普城沙雷氏菌ACCC 02146产灵菌红素调控基因的鉴定

丁霞飞; 贾宪波; 林陈强; 庄振宏; 陈济琛

doi:10.19303/j.issn.1008-0384.2023.04.013

普城沙雷氏菌ACCC 02146产灵菌红素调控基因的鉴定

doi: 10.19303/j.issn.1008-0384.2023.04.013

1.
福建农林大学生命科学学院，福建　福州　350001
2.
福建省农业科学院土壤肥料研究所，福建　福州　350003

基金项目: 福建省科技计划公益类专项（2020R1025002）；福建省农业科学院科技创新团队建设项目（CXTD2021002-3）

详细信息

作者简介:
丁霞飞（1995−），女，硕士，研究方向：生物化学与分子生物学（E-mail：dxf925@163.com）

通讯作者:
陈济琛（1964−），男，研究员，研究方向：农业微生物（E-mail：chenjichen2001@163.com）

中图分类号: Q81
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出版历程
- 收稿日期: 2022-11-10
- 修回日期: 2023-03-01
- 网络出版日期: 2023-05-09
- 刊出日期: 2023-04-28

Prodigiosin-producing Genes in Serratia plymuthica ACCC 02146

1.
College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350001, China
2.
Institute of Soil and Fertilizer, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350003, China

摘要

摘要: 目的鉴定影响普城沙雷氏菌ACCC 02146产灵菌红素能力的基因，构建ACCC 02146的转座子突变体文库，为进一步研究普城沙雷氏菌ACCC 02146灵菌红素合成机制奠定基础。方法采用平板划线法从菌种保藏中心购买菌株和实验室保藏菌株中获得15株产灵菌红素的菌株。采用16S rRNA基因测序法鉴定各菌株，邻接建树将其分类，并比较不同类别菌株灵菌红素合成基因簇启动子序列差异，观察各菌株产色能力。对16S rDNA序列和灵菌红素合成基因簇启动子序列均有异于其他菌株的普城沙雷氏菌ACCC 02146合成灵菌红素的调控基因展开研究。构建ACCC 02146的转座子突变体文库，筛选出产色能力明显改变的克隆子并鉴定出对应的转座子插入突变基因。结果该突变体文库中有74个突变体表现出产灵菌红素能力的变化。其中25个突变体转座子插入发生在pigA、pigB、pigC、pigD和pigH 等5个灵菌红素合成簇基因上，49个突变体转座子插入基因为灵菌红素合成基因簇之外的基因。在鉴定到的产色能力改变的突变体中，麦芽糖O-乙酰基转移酶基因突变菌株有6个，二氢乳清酸脱氢酶基因突变菌株有4个，MarR家族转录因子SlyA基因突变体有3个，winged helix家族的双组分转录调控因子RstA基因突变体有3个，H-醌氧化还原酶亚基I基因突变菌株有3个，NADH-醌氧化还原酶G链基因突变菌株有3个，肽基脯氨酰异构酶B基因突变菌株有3个，其他突变基因对应的克隆子数量为1～2个。结论在沙雷氏菌中，除了灵菌红素合成簇基因调控灵菌红素合成，推测灵菌红素合成簇外编码相关酶、转录调控因子和一些结构蛋白的基因通过直接或间接途径在不同程度上调控了灵菌红素的合成。
- 沙雷氏菌 /
- 灵菌红素 /
- 转座子 /
- 突变体库 /
- 调控基因
Abstract: Objective Genes relating to prodigiosin synthesis in Serratia plymuthica ACCC 02146 were identified, and a transposon mutant library on the strain constructed. Methods Fifteen prodigiosin-producing microbes were obtained from a conservation center and a laboratory. After identification by 16S rRNA gene sequencing, they were classified according to the neighbor-joining trees. Promoter sequences of prodigiosin synthesis gene were analyzed, and color producing capacity of the individual strains evaluated. Selected strain was cloned and further studied to establish a library on the transposon mutants. Results Differed from other strains in terms of 16S rDNA and promoter sequences of the prodigiosin biosynthesis gene clusters, S.plymuthica ACCC 02146 was selected to clone the candidate gene for further investigation. A transposon mutant library was constructed subsequently. In the library, of 74 mutants showing significant variations in prodigiosin-producing ability, 25 had the insertions in pigA, pigB, pigC, pigD, and pigH, while 49 in the genes outside the cluster. On color formation, 6 strains with the mutation on maltose o-acetyltransferase gene, 4 on dihydroorotate dehydrogenase gene, 3 on MarR family of transcription factor SlyA genes, 3 on two-component transcriptional regulator RstA of the winged helix family, 3 on NAD(P)H-quinone oxidoreductase subunit I gene, 3 on NADH-quinone oxidoreductase, chain G gene, 3 on peptidylprolyl isomerase B gene, and one to two on other genes were found possibly related to significant alterations on the prodigiosin production as well. Conclusion Aside from the identified specific clusters of prodigiosin synthesis-associated genes, additional factors in the forms of enzymes, transcriptional regulators, and/or structural proteins were now speculated to also directly or indirectly contribute in varying degrees to the prodigiosin synthesis in Serratia sp.
- Serratia marcescens /
- prodigiosin /
- transposon /
- mutant library /
- regulatory gene

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图 1 菌株系统发育树

Figure 1. Phylogenetic tree of Serratia sp.

