Differential gene expression in the culm of sugarcane during development, with special emphasis on the storage parenchyma cells

[摘要] ENGLISH ABSTRACT:For the expression of transgenes in plant cells, appropriate promotersequences have to be introduced upstream of the gene to ensure efficienttranscription. While to date the maize ubiquitin (Ubi1) promoter has been themost effective transgene promoter for sugarcane, there is a high demand fortissue and stage specific promoters for localised transgene expression in themature culm. The present study sought to characterise genes preferentiallyexpressed in the core and peripheral tissues of the mature culm, which canfurther be used as research tools for specific promoter isolation.cDNA expression arrays containing 3840 clones from a late stage cDNAlibrary representative of the core and peripheral tissues of the mature culmwere prepared. The cDNA expression arrays were then differentiallyscreened in independent hybridisation experiments with radioactively-labeledcDNA representations of core and peripheral tissues of internode 7, andperipheral tissues of internode 10. Comparison of the expression profiles ofthe arrayed cDNA targets in the three probes led to the identification of 60tissue-specific, 17 stage-specific and 50 selectively expressed cDNAs withinthe mature sugarcane culm.~ESTs of 33 chosen selectively expressed cDNAs with a relatively strongerpattern of expression in the core than in the peripheral tissues revealedsequence homology to a diverse collection of genes in the mature culm.These included genes associated with general cellular metabolism such asprotein synthesis, protein modification and structural protein. Also identifiedwere stress-responsive genes. The putative translational products of some ofthese clones had homologs that are involved in cell-wall structure in otherspecies. These included the [acalin homolog, a lectin, hydroxyproline richglycoprotein and structured polyprotein C. Many of the cDNAs thought to beinvolved in cell wall structure or stress related responses also accumulate ina developmental manner in other plants. These may indicate that specificmature culm mRNAs accumulate in response to stresses such as rapid cellexpansion or as part of the late developmental program. An unexpectedobservation was that only one gene associated with sucrose metabolism wasidentified, namely sucrose synthase. These results confirmed that culmmaturation was not controlled by sucrose metabolism despite its distinctphysiological characteristic of storing high levels of sugars.ESTs analysis further revealed that sequence homology was not obtained forall the cDNAs exhibiting stage and tissue specific expression in the core andperipheral tissues of the mature culm. These could represent novel genes notonly from sugarcane but all plants.Northern analysis demonstrated that 9 putatively identified selectivelyexpressed genes tested so far accumulated specifically in the core andperipheral tissues of the mature culm. No expression was detected in root,leaf, leafroll and internode 3. However, their selective expression in a singleinternode as observed on the arrays (i.e hybridisation signal intensity beinghigher in the core than in the peripheral tissue) was not detected on thenorthern blots. These showed that cDNA expression arrays were not a highcapacitygene expression assay since they were prone to false expressionanalysis. The validity of results obtained through array screening shouldalways be verified in an independent manner, preferably by the northernhybridisation analysis.Hence, the present study shows that the combination of differentialscreening, northern blot and DNA sequence analysis permits the rapidcharacterisation of differentially expressed genes in the core and peripheraltissues of the mature sugarcane culm. These can further be used asresearch tools for mature culm - specific promoter isolation in the sugarcane.

[发布机构] Stellenbosch University