| The old trees are the unique landscape resources, which are the comprehensive carriers of human landscape and natural landscape in cities. But varying degrees of senility or death threaten the old trees. So protecting old trees can give full play to its improtant role in landscaping. To protect the resource of old trees, we used the thousands years old of Platycladus orientalis in the Huangdi Mausdeum of Shanxi Province as experimental material in this study. We cloned the old P. orientalis by tissue culture which can reserve parental traits. At the same time, we cloned the full-length genes of telomere related to decipher the aging at the molecular level, which can lay the foundation for rejuvenation of old trees. The main results of this study were as follows:1. To select the sterilization method: by testing different sterilization methods, we could find the optimal method for P. orientalis. When disinfected with 75% alcohol for 30 s, washed five times with sterile water and then disinfected by 0.1% HgCl2 for 8 min, both the contamination rate and the mortality rate were the lowest, while the survival rate was the highest. The result showed this method initially solved the problem of sterilization.2. To screen out primary culture medium: we used five kinds of media on the ancient oriental trees for tissue culture. After one-month culture, the results demonstrated that 1/2MS medium had a good effect, while B5 medium-induced effects were somewhat less induction rate than that of 1/2MS, MS, and WPM and DKW medium effect on stem culture had little difference which might be related to the medium itself nutrition.3. To screen out propagation culture medium: we explored different proliferation media(MS, 1/2MS, WPM, B5, DKW), and different hormones(6-BA, KT, NAA). The result showed that the optimum medium for proliferation was 1/2MS + 0.05 mg/L 6-BA + 0.2 mg/L NAA + 0.1 mg/L KT.4. To screen out rooting medium: it plays a decisive role for the matching relationship between internal and external hormones in the adventive root formation. The tests showed that in rooting culture, contrary to the former two media above, the WPM effect was better than that of 1/2MS for rooting stage, and the optimal rooting medium was WPM+0.5mg/L NAA+ 0.5mg/L IBA.5. Three telomeres genes cloning: three telomere related full-lenght genes, PoTRF1, PoPOT1 and PoKu70 were cloned by RACE from the P. orientalis.Bioinformatic analysis showed that, the length of PoTRF1 gene is 1464 bp, including 176 bp 5 ’- untranslated region(UTR, untranslated region), 361 bp 3’- UTR is 26 bp polyA tail, 927 bp ORF encoding 308 amino acids(molecular weight of protein is 119 kDa, and the isoelectric point is 5.04). The length of PoPOT1 gene is 1337 bp, including 163 bp 5 ’-UTR, and 556 bp 3’-UTR, 28 bp polyA tail, 618 bp ORF encoding 205 amino acids(molecular weight of protein is 110 kDa, and the isoelectric point is 4.98). The length of PoKu70 gene is 1010 bp, including 81 bp 5 ’-UTR, and 368 bp 3’-UTR, 24 bp polyA tail, 561 bp ORF encoding 186 amino acids(molecular weight of protein is 82 kDa, and the isoelectric point is 5.02). Bast analysis showed that the protiens encoded by PoTRF1、PoPOT1 and PoKu70, had the similarities of 54% and 43%, 54% and 44%, 70% and 66% to the homologs in Arabidopsis and Populus, respectively。... |
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