Cloning of three members of ACC synthase gene family in sugarcane

To clone the members of ACC synthase gene family in sugarcane, two degenerate oligonucleotide primers were designed, coding for two conservative acid regons in ACC synthase protein family. PCR amplification was performed on sugarcane DNA template, which produced three fragments that were 1041bp (Sc-ACS1), 1345bp (Sc-ACS2) and 1707bp (Sc-ACS3), respectively. Analyses by using the program of BLAST on NCBI GenBank database showed that the polypeptide sequences of their coding regions highly matched with the family of ACC synthase genes from grass plants, the identity of their nucleotide sequence reached 88% to 98%. The nucleotide sequence of both Sc-ACS1 and Sc-ACS3 in opposite directons shared a homology of 98% but they were less similar to Sc-ACS2 (49%). Alignment and phylogenetic analyses of the amino acid sequence deduced from these fragments and sequences of ACC synthase from other plants in the GenBank. According to the ACC synthase protein family, the intron was excluding and the three sequences coded 326,242 and 310 amino acids, respectively, which spanned 67%, 50% and 64.6%, respectively, of the putative whole sequence in length. The putative amino acid sequence of both Sc-ACS1 and Sc-ACS3 shared significant identity to other ACC synthases (53-96%) and they were more similar to the sequences in grass plant species (80% with Zea mays Zm-ACS6, 75% with Asparagus officinaland and Phyllostachs edulis, and 73% with Oryza sativa OS-ACS1) than to Sc-ACS2 (45-49%). The amino acid sequence of both Sc-ACS1 and Sc-ACS3 in opposite directions shared a homology of 96%. Based on the Northern analysis of the three ACC synthase gene members isolated it was concluded that accumulation of Sc-ACS1 RNA increased moderately in the sugarcane leaves treated with dark-growing condition, ethrel, IAA, LiCl and cold-stress, but not expressed while treated with BA and light- growing condition. The Sc-ACS2 was only expressed in the ethrel treatment, whereas Sc-ACS3 did not express in any treatment. The present study provided a new evidence of ethylene regulation in sugarcane at molecular level, as part of the regulation mechanism of ethylene to the growth of sugarcane plants.