Isolation And Functional Analysis Of Root-Specific Promoters In Rice

Tissue-specific promoters activate the expression of exogenous genes only in the specific organs and tissues, which avoids the unnecessary waste of nutrition in plant and reduces the adverse effects on plant growth. Therefore, the use of such promoters for specific expression of exogenous genes in corresponding tissues can adapt more to the nature of rice growth and make the expression of foreign genes in rice more oriented, safe and efficient. Roots are the major organs for absorbing water and nutrients from outside the plants. Root-specific expression system can thus be used to study the problem such as the permeability of plant stress tolerance, plant repairing and rhizosphere secretion.In this study, RT-PCR was used to identify root-specific genes in rice. The upstream promoter reigon of the selected root-specific genes were isolated, cloned, and fused with the gus report gene. Then the plasmids containing the fusion constructs were transferred into japonica rice Zhonghua 11 by Agrobacterium-mediated transformation. GUS histochemical analysis and quantitative analysis were used to detect the expression pattern and level for each promoter. This research provides a useful resource for the future transgenic breeding.The main results are as follows:1. Based on our lab's database, gene expression pattern was analysised and 18 root-specific genes were selected, among which 6 genes were proved root-specific after RT-PCR identification.2. Promoter sequence of each gene was obtained by bioinformatics.4 promoters named P6, P9, P11, P12, with length of 1971 bp,1461 bp,1951 bp,1950 bp respectively, were obtained from MingHui 63 genomic DNA by PCR. The obtained promoters were cloned into DX2181b and fused with gus gene to construct expression vectors.3. Each expression vector was introduced into rice (Oryza sativa ssp. Japonica) by Agrobacterium-mediated transformation.4. After GUS histochemical analysis and quantitative analysis, two root-specific promoters (P9 and P12) were obtained. Moreover, P12 was temporally specific. It droves gus expression in young tissues, but not in mature tissues.5. Deletion analysis was used to analyze the P12 promoter. P12 promoter deleted at the 5'end with different length were fused to gus gene and transformed into rice respectively. Quantitative analysis of GUS activity showed that the expression level of gus gene in root was markedly reduced after deletion of the -957～-717bp region, which implied that there were some cis-activating elements in this region.