Identification And Functional Analysis Of Differentially Expressed Genes In Differentiating Xylem Of Chinese Fir (Cunninghamia Lanceolata) By Suppression Subtractive Hybridization

Wood is the most important natural and endlessly renewable source of energy and therefore has a major future role as an environmentally cost-effective alternative to burning fossils fuels. It is also ecologically significant, for being a long-term sink for atmospheric carbon and being a crucial process to achieve reequilibrium of various matters in the biosphere.Wood is an important raw material for global industries with rapidly increasing demand. It is the main products of forest trees. Because of the economic and ecological significance of woody plants, plant biologists have being paid more and more attention to the forest research. Wood formation (xylogenesis) is a crucial developmental process for both scientific interest and applicable value. It originates from a highly specialized tissue called the vascular cambium and includes 5 steps: cell division, cell enlargement, cell wall thickening, lignification and programmed cell death.Thanks to application of new genomic technologies to forest trees and the knowledge about evolutionarily conserved mechanisms from model annuals, our current understanding of the molecular mechanisms and genetic control underlying wood formation has been largely extended. But it is still limited. Recently, most of the research progress concerning wood formation has been based on EST sequencing strategies derived from wood-forming tissues, separation of proteins isolated from wood tissues, and cDNA microarray analysis.Part I Construction and analysis of the forward and the reverse subtractive cDNA libraries of Chinese fir during wood formationTo isolate the differentially expressed genes in xylogenesis of Chinese fir (Cunninghamia lanceolata (Lamb) Hook),we used a novel system. A forward and a reverse subtractive cDNA libraries were constructed using the suppression subtractive hybridization (SSH) method, which was performed using the cDNA from the mutant Dugansha as the tester and the cDNA from the wild type Jurong 0 as the driver, the wild type Jurong 0 as the tester and the mutant Dugansha as the driver, respectively. Transcriptional profiling was performed using macroarray with four DIG-labeled probes. We obtained 618 and 409 clones from the forward and the reverse subtractive library respectively.A total of 405 unique ESTs were obtained, 40% of the ESTs exhibiting homologies with proteins of known function and falling into 4 major classes: metabolism, cell wall biogenesis and remodeling, signal transduction and stress. Real-time PCR was performed to confirm the results. The expression levels of 11 selected uniESTs were consistent with both macroarray and real-time PCR.The systematic analysis of genes involved in wood formation in Chinese fir provides valuable insights into the molecular mechanisms involved in xylem differentiation, and is an important resource for forest research that can be directed toward understanding the genetic control of wood formation and future endeavors to modify wood and fiber properties for industrial use.Pa rt II Cloning and functional analysis of genes involved in cell wall expansionProteins in the cell wall are believed to play important roles in regulation of cell wall extensibility, which is a key parameter in determining cell expansion. Among the cell wall proteins studied to date, expansins are unique in their ability to induce immediate cell wall extension in vitro and cell expansion in vivo, without any activate protein at a certain pH condition.We successfully cloned 3 full-length expansin genes using RLM-RACE. Sequence analysis showed that ClEXP1 cDNA was 1033bp in length capable of encoding 247 amino acids, ClEXP2 cDNA was 1374bp in length capable of encoding 268 amino acids, and ClELP1 cDNA was 1023bp in length capable of encoding 272 amino acids. The GenBank accession numbers are EF102108, EF158813 and EF192940, respectively. Phylogenetic reconstruction indicated that ClEXP1 and ClEXP2 belonged toα-expansin (EXPA); whereas ClELP1 belonged toγ-expansin (EXLA). The intron-extron pattern of ClEXP1 is similar to the model of Arabidoposisα-expansins (EXPA), it was disrupted by intron A and B; while genomic sequence of ClEXP2 contains three introns (A, C, and F), more like Arabidoposisβ-expansins (EXPB).Expression patterns of ClEXP1 and ClEXP2 were similar: high transcript levels were detected in cambium region, and intermediate levels were observed in needle and shoot tip; in mature xylem mRNA levels were very low, and in root and pollen, the transcript levels were barely detectable. Whereas for ClELP1, high transcript levels were observed in cambium region and needle; intermediate levels were detected in shoot tip and xylem; low expression levels in pollen and barely detectable in root. These isolated genes will provide an important pool to systematically study the expression, function and regulation mechanism of expansins during wood formation.Spatial expression pattern suggested that both ClEXP1 and ClEXP2 are preferentially expressed in cambium region. To investigate in vivo functions of both genes during wood formation in plants, we carried out ectopic expression of Chinese fir expansin genes in tobacco. Overexpression of ClEXP1 and ClEXP2 in tobacco plants yielded pleiotropic phenotypes in plant height, stem diameter, leaf number, flowering time, phyllotaxy, and seed set. The height and diameter growth of both 35S::ClEXP1 and 35S::ClEXP2 transgenic plants increased significantly, with an enlargement of pith parenchyma cell area. The crystalline cellulose contents of 35S::ClEXP1 and 35S::ClEXP2 transgenic plants were increased by 50% and 30%, respectively, acompanied by partially thickening of the cell wall in the wood-forming region.Combined together, both ClEXP1 and ClEXP2 are involved in plant growth, with partially function redundancy; ClEXP1 maybe involved in primary growth, while ClEXP2 may play an important role in secondary growth; and the functional manner or site of ClEXP1 and ClEXP2 may be distinct. These results suggest that expansins take part in the assembly of the cell wall by directly or indirectly affecting either cellulose synthesis or deposition.