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Tobacco mosaic disease is generally a disease of the various tobacco-producing areas of the world, the incidence plots can be cut by 30-50%, resulting in significant economic losses, the selection of resistant varieties is the most effective means of prevention and treatment. The TMV resistance genes from wild species sticky tobacco (N.glutinosa), a single dominant gene N gene, and ordinary the tobacco Ambalema varieties the recessive allele rm1 and rm2, but above resistance genes with undesirable traits gene linkage or gene multi-effect, greater difficulties in the use of commercial varieties, TMV resistance gene markers and screening, has become the subject of today need to be solved. Study TMV highly resistant varieties Longjiang 925 test material, the use of cDNA-AFLP technique responsive genes in tobacco leaves with TMV interaction analysis. Through the screening of 240 primer combinations, the results obtained are as follows: 1, a total of about 9500 transcriptional gene fragments and 20 transcriptional gene fragments can be obtained on selective amplification primers, 190 which is constitutively expressed gene fragments, 42 a induced expression of the gene fragments, 41 to inhibit the expression of gene fragments. After cloning and sequencing, and ultimately won the 15 expressed genes, including the the 12 TMV induced expression of the gene fragment sequences, two constitutive expression of the gene fragment and an inhibition of the expression of gene fragments. 2 To verify the authenticity of the expressed genes using quantitative PCR analysis of an inducible expression fragment (TIF2) 5 point differential gene expression between 0-72 hours, and confirmed that the gene sequence for real-induced expression sequence. This sequence of RACE, finally obtained a full-length cDNA sequence. 3, using the GenBank and other public databases on NCBI transcription gene fragment carried on the obtained bio-information analysis and functional prediction. The results show that: TMV-induced the tobacco disease resistance mechanism involving physiological and biochemical reactions in many ways, the multiple functions of different genes involved in the control. In this study, a total of 15 expressed genes, bioinformatics analysis and function prediction, that 12 induced expression of the gene fragments and nucleic acid metabolism, protein synthesis and modification, energy metabolism, stress response, intracellular transport, glucose metabolism associated ; confirmed that two fragments are constitutively expressed chloroplast DNA, 1 inhibit expression of genes with cell wall metabolism related. To TIF2 sequence-based application RACE obtained cDNA sequence 857bp predicted coding region between 101bp to 613bp. Warp of Blastn Blastp to be of comparative analysis of homologous sequences, no tobacco, determined that the gene is found in tobacco for the first time a new gene associated with resistance to TMV. The above findings lay the material and technical basis for Tobacco molecular breeding, but also to make up for the lack of differential gene expression studies in tobacco and TMV interaction.
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