维氏气单胞菌新型sRNA N155的鉴定及功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2020-1506

摘要/Abstract

摘要：

维氏气单胞菌（Aeromonas veronii,A.veronii）是一种新型病原体,既能感染鱼类,又可以感染包括人在内的哺乳动物,其致病机理尚不完全清楚。基于A.veronii C4野生型菌株转录组数据一个新的sRNA N155被鉴定,进一步利用同源重组等分子生物学手段构建了sRNA N155的缺失菌株。转录组数据和RT-qPCR分析结果表明,sRNA N155独立转录。序列比对分析发现,sRNA N155具有物种特异性,仅存在于气单胞菌属中。生长曲线测定和运动性实验结果表明,sRNA N155的敲除不影响A.veronii生长,但会导致A.veronii运动能力下降。本研究成功构建的ΔsRNA N155敲除菌株,为进一步研究sRNA N155在A.veronii中的功能提供了可靠材料。

关键词: 维氏气单胞菌, 转录组, sRNA, 运动性

Abstract:

Aeromonas veronii is a new type of pathogen that can infect both fish and mammals including humans. and its pathogenic mechanism is not fully understood. A new sRNA N155 in wild type A. veronii C4 was identified based on the transcriptome data,and molecular biological methods such as homologous recombination were used to construct the sRNA N155 deletion strain. The results of transcriptome data and RT-qPCR analysis showed that sRNA N155 was independently transcribed. Sequence alignment analysis indicated that sRNA N155 was species-specific and only existed in Aeromonas. The results of growth curve determination and swimming motility experiments revealed that the knockout of sRNA N155 didn’t affect the growth of A.veronii,but caused the swimming motility of A. veronii reduced. The ΔsRNA N155 strain successfully constructed in this study provides a reliable material for further research on the function of sRNA N155 in A. veronii.

Key words: Aeromonas veronii, transcriptome, sRNA, swimming motility

何金花, 马香, 唐燕琼, 王丹, 李宏, 刘柱. 维氏气单胞菌新型sRNA N155的鉴定及功能研究[J]. 生物技术通报, 2021, 37(11): 267-275.

HE Jin-hua, MA Xiang, TANG Yan-qiong, WANG Dan, LI Hong, LIU Zhu. Identification and Function Study of a New Type of sRNA N155 from Aeromonas veronii[J]. Biotechnology Bulletin, 2021, 37(11): 267-275.

图/表 14

表1 本实验所用菌株、质粒

Table 1 Strains,plasmids used in this study

菌株/质粒 Strain/plasmid	相关属性 Related attributes	来源 Sources
A.veronii C4	野生型菌株,具有氨苄青霉素抗性 Wild type strain,having ampicillin resistance	本实验室 Our lab stock
A.veronii Δhfq	hfq基因敲除型菌株,具有氨苄青霉素抗性 Gene hfq-deleted strain,having ampicillin resistance	本实验室 Our lab stock
A.veronii Δhfq-pBBR-hfq	hfq基因回补菌株,具有氨苄青霉素和卡纳霉素双抗性 Gene hfq-complemented strain,having ampicillin and kanamycin resistances	本研究 This study
A.veronii ΔsRNA N155	sRNA N155敲除型菌株,具有氨苄青霉素抗性 Gene sRNA N155-deleted strain,having ampicillin resistance	本研究 This study
Escherichia coli WM3064	二氨基庚二酸（DAP）营养缺陷型菌株 Diaminopimelic acid（DAP）auxotrophic strain	本实验室 Our lab stock
pBBR-MCS-2	穿梭质粒,具有卡纳霉素抗性 Broad-host-range cloning vector,having kanamycin resistance	本实验室 Our lab stock
pBBR-hfq	pBBR-MCS-2的衍生质粒,包含hfq的完整ORF区域 Derivatives from pBBR-MCS-2,containing full ORF region of hfq	本研究 This study
pRE112	基因敲除质粒,自杀性质粒,表达了用于蔗糖选择的sacB基因,具有氯霉素抗性 Plasmid for gene knock out,suicide plasmid,expressing the sacB gene for sucrose selection,having chloramphenicol resistance	本实验室 Our lab stock
pRE112-ΔsRNA N155	sRNA N155敲除重组质粒 sRNA N155 knockout recombinant plasmid	本研究 This study

