The Functional Analysis Of DREBs Transcription Factors In Plant Stress Tolerance--Isolation Of DREBs Transcription Factors And The Functional Analysis Of Its Stress Tolerance In Peanut (Arachis Hypogaea L.)

Environmental stress conditions such as drought,high salinity and extreme temperature are known as abiotic stresses.They impaire the growth and productivity of plants greatly,and reduce the potential yields as high as 70%in crop plants.Transcription factors(TFs) have been reported to play crucial roles in plant development,they can specially interact with cis-acting element and therefore control the expression of many functional genes downstream.Among them,there are 5 families,such as AP2/EREBP,bZIP,WRKY,MYB and NAC,are reported to play key role in plant stress resistance and tolerance by regulating the expression of stress-inducible genes and participation in stress signal transduction.Here we have isolated a dehydration-responsive element-binding proteins(DREBs)-like gene,PNDREBl(FM955398) by screening a full-length cDNA library of peanut(Arachis hypogaea L.) immature seeds.PNDREB1 shared high protein sequence homology with other DREBs genes,identities of 57%to GmDREB6(EF551166),51%to HvCBF7(AY785864),42.1 and 44.4%to DREB1A and DREB1B(AB007787,AB007788),respectively.The gene encodes 227aa with caculated molecular mass of 24.7 kDa.PNDREB1 contains a putative NLS located near the N-terminus,and a putative acidic activation domain in C-terminal end,with a highly conserved APETALA2(AP2) binding domain about 64aa,the two amino acids of 14^th(valine) and 19^th(glutamic),which have been reported crucial in the determination of DNA-binding specificity,were all be found within this region.In order to confirm the functions of PNDREB1 biochemically and physiologically,the yeast hybrid system and the genetic transcription were carried out.The results showed that PNDREB1 can specially interact with DRE cis-acting element,by which the PNDREB1 AP2 domain could be verified:the C-terminal end which could function as a transcriptional activator was also proved.To analyze the expression patterns of PNDREB1 in peanut,particularly under abiotic stresses treatments,RT-PCR analysis was performed.The results showed that the PNDREB1 transcripts were constitutively detected in root,stem,leaf,flower and seeds.When treated by dehydration(20%PEG) and 4℃low temperature for 2 hrs,the expression of PNDREB1 was all induced,which was almost no response when treated by 250mM NaCl for 2 hrs.Meantime, the binary vectors of pROKâ…¡-PNDREB1 and p35S-1301-PNDREB1 were constructed for tobacco transformation,and finally,about ten tobacco independent resistant lines were regenerated.Given the important function of PNDREB1 in plant stress resistance,we further isolated another two DREBs-like gene in peanut-AHTINY1(FM955394),AHTINY2(FM955395) by homology searching.Sequences analysis showed,AHTINY1 was closely related to Arabidopsis TINY2(NM₁21197),with 82%identity at amino acid level,and AHTINY2 shared 62%protein sequence homology with soybean DREB3(DQ208969).Both of them contained a conserved AP2/EREBP domain,and encode 205aa,209aa,respectively.RT-PCR analysis showed that both AHTINY1 and AHTINY2 were constitutively expressed,and the induced expression pattern showed that AHTINY1 was induced by low temperature,especially ABA,and ethylene,with slight induced by dehydration(20%PEG) and 250mM NaCl.But AHTINY2 was only induced by ethylene.The binary vectors of AHTINY1 and AHTINY2 were constructed for genetic transformation,and the work of transgenics screen and functional analysis are still undergoing.To sum up,we have isolated and verified three DREBs transcription factors in our research, and the physiological study also indicated they could participate and play role in abiotic stress resistance processes.These results will give some clues for peanut germplasm innovation and breeding,and further provide practical and application values for DREBs in crop improvement engineering.