The objective of this research was to evaluate Ag+ toxicity in Trifolium pratense L. seedlings subjected to increasing doses of Ag+ by determining photosynthetic pigment and malondialdehyde (MDA) contents, microstructure and hereditary substance alterations, changes in activities of antioxidase-superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) as well as the content of total Ag absorbed in vivo with evaluation of root growth. Doses of approximately 80 mg L-(1 ) Ag+ severely affected photosynthetic efficiency in Trifolium pratense L. seedlings promoted by damages in photosynthetic apparatus evidenced by downward trend in photosynthetic pigment contents and obvious chlorosis. Alterations in enzymatic activity, lipid peroxidation, genic material damage and the presence of Ag+ in vivo had impacted on photosynthetic machinery as well. A hormesis effect was observed at 60 mg L-1 Ag+ for the photosynthetic pigments and antioxidase for Trifolium pratense L. seedlings. Tissue changes (i.e., roots, stems and leaves) observed in fluorescence microscope with obvious chlorosis, roots blackening and formation of agglomerated black particles, were related to the lesion promoted by excessive ROS in vivo. Asynchronous change of antioxidase activity corresponded to the alteration in the MDA content, indicating the synchronization in the elimination of ROS. The changes occurred in RAPD profiles of treated samples following Ag+ toxicity containing loss of normal bands, appearance of new bands and variation in band intensity compared to the normal plants with a dose-dependent effect. On average, the roots of Trifolium pratense L. immobilized 92.20% of the total Ag absorbed as a metal exclusion response. Root growth was significantly sensitive to Ag+ stress with obvious hormesis, which corresponded to the changes in Ag uptake, demonstrating the functional alterations in plants. To sum up, we suggest that modulating the genotype of Trifolium pratense L. seedlings to bear higher proportion of pollutants is conducive to contamination site treatment.

MSH6在激活细胞周期阻滞和修复镉诱导的DNA损伤等方面具有重要功能.本研究以拟南芥为实验材料,通过生物信息学分析发现,MSH6具有miRNA172b-5p、miRNA172e-5p、miRNA472-3p等microRNAs (miRNAs)的作用靶点.利用烟草双荧光素酶报告系统体外验证检测发现,与阴性对照组相比,miRNA172b-5p、miRNA172e-5p和miRNA472-3p均可显著抑制荧光素酶的相对活性,其比值分别下降了25.5％、47.2％和49.5％.采用拟南芥植株瞬时侵染体内验证结果显示,miRNA172b-5p、miRNA172e-5p和miRNA472-3p分别上升了178.9％、123.6％和37.6％;同时,qRT-PCR(real time quantitative reverse transcript polymerase chain reaction)结果表明,拟南芥幼苗在1.25、2.5、4.0mg·L-1Cd胁迫7d后,与对照组相比,MSH6表达显著降低,分别下降了20.3％、22.7％和38.2％.而miRNA172b-5p、miRNA172e-5p和miRNA472-3p表达显著增加,二者呈显著的负相关关系(r=-0.997,P＜0.01;r=-0.997,P＜0.01;r=-0.952,P＜0.05),表明miRNA172b-5p、miRNA172e-5p和miRNA472-3p是拟南芥Cd胁迫响应的miRNAs,并且靶向MSH6基因.本研究为DNA mismatch repair (MMR)对Cd毒理调控机制的研究提供miRNA理论基础.

DNA mismatch repair (MMR) proteins have been implicated in sensing and correcting DNA damage, and in governing cell cycle progression in the presence of structurally anomalous nucleotide lesions induced by different stresses in mammalian cells. Here, Arabidopsis seedlings were grown hydroponically on 0.5 x MS media containing cadmium (Cd) at 0-4.0 mg L-1 for 5 d. Flow cytometry results indicated that Cd stress induced a G2/M cell cycle arrest both in MLH1-, MSH2-, MSH6-deficient, and in WT roots, associated with marked changes of G2/M regulatory genes, including ATM, ATR, SOG1, BRCA1, WEE1, CYCD4; 1, MAD2, CDKA;1, CYCB1; 2 and CYCB1; 1. However, the Cd-induced G2/M phase arrest was markedly diminished in the MSH2- and MSH6-deficient roots, while a lack of MLH1 had no effect on Cd-induced G2 phase arrest relative to that in the wild type roots under the corresponding Cd stress. Expression of the above G2/M regulatory genes was altered in MLH1, MSH2 and MSH6-deficient roots in response to Cd treatment. Furthermore, Cd elicited endoreplication in MSH2- and MSH6-deficient roots, but not in MLH1-deficient Arabidopsis roots. Results suggest that MSH2 and MSH6 may act as direct sensors of Cd-mediated DNA damage. Taken together, we conclude that MSH2 and MSH6, but not MLH1, components of the MMR system are involved in the G2 phase arrest and endoreplication induced by Cd stress in Arabidopsis roots. (C) 2018 Elsevier Ltd. All rights reserved.

