Biosynthesis of 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (Ectoine) from Marinococcus halophilus into tobacco

摘要

Ectoine (l,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is a compatible solute in many halotolerant bacteria. Its biosynthesis originates from L-aspartate-#beta#-semialdehyde and requires three enzymes: L-2,4-diaminobutyric acid aminotransferase (gene: ect B), L-2,4-diaminobutyric acid acetyl transferase (ect A) and L-ectoine synthase (ect C). Transgenic tobacco lines expressing the three genes, cloned from Marinococcus halophilus, were generated. HPLC, MS and NMR, indicated variation in amounts of ectoine in the transgenic lines ranging from 0.25 to 25 nmol/g fresh weight. Ninety-eight to one hundred percent seeds of T1 generation of both transgenic and SR1 progenitors germinated at NaCl concentrations ranging from 100 to 200 mM in the growth medium. Growth of transgenic seedlings was enhanced at all additional NaCl concentrations as compared to SR1 progenitor lines. Forty to forty-five percent germination at 250 and 25-30% germination at 300 mM NaCl in the growth medium, was seen in transgenic lines as compared to 2% germination at 250 mM and absence of germination at 300 mM NaCl of SR1 controls. Leaves of theseedlings growing at these NaCl concentrations were bleached and deformed. Transfer of these seedlings to media having 100 and 200 mM NaCl, 20 weeks after germination restored growth in 90% of the transgenic seedlings. Cation and amino acid analyses indicated higher amounts of Na~+, K~+, Ca~(2+), Pro, Glu, Gln, Asp, Asn, Ser and Thr in the transgenic seedlings in concert with increasing NaCI concentrations as compared to controls. Likely role of ectoine as an osmoregulator contributing to these effects in seedlings of transgenic tobacco as in several nonhalophylic bacteria is discussed.