二斑叶螨不同发育时期基因组DNA甲基化的F-MSAP分析

DNA甲基化是表观遗传调控的重要机制,在真核生物基因表达调控中发挥重要作用。本研究通过荧光标记的甲基化敏感扩增多态性技术(F-MSAP,fluorescence-labeled methylation-sensitive amplified polymorphism)对二斑叶螨Tetranychus urticae Koch 4个龄期(卵、幼螨、若螨、成螨)基因组DNA中CCGG位点的胞嘧啶甲基化水平和模式进行分析。研究结果显示3种甲基化模式:无甲基化(TypeⅠ),半甲基化(TypeⅡ),全甲基化(TypeⅢ)在4个龄期均有出现,扩增的总甲基化位点共有641个,其中半甲基化率(TypeⅡ)均高于全甲基化率(TypeⅢ),各个龄期的平均总甲基化率(TypeⅡ+TypeⅢ)为16.01%,平均半甲基化率为10.24%,平均全甲基化率为5.77%。F-MSAP分析结果表明不同发育时期的二斑叶螨基因组DNA的甲基化水平和模式存在差异。     

仿刺参“化皮病”体壁组织DNA甲基化的MSAP分析

利用甲基化敏感扩增多态性(Methylation-sensitive amplified polymorphism, MSAP)技术分析了健康仿刺参(Apostichopus japonicus)体壁和“化皮病”仿刺参病变体壁、正常体壁DNA序列中CCGG位点的甲基化情况。结果显示, 健康仿刺参体壁和“化皮病”仿刺参病变体壁、正常体壁总甲基化水平分别为(18.60±5.61)%、(26.70±6.82)%和(19.53±3.34)%, 其中全甲基化水平分别为(13.97±4.86)%、(20.08±5.26)%和(15.42±2.61)%, 半甲基化水平分别为(4.63±3.59)%、(6.62±3.80)%和(4.11±2.08)%。“化皮病”仿刺参病变体壁总甲基化水平和全甲基化水平显著高于健康仿刺参体壁和“化皮病”仿刺参正常体壁(P<0.05), 健康仿刺参体壁与“化皮病”仿刺参正常体壁总甲基化水平和全甲基化水平差异不显著(P>0.05); 三者的半甲基化水平无显著差异(P>0.05)。因此, 推测仿刺参体壁“化皮”与DNA甲基化有关。     

孝顺竹开花过程中DNA甲基化水平动态研究

以孝顺竹为材料,利用HPLC和MSAP技术在其未开花至开花的生长过程中进行甲基化水平的检测,分析竹子开花过程中基因组DNA甲基化的动态,以揭示DNA甲基化水平与竹子开花现象的相关性。结果显示:(1)孝顺竹基因组DNA甲基化率在不同的时间处于动态变化中,进入开花状态的孝顺竹植株其甲基化水平极显著低于开花竹丛中未开花植株和未开花竹丛;开花和未开花植株的总甲基化率分别为9.00%和12.42%,全甲基化率分别为5.06%和7.53%。(2)MSAP位点中有66.83%的位点在开花和未开花材料中甲基化状态保持一致,33.17%的位点在开花和未开花植株中发生甲基化变化:其中22.28%的位点在开花植株中发生完全的去甲基化,1.98%的谱带在开花植株中发生甲基化,8.91%的位点在开花和未开花材料中甲基化水平呈现上升或降低的趋势。研究表明,开花的孝顺竹同时发生甲基化和去甲基化,但发生去甲基化的概率明显大于发生甲基化的概率,最终导致其甲基化水平极显著降低。     

