| Tobacco is one of the important economic crops in China, however the disease is an important factor in tobacco production. Therefore, exploring the disease-resistant genes in tobacco is especially important, as early as in the 1920s has begun. Early tobacco resistant genotypes are mostly on the individual level through the identification of resistance obtained. The early disease-resistant genes of tobacco were mostly obtained through Map-based cloning, and with the development of sequencing technology, more and more plant genome sequences have been completed, disease-resistant genes of abundance plants can be screened by homology comparison. RNA Sequencing(RNA-seq) has been a new important tool to analyse the genes expression and transcriptome. Therefore, the transcriptome of the main organs(root, stem, leaf and flower) of four tobacco species (N.tabacum(2n=48), N.sylvestris (2n=24), N.tomentosiformis( 2n=24), and N.otophora (2n=24))during the flowering period has been sequenced and analyzed in this research and the NBS genes in N.tomentosiformis genome has been analyzed. The number, types, distribution and cluster of NBS like of disease-resistant genes of tobacco have been analyzed. Based on R genes Bioinformatics of N.tomentosiformis, high-throughput RNAi vector libraries of tobacco have been established with GATEWAY expression vector construction method using main part of the genes to design the primers. The main results are as follows:1. The gene expression map in transcriptome level of different species and different organs of N.tomentosiformis is our new study.The expression of four organs of four kinds of tobacco has been analyzed using transcriptome sequencing method for the first time. It was found that about 74% and 59% of the reads could be compared to the genome of the N.tomentosiformis and gene sets as a reference, and 43% and 35% of the reads could be well-matched to the genome, respectively. A total of 28,144 sets were supported by gene transcription, the number accounted for 83.5% of all the gene sets. Many new transcripts and alternative splicing forms were discovered by prediction gene structural optimization of transcriptome data .The quantitative expression of each gene in the transcriptome data were determined by RPKM. The results showed that different tissues of the number of tissue-specific genes varied widely, there was at least 1642 tissue-specific gene expression. Among them, the high distribution of tissue-specific gene expression were the flowers, with genes up to 1478; in the root, stem and leaf tissue, the specific gene were 866, 179 and 165.2. It is the first time to screen 282 disease-resistance genes containing NBS structure from N.tomentosiformis.A total of 282 disease-resistant genes containing NBS structure were found in N.tomentosiformis gene-set, by analyzing the structure domain of the candidate gene's amino acid sequence by pfam to wipe out the candidate gene without NBS structure.All types of R gene NBS transcriptome expression profile showed that 154 gene expression data were supported by transcription group among 282 genes. 145, 134, 129 and 132 genes had expression data in the roots, stems, leaves and flowers, respectively. More than 10 genes in four species had shown high expression, indicating that these R genes may have a broad-spectrum resistance.The transcriptome data also showed that there were 220, 247 and 320 sequences containing NBS domains in N.sylvestris, N.otophora and N.tabacum, respectively.3. The phylogeny tree of the NBS-like tobacco disease-resistant gene have been clusteredAnalyzing phylogeny of NBS-like disease-resistant gene indicated that in the phylogenetic tree the large branched clusters was more obvious apart from a few small branches, basically in accordance with the structure of categories to form larger clusters of branches in the evolution of gene clusters, at the end of the tree together, during the most recent period they may be generated by copying and showed closer genetic relationship; as well as some genes that was a separate branch, with the class differences because their tobacco disease-resistant gene had more complex evolution.136 genes in the chromosome that is physically located close to or adjacent to, formed the gene clusters in the identified 282 NBS-like disease-resistant genes. In the 54 genes in the formation of clusters, each gene cluster contained an average of 2.5 genes, one of the largest gene cluster contained 6 genes. There were 11 homologous genes in, indicating that hm is a multi-gene family.4. The high-throughput tobacco RNAi vector libraries have been established by Gateway expression vector method.Total of 186 candidate genes were verificated for function from the disease genes of the genome of N.tomentosiformis,all type of resistance genes have been reported, and tobacco transcriptome data.A high-throughput RNAi vector libraries of tobacco were constructed by GATEWAY expression vector method. 186 completed R siRNA fragments were connected to all or part of the entry vector pDONR201. Tobacco R gene resistance capacity of RNAi library were evaluated with the number of clones transformed, the result showed that the initial library had clones 4.66×10~4 cfu/μL, a total of RNAi clones 9.32×10~6 cfu in 200μL initial vector library.
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