阿维链霉菌中ECF-Sig16对阿维菌素合成的影响

doi:10.13304/j.nykjdb.2022.0105

摘要/Abstract

摘要：

阿维菌素是阿维链霉菌产生的一种广谱、高效、低毒的杀虫抗生素，在医药、农业和畜牧业领域有着广泛的应用。ECF-Sigma因子在细菌适应生存环境变化和高效响应激活压力中起重要作用。为研究阿维链霉菌中ECF-Sig16因子对阿维菌素合成的影响，通过对sig16基因缺失、回补和过表达，利用摇瓶发酵试验初步证实Sig16能够抑制阿维菌素的合成，但不影响菌株的生长。表型观察试验显示Sig16不影响阿维链霉菌的形态分化。通过凝胶阻滞试验（EMSA）确定Sig16直接结合在支链氨基酸ABC转运体操纵子SAV1190~SAV1194的启动子区，暗示Sig16可能通过影响阿维菌素生物合成的前体物质，抑制阿维菌素的合成。

关键词: 阿维菌素, 阿维链霉菌, ECF-Sigma调控蛋白, Sig16

Abstract:

Streptomyces avermitilis produces avermectin， which is a kind of anthelmintic antibiotics with broad-spectrum， low toxicity activities and high potency and widely used in agricultural and medical fields. Extracytoplasmic function Sigma （ECF-Sigma） regulators play key roles for adaptation to changes of living environment and activating stress responses efficiently. To investigate the function of ECF-Sig16 regulator in avermectin biosynthesis， shaking flask fermentation of sig16 gene deletion， complementary， and overexpression strains were test. The results indicated that Sig16 inhibited avermectin biosynthesis， but exerted no function on cell growth. Phenotype observation experiment showed that Sig16 didn’t affect Streptomyces avermitilis morphological differentiation in some conditions. Electrophoretic mobility shift assay （EMSA） showed that Sig16 binded to the promoter of branched-chain amino acid ABC transporter operon SAV1190~SAV1194， which indicated Sig16 could inhibit avermectin production by affecting avermectin precursor supply.

Key words: avermectin, Streptomyces avermitilis, ECF-sigma regulators, Sig16

中图分类号:

S182

罗帅, 问亚琴, 朱华, 张蓉, 王晓雯, 刘丽丽, 朱建亚. 阿维链霉菌中ECF-Sig16对阿维菌素合成的影响[J]. 中国农业科技导报, 2023, 25(7): 113-121.

Shuai LUO, Yaqin WEN, Hua ZHU, Rong ZHANG, Xiaowen WANG, Lili LIU, Jianya ZHU. Effect of ECF-Sig16 on Avermectin Production in Streptomyces avermitilis[J]. Journal of Agricultural Science and Technology, 2023, 25(7): 113-121.

图/表 7

表1 试验菌株和质粒

Table 1 Plasmids and strains used in the study

类型Type	名称Name	特征Character	来源Source or reference
质粒Plasmid	pKC1139	大肠杆菌-链霉菌穿梭质粒，多拷贝，温敏型E.coli-Streptomyces shuttle vector， multi-copy and temperature-sensitive	［32］
	pSET152	大肠杆菌-链霉菌穿梭质粒，整合型E.coli-Streptomyces shuttle vector， integrative	［32］
	pET-28a（+）	蛋白大量表达载体 Vector for heterologous protein overexpression	Novegen
	pJL117	携带红霉素抗性基因启动子的pIJ2925衍生质粒 pIJ2925 derivative carrying promoter with erythromycin resistance	［33］
菌株Strain	阿维链霉菌 S. avermitilis	野生型阿维链霉菌Wild-type （WT） S. avermitilis	实验室保存 Laboratary stock
大肠杆菌E. coli	JM109	基因克隆宿主菌 General cloning host	Novegen
	ET12567	甲基缺陷型菌株 MetHylation-defective strain	［34］
	BL21（DE3）	蛋白异源表达宿主菌 Host for protein heterologous overexpression	Novegen

