Nuclean plantgen dna kit

Fresh leaves were collected from S. mussotii in Yushu County, Qinghai Province. Total DNA was extracted using the NuClean PlantGen DNA Kit (CWBIO, Beijing, China) and was used to construct an SMRT sequencing library with an insert size of 10 kb. The genome was sequenced using the PacBio RS II platform (Pacific Biosciences, Menlo Park, CA, USA) at the Institute of Medicinal Plant Development of the Chinese Academy of Medical Sciences. We assembled the cp genome of S. mussotii as follows: first, the PacBio reads were error-corrected and assembled to produce the initial contigs using the hierarchical genome assembly process (HGAP) of SMRT Analysis (Pacific Biosciences); then, the coverage for each contig was calculated by mapping the PacBio reads to these initial contigs using BLASR [47 (link)], and contigs either showing similarity to the closely-related cp genome sequences or exhibiting similar coverage were extracted; finally, the complete cp genome was constructed by assembling these contigs. Based on the BLASR results, 3904 PacBio reads were used in the assembly of the complete cp genome, with a total length of 46,037,271 bp, thus yielding a 300× depth of the cp genome. Four junction regions between IRs and LSC/SSC were verified by PCR amplifications and Sanger sequencing. The final cp genome of S. mussotii was submitted to GenBank under the accession number KU641021.

Full length CDSs of GmChlIs, GmChlDs, and GmChlHs tagged with HA at C-terminus were cloned into pSPYCE by replacing the YFP_C fragment, and transformed into the corresponding Arabidopsis mutants, heterozygous chli1/+, chld-2/+, and homozygous gun5-2, through Agrobacterium mediated transformation (Clough and Bent, 1998 (link)). Transgenic lines were screened on MS plates against kanamycin. The genotypes at chli1 or chld-2 loci were evaluated by PCR in GmChlI-HA and GmChlD-HA transformants. Genomic DNA for PCR analysis was extracted from leaves using NuClean PlantGen DNA Kit (CWBIO, China). Transgenic plants with homozygous mutant background would be advanced to next generation for further analysis. Three-week old T₃ plants derived from three independent T₁ transgenic lines were surveyed for phenotypes and photographed. The lines that did not show a full complementation by the transgene were subject to further examinations of protein and chlorophyll content. The protein level of these transgenic lines was examined by western-blot analysis with HA monoclonal antibody (H9658, Sigma-Aldrich, USA).

DNA was extracted from theleaves using a NuCleanPlantGen DNA Kit (CWBIO, Beijing, China). To clone promoter of PsnNAC036 from P. simonii × P. nigra, the promoter sequence of the PtrNAC036 gene (https://phytozome.jgi.doe.gov/jbrowse/index.html accessed on 4 March 2021, Populus trichocarpa v3.0)was taken as a reference for designing primers pF: 5′-GCGAAGCTTGTTAGGTGCCGAATCTCCGGTGTCC-3′ and pR: 5′-CGCGGATCCCGGGTGAAACCGAAATCCCGGTGGC-3′, which contain HindШ and BamHI restriction enzyme sites, respectively (underlined). The promoter sequence was input in PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ accessed on 4 March 2021) for cis-acting element prediction. The promoter sequence was cloned into a pBI121 vector to replace its CaMV35S promoter. Then the recombinant vector was transferred into Agrobacterium tumefaciens EHA105. Using a leaf disc method [57 (link),58 (link)], it was then transformed into tobacco plants. Three homozygous transgenic lines P1, P2, and P3 and WT (control) were treated with 150 mM NaCl, 50 µM ABA, HT (37 °C), cold (4 °C), or drought for 12 h, respectively. Histochemical assays of GUS enzyme activity driven by the PsnNAC036 promoter were conducted as previously described [59 (link)].

We extracted the DNA from fresh tissue dried in silica gel by the NuClean PlantGen DNA Kit (CWBIO, China) and amplified the ITS region with primers ITS1F and ITS4 (White et al. 1990 (link); Gardes and Bruns 1993 (link)).The PCR amplification progress followed Xie et al. (2019) (link) and was sequenced by Sangon Biotech (Shanghai) Co. Ltd. The newly generated ITS sequences have been submitted to GenBank.

Genomic DNA was isolated from transgenic wheat seedlings using a NuClean PlantGen DNA Kit (CWBIO, China). Bisulphite treatment of genomic DNA was conducted using a Methylation Gold Kit according to the manufacturer's instructions (ZYMO Research Corporation). The bisulphite‐treated DNA was then subjected to PCR amplification using Hot‐Start Taq DNA polymerase (R110A, TaKaRa), using the primers listed in Table S2. Sequenced data were analysed using KISMETH (Gruntman et al., 2008). At least 10 independent clones from each PCR product were sequenced.