Screening And Identification Of Interacting Proteins Of Two Cotton14-3-3Proteins

In our previous research, we found that two14-3-3proteins were differentially expressed in the wild type and a cotton fiber developmental mutant. One (EST: gi|78344446) was up-regulated in the-3DPA ovules of a fuzzless-lintless mutant, while another (EST:gi|783240560) was repressed in the0DPA ovules of this mutant compared to its wild parental line of Xuzhou142. Thus14-3-3protein may play a critical role in regulating cotton fiber development.14-3-3proteins are highly conserved regulatory proteins that ubiquitously exist in eukaryotes, because they exert their functions mainly through interacting with other proteins, therefore, it is necessary to investigate the interactome of14-3-3protein for unraveling their function.In this study, we first molecularly clone the full length of above two14-3-3protein encoding genes, and then cloned into pET30prokaryotic expression vector and transformed into E.coli BL21(DE3). The recombinant6×His-Tagged proteins were expressed under IPTG induction and subsequently purified with Ni-NTA resin. Proteins were extracted from-3,-1,0DPA ovules and3and6DPA fibers of the wild-type cotton cv. Xuzhou142, Xuzhou142fuzzless-lintless mutant, TM-1and Ligon lintless mutant (Li-1) respectively and mixed equally. Using this protein mixture as protein source that contains putative "prey" proteins, two14-3-3proteins as "bait" proteins, pull-down assays were conducted respectively. Cotton14-3-3protein interacting proteins were further isolated by2-DE and identified by MALDI-MS/MS analysis. Our results pave the way for better understanding of the role of14-3-3proteins in cotton fiber development.The major findings are as follows:1. According to the EST sequences of gi|78344446and gi|783240560, the full lengths of genes encoding two14-3-3protein were cloned by means of RACE-PCR from cotton. Sequence alignment shows that one is a new cotton14-3-3protein family gene, which was nominated as Gh14-3-3-like protein2(GenBank Accession No. JF320796), the other is highly homologous to Gh14-3-3b (GenBank Accession No. EU189221) with only three amino acid alteration, so it is regarded same as protein Gh14-3-3b.2.6×His Tag was added to the3’end to the two full-length Gh14-3-3protein coding genes and were cloned into pET30prokaryotic expression vector. Two proteins were over-expressed under1mM IPTG induction. The molecular weight of both proteins was about30kD determined by SDS-PAGE analysis, which is consistent to the theoretical value. The recombinant proteins were successfully purified with Ni-NTA resin.3. Using the two Gh14-3-3proteins as "bait" proteins, the interacting proteins of each cotton14-3-3protein were pulled down from the protein source by equally mixing protein samples from-3,-1,0DPA ovules and3and6DPA fibers. The pull-down protein samples were further isolated by2-DE, and the14-3-3interacting proteins were identified by MALDI-TOF-TOF MS/MS analysis.23proteins were detected to interact with Gh14-3-3-like protein2, and7of them were identified. The putative functions involve stress responses, protein and nucleic acid metabolism, cytoskeletal organization, transmembrane transportation, carbon/energy metabolism, and signal transduction.29proteins were found to interact with Gh14-3-3b,16of them were identified. They are implicated in stress responses, protein transport and subcellular localization, protein synthesis, protein construction, cytoskeleton organization, and other biological function. The identification of these cotton14-3-3interacting proteins throw light on further understanding of the regulation mechanisms of14-3-3proteins in early cotton fiber development.