【作者】 聂丽娟；

【导师】 王子成；

【作者基本信息】 河南大学 ， 遗传学， 2008， 硕士

【摘要】 DNA甲基化参与植物基因表达的调控,在植物生长发育过程中起着非常重要的作用。DNA甲基化抑制剂(5-azaC)已被用于因DNA甲基化引起的植物表观遗传研究中,它可以代替低温促进植物开花,提早开花时间。本研究以菊花(Dendranthema grandiflorum)品种‘墨麒麟’为材料,应用甲基敏感扩增多态性(MSAP)技术,对其组织培养继代过程中的甲基化变化进行研究,并通过形态观察和分子分析研究5-azaC对其表型及花期的影响。主要研究结果如下:1.采用MSAP方法对菊花组织培养继代过程中的DNA甲基化情况进行分析。三个单芽系的组培苗较田间材料均有DNA甲基化增加和去甲基化现象,各单芽系DNA甲基化变异率分别为8.929%、8.902%、8.986%。继代材料较母体均有甲基化变异发生,且变异类型中DNA甲基化减少的比例高于甲基化增加的比例,随着继代次数的增加,两种变异间的差异逐渐减小,比例相近。同时在同一单芽系内的不同次继代个体间有DNA甲基化变化现象。2.对5-azaC处理的材料与对照的生长发育进行形态观察比较。结果表明,5-azaC处理过的材料茎尖的丛生芽分化数量都少于对照,100μmol/L以上高浓度的5-azaC还影响根的形态发生。5-azaC对丛生芽的抑制作用具有时间累加效应和剂量累计效应,同时,5-azaC对丛生芽形成的抑制效应具有稳定性,在去处理后的连续继代过程中,各种浓度条件下的丛生芽分化数量虽然有所上升但仍不及对照。同时,5-azaC处理对开花时间也有影响,各种低浓度处理均能使菊花提前开花,但提前的时间与浓度密切相关,且这种对花期的影响在材料的田间生长过程中具有稳定性。3.应用扩增片段长度多态性(AFLP)技术对处理组及对照组的基因组DNA进行分析。从不同浓度5-azaC处理30天材料及对照的叶片中分别提取DNA,EcoRⅠ和MseⅠ对基因组DNA进行双酶切,对处理材料的基因组进行序列分析。25对引物组合扩增产生1267条可统计的条带,这些条带在各种浓度5-azaC的处理材料中没有表现出特异性。表明在5-azaC处理过程中基因组DNA序列没有发生变化。4.运用甲基敏感扩增多态性(MSAP)标记分析了处理样品与对照组样品间及其去处理后的甲基化变化情况。5-azaC处理引起基因组的DNA甲基化减少,且基因组DNA甲基化水平随抑制剂的浓度增加而减少,同一浓度5-azaC处理条件下,DNA甲基化减少量随处理时间的延长而增加,这些低甲基化水平在去除抑制剂处理后仍能稳定持续。四种低浓度5-azaC处理30天的材料移至田间后,3个位点发生了DNA甲基化增加,8个位点发生甲基化减少,且模式一致。更多还原

【Abstract】 DNA methylation is critical for regulation of plant gene expression, and plays an essential role in plant development. DNA methylation inhibitor (5-azaC) has been used in the studies of plant epigenetics that based on DNA methylation. 5-azaC could substitute the low temperature and promoted plant flowering, ahead of flowering time. Methylation-sensitive amplification polymorphism (MSAP) technique was used to study the variation of DNA methylation during continued generation of tissue culture in the‘moqilin’variety of Chrysanthemum(Dendranthema grandiflorum). Also, the effect of 5-azaC to the morphogenesis and flowering time has been evaluated by developmental phenotypes and DNA molecular marker techniques. The main results are as follows:1. The DNA methylation status during the successive transfer culture of Chrysanthemum was analysised by MSAP technique. The results indicated that three single shoot system of the tissue culture seedling had distinct DNA hypermethylation and demethylation compared to the field materials, the mutation rate were 8.929%、8.902%、8.986% respectively. All the successive transfer materials had DNA methylation variety compared to the parent materials. During the variation, demethylation were more than hypermethylation. Along with the successive transfer culture, the diversity between the two variety pattern was smaller, and the proportion allmost similar.Meanwhile, the different individual of each single shoot system also existed DNA variation phenomenon.2. The development of the samples with 5-azaC treatment and the controls were observed. The results indicated that, The number of multiple buds differentiated from treated shoot-tips was fewer than control’s. The concentration of 5-azaC higher than 100μmol/L also affect root’s morphogenesis. Research indicated that, the inhibiting effect of 5-azaC has time and dose adding domino offect. Meanwhile, the role of 5-azaC to the inhibition of multiple bud was stability. During the successive transfer culture after removed 5-azaC, the number of multiple buds differentiation of all treatment were ascend, but also less than control. Meanwhile, 5-azaC even affected the flowering time of Chrysanthemum, of all low concentration 5-azaC treatment promoted flowering. while the time that aheaded was correlated to the concentration, and these effect to the flowering time were stability when treated materials growth in the field.3. The genome DNA of treatments and controls were analyzed by amplified fragment lenth polymorphism (AFLP) technique. The DNA were distilled from the leaves of treatments treated by different concentration and controls after growthed 30 days. The DNA were digested by EcoRⅠand MseⅠ, and 1267 bands were scored using 25 pairs of primers combination. No variation of bands was found during the samples, which suggested that no nucleotide sequence had altered during 5-azaC treatment.4. Methylation-sensitive amplification polymorphism (MSAP) technique was used to investigate the DNA methylation status of the treated samples, the materials after removed 5-azaC and the control. 5-azaC caused the genomic DNA methylation decrease. The decrease was increased when 5-azaC concentration was added, and when the materials treated by the same concentration of 5-azaC, the decrease was increased with the treated time extend. The demethylation status was stability after 5-azaC was removed. The materials treated by four kinds lowness 5-azaC had 3 sites DNA hypermethylation and 8 sites DNA demethylation when they were growth in field, and the patterns were consistent.更多还原