tissue: anthers of middle buds development stage: from meiosis to the early-uninucleate-microspore stage
Treatment protocol
The experimental plot contained about 2400 plants grown in 120 rows (2 m long each) at a density of 50 cm space between rows and 10 cm between plants within a row. When the rapeseed plants were at the bolting stage with the longest floral bud≤2mm, the plot was divided into two groups, the Mes-treated group and Mock-treated group, each containing 60 rows. Mes-treated group plants were foliar sprayed with 0.1μg mL-1 Mes solution containing 50ppm DMF and 5ppm Tween 80 for about 15 ml per plant (about 1% of the concentration that required for it acting as a herbicide in wheat fields to control broadleaf weeds) to induce male sterility during the entire flowering period without affecting the growth and development of other tissues of rapeseed plants. Meanwhile, Mock-treated group plants were foliar sprayed with the same amount of solution only containing 50ppm DMF and 5ppm Tween 80 as control.
Growth protocol
The rapeseed cultivar “Zhongshuang No. 9”, developed by the Oil Crops Research Institute of Chinese Academy of Agricultural Sciences (Wuhan) and selfed for eight generations, was planted in the experimental field of Northwest A&F University, Yangling, Shaanxi, China (longitude 108°E , latitude 34°15’N), during a natural growth season in 2009 to 2010.
Extracted molecule
total RNA
Extraction protocol
Total RNAs from four different tissues of Mes-treated and Mock-treated plants were extracted using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, US) and purified with QIAGEN RNeasy® Mini Kit (QIAGEN, GmBH, Germany).
Label
Cy3
Label protocol
Total RNA was amplified and labeled by Low RNA Input Linear Amplification kit (Cat#5184-3523, Agilent technologies, Santa Clara, CA, US), 5-(3-aminoallyl)-UTP (Cat#AM8436, Ambion, Austin, TX,US), Cy3 NHS ester (Cat#PA13105,GE healthcare Biosciences, Pittsburgh, PA,US) followed the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany).
Hybridization protocol
Each Slide was hybridized with 1.65μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat#5188-5242, Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (Cat#G2545A, Agilent technologies, Santa Clara, CA, US), according to the manufacturer’s instructions. After 17 hours hybridization, slides were washed in staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit(Cat#5188-5327, Agilent technologies, Santa Clara, CA, US), Stabilization and Drying Solution (Cat#5185-5979, Agilent technologies, Santa Clara, CA, US)followed the manufacturer’s instructions。
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Cat#G2565BA, Agilent technologies, Santa Clara, CA, US) and Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US) with default settings，Scan resolution=5μm, PMT 100%，10%. Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
Description
Gene expression in anthers of middle buds treated with Mes
Data processing
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) and normalized and log2 transformed using Quantile algorithm using Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
