Regulation Analysis Of Stress-Related E3 Ubiquitin Ligase SINA2 In Arabidopsis And Cloning Of Homozygous SINA Gene In Wheat

Abiotic stresses,such as drought,salt and extreme temperature,have adverse effects on plant growth and development.Plants have evolved complex and elaborate signaling networks that perceive signals from their surroundings and respond appropriately.Under abiotic stresses,plants can modulate the expression of stress-related genes,resulting in adapting to changing environment.It is essential to investigate the mechanism of plant response to abiotic stresses in the effort to improve plant resistance.E3 ligases in plant are involved in several signaling pathways.SEVEN IN ABSENTIA(SINA)is a ubiquitin ligase involved in Drosophila eye formation.SINA contains a conserved RING domain,which is crucial for the activity of E3 ubiquitin ligase.Of 18 SINA-like proteins identified in Arabidopsis,SINA2 lacks a canonical RING domain which is found in other SINAs.In this study,the role of SINA2 in Arabidopsis was investigated under abiotic stresses.Recombinant SINA2 was expressed in E.coli and the activity of E3 ubiquitin ligase of SINA2 was analyzed in vitro;Yeast two hybrid screening was performed to identify SINA2-interacting proteins;the expression and function of SINA2-interacting protein were charactered.In addition,TaSINA1 homologue gene was cloned in wheat.The main results are described as follows.1.Multiple sequence alignment showed that SINA2 lacks a canonical RING domain,contains a modified B-box2 domain.Phylogenetic tree showed that SINA2 has a far evolution relationship with other SINAs in Arabidopsis.Recombinant SINA2 was expressed in E.coli as a fusion protein with a 6×His epitope tag,and the recombinant SINA2 catalyzed auto-ubiquitination in vitro,with a preference for UBCH5 a.2.SINA2 was localized in nucleus and cytoplasm.SINA2 was expressed in every organ,with highest expression in flower and capsule.The expression of SINA2 was induced by drought,salt and ABA treatment.sina2 mutants are sensitive to drought,salt and ABA treatment,the reduced rate of seed germination and cotyledon greening were more severely in mutant lines than in WT.3.CDKG1 was identified to interact with SINA2 by yeast two hybrid screening.MBP-CDKG1 fusion protein was expressed in E.coli,and was able to pull down His-SINA2 in vitro.Vectors that express YFPN-SINA2 and YFPC-CDKG1 were co-transformed into N.benthamiana leaf epidermal cells by agroinfiltration.Strong fluorescence signals were observed in nuclei of cells that transiently co-express YFPN-SINA2 and YFPC-CDKG1.4.CDKG1 has Ser/Thr phosphorylation activity in vitro.Recombinant His-SINA2 was phosphorylated in E.coli,λ protein phosphatase can remove the putative E.coli-mediated phosphorylation,while CDKG1 increases the phosphorylation of de-phosphorylated His-SINA2.Phosphorylation of SINA2 by CDKG1 promotes its ubiquitin ligase activity.HIS-SINA2 that was dephosphorylated by treatment with λ protein phosphatase showed very weak auto-ubiquitination activity,while CDKG1-dependent rephosphorylation of this dephosphorylated His-SINA2 rescued its activity to a considerable extent.5.CDKG1 contains a conserved Ser/Thr-type protein kinase domain,and has a close relation with CDKG1 homologue proteins in other plants.CDKG1 was localized in nuclei.CDKG1 is ubiquitously expressed in seedlings and in all organs examined,including roots,stems,leaves,flowers and siliques of Arabidopsis plants.Highest expression of CDKG1 mRNA was detected in flowers and siliques of mature plants.Under drought,salt stress and ABA treatment,the proportion of seed germination and seedlings with green cotyledons remained somewhat higher for 35S::CDKG1 lines than for WT,while cdkg1 mutants are more sensitive to those stresses,with severe inhibition of germination and cotyledon greening.6.Three SINA homologue genes were identified in wheat,which named as TaSINA1,TaSINA2 and TaSINA3.Amino acid sequence alignment showed that three TaSINAs contains a conserved RING domain.Of three SINA homologues in wheat,TaSINA1 was located in cytoplasm and nuclei.The expression of TaSINA1 was induced by drought,salt stress and ABA treatment.