Cloning And Expression Analysis Of An1-aminocyclopropane-1-carboxylate Synthase Gene From Oncidium

The tropical orchids industry is an important part of the high-tech agriculture production. It is one of the most dynamic industries in the word. Ethylene is a major regulator during the senescence of the Orchidaceae flower. In higher plant, the step converting SAM to ACC catalyzed by ACC synthase (ACS) is considered as a rate-limitig step among the ethylene biosynthesis, and ACS is a rate-limiting synthase. The research on flower senescence mechanism and delaying of Oncidium flower senescence through the manipulation of ethylene biosynthesis related genes has a great significance to the current tropical orchid industry. In this study, we cloned the ACS gene using the cut flowers Oncidium Golden NO.2as experimental material, which was bred from the O.Gower Ramsey by Taiwan Provincial Orichid Park. We isolated a member of1-aminocyclopropane-1-carboxylate synthase gene family, OnACS2(GeneBank Accession No.KJ004563), with RACE method. In this thesis, a series of studies have been conducted on the isolation, sequence and expression analysis, fuction identification of OnACS2. The main results are as follows:1、RNA was extracted from differential tissue of Oncidium with CTAB-LiCl method. Gel electrophoresis analysis showed that total RNA was in good integrity with no degradation and met with the requirements of Molecular Cloning. According to the conserved sequence of ACS gene in plant, specific primers were designed respectively. Then we got one segment of OnACS2gene by PCR, and late,5’and3’sequences of the gene were cloned from the Oncidium flower by RACE method. The full-length cDNA of the OnACS2gene is1557bp, and encoding a49.1kDa, PI7.51protein with435amino acid residues. We obtained the full-length DNA of the OnACS2with specific primer by LA-PCR, which containing three introns and four exons. Amino acid comparison indicated that the OnACS2gene has the major characterization of ACC synthases such as seven conserved regions, eleven invariant amino acid residues and active site of serine.2、For prokaryotic expression analysis, we successful constructed the pET-OnACS2expression vector, which then expressed in BL21(DE3). SDS-PAGE of pET-OnACS2protein induced by IPTG showed that a strengthened protein bands similar to that of the expected protein. For further research the characteristics of ACC synthase, we extracted ACS protein from bacteria containing the expression vector pET-OnACS2and determined the activity of ACCsynthase protein by the enzymatic reaction. The results showed that it have similar biological activity to that of the ACS protein in vivo.3、Organ-specific expression of the OnACS2gene in Oncidium tissues were investigated, with the Real-time PCR method. The organ-specific expression pattern of the OnACS2gene showed that the highest expression was observed in gynostemium of the Oncidium flower, and the expression levels in petals, sepals, lip and other organizations were very low. 4、We analyzed the expression patterns of the OnACS2gene and determined the rate of ethylene release in order to find the relation between the expression patterns of theOnACS2gene and the rate of ethylene release during flower senescence. The results showed that mRNA of the OnACS2gene accumulated in flower tissues to accelerate the rate of ethylene release.