Functional Characterization Of Tobacco WRKY Transcription Factors And Quantification Of T-DNA Tandem Repeats In Transgenic Plants Using Agrobacterium Mediated Transgenic Approach

WRKY transcription factors had previously reported to involve in plant abiotic defense.A WRKY gene,which dramatically induced by tobacco mosaic virus(TMV),was cloned in this study.BLASTX analysis revealed that it was a homologous of Arabidopsis WRKY40 transcription factor,thus it was named Nt WRKY40.To verify its function,NtWRKY40 overexpressed and RNA interference mediated suppression tobacco plants were employed.After TMV infection,the transcription levels of NtWRKY40,coat protein(CP)and moving protein(MP)genes were quantified using quantitative real time PCR at three stages(6h,3d,9d).Results showed that in the wild type control,NtWRKY40 expression induced not much(one or two times compared to0 h control)after 6h and 3d infection,but rapidly increased to one 150 fold after 9 h infection.However,NtWRKY40 expression were not significantly changed after infection at all three stages.To analyze the relationship between NtWRKY40 expression and TMV growth,the expression level of each gene at 6 h after infection were used as control respectively,for analyzing the expression level at 3 d and 9 d.Combined the qRT-PCR results and the tobacco plant growth,we found that NtWRKY40 overexpressed tobacco plants were more sensitive to TMV compared to the wild type control plants.Thus we hypothesized that NtWRKY40 is a negative regulator in plant abiotic stresses.However,RNAi-NtWRKY40 are now not available,further studies combined with RNAi and overexpression can verify a more clear function of NtWRKY40.Agrobacterium mediated transgenic technology often inserted multiple T-DNA copies into a single locus or multiple loci.Further,tandem repeat structures and multiple transgene insertions at different loci often lead to co-suppression of gene expression,and even gene silencing.Therefore,screening for single-copy transformation events are important for simplification of homozygote offspring selection with stable expression of transgenic,both for genetic improvement of crop traits and for functional characterization studies.An efficient,accurate,real-time qPCR assay was developed for high throughput screening and quantification ofT-DNA tandem repeats present in transgenic Arabidopsis mutants.Moreover,we combined the assay with multiplex PCR to quickly clone and analyze the junctions and filler DNA linking these tandem repeats.The sequence analysis revealed variability in the presence and nature of filler DNA and residual T-DNA border sequences of directly repeated junctions.