佛手低温胁迫相关基因的差异表达
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浙江师范大学 化学与生命科学学院,浙江师范大学 化学与生命科学学院,浙江师范大学 化学与生命科学学院,浙江师范大学 化学与生命科学学院,浙江师范大学 化学与生命科学学院,浙江师范大学 化学与生命科学学院

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浙江省自然科学基金(Y307472);浙江省科技计划项目(2008C22002)


Analysis of cold-regulated gene expression of the Fingered Citron(Citrus medica L. var. sarcodactylis Swingle)
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College of Chemistry and Life Science, Zhejiang Normal University,,,,,College of Chemistry and Life Science, Zhejiang Normal University

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    摘要:

    佛手(Citrus medica L. var. sarcodactylis Swingle)是药用及观赏价值都很高的经济植物,但它对低温反应敏感,容易发生冻害现象。因此,了解其冷敏感机制对提高抗寒性起着重要的作用。以佛手为试材,-4℃ 低温处理24 h后,采用mRNA差异显示技术(DDRT-PCR)和半定量RT-PCR技术,分析和鉴定与冷敏感相关的差异表达基因。DDRT结果获得差异片段121个,经生物信息学分析,差异表达序列中有33条为功能已知序列,88条为未知序列(其中5条具有开放性阅读框);半定量结果获得34个阳性基因片段,其中上调基因片段29个,下调基因片段5个。除了3个基因功能未知外,其余基因主要涉及植物防御/应激反应,细胞壁的修饰,信号转导,代谢,氨基酸转运,氧化损伤,转录和蛋白质的合成,其中与植物防御/应激反应和光合作用有关的基因可能是造成佛手寒敏感的主要原因。

    Abstract:

    Low temperature is one of the most common environmental stresses causing severe economic losses of most crops. Plants adapation to low temperature is mediated by differential gene expression, Investigation of the transcriptome profiling stressed by low temperature is important to unveil the mechanism of plant adapation to the stress. Multiple mechanisms are involved this process including differential expression of dehydrins and antifreeze proteins, chaperones and detoxification enzymes, as well as variations of biosynthetic enzymes producing the low molecular weight compounds. Regulatory proteins such as transcription factors, protein kinases and phospholipases are known to be differentially regulated under cold stress. However, few literatures were available on gene express correlated with cold-sensitivity in fingered citron.
    Fingered citron(Citrus medica L. var. sarcodactylis Swingle)has high values due to its medicinal properties and ornamental potential. However, high sensitivity of fingered citron to low temperature resulted in significant reduction of its production. Fingered citron's maximum freezing tolerance, considered as the semilethal temperatures (LT50), is-4℃, and 24 h was considered as a critical period for cold stress. It is thus important to understand possible mechanisms involved in cold sensitivity of this plants species, as to promote its tolerance of cold stress.
    In the present study, the mRNA differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) was used to analyze gene expression patterns involved in the cold sensitivity of Fingered Citrons. The plants were subjected to low temperature (-4℃) for 24 h, and alterations of gene expression patterns were analyzed. One hundred and twenty-one putative differentially expressed DNA fragments were cloned, sequenced, and analyzed. Thirty-eight genes with changed transcript levels were left after the exclusion of repeated sequences, 33 of which are genes of known functions and 5 unknown through BLAST analyses. Expression analysis by semiquantitative RT-PCR (sqRT-PCR) showed that twenty-nine genes were up-regulated and five genes were down-regulated under cold stress. No significant variation of remaining four genes were observed,and considered to be false positives.
    Among the 34 differentially expressed fragments, eight genes were related to plant defense/stress responses, including hepicidin-like precursor, DNA-damage-repair/toleration protein (DRT), 26S proteasome regulatory subunit, Retrotransposon-like protein, Avirulence protein (Avr), Ac-like transposase protein, Rhodanese domain protein and β-1,3-glucanase; Eight of nine metabolism-related genes are photosynthesis-related genes includes Abl interactor-like protein-2、3, cytochrome f, P700, plastocyanin-like protein, γ-type carbonic anhydrase protein, ATP synthase CF0 subunit I, Chlorophyllase-1 and phototropins; and 17 other genes are associated with cell wall modification, signal transduction, metabolism, amino acid transport, oxidative damage, transcription and protein synthesis. These data provide a new insight into molecular mechanisms underlying cold regulation of plant defense/stress responses and photosynthesis in Fingered Citrons, which is important to improve the cold tolerance of the citrus species.

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陈文荣,叶杰君,李永强,张真真,曹诣斌,郭卫东.佛手低温胁迫相关基因的差异表达.生态学报,2013,33(5):1594~1606

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