Cloning And Expression Characters Of 1-aminocyclopropane-1-carboxylate Synthase Gene FhACS1 From Freesia Hybrida

Freesia hybrida were used as materials in this study to isolate the 1-aminocyclopropane-1-carboxylate synthase gene FhACS1 (GenBank Accession No.HQ833204) with Genome-Walking method and to investigate the expression characters of this gene in different developmental stages and floret organs, the expression patterns induced by different chemical inducements, drought and cold environment with real-time PCR. The main results are as follows:1. The new gene, FhACS1, was isolated from Freesia hybrida for the first time. The full length of FhACS1 was 2576 bp long, including a 433 bp 5'UTR and a 373 bp 3'UTR, containing 3 introns and 4 exons. Several typical elements such as CAAT-box, TATA-box and putative cis-elements responding to phytohormones and stresses were found in its promoter area. Southern blotting suggested only low copies of FhACS1 existed in freesia genome. The full length of CDS is 1371 bp, encoding a 51.2 kDa, pI 8.03 protein with 457 amino acid residues. FhACS1 contained the typical seven conserved domains, invariant amino acid residues, and active sides of ACS, and was highly homologous to ACS of Musa acuminata with amino acid sequence homology of 85%. Phylogenetic analysis showed that FhACS1 was closely related to bamboo, rice and banana.2. Organ-specific and time-course expression of FhACS1 in freesia florets were investigated. The organ-specific expression pattern of FhACS1 showed that the highest and the lowest expression were observed in stamens and pistil, respectively, and the expression level in petals was between them. Expression level in stamens was 5.7-fold and 2.6-fold the expression in pistil and petals, respectively. The expression pattern of FhACS1 in florets at different developmental stages suggested that the highest and the lowest expression was observed in floret fully open (stage 0) and florets at stage 4, respectively, and the former was 32.6-fold of the latter. To study the time-course expression pattern of FhACS1 in Freesia florets, the 3 rd and the 6 th bud on inflorescence were evaluated. The results showed that expression level in the 6 th bud was significantly higher than that in the 3 rd bud. Expression patterns of FhACS1 in petals, stamens and pistil during vase life indicated that:for the 3 rd bud, the expressions in petals, stamens and pistil were all first increased and then decreased, but the highest expression in each floral organ occurred at different time, on the 6 th,4 th and 7 th day of vase life, respectively, and the expression pattern in entire bud was consistent with that in petal. For the 6 th bud, expressions of FhACS1 continued to rise in petals, and first increased and then decreased in stamens and pistil. The highest expressions in stamens and pistil occurred at the 3 th and 6 th day, respectively. The expression pattern in entire bud was similar with that in petals.3. Expression patterns of FhACS1 during 8 day's vase life after induced by different chemical substances for 24 h were analyzed. The results showed:The expression pattern of FhACSl in the 6 th floret was significantly different from pattern in the 3rd floret. Low and high concentration ethephon, and IAA inducement could accelerate the growth, and trigger the senescence, and speed up the senescence process of florets. The effects of higher concentration ethephon were more significant than effects of lower concentration ethephon. 6-BA, STS and sucrose could retard the start of senescence of florets, and the expression of FhACSl was restrained from increasing, and expression level was lowered. However, 6-BA obviously inhibited the growth and flowering processes of florets, while STS impacted no effect on these processes, and sucrose contributed to speed up these processes.4. Expression characters of FhACSl in florets responsing to drought and low temperature were explored. The expression of FhACS1 in drought was dramatically increased after a slight rise, followed by a decrease, and then increased again in the end of vase life. Low temperature could remarkably lessen the increase of FhACS1 expression, and inhibited the expression from increasing.Results mentioned above indicated that induce signals could influence the developmental and senescence process of freesia florets though impacting on the expression level of FhACS1. That's to say, the expression level of FhACS1 could directly regulate the senescence of freesia florets, and the senescence of freesia florets could be retarded though inhibiting FhACSl expression.