استفاده از رونويسي معكوس و واكنش زنجيره اي پليمراز در تكثير و شناسايي ژن هاي القاءشونده در معرض تنش شوري در AGROPYRON ELONGATUM

Application of RTand PCR techniques for amplification and characterization of satt-inducible genes in Agropyron elongation.

Total RNA isolated from A. elongatum treated with salt (150 mM NaCI), was reverse transcribed. The resulting cDNAs were amplified by two-step PCR, using one specific primer and oiigo-dT. Four distinct bands of DNA were displayed in agarose gel electrophoresis, with approximately 501, 446, 398 and 356 base pairs. These were then excised, reamplified and the DNA used for hybridization, cloning and sequencing. Using southern blot analysis with the common oligonucleotide probe for non-specific lipid transfer preoteins (nsLTPs), the band of the largest size was hybridized. After elution of the PCR bands, the cDNAs were cloned in PCR plasmid vector, and two of the clones (Les11 and Les12) with inserts were sequenced. The two clones were shown to be similar in nucleotide sequence and with a longer sequence for Les11. The nucleotide sequence of Les11 showed 45% similarity to the oligonucleotide used as probe in the southern hybridization. It also showed a similarity of 41.5% to a nsLTP gene (blt4.9). A new sequence was, however, obtained which included a possible expressed region.