小麦易位系基因组DNA甲基化分析中MSAP体系建立及应用

DNA甲基化在真核生物生长发育过程中起着重要调控作用.依据对DNA甲基化敏感程度不同的同裂酶,在AFLP(amplified fragment length polymorphism)基础上发展而来的MSAP(methylationsensitive amplification polymorphism)技术可以方便的检测全基因组DNA胞嘧啶甲基化模式及程度.本文以一套高代分离的小麦黑麦1RS/1BL易位系及其亲本材料为研究对象,采用EcoRⅠ和HpaⅡ(或MspⅠ)双酶切建立适合于小麦易位系基因组的MSAP技术体系,针对研究中出现的问题提出解决方法,并对酶切位点的甲基化模式进行分析,检测易位系及亲本材料间的甲基化多态性,为进一步的基础研究和育种应用提供理论依据.     

MS-RDA的改进及在白背飞虱的应用研究*

甲基化敏感性代表性差异分析方法（MS-RDA）适用于特异、低丰度基因组DNA甲基化的筛查与鉴定，已大量应用于哺乳动物的疾病发生和植物发育机理方面的研究，但昆虫中尚未发现该方法的应用。从DNA提取、酶切、扩增子制备、消减杂交等方面对该方法进行了改进，并在水稻重要害虫白背飞虱上得到成功应用，获得了4条与雌、雄性别相关联的特异性甲基化差异条带，其中有3条序列已被GenBank收录，登陆号分别为JX847621、JX624161、JX472453，经BLAST比对，有3条未发现同源性序列，属于功能未知的新基因；同时还获得了2条与长短翅型相关联的特异性甲基化差异条带，也已被 GenBank 收录，登陆号分别为 JX514031、JX472454，同源性分析发现与多种动物的18S核糖体和28S核糖体RNA基因序列高度相似，因此有关核糖体基因的甲基化可能对白背飞虱的翅型分化起到一定的调控作用。     

慢性肾功能衰竭大鼠肾皮质基因组DNA表观遗传学变化的MSAP分析

以灌胃腺嘌呤诱导的慢性肾功能衰竭Wistar大鼠为实验材料,用高效液相色谱法(HPLC)检测了大鼠血清Hcy浓度的变化；应用甲基化敏感扩增多态性(methylation-sensitive amplification polymorphism,MSAP)技术,检测了慢性肾衰大鼠肾皮质基因组DNA甲基化程度的变化,并对M...     

长筒石蒜组织培养中器官发生的MSAP分析

采用甲基化敏感扩增多态性技术,从64对MSAP选扩引物中,选出扩增清晰、可辨且可重复的51对MSAP引物组合,扩增获得长筒石蒜种子胚、愈伤组织、苗等3个不同发育时期的DNA胞嘧啶甲基化修饰水平及MSAP扩增图谱。结果表明,种子胚基因组中有68.0%(全甲基化50.2%、半甲基化49.8%)、愈伤组织60.0%(全甲基化52.2%、半甲基化47.8%)、苗64.3%(全甲基化40.8%、半甲基化59.2%)的CCGG/GGCC位点发生胞嘧啶甲基化。3个不同时期甲基化的水平互有差异,依据超甲基化/去甲基化的修饰机理分析,甲基化水平的变化或许导致了长筒石蒜不同的器官发生。     

DNA甲基化敏感扩增多态性技术及其在作物遗传研究中的应用

DNA甲基化是表观遗传修饰的基本方式之一,在调控植物基因表达、抵御逆境胁迫、防御外源基因侵入等方面具有重要作用,随着对DNA甲基化研究的深入,在AFLP技术基础上衍生的DNA甲基化敏扩增多态性技术（MSAP）,以其高通量、高多态性、低成本、易操作等优点,在作物遗传育种研究的各个领域得到广泛应用。本文综述了该技术的基本原理与操作,及其在作物遗传研究中的应用现状。     

不同倍性(2×、3×、4×)鸭梨基因组DNA甲基化水平与模式分析

以鸭梨(Pyrus.bretschneideri Rehd.cv.‘Yali')多倍体(2×、3×、4×)为材料,应用甲基化敏感扩增多态性(methylation sensitive amplified polymorphism,MSAP)技术研究不同倍性鸭梨基因组DNA甲基化水平及模式变化.共筛选22对引物,平均每对引物产生约50条带,统计100～500 bp的扩增带,共计954个位点.分析表明:鸭梨2×、3×、4×分别检测到831、886、852个位点,发生甲基化位点分别占15.5%、20.0%、16.8%,其中全甲基化占9.7%、11.9%、9.7%.分析这些位点甲基化模式表明:甲基化模式在三个倍性间类型相同的占所检测位点的69.2%,仅两种倍性相同的为25.4%,各不相同的占5.5%,其中三倍体41.4%甲基化位点发生改变,显示出独特的DNA甲基化特点.     

