2009 Annual Report
1a.Objectives (from AD-416)
Sequencing and profiling of functional transcripts constructed in expressed sequence tags (ESTs) of Coptotermes formosanus to fulfill the following objectives: a) Identifying and annotating of genes specifically associated with post-embryonic polyphenism (caste differentiation and development); b) Discovering of genes specifically responding to environmental cues and internal signals (pesticides, food sources, juvenile hormones, etc.); and c) Characterizing of genes uniquely involved in critical physiological pathways (digestion, molting, immunity, etc.). The fulfillment of these objectives would directly lead to achieving the following goals: a) providing biochemical/physiological/molecular bases for disruption of colony formation, development and survival; b) Discovering novel target site(s) that would be developed into new control strategies that could be incorporated into effective area-wide integrated management of Formosan subterranean termites.
The overall objectives of this cooperative research are to determine genes that are unique to termites - specifically reproductive and development associated genes e.g. nymph or soldier formation mechanisms with specific emphasis on Coptotermes formosanus while also examining cellulosic material hydrolyzing enzymes for agriculture and industry. We will also concentrate on private and public database and website development strategies.
1b.Approach (from AD-416)
A cDNA library representing expressed genes in each different developmental stage of Coptotermes formosanus has been constructed. To facilitate transcriptome analysis and rare gene discovery, repeated transcripts were proportionally removed from the cDNA library using the procedure of cDNA normalization. The cDNA library would contain approximately 400,000 independent clones, an estimate of at least 16X coverage of the entire expressed genes, provided that the protein-coding capacity of invertebrate genomes is in the range of 16,000 to 25,000 genes. The sequencing and gene data assembly of the cDNA library will be cooperatively conducted in JCVI. We will continue Sanger sequencing of EST clones and perform SoLID sequencing to determine differential gene expression. EST sequences will be compared against existing databases and annotated using the Basic Local Alignment Search Tool (BLAST). Batches of sequences will be sequentially released to GenBank for public access. Unique genes and singletons would be selected and gene expression analysis. Differentially-expressed genes or development-stage specific genes will be preferentially analyzed quantitatively (such as real-time PCR) or qualitatively (such as gene silencing by RNA interference).
Last year, J. Craig Venter Institute (JCVI) completed determining the sequence of a portion of the genetic material obtained from samples from virtually all life stages of the Formosan subterranean termite, Coptotermes formosanus. The 132,000 cDNA (complementary deoxyribonucleic acid) sequencing reads were determined to represent 25,000 unique sequences corresponding to individual genes from the termites and microorganisms living in the termite gut. Over 25% of these unique transcripts showed similarity to known genes from C. formosanus, other termite species, other insect species, or protozoan symbionts (mutually beneficially microrganisms known to inhabit the gut of C. formosanus). These lists of characterized genes included lipopolysaccharide binding protein, trypsin binding protein, chitin binding protein, an actin binding protein, and a zinc binding dehydrogenase, as well as cellulases, glucosidases, hexamerins, and several odorant binding proteins. Unexpectedly, many sequencing reads showed no similarity to any known protein suggesting that they represent non-protein coding regions of the isolated genetic material.
Further computational analysis performed by the JCVI team demonstrated that the
additional analysis is required to determine all of the genes expressed during the development of the termite and its symbionts. Our planned strategy is to characterize all individual stages of the termite by using an instrument that can generate 75,000,000 genetic material sequence reads, while only 10,000,000 reads are believed to be sufficient to determine all of the genes that should be expressed. We will use this approach to perform analysis of 7 individual termite stages and identify genes that may regulate processes associated with differentiation in order to determine possible biologically-based control measures for the termite. This project was monitored through telephone calls, emails and meetings.