Functional Analysis Of Brassica Napus Transcription Factor Bna.A07.WRKY70 On Leaf Senescence In Arabidopsis Thaliana

Rapeseed（Brassica napus L.）is a major oilseed crop in our country even in the world.There is important social and economic significance for studying its associated biological processes.Leaf senescence is the final stage of leaf development and is essential for storage properties and crop productivity.Previous studies have revealed that WRKY transcription factors play crucial roles in leaf senescence.In Arabidopsis thaliana,WRKY70 negatively regulates leaf senescence.However,the functions of Brassica napus WRKY70 transcription factors in leaf senescence remain unclear.In the present study,the full-length coding domain sequence（CDS）of Bna.A07.WRKY70 was cloned from the B.napus cultivar“Zhongshuang11（ZS11）”.The physicochemical properties,sequence structure of Bna.A07.WRKY70 protein and its genetic relationship with other plant species were systematically analyzed.The subcellular localization and transcriptional self-activation assays were performed to study the transcription factor characteristics of Bna.A07.WRKY70.In addition,expression pattern of Bna.A07.WRKY70 was analyzed by real-time quantitative reverse transcription PCR（RT-q PCR）and GUS histochemical staining assays.On this basis,i conducted the recombinant vector35S:Bna.A07.WRKY70-GFP and transformed into the Arabidopsis thaliana mutant wrky70to explore the functions of Bna.A07.WRKY70 in leaf senescence.These results improve theoretical basis on the functions of Bna.A07.WRKY70 in Brassica napus and provide a novel strategy for breeding the new stay-green cultivars in rapeseed through genetic manipulation.The main findings for the experiment were listed as follows:1.In the B.napus cultivar“Zhongshuang11（ZS11）”,six paralogs of Bna.WRKY70were blasted and named respectively Bna.A07.WRKY70,Bna.C06.WRKY70,Bna.A04.WRKY70,Bna.C08.WRKY70,Bna.A09.WRKY70,and Bna.C04.WRKY70.The results of protein sequences alignment showed that six WRKY70 proteins from B.napus possessed highly conserved WRKY domains and had a high similarity with the protein sequence of At WRKY70.Among them,Bna.A07.WRKY70 was predicted to share the highest identity in the amino acid sequence with the At WRKY70 protein（66.01%）.Meanwhile,the phylogenetic analysis of WRKY70 form multiple plant species indicated that Bna.A07.WRKY70 is closely related to the WRKY70 protein from A.thaliana and it might have similar functions as At WRKY70.2.The physicochemical properties and protein structure analysis of Bna.A07.WRKY70protein showed that Bna.A07.WRKY70 encodes 283 amino acids,and it is a faintly acid,hydrophilic,and unstable protein without signal peptide and transmembrane structure.The secondary structure of Bna.A07.WRKY70 protein containsα-helices,β-angles,elongated chains and random coils,in which,the random coils andα-helices in a large proportion are49.12%and 39.22%respectively.Its tertiary structure is also mainly composed of random coils andα-helices.The post-translational phosphorylation sites of Bna.A07.WRKY70 were44,among which serine residues sites were the most,with 26.3.The subcellular localization showed that the fluorescence signal was detected in the nucleus,and transcriptional self-activation assays demonstrated that Bna.A07.WRKY70could activate the expression of the reporter genes,which suggested that Bna.A07.WRKY70 might function as a transcription activator.4.Analysis of Bna.A07.WRKY70 expression pattern.The results of RT-q PCR showed that the expression level of Bna.A07.WRKY70 was the highest in leaves of B.napus.Meanwhile,the p Bna.A07.WRKY70:GUS transgenic positive plants were used for GUS activity staining assay.The result showed that GUS staining was predominantly detected in rosette leaves of A.thaliana.These findings suggested that Bna.A07.WRKY70 might regulate a significant function during leaf development.5.Phenotypes of T₃ homozygous transgenic lines 35S:Bna.A07.WRKY70-GFP were observed and analyzed that ectopic expression of Bna.A07.WRKY70 fully restored the rate of leaf senescence and chlorophyll content to wild-type levels in the A.thaliana mutant wrky70 background.Meanwhile,using the RT-q PCR to detect the transcript levels of three senescence-related marker genes At SAG13（senescence-associated gene 13）,At SEN1（senescence-associated gene 1）,At CAB1（chlorophyll a/b-binding protein）of A.thaliana wild-type,the wrky70 mutant and wrky70 35S:Bna.A07.WRKY70-GFP transgenic plants,it was found that the expression levels of three senescence-related marker genes of wrky70mutants were restored to wild-type levels by ectopic expression of Bna.A07.WRKY70.These results demonstrated that Bna.A07.WRKY70 may negatively regulate the leaf senescence by adjusting the expression of senescence genes in A.thaliana.