Establishment Of Agrobacterium-mediated Brassica Rapa Transformation System And Verification Of Protein Interaction Between WRKY Ⅱa Transcription Factors

Rapid-cycling Brassica rapa(RCBr)are small robust plants with a short lifecycle that are widely used in biology teaching.RCBr have been used for decades but there are no published reports of RCBr genetic transformation.Arabidopsis can be transformed via Agrobacterium-mediated floral dip,and‘49 Caixin’ can be transformed via vacuum infiltration.Three different aged plants of RCBr were inoculated with Agrobacterium via vacuum infiltraton or floral dip,and GFP-positive plants were obtained.In addition,transformants were confirmed by PCR and Southern blot.A transformation system for RCBr was established in this study.This will promote development of new biology teaching tools as well as basic biology research on Brassica rapa.WRKY transcription factors are one of the largest families of transcriptional regulators and play an important role in defense response against pathogens in plants.According to the amino acid sequences of Arabidopsis WRKY Ⅱa transcription factors and the existing WRKY gene family analysis of Brassica rapa,the WRKY Ⅱa transcription factors of non-heading Chinese cabbage were cloned.It was found that there were six genes,and the newly discovered two sequences were named as BcWRKY98 and BcWRKY104.By phylogenetic analysis,it was found that BcWRKY1,BcWRKY18 and BcWRKY60 were homologous to AtWRKY18,and BcWRKY40,BcWRKY98 and BcWRKY104 were homologous to AtWRKY40.Infection experiments of downy mildew showed that the relative expression of WRKY Ⅱa transcription factors increased at 48 hpi and decreased at 72 hpi in the resistant cultivar ’Suzhouqing’.Therefore,to understand the function of WRKY Ⅱa transcription factors would contribute to the development of disease-resistant breeding of non-heading Chinese cabbage.Through yeast two-hybrid experiments,it was found that there was a complex interaction between WRKY Ⅱa transcription factors in non-heading Chinese cabbage.Yeast two-hybrid library screening using BcWRKY1 as a bait found that some candidate proteins were involved in plant signal transduction and stress response.Considering the problem of protein function redundancy,CRISPR/Cas9 vectors targeting one,several or all WRKY Ⅱa genes were constructed and transformed into Brassica rapa.No mutation was found in target sites in transgenic Brassica rapa,but abnormal phenotypes were still appeared.These might be caused by T-DNA insertional mutagenesis in transgenic plants.TAIL-PCR was performed to amplify T-DNA flanking sequences in transgenic Brassica rapa,and the products of third round PCR were sequenced.The 3’ end of gene Brara.03841 was found to be a T-DNA insertion site in FPsc transgenic plant.