酿酒酵母TAP42基因在细胞壁应激反应中的作用

doi:10.13560/j.cnki.biotech.bull.1985.2021-0036

摘要/Abstract

摘要：

研究蛋白质磷酸酶2A调节亚单位Tap42p（type 2A associated protein-42kD）在酵母细胞壁应激反应中的作用。采用一步基因置换法构建TAP42基因缺失（tap42Δ）酵母菌株;观察在细胞壁应激反应条件下tap42Δ菌株的克隆形成能力;通过全自动生长曲线分析仪检测细胞分裂增殖活力;RT-PCR检测细胞壁应激反应通路中转录因子Swi4p、Swi6p、Ssd1p和Mpt5p的表达水平。在正常培养条件下,与野生型酵母细胞（BY4743）比较,tap42Δ菌株形成较小克隆,细胞增殖活力较差;在细胞壁应激反应条件下,tap42Δ菌株的克隆大小和增殖活力都与BY4743菌株相似,菌株对应激反应诱导剂有一定的抗性;且缺失TAP42基因上调SWI4、SWI6、SSD1和MPT5等基因的转录表达水平。因此,缺失TAP42基因影响酵母细胞的分裂增殖能力,增强对细胞壁应激反应的抵抗性,上调细胞壁应激反应通路中转录因子的表达水平。

关键词: 酿酒酵母, 细胞壁应激, TOR, Tap42

Abstract:

We herein aim to explore the roles of the protein phosphatase 2A（PP2A）regulatory subunit Tap42p（type 2A associated protein-42kDa）in the cell proliferation of Saccharomyces cerevisiae. TAP42 -deficient strain（tap42Δ）was constructed through polymerase chain reaction（PCR）-mediated one-step gene disruption. The colony-forming ability was observed on YPD（yeast extract peptone dextrose）culture plate under cell wall stress condition,and cell proliferation assay of the tap42Δ strain was performed using a Bioscreen C MB instrument. The expression levels of transcription factors including Swi4p,Swi6p,Ssd1p and Mpt5p in the cell wall stress response signaling pathway were determined by RT-PCR. The tap42Δ formed smaller clones and had poor proliferation compared with wild-type S. cerevisiae BY4743 under the physiological condition. The clone-forming ability and proliferation of the tap42Δ strain presented a similarity to those of the BY4743 under cell wall stress condition,indicating that the tap42Δ strain showed a weak resistance to cell wall stress response. And TAP42 deficiency enhanced the transcriptional expression levels of SWI4,SWI6,SSD1 and MPT5. Conclusively,TAP42 deficiency impairs the ability of yeast cell proliferation,and enhances the resistance to the cell wall stress response and the transcriptional expression levels of SWI4,SWI6,SSD1 and MPT5 in the cell wall stress response signaling pathway.

Key words: Saccharomyces cerevisiae, cell wall stress, TOR, Tap42

崔新刚, 孙雅欣, 崔晓静, 邓雁文, 孙恩浩, 王俊芳, 崔红晶. 酿酒酵母TAP42基因在细胞壁应激反应中的作用[J]. 生物技术通报, 2021, 37(10): 57-62.

CUI Xin-gang, SUN Ya-xin, CUI Xiao-jing, DENG Yan-wen, SUN En-hao, WANG Jun-fang, CUI Hong-jing. Roles of Gene TAP42 in the Cell Wall Stress Response of Saccharomyces cerevisiae[J]. Biotechnology Bulletin, 2021, 37(10): 57-62.

