Cloning and characterization of a fructosyltransferase gene in the native graminean species Bromus pictus

FUSARI CM; DEL VISO F; NAZER E; HEINZ RA; PUEBLA AF; HOPP E

La Havana

Simposio; Fructan 2004. Fifth Internacional Fructan Symposium; 2004

Center for Genetic Engineering and Biotechnology (CIGB)

Abiotic stresses induce complex responses in plants, affecting productivity of agronomically important crops. Acclimation to low temperatures involves biochemical and physiological processes that are associated to cold tolerance. Accumulation of compatible solutes have been found during cold acclimation in Graminean species. Fructans are produced in the vacuole by the action of fructosyltransferases and might be related to low temperatures tolerance in Gramineae species. The aim of this work was to isolate and characterize genes codifying for fructosyltransferases from both native Gramineae species Bromus pictus and an agronomically interesting species, such as wheat.A genomic BAC library of T. monococcum was screened with a fructosyltransferase cDNA of B. pictus. Positives clones were subcloned and sequenced. The 5` end of a fructosyltransferase cDNA from B. pictus previously isolated was obtained by PCR techniques. The fragments amplified were cloned and sequenced. Bioinformatic analysis demostrated that 5` end is less similar to other fructosyltransferase genes than the 3` end of the same sequence. The resulting cDNA was used to transform tobacco plants. Transgenic and wild type plants were analyzed by genomic PCR, RT-PCR and the enzyme activity was measured to evaluate the expression and functionality of the transgene. TLC runs of sugar extracts were evaluated to estimate the fructan contents of both transgenic and wild type tobaccos. Gene expression studies of Bromus cDNA were done by real-time RT-PCR. Preliminary results suggests that low temperatures in Bromus pictus would not modify the expression of this fructosyltransferase gene.