Analysis Of Gene Expression Profiles Responsed To Fusarium Oxysporum In Lilium Regale

Lily (Lilium L.) is one of the major cut flowers in the world, which was generally called with the king of flower bulbs. However, as a kind of valuable flowers, lily is a susceptible flower. With the ampliative area of lily production, the diseases of lily have become the major factor to limit the development of lily production industry. The Fusarium wilt, caused by the soilborne pathogen Fusarium oxysporum, is the major disease of lily, which cause large economic losses to the productions of fresh cut flowers and bulbs. Lilium regale is a special and wild lily species of China, and it distributes in the river basin of Minjiang of Si Chuan province. L. regale was found to have extremely high resistance to F. oxysporum. Nonetheless, the molecular mechanisms on resistance of L. regale against F. oxysporum are still unclear. In this study, the suppression subtractive hybridization (SSH) combined with gene chip technology were developed to better understand the molecular mechanism in L. regale defense response to F. oxysporum.The main results of this study were shown as follow:A SSH based cDNA library was constructed from L. regale inoculated F. oxysporum. The RNA samples from infected root recovered at different time points (6,12,24,36and72hpi) were mixed together in equal proportions as the tester, and RNA from water-treated root at the same time points was as the driver. The cDNA library consisted of3168positive transformants through randomly picking. The length of the cDNA inserts ranged from200to1000bp with average length of400bp. In total,1101clones were successfully sequenced, and there were585unique sequences including194contigs and391singlets through sequence analysis. The585unique sequences were then performed homology analysis with BLASTx tool in GenBank. As a result,342unique sequences were assigned to no significant homology. The functional categories of the remaining243ESTs which had significant homology with known proteins were assigned and classified into14putative cellular functions according to categories established in Arabidopsis, including disease/defense (33%), metabolism (13%), protein synthesis (9%), transporters (8%), secondary metabolim (5%), transcription (5%), and so on. Analysis of gene expression profiles of the585unique ESTs responsed to F. oxysporum in the resistant species (L. regale) and the susceptible cultivar (’Siberia’) were performed with oligonucleotide microarray. The microarray experiment was conducted with the four selected samples (0,2,12and24hpi) in L. regale and ’Siberia’, respectively. The results of microarrays hybridization showed that there was a big difference in the gene expression profiles between incompatible reaction and compatible reaction. There were152genes up-regulated in incompatible reaction, and157genes were down-regulated. While in compatible reaction, the numbers of up-and down-regulated genes by F. oxysporum infection were85and115, respectively. In the comparative study of gene expression profiles between L. regale and’Siberia’, there were221genes up-regulated and260genes down-regulated in L. regale, respectively. The functional categories of these differently expression genes were performed, and there were33genes belong to category of disease and defense, which was the major part of the genes having significant homology with known proteins. The result of detailed gene expression profiles in L. regale and ’Siberia’ response to F. oxysporums showed that some genes encoding pathogenesis-related proteins, oxidative stress-related genes, secondary metabolim related genes, signalling-related genes or transcription factors as well as some genes encoding unkown proteins were involed in L. regale defence responses. Both types of lilium plants showed an increased level of these genes after inoculation with F. oxysporum, but the increase was greater and faster in L. regale. This may be the major reason of the different susceptibility to F. oxysporum in L. regale and’Siberia’.A novel bZIP transcription factor was isolated from L. regale using the rapid amplification of cDNA ends (RACE) which was named as LrbZIPl. This bZIP transcription factor had a basic region that binds DNA and a leucine zipper dimerization motif. LrbZIPl was979bp in length with an ORF of429bp, a5’-UTR of305bp, and a3’-UTR of245bp, which encoded a predicted polypeptide of142amino acids. Expression analyses of the LrbZIP1in L. regale through qPCR was conducted, as a result, LrbZIPl was induced by F. oxysporum, SA, JA, ET, and H2O2.