Roles of Gene TAP42 in the Cell Wall Stress Response of Saccharomyces cerevisiae

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

Abstract

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

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.

Figures/Tables 5

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

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.

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

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

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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