Cloning And Functional Analysis Of Haloxylon Ammodendron SUMO E3 Ligase Gene HaSIZ1

Drought,soil salinity and extreme temperatures are important abiotic stresses limiting plant growth and crop production.SUMOylation,as a post translational modification,plays an essential role in regulating plant growth,development and stresses response.E3 ligase can directly affect the SUMOylation under abiotic stresses by mediating the binding of SUMO to target proteins,thus regulate plant responses to various stresses.At present,researches on SIZ1-mediated SUMO mainly focuses on glycophytic plants such as Arabidopsis and rice without any reports on desert plants.In current work,a desert shrub Haloxylon ammodendron was used as the plant material to analyze the responses of SUMO in the process of osmotic stress,salt,heat and chilling treatments.H.ammodendron HaSIZ1,the gene encoding SUMO E3 ligase,was cloned and its expression pattern under various stresses was analyzed,and we constructed the overexpression vector of HaSIZ1 and transformed it into WT and siz1-2 mutant of Arabidopsis.The main results are as follows:1.The total cDNA length of HaSUMO1 is 632 bp with its ORF 291 bp,encoding 96 amino acids;the molecular weight of HaSUMO1 is about 10.9 kDa,and the amino acid homology with AtSUMO1 is about 86%.Anti-AtSUMO1 antibody can be used to detect SUMO protein of H.ammodendron.2.The high temperature of 50 ℃ induced rapid accumulation of SUMOconjugated proteins to a high level,however,-0.75 MPa osmotic stress,350 mM NaCl and the low temperature of 4℃ induced only a small accumulation of SUMOconjugated protein in H.ammodendron seedlings.3.H.ammodendron HaSIZ1 with full-length 3148 bp and ORF 2598 bp was cloned.Tissue-specific analysis showed that HaSIZ1 can express in the whole plant of H.ammodendron.-0.75 MPa osmotic stress,350 mM NaCl and the low temperature of 4℃ significantly induced the expression of HaSIZ1,while 50℃ reduced its expression.4.SIZ1 was localized in the nucleus,containing SAP,SP-RING ZINC FINGER and PHD domains,and identified as the SUMO E3 ligase of H.ammodendron.Phylogenetic tree analysis showed that HaSIZ1 belongs to SIZ1 family,is closer to Arabidopsis AtSIZ1 and further to rice OsSIZ1.5.The 35S::HaSIZ1 vector was constructed and then was transformed into Arabidopsis WT plants and siz1-2 mutant plants,respectively.The homozygous line with high expression level of HaSIZ1 was obtained.Above results preliminarily indicate that HaSIZ1 as a SUMO E3 ligase plays important roles in regulating the SUMOylation of H.ammodendron seedlings in response to drought,salinity,heat and chilling stresses.