGTTGGCGATG	173
STM1252-F	ATGGCGAATCATGGCTACCG	85
STM1252-R	ATGCCGCCCTCGCTAATAAT	85
dcoC-F	CGGTGCTATTAGGTGAAGGCT	87
dcoC-R	GGATGGCGAAAATCAGCAGG	87
GAPDH-F	CCCAGATGGGATTAGCTAGTTG	133
GAPDH-R	ATTCCCCACTGCTGCCTCCCGT	133

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表 2 TargetRNA2软件预测结果

Table 2. Prediction by TargetRNA2

排名 Rank	基因ID Gene ID	符号 Symbol	能量 Energy	P值 P value
1	STM1777	hemA	−16.51	0.000
2	STM0951	—	−15.39	0.000
3	STM3373	mreC	−14.91	0.001
4	STM1252	—	−12.84	0.004
5	STM0766	dcoC	−11.91	0.008
6	STM3127	—	−11.74	0.009
7	STM4500	yjhP	−11.69	0.009
8	STM0715	—	−11.03	0.013
9	STM1088	pipB	−10.89	0.015
10	STM4583	trpR	−10.75	0.016
11	STM4599	yjjY	−10.68	0.016
12	STM1625	ydcI	−10.52	0.018
13	STM0691	—	−10.52	0.018
14	STM3543	gntR	−10.2	0.021
15	STM0317	gpt	−10.2	0.021
16	STM3533	—	−10.1	0.022
17	STM0246	metI	−10.08	0.022
18	STM1445	slyB	−9.89	0.025
19	STM0720	—	−9.7	0.027
20	STM4520	—	−9.67	0.027
21	STM3888	yieP	−9.66	0.027
22	STM2193	folE	−9.22	0.034
23	STM1440	sodC	−9.2	0.034
24	STM4089	menG	−9.14	0.035
25	STM0529	fdrA	−8.69	0.042
26	STM3203	ygiM	−8.66	0.043
27	STM3803	yidF	−8.54	0.045
28	STM3563	livH	−8.52	0.046
29	STM0051	rihC	−8.43	0.047

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表 3 靶位点预测结果

Table 3. Predicted target sites

mRNA	功能　　　 Function	靶位点预测 Target site prediction
hemA	谷氨酰-tRNA还原酶 Glutamyl-tRNA reductase	SdsR	59
hemA	谷氨酰-tRNA还原酶 Glutamyl-tRNA reductase	hemA	20
STM0951	细胞质蛋白 Cytoplasmic protein	SdsR	61
STM0951	细胞质蛋白 Cytoplasmic protein	STM0951	−65
mreC	棒状决定蛋白质 MreC Rod shape-determining protein MreC	SdsR	63
mreC	棒状决定蛋白质 MreC Rod shape-determining protein MreC	mreC	20
STM1252	细胞质蛋白 Cytoplasmic protein	SdsR	58
STM1252	细胞质蛋白 Cytoplasmic protein	STM1252	19
dcoC	草酰乙酸脱羧酶γ亚基 Oxaloacetate decarboxylase subunit gamma	SdsR	75
dcoC	草酰乙酸脱羧酶γ亚基 Oxaloacetate decarboxylase subunit gamma	dcoC	−45

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表 4 预测靶基因GO、KEGG和eggNOG数据库注释结果

Table 4. Annotation of predicted target genes by GO, KEGG, and eggNOG databases

基因ID Gene ID	长度 Length/bp	log₂变化倍数 log₂FC	符号 Symbol	GO富集分析 GO	KEGG富集分析 KEGG	直系同源蛋白分组比对 eggNOG	eggNOG 蛋白功能分类 eggNOG Class
STM1777	1276	—0.236124007	hemA	生物过程（原卟啉原IX生物合成过程、氧化还原过程）；细胞成分（−）；分子功能（槲皮素2,3-双加氧酶活性）；	卟啉与叶绿素代谢	proNOG01837	H：辅酶转运与代谢
STM0951	868	0.509384983	STM0951	生物过程（氧化还原过程）；细胞成分（−）；分子功能（槲皮素2,3-双加氧酶活性）；	—	proNOG03931	R：仅适用于一般功能预测
STM3373	1069	—0.391500147	merC	生物过程（细胞形状的调节）；细胞成分（膜的组成部分；质膜）；分子功能（−）；	—	COG1792; proNOG06657	M：细胞壁/ 膜/包膜生物发生
STM1252	1049	0.346239576	STM1252	生物过程（−）；细胞成分（−）；分子功能（−）；	—	proNOG13117	G：碳水化合物运输和代谢
STM0766	245	1.855584795	dcoC	生物过程（脂质代谢过程）；细胞成分（质膜；膜的组成部分）；分子功能（钠离子跨膜转运蛋白活性、草酰乙酸脱羧酶活性、水解酶活性）；	丙酮酸代谢	proNOG71341	C：能源生产和转换

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