Analysis of the genic expression in truffle life cycle by mRNA differential display.

ZEPPA, SABRINA¹, LUCIA POTENZA¹, DEBORAH AGOSTINI¹, EMANUELA POLIDORI¹, ANTONELLA AMICUCCI¹ & VILBERTO STOCCHI¹.

¹Istituto di Chimica Biologica "Giorgio Fornaini", Università di Urbino, via Saffi, 2 - 61029 Urbino (PS) - ITALY.

Differential display has been developed as a tool to detect and characterize expressed genes in the sporal and vegetative phases of Tuber borchii life cycle. This technique involves the reverse transcription of mRNA population with an anchored oligo (dT) primer (T₁₂MN) followed by the polymerase chain reaction using a combination of arbitrary 10mers and T₁₂MN. Recovered complementary DNA fragments corrisponding to several apparently expressed mRNA were screened by dot blot differential hybridization to avoid false positive clones. The procedure consisted of imobilizing the amplified fragments on nylon membranes using a dot blot apparatus and hybridizing the membranes to ³²P-labelled cDNAs from two phases tissue. One hundred clones were selected from denaturing polyacrylamide gels obtained using 20 primers combinations. These clones were screened with dot blot differential hybridization. Clones which confirm differential regulation were sequenced and introduced in the gene databases to search for homology.
The identification of genes differentially regulated is important to know the biochemical and physiological modification observed during the life cycle of truffle.

Key words: differential display, Tuber borchii, fruitbody, mycelium, reverse transcriptase PCR