裂殖酵母中不稳定遗传耐药菌株的筛选及其运用

doi:10.16288/j.yczz.24-266

遗传 ›› 2025, Vol. 47 ›› Issue (5): 589-599.doi: 10.16288/j.yczz.24-266

裂殖酵母中不稳定遗传耐药菌株的筛选及其运用

但露凤¹^,²(), 褚以文¹^,², 王欣荣¹^,², 何向伟³()

1.成都大学药学院，四川抗菌素工业研究所，成都 610100
2.成都大学抗生素研究与再评价四川省重点实验室，成都 610106
3.浙江大学生命科学研究院，杭州 310000

收稿日期:2024-10-14 修回日期:2024-12-18 出版日期:2025-05-20 发布日期:2025-01-07
通讯作者: 何向伟，博士，教授，博士生导师，研究方向：研究染色体结构表观遗传稳定性的分子机理。E-mail: 1187853534@qq.com
作者简介:但露凤，博士，讲师，研究方向：微生物遗传、表观遗传的稳定性。E-mail: danlufeng@zju.edu.cn
基金资助:
国家自然科学基金项目(32400477);四川省自然科学基金青年基金(2023NSFSC1231)

Screening and application of unstable genetically resistant strains in fission yeast

Lufeng Dan¹^,²(), Yiwen Chu¹^,², Xinrong Wang¹^,², Xiangwei He³()

1. School of Pharmacy, Sichuan Industrial Institute of Antibiotics, Chengdu University, Chengdu 610106, China
2. Antibiotic Research and Re-Evaluation Key Laboratory of Sichuan Province, Chengdu University, Chengdu 610106, China
3. Institute of Life Sciences, Zhejiang University, Hangzhou 310000, China

Received:2024-10-14 Revised:2024-12-18 Published:2025-05-20 Online:2025-01-07
Supported by:
National Natural Science Foundation of China(32400477);Natural Science Foundation of Sichuan Province(2023NSFSC1231)

摘要/Abstract

摘要：

发生在DNA序列水平或表观遗传水平的可逆遗传突变可以调控不稳定遗传的表型，其可逆性使得生物体能更好更快地适应外界多变的环境，但也正是因为其遗传的不稳定性，在传统研究(尤其是针对耐药性调控的研究)中往往被忽略。本研究利用雷帕霉素(+咖啡因)对野生型裂殖酵母菌株进行了耐药突变株的分离，共得到173株耐药突变株，经传代培养和子代耐药性试验，发现14株耐药株存在遗传不稳定现象。进一步研究表明，其中部分菌株的不稳定遗传耐药性受到ssp1位点可逆的DNA序列改变调控。本研究对雷帕霉素作为临床抗肿瘤药物治疗过程中易产生不稳定耐药性的分子机制提供了新的思路和相关的科学依据，并为解决其耐药性问题提供了可能的新作用靶点。

关键词: 耐药性, 不稳定遗传, 可逆突变, 雷帕霉素

Abstract:

Reversible alterations at DNA sequence or epigenetic levels can result in phenotypes that are unstably inherited. The reversibility of these inheritable changes might be uniquely beneficial for adaption to possible fluctuations in environment. However, unstable changes are always ignored for the genetic instability in traditional studies, especially in the cause of drug resistance. In this study, we conduct a specific genetic screen in fission yeast using rapamycin (+caffeine) and obtain 173 resistant isolates. In contrast to the common strategy of isolating stable genetic mutants, we passage the cell culture with rapamycin resistance on drug free condition and test the resistance of offspring every five days, and obtain 14 strains that exhibit unstable resistance to rapamycin (the drug resistance is lost randomly among the cell progenies without drug selection pressure). Further studies show that the unstable genetic resistance of some strains is regulated by reversible DNA sequence alterationat the ssp1 gene locus. This study provides new insights and relevant scientific basis for the regulatory mechanism of unstable drug resistance in the process of rapamycin as a clinical anti-tumor drug, and a new possible target for solving the problem of drug resistance.

Key words: drug resistance, genetic instability, reversible mutation, rapamycin

但露凤, 褚以文, 王欣荣, 何向伟. 裂殖酵母中不稳定遗传耐药菌株的筛选及其运用[J]. 遗传, 2025, 47(5): 589-599.