图/表 5

表1 本实验所用引物序列

Table 1 Nucleotide sequence of primers used in this study

引物名称Primer name	引物序列Primer Sequence（5'-3'）	产物大小 Amplicon size/bp	用途Purpose
TAP42-F	AAGAAGAGGAAGAAAGTAATAACGAGGAGAGATCATAAACCTGTGCGGTATTTCACACCG	1 232	扩增基因破坏元件
TAP42-R	TCTAATAGATACTTATGTGTACGTATATAGGTATTTTTTTAGATTGTACTGAGAGTGCAC	1 232	扩增基因破坏元件
V-TAP42-F	TGGTGGTGTTGTAGCGGTAAATG	1 678	扩增基因破坏片段
V-TAP42-R	TCTCGGAAAGAAAAGAGCCTGTG		扩增基因破坏片段
TAP42-int-F	CCCTCAGCTTAGCGAATTGTAC	164	扩增目的基因内部片段
TAP42-int-R	CGTGGTCCTCGTCGTTGTTT		扩增目的基因内部片段
TAP42-int-F	CCCTCAGCTTAGCGAATTGTAC	712	扩增破坏元件部分片段
URA-int-F	GGAACCTAGAGGCCTTTTGATGT		扩增破坏元件部分片段
SWI4-int-F	AGGTGGGTATGGTAGGT	254	定量引物
SWI4-int-R	TTGTAGCCGATGTTGAT		定量引物
SWI6-int-F	GCAAGCCTAACGATGAT	214	定量引物
SWI6-int-R	CGTTGTAAGAATGCCTCTA		定量引物
SSD1-int-F	CTGTTGATAGGCGTGCGAGA	143	定量引物
SSD1-int-R	AACAACCGACAGCGTGGC		定量引物
MPT5-int-F	ACGCATCTCAGGCCACCTG	194	定量引物
MPT5-int-R	GATGCTGAGTCGGCAGATGG		定量引物

图1 TAP42基因缺失酵母菌株的验证图 M:DNA marker;1和3:破坏元件的全长和部分片段;2:TAP42基因内部片段,模板为tap42Δ菌株基因组DNA

Fig. 1 Agarose gel electrophoresis of PCR products on verifying the TAP42 deletion strain M represents DNA marker;the bands of lane 1 and 3 are the full or part length PCR products of disruption module as template of tap42Δ genomic DNA;and the band of lane 2 is PCR products of an internal fragment from the TAP42 gene.

图2 酵母细胞的分裂增殖能力 BY4743和tap42Δ菌株在YPD培养条件下的克隆形成情况（A）和生长曲线图（B）,BY4743为野生型酵母菌株

Fig. 2 Cell proliferation ability of the yeast strains The colony-forming ability (A) and cell proliferation activity (B) of BY4743 and tap42Δ strains. BY4743 is the control wild strain

图3 酵母菌株的细胞壁应激反应抗性 BY4743和tap42Δ菌株在YPD或细胞壁应激反应（含10 μg/mL CFW）培养条件下的克隆形成情况（A）和生长曲线图（B）,CFW（荧光增白剂）为细胞壁应激反应诱导剂,BY4743为野生型酵母菌株

Fig.3 Resistance to the cell wall stress response of the yeast strains The colony-forming ability (A) and cell proliferation activity (B) of BY4743 and tap42Δ strains without or with 10 μg/mL calcofluor white stain (CFW) treatment. BY4743 is the control wild strain

图4 酵母菌株中 SWI4、SWI6、MPT5 和 SSD1 基因定量表达水平应激反应抗性 BY4743 和 tap42Δ 菌株中 SWI4、SWI6、SSD1 和 MPT5 基因表达情况,数据表示为均数 ± 标准差（n=3）,*P < 0.05,**P < 0.01 vs BY4743,CFW（荧光增白剂）为细胞壁应激反应诱导剂,BY4743 为野生型酵母菌株

Fig.4 Expression levels of SWI4, SWI6, MPT5 and SSD genes in yeast strains The relative mRNA expression levels of SWI4, SWI6, SSD1 and MPT5 genes for BY4743 and tap42Δ strains. Data are in the form of mean±standard error. *: P < 0.05, **: P<0.01 vs BY4743; CFW is calcofluor white stain as an inducer of cell wall stress response. BY4743 is the control wild strain

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