Study On The Mechanisms Of PtAOS1 And PtNAC72 Responding To Drought Stress In Poncirus Trifoliata(l.) Raf.

Drought stress is a main abiotic stress affecting citrus yield and quality,but the signal transduction and reg?lating mechanism of citrus responding to drought stress is still not known clearly.Jasmonic acid(JA)is an important reg?latory substance in plants,which plays an important and positive role in drought stress tolerance and fruit quality.Trifoliate orange(Poncirus trifoliate)is a widely used rootstock in citrus planting because of its resistance to citrus foot rot,citrus tristeza virus and tolerance to cold and drought stresses.In this study,the allene oxide synthase(AOS)gene family(PtAOS1 and PtAOS2)were cloned and isolated from trifoliate orange,and their expression patterns under drought stress were revealed.Promoters of AOS family genes were cloned and cis-acting elements and transcriptional activation core region were analyzed.Yeast-one-hybrid method was used to screen the potential reg?latory factors of AOS promoter.The gene PtNAC72 was overexpressed in Dahongtiancheng(Citrus sinensis),and the role of PtNAC72 in drought stress was explored.The main res?lts are as follows:1.The PtAOS gene family were divided into two different branches in evolutionary analysis.The evolutionary position of PtAOS1 was close to AOS1 protein of sweet orange and pistachio,while PtAOS2 was close to AOS3 protein of clementine orange and pistachio.Drought treatment induced PtAOS1 gene expression specifically.Exogenous JA induced up-reg?lated expression of PtAOSs,in which PtAOS1 expressed significantly higher than PtAOS2.These res?lts suggested that PtAOS1 might be a dominant gene in JA biosynthesis of trifoliate orange.2.Promoters of PtAOS genes were cloned and isolated.Analysis showed that there were m?ltiple stress response cis-acting elements,light response elements and hormone response elements in the promoter regions of PtAOS1 and PtAOS2.The main hormone response elements in PtAOS1 promoter are methyl jasmonate response element(CGTCA-motif)and abscisic acid response element(ABRE),and PtAOS2 promoter contains abscisic acid response element(ABRE),auxin response element(TGA-element)and gibberellin response element(P-box).Transient expression analysis in tobacco showed that PtAOS1 promoter had transcriptional activation activity strongly,while PtAOS2 promoter worked weakly.Basing promoter deletions at 5' end of PtAOS1 promoter,-532 to-265 bp was determined as a core region affecting promoter activity.Deletion of this region led to the losses of PtAOS1 promoter responding to JA and ABA and activating transcription.It ws concluded that the CGTCA-motif and ABRE elements in-532 to-265 bp probably were the core elements affecting the transcriptional activation of PtAOS1 promoter.3.With a bait vector of PtAOS1 promoter,a c DNA library which was constructed using the drought-stressed trifoliate orange leaves was screened by yeast-one-hybrid system.Five proteins interacting with PtAOS1 promoter,PtDUF886,PtDUF1685,PtPUP1,PtRBP1 and PtRAP2.5,were obtained finally.Yeast-one-hybrid system and dual luciferase activity analysis confirmed that the five proteins interacted with PtAOS1 promoter and enhanced its transcriptional activation activity.4.Real-time PCR analysis showed that PtDUF886,PtDUF1685,PtRBP1 and PtRAP2.4 were all up-reg?lated in response to drought stress while the expression of PtPUP1 changed slightly and decreased at the later stage of drought.Homologous gene mutants in Arabidopsis thaliana showed that duf886,duf1685 and rap2.4 had no obviously phenotypic difference comparing to wild type except lower seed germination percentage.With a dehydration treatment,duf886,duf1685 and rap2.4wilted faster than wild type along with the proline content decreased significantly while MDA did not change.In dehydration treatment,duf886,duf1685 and rap2.4exhibited higher leaf water loss rate than wild type.In addition,the proline content of duf886,duf1685 and rap2.4 mutants under salt stress was significantly lower also than that in wild type.And the proportion of yellowing leaf in mutant plants was higher than in wild type indicating that duf886,duf1685 and rap2.4 were more sensitive to salt stress.These res?lts implied that DUF886,DUF1685 and RAP2.4might play positive roles in plant abiotic stress response.5.The expression of trifoliate orange PtNAC72 was significantly up-reg?lated by drought stress.The content of proline and peroxidase and catalase activities were all lower in overexpressed PtNAC72 Dahongtiancheng than those in wild-type except MDA content changed seldom,indicating that PtNAC72 was likely to be a negative reg?latory factor in drought stress response.Transcriptome analysis suggested that4886 genes were differentially expressed between overexpressed PtNAC72 Dahongtianheng and wild type under drought stress,of which 3620 genes were up-reg?lated and 1266 genes were down-reg?lated.Differently expressed genes in plant-pathogen interaction,MAPK signal pathway and plant hormone signal transduction accounted for 14.02%,20.93% and 7.8% of the total,respectively,implying PtNAC72 probably functioned in these pathways.