玉米杂交种及其亲本基因组DNA胞嘧啶甲基化水平研究

基因组DNA甲基化对基因表达起着重要的调控作用.本研究采用MSAP方法,对2个玉米杂交种及其相应亲本DNA 5'-CCG G位点胞嘧啶的甲基化水平进行分析,比较了2个玉米杂交种与其相应亲本的甲基化类型与差异.研究发现,2个玉米杂交种Mo17×Suwan 5和Suwan 1×太3221的F1代的甲基化敏感扩增多态性(MASP)比率分别为39.1%和40.1%,均略低于其相应的双亲(39.8%和39.7%、44.6%和43.2%).2个杂交种的全甲基化(双链CmCGG)率分别为24.4%和23.3%,而其相应亲本Mo17、Suwan 5、Suwan 1和太3221分别为17.1%、24.4%、24.6%和21.6%.4个玉米亲本的MASP比率变化范围为39.7%～44.6%,平均为41.8%,全甲基化比率为17.1%～24.6%,平均为21.9%.杂交种与其相应亲本比较有4种类型的变化A型,F1与其亲本甲基化模式相同;B型,去甲基化;C型,超甲基化;D型,次甲基化.杂交种F1代DNA的甲基化模式与其双亲比较,发生了较大的改变与调整.F1代基因组的杂合性与基因组DNA甲基化模式的重新调整有关.     

高温胁迫对不同热敏型辣椒同工酶及DNA甲基化的影响

分别以耐热型晚熟紫色辣椒‘7036’和热敏型早熟绿色辣椒‘9050’为材料,研究了40℃高温胁迫对其POD同工酶以及DNA甲基化表达的影响。结果显示:(1)在POD同工酶表达方面,未经高温胁迫的辣椒‘7036’比其它处理多出1条带,而同工酶活性在2种辣椒中呈现不同的变化趋势。(2)随着高温胁迫时间延长,辣椒‘7036’DNA无甲基化和全甲基化比率增加,半甲基化和总甲基化比率降低,而辣椒‘9050’只有高温处理7d(T_7)的甲基化水平变化与之类似,但半甲基化和全甲基化比率的绝对值远低于辣椒‘7036’。(3)高温处理7d的DNA甲基化模式中,辣椒‘7036’去甲基化C型条带比率较高,辣椒‘9050’以超甲基化B型条带为主。实验表明,高温胁迫下紫色辣椒‘7036’的POD同工酶活性恢复,半甲基化水平大幅下降和全甲基化水平快速上升以及去甲基化等变化可能与其耐高温胁迫特性相关。     

镉胁迫下萝卜基因组DNA甲基化敏感扩增多态性分析

应用甲基化敏感扩增多态性(MSAP)技术分析了重金属镉(cd)胁迫处理后萝卜基因组DNA甲基化程度的变化。结果表明,经50、250和500mg/L CdCl_2处理后,MSAP比率分别为37%、43%和51%,均高于对照(34%);全甲基化率(双链C~mCGG)分别为23%、25%和27%,而其对照为22%,表明重金属CdCl_2胁迫后,某些位点发生了重新甲基化。萝卜叶片DNA中总甲基化水平的增加与CdCl_2处理浓度呈显著正相关。甲基化变异可分为重新甲基化、去甲基化、不定类型以及与对照相同的甲基化模式等类型,Cd胁迫处理引起的植株基因组DNA甲基化程度的提高主要是重新甲基化。     

缺氮胁迫下雨生红球藻虾青素积累过程中的基因组MSAP分析

虾青素具有多种生物学活性,雨生红球藻为天然虾青素的最佳来源,缺氮胁迫会导致雨生红球藻积累虾青素。为了解缺氮条件下雨生红球藻虾青素积累的分子机制,该研究通过对雨生红球藻进行缺氮胁迫,结合MSAP法,研究了雨生红球藻在缺氮胁迫下虾青素积累过程中基因组甲基化水平的变化,结果表明:缺氮胁迫0~72 h期间,雨生红球藻生长速度减慢,而虾青素积累主要发生在缺氮处理12~24 h期间,随后积累速度减慢。同时,对缺氮胁迫0、24、72 h的雨生红球藻基因组DNA进行甲基化敏感扩增多态性分析,共得到了291个甲基化多态性位点,其中发生甲基化变化的位点在0~24 h和24~72 h分别占总位点的29.90%和53.95%。在缺氮胁迫24 h处DNA半甲基化率最大(为12.71%),全甲基化率最低(为26.80%);缺氮胁迫72 h处DNA全甲基化率最高(为28.52%),半甲基化率最低(为1.72%)。这表明DNA甲基化调节方式的改变是虾青素积累过程中的一种重要调控模式。     

