运用MSAP技术测定谷子基因组DNA胞嘧啶甲基化水平

DNA甲基化是真核生物一种重要的表观修饰形式。为了探讨谷子基因组DNA胞嘧啶甲基化的水平和模式,以谷子Setaria italica的两个品种朝谷58号和豫谷1号为实验材料,利用Eco RⅠ和HpaⅡ/MspⅠ双酶切建立适合于谷子基因组的甲基化敏感扩增多态性(MSAP)分析体系。结果表明,从100对MSAP选扩引物中,筛选出32对MSAP引物组合,在朝谷58号和豫谷1号中分别扩增产生1 615、1 482条清晰可辨且可重复的DNA条带,其中包括3种类型的甲基化条带,朝谷58号和豫谷1号的基因组中CCGG序列胞嘧啶甲基化水平分别为6.93%和8.77%。这种谷子不同品种间甲基化水平和分布位点的差异为从表观遗传学的角度培育新品种提供了初步的理论依据和参考。     

湿地松×洪都拉斯加勒比松及亲本DNA胞嘧啶甲基化的初步研究

以湿地松×洪都拉斯加勒比松（Pinus elliottii×P.caribaea var.hondurensis）及亲本为实验材料,采用甲基化敏感扩增多态性技术对其基因组中CCGG位点的甲基化相对水平及遗传变异模式进行了初步分析。结果表明,杂种及亲本CCGG总甲基化相对水平介于77.74%~81.75%,CG甲基化相对水平略低于CNG甲基化水平,CG/CNG甲基化相对水平高于亲本。杂种遗传自亲本的CG与CNG甲基化位点数之比接近1：1,遗传自母本的甲基化位点数与遗传自父本的CCGG甲基化位点数比例为1：1;杂种产生的全新甲基化与完全去甲基化位点数之比接近7：1,初步推测大量甲基化变异促进了杂合体的生长发育。     

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(Metyhfion 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牛频繁,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甲幕化模式也存在较大差异,共榆测到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较少,只占25.6%,归为Ⅰ型;大部分榆测位点(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,大化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.     

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。     

甘露醇对拟南芥基因组DNA甲基化的影响

以拟南芥（Arabidopsis thaliana）为材料,研究不同浓度甘露醇处理下拟南芥幼苗生长发育及其基因组DNA的甲基化水平和变化模式。结果表明,用50、100、150和200mmol·L―1甘露醇处理拟南芥种子会对拟南芥幼苗的形态特征和生长态势产生影响;甲基化敏感扩增多态性（methylation-sensitive amplification polymorphism,MSAP）分析表明,经50、100、150和200mmol·L―1甘露醇处理后,基因组DNA甲基化比率分别为17.75%、21.15%、15.49%和46.10%。甘露醇处理使拟南芥发生基于DNA甲基化水平和模式改变的表观遗传变异。与对照相比,在50、100、150和200mmol·L-1甘露醇处理下拟南芥幼苗基因组DNA的甲基化和去甲基化比率分别为5.78%、15.48%、10.71%、33.73%及10.98%、5.36%、8.33%、7.69%。由此推测,5-甲基胞嘧啶百分含量随着甘露醇胁迫的增强而发生不同程度的变化。     

