Cloning And Characterization Of 1-Aminocyclopropane-1-Carboxylate Acid Synthase, GhACS1, From Cotton(Gossypium Hirsutum L.)

The gaseous plant phytohormone ethylene as one of the internal signal molecules plays a pivotal part in plant growth and development and extensively affects the seed germination, root hair development, flower senescence, abscission and fruit ripening. It can also respond to many biotic (e.g. pathogen attack) or abiotic stimuli such as wounding, chilling, ozone, flooding, Li+ and so on. In higher plants, the step converting SAM to ACC catalyzed by ACC synthase (ACS) is considered the rate-limiting step among the ethylene biosynthesis, and the ACS is the rate-limiting synthase.Many ACS genes have been isolated and identified from different plant species these years, and the expression patterns have also been studied extensively. The transgenic plants obtained using the anti-sense method all display an evident character of inhibiting the production of ethylene. Cotton (Gossypium hirsutum) is an economically important species in China, its ACSs, however, have not been reported before. In this thesis, a series of studies have been conducted on the isolation, sequence and expression analysis, function identification of cotton ACS (GhACS1). The main results are as follows:1. A novel wound-inducible ACS gene, termed Gossypium hirsutum ACS (GhACS1), was isolated from cotton, and the accession number is DQ355792. The full-length cDNA of GhACS1 is 1802bp long, has a 1428bp ORF, and encodes for a 476 amino acid protein. Amino acid comparison indicated that GhACS1 has the major characterization of ACC synthases such as seven conserved regions, 11 invariant amino acid residues and the active site. The gene displays high homology to ACC synthases from citrus, tobacco, potato, soybean, mungbean and Arabidopsis. GhACS1 has more closely relationship with some wound-inducible ACS genes, such as CsACS1, CsACS2, NtACS2, GmACS1 and LeACS2.2. The results of Southern blotting indicate that there might exist other ACS genes that share some homology with GhACS1. These results raise the possibility that a multiple gene family may also encode ACC synthase in cotton. After comparison the genomic sequence with the cDNA sequence of GhACS1, we found there are two introns and all of them are located in the 5′-end nearby of the gene, which is consistent with the characterization of the introns in ACS genomic sequence.3. The results of RT-PCR indicate that GhACS1 is an inducible gene. It can transiently expressed by the induction of wounding, ABA and Cu2+, but it can not be induced by the pathogen, chilling, IAA, LiCl and NaCl. The results revealed that GhACS1 is an inducible gene but not multi-responsive, it can just respond to certain stimuli and participate in certain signal transduction of ethylene biosynthesis.4. We constructed the pET-GhACS1 expression vector, expressed in BL21 (DE3), and examed a very light band. The results might be due to the transient expression nature of GhACS1.5. We constructed the pBI122-GhACS1 expression vector, and transformed it into the tobacco plant (NC89). We also transformed the empty vector into tobacco at the same time as control, and selected some transgenic plants.6. We obtained a partial promoter sequence by LA PCR. The sequence analysis revealed a group of some putative cis-acting elements and promoter core elements.