拟南芥MLH1基因启动子甲基化作为镉胁迫生物标记物潜力评估

应用重亚硫酸盐测序技术,研究了镉(Cd)胁迫21 d后拟南芥幼苗错配修复基因Mut L-homologue1(MLH1)启动子甲基化的变化趋势。结果显示,拟南芥MLH1启动子区域为-346~+42(391 bp),含有71个胞嘧啶:16个Cp G、6个CHG(H为C、A或T)和49个CHH位点。对照植株Cp G、CHH和CHG位点的甲基化率分别为44.8%、40.5%和52.0%。随Cd胁迫浓度的增加,MLH1启动子区胞嘧啶位点发生超甲基化和去甲基化的位点数逐渐增加;这些位点的甲基化率均呈上升趋势,且均高于对照组(除CHG位点外)。MLH1启动子区域71个胞嘧啶中,Cp G6、Cp G9、CHH44和CHG4位点的甲基化多态性对Cd胁迫更敏感,且具有剂量-效应关系;其中0.25 mg·L-1Cd胁迫下,Cp G9位点为超甲基化,其甲基化变化率为20.0%,其他3个位点为去甲基化,其甲基化变化率分别为12.0%、20.0%和20.0%。相对于幼苗的其他生物学性状(叶片数、生物量及叶绿素含量),Cd胁迫下MLH1启动子甲基化变化更显著且敏感,上述4个胞嘧啶热点的甲基化多态性可作为检测Cd胁迫对植物遗传毒性效应潜在的、有效的生物标记物。     

海州香薷(Elsholtzia haichowensis Sun)细胞壁转化酶基因启动子(EhcwINVP)的克隆及活性分析

以海州香薷基因组DNA为模板,通过hiTAIL-PCR和walking技术扩增得到其细胞壁转化酶基因启动子(Ehcw INVP)片段,长度为1727 bp。生物信息学分析结果表明,该启动子片段中含有多个对脱落酸、赤霉素、细胞分裂素等激素以及对干旱、低温、重金属铜等逆境胁迫响应相关的顺式作用元件。将通过克隆得到的Ehcw INVP序列替换p CAMBIA1301载体上驱动GUS报告基因表达的Ca MV35S启动子序列,构建Ehcw INVP融合GUS的植物表达载体Ehcw INVP::GUS。转基因拟南芥植株的组织化学分析结果表明,海州香薷细胞壁转化酶基因启动子序列具有驱动GUS基因表达的功能,且在10μmol/L铜胁迫下,转基因拟南芥植株叶和根中的GUS活性分别约是对照组的1.7倍和1.5倍。     

海州香薷不同抗性种群液泡转化酶基因EhvINV序列趋异及表达差异分析

根据GenBank中海州香薷(Elsholtzia haichowensis)液泡转化酶基因EhNvINV(JX500755)和EhCvINV(JX500756)的序列设计特异性引物,克隆cDNA全长序列,并通过生物信息学分析基因及推导的蛋白序列,利用SWISS-MODEL进行同源建模,并与蔗糖分子进行模拟对接,实时荧光定量PCR分析EhNvINV和EhCvINV在铜胁迫下的转录表达。结果表明,海州香薷非抗性和抗性种群液泡转化酶蛋白EhNvINV和EhCvINV在114和346处存在氨基酸趋异位点,模拟3D结构相似,仅在趋异位点Glu114/Gln114和Leu346/Pro346处有差别。EhNvINV、EhCvINV和模拟突变体(EhNvINV-E114Q和EhNvINV-L346P)与蔗糖分子对接形成的活性中心构象基本一致,但在空间位置上存在细微差异。实时荧光定量PCR结果表明铜胁迫7 d后,EhCvINV的表达受铜的诱导,而EhNvINV受铜的抑制。     

