| Circadian clock is an internal autonomous timer prevalent in all organisms, which make itsgenetic, physiological and metabolic processes persist with the approximately24-hour periodicrhythm. The circadian clock regulates diverse aspects of plant growth and development,including photosynthesis, stem growth, odour sending, the transition of vegetative growth toreproductive growth, dormancy and other seasonal processes. Meanwhile, the circadian clockis also involved in other signaling pathways. It was reported that the circadian clock in higherplants consists of multiple interconnected transcription feedback loops, and multiple types ofepigenetic modification play a critical role in regulation of the circadian network.Poplars arenot only a kind of tree species with economic and ecological importantance, but also the modeltree for genetic engineering. In this study, the CCGG sites methylation status in mature leavesof one-year-old Populus nigra N46were detected by capillary electrophoresis MSAP(Amplified fragment length polymorphism) during the diurnal changes, in order to determinewhether the circadian clock is regulated by DNA methylation and explore the epigeneticmechanisms of the circadian clock regulation. The main results are as follows:1. The MSAP selective amplification system in poplar was optimized. Selectiveamplification system of MSAP technique based on capillary electrophoresis in poplar wasoptimized through orthogonal experimental design method. The optimal concentration of eachcomponents in the reaction system was dNTP0.5mM, selective primers0.075μM, exTaq0.625U/μL, Mg2+1.5mM and DNA22.5ng/μL, respectively.2. The DNA methylation pattern in the leaves of P. nigra was revealed. Twenty-six primerpairs were selected from72pairs of selective amplification primers, to analyze the DNAmethylation pattern in the leaves of P. nigra by MSAP. In each plant,2065CCGG sites weredetected, among which55.51%were methylated, with44.89%total methylation sites and4.65%half methylation sites. Therefore, the total methylation ratio of CCGG sites is muchhigher than the hemi-methylation ratio in genome of P.nigra leaves. 3. The methylation status of2065CCGG sites in mature leaves of P.nigra every4hduring24h light-dark period and a followed12h dark period were detected by MSAP, and allbut2CCGG sites in the8h dark treatment were found to have different methylation status,then the methylation status restored in16h dark treatment. That is, the methylation patterns ofCCGG sites in poplar didn’t not have characteristics of circadian oscillator. So we speculatedthat although the epigenetic modification plays an important role in the circadian clockregulation process, the DNA methylation is not the main regulation mechnism for the rhythmicexpression of clock genes and their downstream genes. |
