Cloning And Expression Analysis Of Three Genes Encoding Ethylene Biosynthestic Enzymes From Hosta Plantaginea Flowers

Ethylene, one of several plant hormones, plays an important role during the life cycle of plants such as seed germination, sex differentiation, floral senescence, organ abscission, fruit ripening and so on. It is also associated with responses to stresses including mechanical damage, cold and waterlogging. In addition, it also involves in programmed cell death and DNA degradation of plants.As an ornamental plant for both flowers and leaves, Hosta plantaginea has been more and more widely used in garden landscaping, city afforestation and home decoration. However, its flower senesces shortly after blooming like many fresh flowers, which reduces its ornamental value to a great extent. Our previous results showed that the expression of1-aminocyclopropane-l-carboxylic acid oxidase (HpACO) which encodes the key enzyme involving in ethylene biosynthesis was related to flower senescence of H. plantaginea, indicating its probable role of ethylene in this event. In this study, three full-length genes encoding the enzymes of ethylene biosynthesis were cloned from H. plantaginea flowers. Furthermore, spatial and temporal expression of the three genes was analyzed. The main results are summarized as follows:1. Degenerate primers were designed based on homologous sequences for cloning cDNA fragments of S-adenosylmethionine synthetase (HpSAMS) and1-aminocyclopropane-l-carboxylic acid synthase (HpACS). On the basis of above two cDNA sequences and previously cloned HpACO cDNA sequence, special primers were designed for3’and5’RACE method. The full-length sequences were obtained successfully by RT-PCR.(1) The full-length cDNA sequence of HpSAMS was1704bp, which contained an ORF of1191bp encoding396amino acids.5’UTR and31UTR were119bp and 394bp respectively. There were no difference between the both sequences of cDNA and DNA, suggesting no introns inserted. The putative molecular weight was43.2kD and the pI was5.48.(2) The full-length cDNA sequence of HpACS was1562bp containing an ORF of1347bp which encoded448amino acids.5’UTR and3’UTR were119bp and394bp in length, respectively. By contrast, the full-length cDNA sequence of1915bp in length was inserted by two introns. The molecular weight and the pi were49.9kD and5.81, respectively.(3) The full-length cDNA sequence of HpACO was1152bp and contained an ORF of957bp which encoded448amino acids,5’UTR of119bp and3’UTR of394bp in length, respectively. The full-length cDNA sequence was1611bp and inserted by two introns accordingly. The molecular weight and the pI were calculated to be49.9kD and5.81, respectively.2. The expression patterns of three genes in the flowers of H. plantaginea at the stages of small bud, big bud, blooming and senescing were analyzed. HpSAMS showed a significant decrease at the senescing stage, while HpACS increased sharply at blooming stage and then decreased slightly. The expression of HpACO appeared to be a gradual increase before a decline at the senescing stage. There were differences among expression patterns of the three genes in roots, stems and leaves. The expression of HpSAMS was much higher in roots than in stems. HpACS expressed in stems while HpACO had a strong expression in leaves.3. In the mechanically wounded leaves of H. plantaginea, HpSAMS expressed with a sharp increase within2h and then reduced slightly in the following4h. The transcript level went on rising until the end of treatment with3times as much as control level. The expression of HpACO declined throughout the experiment and reached the significant level after12h. No expression of HpACS was detected during the period, which suggested that HpACS was not induced by wounding.