Isolation, Functional Characterization And Application Of The Tissue Specific Expression Promoters From Rice

The growth and development of plant are controlled by specific expression of certain genes at appropriate times, in particular tissues and at appropriate abundance. Through interaction with transcription factors, promoters regulate gene expression under particular environmental conditions and at specific concentrations within cells or tissues. To study of the promoter's structure, billogical function and expression profile are very important for molecular biology theoretical research. The start point and ultimate goal for plant genetic engineering research is realization of the directional, stable and efficient expression of target gene in plants. In plant genetic engineering, using tissue-specific expression promoters to control the expression of target gene is an effective way to avoid potential negative effects of using constitutive promoter, such as metabolic burden and so on. However, until now, there is few tissue-specific promoters with strong and reliable expression that could be used in crop biotechnology application.Rice is one of the most important food crops in the world and an excellent model for genomic research in cereals. In this study, our aims are isolation and functional characterization of the tissue-specific expression promoters and the related cis-acting elements from rice, and use these promoters to development transgenic rice. Furthermore, the cis-acting elements will serve as useful tools in "promoter design" in future. The main results in this study are as follows:1. Constructed five novel vectors for promoter cloning and expression:DX2181, DX2181S, DX2181G, DX2181Smini46and DX2181Smini89.2. Based on microarray and RT-PCR data, we identified a rice endosperm specific expression gene En2, The expression pattern of En2gene promoter (EnP2) was examined by using the GUS reporter gene and analyzed in transgenic rice plants in different tissues. The result verified that EnP2is an endosperm specific expression promoter.5'end deletion analyses indicate that,155bp length promoter fragment (EnP2-155) is sufficient for maintain the endosperm specific expression model.3. Based on microarray and RT-PCR data, we identified a rice endosperm specific expression gene En3, The expression pattern of En3gene promoter (EnP3) was examined by using the GUS reporter gene and analyzed in transgenic rice plants in different tissues. The result verified that EnP3is an endosperm specific expression promoter.5' end deletion analyses indicate that,126bp length region (from EnP3-292to EnP3-166) is essential for maintain the endosperm specific expression model. Site-directed mutagenesis analysis found that two c/s-elements (2SSEEDPROTBANAPA and GCN4-like motif) were function as negative cis-regulatory elements in the regulation of green tissue-specific gene expressioa4.Based on microarray and RT-PCR data, we identified a rice green tissue specific expression gene DX1, The expression pattern of DX1gene promoter (PDX1) was examined by using the GUS reporter gene and analyzed in transgenic rice plants in different tissues. The result verified that Pdxi is a green tissue specific expression promoter.5' end and3' end deletion analyses revealed two positive regulatory regions (-195to-22and+1to+86) were responsible for the basal activity of Pdxi, two novel tissue-specific cis-acting elements (GSE1and GSE2) were identified in these two region by electrophoretic mobility shift assay. Site-directed mutagenesis analysis found that, GSE1functions as a positive regulator in all green tissues (leaf, sheath, stem and panicle), and the regulation effect is strong. Compared with GSE1, GSE2functions as a rather weak positive regulator, but is specific in sheath and stem tissues.5.The resistant gene, cry1C*, driven by the rice rbcS promoter was introduced into rice Zhonghual1by Agrobacterium-mediated transformation. By combining southern blotting analysis, homozygous line selective, field tests for insect resistance, agronomic performance investigation and CrylC*protein quantification, an elite (single copy insertion, high resistance to lepidopteran pests, normal agronomic performance and CrylC*-free endosperm) transgenic line RJ5was selected. All results indicated that RJ5has the potential for widespread utility in rice production.