Identification And Characterization Of Drought Stress Responsive Genes In Populus Hopeiensis

Populus hopeiensis Hu et Chow is a native species in Section Leuce,which is mainly distributed in northwest of China and north China.It's a drought- and chilling-tolerant tree species,therefore being an ideal material to study the mechanism of drought tolerance of woody plants.However,previous studies in P.hopeiensis mainly focused on in vitro culture,screening of somatic stress-resistant mutants,hybridization,and so on.In present study,drought stress responsive genes were screened with cDNA-AFLP in P.hopeiensis. Meanwhile,two pairs of highly similar DREB-like genes,PhCBF4a/4a and PhDREB2a/2b, were isolated from drought-stressed in vitro cultured plantlets,and their tissue-specific expression pattern and expression profile under various stress conditions were also studied. In addition,by transient expression assay and transgenic Arabidopsis assay,their function differences were characterized.The major results and conclusions are described as follows:1.Drought stress responsive genes were screened with cDNA-AFLP,and 484 differential expressed TDFs were excised from PAGE gels.457 TDFs were successfully cloned,among which,454 TDFs were found homologs in NCBI protein Database,or in Populus EST Database,or in Genome Database of P.tricocarpa,but the rest 3 TDFs, TDF45-1,TDF183,TDF280-1,found no homologs,meaning that they are probably specific genes in P.hopeiensis.Based on the expression patterns of TDFs in the different drought-stressed plantlets,the TDFs were classified into 12 clusters.And the Cluster8 and Cluster9 contained 118 and 106 TDFs respectively,occupying 49.0%of all obtained TDFs, suggesting their important roles in response to drought stress in P.hopeiensis.Meanwhile, some genes or gene families were represented by more than one TDF,also suggesting their roles in regulation of drought stress tolerance in P.hopeiensis.2.Using homolog analysis,the putative core motif of CRT/DRE cis-acting element was identified as A/GCCGACA/G in Populus.Corresponding to this motif,8 of the cloned 457 TDFs were presumable target genes of DREB-related genes of P.hopeiensis.Among them,4 TDFs shared high identity with LEA or ERD genes in other plants,and the rest 4 TDFs were probably involved in gene transcription or translation.3.By RT-PCR and RACE-PCR,PhCBF4a/4b and PhDREB2a/2b were isolated from drought stressed plantlets in P.hopeiensis.Although indel fragment and substitution of amino acids residue were found in their coding region,PhCBF4a and PhCBF4b, PhDREB2a and PhDREB2b,shared a 94.0%and 97.7%overall amino acid sequence identity each other respectively,suggesting that PhCBF4a and PhCBF4a,PhDREB2a and PhDREB2b,were two pairs of alleles,i.e.,in terms with PhCBF4a/4b and PhDREB2a/2b, P.hopeiensis was heterozygous.It was further characterized by studying the occurrence patterns of their orthologous genes in other Populus or cross population.In addition,the expression profiles of PhCBF4a/4b and PhDREB2a/2b were characterized with semi-quantitative PCR.Expression of PhCBF4a/4b was induced by drought,cold and NaCl stresses,whereas drought,heat and NaCl stresses upregulated expression of PhDREB2a/2b.Meanwhile,the expression amounts of PhDREB2a/2b were higher than those of PhCBF4a/4b under stress conditions.Moreover,PhCBF4a and PhCBF4b, PhDREB2a and PhDREB2b,showed similar but not identical expression patterns.All these revealed their similar but not identical roles in response to stresses in P.hopeiensis.4.A comprehensive phylogenic analysis revealed that the PhCBF4a/4b-like genes underwent complex evolution events in plants.In the eudicot subfamily,the existence of multiple subclades,each of which includes CBF gene members from Arabidopsis and P. tricocarpa,suggested that CBF genes were duplicated multiple times and diverged before or during speciation from the common eudicot ancestor.Meanwhile,clustering of the CBF homologs partly parallels the taxonomic classification,suggesting that duplication and divergence of some CBF genes occurred after speciation.5.Characterization of trans-activation of PhCBF4a/4b and PhDREB2a/2b was performed with transient expression assay.The results showed that PhCBF4a/4b and PhDREB2a/2b could activate expression of GUS gene,thus were trans-active.At the same time,significant differences of trans-activity of PhCBF4a and PhCBF4a,PhDREB2a and PhDREB2b were found,confirming that the indel and substitutions of amino acid resulted in the different trans-activity,thereby presumably affected their functions in regulating responsiveness to stresses in P.hopeiensis.6.Compared with the Control plants,overexpression of PhCBF4a/4b,PhDREB2a/2b in Arabidopsis not only induced strong expression of DREB target genes under unstressed conditions but also caused dwarfed phenotypes,later flowering in the transgenic plants.In addition,the transgenic plants also revealed elevated drought tolerance.All these also confirmed the trans-activation of PhCBF4a/4b and PhDREB2a/2b.7.With over-lapping PCR,two PEST fragments in 3' coding region of PhDREB2a/2b were deleted.However,the subsequent transient expression assay and transgenic Arabidopsis assay revealed that trans-activity of the obtained PhDREB2bP12d wasn't promoted.On the contrary,its trans-activity was reduced,so it could be preliminarily concluded that the post-translation regulation in AtDREB2A doesn't exist in P.hopeiensis.Taken together,the obtained results and conclusions gave new insights not only into elucidating the molecular mechanism of drought tolerance in P.hopeiensis but also into the genetic improvement of drought tolerance of woody plants.