Gene discovery and expression analysis in sugarcane leaf and culm

[摘要] ENGLISH ABSTRACT:Sugarcane (Saccharum spp. hybrids) is a commercial crop plant capable of storing upto 20% sucrose on a fresh mass basis in the culm. Knowledge about gene expressionduring sugarcane growth and maturation is limited. The aim of this study was to assesswhether an Expressed Sequence Tag (EST)-based approach towards analysis ofsugarcane would reveal new information about gene expression and metabolicprocesses associated with sugarcane growth and development. The specific objectiveswere two-fold: firstly, to develop an EST database for sugarcane and secondly, toidentify and analyse genes that are expressed in different sugarcane tissue types anddevelopmental stages, with a specific focus on leaf and culm.An EST database for sugarcane was initiated to obtain information on sugarcane genesequences. A total cDNA library was constructed from sugarcane immature leaf (leafroll: meristematic region) tissue and 250 clones randomly selected and subjected tosingle-pass DNA sequence analysis. Sugarcane ESTs were identified by sequencesimilarity searches against gene sequences in international databases. Of the 250 leafroll clones, 26% exhibited similarity to known plant genes, 50% to non-plant geneswhile 24% represented new gene sequences. Analysis of the identified clones indicatedsequence similarity to a broad diversity of genes. A significant proportion of genesidentified in the leaf roll were involved in processes related to protein synthesis andprotein modification, as would be expected in meristematic tissues. Submission of 495sugarcane gene sequences to the dbEST database represented the first sugarcane ESTsreleased into the public domain.Two subtracted cDNA libraries were constructed by reciprocal subtractivehybridisation between sugarcane immature and maturing internodal tissue. To exploregene expression during sugarcane culm maturation, partial sequence analysis ofrandom clones from maturing culm total and subtracted cDNA libraries wasperformed. Database comparisons revealed that of the 337 cDNA sequences analysed,167 showed sequence homology to gene products in the protein databases while 111matched uncharacterised plant ESTs only. The remaining cDNAs showed no databasematch and could represent novel genes. The majority of ESTs corresponded to a variety of genes associated with general cellular metabolism. ESTs homologous tovarious stress response genes were also well represented. Analysis of ESTs from thesubtracted library identified genes that may be preferentially expressed during culmmaturation.The expression patterns of sugarcane genes were examined in different tissue sourcesand developmental stages to identify differentially expressed genes. cDNA arrayscontaining 1000 random clones from immature leaf and maturing culm cDNA librarieswere hybridised with poly (At RNA from immature leaf, mature leaf, immature culmand maturing culm. All cDNAs examined hybridised to all four probes, but differencesin signal intensity were observed for individual cDNAs between hybridisation events.No cDNAs displaying tissue- or developmental-stage specific expression weredetected. Comparisons between hybridisation patterns identified 61 cDNAs that weremore abundantly expressed in immature and mature leaf than the culm. Likewise, 25cDNAs preferentially expressed in immature and maturing culm were detected. ESTsestablished for the differentially expressed cDNAs revealed sequence homology to adiverse collection of genes in both the leaf and the culm. These included genesassociated with general cellular metabolism, transport, regulation and a variety ofstress responses. None of the differentially expressed genes identified in the culm werehomologous to genes known to be associated with sucrose accumulation.To examme differences at the level of gene transcription between low sucroseaccumulatingand high sucrose-accumulating tissues, subtracted cDNA libraries wereutilised. To isolate cDNAs differentially expressed during culm maturation, cDNAarrays containing 400 random clones (200 from each library) were screened with totalcDNA probes prepared from immature and maturing culm poly (At RNA. Resultsindicated that 36% and 30% of the total number of cDNAs analysed werepreferentially expressed in the immature and maturing culm, respectively. Northernanalysis of selected clones confirmed culm developmental stage-preferentialexpression for most of the clones tested. ESTs generated for the 132 differentiallyexpressed clones isolated exhibited homology to genes associated with cell wallmetabolism, carbohydrate metabolism, stress responses and regulation, where thespecific ESTs identified in the immature and maturing culm were distinct from each other. No developmentally regulated ESTs directly associated with sucrose metabolismwere detected.These results suggest that growth and maturation of the sugarcane culm is associatedwith the expression of genes for a wide variety of metabolic processes. In addition,genes encoding enzymes directly involved with sucrose accumulation do not appear tobe abundantly expressed in the culm.

[发布机构] Stellenbosch University