2.1 Sequence mining
Two SuSy sequences from Anabaena sp. PCC 7119 (An SuSy,
CAA09297) and Melioribacter roseus (Mr SuSy, AFN74551) were
used as templates for BLAST search in NCBI
(https://www.ncbi.nlm.nih.gov/Blast.cgi). The resulting 20,000 sequences
were downloaded for further analysis (in April 2020). Multiple sequence
alignment was performed using MAFFT-7.037 or ClustalW
(https://www.genome.jp/tools-bin/clustalw) with default
parameters.[23] After removing redundancy, three
putative SuSys from algae, including Micractinium conductrix(Mc SuSy, PSC73946) and Chara braunii (Cb SuSy1,
GBG73881; Cb SuSy2, GBG70160), were selected from the sequences
with conserved residues G302, G303, H438, R580, L581, K585, Q648, N654,
E675 and E683,[11, 22, 24] and used as candidates
in the subsequent experiments. The residue number refers to the sites of
SuSy from Arabidopsis thaliana (At SuSy1, CAA50317) in the
multiple sequence alignment.
The translated protein sequences of Mc SuSy, Cb SuSy1, andCb SuSy2 were used to construct a phylogenetic tree using MEGA 7.0
with the known SuSys from G. max (Gm SuSy, AAC39323),A. thaliana (At SuSy1,CAA50317; At SuSy3,CAB80721),S. tuberosum (St SuSy1, AAA33841), D. acetiphilus(Da SuSy, ADD69694), A. caldus (Ac SuSy, AIA55343),N. europaea (Ne SuSy, CAD85125), M. roseus , T.
elongatus (Te SuSy, BAC08600), and Anabaena sp. PCC 7119
by using the neighbor-joining method.[25, 26]Motifs were found by MEME according to the result of sequence alignment
and displayed by WebLogo
(http://weblogo.threeplusone.com/).[27, 28]
To predict the phosphorylation sites of SuSys, protein sequences ofMc SuSy, Gm SuSy, and the SuSy from Zea mays(Zm SuSy) were submitted to NetPhos 3.1 Server
(http://www.cbs.dtu.dk/services/NetPhos/).[29]