With the advent of automated capillary-based DNA sequencers, it is now possible and cost-effective for a single automated machine to sequence hundreds of cDNA fragments in a day. These cDNA fragments are generated from mRNAs being expressed in a particular tissue at the time of extraction. Many short sequences of such cDNA which are unique for specific expressed genes are called ESTs (Expressed Sequence Tags) for those expressed genes. A number of large EST libraries now exist for several plants and the list grows longer each day because the cost of producing such libraries is relatively low. This cost effectiveness of generating ESTs from a broad range of plant tissues can be exploited by most researchers to rapidly identify and engineer a trait from an exotic species into a commercially produced one. In the June 2000 issue of Current Opinion in Plant Biol. 3(3):224-8, Drs. John Ohlrogge and Christoph Benning describe several recent examples in which ESTs have been successfully used to rapidly identify and isolate several exotic but commercially important genes from largely genetically not a characterized plant species.
First in their list of EST sequencing as a gene discovery tool is the gene for castor hydroxylase, the crucial enzyme in the synthesis of ricinoleic acid, a valuable industrial oil, which occurs in abundance in castor seeds but very few other plants. From a cDNA library constructed from mRNA extracted from castor-bean endosperms and enriched by hybridizing against leaf and storage protein cDNAs, 468 clones were produced. Two of these showed similarity to a membrane-bound desaturase from Arabidopsis. Expression of the full length cDNA in tobacco, confirmed that the gene for castor hydoxylase had been identified. A second example is outlined which concerns the use of an EST library to identify genes for enzymes involved in isoprenoid biosynthesis of menthol in the oil glands of the peppermint plant. A third example is the use of ESTs from developing cotton fibers in idenifying genes encoding plant cellulose synthase, the enyzme reponsible for cellulose synthesis.
Ohlrogge and Benning then turn their attention to describing how EST data sets could also be used to provide information on gene expression in a section they call Transcription profiling and metabolic networks. “This aspect of EST analysis is based on the rationale that if gene X is highly expressed, (leading to high mRNAx levels), the cDNAs corresponding to mRNAx will be abundant in a cDNA library made from the tissue in which the gene X is expressed.” A simple count of the numbers of specific ESTs in random samples should give an estimate of relative gene expression in what the authors dub “electronic or digital northerns”. The advantages and disadvantages of this approach relative to conventional northern analysis and microarray analysis are discussed.