Cdna arrays and PCR-select applied for characterization of agricultural features in plants

Identification of genes activated or inhibited during the analyzed process is the base for plant biotechnology. PCR-select (suppression subtraction hybridization) - technique of isolation of genes specific for a process of interest - was applied for analysis of symbiotic interactions between Lupinus angustifolius and nitrogen fixing bacteria. The subtraction, with RNA from the early stages of symbiosis of Lupinus angustifolius - nitrogen fixing bacteria as a tester and RNA from uninfected roots as a driver, enriched the pool of cDNA molecules with ones specific for symbiosis. ENOD40 and enolase were used to evaluate efficiency of PCR-select. The pool of subtracted cDNA molecules was cloned and a set of 768 randomly picked up clones was organized in an array. SNF (Symbiotic Nitrogen Fixation)-specific genes (cDNAs) were identified with differential hybridization using cDNA array. Eighteen marker cDNAs representing genes of well characterized expression pattern and/or function (carbon and nitrogen metabolism, SNF development, cytoskeleton) were cloned in advance and used as references in expression profiling.

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