海州香薷(Elsholtzia haichouensis Sun)不同生态型Cu吸收和酸性磷酸酶活性差异

以海州香薷铜矿区种群（大冶铜绿山）和非矿区种群（红安）为对象,通过水培实验,比较分析了两个种群根中铜的吸收、MDA含量、根伤流液量和酸性磷酸酶活性变化的差异。结果显示, 红安非矿区种群根中的MDA含量随处理浓度的增加而显著升高,铜绿山矿区种群则没有明显的变化;在≥20 μmol/L Cu处理时,非矿区种群根伤流液量急剧下降而矿区种群反而增大。红安种群根中的Cu吸收量明显比铜绿山种群的高,如20 μmol/L Cu处理时,红安种群根中的铜含量在处理后的第1天和第5天分别是铜绿山种群的3倍和4倍;80 μmol/L Cu处理时,第1天和第5天的Cu含量则分别是铜绿山种群的20倍和5倍。然而,铜绿山种群根分泌的和组织内的酸性磷酸酶活性则明显高于红安种群,如在80 μmol/L Cu处理的第3天和第5天,铜绿山种群根组织内的酸性磷酸酶活性都约为红安种群的3倍,根系分泌的酶活性则分别为红安种群的1.6倍和1.8倍。两个种群也表现出不同的变化趋势：铜绿山种群在低浓度处理时,根分泌的和组织内的酸性磷酸酶活性有所增强,高浓度处理时下降,而红安随处理浓度的增加则显著下降。总之,铜胁迫下,海州香薷红安种群根系过氧化损伤明显,而铜绿山种群根系没有产生明显的过氧化损伤;铜绿山种群根系增强酸性磷酸酶的分泌以及保持组织内的高活性,可能分别在减少其铜的吸收及胁迫条件下维持体内正常的磷生理代谢方面具有重要作用。     

海州香薷(Elsholtzia haichouensis Sun)不同生态型Cu吸收和酸性磷酸酶活性差异

以海州香薷铜矿区种群(大冶铜绿山)和非矿区种群(红安)为对象,通过水培实验,比较分析了两个种群根中铜的吸收、MDA含量、根伤流液量和酸性磷酸酶活性变化的差异。结果显示,红安非矿区种群根中的MDA含量随处理浓度的增加而显著升高,铜绿山矿区种群则没有明显的变化;在≥20μmol/LCu处理时,非矿区种群根伤流液量急剧下降而矿区种群反而增大。红安种群根中的Cu吸收量明显比铜绿山种群的高,如20μmol/LCu处理时,红安种群根中的铜含量在处理后的第1天和第5天分别是铜绿山种群的3倍和4倍;80μmol/LCu处理时,第1天和第5天的Cu含量则分别是铜绿山种群的20倍和5倍。然而,铜绿山种群根分泌的和组织内的酸性磷酸酶活性则明显高于红安种群,如在80μmol/LCu处理的第3天和第5天,铜绿山种群根组织内的酸性磷酸酶活性都约为红安种群的3倍,根系分泌的酶活性则分别为红安种群的1.6倍和1.8倍。两个种群也表现出不同的变化趋势:铜绿山种群在低浓度处理时,根分泌的和组织内的酸性磷酸酶活性有所增强,高浓度处理时下降,而红安随处理浓度的增加则显著下降。总之,铜胁迫下,海州香薷红安种群根系过氧化损伤明显,而铜绿山种群根系没有产生明显的过氧化损伤;铜绿山种群根系增强酸性磷酸酶的分泌以及保持组织内的高活性,可能分别在减少其铜的吸收及胁迫条件下维持体内正常的磷生理代谢方面具有重要作用。     

