Study On Salt Tolerance Function Of PdbRAP2-12 Gene In Populus Davidiana×p.bolleana

Transcription factor(TF)is a type of DNA binding protein that can specifically bind to cis-acting elements on gene promoters.It can regulate a series of downstream genes expression by binding to cis-acting elements on the promoters.So it is extremely important that transcription factors with excellent salt tolerance were screened from stress-resistant plants in genetic engineering breeding.In this study,a PdbRAP2-12 gene were cloned and obtained from Populus davidiana×P.bolleanaPopulus davidiana×P.bolleana.The expression patterns of PdbRAP2-12 gene under three abiotic stresses and ABA treatment were analyzed.Further,the salt tolerance function of PdbRAP2-12 was identified by transit expression poplar.The specific results were as follows:A RAP2-12 gene(named PdbRAP2-12)with complete open reading frame(ORFs)was screened by searching the transcriptome sequences established in the previous period under salt stress.The coding regions lengths of PdbRAP2-12 gene were 1191 bp,with the numbers of coding amino acid residues 396.The predicted relative molecular weight was 43.9 kDa,and the theoretical isoelectric point(pI)was 4.93.The results of multiple sequence alignment showed that the PdbRAP2-12 transcription factor had the typical structural characteristics of the ERF family of AP2/ERF.The results of subcellular localization and transcriptional activation analysis of PdbRAP2-12 showed that PdbRAP2-12 was located in the nucleus and had transcriptional activation activity,and the transcriptional activation domain was located in 178-397aa(without the intermediate region of the conserved domain).The expression patterns of PdbRAP2-12 gene in the roots,stems and leaves of Populus davidiaua×P.bolleana treated with abiotic stress(high salt,drought,heavy metal)and hormone treatment(ABA)were monitered by qRT-PCR.The results showed that the expression levels of PdbRAP2-12 gene changed significantly at most time points after the four stresses,and may be involved in at least one kind of stress resistant response of Populus davidiana ×P!bolleana.Especially under salt stress,the expressions of PdbRAP2-12 gene changed significantly at μmost time points in Populus davidiana×P.bolleana leaves,which may play an important role in the salt stress response process of Populus davidiana×P.bolleana.Recombinant vector pROKII-PdbRAP2-12 was constructed to transform Populus davidiana×P.bolleana.Meanwhile,pROKII was used as control to infect Populus davidiana×P.bolleana.The trasncripts of PdbRAP2-12 gene in two transient transgenic poplar strains co-cultured at different time points was analyzed.The results showed that the expressions of PdbRAP2-12 gene after co-cultured for 48 h was the highest in transient overexpression(OE)lines.Determination and comparison of physiological indexes in the two transient transformation lines before and after salt stress,the results show that the transeint overexpression PdbRAP2-12 gene imporved salt tolerance through higher the protective enzyme activity,enhance the capacity of ROS in cells,prompting O2-and H2O2 accumulation is reduced under NaCl stress,thus reducing the damaged cells or cell death,indicated that PdbRAP2-12 gene may be a good salt tolerance candidate genes.At the same time,the overexpression vector of PdbRAP2-12 gene was stably transformed into the poplar,and the resistant seedlings were obtained,which laid a foundation for further analysis of the salt-tolerant function of PdbRAP2-12 gene.The possible cis-acting elements of PdbRAP2-12 TF were further analyzed by using the TF-Centered Y1H technique(a yeast single hybrid technique centered on transcription factors).RandomLy sequencing analysis of selected elements showed that GCC-box element(AGCCGCC)and unknown element W1(AAACACTGTT)appeared the most frequently.The deletion and mutation analysis of unknown element bases showed that"CTGT"was the core region of the recognition of PdbRAP2-12.The"CTGT" element was found in promoter regions of several salt stress related genes.Real-time quantitative RT-PCR analysis showed that the expression of three genes(PdaPOD12、Pd3POD73、PdaPOD5)was significantly increased in the overexpression lines of poplars.In the following studies,To clarify the salt tolerance regulation mechanism of PdbRAP2-12,whether these genes are downstream target genes directly regulated by PdbRAP2-12 will identify,...