Study Of Drought Tolerance Function Of PubZIP43 In Populus Ussuriensis

Drought,as one of the main environmental stresses affecting plant growth and development,which seriously threatens forest health.Under the current severe situation,it is of great significance to cultivate trees that have stronger resistance to drought environment.Transcription factors can participate in regulation and expression of genes related to biological and abiotic stress;transcription factors of plant genetic engineering may be an effective strategy to get the plant stress tolerance.As one of the largest and most diverse transcription factor families in higher plants,basic-region leucine zipper(bZIP)transcription factors are widely involved in plant growth and development.It also plays a vital role in biotic and abiotic stress responses.In this study,Populus ussuriensis was used as the material,and PubZIP43,one of the transcription factors with higher expression level,was cloned and the genetic transformation lines was obtained,and the drought stress resistance of the obtained transgenic line and the wild-type P.ussuriensis were compared.The main results are as follows:(1)The PubZIP43 was cloned from P.ussuriensis.The full-length cDNA of PuhZIP43 was 603 bp which encoding 200 amino acids and contains a bZIP conserved domain.The amino acid sequence comparison and construction of a phylogenetic tree revealed that PubZIP43 is closely related to Arabidopsis thaliana AtbZIP48,AtbZIP3 and AtbZIP8,and belongs to the S group member of the bZIP family.(2)The results of the subcellular localization experiment of poplar protoplasts indicated that PubZIP43 protein was localized in the nucleus.Transcription autoactivation assay demonstrated that the gene has autoactivation activity and the transcription activation region is located at the C-terminal region(145-200 aa).(3)The results of qRT-PCR found that PubZIP43 was induced by 6%PEG6000 stress and ABA treacment.The expression level of PubZIP43 in leaves under 6%PEG6000 stress was relatively stable and highest at 7 d of stress,with a 2.1-fold increase in expression compared to 0 h.expression was higher in roots than in leaves and was highest at 7 d of stress.with a 4.4-fold increase in expression compared to 0 h.Under 200 μM ABA treatment.PubZIP43 expression was also higher in roots than in leaves,with a 4.1-fold increase in expression compared with 0 h.The above results suggest that PubZIP43 is drought-induced.more sensitive to drought stress in roots than in leaves.(4)Analysis of cis-elements found that the promoter of PubZIP43 contains cis-acting elements that respond to low temperatures,cis-acting elements involved in the anaerobic induction response and cis-acting elements associated with gibberellin,methyl jasmonate and ethylene,as well as some cis-acting elements related to stress resistance such as ABRE.MBS and DRE.The ProPubZIP43::GUS vector were constructed.Clone the PubZIP43 promoter and construct the ProPubZIP43::GUS vector,and perform GUS histochemical staining analysis on the transiently infected P.ussuriensis,the results indicated that ProPubZIP43::GUS was mainly induced by drought stress in roots,which was consistent with the results of PubZIP43 gene expression pattern analysis.(5)The PubZIP43 overexpression vector(PubZIP43-OE)and PubZIP43 fused with a repressor(PubZIP43-SRDX)were constructed,and we got 11 PubZIP43-OE lines and 10 PubZIP43-SRDX lines.The wild-type(WT)and PubZIP43-OE lines were subjected to 6%PEG6000 stress,and the drought resistance function of PubZIP43 was identified by phenotypic observation and physiological index determination 7 days later.The results of growth trait comparison and physiological index measurements showed that the PubZIP43-OE lines grew well and had higher roots,stems,and leaves biomass,which was significantly better than WT.Compared with WT,the superoxide dismutase activity in the roots and leaves of PubZIP43-OE lines under drought stress and peroxidase activity is higher than WT,hydrogen peroxide content,malondialdehyde content and electrolyte leakage are lower than WT.This study clarified the drought resistance mechanism of PubZIP43.provided data and materials for the molecular breeding of poplar,and has important reference significance for the research on the resistance of P.ussuriensis.