不同倍性西瓜基因组DNA甲基化水平与模式的MSAP分析

DNA甲基化是表观遗传修饰的主要方式之一,在基因表达调控中发挥重要作用。本研究以不同倍性（2x、3x、4x）西瓜为试材,采用基于DNA甲基化敏感酶的扩增多态性分析（Methylation-Sensitive Ampliftcation Polymorphism,MSAP）方法,在全基因组水平上探究西瓜同源多倍化过程中DNA序列中CCGG位点的甲基化水平及模式变化特征。研究中选用23对选扩引物,共检测到1883个基因位点。二倍体、三倍体、四倍体中检测到的位点数分别为647、655和581;其中发生甲基化的位点数分别为181、150和159。相应的扩增总甲基化率分别为28．0％、22．9％和27．4％：全甲基化位点数分别为121、80和82,相应的全甲基化率分别为18．7％、12．2％和14．1％。进一步对不同倍性西瓜DNA甲基化模式的变化特征进行分析,结果显示：四倍体西瓜与二倍体西瓜相比有超过半数的位点（54．4％）DNA甲基化模式发生了变化,其与三倍体西瓜相比也有近一半的位点（45．4％）DNA甲基化模式发生了变化,并且变化趋势都以四倍体西瓜甲基化程度升高为主：而三倍体西瓜与二倍体西瓜相比．虽然也有41．6％的位点DNA甲基化模式发生了改变,但变化趋势以三倍体西瓜甲基化程度降低略占优势：与之相似,三倍体西瓜与四倍体相比较。甲基化的变化趋势也是以三倍体西瓜甲基化程度降低为主。以上结果表明：不同倍性西瓜中DNA甲基化事件虽均有发生．但不论是从总甲基化率还是全甲基化率来看,DNA甲基化水平与倍性高低关系不大．三倍体西瓜表现出较为显著的低甲基化水平特征。DNA甲基化模式的分析也表明。与二倍体及四倍体西瓜相比．三倍体西瓜DNA甲基化模式的调整主要以去甲基化为主。显示出三倍体西瓜基因组独特的DNA甲基化特征。本研究为进一步从表观遗传学的角度探讨西瓜的三倍体优?     

土霉素胁迫下拟南芥基因组DNA甲基化的MSAP分析

以拟南芥 (Arabidopsis thaliana)为材料,研究不同土霉素浓度下拟南芥幼苗生长发育及基因组DNA的甲基化水平和变化模式。结果表明,3、5、7\,9 μmol/L土霉素胁迫对拟南芥幼苗的根长和株高有显著抑制作用;但对拟南芥幼苗的侧根数量有显著促进作用。甲基化敏感扩增多态性 (methylation-sensitive amplification polymorphism, MSAP)分析表明,经3、5、7\,9 μmol/L土霉素处理后基因组DNA甲基化比率分别为17.91%、12.50%、11.81%和14.62%,均低于对照 (18.18%)。结果表明,拟南芥经土霉素胁迫后存在基于 DNA甲基化水平和模式改变的表观遗传变异,5-甲基胞嘧啶百分含量的变化无统一趋势或规律。与对照相比,3、5、7\,9 μmol/L土霉素胁迫下拟南芥幼苗基因组DNA的甲基化和去甲基化分别为13.29%、9.22%、8.03%、12.59%和2.80%、4.26％、5.11%、4.90％。由此推测,DNA甲基化可能是植物适应土霉素胁迫机制的机制之一。     

马哈利樱桃矮化砧的DNA甲基化水平及模式分析

该研究利用MSAP技术,对25株矮化马哈利樱桃和25株半矮化马哈利樱桃进行甲基化水平和模式分析,以探讨其矮化的表观性状与其基因组甲基化修饰的关系。结果表明:(1)从64对引物中筛选出15对引物,在半矮化组中共扩增4 577个条带,其中半甲基化336个,全甲基化1 274个;在矮化组中共扩增4 444个条带,其中半甲基化349个,全甲基化1 383个;t检验和方差分析表明,矮化组与半矮化组在总甲基化水平和全甲基化水平上差异极显著,在半甲基化水平上差异显著,矮化组甲基化水平高于半矮化组。(2)半矮化组单态性位点23个,多态性位点136个;矮化组单态性位点17个,多态性位点142个,表明矮化组多态性高于半矮化组。(3)多态性类型分析表明,矮化组出现A4类型的频率较半矮化组高,A2类型的频率较半矮化组低,即矮化组中发生超甲基化的位点多于半矮化组,且‘马哈利’基因组甲基化多态性位点主要发生在双链内侧甲基化位点以及超甲基化位点上。研究认为,马哈利樱桃矮化和半矮化的基因组甲基化水平及模式存在差异,马哈利砧木的矮化性状与其基因组甲基化修饰有关。     

