Cloning And Functional Analysis Of Rubisco Activase Tissue-specific Promoter In Potato

Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) is an important enzyme to catalyze the first major step of carbon fixation. In vivo, Rubisco activase (RCA), a newly found enzyme recently, is essential for regulate the activity of Rubisco and keep it in a high activation level. RCA is not only had the ability of activating Rubisco, but also had the activity of hydrolysis of ATP. It is thought that Rubisco activase promotes the dissociation of ribulose bisphosphate and other inhibitory sugar phosphates from the Rubisco active site in a process requiring the hydrolysis of ATP. Transcription of rca gene is light-regulated and green tissue specific respectively. In this work, rca gene and its upstream cis-acting elements of potato have been cloned, followed by expression and promoter analysis, the results showed below.1. Isolation and sequence analysis of potato rca geneThe potato (Solanaum tuberosum) species "Atlantic" was used as a material to isolate rca gene by PCR and RACE technique. The cloned 1630bp full-length cDNA, contains open reading frame of 1614bp, encoding a protein of 537 amino acid residues. Alignment analysis of potato rca sequence with Arabidopsis, spinach and tomato indicated that the gene we cloned is high degree of similarity to those of Arabidopsis, spinach and tomato, which is 75% identical to Arabidopsis and spinach, 98% to the same family of tomato. The alignment of the deduced amino acid sequence of potato RCA with those three plants are 76%, 79% and 96%, respectively.2. Characterization of rca gene in tissue specific and light responsive expressionTo determine the expression of rca, Northern blotting analysis was performed using total RNA extracted from various light treatments of potato leaves. As a result, the expression of rca gene was light responsive and in a tissue specific manner. It has a positive correlation with light exposing time while cultivated in the same condition. From the accumulation of endogenous rca transcripts 8h, 16h and 24h of continuous light irradiation, the enhancing of rca expression was greater at 24h than at 8h and 16h, no band found in complete darkness treatment. On the other hand, the expression of rca gene in caudexes is far lower than that in leaves under a regular day/night cycle.3 Amplify and function analysis of rca upstream cis-acting elementsOn the basis of rca cds, parts of upstream sequence have been cloned by chromosome walking. For the identification of putative cis-acting elements in rca upstream sequence, PLACE database was used to find some CAAT box, TATA box, and G box. Gus assays of transgenic tobacco exhibited the driven function of this fragment.