Functional Analysis Of SUMO E3 Ligase CpSIZ1 From Wintersweet (Chimonanthus Praecox (L.) Link)

SUMO modification is an important one of protein post-translational modification regulation,which is an important way for organisms to participate in self-regulation when responding to changes in the external environment.SUMO protein-mediated SUMO modification can be linked to the target protein through an isopeptide bond,thereby regulating the plant’s adaptability to growth and development and abiotic stress.The process of SUMO modification involves a series of enzymes,including E1activation,E2 conjugation and E3 ligation enzymes.Among them,SUMO E3 ligases promote the SUMO protein targeting the substrate and enhance the specificity of the substrate.The SIZ1 gene has been extensively studied in plants.Some studies indicated that the SIZ1 gene was widely involved in the regulation of plant growth and abiotic stress responses.Wintersweet（Chimonanthus praecox）is a unique winter flowering plant in China.Its unique flowering characteristics under cold stress may involve the regulation of a large number of proteins.To date,SUMO modification has not been reported in wintersweet.In this paper,the CpSIZ1 was first isolated from wintersweet.Then,the sequence characteristic,expression pattern and biological function of CpSIZ1were carried out to investigate the regulatory role of CpSIZ1 in growth and stress response in wintersweet.The main results are as follows:（1）Cloning and molecular characteristics of CpSIZ1 gene from wintersweet.Based on the sequence obtained from the transcriptome database of flower development,a 5’RACE and 3’RACE was performed to get the cDNA sequence of CpSIZ1.Sequence analysis showed that the 4299 bp full-length cDNA of CpSIZ1（Gene bank accession number:MH454597）contains a 2523 bp open read frame（ORF）,a 540 bp 5’-untranslated region（UTR）,and a 1236 bp 3’-UTR.The gDNA sequence of CpSIZ1 was cloned from the genomicDNA of wintersweet,and it was 6655bp.Compared with the cDNA sequence of CpSIZ1,the gDNA sequence contained four introns with lengths of1073 bp,1043 bp,137 bp,and 103 bp,respectively.All the introns had the standard GT/AG splicing site.（2）Characterization of the deduced CpSIZ1 protein.The deduced CpSIZ1 protein was analyzed by the online software Prot Param 10.0,the resuts showed that the peptide had a molecular forum of C₄₀₂₃H₆₃₀₇N₁₁₂₅O₁₂₈₉S₃₉,a calculated molecular mass of 92.32kD and a theoretical isoelectric point of 4.58.The instability coefficient of CpSIZ1protein is 38.81,which indicating that the CpSIZ1 protein is a stable hydrophilic protein.According to BLAST search tool in NCBI and lasergene 7.0,CpSIZ1 protein belongs to SIZ/PIAS type of SUMO E3 ligase and contains five typical conserved domains:SAP,PHD,PINIT,SP-RING and SXS domains.A CpSIZ1 subcellular localization expression vector containing a GFP tag protein was constructed and transiently expressed in tobacco.It was found that the CpSIZ1 protein can be located in both the nucleus and the cell membrane,which is different from the localization of other plant SIZ1 homologous proteins.（3）Expression analysis of CpSIZ1 in wintersweet.The q RT-PCR was performed to detect the expression pattern of CpSIZ1 in different tissues and organs and flowers at different developmental stages.The results showed that the expression of CpSIZ1 gene was highest in flowers,followed by leaves,and lowest in other vegetative organs such as roots and stems.Among the various stages of flower development,the stage 6showed the highest expression of CpSIZ1.In mature leaves of adult wintersweet plants at different months of the year,the expression of CpSIZ1 in mature leaves was significantly up-regulated in November to January.The expression pattern of CpSIZ1under different abiotic stresses and hormone treatments was detected by real-time quantitative PCR.The results showed that the expression of CpSIZ1 was induced by abiotic stresses like low temperature（4℃）,drought（PEG stress）and salt（NaCl Stress）,but it was not related to the high temperature（42°C）stress.Meantime,the expression of the CpSIZ1 was induced by the various hormones like IAA,GA and ABA,but it has little to do with SA hormones.Real-time quantitative PCR was used to detect the expression of CpSIZ1 gene in wintersweet flowers under various hormone treatments.The results showed that,during the ABA hormone treatment from 12h to 72 h,the CpSIZ1 in wintersweet flowers showed significant up-regulated expression comparing to the control flowers.Only GA hormone treatment for 12h can induce the significant up-regulation of CpSIZ1 gene in wintersweet flower.After the treatment time is extended to 24h～72h,the expression of CpSIZ1 gene in wintersweet flower gradually increases from no significant up-regulation to almost consistent with the control.