Recognize BnSP especially and get autophosphorylated, then phosphorylated SRK would phosphorylate ARC which could cause ubiquitination of its target proteins (e.g ExoA).An insertion of a DNA fragment having a length of bp in the promoter region of BnSP was accountable for the selfcompatibility (SC) of B.napus line “Westar” (Okamoto et al Tochigi et al).By complementing the function of BnSP in “Westar,” we obtained the transgenic line “W” which showed powerful SI (Gao et al).When pollen of “Westar” lands on its own stigma, compatible interaction happens and regular pods are set; when pollen of “W” lands around the stigma of “Westar,” selfincompatible reaction happens and pods seldom set seeds.They are excellent materials for us to shed further light around the complicated responses of compatibleincompatible pollenstigma interactions working with nextgeneration RNAseq coupled with a extensive timecourse experiment.Supplies AND Procedures Plant Material and Growth ConditionsThe wild sort selfcompatible B.napus line “Westar” and transgenic selfincompatible line “W” (Gao et al) have been grown in the greenhouse with h daynight photoperiod and temperatures of C.Floral buds of wild form selfcompatible B.napus line “Westar” had been emasculated day prior to anthesis to avoid pollen contamination.The next day, stigma samples had been collected by cutting the pistil just below the base of your stigma and immediately frozen in liquid nitrogen.By this system, stigma samples of “Westar” with no pollen pollinated (UP) and pollinated Technical Information stigmas (Pc, Computer, Pc, Pc, Pc selfpollinated stigmas of “Westar” at , , , , and min; PI, PI, PI, PI, PI stigmas of “Westar” pollinated with the incompatible pollen of “W” at , , , , and min) have been collected.Transmission Electron MicroscopyThe pollinated stigmas (PI and Pc) have been vacuuminfiltrated and prefixed inside a remedy of .glutaraldehyde adjusted to pH .with .M phosphate buffer, fixed in OsO in the very same buffer, and after that dehydrated and embedded in epoxyFrontiers in Plant Science www.frontiersin.orgMay Volume ArticleZhang et al.PollenStigma Interactions in Brassica napus L.resin and SPI (Structure Probe, Inc www.spi.com), respectively.Ultrathin sections were obtained employing a Leica UC ultramicrotome (www.leica.com) and have been stained with uranyl acetate and subsequently with lead citrate.The observations and recording of pictures had been performed using a Hitachi H transmission electron microscope (www.hitachihitec.com) at kV in addition to a Gatan CCD camera (www.gatan.com).cDNA synthesis was performed making use of a SuperScript kit (Gibco BRL, www.invitrogen.com).Realtime RTPCR was also performed applying a BioRad IQ with SYBR Green detection (www.biorad.com).Actin (GeneBank accession no. AF) was used as an internal manage to normalize transcript levels for all expression analyses.Supplemental Table S lists the specific primers employed to test the genes.cDNA Library Construction and SolexaIllumina SequencingTotal RNA was extracted using the DNARNA isolation kit (Qiagen).The good quality and quantity of purified RNA had been determined by measuring absorbance at nm nm (AA) making use of a SmartSpec plus (BioRad).In total, RNA samples (UP, Pc, Computer, Computer, Computer, Pc, PI, PI, PI, PI, and PI, every single with two biological replicates) have been subjected to library building using an Illumina R TruSeqTM RNA Sample Preparation Kit following the manufacturer’s instructions.All samples had been sequenced applying an Illumina HiSeq sequencer in the National Key PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21541725 Laboratory of Crop Genetic Improvement, Huazhong Agricultura.
