Isolation And Functional Analysis Of Tissue Specific Expression Promoters In Rice

Genetic engineering has been widely applied to agricultural crop improvement and has the potential to significantly increase crop production. An important component in genetic engineering is the use of different gene expression control elements that direct the expression of the introduced genes, and promoters are the most important one among the elements. The availability of tissue or organ specific promoters with diverse specificities would allow precise spatial control of transgene expression for the biotechnological improvement of crop plants and limit the unintended impacts on plant physiology. Since there are a few novel tissue or organ specific promoters can be used currently, isolation of some new tissue specific promoters is needed.Rice is an important food crop, utilizing novel promoters with reliable tissue specific expression to improve specific traits of rice has great economic benefits. Therefore, the target of this study is to isolate tissue specific promoters and analysis the functional characterization of the cloned promoters in order to provide a useful resource for the transgenic breeding in the future. The main achievements of this study are showed as follows:1. Based on microarray and RT-PCR data,3green tissue specific genes,1root specific gene and1endosperm specific gene were selected and analysed.5promoters corresponding to each gene named PYY2,PYY4, PYY7, PYY6and DXCP35were cloned from rice genomic DNA by PCR.2. Each promoter was fused to the upstream of gus reporter gene and transformed into rice respectively. The expression pattern of each promoter was identified by histochemical staining. The results verified that PYY2, PYY4, PYY7were green tissue specific promoters; DXCP35was an endosperm specific promoter; PYY6did not meet the predicted result, cannot drive gus gene expression in any tissues.3. DXCP35promoter was detailed study by5’end deletion analysis. Different length of5’end deletions of DXCP35were fused to the upstream of gus reporter gene and transformed into rice respectively. The result of deletion analysis indicated that the region between DXCP35-308and DXCP35-112was essential for maintain the promoter activity.4. PYY2promoter was detailed study by5’end deletion analysis. Different length of5’ end deletions of PYY2were fused to the upstream of gus reporter gene and transformed into rice respectively. Quantitative analysis of GUS activity indicated that592bp length promoter fragment can maintain the sheath and leaf specific expression model with the highest activity.