加勒比松种源苗期DNA甲基化多样性分析

为了解加勒比松(Pinus caribaea)种源的遗传多样性,利用甲基化敏感扩增多态性技术对加勒比松3个变种17个种源的DNA甲基化多样性进行了研究。结果表明,56对引物组合共扩增出425条谱带,其中多态性谱带422条,多态性百分率为99.25%。加勒比松种源幼苗半甲基化比率比全甲基化比率稍高,洪都拉斯加勒比松、古巴加勒比松和巴哈马加勒比松的DNA甲基化率分别为22.39%、22.29%和22.35%,差异不显著。加勒比松的DNA序列遗传多样性(H=0.4376)高于DNA甲基化多样性(H=0.3274),Mantel检验表明,基因组遗传变异与表观遗传变异不存在相关性(r=-0.171,P=0.16)。表观聚类与遗传聚类间存在较大差异,两种聚类分析结果均未将3个加勒比松变种分开。这表明加勒比松变种间的表观遗传变异极为丰富,能为加勒比松遗传改良提供优良种质资源。     

利用 MSAP 分析18个芥蓝齐口期的表观遗传多样性

本研究利用MSAP检测18个芥蓝齐口期DNA甲基化水平,分析了表观遗传多样性,探讨DNA甲基化模式对齐口期的影响。结果表明,18个芥蓝齐口期平均为50d,叶片数平均为10片,齐口期和叶片数不相关(相关系数为0.296);变异系数分别为21%和18%;遗传距离分布在0~40,平均值为12.2276,在10.62处分为3类。MSAP分析表明,5对引物组合扩增得到432条多态性条带,201条片段表现出多态性,多态性比率为47%;Nei遗传距离分布在0.004~0.467,平均值为0.0958,表明遗传多样性水平较低;在0.04处分为3类。Mantel测验表明两种分析的遗传距离相关系数为-0.1366,显示齐口期、叶片数与DNA甲基化多态性没有相关性。DNA甲基化模式分析表明,非甲基化片段为110条,甲基化多态性片段为322条,分为3种带型,类型一为非甲基化带型(110条),类型二为甲基化带型(110条),类型三为半甲基化带型(152条),非甲基化片段和半甲基化片段在不同品种之间呈现多态性,甲基化片段在不同品种之间呈现多态性与单态性相差不大,显示MSAP多态性主要来源于非甲基化和半甲基化片段,芥蓝甲基化模式以半甲基化为主。本文推测DNA甲基化水平降低参与芥蓝齐口期调控,MSAP分析既可用于基因组结构研究,又可用于基因组水平上性状的功能研究。     

中华菊头蝠不同组织基因组DNA甲基化分析

DNA甲基化在动物组织发育和分化等生物过程中发挥至关重要的作用.本研究采用荧光标记甲基化敏感扩增多态性(fluorescence-labeled methylation sensitive amplified polymorphism,F-MSAP)技术分析了中华菊头蝠(Rhinolophus sinicus)脑、心、肾、肝、肌肉、棕脂和白脂7种组织的基因组DNA甲基化模式和水平,及其组织差异性.结果表明,中华菊头蝠具有较高的总甲基化水平(平均58.5％),且组织间具有差异显著性(P=0.002);全甲基化为主要甲基化模式(平均36.8％),半甲基化也占据比较高的比例(平均21.7％),表明这两种甲基化模式在组织分化中可能均起到重要作用.相对于心、肌肉、棕脂和白脂4个组织,脑、肾和肝与之具有较显著的表观遗传差异(P＜0.001),其潜在功能有待进一步深入研究.本研究为进一步深入研究翼手目动物组织发育和分化、冬眠、免疫和环境适应性的内在表观遗传机制提供了基础数据.     

