Rice (Oryza sativa) and rapeseed (Brassica napus) are important crops for food resource. The yield and quality of them directly effect human life and healthy. Our group provides strong support to the rice and rapeseed functional genomics studies, focusing on seed development. We hope to isolate and functional analyze genes determining important agri-traits. The rice and rapeseed mutant populations via EMS mutagenesis have been generated. Rice T-DNA insertion population has also been generated with detailed flanking sequences analysis. All the mutant information contributes to the establishment of a web-based database. The collection is also used to identify important rice genes involved in growth, development and responses to environmental stress on a genome-wide level. The expression profiling of transcription factors and receptor-like kinase during seed development were in-depth studied. Through large-scale sequencing, 8,369 uni-ESTs related to seed development were obtained and classified. The glass-based cDNA chip was then generated and the expression profiles of the relevant clones were studied. These studies not only give foundation for further gene research, but also supply precious resource for world scientific research in rice and rapeseed.
1.Generation mutant population of rice and rapeseed
T-DNA tagging in rice:Currently, a collection of rice mutant lines harbouring T-DNA insertion has been generated. Around 60,000 transgenic lines with T-DNA insertion have been obtained, in which 8,000 homozygous lines have been isolated. High-throughput thermal asymmetric interlaced (TAIL)-PCR has been set up to efficiently recover the flanking sequence of the T-DNA left borders. Now nearly 5,000 mutants are linked with the FST (flanking sequence tag) that can be located on the rice genome sequence. All the FST are analyzed in the bioinformatics pipeline against the TIGR Rice Pseudomolecules (virtual contigs). All information has been settled into the web based mutant database. (http://www.plantsignal.cn/ship).
Plants showing mutation related with flower and seed development are screened and studied. Screening for rice root growth mutants and plant hormone (Auxin, Brassinosteroid) response mutants has been performed. Using the information of the FST analysis, insertion mutants of rice transcription factors or auxin related have been isolated and planted in the field for further researches.
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Fig. A: Rice mutant population in field,
Fig. B: Database of rice mutants population
Fig. C: Rice 2003.985 mutant displays the extra Lemma and pelea
Fig. D: Rice IAA insensitive mutant
Fig. E: Rice mutants of flower and seed development
Rice and Rapeseed EMS mutant population: With the collaboration of Plant Research International (Netherlands), rice mutant population (around 60,000 lines) via EMS mutagenesis was generated and used for phenotype screening; Alternations of rice growth, including height, tillering, leaf development, floral development and seed development, were observed. Map-base cloning of mutation gene and further phenotype analysis were performed.
Rapeseed EMS mutant population (about 20,000 lines) was also generated for functional genomics. Now seed quality of total 6,000 mutant lines has been analyzed using the FOSS Near-Infrared system. We also have set up the TILLING (Targeting Induced Local Lesions IN Genomes) screening system based on the DHPLC technology for isolating the EMS mutants of the genes in the fatty acid metabolic pathway.
Rapeseed EMS mutant population in fields
2.Studies on transcription factors in rice development
Expression profiling during seed development: Transcription factors (TFs) are crucial factors for gene expression regulation in plant development. 325 genes encoding rice transcription factors from 12 families including Myb, Homeobox, Zinc-finger and so on were used for expression profiling analysis at 8 continuous seed developmental stages via cDNA chips technology. Cluster analysis based on the temporal expression patterns grouped them into 12 types, each of which contained members of various families showing common unique expression patterns. The results showed that many of the seed-preferential TFs were also involved in hormone and/or abiotic stress effects, suggesting the potential existence of uncharacterized transcriptional networks, or cross talk between hormone and abiotic stress signaling and seed development. Analysis on the cis-elements locating in promoter region of seed preferential TF genes suggested that Dof proteins play essential roles in hierarchical regulation of gene expressions during rice seed development. Several TF genes with specific expression pattern were further studied using the anti-sense transgenic rice.
(New insights into the complex and coordinated transcriptional regulation networks underlying rice seed development through cDNA chip-based analysis. Plant Molecular Biology. 2005)
Fig. upper: Different development stages of rice seed and embryo
Fig. bottom: Cluster analysis of the TF’s expression
Transcription proteins in rice seed development stages:
An efficient yeast-based system was developed for the isolation of plant cDNAs encoding transcription factors (TFs) and proteins with transcription activation functions (co-activators) in rice embryo development. The system is based on the trans-activation domain screening and ß-galactosidase (lacZ) reporter gene system; TFs during rice embryo development are isolated through this system. Approximately 200 confirmed positive colonies were obtained from screening 106 yeast colonies, including 75 independent cDNAs: 33 of which encoded plant Transcription activators belonging to different gene families. Expression pattern of selected TF-encoding genes was analyzed via RT-PCR. This work provides informative hints for further study of the regulatory mechanism of rice seed development, and illustrates an identification strategy that will be of practical value for the isolation of TFs and co-activators associated with specific plant developmental processes. (Development of an efficient method for the isolation of factors involved in gene transcription during rice embryo development.The Plant Journal. 2004)
Fig. left: Trans-activation system
Fig. right: Protein structure of TFs isolated via screening
3.Function of receptor like kinase in rice seed development
Receptor-like kinase (RLK) genes, which encoding an extracellular receptor domain and an intracellular kinase domain, are one of the most important gene families in plants. They take function in an extensive range from plant development to hormones or stresses response. We have obtained and analyzed the expression patterns of 179 RLKs during seed development and hormone and/or abiotic stress through cDNA chip. Our work focuses on the functions of RLKs during seed development. Transformed rice with sense/anti-sense construct and T-DNA insertion lines are used to further investigate the functions of these genes. Moreover, we are detecting the interactions between RLKs through a high-throughput yeast two hybrid method thus we may find out the mechanisms of this gene family.
Structure of receptor-like kinase in plant
4. Improvement of rapeseed seed quality and studies of the fatty acid metabolism pathway
Brassica napus is one of the main food-oil suppliers in the world. We started the functional genomics of rapeseed to study seed development. Through large-scale sequencing ESTs related of seed development, 8,369 uni-ESTs were obtained in total ~22,000 independent sequences. All these ESTs, including 3,355 full-length cDNA have been detailed annotated and classified comparing against the Arabidopsis protein sequences in NCBI database. SAGE analysis indicated the transcriptome scale of approximately 35,000 transcripts in the developing seeds. The glass-based cDNA chip was then generated and the expression profiles of the relevant clones were studied. We are mainly interested in the genes involved in fatty acid metabolism pathway. The functions of these genes will be further studied in the transgenic rapeseed to improve seed quality finally.
Classification of 8369 rapeseed ESTs