应用MSAP方法检测鸡不同组织基因组的甲基化状态

以白洛克肉鸡和白来航蛋鸡及其杂交F1代基因组为实验材料,应用甲基敏感扩增片段多态性方法(Methylation sensitive amplified polymorphism,MSAP)检测了鸡肌肉、心脏、肝脏和肾脏4个不同组织基因组在CCGG位点的甲基化状态,分析了不同组织的DNA甲基化水平及组织特异性甲基化模式。研究发现:肌肉组织的甲基化水平约为29.7%,肝脏组织约为27.5%,心脏组织约为27.5%,肾脏组织约为26.1%;在鸡3个不同群体及其中3个不同组织间,基因组甲基化程度差异显著(P<0.05);在检测的4个组织中,CCGG序列的全甲基化位点少于半甲基化位点,与植物的相关研究不一致;分离及鉴定了2个组织特异的甲基化片段。结果表明:鸡不同组织基因组的甲基化状态是不同的,同一组织的甲基化水平在不同的群体是不同的,而不同组织甲基化水平的排序在不同的群体是不一致的。这些结果揭示遗传效应可能影响个体的组织甲基化水平。     

不同生理年龄毛竹DNA甲基化的MSAP分析

为分析竹子年龄变化与基因组DNA甲基化之间的相关性,以5年、31年和>60年起源(从种子萌发年龄算起)的毛竹当年生叶片为材料,采用35对引物对其进行MSAP检测。结果表明:3个年龄段的总甲基化率和全甲基化率分别为24.44%、28.21%、32.12%和16.57%、19.41%、21.23%;发生DNA甲基化的变异位点为52.3%,去甲基化变异位点为10.3%。可以看出,随着年龄的增加,毛竹基因组DNA甲基化敏感多态性呈上升趋势。总甲基化率单因素方差分析的结果表明相同年龄的毛竹个体间没有差异(P=0.307>0.05),而不同年龄间的差异达极显著水平(P<0.001)。同时,对所用引物组合进行分析后发现有6对引物(E3/HM2、E3/HM6、E3/HM7、E4/HM5、E4/HM6和E5/HM5)扩增出的位点与总趋势显著相关,为进一步开展深入研究奠定基础。     

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.     

Temperature-independent genome-wide DNA methylation profile in turbot post-embryonic development

The morphological and biological characteristics of ectothermic vertebrates are known to be strongly influenced by environmental conditions, particularly temperature. Epigenetic mechanisms such as DNA methylation have been reported to contribute to the phenotypic plasticity observed in vertebrates in response to environmental changes. Additionally, DNA methylation is a dynamic process that occurs throughout vertebrate ontogeny and it has been associated with the activation and silencing of gene expression during post-embryonic development and metamorphosis. In this study, we investigated genome-wide DNA methylation profiles during turbot metamorphosis, as well as the epigenetic effects of temperature on turbot post-embryonic development. Fish growth and rates of development were greatly affected by rearing temperature. Thus, turbot raised at ambient temperature (18 °C) achieved greater body weights and progressed through development more quickly than those reared at a colder temperature (14 °C). Genome-wide DNA methylation dynamics analyzed via a methylation-sensitive amplified polymorphism (MSAP) technique were not significantly different between animals reared within the two different thermal environments. Furthermore, comparisons between phenotypically similar fish revealed that genome-wide DNA methylation profiles do not necessarily correlate with specific developmental stages in turbot.     

逆境处理和DNA甲基化影响柑橘体细胞胚发生

对15种柑橘胚性愈伤组织进行体细胞胚诱导,发现逆境处理有利于体细胞胚发生,并可以恢复部分品种的体细胞胚发生能力.对具有和失去体细胞胚发生能力的两种纽荷尔脐橙( Citrus sinensis Osb.)愈伤组织进行随机扩增多态性DNA (RAPD) 分析没有检测到带型的差异,而对它们的甲基化敏感扩增多态性 (MSAP) 进行分析则发现两种愈伤组织间具有明显的DNA甲基化差异,具体细胞胚发生能力的愈伤组织的甲基化水平较失去体细胞胚发生能力的低.     

