Seamless cloning kit

SIRT1 cDNA was made from pCMV-SIRT1-t1-Flag (purchased from Sino Biological) via PCR amplification. SIRT1 cDNA was cloned into the FUGW vector using Seamless Cloning Kit (Beyotime, D7010M) and confirmed by DNA sequencing. HMC3 cells were plated into 24-well or 6 cm dish at appropriate intensity and cultured overnight. We transfected 150 ng plasmid per well into 24-well or 1.5 μg plasmid per well into 6 cm dish. SIRT1 plasmid or FUGW plasmid was transfected using ViaFect reagent (Promega, #E4981) according to manufacturer's instructions. After transfection for 24 h, cells were stimulated by Aβ for 72 h. The knockdown of SIRT1 was performed by the transfection with specific siRNA (Tsingke Biotechnology Co., Ltd.) using ViaFect reagent. The cloning primers used were as follows: SIRT1 (forward: 5′-TGGGCTGCAGGTCGACTCTAGAATGGCAGATGAAGCAGCTCTC-3′ and reverse: 5′-TTG ATATCGAATTCTAGACTATGATTTGTTTGATGGATAGTTCATGTCT-3′). The siRNA primers were as follows: siSIRT1-1 (forward: 5′-CACCUGAGUUGGA UGAUAUTT-3′ and reverse: 5′-AUAUCAUCCAACUCAGGUGTT-3′) and siSIRT1-2 (forward: 5′-GUCUGUUUCAUG UGGAAUATT-3′and reverse: 5′-UAUUCCACAUGAAACAGACTT-3′).

The SVA VP2 gene (852 bp, encoding 284 amino acids [aa]) was amplified by specific primers (Sangon, Shanghai) and inserted into the Xho-I and Sal-I restriction sites of the pEGFP-gI28K vector. The eukaryotic transfer vector pEGFP-gI28K-SVA-VP2 was constructed using the seamless cloning kit (Beyotime, Shanghai) and was extracted using the end-free plasmid minikit II (Omega). The accuracy of the plasmid was verified by enzyme digestion and sequencing.

Details of wheat cell-free reaction have been described previously [39 (link)]. Cell-free protein expression was performed using the TNT SP6 Wheat Germ Master Mix Kit (Promega, Madison, WI). The primers for the irr gene are as follows: irr forward, 5΄-CCATATGATGCATTCTTCACATACCCA-3΄; irr reverse, 5΄-CTCTAGATCAGCGGGCCTGACGGCG-3΄. The polymerase chain reactions (PCRs) were incubated at 95°C for 5 min. Then 35 cycles were performed as follows: 30 s at 95°C, 40 s at 57°C, and 7 min at 72°C. The amplified PCR product was subcloned into a pDAP-Halo-Kan vector (Zoobio, Nanjing, China) using the Seamless cloning kit (Beyotime, Shanghai, China), and the recombinant plasmid was used as a transcription template. The protein expression volume was 50 μL, comprising 30 μL Wheat Germ Master Mix, 1 μg pDAP-Halo-Kan-irr plasmid, and nuclease-free water to make 50 μL; this was incubated at 25°C for 2 h. The expressed protein was confirmed using western blot analysis and an anti-Halo tag antibody (Promega, Madison, WI).

Seamless Cloning Kit and SUMO protease were purchased from Beyotime Biotechnology (Shanghai, China). The expression vector pET-21a-His-SUMO and Escherichia coli strain BL21(DE3) were stored by Institute of Biomedicine, Jinan University, while primers were synthesized by Sangon Biotech (Shanghai, China). Ni-Sepharose 6 Fast Flow column was purchased from GE Healthcare (Marlborough, MA, USA). Isopropyl-1-thio-β-galactopyranoside was purchased from Genebase (Guangzhou, China). Restriction endonuclease and Agarose Gel Extraction kits were purchased from TAKARA Bio. Plasmid extraction kits were purchased from TIANGEN (Beijing, China). Other chemical reagents were purchased from Sigma-Aldrich (Saint Louis, MO, USA), were reagent grade, and could used without further purification.

The pCAMIBIA1300-EGFP-MCS vector was ligated with the ORFs of sensitive transcription factors using the Seamless Cloning Kit (Beyotime, Shanghai, China). A. tumefaciens GV3101 receptor cells transformation. Segments 1 cm × 1 cm were cut from fresh mature E. senticosus leaves. A solution containing 100 µmol/L acetosyringone, 10 mmol/L MES, and 50 mmol/L MgCl₂ was added to the converted A. tumefaciens GV3101, and the OD₆₀₀ value was measured to be 0.6. After 10 min of 400 Pa vacuum filtrations, the chopped E. senticosus leaves were submerged in the above liquid. After spreading sterile three-layer filter paper onto a sterile petri dish and moistening it with sterile water, the filtered leaves were cultured for 72 h at 23°C. Using reverse transcription into cDNA from isolated RNA. Real-time PCR was used to identify the expression of sensitive transcription factors and EsFPS, EsSS, and EsSE [18 ]. Table 1 displays the primers that were utilized. Using HPLC, the amount of oleanolic acid in the leaves was ascertained.

E. coli DH5α was used as a host strain for plasmid construction. Target gene cloning was performed by designing primer probes using the NCBI database genome sequence. The primers used in this study are listed in Supplementary Table 12. All the enzymes used in this study were constructed into pET-22b( + ) by the Seamless Cloning Kit (Beyotime, China). Ligation between the target fragment and pET-22b( + ) vector was incubated for 30 min at 50 °C, then adding 10 µL reaction mix to 100 µL of E. coli DH5α competent cells and rested for 30 min. They were coated on LB plates after heat shock for 60 s. All microorganisms and plasmids used in this study were complemented in the supplementary information.