|Public on Sep 08, 2008
|Arabidopsis growing pollen tubes rep2
|Growing pollen tubes
|Ecotype: Arabidopsis thaliana (Columbia)
Age: 50 days
Tissue: growing pollen tubes
|Arabidopsis thaliana (ecotype Columbia) plants were grown in mixed soil in a growth chamber. The light intensity was 120-150 umol m-2 s-1 for a 16 h daily light period and day/night temperatures were 22℃±2℃ and 18℃±2℃, respectively. Plants were watered once every 5 days with tap water and the relative humidity in the growth chamber was kept near 70%.
|The total RNA was extracted using the TRIzol reagent (Invitrogen). RNA concentration, purity and integrity were determined and confirmed by using an Eppendorf Biophotometer (Eppendorf) and electrophoresis. The total RNA was further purified using RNeasy Mini Kit (Qiagen).
|Eight ug of purified total RNA was used to generate first-strand cDNA in a reverse transcription reaction (One-Cycle Target Labeling and Control Reagents, Affymetrix). After second-strand synthesis, the double-stranded cDNA were used to generate cRNA via in vitro transcriptional reaction. The cRNA was labeled with biotin and 20 ug of this cRNA was fragmented. Size distribution of the cRNA and fragmented cRNA was assessed using an Eppendorf Biophotometer (Eppendorf) and electrophoresis.
|Fifteen ug of fragmented cRNA was added in 300 ul hybridization solutions and 200 ul of this mixture was used for hybridization on Arabidopsis ATH1 Genome Arrays for 16 h at 45 ℃. The standard wash and double-stain protocols (EukGE-WS2v5-450) were applied using an Affymetrix GeneChip Fluidics Station 450.
|The arrays were scanned on an Affymetrix GeneChip scanner 3000. The scanned arrays were analyzed with Affymetrix GCOS 1.0 (MAS 5.0) software to generate both the CEL and CHP files.
|In order to get enough pollen tubes, the “thin liquid layer” germination methods were developed. First, the freshly anther-dehisced flowers were collected into 1.5 ml microfuge tubes filled with 800 ul of basic medium (the basic medium was composed of 1 mM KCl, 5 mM CaCl2, 0.8 mM MgSO4, 1.5 mM boric acid, 15% (w/v) sucrose, 0.05% (w/v) lactalbumin hgdrolysate, 10 uM myo-inositol, 5 mM MES, and the pH was adjusted to 5.8 with Tris). The microfuge tubes were strongly vortexed for 2 min to release the mature pollen grains into the medium. The mixture was then transferred into new microfuge tubes and centrifuged at 11,000 rpm for 1 min. The pollen pellet was resuspended with 30 ul of the basic medium and subsequently cultured in the Petri dishes. It was noticed that making a well-spreaded thin layer of pollen grains is a key point to get high rates (70%-75%) of pollen germination. In order to well spread the tiny drop of pollen grains on the Petri dish (35 mm diameter) surface, a steel-wire net (D=80 um) was placed to form a thin liquid layer. Then, the covered Petri dishes were transferred to a chamber and incubated at 25℃. For the collection of the pollen tube samples, the pollen grains were incubated for 4 h, which resulted in an averaged pollen tube length about 150 um. After removing the steel-wire net, the culture mixture was collected and filtered through 50 um nylon mesh to remove the ungerminated pollen grains. The pollen tube samples were collected from the nylon mesh for total RNA extraction.
|To ensure the reproducibility and reliability, two biological replicates were conducted and the RNAs that were used for microarray experiments were extracted from two independent plant populations. The scanned arrays were analyzed first with Affymetrix GCOS 1.0 (MAS 5.0) software to generate detection calls. All the 6 datasets (3 different samples from two biological replicates) were normalized using Affymetrix GCOS software, and the TGT value was set to 100.
|Jan 10, 2007
|Last update date
|Aug 28, 2018
|China Agricultural University
|College of Biological Sciences
|Wu Wei-Hua's Lab
|No.2 Yuanmingyuan West Road
|Transcriptome analyses show changes in gene expression to accompany pollen germination and tube growth in Arabidopsis