鞍带石斑鱼、棕点石斑鱼及其杂交子代DNA甲基化的MSAP分析

为了探究海洋鱼类杂交优势的产生机制,以鞍带石斑鱼、棕点石斑鱼及其杂交子一代石斑鱼为研究对象,采用甲基化敏感扩增多态性(MSAP)技术对三个群体的基因组DNA的胞嘧啶甲基化修饰水平进行了研究.实验结果显示,棕点石斑鱼、鞍带石斑鱼、杂交子一代石斑鱼的基因组DNA的总甲基化率分别为57.18%,63.16%,54.76%,在石斑鱼亲本的基因组中,其DNA的甲基化程度较高,而在杂交子代中,其DNA的甲基化程度较低.三个群体的DNA全甲基化率分别为31.66%,39.71%,40.00%,半甲基化率分别为25.52%,23.44%,14.76%,杂交子代的半甲基化率显著低于亲本的半甲基化率.研究表明,鞍带石斑鱼与棕点石斑鱼的杂交子代在基因组层面上和双亲相比发生了较大的甲基化水平的调整,于不同位点,DNA甲基化的增强或减弱对石斑鱼杂种优势可能会产生影响.     

分子标记及AFLP技术

分子标记类型可以基因表达的结果为基础,对基因的间接反映;分子标记则是DNA分子碱基序列变异的直接反映.早期利用限制性内切酶,酶切生物体DNA后来检测不同遗传位点等位变异(RFLP)和以一个碱基顺序随机排列的寡核苷酸序列为引物,利用对基因组DNA随机扩增来鉴别DNA多态性(RAPDs).真核生物基因组中普遍存在的重复序列产生了微卫星(microsatellites)标记技术.而RFLP与RAPDs有机结合形成了有着更为广阔的应用前景的AFLP技术.SNP标记利用大多数基因位点上都会有若干个等位型(alleles)为DNA芯片技术应用于遗传作图提供了基础.     

草鱼基因组随机扩增多态性引物及多态性位点的筛选

为了建立草鱼基因组DNA多态性分析的指标体系,应用RAPD技术,从180条10碱基随机引物中筛选出了31条能扩增出多态性DNA片段的引物。用这31条多态性引物共扩增出了327条重复性好、带型清晰、分辨率高的谱带。扩增产物的片段大小范围在400—2000bp之间。单一引物扩增条带为5—14条。用这些多态性引物在草鱼基因组DNA中检测到了93个多态性位点,并对这些多态性位点上的等位基因频率进行了统计分析。这31条多态性引物和所检测到的93个多态性位点初步为草鱼基因组的多态性分析提供了可靠的分析指标体系。     

黄鳝性别决定与SRY基因不相关

以人和鼠的SRY片断为探针与黄鳝基因组DNA进行杂交,发现雌雄黄鳝中均有相同的杂交带.用一对SRY基因保守区HMGbox的引物,在雌雄黄鳝的基因组DNA中均扩增出约200bp片段.将此片断克隆测序后发现雌雄个体中此片断仅相差1个bp,且与人的SRY基因HMG盒基因极相似.以该片段与雌雄个体的RNA进行Northern杂交未能检测到杂交带,RT-PCR反应扩增出一条微弱的200bp片段.以上结果表明:在雌雄黄鳝的基因组DNA中均存在SRY同源片断;黄鳝中的SRY同源片断可能与其性别无直接关系,而是具备其他功能,由此推测低等脊椎动物的性别决定可能由其他基因控制.     

F-MSAP： A practical system to detect methylation in chicken genome

By replacing radiation with fluorescent system in the technique of methylation sensitive amplified polymorphism （MSAP） and optimizing reaction conditions, a modified technique to detect DNA methylation called F-MSAP （fluorescent labeled methylation sensitive amplified polymorphism） was developed. In the present study, cytosine methylation patterns of genomic DNA were investigated in two inbred chickens and their F1 hybrids. Three types of methylation patterns were observed in each individual, namely fully methylated, hemi-methylated or not methylated types. The average incidence of methylation was approximately 40%. The percentage that the F1 hybrid individual inherits the methylation for any given sites from either/both parent amounted to 95%, while the percentage of altered methylation patterns in F1 individual was only 5%, including 14 increased and 12 decreased methylation types, demonstrating that F-MSAP was highly efficient for large-scale detection of cytosine methylation in chicken genome. Our technique can be further extended to other animals or plants with complex genome and rich in methylation polymorphism.     

Detection of DNA methylation changes during seed germination in rapeseed （Brassica napus）

DNA methylation is a common yet important modi- fication of DNA in eukaryotic organisms. DNA methy- lation, especially methylation of cytosine (m5C), have both epigenetic and mutagenic effects on various cellu- lar activities such as differential gene exp…     

高活力水稻种子萌发过程中DNA甲基化变化的MSAP分析

DNA甲基化是基因组DNA的一种主要表观遗传修饰形式,是调节基因组功能的重要手段。本实验采用MSAP方法分析,旨在研究高活力水稻种子萌发过程中的甲基化与去甲基化变化的规律,为研究种子的老化机理和种子的长期保存提供依据。结果表明:水稻种子萌发过程中,同时发生了甲基化与去甲基化作用,且去甲基化作用先于甲基化作用发生。发生去甲基化可能与基因活化有关,发生甲基化可能与组织特异性有关。     

