Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan.
BMC Plant Biol. 2011 Apr 7;11:60. doi: 10.1186/1471-2229-11-60.
Orchids comprise one of the largest families of flowering plants and generate commercially important flowers. However, model plants, such as Arabidopsis thaliana do not contain all plant genes, and agronomic and horticulturally important genera and species must be individually studied.
Several molecular biology tools were used to isolate flower-specific gene promoters from Oncidium 'Gower Ramsey' (Onc. GR). A cDNA library of reproductive tissues was used to construct a microarray in order to compare gene expression in flowers and leaves. Five genes were highly expressed in flower tissues, and the subcellular locations of the corresponding proteins were identified using lip transient transformation with fluorescent protein-fusion constructs. BAC clones of the 5 genes, together with 7 previously published flower- and reproductive growth-specific genes in Onc. GR, were identified for cloning of their promoter regions. Interestingly, 3 of the 5 novel flower-abundant genes were putative trypsin inhibitor (TI) genes (OnTI1, OnTI2 and OnTI3), which were tandemly duplicated in the same BAC clone. Their promoters were identified using transient GUS reporter gene transformation and stable A. thaliana transformation analyses.
By combining cDNA microarray, BAC library, and bombardment assay techniques, we successfully identified flower-directed orchid genes and promoters.
兰花是开花植物中最大的科之一，能产生具有商业价值的花卉。然而，拟南芥等模式植物并不包含所有植物基因，因此必须单独研究农业和园艺上重要的属和种。
我们使用了几种分子生物学工具，从“Gower Ramsey”蝴蝶兰（Onc. GR）中分离出花特异性基因启动子。利用生殖组织的 cDNA 文库构建了一个微阵列，以比较花和叶中的基因表达。有 5 个基因在花组织中高度表达，并用带有荧光蛋白融合构建体的脂瞬变法确定了相应蛋白的亚细胞定位。克隆了这 5 个基因以及 Onc. GR 中之前发表的 7 个与花和生殖生长特异性相关的基因的 BAC 克隆，以克隆其启动子区域。有趣的是，这 5 个新的花高丰度基因中的 3 个是假定的胰蛋白酶抑制剂（TI）基因（OnTI1、OnTI2 和 OnTI3），它们在同一个 BAC 克隆中串联重复。通过瞬时 GUS 报告基因转化和稳定的拟南芥转化分析鉴定了它们的启动子。
通过结合 cDNA 微阵列、BAC 文库和轰击分析技术，我们成功地鉴定了定向于花的兰花基因和启动子。
