Construction Of CDNA Library And Identification Of Genes Involved In Floral Development In Male Floral Buds Of Populus Deltoides

Populus deltoides Marsh., belonging to the family of Salicaceae, is distributed worldwide and plays an important role in the genetic improvement for the Populus. But the fact that many of the traits of poplars are controlled by multiple genes, pulsed high heterozygosity, and a long life cycle makes conventional breeding very difficult, moreover the risks associated with the use of engineered plants include the potential effects on non-target organisms and environment, and causing allergic reaction to human due to the escape of transgenes through pollen dispersion. In order to shorten the breeding cycle and to reduce the pollution derived from pollens, a cDNA library for the male buds of P. deltoides was constructed and several genes involved in flower development were isolated based on EST sequences in databases in this study. In addition, many transgenic tobaccos with sense and anti-sense genes were produced via Agrobacterium tumefaciens-mediated transformation. The results are as follows:1. A full-length cDNA library for the male floral buds of P. deltoides was constructed by using SMATR (the Switch Mechanism At the 5' end of RNA Templates) technique. The quality detection indicated that the library had a starting titer of 6.8×10⁶ pfu·mL^-1 and a final titer of 7×10¹²pfu·mL^-1 after amplification. It also had a high ratio of recombination (up to 95%) with the inserts at an average size of approximately 1 kb. These results showed that this library was highly quality, which is suitable for the isolation and expression of target genes and provide an important foundation for the studies on the molecular mechanisms involved in the flower development of forest trees.2. By large-scale ESTs sequencing of 4 200 randomly selected clones from the constructed library of male floral buds of P. deltoides, 3 092 raw sequences were obtained. About 3 086 valid ESTs were generated after cleaning of inserts that is shorter than 150 bp, and the contaminants were phylogenetically classified into 416 clusters. An assembly of sequences within independent clusters finally 451 contigs and 1 104 singletons were identified. BLAST analyses within these 1 555 unique sequences against NCBI database demonstrated that 540 unique sequences (34.7%) did not have homologous proteins or nucleotide sequences in the database, suggesting that they might be new genes involved in the floral development. In addition, eight MADS-box genes, including three full-length genes that have not been reported, were obtained.3. Multiple alignment analyses showed that PdPI gene of male floral buds of P. deltoides shared high similarity (over 80%) with the MADS genes from Litchi chinensis, Betula pendula, Cucumis sativus and Malus domestica, while PdAGL gene did not have homologous nucleotide sequences in the database. Further analyses indicated that there were a highly conserved MADS domain, a middle level of conserved K domain and the variable I and C domains present in both PdPI and PdAGL genes. In addition, a highly conserved PI motif was found in the C domain of PdPI gene. The results of phylogenetic analyses showed that PdPI and PdAGL genes were belonging to the subfamilies PI and DEFH 7 respectively.4. The expression pattern of PdPI and PdAGL was analyzed by using quantitative real-time PCR analyses. A dramatic increase in the expression of the PdPI gene was detected in a reproductive organ, including the catkins and roots, It can be suggested that PdPI gene might be involved in the processes of pollen maturation and root development of P. deltoides. The results showed that the PI homo log of Arabidopsis was expressed not only in reproductive organs, but also in vegetative organs Although PdAGL was closely related to the DEFH 7 from Antirrhinum, the expression pattern of PdAGL gene observed in P. deltoides was significant difference from that of DEFH 7.5. A sense and an anti-sense plant expression vectors for PdPl and PdAGL were constructed and were transferred into tobacco via Agrobacterium tumefaciens-mediated transformation based on the establishment of a high efficient genetic transformation system. PCR analysis with NPT II specific primers confirmed the integration of foreign genes into the genome of transgenic tobaccos. The observation of phenotype revealed the pre-mature transgenic tobaccos with sense gene and anti-sense-gene-containing tobaccos with abnormal leaf, delayed flowering and reduced amount of flowers.