CDNA-AFLP Analysis Of Differentially Expressed Genes In Tobacco Infection By Tobacco Mosaic Virus

Tobacco mosaic virus (TMV) is one of the main tobacco diseases in the world , and cause serious economic losses every year. Generally, the tobacco yield losses caused by TMV infection could reach up to 30%-50%. Identification of tobacco resistance gene is always an important object and one of crucial key topic in tobacco production.The variety of Long Jiang 925 which was TMV high resistant was taken as tested plant material in this study. cDNA amplified fragment length polymorphism analysis (cDNA-AFLP) was used to display transcripts whose expression is rapidly altered during the treatment of TMV to tobacco leaf. Two hundred and forty primer combinations were used for selective amplification. The main results were as follows:1. The results showed that there existed over 9500 expression bands appeared at different treatment times, and 20 expression bands were amplified by each primer pair on average. Among these, 190 fragments were constitutively expressed, 42 fragments were inducible expressed, 41 fragments were suppressive expressed. By clone and sequencing, the sequences of 15 expression bands were obtained, including two constitutively expressed fragments and one suppressive expressed fragment.2. In order to validate the functions of these differentially expressed gene sequences obtained, the TIF2 fragments was investigated by Real-time PCR. The mRNA samples isolated from tobacco leaf infected by TMV, collected at 5 time points during 0-72 h time period after infection, were taken for Real-time PCR analysis. Supported by the result of Real-time PCR, the genes isolated in this study were resistance related. Then, both 5′-and 3′-rapid amplification of cDNA ends(RACE) were preformed, we got full length cDNA sequence of TIF2.3. Then 15 of ESTs and one full length cDNA sequence were analyzed using methods of bioinformatics. Results of Blast analysis in GenBank showed that the TMV induced resistance involves many processes of tobacco biochemistry and physiology and are controlled by multi different genes in tobacco. According to the functions of homology genes, the twelve inducible expressed gene fragments functions involves in the Nucleic Acid Metabolism, protein synthesis and modulation, energy metabolism, stress responding, intracellular transport, Metabolism of Carbohydrates, etc. Two constitutively expression fragment were chloroplast DNA and one suppressive expression fragment was related to cell wall metabolism gene.4. By RACE, we got full length cDNA sequence of TIF2, 857bp,and putative reading frame from 101bp to 613bp. Analysis of Blastn and Blastp showed that the gene was most probably the anti-TMV related novel gene.Under the study, the substance and technique basis for advanced tobacco molecular breeding resistant to TMV was established. And part of the shortage in the area of gene differentially expressed research in tobacco-TMV interaction has been filled up.