黏质沙雷氏菌灵菌红素合成基因簇异源表达及其潜在的温度调控机制

刘方晨; 贾宪波; 吴良泉; 方宇; 赵恪; 林俊杰; 陈龙军; 张慧; 林陈强; 陈济琛

doi:10.19303/j.issn.1008-0384.2021.03.013

黏质沙雷氏菌灵菌红素合成基因簇异源表达及其潜在的温度调控机制

doi: 10.19303/j.issn.1008-0384.2021.03.013

1.
福建农林大学资源与环境学院，福建　福州　350002
2.
福建省农业科学院土壤肥料研究所，福建　福州　350003
3.
福建农林大学生命科学学院，福建　福州　350002

基金项目: 国家自然科学基金青年基金项目（31800068）；福建省农业科学院国家自然科学基金项目延伸研究专项计划（AGY2018-1）；福建省科技厅区域发展项目（2019Y3005）

详细信息

作者简介:
刘方晨（1996−）男，硕士，研究方向：环境微生物（E-mail：liufc1996@163.com）

通讯作者:
贾宪波（1986−）男，博士，助理研究员，研究方向：环境微生物（E-mail：xbj2011@163.com）

中图分类号: S 182
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出版历程
- 收稿日期: 2020-11-24
- 修回日期: 2021-01-28
- 网络出版日期: 2021-03-27
- 刊出日期: 2021-03-31

Heterologous Expression and Temperature Regulation of Prodigiosin-synthesis Gene Cluster in Serratia marcecens

1.
College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou, Fujian 　350002, China
2.
Institute of Soil and Fertilizer, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 　350003, China
3.
College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian 　350002, China

摘要

摘要: 目的构建黏质沙雷氏菌FZSF02灵菌红素合成基因簇的异源表达菌株，探究其合成灵菌红素可能的温度调控机制。方法构建3种携带不同启动子的异源表达大肠杆菌菌株，并利用实时荧光定量PCR（RT-qPCR）检测FZSF02中pig基因簇pigA、pigF、pigN基因在37 ℃与28 ℃培养条件下的转录水平差异。结果在28 ℃低温条件下，3种不同的启动子均能在大肠杆菌中启动各自重组质粒所携带的pig基因簇表达并合成灵菌红素，其中携带T7强启动子的重组菌株经IPTG诱导后可以在37 ℃少量合成色素，其余菌株37 ℃不能合成色素。RT-qPCR结果显示在黏质沙雷氏菌中上述基因转录水平在37 ℃条件下较28 ℃出现下调。结论黏质沙雷氏菌FZSF02之所以能够在28 ℃合成灵菌红素而在37 ℃无法合成灵菌红素，一方面由于pig基因簇的编码基因转录水平下调，另一方面是由基因簇编码的合成灵菌红素所需前体MBC和MAP的一种或者几种关键酶在相对较高的温度（37 ℃）条件下活性受到抑制导致。
- 异源表达 /
- 实时荧光定量PCR /
- 温度调控 /
- 启动子 /
- 黏质沙雷氏菌
Abstract: Objective Strains of Serratia marcescens FZSF02 that had heterologous expression and could produce prodigiosin with relevant synthesis gene cluster were constructed to study the mechanism relating to temperature regulation in the biological process. Methods Three Escherichia coli strains with heterologous expression carrying different promoters were constructed. Real-time fluorescent quantitative PCR (RT-qPCR) was used to determine the transcriptional differences of pigA, pigF and pigN in the pig gene cluster at 37 ℃ and 28 ℃. Results At 28 ℃, all 3 promoters initiated the expression of the pig gene cluster in E. coli, and prodigiosin was produced in the 3 recombinant strains. In contrast, only the recombinant strain carrying the strong T7 promoter delivered a small amount of the pigment after IPTG induction at 37 ℃. The RT-qPCR results showed the transcription levels of the above genes in S. marcescens FZSF02 were down-regulated at 37 ℃ in comparison to 28 ℃. Conclusion The reason why S. marcescens FZSF02 was able to produce prodigiosin at 28 ℃ but failed at 37 ℃ might be, at higher temperatures, that the pig gene cluster was down-regulated and/or that the activities of one or several key enzymes encoded by the pig genes for the biosynthesis of precursors MBC and MAP were somehow inhibited.
- heterologous expression /
- real-time fluorescent quantitative PCR /
- temperature regulation /
- promoter /
- Serratia marcescens

HTML全文

图 1 pig基因簇、pig基因簇和pig启动子序列以及3种重组质粒所连片段的验证PCR扩增结果

注：M：DNA maker。A：1，pig基因簇；2，pig基因簇启动子与pig基因簇。B：（1，2）M13引物PCR扩增产物；3，T7引物PCR扩增产物。

Figure 1. PCR amplification results on pig gene cluster, pig gene cluster and pig promoter, and validation on sequences of 3 recombinant plasmid segments

Note：M: DNA maker. A: pig gene cluster (1); pig gene cluster and pig promoter (2). B: M13 primer PCR amplification product (1 and 2); T7 primer PCR amplification product (3).

