Identification and characterization of low temperature stress responsive genes in Poncirus trifoliata by suppression subtractive hybridization
Identification and characterization of low temperature stress responsive genes in Poncirus trifoliata by suppression subtractive hybridization
复制标题
抑制消减杂交鉴定和表征枳壳低温胁迫响应基因
DOI：
10.1016/j.gene.2011.10.025
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发表时间：
2012-01-15
期刊：
影响因子：
3.5
通讯作者：
Liu, J. H.
中科院分区：
文献类型：
--
作者：
Peng, T.;Zhu, X. F.;Liu, J. H.
枳（Poncirus trifoliata（L）Raf.）当完全驯化时是非常寒冷的哈代，但是到目前为止，与驯化背后的分子事件相关的知识仍然有限。本研究以枳橙子为材料，构建了正向（4 ℃/25 ℃）和反向（25 ℃/4 ℃）抑制性消减杂交（SSH）文库，以鉴定与冷驯化相关的基因。经反向北方印迹分析和测序后，从正向和反向文库中分别获得了105个和117个非冗余差异表达序列标签（EST）。Blast2go分析表明，91个ESTs，31个来自正向文库，60个来自反向文库，与已知或推定功能的基因显示出显著的序列同源性。它们被分为不同的功能组，包括催化活性、结合蛋白、结构分子、酶调节剂、分子转换器、电子载体和转运活性/转录调节。用逆转录聚合酶链反应对筛选出的EST进行表达分析，结果与差异筛选结果一致。此外，这些基因的时程表达模式进一步证实了它们对低温处理有响应。在已知功能的基因中，许多与维持细胞壁完整性、调节渗透势和维持活性氧稳态有关，这意味着这些生理过程可能在提供抗低温胁迫的保护机制中具有重要意义。这些结果为进一步揭示枳橙子低温驯化过程中的分子变化规律提供了参考，也为进一步发掘具有潜在应用价值的基因工程候选基因，创造具有较强低温抗性的新种质提供了参考。(C)2011爱思唯尔有限公司版权所有。
Trifoliate orange (Poncirus trifoliata (L) Raf.) is extremely cold hardy when fully acclimated, but knowledge relevant to the molecular events underlying the acclimation is still limited so far. In this study, forward (4 degrees C over 25 degrees C) and reverse (25 degrees C over 4 degrees C) suppression subtractive hybridization (SSH) libraries were constructed in order to identify the genes involved in cold acclimation in trifoliate orange. After reverse northern blotting analysis and sequencing, a total of 105 and 117 non-redundant differentially expressed sequence tags (ESTs) were obtained from the forward and reverse libraries, respectively. Blast2go analysis revealed that 91 ESTs, 31 from the forward library and 60 from the reverse library, displayed significant sequence homology to the genes with known or putative functions. They were categorized into various functional groups, including catalytic activity, binding protein, structural molecule, enzyme regulator, molecular transducer, electron carrier, and transport activity/transcription regulation. Expression analysis of the selected ESTs by reverse transcriptase polymerase chain reaction was consistent with the results of differential screening. In addition, time-course expression patterns of the genes further confirmed that they were responsive to low temperature treatment. Among the genes of known functions, many are related to maintenance of cell wall integrity, adjustment of osmotic potential and maintenance of reactive oxygen species homeostasis, implying that these physiological processes might be of paramount significance in rendering protective mechanisms against the low temperature stress. The data presented here gain an insight into the molecular changes underlying the cold acclimation of trifoliate orange, and the results can be of reference for unraveling candidate genes that hold great potential for genetic engineering in an effort to create novel germplasms with enhanced cold stress tolerance. (C) 2011 Elsevier B.V. All rights reserved.