Functional Analysis Of Brassica Napus BnWRKY41-1 Transcription Factor On Anthocyanin Accumulation In Arabidopsis Thaliana Leaves

Rapeseed?Brassica napus?has the largest planting area among oil-producing crops in China and provides the third most consumed vegetative oil in the world,so it plays an important role in safety of food production.Anthocyanin has positive effects on the improvement of rapeseed agronomic traits,including canola oil quality,trait marker in breeding,tolerance to stresses and colorful rapeseed.To optimize the use of anthocyanin in rapeseed,constantly completing the relevant gene network should be done.In the present study,we found that BnWRKY41-1 transcription factor inhibited anthocyanin accumulation in A.thaliana leaves via repressing the expression of five anthocyanin biosynthetic genes.The study would pave a way for further understanding of the function of BnWRKY41 in rapeseed.The main results are showed as follows.1.There are two WRKY41 homologs in rapeseed,BnWRKY41-1?XP₀13686534.1?and BnWRKY41-2?XP₀13695247.1?.BnWRKY41-1was cloned successfully from Westar.Protein sequence alignment and phylogenetic analysis showed that BnWRKY41-1 and AtWRKY41 had close evolutionary relationships,and they shared 92% similarity in functional domain,therefore the genes and the phenotypes that they affected could resemble each other.2.pGreen-35S: BnWRKY41-1-6HA was transformed into A.thaliana wrky41-2 mutant?SALK₀28449.34.95.x?,and homozygous T3 transgenic plants were used for further study.At 15,20 and 25 days after germination?DAG?,leaves anthocyanin content?LAC?in wrky41-2 was significantly higher than that in Col-0,whereas the introduction of BnWRKY41-1 into A.thaliana wrky41-2 mutant could recover LAC.Thus AtWRKY41 and BnWRKY41-1 could have similar biological function on anthocyanin accumulation.3.At fastest increasing time of LAC?20 DAG?,rosette leaves were sampled for experiments bellow.Firstly,RNA-seq and qRT-PCR revealed five significantly up-regulated ACR-DEGs?anthocyanin content related differentially expressed genes?in wrky41-2 compared to Col-0 wild type,including AtMYB75,AtMYB111,AtMYBD,GSTF12 and AT1G68440.Moreover,expression levels of ACR-DEGs in wrky41-2 were rescued to wild type levels by ectopic expression of BnWRKY41-1.Finally,ChIP-qPCR showed that AtWRKY41 and BnWRKY41 directly bound to some of ACR-DEGs,so they could directly or indirectly affect the expression levels of the five ACR-DEGs.