Than 24 h. Variable-to-maximum fluorescence (Fv /Fm = (Fm – F0 )/Fm ) was
Than 24 h. Variable-to-maximum fluorescence (Fv /Fm = (Fm – F0 )/Fm ) was measured applying a FC1000-H fluorescence imaging method (Photon Systems Instruments, Czech Republic) to ascertain the growth status from the GSK2636771 Technical Information thallus (Table S3). For dehydration remedies, the samples had been placed inside a dry petri dish inside the Dynasore In Vivo darkness for 2, 4, 6, eight, and ten h at 20 C; for high-temperature stress, the samples had been placed inside the darkness at 30 C for 1, 2, three, 4, and 5 h; the samples have been placed in 12, 60, and 300 mM NH4 Cl for two h inside the darkness at 20 C for ammonium salt strain.Molecules 2021, 26,12 ofFigure 9. The sampling sites of P. haitanensis. The thallus was collected from Putian (25 28 N, 119 02 E), Dongtou (27 51 N, 121 08 E), Cangnan (27 30 N, 120 24 E), and Yancheng (33 24 N, 120 09 E).four.two. Cloning and Sequence Evaluation of PhGDH1 and PhGDH2 Total RNA was extracted using the Plant RNA Kit (OMEGA, China) and converted into cDNA with all the Transcriptor Very first Strand cDNA Synthesis Kit (Takara, Japan) as outlined by the manufacturers’ guidelines. Sequences annotated as GDH in the transcriptome of P. haitanensis (accession: PRJNA428906, accessed on eight January 2018) had been BLAST against the NCBI nucleotide database, after which two GDH sequences (PhGDH1 and PhGDH2) with all the highest identities were chosen. The PhGDH coding sequences are listed in Table S4. The open reading frames (ORFs) of PhGDH1 and PhGDH2 have been amplified with primers of PhGDH1-F, PhGDH1-R, PhGDH2-F, and PhGDH2-R (Table S5) and 2Phanta Master Mix (Vazyme, China). PCR program was as follows: 98 C for five min; 35 cycles of 98 C for ten s, 55 C for 15 s, and 72 C for 90 s; and 72 C for 10 min. The obtained PhGDH1 and PhGDH2 coding sequences had been translated into amino acid sequences with ORF Finder [39], which have been then aligned with other GDH proteins by CLUSTALW (https://www.genome.jp/tools-bin/clustalw, accessed on 25 February 2021). The physical and chemical parameters (molecular weight, isoelectric point) of PhGDH1 and PhGDH2 were predicted with ProtParam [40], plus the motifs of PhGDH1 and PhGDH2 had been analyzed by the MOTIF tool (http://www.genome.jp/tools/motif/, accessed on 25 February 2021). The subcellular localization of PhGDHs was predicted by TargetP v1.1 [41], and the SignalP v4.1 Server (http://www.cbs.dtu.dk/services/SignalP-4.1/, accessed on 25 February 2021) was employed to predict signal peptides [41]. The transmembrane helices had been predicted with the TMHMM Server v2.0 (http://www.cbs.dtu.dk/services/ TMHMM/, accessed on 25 February 2021). The tertiary structures of PhGDH1 and PhGDH2 had been predicted by SWISS-MODEL [42], plus the secondary structures have been illustrated by ESPript [43]. four.3. Expression and Purification of PhGDH1 and PhGDH2 The pET and pCold systems have been used to express PhGDH1 and PhGDH2 in vitro, respectively. The full-length ORF of PhGDH1/PhGDH2, which was amplified as an EcoR I/Hind III fragment by PCR, was cloned in to the vectors pET-32a and pCold-I with His-Molecules 2021, 26,13 oftagged. PCR program was as follows: 98 C for 5 min; 35 cycles of 98 C for ten s, 55 C for 15 s, and 72 C for 90 s; and 72 C for 10 min. The Escherichia coli cells (BL21 (DE3) pLysS and Transetta (DE3)) had been transformed using the recombinant expression plasmids (pET-PhGDH1 and pCold-PhGDH2). The transformed E. coli cells were then incubated in 1 L of Luria ertani (LB) medium with one hundred L of ampicillin and 20 L of chloramphenicol at 37 C. When OD600 reached 0.six, 0.1 mM IPTG was suppleme.
