| The experiment was carried out in the horticulture experimental station of ShandongAgricultural University and central laboratory of College of Horticulture Science andEngineering during2008.8~2010.12. In the study, floral buds of ten-year-old field culturedand three-year-old potted nectarine (Prunus persica var. nectariana cv. Shuguang) trees wereused to identify and characterize regulatory genes of dormancy release. Expression changes ofthe genes in the dormancy release process under low temperature, short day and hormonetreatments were also detected. The study could be supplementary of the bud dormancymechanism and have important theoretical and practical value in the dormancy control ofprotected production of fruit trees. The main results were as follow:The cDNA library of genes associated with bud dormancy release was established usingsuppression subtractive hybridization (SSH) procedure. The RNA of dormancy-released budswas used as Tester and the RNA from dormant buds was used as Driver.180up-regulatedpositive cDNA clones were detected and128of them were successfully sequenced.28uniquegenes related to dormancy release were screened after the elimination of redundant sequences.The results of BLAST analysis and molecular function prediction showed that genes involvedin defense and metabolism pathway made up lager percentages, accounting for25%and17.86%respectively. Genes associated with oxidation reduction and signaling accounted for14.29%and7.14%respectively. And genes related to activation of structure or function,unknown protein, un-match protein and transport activator protein accounted for35.71%.According to functional predict of candidate genes and research progress of plantdormancy,8doemacy related genes, PpMt, PpDhn, PpHis, PpPod, PpSenescence-associatedprotein, PpCyp450, PpATP-binding cassette transporter protein and unknown, wereanalysised with qRT-PCR. The results showed that expression patterns of the genes wereup-regulated during dormancy release. Complex metabolism and energy consumption wereassociated with dormancy release, and the genes may be mainly involved in growth,development and tolerance to stress. The expression of ribosomal proteins induced by lowtemperature may be related to dormancy release of floral buds.PpDfn、PpDhn were full-length cDNA sequence screened from the library. Amino acidsequences of PpDFN、PpDHN were highly homologous with defensins (DFN, DHN) of other species. Analysis of signal peptide, trans-membrane domain and hydrophobicity of the twogenes suggested that PpDFN was a secretory and hydrophobic protein containning a signalpeptide and a trans-membrane domain. PpDHN was hydrophilic protein without signalpeptide and trans-membrane domain. Cladogram analysis of amino acid sequences showedthat PpDFN of ‘Shuguang’ nectarine had closer genetic relationship with those of grape andolive and PpDHN had closer genetic relationship with that of Armeniaca. Expression analysisof PpDFN and PpDHN showed that expression of the two genes were up-regulated along withchilling accumulation during dormancy release, and peaked at the2thweek of chillingtreatment. Short day showed no obvious effect on the expression of the two genes. PpDFNmight be involved in ethylene signaling pathway and PpDHN might related tofreeze-resistance, eliminating free radicals and protecting bio-membrane from injury.At stage of dormancy and dormancy release, transcripts of PIP1;1increased consistently.The high expression on15thJanuary led to outflow of water through vacuolar membrane andcytoplasm membrane, which caused water content decrease in buds and prevented frozeninjury. Contents of soluble sugars, proteins and proline peaked at the same time, whichprevented cell from dehydration injury. The high level expression after2weeks of lowtemperature treatment suggested that PIP1;1was a cold-inducible gene. Transcription ofδTIP1fluctuated during dormancy but increased significantly at stage of dormancy release.The increase might be related to dormancy release signal and plant activity enhancement.Expression of δTIP1did not increase after2weeks of low temperature treatment, indicatingthat δTIP1was not a cold-inducible gene.GA3and6-BA were used at15thNovember (endo-dormancy) and15thDecember (latestage of endo-dormancy) to study effects of hormone on dormancy release. Application ofGA3and6-BA at late stage of dormancy increased H2O2level and expression of POD andSOD, but decrease the expression of CAT. Application of GA3and6-BA duringendo-dormancy did not increase budburst of dormant buds. However, at the late stage ofendo-dormancy, budburst time was brought forward by8and3days respectively by GA3and6-BA treatment, budburst rate were also increased by37%and30%respectively. Effect ofGA3on dormancy release was more significant than6-BA. |
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