由于佳乐麝香(HHCB)被广泛应用于日用化工产品中,被持续不断地释放到环境中,所产生的生态风险已引起越来越多的重视.为探究HHCB的生态毒性效应,在水培条件下考察了不同浓度HHCB对萝卜的表观生长指标(发芽率、根伸长抑制率、芽伸长抑制率)和基于随机引物扩增多态性(RAPD)图谱的根尖DNA损伤状况.研究结果显示:低剂量(≤25 mg·L-1)胁迫对萝卜发芽无显著影响(P＞0.05);高剂量(≥50 mg· L-1)胁迫可以显著抑制萝卜发芽率(P＜0.05).萝卜的根长和芽长抑制率随HHCB浓度增加而呈上升趋势,且根伸长对HHCB胁迫较芽伸长更敏感,更适宜指示HHCB对植物的生态毒性效应.萝卜根尖基因组DNA的RAPD分析结果表明:大于或等于5 mg·L-1的HHCB即可明显导致萝卜根尖基因组DNA损伤,且随着HH-CB浓度的升高,根尖基因组DNA含量呈线性降低,DNA多态率增加,基因组模板稳定性(GTS)减小,遗传相似性变远.这表明较低剂量的HHCB胁迫就能够导致萝卜根尖基因组DNA损伤,且随浓度升高而损伤严重.因此,利用RAPD技术获得的萝卜DNA多态性变化可作为检测HHCB遗传毒性效应的敏感生物标记物,为化学品污染生态毒理早期诊断提供科学依据.

DNA damage assay based on RAPD, ELISA-based global methylation analysis, and expression analysis of genes related to DNA damage, repair, and cell cycle were used to study genomic DNA damage and stress response of DNA repair and cell cycle in Arabidopsis plantlets exposed to 0,0.125,0.25,1.0 mg·L-1 and 2.5 mg·L-1 cadmium(Cd)for 5 d. Compared with the control, DNA damage and global methylation rate were increased significantly with the increased Cd dose , and expression of cell division genes(PCNA 1 and PCNA 2), MMR genes(MLH1, MSH2 and MSH6), homologous recombination genes(RAD51 and BRCA1), and non- homologous end joining genes(KU70 , MRE11 and GR1)were followed an obvious inverted U- shaped dose- response effect of Cd exposures and a MMR>HR>NHEJ sensitivity rule. The results showed that low-dose Cd result in easy-repaired mismatch damage, and high-dose Cd stress cause diffi-cult-repaired chromosome damage and double strand break, which could accumulate and aggravate with the stress duration. In addition, the expression of MSH6 and MLH1 is the most sensitive indicator which could be a sensitive biomarker, applied to early diagnosis and risk as-sessment of genotoxic effects of Cd pollution in ecotoxicology.