DNA methylation changes detected by methylation-sensitive amplified polymorphism in two contrasting rice genotypes under salt stress

DNA methylation,one of the most important epigenetic phenomena,plays a vital role in tuning gene expression during plant development as well as in response to environmental stimuli.In the present study,a rnethylation-sensitive amplified polymorphism (MSAP) analysis was performed to profile DNA methylation changes in two contrasting rice genotypes under salt stress.Consistent with visibly different phenotypes in response to salt stress,epigenetic markers classified as stable inter-cultivar DNA methylation differences were determined between salttolerant FL478 and salt-sensitive IR29.In addition,most tissue-specific DNA methylation loci were conserved,while many of the growth stage-dependent DNA methylation loci were dynamic between the two genotypes.Strikingly,salt stress induced a decrease in DNA methylation specifically in roots at the seedling stage that was more profound in IR29 than in the FL478.This result may indicate that demethylation of genes is an active epigenetic response to salt stress in roots at the seedling stage,and helps to further elucidate the implications of DNA methylation in crop growth and development.     

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。     

Age group DNA methylation differences in lemon sharks (Negaprion brevirostris): Implications for future age estimation tools

Age information is often non‐existent for most shark populations due to a lack of measurable physiological and morphological traits that can be used to estimate age. Recently, epigenetic clocks have been found to accurately estimate age for mammals, birds, and fish. However, since these clocks rely, among other things, on the availability of reference genomes, their application is hampered in non‐traditional model organisms lacking such molecular resources. The technique known as Methyl‐Sensitive Amplified Polymorphism (MSAP) has emerged as a valid alternative for studying DNA methylation biomarkers when reference genome information is missing, and large numbers of samples need to be processed. Accordingly, the MSAP technique was used in the present study to characterize global DNA methylation patterns in lemon sharks from three different age groups (juveniles, subadults, and adults). The obtained results reveal that, while MSAP analyses lack enough resolution as a standalone approach to infer age in these organisms, the global DNA methylation patterns observed using this technique displayed significant differences between age groups. Overall, these results confer that DNA methylation does change with age in sharks like what has been seen for other vertebrates and that MSAP could be useful as part of an epigenetics pipeline to infer the broad range of ages found in large samples sizes.     

逆境处理和DNA甲基化影响柑橘体细胞胚发生

对 1 5种柑橘胚性愈伤组织进行体细胞胚诱导 ,发现逆境处理有利于体细胞胚发生 ,并可以恢复部分品种的体细胞胚发生能力。对具有和失去体细胞胚发生能力的两种纽荷尔脐橙 (CitrussinensisOsb .)愈伤组织进行随机扩增多态性DNA (RAPD)分析没有检测到带型的差异 ,而对它们的甲基化敏感扩增多态性 (MSAP)进行分析则发现两种愈伤组织间具有明显的DNA甲基化差异 ,具体细胞胚发生能力的愈伤组织的甲基化水平较失去体细胞胚发生能力的低     

DNA methylation and tissue culture-induced variation in plants

Summary Plant cells growing in an artificial culture environment make numerous genetic mistakes. These alterations are manifested as increased frequencies of single-gene mutations, chromosome breakages, transposable element activations, quantitative trait variations, and modifications of normal DNA methylation patterns. Evidence is presented that indicates a high frequency of DNA hypomethylation as the result of the tissue culture process. Fifteen percent of the methylation changes appear to have been homozygous in the original regenerated plants. A hypothesis is advanced that relates DNA methylation to the variety of genetic alterations found among maize tissue culture regenerants and their progenies. The epigenetic nature of DNA methylation raises questions concerning the stability of tissue culture-induced changes in self-pollinations and crosses. Presented in the Session-in-Depth Exploitation of Plant Cell Culture Variants at the 1992 World Congress on Cell and Tissue Culture, Washington, DC, June 20&#x2013;25, 1992.