铜胁迫条件下AMF对海州香薷光合色素含量、抗氧化能力和膜脂过氧化的影响

以盆栽海州香薷为研究对象,模拟Cu胁迫条件下,接种丛枝菌根真菌(AMF)对海州香薷叶片光合色素含量、抗氧化酶活性、抗氧化剂含量、膜脂过氧化程度的影响。结果表明:(1)与对照相比,Cu胁迫使海州香薷叶片叶绿素a(Chl a)、叶绿素b(Chl b)、总叶绿素(Chl(a+b))、类胡萝卜素(Car)含量以及叶绿素a/b(Chl a/b)均显著降低,抗氧化酶活性和抗氧化剂含量也显著下降,质膜相对透性(MRP)和丙二醛(MDA)含量显著增大。(2)与Cu胁迫相比,Cu胁迫下接种AMF可使海州香薷叶片叶绿素含量显著增加;超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)活性显著提高;还原型谷胱甘肽(GSH)、抗坏血酸(As A)含量显著增加;MDA含量、MRP显著下降。总之,接种AMF可提高Cu胁迫下海州香薷叶片光合色素含量和抗氧化能力,降低膜脂过氧化水平,从而缓解Cu胁迫对植株造成的伤害,增强海州香薷对Cu胁迫的适应性,提高了植株的生物量。     

接种土壤微生物对铜胁迫下海州香薷生长及光合生理的影响

采用框栽试验方法,模拟Cu胁迫条件下,探讨接种土壤微生物对海州香薷(Elsholtzia splendens)生长和光合生理的影响。结果表明:(1)在Cu胁迫下,海州香薷株数、株高、基径、生物量、茎重比均显著低于对照;与Cu胁迫相比,接种土壤微生物能显著缓解Cu胁迫对海州香薷生长的抑制作用,使植株的株数、株高、生物量、茎重比显著提高。Cu胁迫下,接种土壤微生物均降低了植株体内不同器官Cu含量,茎和叶Cu的累积量显著减少,但对其它器官的Cu含量影响不显著。(2)秋季,各处理的海州香薷的净光合速率(Pn)日变化均呈"单峰"曲线,接种土壤微生物显著提高了Cu胁迫下海州香薷的日均Pn、日均蒸腾速率(Tr),而日均气孔导度(Gs)、日均胞间CO2浓度(Ci)显著降低。(3)Cu胁迫下,接种土壤微生物显著提高了植株的最大净光合速率(Pnmax)、光饱和点(LSP)、表观量子效率(AQY)、最大羧化速率(Vcmax)、最大电子传递速率(Jmax)、磷酸丙糖利用率(TPU),且使光补偿点(LCP)显著降低。表明接种土壤微生物通过提高光能利用率、利用弱光和碳同化能力来增强光合作用能力及有机物的积累,缓解Cu胁迫对海州香薷的毒害。因此,接种土壤微生物可促进Cu胁迫下海州香薷的生长,在重金属污染土壤的植物修复中具有较好的应用潜力。     

小鼠骨髓细胞中p16和Ralb基因启动区CpG岛的甲基化位点分析

抑癌基因p16和白血病致癌因子Ralb与白血病的发生密切相关,其启动子区CpG岛的甲基化对基因表达具有重要作用.本文旨在分析p16、Ralb基因启动子区CpG岛甲基化位点信息,并比较这两个基因在小鼠骨髓细胞和原代培养的骨髓细胞中甲基化状态的差异.运用"MethPrimer"软件预测p16、Ralb基因启动子区的CpG岛,设计甲基化特异性引物.利用重亚硫酸盐测序法(BSP)检测甲基化位点信息.结果显示,p16有1个CpG岛,岛上21个CpG位点全部未发生甲基化;Ralb有2个CpG岛,CpG岛1上的5个CpG位点全部呈甲基化状态,而CpG岛2上的17个CpG位点全部呈非甲基化状态,且小鼠骨髓细胞和体外原代培养的骨髓细胞中两基因的甲基化状态一致.表明p16、Ralb基因甲基化状态未受外界培养条件的影响而改变,提示在与两基因甲基化相关的研究中体外试验可替代体内试验.     

