Cloning And Expression Of S.lycopersicum SYTA And Screening Of Its Interacted Protein

Synaptotagmin(SYT) is generally existed in the genome of annimals and arabidopsis thaliana, which contains a C2 domain struture. Now, many proteins containing C2 domain had been reported, most of which involves in the cell signaling transduction and membrance transport. All SYT contains five domain strutures: a short uncleaved N–terminal signal peptide that overlaps a transmembrane(TM) domain, followed by a cytosolic variable region and two C–terminal C2 domains, C2 A and C2 B. Whereas C2 A and C2 B are essential for activity. There are six SYT homologs in arabidopiss, which named SYTA to SYTF one by one. It had been reported that arabidopsis SYTA played a important role in the regulation of the signaling response to the salt stress. Recent report showed that the arabidopsis SYTA interacted with the movement protein of TMV. In the SYTA silenced mutants, the cell to cell spread of movement protein was significantly inhibited.Solanum lycopersicum, as a member of Solanaceae, is typical plant for studying the development and mature of fruit. Based on the published genone of Solanum lycopersicum in 2012, we found the homolgous of SYTA in S. lycopersicum. In order to definite the sublocation of the S. lycopersicum SYTA and screen the protein that interacted with it, we cloned the S. lycopersicum SYTA and inserted it into the plant expression vector and analyzed the sublocation of it. Further more we screened the cDNA library of tobacco by yeast two hybrid systerm with S. lycopersicum SYTA as the bait and found that ferredoxin I(Fd–I) could ineracted the it directly in the yeast. Detailed results are as fellowing:1. We cloned the S. lycopersicum SYTA, 1620 bp in length, with the specific primer pair deisgned based on the genome of S. lycopersicum. Sequence analysis showed that it was 99.9% identical to that of the genome of S. lycopersicum, and only one amino acid was mutated from Lys to Glu. S. lycopersicum, and were 71.1%, 91.1%, 91.1%, 71.2%, 83.1%, 80.9%and 79.4% identical in nucleotide to that of Arabidopsis thaliana, Nicotiana sylvestris, Nicotiana tomentosiformis,Cucumis sativus, Sesamum indicum, Theobroma cacao and Populus euphratica, respectively. The amino acid sequence S. lycopersicum SYTA were 73.6%, 91.2%, 91.2%, 71.6%, 83.3%, 81.1% and 79.6% identical to that of Arabidopsis thaliana, Nicotiana sylvestris, Nicotiana tomentosiformis,Cucumis sativus, Sesamum indicum, Theobroma cacao and Populus euphratica, respectively.Prediction of the secondary struture of S. lycopersicum SYTA online showed that the secondary struture of S. lycopersicum SYTA was similar to that of the arabidopsis, which contained a short uncleaved N–terminal signal peptide that overlaps a transmembrane(TM) domain, followed by a cytosolic variable region and two C–terminal C2 domains.2. The recombined vector Ppzp–SYTA–EGFP was constructed by insetting the S. lycopersicum SYTA into a plant expression vector. The fused protein EGFP–SYTA was expressed in the Nicotiana benthamiana by agroinfiltration. The green fluoresence was detected in the leaf cells of Nicotiana benthamiana and the EGFP–SYTA was mainly located in the cell membrance, some in the periphery of the cytoplasm.3.We constructed the pSTT91–SYTA by insertting S. lycopersicum SYTA into the pSTT91 and used it as a bait to screen the cDNA library of tobacco by yeast two hybrid systerm. The yeast cells which could grow on the plate were tested by the X–gal to confirm the interaction. Sequence analysis showed that ferredoxin I(Fd–I) was screened out to interacted with S. lycopersicum SYTA. The we tested the interaction between S. lycopersicum Fd–I and S. lycopersicum SYTA, and found that they could interacted with each other in the yeast cells. In previous study, we had confirmed the interaction of S. lycopersicum Fd–I ToMV coat protein(CP). Taken together, we may got an intresting intraction of ToMV CP– S. lycopersicum Fd–I– S. lycopersicum SYTA– ToMV MP. The results of further study on biological effect of the interaction may shed light on the pathological mechanism of ToMV.