Patterns of DNA cytosine methylation between haploids and corresponding diploids in rice

In higher plant, about 30% cytosines are methy-lated[1], among which about 90% methylated sites lie in CpG dinucleotide and CpNpG trinucleotide[2]. The me-thylated DNA has inducing and epigenetic effects on cell biological procedures such as gene differen…     

抗白粉病小麦近等基因系DNA甲基化的MSAP分析

应用甲基化敏感扩增多态性（MSAP）技术检测小麦抗白粉病代换系6A／6V与感病品系京411的近等基因系NILs,及其亲本6A／6V和京411的基因组DNA甲基化变化．结果表明,NILs的整体甲基化水平（22．9％）略高于6A／6V（21．2％）,低于京411（23．4％）．分析其甲基化模式发现,NILs相对京411甲基化水平降低条带比例（2．89％）明显高于甲基化提高条带（0．55％）,而相对抗病亲本6A／6V,NILs甲基化水平降低条带比例（2．96％）明显低于甲基化提高条带（4．20％）,说明NILs的整体基因表达水平低于6A／6V,高于京411．     

糖尿病大鼠肾、胰基因组DNA甲基化状态的变化

从正常大鼠，糖尿病大鼠，中药糖微康治疗的糖尿病大鼠，西药开搏通治疗的糖尿病大鼠的肾，胰等组织中提取出其基因组DNA，应用对DNA甲基化作用敏感的限制性核酸内切酶HpaⅡ和MspⅠ（为一组同裂酶（进行了限制性酶切图谱分析，结果表明，DNA化作用的主要位点在CpG二核苷酸处，且“糖微康”在关闭患糖尿病后肾组织中被异常活化的基因的同时，还能活化糖尿病动物胰组织中处于静息状态的基因，由此可见糖尿病的发病和治疗都与DNA甲基化作用密切相关。     

Analysis of DNA methylation variation in wheat genetic background after alien chromatin introduction based on methylation-sensitive amplification polymorphism

During the process of alien germplasm introduced into wheat genome by chromosome engineering, extensive genetic variations of genome structure and gene expression in recipient could be induced. In this study, we performed GISH （genome in situ hybridization） and AFLP （amplified fragment length polymorphism） on wheat-rye chromosome translocation lines and their parents to detect the identity in genomic structure of different translocation lines. The results showed that the genome primary structure variations were not obviously detected in different translocation lines except the same 1RS chromosome translocation. Methylation sensitive amplification polymorphism （MSAP） analyses on genomic DNA showed that the ratios of fully-methylated sites were significantly increased in translocation lines （CN12, 20.15%; CN17, 20.91%; CN18, 22.42%）, but the ratios of hemimethylated sites were significantly lowered （CN12, 21.41%; CN17, 23.43%; CN18, 22.42%）, whereas 16.37% were fully-methylated and 25.44% were hemimethylated in case of their wheat parent. Twenty-nine classes of methylation patterns were identified in a comparative assay of cytosine methylation patterns between wheat-rye translocation lines and their wheat parent, including 13 hypermethylation patterns （33.74%）, 9 demethylation patterns （22.76%） and 7 uncertain patterns （4.07%）. In further sequence analysis, the alterations of methylation pattern affected both repetitive DNA sequences, such as retrotransposons and tandem repetitive sequences, and low-copy DNA.     

AFLP的一种改进方法

扩增片段长度多态性技术是一种兼具稳定性及多态检出率高的ＤＮＡ指纹分析技术,具有广泛应用前景,但该技术费用高,操作较复杂,本文对ＡＦＬＰ技术从限制性内切酶水解,扩增条件,检测方法三方面作了一些改进,建立了一磋稳定的和简便易行的实验条件,同时对这种改进的方法对家蚕基因组ＤＮＡ的多态性进行了初步研究,得到了良好的ＤＮＡ的多态性检测效果,不仅分辨力较高,而且带型稳定。     

Habitat-induced reciprocal transformation in the root phenotype of Oriental ginseng is associated with alteration in DNA methylation

Oriental ginseng is an important medicinal plant that grows in 2 major forms or ecotypes, wild and domesticated. Each form differs conspicuously in root phenotype, but can be converted from one type to another by habitat. Here we show that the habitat-induced transformation of ginseng root phenotype was accompanied by alteration in cytosine methylation at a large number of 5′-CCGG-3′ sites detected by the methylation-sensitive polymorphism (MSAP) marker. The collective CG and CHG methylation levels of all 4 landraces of the domesticated form were significantly lower than those of the wild form. Interestingly, artificially transplanted ginseng plants recreated in both directions the methylation levels (at least in CHG) of their natural counterparts. The methylation differences between the 2 ginseng ecotypes were validated at 2 isolated MSAP loci bearing homology to a 5S rRNA gene or a copia retrotransposon. Our results implicate a link between epigenetic variation and habitat-induced phenotypic flexibility in Oriental ginseng.