铜胁迫条件下土壤微生物对海州香薷光合特性和叶绿素荧光参数的影响

为探讨铜（Cu）胁迫条件下土壤微生物对海州香薷（Elsholtzia splendens）光合生理和叶绿素荧光参数的影响,实验设置添加Cu（Cu胁迫）、接种土壤微生物、添加Cu与接种土壤微生物等3个处理,以不添加Cu与不接种土壤微生物为对照（CK）。结果表明：接种土壤微生物处理的植株相对叶绿素含量、净光合速率（P_n）、水分利用效率（WUE）均显著高于CK;且对初始荧光（F_o）和最大光化学效率（F_v/F_m）均有显著性影响。与CK相比,添加Cu降低了海州香薷的P_n和气孔导度（G_s）,但胞间CO₂浓度（C_i）的变化与P_n相反,表明其对光合作用的影响主要是非气孔限制因素。添加Cu的植株相对叶绿素含量显著下降,但Cu胁迫下接种土壤微生物提高了植株相对叶绿素含量,差异显著。在Cu胁迫条件下,接种土壤微生物的植株具有较高的F_v/F_m及较低的F_o,显著提高了海州香薷的WUE、P_n、G_s。说明接种土壤微生物可通过提高相对叶绿素含量、改善叶绿素荧光和光合作用来减轻Cu胁迫对海州香薷植株造成的伤害,从而提高海州香薷耐受Cu胁迫的能力。     

外源GSH对海州香薷铜毒害的缓解作用

采用溶液培养的方法,研究了外源GSH对海州香薷铜毒害的缓解作用。结果表明,100μmol·L~（-1）Cu处理6天可以抑制海州香薷根系伸长生长,降低叶片中叶绿体色素含量,增加植株中膜脂过氧化物丙二醛（MDA）的含量。100μmol·L~（-1）Cu处理下施加GSH可以缓解Cu对海州香薷的毒害作用,促进根系伸长生长,增加生物量,提高叶绿体色素含量,降低MDA含量,减少植株中Cu含量。     

Transgenerational maintenance of transgene body CG but not CHG and CHH methylation

In plants, RNA-directed DNA methylation (RdDM) can target both transgene promoters and coding regions/gene bodies. RdDM leads to methylation of cytosines in all sequence contexts: CG, CHG and CHH. Upon segregation of the RdDM trigger, at least CG methylation can be maintained at promoter regions in the progeny. So far, it is not clear whether coding region methylation can be also maintained. We showed that the body of Potato spindle tuber viroid (PSTVd) transgene constructs became densely de novo methylated at CG, CHG and CHH sites upon PSTVd infection. In this study, we demonstrate that in viroid-free progeny plants, asymmetric CHH and CHG methylation was completely lost. However, symmetric CG methylation was stably maintained for at least two generations. Importantly, the presence of transgene body methylation did not lead to an increase of dimethylation of histone H3 lysine 9 or a decrease of acetylation of H3. Our data supports the view that CG methylation can be maintained not only in promoters but also in the body of transgenes. They further suggest that maintenance of methylation may occur independently of tested chromatin modifications.     

Transgenerational maintenance of transgene body CG but not CHG and CHH methylation

In plants, RNA-directed DNA methylation (RdDM) can target both transgene promoters and coding regions/gene bodies. RdDM leads to methylation of cytosines in all sequence contexts: CG, CHG and CHH. Upon segregation of the RdDM trigger, at least CG methylation can be maintained at promoter regions in the progeny. So far, it is not clear whether coding region methylation can be also maintained. We showed that the body of Potato spindle tuber viroid (PSTVd) transgene constructs became densely de novo methylated at CG, CHG and CHH sites upon PSTVd infection. In this study, we demonstrate that in viroid-free progeny plants, asymmetric CHH and CHG methylation was completely lost. However, symmetric CG methylation was stably maintained for at least two generations. Importantly, the presence of transgene body methylation did not lead to an increase of dimethylation of histone H3 lysine 9 or a decrease of acetylation of H3. Our data supports the view that CG methylation can be maintained not only in promoters but also in the body of transgenes. They further suggest that maintenance of methylation may occur independently of tested chromatin modifications.     