表2 本实验所用引物序列

Table 2 Primer sequence used in this study

引物 Primer	引物序列Primer sequence（5'-3'）	注释 Note
F0	GAGCTGGTCTTTATGCGCC	验证sRNA N155敲除 Validation of sRNA N155 knockout
R0	GGAAGAAAAAGCTGACGAAG	验证sRNA N155敲除 Validation of sRNA N155 knockout
F1	CGAGCTCCCTGTCTATTGGCACTGC	扩增sRNA N155上游同源序列 Amplification of sRNA N155 upstream homologous sequence
R1	CCGGAATTCGAACAGGTTGCAGAAGTC
F2	CGGAATTCTTTACTGCGCAGTGTTGAATC	扩增sRNA N155下游同源序列 Amplification of sRNA N155 downstream homologous sequence
R2	CGGGGTACCAATCGCAGCTGTTACAAAG
pRE112 F	ACATAGCCCCACTGTTCGT	验证pRE112载体 Verification of pRE112 vector
pRE112 R	TTTTCGTCTCAGCCAATCC	验证pRE112载体 Verification of pRE112 vector
GyrB F	TGGTTGTGGTATCGGTCGTG	内参基因引物 Primers for reference gene
GyrB R	CTGTTCCTGCTTGCCTTT	内参基因引物 Primers for reference gene
RT-F	TTATCCGGTAAGGAGACG	sRNA N155 RT-qPCR引物 sRNA N155 RT-qPCR primers
RT-R	CACGCGGTAAAAGAACAC	sRNA N155 RT-qPCR引物 sRNA N155 RT-qPCR primers

图1 sRNA长度分布图

Fig. 1 Length distribution of sRNA

图2 A.veronii C4野生型中sRNA的表达

Fig. 2 Expressions of sRNA in A.veronii C4 wild type

图3 sRNA N155的二级结构

Fig. 3 Secondary structure of sRNA N155

图4 sRNA N155及其上下游基因的表达量

Fig. 4 Expressions of sRNA N155 and its upstream and downstream genes

图5 sRNA N155的RT-qPCR分析

Fig. 5 RT-qPCR analysis of sRNA N155

图6 cDNA琼脂糖凝胶电泳 M:DL5000 DNA marker;1-2为WT+pACYC的大肠杆菌内参基因GyrB的条带;3-4为WT+ pACYCDuet-1∷psRNAN155-sRNA N155的内参基因GyrB的条带;5-6为WT+pACYC的sRNA N155的RT-qPCR产物;7-8为WT+ pACYCDuet-1∷psRNAN155-sRNA N155的sRNA N155的RT-qPCR产物,约110 bp

Fig. 6 cDNA agarose gel electrophoresis M:DL5000 DNA marker. 1-2 are the bands of internal reference gene GyrB of Escherichia coli of WT+pACYC. 3-4 are the bands of internal reference gene GyrB of WT+ pACYCDuet-1∷psRNAN155-sRNA N155. 5-6 are RT products of sRNA N155 of WT+pACYC. 7-8 are RT products of sRNA N155 of WT+pACYCDuet-1∷psRNAN155-sRNA N155,about 110 bp

图7 sRNA N155基因多序列比对

Fig. 7 Multiple sequence alignment of sRNA N155 gene

图8 sRNA N155基因敲除载体pRE112-ΔsRNA N155的PCR验证 M:DL5000 DNA marker;1为阴性对照;2为阳性对照;3-8为pRE112-ΔsRNA N155基因敲除质粒PCR产物,目的条带约1 200 bp

Fig. 8 PCR verification of sRNA N155 gene knockout vector pRE112-ΔsRNA N155 M:DL5000 DNA marker. 1 is a negative control. 2 is a positive control. 3-8 are plasmid PCR products of pRE112-ΔsRNA N155 gene knockout,the target band is about 1 200 bp

图9 22%蔗糖板筛选 M:DL5000 DNA marker;1:A.veronii C4基因组阳性对照;22:pRE112质粒阳性对照;2-13:敲除菌株验证,其中2-3、10-11与预期条带相符,均低于野生型;14-21为pRE112特异性引物验证,表示pRE112质粒成功丢失

Fig. 9 22% sucrose plate screening M:DL5000 DNA marker. 1:A. veronii C4 genome positive control. 22:pRE112 plasmid positive control. 2-13:Knockout strain verification,where 2-3,10-11 are the same as expected,all are lower than the wild type. 14-21 are pRE112-specific primer verification,indicating that the pRE112 plasmid has been successfully lost

图10 A.veronii sRNA N155敲除菌株PCR验证 M:DL5000 DNA marker;1:A.veronii C4基因组阳性对照;2-5:sRNA N155敲除菌株PCR验证

Fig. 10 PCR verification of A.veronii sRNA N155 knockout strain M:DL5000 DNA marker. 1:A.veronii C4 genome positive control. 2-5:sRNA N155 knockout strain PCR verification

图11 A.veronii C4野生型及ΔsRNA N155生长曲线测定

Fig. 11 Determination of the growth curve of A.veronii C4 wild type and ΔsRNA N155

图12 敲除sRNA N155基因对细菌运动性的影响 1:A.veronii C4野生型菌株;2:Δhfq敲除株;3:ΔsRNA N155敲除株;4:Δhfq-pBBR-hfq回补株

Fig. 12 Effect of knocking out the sRNA N155 gene on bacterial motility 1:A.veronii C4 wild-type strain. 2:Δhfq knockout strain. 3:ΔsRNA N155 knockout strain. 4:Δhfq-pBBR-hfq complement strain

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