Analysis Of Functional Domains Of AtSUA41

AtSUA41（SUMO substrate in Arabidopsis41）is a nucleoporin and also called as Nup96/MOS3/SAR3. It is involved in mRNA export from the nuclus and required for basal defense,constitutive resistance responses. sua41mutant displays an early flowering phenotype. AtSUA41wasinitially identified as a SUMO target in our lab. This study aims to verify the sumoylation of SUA41and to explore the function of its domain. We predicted the putative sumoylation sites and analyzed theconservation among different species bioinfomatically. We identified the main site as K534. Next, weanalyzed the interaction of SUA41with SUMOs and sumoylation enzymes by yeast two-hybrid. We gotthe mutant SUA41(K534R) gene (SUA41-9), and then constructed35S:SUA41-9. We over-expressedthe SUA41-9in sua41mutant and analyzed the phenotype of transgenic lines. At the same time, in orderto well study the sumoylation of proteins in plants, we constructed a series of vectors Fu40for identifyand verify SUMO targets. We used the known SUMO target MYB30to verify the BiFC (BimolecularFluorescence Complementation) vector, and confirmed the interaction of SUA41with SUMO1. Inaddition, we predicted major functional domains in SUA41and cloned eight deletion mutated SUA41genes (SUA41-1~8). Then, we over-expressed these mutated genes in sua41mutant to explore thefunction of these functional domains. The main results were as follows:1. We got3putative sumoylation sites in SUA41by bioinfomatics, K534, K539, and K698. Weconstructed a series of vector with these site mutated.2. We verified the sumoylation of SUA41. SUA41interacted with SUMO1, SUMO2, SUMO3, andSUMO conjuction enzyme E2in yeast. But none of the specific proteases (among ESD4, ELS1, OTS1,and OTS2) interacted with SUA41.3. We got the mutant SUA41(K534R) gene (SUA41-9) and constructed35S:SUA41-9. Weover-expressed the SUA41-9in sua41mutant, and found out that over-expressed SUA41-9can’t rescuethe early flowering phenotype of sua41mutant. The results indicated that early flowering phenotype insua41may be related to its sumoylation on the K534site.4. We constructed a series of vectors Fu40to identify and verify SUMO targets. It would used forBiFC, Co-localization, western, and Co-IP (Co-Immunoprecipitation) experiment. Then we verified theBiFC vector with the known SUMO-target MYB30the Arbidopsis protoplasts. With this system, wefound out that SUA41indeed interacted with SUMO.5. We cloned eight deletion mutated SUA41genesand got the T0generation oeverexprssing theSUA41-1mutated gene in sua41mutant.In summary,our paper found the key sites K534of SUA41that is one of the NPCs.We confirmedthat early flowering phenotype of sua41is relevant to sumoylation of SUA41.We also cloned8deletionmutated SUA41genes, which will be helpful in further study.