Accumulating evidence demonstrates that the aberrant expression of cell cycle regulation and DNA repair genes can result in abnormal cell proliferation and genomic instability in eukaryotic cells under different stresses. Herein, Arabidopsis thaliana (Arabidopsis) seedlings were grown hydroponically on 0.5 × MS media containing cadmium (Cd) at 0-2.5mgL-1 for 5d of treatment. Real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed that expression of DNA damage repair and cell cycle regulation genes, including BRCA1, MRE11, WEE1, CDKA;1 and PCNA1, showed an inverted U-shaped dose-response. In contrast, notably reduced expression was observed for G1-to-S transition-related genes, Histone H4, E2Fa and PCNA2; DSB end processing, GR1; G2-to-M transition-related gene, CYCB1;1; and DNA mismatch repair, MSH2, MSH6 and MLH1 genes in root tips exposed to 0.125-2.5mg/L Cd for 5d. Flow cytometry (FCM) analysis revealed significant increases of cells with a 2C nuclear content and with a 4C and 8C nuclear content under Cd stresses of 0.125 and 1-2.5mgL-1, respectively. Our results suggest that 0.125mgL-1 Cd-induced DNA damage induced the marked G1/S arrest, leading to accelerated growth in root tips, while 1.0-2.5mgL-1 Cd-induced DNA damage caused a notable G2/M arrest in root tips, leading to reduced growth in root tips. This may be a protective mechanism that prevents cells with damaged DNA from dividing under Cd stress.

Microsatellite instability (MSI) analysis, random-amplified polymorphic DNA (RAPD), and methylation-sensitive arbitrarily primed PCR (MSAP-PCR) are methods to evaluate the toxicity of environmental pollutants in stress-treated plants and human cancer cells. Here, we evaluate these techniques to screen for genetic and epigenetic alterations of Arabidopsis plantlets exposed to 0-5.0 mg L(-1) cadmium (Cd) for 15 d. There was a substantial increase in RAPD polymorphism of 24.5, and in genomic methylation polymorphism of 30.5-34.5 at CpG and of 14.5-20 at CHG sites under Cd stress of 5.0 mg L(-1) by RAPD and of 0.25-5.0 mg L(-1) by MSAP-PCR, respectively. However, only a tiny increase of 1.5 loci by RAPD occurred under Cd stress of 4.0 mg L(-1), and an additional high dose (8.0 mg L(-1)) resulted in one repeat by MSI analysis. MSAP-PCR detected the most significant epigenetic modifications in plantlets exposed to Cd stress, and the patterns of hypermethylation and polymorphisms were consistent with inverted U-shaped dose responses. The presence of genomic methylation polymorphism in Cd-treated seedlings, prior to the onset of RAPD polymorphism, MSI and obvious growth effects, suggests that these altered DNA methylation loci are the most sensitive biomarkers for early diagnosis and risk assessment of genotoxic effects of Cd pollution in ecotoxicology.

The present study was to explore design methods for short and long primers in plant methylation research and their corresponding PCR conditions employing large number of designed primers and to analyze the effects on methylation rates using three different Taq poly-merases. Results showed:a Touch-down PCR was suitable when using short primers of 17 ~ 30 bp, however, low specificity and success rates were observed; PCR performed the best when using long primers of 31~50 bp, provided that length and Tm of forward primers﹥those of reverse primers and 55 ℃ and 60 ℃ served as annealing and prolonging temperature, respectively; Super-Fidelity Taq polymerase like Prime STAR R HS isn't suitable for methylation research because of its 3'→5'exonuclease activities. However, PCR performed the best when using EpiTaqTM HS.

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MutL-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CpG、6个CHG(H为C、A或T)和49个CHH位点.对照植株CpG、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,CpG6、CpG9、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,CpG9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.

全基因组DNA甲基化分析是植物胁迫表观遗传损伤研究的主要方向之一,目前可用手段虽多,然而多数较繁琐,不利于表观遗传损伤的快速诊断.为此应用MSAP-PCR法研究Cd胁迫下拟南芥基因组甲基化变化,并通过优化实验条件、筛选引物(共5条,包括通用引物MLG2和本实验室设计引物AP-1、AP-2、AP-3、AP-4)以及检验多态性和敏感性,综合评估该方法在植物甲基化研究中的应用前景.研究结果发现:该方法模板量选择在150～250 ng并使用4％聚丙烯酰胺凝胶电泳(含50％尿素)分辨PCR产物多态性最佳;该方法对镉敏感性高,在0.2 mg·L-1Cd2+水平就可检测约30个位点的甲基化多态性;引物AP-4对CpG位点甲基化变化最敏感,而AP-3对CHG位点甲基化变化敏感性最高.该方法操作简便、成本低廉、结果准确且敏感性高,可作为植物全基因组甲基化研究的理想方法,以及植物抗逆研究和环境污染早期诊断的生物胁迫标记物.