Cloning Of Resistance Gene And Analysis Of MicroRNA Expression Profile Under Tobacco Mosaic Virus Infection In Tobacco CV58

Tobacco is a model pant and an important economic crop.Tobacco mosaic virus(TMV)seriously infects tobacco and affects the quality and yield of tobacco.Therefore,it is important to screen of high TMV-resistant tobacco material and identify its resistance genes.On the other hand,microRNA(miRNA)plays an important role in the process of post transcriptional gene silencing,which can mediate the cleavage of the target gene or inhibit its translation.Nicotiana tabacum cv.CV58 is an important germplasm of high resistance to TMV,and the identification of miRNA in TMV-infected Nicotiana tabacum cv.CV58 will reveal its resistant pathway.The main research results are as following:1.In the resistant screening experiment,tobacco CV58 shows N-like HR(hypersensitive response)with necrotic lesions.With the aim to identify this resistance gene,homology-based cloning was performed.The CDS length of the resistance gene in CV58 was 3435 bp,showing 99%of homologies in nucleotide sequence with N gene,and its coding proteins belonged to TIR/NBS/LRR class.Then the CRISPR/Cas9 system was used to construct the gene knockout vector,and three different mutant plants of gene deletion were obtained after agrobacterium-mediated transformation.No HR was observed on TMV-inoculated mutants,and the temperature sensitivity was disappeared too.Last,the resistance gene was transferred into tobacco K326 using the agrobacterium-mediated method.Transgenic tobacco K326 showed typical HR and temperature sensitivity after TMV inoculation.So the resistance in tobacco CV58 comes from N gene.2.The total RNA was detected by Illumina Hiseq2000 sequencing system for both TMV infected CV58 plants and blank controls.The differential expressed miRNAs in the two samples were screened out.Then Target-Scan S.1 and PicTar2 were used to analyze differential expressed miRNAs,and identify the possible biological pathways via GO and KEGG analysis.The results showed that TMV infection can change the expression level of endogenous miRNA.329 miRNAs showed differential expression,in which 256 miRNAs showed significant fold change>1(184 up-regulated,72 down-regulated,p<0.05).GO enrichment analysis showed that target genes of the 184 up-regulated miRNAs were enriched in 7 pathways,such as Plant-pathogen interaction,Cell cycle,MAPK signaling pathway,Fc gamma R-mediated phagocytosis and others.These results put forward to study the roles of miRNAs in plant resistant to virus.3.Nine mature miRNAs and five traditional housekeeping genes were selected as candidate reference genes.Their expression stability was evaluated by using GeNorm and NorFinder procedures with TMV-infected.The results showed that L25 and miR159 were the most suitable reference gene for qPCR of mature miRNA.And L25/Ntubc2 were the most suitable reference gene pairs in roots and leaves.