表 2 试验用引物

Table 2 Primer sequences in this study

引物 Primer	引物序列 Primer sequence （5’-3’）	用途 Usage
LS63	CCCAAGCTTATGCTGGAGATCGACGACGT， HindⅢ	缺失sig16基因 Deletion of sig16
LS64	ATGGCCGTCGGTGTCGAGAGTGAACCGCTACACGCTCA
LS65	TGAGCGTGTAGCGGTTCACTCTCGACACCGACGGCCAT
LS66	GGAATTCGAACCACGACGAACAGCAGG， EcoRⅠ
LS67	CCCAAGGTGCAGGAGGTGTA	检测sig16基因是否缺失成功 Confirmation of sig16 deletion
LS68	GCATCACGTCGTTGAGCCC
LS69	TCCAGGACGCCTTCACC
LS70	GAAGATGAGGTAGACGACGG
LS81	CCCAAGCTTCGAACAGCACGTGGGCTT， HindⅢ	扩增sig16基因开放阅读框用于构建回补和过表达菌株 Amplification of sig16 open reading frame
LS82	GAAGATCTGCGGGATCTGCCTCATCAA， BglⅡ
LS89	GGAATTCCATATGGCCGTCGGTGTCG， NdeⅠ	扩增sig16基因开放阅读框用于大量表达His₆-Sig16蛋白 Amplification of sig16 open reading frame for overexpression of His₆-Sig16
LS90	CGGGATCCTCGAACAGCACGTGGGC， BamHⅠ
GJ105	GGTATTCCATTCGGTGTTGC	探针aveRp
GJ106	TGTTATGAATTTGCCCTGGTG	Probe aveRp
LS52	ATGGTCGGGAACCTCCGCAA	探针aveA1p
LS53	CTGTGTCCTCACCGCTAGGC	Probe aveA1p
GJ91	CCAAGGGCTACAAGTTCTCC	探针hrdB
GJ92	TTGATGACCTCGACCATGTG	Probe hrdB
LS207	CGCCTCGATACTGCTCGC	探针 SAV1196p
LS208	TGTGTCCTCCTCGGTGATCT	Probe SAV1196p
LS209	ACCGTGAACCAGTCGGGC	探针 SAV1188p
LS210	GGCATCCTGCTCCTCATCC	Probe SAV1188p
LS211	TCCACAGTTCCCGCCCTT	探针 SAV1190p
LS212	CGCTGGGACCGCTGGACA	Probe SAV1190p

图1 Sig16的保守结构域及 sig16 在野生型阿维链霉菌基因组中的位置A：Sig16的保守结构域；B：sig16在野生型阿维链霉菌基因组中的位置

Fig. 1 Conservative domain of Sig16 and organization of sig16 and its adjacent genesA： Conservative domain of Sig16； B： Organization of sig16 and its adjacent genes； Black bars at bottom is transcriptional units

图2 WT与sig16突变株中的阿维菌素产量注：不同小写字母表示不同菌株间在P<0.05水平差异显著。

Fig. 2 Avermectin production in WT and sig16 mutant strainsNote： Different lowercase letters indicate significant differences between different strains at P<0.05 level.

图3 WT和sig16突变株的阿维菌素产量曲线和生长曲线A：产量曲线；B：生长曲线

Fig. 3 Avermectin yield curve and growth curve of WT and sig16 mutant strainsA： yield curve； B： growth curve

图4 His6-Sig16与探针 aveRp、aveA1p、SAV1188p、SAV1190p 和 SAV1196p 的体外EMSA试验A：EMSA检测His6-Sig16与Probe aveRp与Probe aveA1p的相互作用；B：EMSA检测His6-Sig16与Probe SAV1188p、Probe SAV1190p与Probe SAV1196p的相互作用；每个反应体系包含0.15 nmol·L-1的地高辛标记探针，约100倍未标记的特异性探针用于竞争试验，探针hrdB和BSA分别作为探针与蛋白的负对照。箭头为游离探针；方括号为Sig16-DNA复合物

Fig. 4 EMSA assays of His6-Sig16 with probes aveRp， aveA1p， SAV1188p， SAV1190p and SAV1196pA： EMSA assays of the interaction of Probe aveRp， Probe aveA1p with His6-Sig16； B： EMSA assays of the interaction of Probe SAV1188p， Probe SAV1190p and Probe SAV1196p with His6-Sig16； Each reaction contained 0.15 nmol·L-1 DIG-labeled probe， ~100 fold excess of unlabeled specific probe was used for competition assays. Labeled probe hrdB or BSA protein was used as a negative control respectively for probe or His6-Sig16. Concentration of His6-Sig16 for probe hrdB was 0.5 μmol·L-1； arrowhead shows free probes； bracket shows Sig16-DNA complex

图5 sig16 突变株在YMS和MM固体培养基上的形态观察注：WT—野生型菌株；D—sig16基因缺失菌株；C—sig16基因回补株。Note： WT—Wild type； D—Sig16 deletion strain； C—Sig16 complemented strain.

Fig. 5 Morphologic observation of sig16 mutant strains incubated on YMS and MM solid medium

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