大花蕙兰子房授粉前后基因组DNA 胞嘧啶甲基化状态的MSAP 分析

应用甲基化敏感扩增多态性(Methylation sensitive amplified polymorphism, MSAP) 技术分析了大花蕙兰( Cymbidium hybridium) 授粉前后子房DNA 甲基化状态的变化(甲基化水平和甲基化差异模式) 。采用72 对引物进行选择性扩增, 共得到5892 条带, 其中748 条带为甲基化多态性带。结果显示DNA 甲基化在大花蕙兰子房发育过程中发生频繁, 从授粉前后子房的总扩增位点甲基化水平(14%和11. 4%) 和全甲基化率(9.5%和7.8% ) 来看, 授粉后都略低于未授粉子房, 表明子房在授粉后的发育过程中在某些位点发生了去甲基化。除甲基化水平有变化外, 大花蕙兰子房授粉前后的DNA 甲基化模式也存在较大差异, 共检测到14 种带型, 分为两大类( Ⅰ 和Ⅱ 型)。其中, 授粉前后DNA 甲基化状态保持不变的位点少, 只占25.6% , 归为Ⅰ型; 大部分检测位点( 占74.4% , 归为Ⅱ型) 的DNA 甲基化模式在授粉前后存在显著差异。上述结果表明, 大花蕙兰子房发育过程中以DNA 甲基化为代表的表观遗传调控起重要作用。本研究的开展将促进对与大花蕙兰子房发育相关的甲基化差异片段及受DNA 甲基化调控的关键基因的克隆, 进而为从表观遗传学这一新角度揭示大花蕙兰子房发育的分子机制奠定基础。     

Global DNA methylation levels in white blood cell DNA from sisters discordant for breast cancer from the New York site of the Breast Cancer Family Registry

Lower global DNA methylation is associated with genomic instability and it is one of the epigenetic mechanisms relevant to carcinogenesis. Emerging evidence for several cancers suggests that lower overall levels of global DNA methylation in blood are associated with different cancer types, although less is known about breast cancer. We examined global DNA methylation levels using a sibling design in 273 sisters affected with breast cancer and 335 unaffected sisters from the New York site of the Breast Cancer Family Registry. We measured global DNA methylation in total white blood cell (WBC) and granulocyte DNA by two different methods, the [³H]-methyl acceptance assay and the luminometric methylation assay (LUMA). Global methylation levels were only modestly correlated between sisters discordant for breast cancer (Spearman correlation coefficients ranged from -0.08 to 0.24 depending on assay and DNA source). Using conditional logistic regression models, women in the quartile with the lowest DNA methylation levels (as measured by the [³H]-methyl acceptance assay) had a 1.8-fold (95% CI = 1.0–3.3) higher relative association with breast cancer than women in the quartile with the highest DNA methylation levels. When we examined the association on a continuous scale, we also observed a positive association (odds ratio, OR = 1.3, 95% CI = 1.0–1.7, for a one unit change in the natural logarithm of the DPM/μg of DNA). We observed no association between measures by the LUMA assay and breast cancer risk. If replicated in prospective studies, this study suggests that global DNA methylation levels measured in WBC may be a potential biomarker of breast cancer risk even within families at higher risk of cancer.     

Global DNA methylation levels in white blood cell DNA from sisters discordant for breast cancer from the New York site of the Breast Cancer Family Registry

Lower global DNA methylation is associated with genomic instability and it is one of the epigenetic mechanisms relevant to carcinogenesis. Emerging evidence for several cancers suggests that lower overall levels of global DNA methylation in blood are associated with different cancer types, although less is known about breast cancer. We examined global DNA methylation levels using a sibling design in 273 sisters affected with breast cancer and 335 unaffected sisters from the New York site of the Breast Cancer Family Registry. We measured global DNA methylation in total white blood cell (WBC) and granulocyte DNA by two different methods, the [³H]-methyl acceptance assay and the luminometric methylation assay (LUMA). Global methylation levels were only modestly correlated between sisters discordant for breast cancer (Spearman correlation coefficients ranged from -0.08 to 0.24 depending on assay and DNA source). Using conditional logistic regression models, women in the quartile with the lowest DNA methylation levels (as measured by the [³H]-methyl acceptance assay) had a 1.8-fold (95% CI = 1.0–3.3) higher relative association with breast cancer than women in the quartile with the highest DNA methylation levels. When we examined the association on a continuous scale, we also observed a positive association (odds ratio, OR = 1.3, 95% CI = 1.0–1.7, for a one unit change in the natural logarithm of the DPM/μg of DNA). We observed no association between measures by the LUMA assay and breast cancer risk. If replicated in prospective studies, this study suggests that global DNA methylation levels measured in WBC may be a potential biomarker of breast cancer risk even within families at higher risk of cancer.     

