Peng Fred Y, Reid Karen E, Liao Nancy, Schlosser James, Lijavetzky Diego, Holt Robert, Martínez Zapater José M, Jones Steven, Marra Marco, Bohlmann Jörg, Lund Steven T
Wine Research Centre, Faculty of Land and Food Systems, University of British Columbia, 2205 East Mall, Vancouver, British Columbia, Canada V6T 1Z4.
Gene. 2007 Nov 1;402(1-2):40-50. doi: 10.1016/j.gene.2007.07.016. Epub 2007 Jul 31.
We report the generation and analysis of a total of 77,583 expressed sequence tags (ESTs) from two grapevine (Vitis vinifera L.) cultivars, Cabernet Sauvignon (wine grape) and Muscat Hamburg (table grape) with a focus on EST sequence quality and assembly optimization. The majority of the ESTs were derived from normalized cDNA libraries representing berry pericarp and seed developmental series, pooled non-berry tissues including root, flower, and leaf in Cabernet Sauvignon, and pooled tissues of berry, seed, and flower in Muscat Hamburg. EST and unigene sequence quality were determined by computational filtering coupled with small-scale contig reassembly, manual review, and BLAST analyses. EST assembly was optimized to better discriminate among closely related paralogs using two independent grape sequence sets, a previously published set of Vitis spp. gene families and our EST dataset derived from pooled leaf, flower, and root tissues of Cabernet Sauvignon. Sequence assembly within individual libraries indicated that those prepared from pooled tissues contributed the most to gene discovery. Annotations based upon searches against multiple databases including tomato and strawberry sequences helped to identify putative functions of ESTs and unigenes, particularly with respect to fleshy fruit development. Sequence comparison among the three wine grape libraries identified a number of genes preferentially expressed in the pericarp tissue, including transcription factors, receptor-like protein kinases, and hexose transporters. Gene ontology (GO) classification in the biological process aspect showed that GO categories corresponding to 'transport' and 'cell organization and biogenesis', which are associated with metabolite movement and cell wall structural changes during berry ripening, were higher in pericarp than in other tissues in the wine grape studied. The sequence data were used to characterize potential roles of new genes in berry development and composition.
我们报告了从两个葡萄（Vitis vinifera L.）品种赤霞珠（酿酒葡萄）和巨峰（鲜食葡萄）中总共生成并分析了77,583个表达序列标签（EST），重点是EST序列质量和组装优化。大多数EST来自标准化的cDNA文库，这些文库代表浆果果皮和种子发育系列、赤霞珠中包括根、花和叶的非浆果组织集合，以及巨峰中浆果、种子和花的组织集合。通过计算过滤结合小规模重叠群重新组装、人工检查和BLAST分析来确定EST和单基因序列质量。利用两个独立的葡萄序列集优化EST组装，以更好地区分密切相关的旁系同源物，一个是先前发表的葡萄属基因家族集，另一个是我们从赤霞珠的叶、花和根组织集合中获得的EST数据集。各个文库内的序列组装表明，从组织集合中制备的文库对基因发现贡献最大。基于对包括番茄和草莓序列在内的多个数据库的搜索进行注释，有助于确定EST和单基因的推定功能，特别是在肉质果实发育方面。对三个酿酒葡萄文库的序列比较鉴定出许多在果皮组织中优先表达的基因，包括转录因子、类受体蛋白激酶和己糖转运蛋白。生物学过程方面的基因本体（GO）分类显示，与“运输”和“细胞组织与生物发生”相对应的GO类别，与浆果成熟期间的代谢物移动和细胞壁结构变化相关，在所研究的酿酒葡萄的果皮中比在其他组织中更高。这些序列数据用于表征新基因在浆果发育和成分中的潜在作用。