DNA methylation of retrotransposons,DNA transposons and genes in sugar beet (Beta vulgaris L.)

The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses the activity of transposable elements (TEs), affects gene expression and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%), in particular satellite DNA, retrotransposons and DNA transposons. Genome‐wide cytosine methylation in the sugar beet genome was studied in leaves and leaf‐derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences, and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H = A, C, and T) and CHH sites, whereas the TE pattern differed, depending on the TE class (class 1, retrotransposons and class 2, DNA transposons). Along genes and TEs, CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing to a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome‐wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared with leaves.     

Identification of differential genomic DNA Methylation in the hypothalamus of pubertal rat using reduced representation Bisulfite sequencing

Background

There are many variables affecting the onset of puberty in animals, including genetic, nutritional, and environmental factors. Recent studies suggest that epigenetic regulation, especially DNA methylation, plays a majorrole in the regulation of puberty. However, there have been no reports on DNA methylation of the pubertal genome.

Methods

We investigated DNA methylation in the female rat hypothalamus at prepuberty and puberty using reduced representation bisulfite sequencing technology. The identified genes and signaling pathways exhibiting changes to DNA methylation in pubertal rats were determined by Gene Ontogeny and Kyoto Encyclopedia of Genes and Genomes analysis.

Results

The distribution of the three types of methylated C bases in promoter and CpG island (CGI) regions in the hypothalamus was as follows: 87.79% CG, 3.05% CHG, 9.16% CHH for promoters, and 88.35% CG, 3.21% CHG, 88.35% CHH for CGI in prepubertal rats; and 90.78% CG, 2.13% CHG, 7.09% CHH for promoters, and 88.59% CG, 88.59% CHG, 8.35% CHH for CGI in pubertal animals. CG showed the highest percentage of methylation, and was the highest methylation state in CGI. Compared to prepubertal hyoyhalamus samples, we identified ten genes with altered methylation in promoter regions in the pubertal hypothalamus samples, and 43 genes with altered methylation in the CGI. Changes in DNA methylation were found in gonadotropin-releasing hormone signaling pathways, and the oocyte meiosis pathway.

Conclusion

Our results demonstrate changes in DNA methylation occur in female rats from prepuberty to puberty suggestng DNA methylation may play a crucial role in the regulation of puberty onset. This study provides essential information for future studies on the role of epigenetics in the regulation of puberty.

     

Virus‐induced gene silencing in transgenic plants: transgene silencing and reactivation associate with two patterns of transgene body methylation

We used bisulfite sequencing to study the methylation of a viral transgene whose expression was silenced upon plum pox virus infection of the transgenic plant and its subsequent recovery as a consequence of so‐called virus‐induced gene silencing (VIGS). VIGS was associated with a general increase in the accumulation of small RNAs corresponding to the coding region of the viral transgene. After VIGS, the transgene promoter was not methylated and the coding region showed uneven methylation, with the 5′ end being mostly unmethylated in the recovered tissue or mainly methylated at CG sites in regenerated silenced plants. The methylation increased towards the 3′ end, which showed dense methylation in all three contexts (CG, CHG and CHH). This methylation pattern and the corresponding silenced status were maintained after plant regeneration from recovered silenced tissue and did not spread into the promoter region, but were not inherited in the sexual offspring. Instead, a new pattern of methylation was observed in the progeny plants consisting of disappearance of the CHH methylation, similar CHG methylation at the 3′ end, and an overall increase in CG methylation in the 5′ end. The latter epigenetic state was inherited over several generations and did not correlate with transgene silencing and hence virus resistance. These results suggest that the widespread CG methylation pattern found in body gene bodies located in euchromatic regions of plant genomes may reflect an older silencing event, and most likely these genes are no longer silenced.