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Microarray Analysis Of Gene Expression Patterns And Putative Functions Of Differentially Expressed Genes In Mangrove Kandelia Candel Under High-salinity Stress

Posted on:2011-02-12Degree:DoctorType:Dissertation
Country:ChinaCandidate:P C HouFull Text:PDF
GTID:1100360305964619Subject:Botany
Abstract/Summary:PDF Full Text Request
In this dissertation, a full-length cDNA library of Kandelia candel (L.) Druce leaves was successfully constructed with a capacity of 1.7-2.9×107plaques/library,97% average rate of recombination, and the average size of the inserts was about 1 kb. In our study, a modified sodium borate method was adopted to reduce the interference of polyphenolics and polysaccharides during total leaf RNA isolation.Gene chips were made based on the probes from the designed cDNA library. By means of cDNA microarray, the transcription profiles of K. candel leaves were investigated under short-term (ST) and long-term (LT) of high salinity (500 mM NaCl). We identified a variety of genes that were differentially expressed under ST and LT stress. The transcriptional level of 301 genes among 396 differentially expressed genes increased while the other 95 genes were down-regulated in the ST treatments. Under LT stress conditions,710 genes examined were differentially expressed, among which 507 genes were up-regulated and 203 genes were down-regulated. Moreover, the transcript of 85 genes were found to increase under both ST and LT salt stress.To elucidate the molecular mechanism of salt-tolerant K. candel, expression patterns of up-regulated genes were analyzed combined with gene ontology using Gene Set Enrichment Analysis (GSEA). Salt-induced expression of many organelles and related components, as well as photosynthesis-related genes, were thought to be involved in photosynthetic response and remodeling, which contributing to the salt tolerance. Moreover, genes involved in ROS (active oxygen species) metabolism and homeostasis were highly expressed, indicating K. candel has developed an effective mechanism of ROS scavenging. Meanwhile, ROS may also function as a signal molecular and plays a crucial role in plant salt adaptation. Up-regulation of water channel protein (MIP) genes suggests that salt-stressed K. candel is able to control the transport of water to adjust the intracellular osmotic potential. Some genes involved in protein repairing and degradation of misfolded or incompletely synthesized proteins were also salt inducible, showing a great capacity of protein repairing at the post-translational stage in this mangrove. In addition, the increased expression of genes related to signal transduction and abiotic stimulus indicates that K. candel has a sophisticated defense system upon salt exposure, e.g. stress signal perception, transduction, and down-stream genes activation. Interestingly, the genes controlling ion transport and homeostasis were up-regulated, among which 6 probes containing ATPase (H+-ATPase gene) domain were induced by both short- and long- term treatment, suggesting that H+-ATPase may play an important part in ion transport in salinised K. candel.Real-time PCR confirmation showed a consistent tendency of gene expression with the microarray data. This indicates that the cDNA microarray was reliable to monitor gene expression and subsequent data analysis.By means of open reading frame prediction, three dimentional structure and function prediction, amino sequence alignment, and phylogenetic analysis, we preliminary predicted and analyzed the structure and function of a series of salt regulated genes classified to four gene families in K.candel, including water channel and transporter (plasma and tonoplast membrane), SOD gene family, chlorophll a/b binding protein, and function of unknown genes homology to BURP gene family in plant.
Keywords/Search Tags:Kandelia candel, cDNA library, Expression profile, Gene function
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