De novo assembly, annotation and analysis of transcriptome sequences of callus culture from Aquilaria malaccensis Lam.
Aquilaria malaccensis is a major source of agarwood, a rare and highly priced wood product. Due to its high demand, it is endangered and listed in the Appendix II of Convention on International Trade in Endangered Species of Wild Fauna and Flora. Because of insufficient genomic and transcripto...
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| Format: | Thesis |
| Language: | English |
| Published: |
2015
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/71105/1/FH%202015%2018%20IR.pdf |
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| Summary: | Aquilaria malaccensis is a major source of agarwood, a rare and highly priced wood
product. Due to its high demand, it is endangered and listed in the Appendix II of
Convention on International Trade in Endangered Species of Wild Fauna and Flora.
Because of insufficient genomic and transcriptomic data available in public database for
understanding the molecular basis of agarwood formation, the goal of this study was to
obtain A. malaccensis expressed gene sequences using the transcriptome sequencing by
next-generation sequencing (NGS). To obtain sufficient data from NGS sequencing, a
high quality RNA sample is required. The RNA yield, purity, and integrity of six
different extraction methods were compared. Conventional methods yielded RNA with
good purity but the RNA integrity was poor. When using modified RNeasy Plant Mini
kit, the highest yield was obtained while maintaining the integrity of RNA. This method
was used to extract total RNA from callus samples and sent for transcriptome sequencing.
Callus tissues were treated under stress condition by nutrient shortage of 1.1 μm 1-
naphthaleneacetic acid (NAA), 2.2 μm 6-benzylaminopurine (BAP), and 15 g/L sucrose;
collected during death phase when it producing brownish exudates and agarwood scent.
Two cDNA libraries were constructed from mRNAs of untreated and treated callus
tissues and sequenced using paired-end libraries on an Illumina HiSeq2000 platform.
After filtering and trimming, a total of 200,062,275 and 166,544,202 clean reads were
obtained for untreated and treated callus libraries, respectively. De novo assembly was
carried out using SOAPdenovo-Trans and TGICL. A total of 231,594 unigenes were
generated from the assembly. Assembled sequences were annotated using BLASTX
against the NCBI non-redundant protein database where 41.5% unigenes showed
significant alignment. The differential gene expression value between untreated and
treated samples were calculated using DEGseq. A total of 14,029 genes were identified
as differentially expressed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and
Genomes (KEGG) annotations were reported using BLAST2GO software. Out of the
107,593 unigenes that showed significant alignment with non-redundant protein
database, 96,743 unigenes were successfully annotated with at least one GO term. The annotations were classified into the three main GO categories: biological processes
(50.7%), molecular functions (24.0%) and cellular components (25.3%). A total of 144
KEGG pathways were identified from 46,076 unigenes. Enrichment analysis showed that
the genes were enriched in the stress responses and sesquiterpene biosynthesis activity.
This is the first comprehensive de novo transcriptome assembly and profiling for A.
malaccensis. These transcriptome libraries provide valuable transcriptomic reference
resource for future research. |
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