*4.2. Sequence Alignment and Subcellular Localization*

The open reading frame (ORF) was amplified using Takara LA Taq (Takara, Dalian, China) based on the sequence obtained from transcriptome data (Table 1), and the PCR product was purified and cloned into the pMD19-T vector (Takara) for sequencing verification. Protein sequence was used for alignment using ClustalW (v. 2.1) software, and a phylogenetic tree was constructed using MEGA 7.0 with the neighbor-joining method following 1000 bootstrap replications.

**Table 1.** Primer sequences of the genes for full-length open reading frames (ORFs) and semiquantitative PCR and quantitative real-time PCR.


A ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China) was selected to construct the recombinant plasmid vector containing the target sequence without the stop codon and the pBI121–GFP vector. The recombinant plasmids were transformed into *A. thaliana* protoplasts, which were isolated with enzymatic hydrolysis [44,45] using the PEG transformation method. Confocal laser scanning microscopy (Zeiss LSM 710 META, Jena, Germany) was used to determine the localization of gene expression through observing GFP fluorescence [46].
