Isolation and Characterization of SNAP33 Protein

Total RNA was isolated from cotton seedlings using a kit (Promega, Madison, WI, United States) according to the manufacturer’s instructions. The PolyATract mRNA Isolation System (Promega) was used to generate polyadenylated mRNA. The cDNA library was prepared as previously described (Wang et al., 2011b (link); Liu et al., 2017 (link)) and propagated on 140 mm plates to obtain about 10⁶ clones. The conserved region of SNAP33 (Heese et al., 2001 (link); Wang et al., 2017 (link)) was used as a probe to screen for positive clones (Liu et al., 2016 (link); Wang et al., 2017 (link)).
The theoretical isoelectric point (pI) and molecular mass were calculated with ProtParam¹. A transmembrane hidden Markov model (TMHMM) analysis of the transmembrane domain was performed using the TMHMM online tool². Multiple amino acid sequence alignment was performed with Clustal Omega³, and the multiple alignment file was shaded with BoxShade⁴. A motif analysis was performed using the National Center for Biotechnology Information (NCBI) conserved domain search program⁵. A phylogenetic tree was constructed with the neighbor-joining method using MEGA 6, with bootstrap values from 1000 replicates indicated at the nodes.