Genome-wide Analysis Of The WRKY Transcription Factors In Brassica Napus L.and Functional Characterization Of Genes Involved In Response To Abiotic Stress

Brassica napus L.is one of the important oil crop,and its production is seriously influenced by the change of climate and depravation of environment.With the deepening of plant genome research,the continuous revealing of stress-resistance-related genes and stress-resistance mechanisms,the current research hotspot is to cope with changes in climate and environment by improving crop resistance.During the long-term evolution,plants set up a complex and highly effective stress control network.Under the participation of various transcription factors and downstream stress-responsive genes,plant adaptation to unfavorable environmental factors is systematically controlled.WRKY transcription factor is a unique type of transcriptional regulatory factor in higher plants involved in various stress responses in plants.The completion of rapeseed genome sequencing facilitates the mining of rapeseed stress resistant genes.In this study,all WRKY family members in rape were identified and characterized based on rapeseed whole genome information.Some potential stress resistant genes were screened out and cloned,and transgenic materials were constructed to further study their functions.Based on rapeseed whole genome sequence and transcriptome in different stress treatments of,Zhongsuan 11 rape cultivars,some progress was made in the evolution of the Bn WRKY family and its function in response to stresses.(1)Baed on the genome sequences of Arabidopsis thaliana,Brassica napus,Brassica rapa and Brassica oleracea,WRKY members were identified and characterized according to the basic structure of WRKY protein in Brassica napus,Brassica rapa and Brassica oleracea,The results showed that there were 278 Bn WRKY members,145 BrWRKY members and 148 Bo WRKY members.The Bn WRKY members are divided into three sub-groups,49 belong to Group Ⅰ,183 belong to Group Ⅱ,and 46 belong to Group Ⅲ.Group Ⅱ is further divided into a,b,c,d,and e sub-family.(2)According to the transcriptome data and the expression level of differentially expressed genes in different stress conditions,16 differentially expressed Bn WRKY genes were screened out.The promoter sequce and stress indused expression patterns of these 16 genes were analyzed.The results showed that these 16 BnWRKY genes’ promoter contain a variety of cis-acting elements associated with stress response,such as ABRE,MBS,HSE,LTR,etc.The results of qRT-PCR showed that BnWRKY genes were induced by abiotic stresses such as ABA,NaCl and PEG6000.(3)Three representative BnWRKY transcription factors were selected for subcellular localization and transcription activity analysis.The results showed that three BnWRKY transcription factors were mainly localized in the nucleus and one of which were also distributed on the cell membrane.One BnWRKY transcription factor had a certain transcriptional activity and the other two BnWRKY transcription factors have transcription inhibitory activity(4)11 BnWRKY genes were cloned and transformed into B napus and Arabidopsis.Five homozygous Arabidopsis lines overexpressing Bn WRKY157 and one overexpressing BnWRKY184 were obtained.(5)Overexpression BnWRKY157 Arabidopsis was more sensitive to ABA,Mannitol,and NaCl than wild type and the rosette leaves curled inwards and could not stretch normally.The density of stomata was significantly higher than that of the wild type.The water loss rate of the isolated leaves was also higher than that of the wild type,indicating that the over-expression of Bn WRKY157 results in Arabidopsis more sensitive to adverse stresses.