【作者】 杜丽丽；

【导师】 卢龙斗；

【作者基本信息】 河南师范大学 ， 遗传学， 2011， 硕士

【摘要】 菠菜(Spinacia oleracea L.)为藜科(Chenopodiaceae)菠菜属(Spinacia)一年生或两年生草本植物。菠菜作为一种常见的园艺蔬菜作物已经被广泛栽培,且菠菜一般为雌雄异株,极少雌雄同株,其染色体组为2n=2x=24(XY)。因此,对于菠菜的研究目前主要集中在新品种选育、栽培技术优化以及性别决定及分化机制三个方面,对其他方面研究尤其是菠菜表观遗传特征尤其是性别发育的表观调控机制的研究鲜有报道。DNA甲基化作为最早发现的基因组表观遗传修饰途径之一,已有证据表明该机制可以植物基因的表达进行表观调控,从而影响植物的生长发育。为了探索DNA甲基化在菠菜营养生长阶段及性别发育阶段的作用,本文以甲基化抑制剂5-azaC处理及未处理的菠菜为材料,通过比较观察,分析菠菜的生长发育过程及性比的变化,同时,采用MS-ISSR、MS-RAPD和HPLC法对基因组DNA甲基化水平进行检测,分析菠菜表型变化和基因组DNA甲基化水平之间的相关关系,从而尝试揭示DNA甲基化对于菠菜生长发育及性别过程中的作用机制。主要研究结果如下:1.使用不同浓度的5-azaC对菠菜种子进行处理,分析甲基化抑制剂5-azaC对于菠菜发芽势、发芽率、株高、叶长、根长、开花时间及性比的影响。结果表明,5-15μM的5-azaC对菠菜处理后,使发芽率、发芽势、根长、苗长、叶长、株高与对照相比,随着浓度的增高而降低,开花时间提前,但是与对照相比相差不显著;菠菜对30μM浓度处理不敏感,各项生长指标与对照相差很少;50-1000μM的5-azaC对菠菜处理后,随着浓度的增加与对照相比各项生长指标都降低,其中高浓度有明显的抑制作用,如株高与对照相比明显变矮,开花时间内提前1d以上。但对菠菜性比和千粒重影响不大,表现不显著。2.以浓度为30、100、500μM的甲基化抑制剂5-azaC处理和未处理的菠菜单株叶片基因组为材料,经过甲基化限制性内切酶HpaII和MspI进行酶切后,采用MS-RAPD和MS-ISSR技术对基因组DNA变化进行分析。结果发现,从400条RAPD引物和65条ISSR引物中筛选出20条MS-RAPD特异引物和35条MS-ISSR特异引物,从12个样品共扩增出667条可统计的清晰条带,多态性位点的百分率分别为86.88%和86.24%。通过对特异酶切位点的分析发现,在30、100、500μM处理中发生了甲基化和去甲基化作用,去甲基化作用稍占主导地位。3.以浓度为30、100、500μM的甲基化抑制剂5-azaC处理和未处理的菠菜单株基因组为材料,利用高效液相色谱法检测不同器官组织的菠菜总DNA甲基化的水平。结果表明,经过不同浓度处理,当5-azaC浓度超过30μM后,随着浓度的增加基因组甲基化水平显著性的降低,当5-azaC浓度超过500μM时,基因组甲基化水平变化不显著。在不同器官组织检测中,成熟叶片的DNA甲基化水平最高,其次是幼叶、子叶、下胚轴,胚根最低(平均值在4.61%)。同样可以看出随着处理浓度的增加DNA甲基化水平降低。更多还原

【Abstract】 Spinach (Spinacia oleracea L , 2n=2x=24 (XY)) is an annual (rarely biennial) flowering plant in the family of Amaranthaceae. It was cultivated widely as a common vegetable gardening. Spinach is a diploid dioecious plant, which possesses a pair of heteromorphic sex chromosome X and Y. Breeding of new variety, optimizing cultivation technology and the mechanism of sex determination were being studied in the past several decades. The research that focused on epigenetics characters especicially involved in epigenetics regulating mechanism of sexual development was rarely been reported. Plant DNA methylation was one of the first discovering genome epigenetic modification ways, and it was proved to succeed in regulating the plant gene expression. In order to explore the affection on DNA methylation in spinach growth and sex development stage, the following experiments were performed. The epigenetics phenotype and sex ratio were been investigated after treating use the methylation inhibitor 5-azaC. Meanwhile, the DNA methylation pattern and methylation level were detected by MS-ISSR、MS-RAPD and HPLC methods. The result showed as follow:1. Different 5-azaC concentration was used to treat the spinach feed and its affection on germination energy, germination percentage, Plant height, leaf length, root length, flowering time and sex ratio of spinach was explored. The result showed germination energy, germination percentage, root length, Plant height and leaf length are negative correlated with the 5-azaC concentration among 5-15μM compared with the control plants, and the flowering a little earlier than control plants. The spinach was not sensitive to 30μM 5-azaC and every growth indices was almost same as the control group. The spinach was treated by 5-azaC whose concentration was 50-1000μM and its growth indices decreased as the concentration increased. The high concentration inhibited obviously growth indices, for example the plant height was lower than the control group and the the flowering earlier more than one day. However it has little affection on spinach sex ratio and 1000- grain weight.2. The spinach was treated by 30、100、500μM methylation inhibitor, then its leaf and the normal one gene group were digested by methylation-sensitive restriction endonuclease. The change between them was investigated by MS-RAPD and MS-ISSR technology. The result showed that 20 polymorphism MS-RAPD primers were selected from 400 primers and 35 polymorphism MS-ISSR primers from 65 primers. 667distinct bands were obtained from 12 samples. The percentage of polymorphic loci were respectively 86.88% and 86.24%, respectively.Through the analysis on specific endonuelease sites, it found methylation and demethylation appeared during 30、100、500μM treat and demethylation play a important role.3. The extend of total DNA methylation of different spinach tissue were detected by HPLC method, the material was obtained from the treat plant by 30、100、500μM methylation inhibitor and the untreated control plants. The result showed that extend of gene methylation decreased obviously with the concentration increased in 30μM. However the changed was not obviously when the concentration exceed 500μM. Among the different tissue detection, the mature leaf had the highest DNA methylation extend, then spire, cotyledon and hypocotyl in sequence, and radicle has the lowest (the average value was 4.61%).The same trend that the gene methylation extend decreased as the concentration increased were observed.更多还原