Transcriptional Profile of Cotton Fiber Development

Stems, roots and the shoot (meristematic region) harvested from field grown DES119 cotton plants (2004) were frozen in liquid nitrogen and ground in liquid nitrogen in a Waring blender (Torrington, CT). Flowers from fields grown (2005) cotton plants (DES119 and ST4793R) were tagged with the date of anthesis and harvested 0 dpa, 1 dpa or 10 dpa. Fiber from 10 dpa ovules was dissected from the ovule, quickly frozen in liquid nitrogen and stored at -80°C. Polyribosomal RNA was isolated from 10 dpa fiber and 1 dpa ovules as described elsewhere [46 (link)-48 ]. Polyribosomal RNA was isolated from 1 dpa fiber by freezing freshly harvested 1 dpa ovules from 50 bolls in an excess of liquid nitrogen, adding about 0.1 g glass beads (Sigma, Atlanta, GA) and vortexing for 5 min. After the liquid nitrogen evaporated but before the sample warmed, 20 ml of the first buffer for polyribosomal RNA isolation was added and the intact ovules removed by filtering through cheese cloth. Free-polyribosomal RNA, membrane bound-polyribosomal RNA and total polyribosomal RNA was isolated as usual. Between 25 μg and 65 μg of total polyribosomal RNA was typically recovered. RNA quality was confirmed on a BioAnalyzer (Agilent, Palo Alto, CA).
RNA was separated on a 1.2% agarose gel (Phosphate buffer, pH6.5) and transferred to positively charged Nytran membrane (Roche, Alameda, CA) as described elsewhere [49 ]. The probe was amplified from the 3' end of the selected transcripts using the PCR DIG synthesis Kit (Roche). The blot was hybridized, rinsed and visualized following the instructions in the DIG Wash and Block Buffer Set (Roche). RT-PCR and Semiquantitative PCR were described in Taliercio and Kloth [50 ]. Primer sequences are presented in Table 1.