Yeast Two-Hybrid Screening Of 14-3-3-Interacted Proteins During Early Cotton Fiber Development

As a major source of natural fibers, cotton is an important economic crop playing an important role in the global economy. With the development of textile technology and social demand, it is especially important to breed and grow cotton varieties with high yield production and super fiber quality. Cotton functional genomics have identified a large number of fiber development-related genes, and investigated the molecular mechanisms underlying fiber development and regulation preliminarily. However, the molecular biological mechanism of fiber development is largely unknown. Genes function ultimately through directly or indirectly controlling the signal and/or metabolic pathways of cell. Protein is the product of gene expression that acts as the main function's executor in a cell; data on protein expression, modification, and interaction are very important aspects which can not obtained by transcriptomics.14-3-3 protein initially thought to be unique to animal brain tissue, but with the growing progress achieved,14-3-3 protein is ubiquitous in all eukaryotes, the known functions of this protein currently arrange from regulation of plant hormone synthesis, cell division, apoptosis, transport of other proteins'subcellular localization, regulation of metabolic enzymes, to ion transport and assembly of transcription factors.In our previous study, a differential proteome analysis was performed between the ovules of the Ligon lintless mutant(Li1) and it wild-type (WT), two differentially expressed 14-3-3 proteins were detected and identified. On basis of this result, the full length of the encoding genes was cloned by means of RACE-PCR in the present study, furthermore, 14-3-3s were used as preys to screen 14-3-3-interacted proteins by yeast two-hybrid system, thereby to probe 14-3-3-involved biological processes during the cotton fiber initiation and expansion. The major findings are as follows:1. Two 14-3-3 protein genes were cloned from cotton ovules, and temporarily named as Gh14-3-3-E1, Gh14-3-3-E2, sequence comparison indicates that the two genes encode highly conserved structures, Ghl4-3-3-E1 is a new cotton 14-3-3 protein family gene, Gh14-3-3-E2 is highly homologous to Ghl4-3-3b except for only seven nucleotide changes. The two Gh 14-3-3 genes encode a highly conserved structure showing a high similarity to other plant 14-3-3 proteins.2. Two 14-3-3 protein gene ORFs were cloned into pGBKT7 vector, and the recombinant pGBKT7-E1, pGBKT7-E2 plasmids were successfully constructed. The self-activation test showed that two bait plasmids were unable to autoactivate the reporters, toxicity test demonstrated that pGBKT7-El is toxic to yeast, whaereas pGBKT7-E2 can be used for further screening against the yeast cDNA library.3. By using the CLONTECH company's SMART and LD-PCR techniques, RNA samples were extracted from-3,-1,0 dpa ovules of the upland cotton cv Xuzhou 142, Xuzhou 142 fuzzless-lintless mutant, and Li1 and +4 and +8 dpa fibers of Xuzhou 142 and Li1 rspectively, and mixed equally. The mixed RNA was then reversely transcripted and amplified into dscDNA. The dscDNA and linearized pGADT7-Rec plasmids were co-transformed into yeast strain Y187, and a yeast cDNA library representing cotton fiber initial development was constructed.4. Six proteins which can interact with protein Gh14-3-3-E2 were screened out, the major functional regions including transmembrane, signal transduction and plant transcriptional regulation etc. Follow-up work may use cloning technology such as RACE to obtain these proteins'full-length sequence, and use site-directed mutagenesis or other methods to clarify the key interaction sites or amino acids.