T5 super pcr mix

Isolation of total RNA from treated samples was performed using an RNA extraction kit (Promega, Madison, WI, USA). The cDNA synthesis was performed with total RNA (2 μg) reverse transcribed using All-In-One RT MasterMix (Applied Biological Materials, Zhenjiang, China). RT-PCR analysis was conducted using 2 × T5 Super PCR Mix (Tsingke, Beijing, China) and Taq Master MixTaq mix (Vazyme Biotech, Nanjing, China). All primers used in this study are listed in Supplementary Table S2. Quantification for gel intensity was carried out using Image J software (https://imagej.nih.gov/ij/).

Total DNA was extracted from dried leaf tissue using a CTAB method [13 ]. The amplification of rbcL, matK, trnH-psbA, ITS and ITS2 was carried out with universal primer sets ([14 (link)–19 (link)], Table 1). We amplified DNA in a 25 μL reaction mixture following Zhang et al. [20 (link)] using rTaq DNA polymerase. For those samples that failed to amplify on a first pass, LA or Primer Star DNA polymerase (Takara Biotechnology Co. Ltd.) or 2*T5 Super PCR Mix (Beijing TsingKe Biotech Co., Ltd.) was used as an alternative to rTaq DNA polymerase. Samples showing a clear single band were sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China for bi-directional sequencing. All sequences were uploaded to the GenBank (GenBank accession numbers are given in S1 Table).

The counterselective gene upp was PCR (2×T5 Super PCR Mix, TSINGKE) amplified from the genome of E. coli MG1655 and adapted to upp^6tgg. The upp variant was then cloned into the low-copy pSC101 vector with the P23119 promoter and NGG PAM by Gibson cloning. The negative control plasmid pTTT-UPP^6TGG-MKATE was modified from pNGG-UPP^6TGG by replacing the PAM sequence from NGG to TTT, and expressing an additional red fluorescent protein (Mkate). The PAM library template plasmid pUPP^6TGG-AmpR was derived from pNGG-UPP^6TGG by co-expressing with the ampicillin resistance gene AmpR using p23119 promoter. The BE elements SpdCas9-CDA, xdCas9 3.7-CDA and dCas9 NG-CDA were commercially synthesized by GENERAL BIOL and cloned into the oriRγ plasmid pHK with Ptet promoter. The sgRNA sequence targeting upp^6tgg was inserted into p15A-gRNA via golden gate assembly (Su et al., 2016 (link)). The sgRNA sequence targeting genomic lacZ gene was inserted into pgRNA-bacteria (Qi et al., 2013 (link)) by Gibson cloning. To validate the repression of GFP using various PAM sequences, SpdCas9 was cloned into pBAD24, generating pdCas9. A total of 49 6tgg-GFP expression plasmids containing TTTT, NNGG, GTGT or GTGC PAMs were constructed by Gibson cloning. All the plasmid constructed were confirmed via sanger sequencing. DNA sequences of the core elements used in this study were listed in Table S1.

The total RNA of each transformed yeast strains was extracted using PureLink™ RNA Mini Kit (production ID: 12183018A, ThermoFisher). A yeast actin gene (Genbank No. 850504, primers shown in Table S1) was used as the reference gene for RT-PCR. The template dosage was adjusted to equal amounts. The PCR reaction system was prepared as follows: T5 Super PCR Mix 10 μL (Tsingke, Beijing), AhD2FH primer (10 μM) 0.4 μL, AhD2RH primer (10 μM) 0.4 μL, cDNA (100 ng/μL) 1 μL, and ddH₂O 8.2 μL. The amplification program was as follows: an initial denaturation step that consisted of 3 min at 98 °C, followed by 28 cycles of 5 s at 98 °C, 15 s at 58 °C, 10 s at 72 °C, and extension at 72 °C for 2 min. The PCR products were separated on 1.5% agarose gel and photographed using an AlphaImager EP imaging system (NatureGene Corporation).

Genomic DNA of each strain was extracted from fresh mycelium growing on PDA after 5 days of growth following the rapid “thermolysis” method described in Zhang et al. (2010) (link). For the amplification of ITS, RPB2, and TEF1-α gene fragments, ITS4 and ITS5 for ITS (White et al. 1990 (link)), EF1-728F (Carbone and Kohn 1999 (link)) and TEF1LLErev (Jaklitsch et al. 2005 (link)) for TEF1, and RPB2-5F and RPB2-7R for rpb2 (Liu et al. 1999 (link)) were used. Each PCR reaction consisted of 12.5 μl T5 Super PCR Mix (containing Taq polymerase, dNTP, and Mg²⁺, Beijing TsingKe Biotech Co. Ltd., Beijing), 1.0 μl of forward primer (10 μM), 1.0 μl of reverse primer (10 μM), 0.5 μl DMSO, 3 μl DNA template and 7 μl double sterilized water. PCR reactions were in Eppendorf Mastercycler, following the protocols described by Sun et al. (2016) (link). PCR products were purified with the PCR product purification kit (TIANGEN Biotech, Beijing, China), and sequencing was carried out in both directions on an ABI 3730 XL DNA sequencer (Applied Biosystems, Foster City, California) with primers used during PCR amplification.

DNA restriction enzymes (BamHI, NotI, and SacI) were purchased from Takara Biomedical Technology Co., Ltd (Beijing, China). The Rapid Yeast Genomic DNA Isolation Kit, Plasmid DNA Mini-preps Kit, SDS-PAGE Preparation Kit, DNA Markers, Protein Markers, DNA Gel Extraction Kit, and the primers for α-factor, 5′-AOX, and 3′-AOX were supplied by Sangon Biotech Co., Ltd (Shanghai, China). TS GelRed Nucleic Acid Dye and T5 Super PCR Mix were obtained from Beijing Tsingke Biotech Co., Ltd (Beijing, China). Hemin (CAS: 16009-13-5, purity: 98%) and biliverdin hydrochloride (CAS: 55482-27-4, purity: 97%) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Geneticin (G418) was purchased from Invitrogen Corporation (Carlsbad, CA, USA). All other reagents were commercially available in analytical and biological grades.

For each strain, genomic DNA was extracted from mycelium growing on PDA harvested after 3 days of growth, following the method of Wang and Zhuang (2004) . For the amplifications of ITS, rpb2 and tef1 gene fragments, three different primer pairs were used: ITS4 and ITS5 for ITS (White et al. 1990 (link)), EF1-728F (Carbone and Kohn 1999 (link)) and TEF1LLErev (Jaklitsch et al. 2005 (link)) for tef1 and tRPB2-5F and tRPB2-7R for rpb2 (Chen and Zhuang 2016 (link)). Each 25 μl PCR reaction consisted of 12.5 μl T5 Super PCR Mix (containing Taq polymerase, dNTP and Mg2+, Beijing TsingKe Biotech Co., Ltd., Beijing), 1.25 μl of forward primer (10 µM), 1.25 μl of reverse primer (10 µM), 1μl DNA template, 5 μl of PCR buffer and 4.5 μl sterile water. PCR reactions were run in an Eppendorf Mastercycler following the protocols described by Zhuang and Chen (2016). PCR products were purified with the PCR product purification kit (Biocolor BioScience & Technology Co., Shanghai, China), and sequencing was carried out in both directions on an ABI 3730 XL DNA sequencer (Applied Biosystems, Foster City, California) with primers used during PCR amplification. GenBank accession numbers of sequences generated in this study are provided in Table 1.