Molecular Mechanisms Of Exogenous RdreBIBI Gene Enhanced Cold And Drought Tolerance Of 'Benihoppe' Strawberry

Strawberry(Fragaria x ananassa Duch.),a perennial herb of the Rosaceae family,is an economically important berry.Strawberry can be grown in a wide range of climates,but low temperature is still a limiting factor in production.Genetically modified plants are becoming an increasingly important choice for the horticultural landscape.The dehydration responsive element binding proteins(DREBs)transcription factors control the expression of a number of abiotic stress-related genes,are perfect candidates for genetic modified breeding.The OsDREBIB homolog gene RdreB1BI transgenic strawberry(Fragaria � ananassa Duch.cv.Benihoppe)lines were generated previously.The single copy RdreBIB1 transgenic line that exhibited the highest level of cold tolerance was chosen for the present study.In the present work,digital gene expression(DGE)and two-dimensional gel electrophoresis(2-DE)and subsequent mass spectrometric identification(MALDI-TOF/TOF MS)technologies were used to study the changes in the leaf omics profiles of RdreB1BI transgenic and non-transgenic strawberries exposed to stress conditions.Combine omics,morphological,physiological,and transcription levels to account the mechanism of enhanced low temperature tolerance of transgenic strawberry mediated by RdreB1B1 transgene and the genetic improvement effects of RdreBIBl on transgenic strawberry.The main results were listed as follows:1.To further understand how DREB1B regulates genes expression to promote cold-hardiness,Illumina/Solexa sequencing technology was used to compare the transcriptome of non-transgenic and RdreBIBl transgenic strawberry plants exposed to low temperatures treatment.Approximately 3.5 million sequence tags were obtained from non-transgenic(NT)and transgenic(T)strawberry untreated(C)or low-temperature treated(LT)leaf samples.At control temperature,1119 genes were differentially expressed in NT plants as compared to T plants.In non-transformed plants,1202 DEGs were found following low-temperature treatment.In comparison,1517 DEGs were found in T plants after low temperature treatment.A comparison of the genes that are differentially expressed in response to cold between NT and T plants found that 443 genes were common to both.Analysis of the genes up regulated following low-temperature treatment revealed that the majority are linked to metabolism,biosynthesis,transcription and signal transduction.The expression patterns of DREB and the exclusive DRE cis-acting elements in the promoter regions represent further information on the complex regulatory networks and cross-talk between signaling pathways.2.A proteomic approach based on 2-DE and subsequent MALDI-TOF/TOF MS identification was used to study the changes in the leaf proteome profiles of RdreBIBI transgenic and non-transgenic strawberries exposed to low-temperature condition.More than 300 highly re-producible protein spots(p<0.05)were detected.By comparing the proteomic profiles,we located 21 protein spots that were differently accumulated more than two fold changes between transgenic and non-transgenic plants.These proteins included photosynthetic proteins(RCA1),Cu/Zn-SOD,Lea14-A,eIF5A.In addition,RCA1,Cu/Zn-SOD,and eIF5A were obviously associated with different expressed genes in transcriptome data.They were likely important gene products in the regulatory network of the RdreB1BI gene.This study provides useful information to study the predicted target proteins of DREB 1B transcription factors.3.In the previous transcriptome database,PAL,C4H,4CL,CHI,F3H,FLS,DFR,which encoding critical enzymes in anthocyanin biosynthesis,were up regulated in transgenic plants but not in non-transgenic plats.The biosynthetic pathway is well understood and is conserved among plants and the key enzymes and genes have been characterized and contribute to anthocyanin accumulation.Higher level of anthocyanin content was found in petioles,stolons and fruits of transgenic plants comparing with that of non-transgenic plants during the reproductive period,so as anthocyanin related genes.Both the up-regulated structural genes and regulatory genes in transgenic strawberry indicated that the RdreBIBI transgene contributes to low-temperature tolerance by enhancing the synthesis and accumulation of anthocyanin.4.Based on the previous data,we found many different expressed genes related to drought stress.To further understand the genetic improvement effects of RdreBIB1 on transgenic strawberry,drought stress responses of transgenic plant were detected atmorphological,physiological,and transcription levels.Transactivation assay revealed that RdreB1BI could activate FvPIP2;1 like 1 promoter and regulate the expression of other AQP related genes.RdreBIBI transgenic plants showed enhanced drought tolerance as judged by less electrolyte leakage increase rate,higher relative water content,less stomatal aperture as well as more POD and SOD activities and less MDA accumulation.The transgenic plants also accumulated higher levels of drought related regulatory genes and function genes transcripts in leaves,stolons and fruits,such as PIPs,NAC,RD22,ABI,and NCED.Together,these results demonstrated that RdreB1BI also played an essential role in the regulation of drought stress response.