The majority of plant expressed sequence tags (ESTs) available in the public databases are from nonwoody species such as Arabidopsis, maize, soybean, and rice, with the exception of the model tree species, Populus. In this study, we report the first EST database constructed from the commercially important tree species Acacia. ESTs were generated from pooled RNA extracted from the mature, intermediate, and young inner bark tissues of an Acacia auriculiformis x Acacia mangium hybrid. Finally, 3,182 high-quality ESTs were analyzed representing a total of 1,982 unique transcripts (663 contigs and 1,319 singletons) for the inner bark cDNA library. A total of 1,053 unigenes (1,750 ESTs) showed similarities with protein sequences in public database where 867 were significant matches (E value≤e −10 ). Further analysis performed on unigenes with significant matches and known functions identified 231 genes including genes involved in cellulose, lignin, and hemicellulose biosynthesis. For cDNA library validation, quantitative real-time RT-PCR was performed to study the expression levels of the cellulose and lignin genes identified from this EST database in the phloem tissue of A. mangium, A. auriculiformis, and A. auriculiformis x A. mangium hybrid. All the seven candidate genes were expressed in all the individuals studied confirming the dependability of the EST database. The cDNA library and the EST database constructed are valuable resources for forest tree research aiming towards understanding the genetic control of wood formation and in the future endeavors to modify wood and fiber profile through marker-assisted breeding programs.
ABSTRACT. This study was directed at the understanding of the function of CCoAOMT isolated from Acacia auriculiformis x Acacia mangium. Full length cDNA of the Acacia hybrid CCoAOMT (AhCCoAOMT) was 1024-bp long, containing 750-bp coding regions, with one major open reading frame of 249 amino acids. On the other hand, full length genomic sequence of the CCoAOMT (AhgflCCoAOMT) was 2548 bp long, containing three introns and four exons with a 5' untranslated region (5'UTR) of 391 bp in length. The 5'UTR of the characterized CCoAOMT gene contains various regulatory elements. Southern analysis revealed that the Acacia hybrid has more than three copies of the CCoAOMT gene. Real-time PCR showed that this gene was expressed in root, inner bark, leaf, flower and seed pod of the Acacia hybrid. Downregulation of the homologous CCoAOMT gene in tobacco by antisense (AS) and intron-containing hairpin (IHP) constructs containing partial AhCCoAOMT led to reduction in lignin content. Expression of the CCoAOMT in AS line (pART-HAS78-03) and IHP line (pART-HIHP78-06) was reduced respectively by 37 and 75% compared to the control, resulting in a decrease in the estimated lignin content by 24 and 56%, respectively. AhCCoAOMT was found to have altered not only S and G units but also total lignin content, which is of economic value to the pulp industry. Subsequent polymorphism analysis of this gene across eight different genetic backgrounds each of A. mangium and A. auriculiformis revealed 47 single nucleotide polymorphisms (SNPs) in A. auriculiformis CCoAOMT and 30 SNPs in A. mangium CCoAOMT.
A complementary DNA (cDNA) library was constructed from the developing xylem tissues of Neolamarckia cadamba. A total of 10,368 single-pass sequences was generated through high-throughput 5′-expressed sequence tag (EST) sequencing of the cDNA clones, and 6622 highquality ESTs were obtained after removing the low-quality sequences; this gave approximately 3.17 Mb of data. Clustering of the high-quality ESTs revealed 4728 unigenes, consisting of 2100 consensus and 2628 singletons. A total of 2405 ESTs were successfully annotated with 7753 gene ontology (GO) terms that distributed among three main GO categories, which were biological processes (2333), molecular function (3056) and cellular component (2364). Simple sequence repeat (SSR) mining revealed that the frequency of SSR in the N. cadamba EST database (NcbdEST) was 3.3 %, with the GCT/AGC motif being the most abundant repeat motif. The most abundant transcript with known function found in this database was 60S ribosomal protein followed by 40S ribosomal protein. Some of the important genes involved in xylogenesis and lignin biosynthesis were found in NcdbEST; these include tubulin genes, cellulose synthase (CesA), xyloglucan endotransglycosylase (XET), arabinogalactan, cinnamate 4-hydroxylase (C4H), caffeoylcoenzyme A O-methyltransferase (CCoAOMT) and peroxidase. The data obtained from this study will provide a powerful means for identifying mechanisms controlling wood formation pathways of kelampayan and supply many new cloned genes for future endeavours to modify wood and fibre properties.
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