White, JF 2009, 'Gene expression analysis of malting barley', PhD thesis, Southern Cross University, Lismore, NSW.
Copyright JF White 2009.
Malting barley (Hordeum vulgare) is of significant importance and economic value to the malting and brewing industries. The biochemical and physiological processes of barley seed germination (malting) have subsequently been the focus of substantial research; however the underlying molecular processes require further investigation. The introduction of large-scale gene expression technologies has provided an opportunity to contribute significant genetic data to the understanding of the malting process. Long Serial Analysis of Gene Expression (LongSAGE) was applied to malting barley to characterise the transcriptional profile exhibited during grain germination. Eight LongSAGE tag libraries were constructed, one from whole dry seed and one from each of the time points 0, 12, 24, 48, 72, 96 and 120 hours post-steeping. Approximately 20 000 tags were sequenced from each LongSAGE library, generating 41 909 unique tag sequences. Annotations of the 100 most abundant LongSAGE tags from each library were analysed to reveal the major functional groups expressed in the malting transcriptome. Statistical analysis confidently identified 57 LongSAGE tags with significant changes in tag abundance across time. The genes that correspond to eight of these tags were targeted for validation by real-time RT-PCR. Real-time RT-PCR experiments were conducted using RNA extracted from the same grain sample from four of the time points examined by LongSAGE; whole dry seed, 12 hours post-steeping, 48 hours post-steeping and 96 hours post-steeping. Three of the transcripts show co-ordinated expression demonstrating significant up-regulation at 48 hours post-steeping and remain significantly elevated during malting. These transcripts α-amylase type B, (1-3,1-4)-β-D-glucanase and cysteine proteinase EP-B represent key starch, cell wall and protein modifying enzymes known to play significant roles in the malting process. mRNA abundance levels observed by real-time RT-PCR correlate well with the data obtained from LongSAGE.
Microarray is another large scale gene expression technology with the potential to generate large amounts of genetic data quickly and efficiently. A CombiMatrix 12K ElectraSenseTM Custom Array chip was designed from the unique LongSAGE tags from 24 hours post-steeping. RNA from the malting barley 12 hours post-steeping and 48 hours post-steeping samples was hybridised to the array chips. The CombiMatrix ElectraSenseTM system incorporates electrochemical detection of a redox reaction proximal to probe specific electrodes. 113 probes show a greater than 10 fold difference between 12 and 48 hours post-steeping with 64 probes preferentially expressed at 12 hours post-steeping and 49 probes preferentially expressed at 48 hours post-steeping. The eight genes of interest analysed by real-time RT-PCR were represented by one or more probes on the malting barley CombiMatrix ElectraSenseTM array. Fold changes observed between 12 and 48 hours post-steeping by the CombiMatrix ElectraSenseTM microarray support the fold changes observed by LongSAGE and real-time RT-PCR.