[Cloning and sequencing of ACC oxidase gene from sugarcane]

Abstract

The plant hormone ethylene is not only responsible for the initiation of fruit ripening, senescence and dormancy but also for regulating many other plant developmental processes, such as seed germination, root initiation, growth, floral differentiation, sex differentiation and responding to environment stresses. One of the rate-limiting steps for ethylene biosynthesizing in plant is catalyzed by 1-aminocyclopropane-1-carboxylate (ACC) oxidase. Understanding of ethylene expressive pattern in plant is an entrance to understand the roles of ethylene on plant. In this paper, two degenerate oligonucleotide primers were designed, coding for two conservative amino acid regions in ACC oxidase protein family, the sequences of the two primers were TAGAGCTCGATGC[TA]TG [CT]GA[GA]AA[AC]TGGGG and CGTCTAGAGCTTC[GA]AATCTTGGCTCCTT respectively. A PCR amplification was performed on sugarcane (Saccharum L. Hybrid cv. ROC16) DNA template, and produced a fragment of 940 bp. By using the program of BLAST on NCBI GenBank database, the sequence presented a very high match with the ACC oxidase genes from other plants, 63 searched out sequences were all ACC oxidase genes. After alignment on PCgene program, the identities of the cloned fragment with ACC oxidase genes from rice and bamboo were both reaching about 88%. So we can concluded that the cloned sequence was a member of ACC oxidase genes fragment from sugarcane. The sequence has been submitted to the GenBank database, the accession number is AF442821. According to the ACC oxidase protein family, a 'intron' of 103 bp was excluded and the sequence coded 279 amino acids, which spanned 88% of the putative whole sequence in length. Alignment and phylogenetic analysis of the amino acid sequence deduced from this fragment and the ACC oxidase sequences of other plants retrieved from GenBank were carried out by using PCgene program. The putative amino acid sequence shared a homology of 86% with the ACC oxidases of bamboo and rice, 74.6% with banana, 70% with tomato and potato and 68% with melon and carnation, which showed that the homology of sugarcane ACC oxidase with monocot was higher than with dicot. The results of phylogenetic analysis showed that ACC oxidase from sugarcane and ACC oxidases from rice clustered together firstly, and then came those from banana, ACC oxidases of dicot from potato, tomato, petunia, melon, Arabidopsis thaliana and carnation came subsequently. It indicated that sugarcane ACC oxidase had a closer phylogenetic affinities to the monocot ACC oxidase sequences than to the dicot ACC oxidases sequences. The clustering results of ACC oxidase molecules accorded with morphological classification system.