Cloning And Expression Of PPO Gene During Somatic Embryogenesis In Camellia Nitidissima Chi

Camellia nitidissima Chi., well-known for waxy yellow flowers, is a kind of perennial shrub woody plants from the genus Camellia. In addition to its ornamental value, Camellia nitidissima Chi. is widely used in food, medicine, health products and other industries as additive, compared with its lower ratio of natural propagation ratio, the use of culture tissue technique can quicken its reproduction, also healpful in germplasm conservation and others. In this experiment the repetitive in vitro regenerating system originated from the adult stem-tips was used as the material for synchronization of somatic embryos in Camellia nitidissima Chi., gene cloning of PPO gene from somatic embryos, relative expression of PPO gene by using real-time quantitative PCR technology during somatic embryogenesis in Camellia nitidissima Chi.. Besides, the activities changes of PPO, POD and APX were detected during somatic embryogenesis in Camellia nitidissima Chi., and the changes of POD, APX isoenzymes were analyzed during somatic embryogenesis in Camellia nitidissima Chi.. The main results are as follows:1. Synchronization of somatic embryos in Camellia nitidissima Chi.Camellia nitidissima Chi. somatic embryos were taken as the material for synchronization of somatic embryos. The results indicated that somatic embryos grew well on the MS basal medium with sorbitol, somatic embryos had highly synchronized in globular embryos and cotyledon embryos. The synchronization of somatic embryos were obtained by cultivating and picking means in heart-shape embryos and mature embryos; plantlets and MCFS embryos (embryo with multiple cotyledons and flower-like shape) grew well. Cotyledon embryos and the adult plant leaves were used as the material for callus culture, the results indicated that the pieces cut from cotyledon embryo were cultured on the MS medium with 1.5 mg·L-1 2,4-D and 0.5 mg·L-1 KT, callus could be induced and subcultured well, at 6% sucrose concentration the growth of callus grew well. The adult plant leaves were washed in water for 30 minutes, before putting in 70% ethanol for 30s. Then the adult plant leaves were surface-disinfected for 8 minutes in HgCl2 (0.2%). This kind of treatment could achieve good sterilation effect. the pieces cut from the adult plant leaves were cultured on the MS medium with 1.5mg·L -16-BA, 0.5mg·L-1 IAA and 0.5mg·L-1 NAA, callus could be induced. The highest frequency of callus induction was 98.335%, shading was helpful to the growth of callus.2. Cloning of PPO gene in Camellia nitidissima Chi. somatic embryosThe globular embryos of Camellia nitidissima Chi. were taken as the material, the complete sequence of cDNA of PPO gene was cloned from Camellia nitidissima Chi. somatic embryos. The primers were designed according to the known sequence of PPO gene in genus Camellia and was carried out by trail-PCR. After primer screening, a 1800bp specific band was obtained, then 1788 bp sequence could be got. Sequence analysis revealed that the full length cDNA encoded 595 amino acids. The results showed the PPO gene sequence in Camellia nitidissima Chi. somatic embryos was 74% homologous with other plants reported in GenBank. The deduced amino acid sequence in Camellia nitidissima Chi. somatic embryos was 72% homologous with PPO in other plants.3. PPO gene expression analysis during somatic embryogenesis in Camellia nitidissima Chi. by real-time quantitative PCR18S rRNA gene were used as the control for PPO gene expression analysis by real-time quantitative PCR technology during somatic embryogenesis in Camellia nitidissima Chi.. The results showed that expression of PPO gene showed a tendency to decline, at the stage of globular embryos relative expression levels was the highest and then a small drop appeared at the stage of cotyledon embryos and heart-shape embryos, at the stage of mature embryos relative expression levels was the lowest. Obvious differences of relative expression levels were found between normal cotyledon embryos and MCFS embryo (embryo with multiple cotyledons and flower-like shape).4. The activity changes of PPO during somatic embryogenesis in Camellia nitidissima Chi.The trend of PPO activity changes was detected during somatic embryogenesis in Camellia nitidissima Chi.. The results showed that the PPO activities were lower, its changeable tendency was similar with the expression of PPO gene. 5. The researches of some isozymes activitives during somatic embryogenesis in Camellia nitidissima Chi..The POD, APX activity changes were analysed during somatic embryogenesis in Camellia nitidissima Chi.. The results are as follows: the POD activity first increased then decreased; the APX activity showed high activity and totally had"M"change trend. The analysis of POD and APX isozyme zymogram with polyacrylamide gel electropHoresis during somatic embryogenesis in Camellia nitidissima Chi. indicated that the POD isozyme zymogram was different both in the isozyme band strength and in the number of the band; APX isozyme was stable, and the APX isozyme zymogram at the different stages lines differenate in the isozyme band strength.