Functional Analysis Of Rubisco Activase Promoter In Potato

1,5-bisphosphate carboxylaxe/oxygenase (Rubisco) activase (RCA) is a nuclear-encoded soluble chloroplast protein, it is essential for the activation of Rubisco and hydrolysis of ATP. Reports showed that RCA gene expression is tissue-specific and light-responsive in maize, wheat, spinach, cowpea, tobacco and Arabidopsis. In this study, the cloned 5′upstream sequence of potato RCA gene (StRCA) was used as a template. A series of progressively truncated fragments from within 5′upstream of StRCA gene were amplified and fused to uidA gene, followed by construction of plant express vectors and Agrobacterium-mediated transformation of tobacco. Gene expressions were characterized by Northern blot, GUS histochemical and quantitative assays. Results showed as follows:1. StRCA 5′upstream sequence contains multiple cis-acting elements, such as CAAT box, ATCT, Box I, I-box, GT1-motif associated with light responsive, ethylene-induced ERE.2. Full-length StRCAp731 and progressively truncated fragments, which stand for StRCAp498,StRCAp249,StRCAp175,StRCAp149and StRCAp98, were fused to the uidA gene, followed by construction of plant binary vectors. Tobacco transformants were obtained by Agrobacterium-mediated transformation method and Tansformants were identified by PCR and Southern hybridization. Histochemical GUS assay showed that, the reporter gene expressed in stems and leaves in StRCAp731 transformats seedlings. GUS stain was observed only in leaves of 5'-deletion StRCAp498and StRCAp249.3. Quantitative assay of GUS activity demonstrated that StRCAp731, StRCAp498 and StRCAp249 tobacco transformats were 60%, 22% and 12% as those of CaMV35S, respectively.Northern blot was performed using total RNA extraction from various light treatments of tobacco leaves. As a result, the expression of gus gene fused with StRCAp731 was light responsive. It has a positive correlation with light exposing time. The enhancing of gus expression got the peak for 24hr treatment, and no band found in complete darkness treatment. StRCAp498 and StRCAp249 showed the same results. -249bp of the StRCA promoter is sufficient to confer light responsive and gene expression.