Cubic regression-based degree of correction predicts the performance of whole bisulfitome amplified DNA methylation analysis

Epigenetic mechanisms, including DNA methylation, are important determinants in development and disease. There is a need for technologies capable of detecting small variations in methylation levels in an accurate and reproducible manner, even if only limited amounts of DNA are available (which is the case in many studies in humans). Quantitative methylation analysis of minute DNA amounts after whole bisulfitome amplification (qMAMBA) has been proposed as an alternative, but this technique has not been adequately standardized and no comparative study against conventional methods has been performed, that includes a wide range of methylation percentages and different target assays. We designed an experiment to compare the performance of qMAMBA and bisulfite-treated genomic (non-amplified) DNA pyrosequencing. Reactions were performed in duplicate for each technique in eight different target genes, using nine artificially constructed DNA samples with methylation levels ranging between 0% and 100% with intervals of 12.5%. Cubic polynomial curves were plotted from the experimental results and the real methylation values and the resulting equation was used to estimate new corrected data points. The use of the cubic regression-based correction benefits the accuracy and the power of discrimination in methylation studies. Additionally, dispersion of the new estimated data around a y = x line (R²) served to fix a cutoff that can discriminate, with a single 9-point curve experiment, whether whole bisulfitome amplification and subsequent qMAMBA can produce accurate methylation results. Finally, even with an optimized reagent kit, DNA samples subjected to whole bisulfitome amplification enhance the preferential amplification of unmethylated alleles, and subtle changes in methylation levels cannot be detected confidently.     

MPTP诱导的帕金森病小鼠模型黑质脑组织DNA甲基化研究

为研究DNA甲基化在帕金森病发病机制中的作用,本研究用环境毒素1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)连续腹腔给药诱导小鼠帕金森病(Parkison's disease,PD)模型,应用ELISA检测小鼠黑质脑组织总体甲基化水平,应用实时荧光定量PCR方法检测DNA甲基转移酶表达水平,探讨MPTP诱导的小鼠PD模型黑质部位是否存在DNA甲基化异常.进一步应用甲基化DNA免疫共沉淀结合DNA甲基化芯片方法,构建MPTP诱导的小鼠PD模型黑质脑组织DNA甲基化谱,并寻找DNA甲基化修饰异常的PD相关基因对其进行验证.结果表明,模型组小鼠黑质脑组织DNA总体甲基化水平较对照组显著降低,Dnmt1的表达水平显著增高.利用DNA甲基化芯片在全基因组内筛选出甲基化差异修饰位点共48个,涉及44个基因,这些甲基化差异基因参与信号转导、分子转运、转录调控、发育、细胞分化、凋亡调控、氧化应激、蛋白质降解等生物学过程.在甲基化差异修饰基因中,对Uchl1基因及Arih2基因进行了甲基化水平以及表达水平的验证.结果表明,模型组小鼠黑质脑组织Uchl1启动子区域甲基化水平较对照组增高,m RNA及蛋白质表达水平降低,Arih2启动子区域甲基化水平较对照组降低,m RNA及蛋白质表达水平增高.实验结果进一步证实,DNA甲基化修饰异常在帕金森病发病机制中有重要作用,环境因素(如MPTP)可以通过改变DNA甲基化修饰参与帕金森病的发生发展.     