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图 2 各菌株灵菌红素合成能力比较

Figure 2. Prodigiosin synthesis capabilities of Serratia sp.

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图 3 3种不同转录调控因子突变体菌落

a：突变体F4-4；b：突变体B4-1；c：突变体H8-1。

Figure 3. Colonies of 3 type transcriptional regulatory factor mutants

a: Mutant F4-4; b: Mutant B4-1; c: Mutant H8-1.

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表 1 供试引物

Table 1. Primes applied

引物 Primes	序列 Sequences（5′-3′）	用途 Purpose	来源 Sources
27F	AGAGTTTGATCC TGGCTCAG	16S rDNA 测序	[23]
1492R	ACGGTTACCTTGTTACGACTT	16S rDNA 测序	[23]
LAD1	ACGATGGACTCCAGAG(G/C/A)N(G/C/A)NNNGGAA	高效热不对称交错PCR	[24]
LAD3	ACGATGGACTCCAGAG(T/A/C)N(A/G/C)NNNCCAC	高效热不对称交错PCR	[24]
AC1	ACGATGGACTCCAGAG	高效热不对称交错PCR	[24]
F389	TCAAGCATTTTATCCGTACTCCTG	高效热不对称交错PCR	[24]
F536	CGGTTGCATTCGATTCCTGTTTGTA	高效热不对称交错PCR	[24]
F772	TAGGTTGTATTGATGTTGGACGAG	高效热不对称交错PCR	[24]
KproF	GCAACACTCCGCAATCTATA	菌株ACCC 02146启动子序列	本研究
KproR	CTCTATCTCCATGAAAGAGT	菌株ACCC 02146启动子序列	本研究

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表 2 菌落形态特征

Table 2. Colony morphology

菌株编号 Strain No.	菌落形态特征 Colony morphology characteristics
FZSF02	圆形凸起，湿润黏稠，红色
S1	圆形凸起，湿润黏稠，红色
S2	圆形凸起，湿润黏稠，红色
S3	圆形凸起，湿润黏稠，红色
CICC 23703	圆形凸起，湿润黏稠，深红色
CICC 23838	圆形凸起，湿润黏稠，红色
CICC 24478	圆形凸起，表面干燥粗糙，红色
CICC 10698	圆形凸起，湿润黏稠，橙红色
CICC 20223	圆形凸起，湿润黏稠，橙色
CICC 24369	圆形凸起，湿润黏稠，深红色
CCTCC AB2014323	圆形凸起，湿润黏稠，深红色
CCTCC AB2015384	圆形凸起，湿润黏稠，红色
ACCC 02146	圆形凸起，湿润黏稠，橙红色
ACCC 01294	圆形凸起，湿润黏稠，深红色
ACCC 04168	圆形凸起，湿润黏稠，深红色