Lufeng Dan, Yiwen Chu, Xinrong Wang, Xiangwei He. Screening and application of unstable genetically resistant strains in fission yeast[J]. Hereditas(Beijing), 2025, 47(5): 589-599.

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菌株名称	基因型	来源
446 (野生型)	h^- ade6-210, leu1-32, ura4D	何向伟实验室
447 (野生型)	h⁺, ade6-216, leu1-32, ura4D	何向伟实验室
gaf1∆	h^?, gaf1∆::kanMX6, ade6∆::hphMX6, cnt2::ade6-natMX6, leu1-32, ura4D	基因敲除库
Epm101	h^-, ssp1^MTD-AGCCA, ade-210, leu1-32, ura4D	本研究筛选
Epm102	h^-, ssp1^MTD-GCTTT, ade-210, leu1-32, ura4D	本研究筛选
Epm201-206	h⁺, ssp1^MTD-GTCGTCCG, ade-210, leu1-32, ura4D	本研究筛选
Epm103-108	h^-, ade-210, leu1-32, ura4, 未知改变	本研究筛选
Epm102-R	h^-, ssp1^MTD-GCTTT, ade-210, leu1-32, ura4D	本研究分离
Epm102-S	h^-, ade6-210, leu1-32, ura4D	本研究分离
ssp1∆	h^-, ssp1∆::kanMX6, leu1-32, ura4D	本研究构建
Epm101-wt	h^-, ssp1^wt::kanMX6, leu1-32, ura4D	本研究构建
Epm102-wt	h^-, ssp1^wt::kanMX6, leu1-32, ura4D	本研究构建

咖啡因浓度(mmol/L)	涂布10³个细胞时的菌落数	涂布10⁴个细胞时的菌落数	涂布10⁵个细胞时的菌落数
0 mmol/L(雷帕霉素：0 ng/mL)	10³	10⁴	10⁵
6 mmol/L(雷帕霉素：100 ng/mL)	2~20	10⁴	10⁵
8 mmol/L(雷帕霉素：100 ng/mL)	0	0~2	2~20
10 mmol/L(雷帕霉素：100 ng/mL)	0	0	0~10

菌株	无药处理第1天	无药处理第5天	无药处理第10天	无药处理第15天	无药处理第20天
WT	0%	0%	0%	0%	0%
gaf1△	100%	100%	100%	100%	100%
Epm101	100%	100%	93.5%	9.7%	0%
Epm102	100%	100%	97.5%	35%	0%
Epm201	100%	97%	90.8%	7.8%	0%
Epm202	100%	100%	100%	67%	1%
Epm203	100%	100%	80%	5%	0%
Epm204	100%	100%	99%	74%	1%
Epm205	99.4%	95.4%	55.8%	0.6%	0%

回交	回交比(R∶S )	杂交	杂交比(R∶S )
Epm201×WT	2∶2 (37/42)	Epm101×Epm201	4∶0 (19/19)
Epm203×WT	2∶2 (38/39)	Epm102×Epm201	4∶0 (25/26)
Epm205×WT	2∶2 (46/46)	Epm101×Epm205	4∶0 (20/20)
Epm206×WT	2∶2 (36/36)	Epm102×Epm205	4∶0 (24/24)
Epm103×WT	2∶2 (23/24)	Epm201×Epm205	4∶0 (36/37)
Epm104×WT	2∶2 (37/37)	Epm203×Epm205	4∶0 (26/26)
Epm105×WT	2∶2 (16/17)	Epm106×Epm103	4∶0 (16/16)
Epm106×WT	2∶2 (30/30)	Epm106×Epm104	4∶0 (26/26)
Epm107×WT	2∶2 (26/27)	Epm106×Epm105	4∶0 (17/17)
Epm108×WT	2∶2 (20/20)	Epm106×Epm107	4∶0 (20/20)
Epm103×WT	2∶2 (23/24)	Epm106×Epm108	4∶0 (15/15)
		Epm104×Epm105	4∶0 (11/11)
		Epm206×Epm106	3∶1 (11/17)，2∶2 (6/17)

裂殖酵母中不稳定遗传耐药菌株的筛选及其运用

Screening and application of unstable genetically resistant strains in fission yeast

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