Analysis of DNA methylation in different maize tissues

DNA methylation plays an important role in gene expression regulation during biological development and tissue differentiation in plants. This study adopted methylation-sensitive Amplified fragment length polymorphism （AFLP） to compare the levels of DNA cytosine methylation at CCGG sites in tassel, bracteal leaf, and ear leaf from maize inbred lines, 18 White and 18 Red, respectively, and also examined specific methylation patterns of the three tissues. Significant differences in cytosine methylation level among the three tissues and the same changing tendency in two inbred lines were detected. Both MSAP （methylation sensitive amplification polymorphism） ratio and full methylation level were the highest in bracteal leaf, and the lowest in tassel. Meanwhile, different methylation levels were observed in the same tissue from the inbred lines, 18 White and 18 Red. Full methylation of internal cytosine was the dominant type in the maize genome. The differential methylation patterns in the three tissues were observed. In addition, sequencing of nine differentially methylated fragments and the subsequent blast search revealed that the cytosine methylated 5 ＇ -CCGG-3 ＇ sequences were distributed in repeating sequences, in the coding and noncoding regions. Southern hybridization was used to verify the methylation polymorphism. These results clearly demonstrated the power of the MSAP technique for large-scale DNA methylation detection in the maize genome, and the complexity of DNA methylation change during plant growth and development. The different methylation levels may be related to specific gene expression in various tissues.     

Cubic regression-based degree of correction predicts the performance of whole bisulfitome amplified DNA methylation analysis

Epigenetic mechanisms, including DNA methylation, are important determinants in development and disease. There is a need for technologies capable of detecting small variations in methylation levels in an accurate and reproducible manner, even if only limited amounts of DNA are available (which is the case in many studies in humans). Quantitative methylation analysis of minute DNA amounts after whole bisulfitome amplification (qMAMBA) has been proposed as an alternative, but this technique has not been adequately standardized and no comparative study against conventional methods has been performed, that includes a wide range of methylation percentages and different target assays. We designed an experiment to compare the performance of qMAMBA and bisulfite-treated genomic (non-amplified) DNA pyrosequencing. Reactions were performed in duplicate for each technique in eight different target genes, using nine artificially constructed DNA samples with methylation levels ranging between 0% and 100% with intervals of 12.5%. Cubic polynomial curves were plotted from the experimental results and the real methylation values and the resulting equation was used to estimate new corrected data points. The use of the cubic regression-based correction benefits the accuracy and the power of discrimination in methylation studies. Additionally, dispersion of the new estimated data around a y = x line (R²) served to fix a cutoff that can discriminate, with a single 9-point curve experiment, whether whole bisulfitome amplification and subsequent qMAMBA can produce accurate methylation results. Finally, even with an optimized reagent kit, DNA samples subjected to whole bisulfitome amplification enhance the preferential amplification of unmethylated alleles, and subtle changes in methylation levels cannot be detected confidently.     

转基因白桦不同月份叶片基因组DNA甲基化水平的变异

目的：以转坛￡抗虫基因白桦的不同月份叶片为实验材料,揭示基因组DNA甲基化水平与植物叶片发育及外源基因表达水平之间的相关性。方法：应用DNA-MSAP方法检测叶片基因组DNACCGG位点甲基化状态,利用Northern杂交技术分析其外源基因表达水平。结果：转基因白桦叶片基因组DNA同年5～9月总甲基化水平分别为21．95％、29．62％、25．41％、41.39％和47．24％,除7月份稍有波动外,整体呈现随着叶片的成熟和衰老渐升高趋势;全部扩增位点中,半、全甲基化位点比例分别为17．99％和15．19％,在各月份叶片中变化较大,其中半甲基化位点比例分别为10．37％、19．14％、14．92％、17．2％和28．83％,全甲基化位点比例依次为11．58％、10．49％、10．50％、24．11％和18．40％。同一无性系外源基因在当年5～7月表达量最高,8～9月呈下降趋势,与基因组甲基化状态呈负相关趋势。结论：转基因白桦叶片的成熟和衰老及外源基因表达量降低均可能与基因组DNA甲基化水平的升高相关。