Mangrove Salt Tolerance Gene (aoc) Cloning And Transformation Reyan V Stylosanthes Preliminary Study

Allene oxide cyclase (AOC) catalyzes the stereospecific cyclization of an unstable allene oxide to (9S,13S)-12-oxo-(10,15Z)-phytodienoic acid, which is the precursor next to jasmonic acid. Since the dimeric enzyme is found with two identical N-terminal peptides, the allene oxide cyclase is suggested to be a homodimeric protein assembled through processed N-termination. The homology among the AOC of Bruguiera sexangula (Lour.) Pair and Lycopersicon and Arabidopsis is very high, however, the AOC of Bruguiera sexangula (Lour.) Pair displays salt-tolerance, which the other two have not. By comparing their functional regions, it is found that a special N-terminal 70 amino acids peptide in the AOC of Bruguiera sexangula (Lour.) Pair is essential for the salt-tolerance. As the out of common function, the homologs of mangrove AOC are designated "mangrin.A pair matched primers to the AOC mRNA of Bruguiera sexangula (Lour.) Pair in Gene bank was designed. The gene was isolated by RT-PCR from Bruguiera sexangula (Lour.) Poir. The mangin cDNA is 771bp, which codes 256 amino acids.Two different AOC expressing vectors were constructed in the present work. AOC fused with ipt gene in transcriptional manner down stream double or single constitutive 35S promoters, 晈hich resulted in 35S-35S-0229AOC:::ipt or 35S-0229AOC:::ipt respectively, the two constructs used Bar gene as selection marker.A perfect transgenic regeneration system was established for Stylosanthes guianensis cv. Reyan No.5 through plenty of researches, which was suitable to Bar gene selection system. The 35S-35S-0229AOC:::ipt was transformed to the explants (cotyledon and hytocotyl) of Stylosanthes guianensis cv. Reyan No.5 mediated by Agrobaclerium tumefaciens GV3101. Finally, I get 12 calli that were resistant to 1.5 mg/L Glufosinate.