Construction Of Potato Virus Y-resistance SSH Library And Analysis Of Related Genes In Tobacco

Tobacco Potato Virus Y (PVY) is one of the most serious disease in tobacco production. In recent years, PVY was rapidly spread in mainly tobacco producing region in China and causing serious harm to tobacco production and quality. In this study, we used VAM of burley tobacco germplasm with high PVY-resistance, through suppression subtractive hybridization and cDNA chip technologies, to screen gene fragments induced by PVY, part of the virus resistance related genes were cloned through RACE, analyzed and identified by bioinformatic methods and realtime PCR, also relative expression characters were explored by realtime PCR, and provide theoretical foundation for PVY-resistant tobacco quickly and efficiently breeding. The main contents and conclusions include the following:1. Construction of PVY-resistance SSH Library in tobacco and screening specially expressed gene fragments. In this study, we used burley tobacco varieties VAM with high PVY-resistance to construct PVY-resistance SSH library in tobacco, Results of testing the SSH library showed that the size of the gene fragments in library were between 250bp ~ 2000bp, with an average size of about 750bp, indicated that the library construction was well. The SSH library was screened by cDNA chip technologies, and 951 specially expressed gene clones were totally obtained, part of the clones were sequenced and sequence analyszd. According to the cDNA chip results, some obvious specific expressed gene fragments were selected and cloned the full length sequence.2. Cloning and expression analysis of NtPsaN. The full-length cDNA sequence of NtPsaN was 784 bp in length, the open reading frame (ORF) of the full length cDNA was 507 bp in length, encoding 168 amino acids, accession number of Genbank was GU187328. It had high homology with other PsaN subunit, such as Ricinus communis PsaN, Zea mays PsaN and Arabidopsis thaliana PsaN. The gene possesses conserved domains of PsaN superfamily. The PsaN subunit phylogenetic tree was constructed. The results of realtime PCR showed that the expression of NtPsaN was obviously up-regulated in leaves infected with PVY. The NtPsaN gene may play a critical role in the PVY-resistance of tobacco.3. Cloning and expression analysis of NtDUF300-1. The full-length cDNA sequence of NtDUF300-1 was 1188 bp in length, the open reading frame (ORF) of the full length cDNA was 936 bp in length, encoding 311 amino acids, accession number of Genbank was HM030994. The gene possesses conserved domains of protein family DUF300 with unknown function, and also had higher similarity in the amino acids sequence to some members from DUF300 family. The results of realtime PCR showed that the expression of NtDUF300-1 was obviously down-regulated in leaves infected with PVY. The NtDUF300-1 protein was predicted being a membrane protein which located in the plasma membrane. All the results in this study suggest that NtDUF300-1 may be a new gene related with PVY-resistance of tobacco.