Eveland Andrea L, McCarty Donald R, Koch Karen E
Department of Horticultural Sciences, Plant Molecular and Cellular Biology Program, Genetics Institute, University of Florida, Gainesville, FL 32611, USA.
Plant Physiol. 2008 Jan;146(1):32-44. doi: 10.1104/pp.107.108597. Epub 2007 Nov 16.
Differences in gene expression underlie central questions in plant biology extending from gene function to evolutionary mechanisms and quantitative traits. However, resolving expression of closely related genes (e.g. alleles and gene family members) is challenging on a genome-wide scale due to extensive sequence similarity and frequently incomplete genome sequence data. We present a new expression-profiling strategy that utilizes long-read, high-throughput sequencing to capture the information-rich 3'-untranslated region (UTR) of messenger RNAs (mRNAs). Resulting sequences resolve gene-specific transcripts independent of a sequenced genome. Analysis of approximately 229,000 3'-anchored sequences from maize (Zea mays) ovaries identified 14,822 unique transcripts represented by at least two sequence reads. Total RNA from ovaries of drought-stressed wild-type and viviparous-1 mutant plants was used to construct a multiplex cDNA library. Each sample was labeled by incorporating one of 16 unique three-base key codes into the 3'-cDNA fragments, and combined samples were sequenced using a GS 20 454 instrument. Transcript abundance was quantified by frequency of sequences identifying each unique mRNA. At least 202 unique transcripts showed highly significant differences in abundance between wild-type and mutant samples. For a subset of mRNAs, quantitative differences were validated by real-time reverse transcription-polymerase chain reaction. The 3'-UTR profile resolved 12 unique cellulose synthase (CesA) transcripts in maize ovaries and identified previously uncharacterized members of a histone H1 gene family. In addition, this method resolved nearly identical paralogs, as illustrated by two auxin-repressed, dormancy-associated (Arda) transcripts, which showed reciprocal mRNA abundance in wild-type and mutant samples. Our results demonstrate the potential of 3'-UTR profiling for resolving gene- and allele-specific transcripts.
基因表达的差异构成了植物生物学核心问题的基础，这些问题涵盖了从基因功能到进化机制以及数量性状等方面。然而，由于广泛的序列相似性以及基因组序列数据常常不完整，在全基因组范围内解析密切相关基因（如等位基因和基因家族成员）的表达具有挑战性。我们提出了一种新的表达谱分析策略，该策略利用长读长、高通量测序来捕获信使核糖核酸（mRNA）信息丰富的3'非翻译区（UTR）。所得序列能够独立于已测序基因组解析基因特异性转录本。对来自玉米（Zea mays）卵巢的约229,000个3'锚定序列进行分析，鉴定出至少由两个序列读数代表的14,822个独特转录本。利用干旱胁迫的野生型和胎萌-1突变体植物卵巢的总RNA构建多重cDNA文库。通过将16种独特的三碱基关键编码之一掺入3'-cDNA片段对每个样品进行标记，并使用GS 20 454仪器对混合样品进行测序。通过识别每个独特mRNA的序列频率来定量转录本丰度。至少202个独特转录本在野生型和突变体样品之间表现出丰度上的高度显著差异。对于一部分mRNA，通过实时逆转录-聚合酶链反应验证了定量差异。3'-UTR图谱解析了玉米卵巢中的12个独特的纤维素合酶（CesA）转录本，并鉴定出一个组蛋白H1基因家族中以前未表征的成员。此外，这种方法解析了几乎相同的旁系同源物，如两个生长素抑制、休眠相关（Arda）转录本所示，它们在野生型和突变体样品中显示出相反的mRNA丰度。我们的结果证明了3'-UTR图谱在解析基因和等位基因特异性转录本方面的潜力。
