Yeast genomic DNA extraction kit, total RNA extraction reagent Trizol, Taq PCR premix and YPD medium components peptone, yeast extract, glucose, and agar were purchased from Sangon Biotech Co. Ltd. (Shanghai, China); cell wall interfering agent Calcofluor White (CFW), Congo Red (CR) and sodium dodecyl sulfate (SDS) were obtained from EN Chemical Technology Co. Ltd. (Shanghai, China), BioFroxx and Sangon Biotech Co. Ltd; FastKing one-step reverse transcription-fluorescence quantitative kit (SYBR Green) was purchased from Tiangen Biochemical Technology Co., Ltd(Beijing , China).
Strains and culture conditions
The wild-type S. cerevisiae (WT) has stored in our laboratory; the knock-out yeast library was purchased from Horizon Discovery Co., Ltd. WT yeast can be stably propagated in YPD solid and liquid medium, but for genetic stability, knock-out yeast needs to add 200ug/ml G418 to YPD medium to stably propagate.
Verification of the YLR358C knockout strain
The YLR358C knockout yeast was cultured overnight at 10000rpm/min and centrifuged for 1min to collect the yeast, and the genomic DNA was extracted using the above kit. The obtained genome was used as a template, and A, KanB, KanC and D were used as primers (supplemental Table 1) to verify the YLR358C knockout yeast by PCR, and the successful knockout yeast were named YLR358C△.
The single clones of WT and YLR358C△ yeasts were picked and inoculated into 10ml YPD liquid medium and YPD liquid medium containing 200μg/mL G418, respectively, and cultured overnight in a constant temperature shaker. After centrifugation of the yeast solution, resuspend the cells in fresh YPD medium to OD600=1.0. Then respectively dilute to 10-1, 10-2, 10-3, 10-4, 10-5, take 2.5 μL of diluent was spotted on YPD plates containing different concentrations of CFW, SDS, and CR, and incubated in a constant temperature incubator at 28°C for 2 days to observe the growth.
The WT and YLR358C△ yeast single clones were cultured to the logarithmic phase on a constant temperature shaker at 28°C and 200 rpm/min. After centrifugation, the cells were resuspended in 10 ml YPD medium containing 30 ug/ml CFW. After the yeast is stained with CFW, the cells are resuspended in an appropriate amount of PBS and transferred to the well plate, and the fluorescence is observed under an inverted fluorescence microscope after resting for 10-15 minutes.
Observation of microscopic morphology
The WT and YLR358C knock-out yeasts were cultured overnight at 28°C and 200 rpm/min constant temperature shaker. After the yeast is centrifuged, it is fixed with 1-2ml 3.5% glutaraldehyde, then fixed with 1% osmium acid, ethanol-acetone stepwise gradient dehydration, Epon-618 infiltration, embedding, semi-thin sectioning using Leica-R ultramicrotome (Germany), positioning by light microscope, double staining with lead citrate and uranyl acetate, observation by transmission electron microscope (Gatan Inc, USA).
Transcriptome sequencing and analysis
After centrifuging the yeast, the total RNA is extracted, the mRNA is used as a template to synthesize and purify cDNA, and finally the final cDNA library is obtained by PCR enrichment. The cDNA library is sequenced and the data is filtered to obtain the Clean Data, which is compared with the designated reference genome, and the Mapped Data obtained is subjected to the library quality evaluation such as the length check of the insert and the randomness check. Structural analysis of alternative splicing analysis, discovery of new genes and optimization of gene structure are carried out. According to the expression level of genes in different samples or different sample groups, differential expression analysis, functional annotation and functional enrichment of differentially expressed genes and other expression levels are analyzed.
Extraction of Yeast Total RNA and Real-time Quantitative PCR
After the yeast was collected by centrifugation, total RNA was extracted with Trizol, and real-time quantitative PCR was performed according to the instructions of the FastKing One-Step Reverse Transcription-Fluorescence Quantitative Kit. The primers used are as shown in the supplemental Table 2, TAF10 was used as the internal reference primer, and the results were quantified by the 2-ΔΔCT method.