Functional Identification And Expression Analysis Of Transcription Factor ERF019 Induced By Leptosphaeria Biglobosa In Brassica Napus

Oilseed rape(Rapeseed,canola)is one of the most widely cultivated oil crops in worldwide,which is the main source of human's cooking oil and animal's meals,canola inevitable suffers from many biological and abiotic stresses during the whole period,which resulting in a tremendous yield loss and reduction quality.The researches showed that the gene expression regulated by transcription factors play an important role on plant response to stresses.ERF transcription factors primary express in many kinds of plants.Many studies show that overexpression transcription factors ERF,and the expression of downstream related resistance genes can be activated to enhance the plants tolerance and diseases resistance.We analysed the BnERF019 expression and identifi the functional in this research based on our previous RNA-seq results,the transcription factor Bn ERF019 was up-regulated after infected by Leptosphaeria biglobosa for 4 d in leavesof Brassica napus.The results were as following:1.The total RNA was extracted from leaves of oilseed rape at 4d after inoculation with Leptosphaeria biglobosa,The CDS sequences of BnERF019 transcription factor was amplified,CDS sequence was 453 bp and was 100% homology with transcriptome sequencing(ID=BnaC07g13470D)by DNA sequence data analysis.The amino acid sequence of BnERF019 encoded protein was deduced using CLC sequence viewer 6.0,which encoding 151 amino acids,a molecular mass was16.38 k D and isoelectric point was 6.27,protein secondary structure,phosphorylation site,transmembrane structure,hydrophobicity were predicted.2.The Bn ERF019 expression was analyzed in different tissue and at three different phases after L.biglobosa infection 4 d,5 d and 6 d by quantitative Real-time PCR,the result showed that the BnERF019 gene was transcribed in different tissue organ.The expression of BnERF019 began in 4 days after inoculation,with the relatively highest transcript expression levels in roots and stems on the fifth day,with the relatively highest transcript expression levels in leaves on the sixth day.This suggested that the BnERF019 transcription factor played an important role on oilseed rape resistance to Leptosphaeria biglobosa.3.The result indicated that the BnERF019-GFP fusion protein was localized in nucleus,while the control pYBA-1132-GFP wasdistributed through the Nucleus,cytoplasm and cell membrane.4.In order to characterize the function of BnERF019,the gene was cloned into overexpression vector pYBA-1143-ERF019,and transformed into wild Arabidopsis thaliana by agrobacterium mediated floral dip method.Six transgenic Arabidopsis thaliana were obtained after Kana screen and PCR confirmation.5.Detached leaves of transgenic Arabidopsis thaliana was inoculated Leptosphaeria biglobosa to evaluate the resistance to black leg,The experimental result indicated that transgenic Arabidopsis thaliana demonstrated greatly reduced leaves lesion area and damage with the increased in the number of days in different days treatment,as compared with the wild type.This studies showed that overexpressed the BnERF019 in Arabidopsis thaliana could enhance the resistance to Leptosphaeria biglobosa and also showed the BnERF019 positively regulate the defence responses of plant.