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DNA 指纹图谱分析、端粒分析和抗氧化特性作为监测离体种子寿命的工具。

DNA profiling, telomere analysis and antioxidant properties as tools for monitoring ex situ seed longevity.

机构信息

Dipartimento di Biologia e Biotecnologie L. Spallanzani, Laboratori di Genetica e Microbiologia, Via Ferrata 1, 27100 Pavia, Italy.

出版信息

Ann Bot. 2013 May;111(5):987-98. doi: 10.1093/aob/mct058. Epub 2013 Mar 26.

Abstract

BACKGROUND AND AIMS

The germination test currently represents the most used method to assess seed viability in germplasm banks, despite the difficulties caused by the occurrence of seed dormancy. Furthermore, seed longevity can vary considerably across species and populations from different environments, and studies related to the eco-physiological processes underlying such variations are still limited in their depth. The aim of the present work was the identification of reliable molecular markers that might help in monitoring seed deterioration.

METHODS

Dry seeds were subjected to artificial ageing and collected at different time points for molecular/biochemical analyses. DNA damage was measured using the RAPD (random amplified polymorphic DNA) approach while the seed antioxidant profile was obtained using both the DPPH (1,1-diphenyl, 2-picrylhydrazyl) assay and the Folin-Ciocalteu reagent method. Electron paramagnetic resonance (EPR) provided profiles of free radicals. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to assess the expression profiles of the antioxidant genes MT2 (type 2 metallothionein) and SOD (superoxide dismutase). A modified QRT-PCR protocol was used to determine telomere length.

KEY RESULTS

The RAPD profiles highlighted different capacities of the two Silene species to overcome DNA damage induced by artificial ageing. The antioxidant profiles of dry and rehydrated seeds revealed that the high-altitude taxon Silene acaulis was characterized by a lower antioxidant specific activity. Significant upregulation of the MT2 and SOD genes was observed only in the rehydrated seeds of the low-altitude species. Rehydration resulted in telomere lengthening in both Silene species.

CONCLUSIONS

Different seed viability markers have been selected for plant species showing inherent variation of seed longevity. RAPD analysis, quantification of redox activity of non-enzymatic antioxidant compounds and gene expression profiling provide deeper insights to study seed viability during storage. Telomere lengthening is a promising tool to discriminate between short- and long-lived species.

摘要

背景与目的

尽管种子休眠会带来困难,但发芽试验目前仍是种质库中评估种子活力最常用的方法。此外,不同环境下的物种和种群的种子寿命差异很大,而与这些变化背后的生态生理过程相关的研究在深度上仍然有限。本研究的目的是确定可靠的分子标记,以帮助监测种子的劣化。

方法

将干种子进行人工老化处理,并在不同时间点收集,进行分子/生化分析。使用 RAPD(随机扩增多态性 DNA)方法测量 DNA 损伤,同时使用 DPPH(1,1-二苯基-2-苦基肼)法和 Folin-Ciocalteu 试剂法获得种子抗氧化剂谱。电子顺磁共振(EPR)提供自由基谱。使用定量实时聚合酶链反应(QRT-PCR)评估抗氧化基因 MT2(2 型金属硫蛋白)和 SOD(超氧化物歧化酶)的表达谱。使用改良的 QRT-PCR 方案来确定端粒长度。

主要结果

RAPD 谱突出了两种矢车菊属植物克服人工老化诱导的 DNA 损伤的不同能力。干种子和再水化种子的抗氧化谱表明,高山分类群 Silene acaulis 的抗氧化剂特异性活性较低。仅在低海拔物种的再水化种子中观察到 MT2 和 SOD 基因的显著上调。再水化导致两种矢车菊属植物的端粒延长。

结论

为具有固有种子寿命差异的植物物种选择了不同的种子活力标记。RAPD 分析、非酶抗氧化化合物的氧化还原活性的定量以及基因表达谱分析提供了更深入的见解,以研究储存过程中的种子活力。端粒延长是区分短寿命和长寿命物种的有前途的工具。

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