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表 3 突变体产灵菌红素能力及其菌量

Table 3. Prodigiosin synthesis capabilities and quantity of S.plymuthica mutant

基因登录号 Gene accession number	突变体（基因长度/插入位点） Mutant (gene length/insertion site)/bp	功能 Function	OD₆₀₀	OD₆₀₀ 改变倍数 OD₆₀₀ fold change	OD₅₃₅	OD₅₃₅改变倍数 OD₅₃₅ fold change	OD₅₃₅改变倍数/ OD₆₀₀改变倍数 OD₅₃₅ fold change/ OD₆₀₀ fold change
	普城沙雷氏菌 ACCC 02146		2.540	1.000	0.213	1.000	1.000
AEG27540.1	B5-10 (2 673/1 075), B9-2 (2 673/ 1 274), B6-4 (2 673/1 075), B6-6 (2 673/792), B5-6 (2 673/792), B2-11 (2 673/1 266), B5-9 (2 673/153)	利用磷酸烯醇式丙酮酸酶（PigC）	2.880	1.134↑	0.005	0.023↓	0.020
AEG27539.1	B5-1 (2 601/2 116),B8-1 (2 601/2 096), B7-5 (2 601/1 099), B7-6 (2 601/1 099), B7-11 (2 601/1 338), B9-4 (2 601/2 072), B9-5 (2 601/2 071)	假设蛋白（PigD）	2.644	1.041↑	0.005	0.023↓	0.022
AEG27535.1	B7-7 (1 971/1 905), B7-1 (1 971/1 905)	甘氨酸C-乙酰转移酶（PigH）	2.600	1.025↑	0.015	0.070↓	0.068
AEG27542.1	B5-12 (1 161/195), B5-13 (1 161/794), B6-8 (1 161/35), B7-8 (1 161/35), B9-1 (1 161/35)	异戊酰基-辅酶A脱氢酶（PigA）	2.731	1.075↑	0.004	0.019↓	0.02
AEG27541.1	B5-2 (2 031/1 865), B5-11 (2 031/ 1 865), B6-7 (2 031/1 865), B7-12 (1 161/35)	富马酸还原酶/琥珀酸脱氢酶黄蛋白结构域蛋白（PigB）	2.665	1.049↑	0.012	0.056↓	0.053
AEG30298.1	F5-7 (558/202), F5-4 (558/524), F5-10 (558/273), F5-5 (558/518), F5-6 (558/495), F5-9 (558/189)	麦芽糖O-乙酰转移酶	2.618	1.031↑	0.037	0.174↓	0.169
AEG29267.1	H8-8 (978/910), H8-9 (978/262), H9-8 (978/217）	H-醌氧化还原酶亚基1	2.355	0.927↓	0.245	1.150↑	1.241
AEG27501.1	H7-1 (1 011/842), H7-4 (1 011/425), H9-3 (1 011/378), H9-6 (1 011/234)	二氢乳清酸脱氢酶	1.967	0.774↓	0.589	2.765↑	3.572
AEG29268.1	H9-8 (2 748/217), H7-5 (2 748/2 682), H7-9 (2 748/2 041)	NADH-醌氧化还原酶G链	2.216	0.872↓	0.243	1.140↑	1.307
AEG26904.1	B9-3 (495/290), B7-2 (495/290), B7-3 (495/290)	肽基脯氨酰异构酶B	3.827	1.507↑	0.008	0.038↓	0.025
AEG26553.1	H8-12 (1 800/1 017), H7-3 (1 800/1 236)	亚硫酸盐还原酶（NADPH）黄蛋白α组分	2.646	1.042↑	0.269	1.263↑	1.212
AEG29408.1	H9-5 (1 728/1 484), H7-11 (1 728/1 484)	磷酸烯醇丙酮酸蛋白磷酸转移酶Ptsl	2.660	1.047↑	0.574	2.695↑	2.574
AEG29263.1	H8-4 (1 848/189), H9-1 (1 848/853)	NADH-醌氧化还原酶亚基L	2.473	0.974↓	0.231	1.085↑	1.114
AEG27032.1	H6-5 (390/192)	琥珀酸脱氢酶，细胞色素b556亚基	1.758	0.692↓	0.230	1.080↑	1.561
AEG29294.1	H9-10 (1 518/1 085)	氨基磷酸核糖转移酶	2.317	0.912↓	0.273	1.282↑	1.406
AEG27629.1	H9-9 (1 047/533)	二氢乳清酸酶	2.164	0.852↓	0.231	1.085↑	1.273
AEG27038.1	H8-3 (1 167/336)	琥珀酰辅酶a合成酶（ADP形成）β亚基	2.230	0.878↓	0.282	1.324↑	1.508
AEG27031.1	H8-5 (1 293/402), H8-2 (1 293/0)	柠檬酸合成酶I	2.052	0.808↓	0.333	1.563↑	1.934
AEG29271.1	H8-7 (1 797/618)	NADH-醌氧化还原酶亚基C/D	2.542	1.001↑	0.293	1.376↑	1.375
AEG26076.1	H7-7 (1 284/606)	磷酸核糖胺-甘氨酸连接酶	2.579	1.015↑	0.239	1.122↑	1.105
AEG30581.1	F4-1 (1 785/73)	ATP结合蛋白	2.640	1.040↑	0.052	0.244↓	0.235
AEG26238.1	B5-3 (630/624)	假设蛋白ALQ63_03 824	1.416	0.557↓	0.01	0.047↓	0.084
AEG29272.1	H7-6 (675/317)	H-醌氧化还原酶亚基K	2.342	0.922↓	0.285	1.338↑	1.451
AEG27034.1	H7-8 (1 710/1 665), H9-7 (1 710 /1 352)	琥珀酸脱氢酶或富马酸还原酶黄蛋白亚基	1.603	0.631↓	0.055	0.258↓	0.409
AEG27939.1	B4-1 (435/109), B6-3 (435/109), B6-5 (435/108)	MarR家族转录调控因子SlyA	2.712	1.068↑	0.006	0.028↓	0.026
AEG28365.1	F4-4 (849/38), F4-2 (849/36), F4-3 (849/38)	winged helix家族双组分转录调控因子RstA	2.71	1.067↑	0.039	0.183↓	0.172
AEG30425.1	H8-1 (633/262)	cAMP激活的全局转录调控因子CRP，转录调控因子Crp/Fnr家族	2.243	0.883↓	0.860	4.038↑	4.573
AEF52 157.1	H7-14 (267/3)	FAD装配因子SdhE	2.737	1.078↑	0.259	1.216↑	1.128
AEG27490.1	H7-10 (1 110/1 089)	孔蛋白OmpC	1.460	0.575↓	0.585	2.746↑	4.776
AEG26866.1	F5-12 (1 188/864)	多药外排RND转运蛋白质周适配器亚单位SdeX	2.910	1.146↑	0.022	0.103↓	0.090
AEG29409.1	H3-1 (510/364)	葡萄糖转运蛋白亚基IIA_	2.234	0.880↓	0.051	0.239↓	0.272
下划线标注的为代表性突变体；“↑”代表升高；“↓”代表降低。 Underline indicates mutant; "↑" indicates elevation; "↓" indicates decline.

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