National Center for Gene Research, Chinese Academy of Sciences
Establishment of a Rice Mutant Library by T-DNA Insertion
Changyin Wu1, Wenya Yuan1, Guoxing Chen1, Xiangjun Li1, Dong Guo1, Jian Zhang1, Zhihui Chen1, Caishun Li1, Andrzej Kilian2, Juan Li1, Caiguo Xu1, Shiping Wang1 and Qifa Zhang1
1National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding, Huazhong Agricultural University, Wuhan 430070, China；2Center for the Application of Molecular Biology to International Agriculture, GPO Box 3200, Canberra, ACT 2601 Australia
Rice (Oryza sativa L.) is an important crop worldwide and, with the availability of the draft sequence, a useful model for analyzing the genome structure of grasses. T-DNA tagging is one of the widely-used methods for generation of insertion mutants for gene functional analysis in plants. As a part of our rice functional genomics project, a powerful enhancer trap system carrying a GAL4/VP16 transactivator and a UAS-GUSPlus reporter cassette was employed in a high efficiency Agrobacterium-mediated rice transformation system to generate a library. We have currently obtained more than 35,000 independent transgenic plants. The transformants carried on average 2.0 copies of the T-DNA and 42% of the transformants had single copy insertion in this mutant library. GUS assay of different organs revealed various patterns of the reporter gene expression. Continued screening for GUS activity in leaves, roots and flowers of T1 families confirmed the stable transmission of expression patterns from the T0 to T1 generation, indicating that UAS in rice was not as sensitive to methylation as in tobacco and Arabidopsis. The system GAL4/VP16-UAS-GUSPlus will provide for the first time a powerful tool for the targeted expression of transgenes in an important monocotyledonous crop. The plasmid rescue and Tail-PCR strategies were used to isolate the T-DNA flanking sequence. Analysis of those sequences from about 1400 transformants showed that almost all the sequences had homology with the sequence in the rice genome databases. We are carrying out phenotypic screening of more than 7000 T1 families to identify mutants generated by T-DNA insertion and have obtained some conspicuous morphological mutations. The testing of T-DNA co-segregated with mutant phenotype and isolation the corresponding gene are under way.
This work was supported by a grant from the National Program on High Technology Development of China, and a grant from the National Special Key Project on Functional Genomics and Biochip of China.
Recent Progress of the Rice Functional Genomics Program in China
High throughput isolation and characterization of regulatory genes involved in defense and stress responses in rice
Lizhong Xiong1 and Yinong Yang2
1 National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070 China; 2 Department of Plant Pathology, University of Arkansas, AR 72701, USA Abstract Identification of regulatory genes or signaling components in defense and stress responses is one of most critical steps leading to elucidation of plant defense and stress tolerance mechanism. To this end, a composite suppression subtractive hybridization (CSSH) strategy combined with inhibition of protein synthesis was used to isolate putative regulatory genes or immediate early (IE) genes in defense and stress responses in rice. Compared to traditional differential screening or cDNA microarray technologies, this method allowed us to isolate large number of new regulatory genes or IE genes related to defense and stress responses. A modified CSSH protocol combined with double strand RNA interference technique was developed to characterize these regulatory genes in a high throughput mean. Large overlapping was found for these regulatory genes for their functions in various disease resistance and abiotic stress responses as well as in multiple developmental processes, which was exemplified by functionally characterization of a mitogen-activated protein kinase (OsMAPK5) and a somatic embryogenesis receptor-like kinase (OsSERK1).
Soybean Transcription Factors Responsive to Stresses
1Plant Biotechnology Laboratory, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, PR China；2China Agricultural University
Transcription factors play roles in multiple aspects of plant growth, development and stress responses. Compared with approximate 20% functional genes responsive to stresses, how about transcription factors? In the present research, we cloned three DREB family genes in Soybean and found that two of three DREB genes were induced under drought and high-salinity stresses. We also studied the expression profiles of WRKY gene family in response to various stresses in Soybean. Search of the Soybean ESTs resulted in identification of ≈130 genes encoding proteins characteristic of WRKY DNA-binding transcription factors. We investigated the expression patterns of 60 Soybean WRKY genes with SA, cold, drought and high-salinity treatments using RT-PCR and found that 58%, 57%, 52%, 47% WRKY genes were induced or repressed under four stresses, respectively. Among the 60 WRKY genes, 7 showed little or no change and 4 were induced in all the four treatments. The expression patterns of genes in response to SA treatment were quite different that up to 40% genes were repressed, whereas only one 25% genes were repressed during cold stress and less than 2% genes were down-regulated under drought and high-salinity condition. Eleven SA-induced genes can be categorized into two groups. In one group containing 6 genes, the expression of 6 genes peaked at 0.5h or 1h and then decreased gradually. In another group, the 5 genes were induced slowly and gradually within 12h. The expression of 31 genes and 29 genes increased gradually under drought and high-salinity stresses respectively, indicating a strong correlation between the expression of WRKY genes and abiotic stress responses. In addition, sixty-seven per of the drought-inducible genes were also induced by high-salinity stress, suggesting a strong cross-talk between drought and high-salinity stress signaling processes. Among the 34 cold-responsive genes, 19 genes were induced and 15 genes were repressed. Nine genes were induced by cold, drought and high-salinity stresses. Nine cold-repressed genes were also repressed by SA treatment. These results demonstrate that the WRKY gene family involves in multiple stress responses of plants.