Comparative analysis of differential gene expression in the culms of sorghum

[摘要] Despite numerous attempts involving a variety of target genes, the identity of the keyregulatory genes of sucrose metabolism in sugarcane is still illusive. To date,genomic research into sucrose accumulation in sugarcane has focused on genesthat are expressed in association with stalk development/maturation, with the aim ofidentifying key regulatory steps in sucrose metabolism. The identification of possiblecontrolling points, however, is complicated by the polyploid nature of sugarcane.Although these studies have yielded extensive annotated gene lists and correlativedata, the identity of key regulatory genes remains elusive. A close relative ofsugarcane, Sorghum bicolor, is diploid, has a small genome size and accumulatessucrose in the stalk parenchyma. The main aim of the work presented in this thesiswas to use S. bicolor as a model to identify genes that are differentially expressedduring sucrose accumulation in the stalk of low and high sucrose genotypes.In the first part of the study, a macroarray protocol for identification of differentiallyexpressed genes during sorghum development was established. Firstly, themacroarray sensitivity of probe-target hybridisation was optimised with increasingamounts of target DNA i.e. 0.005-0.075 pmol. The hybridisation signal intensityincreased as expected with increasing amounts of probe until the hybridisationsignals reached maximum levels at 0.05 pmol. As a result, to ensure quantitativecDNA detection, probes were arrayed at 0.05 pmol when 1 μg target cDNA wasused. Secondly, intra-array and inter-array membrane reproducibility was found to behigh. In addition, the protocol was able to detect species of mRNA at the lowestdetection limit tested (0.06%) and permits the detection of an eight-fold variation intranscript levels. The conclusion was therefore that the protocol was reproducible,robust and can reliably detect changes in mRNA levels.In the second part of the study, sugar accumulation levels in the immature andmaturing internodal tissues of sorghum GH1 and SH2 genotypes were comparedduring the boot and softdough stages. Sugars (i.e. fructose, glucose and sucrose)accumulated differently in the immature and maturing internodes in both sorghumgenotypes during the boot and softdough stages, with sucrose being the dominantsugar in both stages. Based on these differences in sugar accumulation patterns,immature and maturing internodal tissues of sorghum genotypes were compared for differentially expressed genes. A number of genes were found to be significantlydifferentially expressed during both stages.In order to validate the reliability of the macroarray analysis, fourteen genes werearbitrarily selected for semi-quantitative RT-PCR. Seven genes (50%) revealed asimilar pattern of transcript expression, confirming the macroarray results. The otherseven genes, however, showed a different expression trend compared with themacroarrays. In this study, ESTs from rice and sugarcane were used for probingsorghum. The probability of cross-hybridisation between the probes and variousisoforms of the homologous sorghum sequences is thus high, potentially leading tothe identification of false positives. In addition, variation in expression patterns couldhave been introduced by technical and biological variation.Lastly, to verify that changes in the levels of a transcript are also reflected in changesin enzyme activity, seven candidates were tested for enzyme activity. Only three i.e.soluble acid invertase (SAI), sucrose synthase (SuSy) and alcohol dehydrogenase(ADH), out of these seven genes showed enzyme activity levels reflective of therelative transcript expression. We concluded that changes in transcript levels may ormay not immediately lead to similar changes in enzyme activity. In addition, enzymeactivity may be controlled at transcriptional and at posttranscriptional levels.In conclusion, sugar accumulation in low (GH1) and high (SH2) sucrose sorghumgenotypes is influenced by differences in gene expression. In addition, the power ofmacroarrays and confirmation with semi-quantitative RT-PCR for identification ofdifferentially expressed genes in sorghum genotypes was demonstrated. Moreover,the transcript and enzyme activity patterns of SAI, SuSy and ADH genes showedexpression patterns similar to those of sugarcane during sucrose accumulation.Therefore, using sorghum as a model promises to enhance and refine ourunderstanding of sucrose accumulation in sugarcane.

[发布机构] Stellenbosch University