Integration of molecular biology tools for identifying promoters and genes abundantly expressed in flowers of Oncidium Gower Ramsey

Abstract

Background: 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.

Results: 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.

Conclusions: By combining cDNA microarray, BAC library, and bombardment assay techniques, we successfully identified flower-directed orchid genes and promoters.

Figure 1

RT-PCR confirmed that genes identified by microarray were highly but variably expressed in reproductive organs according to the developmental stage and tissue. Total RNA was isolated from various organs (R, root; S, stalk; L, leaf; F, flower) during different developmental stages (green bud, showing color, expanding, full bloom), and from various parts of the flower (lip, callus, reproductive column, and sepal and petal). The genes included Oncidium Expansin (OnExpansin), Oncidium Disease Resistant Response Protein (OnDRRP) and Oncidium Trypsin inhibitor (OnTI1, OnTI2, and OnTI3). Each experiment was carried out in triplicate. Ubiquitin was used to measure the amount of RNA used for each RT-PCR reaction.

Figure 2

Alignment of amino acid sequences of OnTI1, OnTI2 and OnTI3. Comparison of the cDNA amino acid sequences of OnTI1, OnTI2 and OnTI3. Amino acids identical in all the proteins are presented in black; those conserved in at least 2 sequences are shaded.

Figure 3

Gene Structure of OnTI. Genes are marked by white boxes. Intergene spaces are denoted by a gray line. Introns are denoted by thin lines. The lengths of the exons, genes and intergene space (in base pairs) are indicated. Red, tandem repeat; orange, conserved regions in the OnTI promoters.

Figure 4

Characteristic features of organelle markers and subcellular location of proteins of flower-abundant genes in Onc. Gower Ramsey. A. Mitochondrial marker: the first 29 amino acids of yeast cytochrome c oxidase IV fused with RFP. B. Plastid marker: the targeting sequence (first 79 aa) of the small subunit of tobacco rubisco fused with GFP. C. CFP peroxisome marker: cytoplasmic tail and transmembrane domain of soybean 1, 2-mannosidase I fused with CFP. D. RFP plasma membrane marker: the full length of AtPIP2A, a plasma membrane aquaporin fused with RFP. E. YFP vacuole marker: γ-TIP, an aquaporin of the vacuolar membrane fused with YFP. F. Nuclear marker: NLS domain of VirD2 fused with mCherry. G. YFP: OnTI1: YFP fused with the N-terminus of OnTI1 protein. H. OnTI1::GFP + Mito-RFP: OnTI1::GFP and Mitochondria RFP marker were co-transformed to the cells. I. YFP::OnTI2: YFP fused with the N-terminus of OnTI1 protein. J. YFP::OnTI3: YFP fused with the N-terminus of OnTI3 protein. K. YFP::OnExpansin: YFP fused with the N-terminus of OnExpansin protein. L. YFP::OnDRRP: YFP fused with the N-terminus of OnDRRP protein.

Figure 5

Promoter study of OnTI1. Plasmids harboring various lengths of OnTI1 promoter fused with GUS were delivered to the lips and leaves of Oncidium Gower Ramsey. (A) The transformed tissues are stained to demonstrate GUS activity. The number on at the top is the length of the promoter. (B) Quantitative analysis of GUS activity. Orange boxes, the conserved regions II, III and IV of the OnTI promoter region. Oncidium alcohol acyl-transferase 500 bp promoter-GUS was used as the positive control, with the negative control being just the vector.

Figure 6

Promoter study of OnExpansin. Plasmids with various lengths of OnExpansin promoter fused with GUS were transformed into Arabidopsis thaliana (A) or delivered to the lips and leaves of Oncidium Gower Ramsey. (B). The number indicates the length of the promoter. The blue box denotes the putative floral-related transcription binding site.

Figure 7

Oncidium promoters that are highly expressed in Oncidium flowers. The Oncidium transient transformation study: clones with a high flower/leaf GUS activity ratio were transformed into Arabidopsis thaliana. The promoters included Oncidium Disease Resistant Response Protein (OnDRRP) and Oncidium Trypsin inhibitor (OnTI1 and OnTI2). The number indicates the length of the promoter.

Figure 8

Strategy for identification of BAC clones containing genes of interest. Genes of interest or partial Oncidium sequences were used for the BAC library screening. Primers were designed and genomic DNA PCR was performed before the screening. The products were sequenced to confirm results. After superpooling, plate PCR, row PCR and spot PCR, BAC clones containing genes of interest were identified. Black box, negative control; white box, genome DNA positive control with genomic DNA; green box, superpool control; red box, plate control; blue box, row control; yellow box, identified clone.