Department of Biochemistry and Molecular Biology, MS330, University of Nevada, Reno, NV 89557-0330, USA.
BMC Plant Biol. 2011 May 18;11:86. doi: 10.1186/1471-2229-11-86.
Abiotic stresses, such as water deficit and soil salinity, result in changes in physiology, nutrient use, and vegetative growth in vines, and ultimately, yield and flavor in berries of wine grape, Vitis vinifera L. Large-scale expressed sequence tags (ESTs) were generated, curated, and analyzed to identify major genetic determinants responsible for stress-adaptive responses. Although roots serve as the first site of perception and/or injury for many types of abiotic stress, EST sequencing in root tissues of wine grape exposed to abiotic stresses has been extremely limited to date. To overcome this limitation, large-scale EST sequencing was conducted from root tissues exposed to multiple abiotic stresses.
A total of 62,236 expressed sequence tags (ESTs) were generated from leaf, berry, and root tissues from vines subjected to abiotic stresses and compared with 32,286 ESTs sequenced from 20 public cDNA libraries. Curation to correct annotation errors, clustering and assembly of the berry and leaf ESTs with currently available V. vinifera full-length transcripts and ESTs yielded a total of 13,278 unique sequences, with 2302 singletons and 10,976 mapped to V. vinifera gene models. Of these, 739 transcripts were found to have significant differential expression in stressed leaves and berries including 250 genes not described previously as being abiotic stress responsive. In a second analysis of 16,452 ESTs from a normalized root cDNA library derived from roots exposed to multiple, short-term, abiotic stresses, 135 genes with root-enriched expression patterns were identified on the basis of their relative EST abundance in roots relative to other tissues.
The large-scale analysis of relative EST frequency counts among a diverse collection of 23 different cDNA libraries from leaf, berry, and root tissues of wine grape exposed to a variety of abiotic stress conditions revealed distinct, tissue-specific expression patterns, previously unrecognized stress-induced genes, and many novel genes with root-enriched mRNA expression for improving our understanding of root biology and manipulation of rootstock traits in wine grape. mRNA abundance estimates based on EST library-enriched expression patterns showed only modest correlations between microarray and quantitative, real-time reverse transcription-polymerase chain reaction (qRT-PCR) methods highlighting the need for deep-sequencing expression profiling methods.
非生物胁迫，如水分亏缺和土壤盐渍化，会导致葡萄生理、养分利用和营养生长发生变化，最终影响酿酒葡萄浆果的产量和风味。本研究大规模生成、整理和分析了表达序列标签（EST），以鉴定与胁迫适应性反应相关的主要遗传决定因素。尽管根系是许多类型非生物胁迫的第一个感知和/或损伤部位，但迄今为止，对暴露于非生物胁迫的酿酒葡萄根系组织的 EST 测序极为有限。为了克服这一限制，本研究对暴露于多种非生物胁迫的根系组织进行了大规模 EST 测序。
从受非生物胁迫的葡萄叶片、浆果和根系组织中获得了 62236 条 EST，与从 20 个公共 cDNA 文库中获得的 32286 条 EST 进行了比较。对 EST 进行了校正注释错误、聚类和组装，将浆果和叶片 EST 与现有葡萄全长转录本和 EST 进行了聚类和组装，共获得了 13278 个独特的序列，其中 2302 个是单序列，10976 个序列映射到葡萄基因模型。在受胁迫叶片和浆果中发现 739 个转录本具有显著差异表达，其中包括 250 个先前未被描述为非生物胁迫响应的基因。在对来自暴露于多种短期非生物胁迫的根系组织的标准化 cDNA 文库中 16452 个 EST 的第二次分析中，根据 EST 在根系中的相对丰度与其他组织相比，确定了 135 个根富集表达模式的基因。
对来自受各种非生物胁迫条件影响的叶片、浆果和根系组织的 23 个不同 cDNA 文库的相对 EST 频率计数进行大规模分析，揭示了独特的组织特异性表达模式、以前未被识别的胁迫诱导基因以及许多具有根富集 mRNA 表达的新基因，这有助于我们理解根系生物学并对酿酒葡萄的砧木特性进行调控。基于 EST 文库富集表达模式的 mRNA 丰度估计值与微阵列和定量实时逆转录聚合酶链反应（qRT-PCR）方法之间只有适度的相关性，这突出表明需要使用深度测序表达谱分析方法。