下载: 全尺寸图片幻灯片

图 2 3种重组质粒的构建过程

注：A: pTOPO-Blunt Simple-ppPig重组质粒；B: pEASY®-Blunt E2-Pig重组质粒；C: pTOPO-Blunt Simple-kpPig重组质粒。

Figure 2. Construction of 3 recombinant plasmids

Note：A: pTOPO-Blunt Simple-ppPig recombinant plasmid. B: pEASY^®-Blunt E2-Pig recombinant plasmid. C: pTOPO-Blunt Simple-kpPig recombinant plasmid.

下载: 全尺寸图片幻灯片

图 3 3种重组菌株在28 ℃和37 ℃培养条件下灵菌红素的合成情况

注：A：28 ℃培养pTOPO-ppPig重组菌株；B：28 ℃培养pTOPO-kpPig重组菌株；C：28 ℃培养pEASY-pig重组菌株；D：37 ℃培养pTOPO-ppPig重组菌株；E：37 ℃培养pTOPO-kpPig重组菌株；F：37 ℃培养pEASY-pig重组株。

Figure 3. Prodigiosin synthesis of 3 recombinant strains cultured at 28 ℃ and 37 ℃

Note: A: pTOPO-ppPig recombinant strains at 28 ℃. B: pTOPO-kpPig recombinant strains at 28 ℃. C: pEASY-pig recombinant strains at 28 ℃. D: pTOPO-ppPig recombinant strains at 37 ℃. E: pTOPO-kpPig recombinant strains at 37 ℃. F: pEASY-pig recombinant strains at 37 ℃.

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图 4 粘质沙雷氏菌在不同温度条件下所选基因的转录水平倍数变化

Figure 4. Expressions of selected genes of S. marcecens at different temperatures

下载: 全尺寸图片幻灯片

表 1 供试菌株和质粒

Table 1. Bacterial strains and plasmids used in this study

细菌 Bacterial strains	菌株 Strain	参考/来源 Reference/source
Serratia marcescens	FZSF02（WT）	This work（本实验）
E.coli	DH5α	This work（本实验）
	BL21（DE3）	This work（本实验）

质粒 Plasmids	注释 Note	参考/来源 Reference/source
pTOPO	CV22-Zero Background pTOPO-Blunt Simple Cloning Kit	Purchased from Aidlab（购自艾伯莱生物公司）
pEASY	pEASY^®- Blunt E2 Expression Kit	Purchase from TransGen Biotech （购自全式金生物公司）
pTOPO-kan	Used for the amplification of universal promoter and Kan resistance genes	This work（本实验）
pTOPO-ppPig	A pTOPO based plasmid containing the pig cluster and pig promoter	This work（本实验）
pEASY-Pig	A pEASY based plasmid containing the pig cluster	This work（本实验）
pTOPO-kpPig	A pTOPO based plasmid containing the pig cluster and Kan promoter	This work（本实验）

下载: 导出CSV

表 2 供试引物

Table 2. Primers used in this study

引物 Primers	序列 Sequences（5′→3′）	用途 purpose
ExpF	ATGGATTTTAACTTATCAAGCGA	Clone a pig gene cluster（克隆pig基簇）
ExpR	TTACAGCACGAAAGGAATGAAACA
CluspigF	TTTTTCCTCCGGAATGCTCCTGC	Clone pig gene clusters and pig promoter （克隆pig基因簇及其启动子）
CluspigR	TTACAGCACGAAAGGAATGAAAC	Clone pig gene clusters and pig promoter （克隆pig基因簇及其启动子）
KanproF2	ATTCCTTTCGTGCTGTAATCAGAA
	TTGGTTAATTGGTTGTA	Linearize the attached pTOPO
KanproR2	TGATAAGTTAAAATCCATAACACC	vector and Kan promoter （线性化pTOPO载体和Kan启动子）
	CCTTGTATTACTGTTA
M13F	TGTAAACGACGGCCAGT	Check the size of the gene attached to the pTOPO vector （验证pTOPO载体所连接的基因大小）
M13R	CAGCAAACAGCTATGACC
16SF	CGTTACTCGCAGAAGAAGCA	Used for qPCR amplification（用于qPCR扩增）
16SR	TCACCGCTACACCTGGAA
PigAF	CGCCATCTTCCACGATTCAA	Used for qPCR amplification（用于qPCR扩增）
PigAR	CATTAGCCGACACTGTTCCA
PigFF	CACGGTATTCGGCGATGAC	Used for qPCR amplification（用于qPCR扩增）
PigFR	CACGGTGTTGCGAGAAGT
PigNF	CGGTTACCCTGGTCTATTG	Used for qPCR amplification（用于qPCR扩增）
PigNR	TGTCAGCACGATGTTCAT

下载: 导出CSV

表 3 菌株在535 nm下的吸光度

Table 3. Absorbance at 535 nm of recombinant strains

菌株 strains	吸光度 Absorbance
菌株 strains	28 ℃	37 ℃
DH5α（pTOPO-ppPig）	0.435	0.002
DH5α（pTOPO-kpPig）	0.468	0.001
DH5α（pEASY-Pig）	0.394	0.026
FZSF02	1.056	0.001

下载: 导出CSV

参考文献(20)

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