Project description:The leaf of Chinese cabbage is the major place of photosynthesis, the mutation of leaf may directly affect the rate of plant growth and development and the formation of leafy head, and ultimately influence the yield and quality of Chinese cabbage. We identified a developmentally retarded mutant (drm) exhibiting stable inheritance, which was derived from Chinese cabbage DH line âFTâ using a combination of isolated microspore culture and radiation treatment (60Co Î³-rays). The drm exhibited slow growth and development at the seedling and heading stages, leading to the production of a tiny, leafy head, as well as chlorophyll-deficient leaves, especially in seedlings. Genetic analysis indicated that the phenotype of drm was controlled by a single recessive nuclear gene. Compared with wild-type line âFTâ, the drmâs chlorophyll content was significantly reduced and its chloroplast structure was abnormal. Moreover, the photosynthetic efficiency and chlorophyll fluorescence parameters were significantly decreased. The changes in leaf color, combined with these altered physiological characters may influence the growth and development of plant, ultimately resulting in the developmentally retarded phenotype of drm. To further understand the molecular regulatory mechanisms of phenotypic differences between âFTâ and drm, comparative transcriptome analysis were performed using RNA-Seq, a total of 338 differentially expressed genes (DEGs) were detected between âFTâ and drm. According to GO and KEGG pathway analysis, a number of DEGs which involved in the chlorophyll degradation and photosynthesis were identified, such as chlorophyllase and ribulose-1,5-bisphosphate carboxylase/oxygenase. In addition, the expression patterns of 12 DEGs, including three chlorophyll degradation- and photosynthesis-related genes and nine randomly selected genes, were confirmed by qRT-PCR. Numerous single nucleotide polymorphisms were also identified, providing a valuable resource for research and molecular marker-assistant breeding in Chinese cabbage. These results contribute to our understanding of the molecular regulatory mechanisms underlying growth and development and lay the foundation for future genetic and functional genomics studies in Chinese cabbage. The RNA from the third true leaves (day 15 to day 24 after the appearance of the third true leaves) of a developmentally retarded mutant (drm) and its wild type âFTâ in Chinese cabbage were sequenced by RNA-Seq, in triplicate.

Project description:To understand signal transduction mechanism by MeJA in rice, we have analyzed transcription profile with 60K Rice Whole Genome Microarray after MeJA treatment. Gene transcripts were extracted from ten individual rice plants treated with 100 uM MeJA for 6 hrs. RNA samples from these plants were used to generate cyanine-3 (Cy3) and Cy5-labeled complementary DNA (cDNA) probes, which were then hybridized to the microarray. Each data set was obtained from three biological repeats independently. Preparation of fluorescence labeled probes and microarray hybridization was performed as procedures provided by Genisphere 3DNA Array Detection Array 50 kit (v. 2, Genisphere, Hatfield, PA). The microarray was scanned with Genepix 4000B (Axon Instruments, Union City, CA) and the quality of the chip data was analyzed with statistical R language and sma package in Bioconductor project (http://www.bioconductor.org/) implemented on Linux platform. Noncorrelation of signal and background intensities were confirmed by plotting base 2 log background intensity in x axis and base 2 log intensity subtracted with background intensity in y axis. Prior to normalization, normal distribution of Cy3 and Cy5 intensities were tested by qqplot function in R statistical language. The spatial effects on the chip during the hybridization process were checked with spatial func in sma package. The variance difference between Cy3 and Cy5 intensities within microarray was tested by the Student's t test under the assumptions firstly uniform and then nonuniform variances. The ANOVA difference of signal intensities between microarrays was performed by one-way ANOVA. Block-by-block Lowess normalization (Yang et al., 2002) and multivariate statistics such as clustering, principal component analysis, multidimensional scaling, etc. were analyzed with Acuity 3.1 (Axon Instruments). Spots with flag of 0 and a diameter greater than 51 pixel size were used for the analysis. Cy3 significant spots were determined when its log ratio is less than –0.67 [2**(–0.67) = 1.6-fold decrease] and its background subtracted intensity is higher than 500. On the contrary, Cy5 significant spots was determined when its log ratio is greater than 0.67 [2**0.67 = 1.6-fold increase] and its intensity is higher than 500. A preliminary microarray experiment using nontreated wild-type RNA labeled Cy3 and Cy5, respectively, gave less than 0.5% false positive signals in the analysis. To assess the reproducibility of the microarray analysis, we repeated the experiment three times with independently prepared total RNA

Project description:Two Chinese cabbage (Brassica rapa ssp. pekinensis) inbred lines, Chiifu (smaple A) and Kenshin (Smaple B), were grown for approximately 4 weeks in a growth chamber at 22degreesC under a 16 h light/8 h dark photoperiod with a photon flux density of 140 umol m-2 s-1. For microarray analysis, plants were exposed to a temperature of -4degreesC for 4 h, and shoots were sampled. Total RNA was isolated from the samples using an Easy-BLUETM Total RNA Extraction Kit (Invitrogen, NY, U.S.A.) and was then purified using an RNeasy MinEluteTM Cleanup Kit (Qiagen, Germany). Mircroarray experiments were carried out using Brassica 24K Oligo Microarray. RNA were prepared from each plant sample and 10 ug of total RNA were used for cDNA synthesis by using Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen, NY, U.S.A.). Following cDNA synthesis, remaining RNA was removed by the addition of RNaseA and the cDNA was precipitated following phenol/chloroform extraction. The cDNA pellet was rehydrated and used for Cy3-labelling. For the synthesis of Cy3-labeled target DNA fragments, 1 ug of double strand cDNA was mixed with 40 ul (1 OD) of Cy3-9 mer primers (Sigma-Aldrich) and the total volume was adjusted to 80 ul with deionized water. After heating at 98degreesC for 10 min, 10 uL of dNTP mix (10 mM each) and 2 ul of Klenow fragment (NEB; 50 units/ul) were added and the reaction was incubated at 37degreesC for 2 h. Finally, the reaction was stopped by the addition of 10 ul of 0.5 M EDTA. Labeled DNA fragments were precipitated with isopropanol and rehydrated with water. The concentration of each sample was determined using spectrophotometer. For hybridization, 13 ug of the labeled DNA fragment was mixed with hybridization buffer (NimbleGen) and then hybridized with the microarray using a MAUI hybridization chamber (Biomicro) at 42degreesC for 16 h. Following hybridization, the microarray was washed with wash solution I, II, and III (NimbleGen), and then dried in a dark desiccator. The microarray slide was scanned using GenePix scanner 4000B (Axon) and the spot intensities were analyzed using a NimbleScan (NimbleGen). The normal distribution of Cy3 intensities was tested by qqline. The data was normalized and processed with cubic spline normalization using quantiles to adjust signal variations between chips and with Rubust Multi-Chip Analysis (RMA) using a median polish algorithm implemented in NimbleScan software (NimbleGen). Two biological replicates were carried out.

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Transcription profiling of chinese cabbage exposed to prolonged cold

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