（4）Sequence cloning,bioimformatic analysis and expression analysis of the promoter of CpSIZ1.A CpSIZ1 promoter with the length of 3398 bp was isolated using the Genome Walking method and named"CpSIZ1pro".Bioinformatics analysis shows that besides the basic promoter elements like TATA-box and CAAT-box,the promoter also has typical stress response elements such as low temperature and ethylene,typical hormone response elements such as abscisic acid,gibberellin,and auxin,and multiple MYB binding sites involved in abiotic stress and hormonal regulation such as drought and ABA.The full-length CpSIZ1pro was constructed to a plant expression vector containing the GUS reporter gene and transformed into tobacco for transient expression analysis.The results showed that the full-length CpSIZ1pro was active.Stable expression analysis in Arabidopsis showed that the full-length CpSIZ1pro promoter can drive GUS gene expression in most tissues and organs of transgenic Arabidopsis,with relatively high expression levels in leaves and flowers.（5）Analysis of Stable Expression of 5′Deletion of CpSIZ1 Promoter.Six 5’deletion fragments of different lengths were constructed,with lengths of 1832bp,1390bp,767bp,729bp,540bp,and 240bp,and were named CpSIZ1pro-P1/P2/P3/P4/P5/P6,respectively.With ATG as the starting position,the transcription start site TSS is located at-664.The stable expression analysis of different promoter fragments showed that the-1832～-1390 region might be the negative regulatory region of the CpSIZ1 promoter.The fragment length of CpSIZ1pro-P3（-767）was only 1/5 of the CpSIZ1pro（-3398）,but its GUS activity accounted for 80%of the GUS activity of CpSIZ1pro.The CAAT-box located in the-767～-729 region may play a key role in the transcription efficiency of the CpSIZ1 promoter.The 5’UTR region of the CpSIZ1promoter is active,and its activity is mainly regulated by the photoperiod.The GUS enzyme activity of the full-length and 5’deletion of CpSIZ1 promoter in transgenic Arabidopsis under various abiotic stress and hormone treatment was measured.The results showed that IAA,GA,ABA hormone and PEG stress can induce the up-regulated expression of GUS enzyme activity in transgenic Arabidopsis seedlings.High temperature treatment（42℃）and low temperature treatment（4℃）could induce the down-regulated expression of GUS enzyme activity in transgenic Arabidopsis seedlings.the auxin cis-acting elements at-1780 and-507 are involved in the regulation of IAA hormones,and the auxin cis-acting elements at-507 played a major role.The gibberellin response element P-box（-1750）located in the negative regulatory region can also participate in the regulation of the promoter GA,but may require the participation of some sequences in-3398～-1832.The MYC cis-acting element at-3398～-729,the ABA response element（ABRE）,and the MYC cis-acting element at-642 may all participate in the regulation of ABA hormones.MYC cis-acting elements in-3398～-1832 can participate in PEG stress.The full length of CpSIZ1 promoter and its 5′deletion fragment did not show significant response to high and low temperature stress.（6）Phenotype observation of Arabidopsis transgenic plants with CpSIZ1 gene.The pCAMBIA2301G-CpSIZ1 overexpression vector was constructed and transferred into the siz1-2 mutant and WT wild-type Arabidopsis,respectively,to obtain the complementation lines（HB）and the overexpression lines（OE）.After resistance screening,PCR amplification identification,and q RT-PCR expression analysis,homozygous OE and HB strains with relatively high expression levels were selected for phenotypic observation.The results showed that the HB lines can restore the dwarf phenotype of the siz1-2 mutant in growth,the early flower phenotype in flowering time,the sensitive phenotype under low temperature treatment,and the sensitive phenotype under ABA hormone treatment.Specifically,the HB lines showed a similar phenotype to the WT plants regarding to the growth and development,flowering time,and ABA hormone treatment.Meantime,it showed a stronger low-temperature resistance phenotype than WT under low temperature stress.The phenotype of OE lines also showed the same phenotype as HB lines compared to WT plants.Not only that,both the statistical data and phenotypic observations showed that the rosette leaves of HB and OE lines aged faster than that of WT.This indicates that CpSIZ1 may be involved in the growth and development of plants,including leaf senescence,flowering regulation,and response to low temperature and ABA stress.（7）Preliminary analysis of the CpSIZ1 interaction protein of wintersweet.The bait plasmid pGBKT7-CpSIZ1^C was constructed for the verification of CpSIZ1 interaction protein,which contained 1-536 amino acids of CpSIZ1.The reported abiotic stress and senescence related genes in wintersweet have been verified by the bait plasmid pGBKT7-CpSIZ1^C,including the genes related to low temperature stress:CpICE1a,CpICE1b and CpDERB1;abiotic stress related MYB family genes:CpMYB1,CpMYBR1,CpMYB5,and etc;and WRKY genes related to aging:Cp34517,Cp12441and Cp12571.The results showed that there were not interactions between these genes and the CpSIZ1 protein.The target proteins of CpSIZ1 needed further research.