Cloning And Preliminary Functional Analysis Of Rubisco Activase Gene OjRCA Of Ophiopogon Japonicus.

Ophiopogon japonicus is an important ground cover plant with high drought, salt tolerant. Because of its extremely shade-tolerance, and the long green period, Ophiopogon japonicus has been widely used in greening.Therefore, research on the genetic resources of Ophiopogon japonicus, in particular, photosynthetic system genes has significant value. Ribulose 1,5-bisphosphate carboxylase/ oxygenase (Rubisco) is a key enzyme in photosynthesis. The activity level of Rubisco has an important influence on the photosynthesis of plants. Rubisco must be activated by an enzyme of Rubisco activase(RCA). Therefore, the study of the function of RCA which would enhance the activity of Rubisco may play an important role in improving the resilience of plants.In this study, PCR is used to clone the full-length cDNA sequence of RCA gene. The length of the cDNA sequence was 1633bp, the 5'UTR contains 41 bp and the 3'UTR contains 284 bp. A complete open reading frame was located between nucleotide 42-1349, which included 1308bp, encoding a deduced protein of 435-amino-acids. The multiple sequence alignment result of the RCA amino acid sequence derived by Dnaman4.0 revealed that there has an up to 77% homelogy among the amino acid sequence of Arabidopsis and other species. The study also cloned partial sequence of Ophiopogon japonicus Rubisco large subunit (rbcL) gene. Analysis showed that the amino acid sequence of Ophiopogon japonicus has an up to 98% homology with tobacco and other species' rbcL gene, indicating that OjRCA may activate rbcL from other species.In this paper, the vector pBI121 was used to construct the plant expression vector of OjRCA gene, named pBI121-OjRCA. The pBI121-OjRCA was transferred into Agrobacterium tumefaciens strain EHA105 by freeze-thaw method, then the RCA gene was transformed into tobacco successfully. The antibiotic selection and PCR molecular detection showed than seven transgenic lines were obtained for studing the function of the RCA gene.The RCA gene expression patterns under drought and salt stress was also analysed by RT-PCT in this test. Preliminary findings showed both drought and salt can affect gene expression of RCA, but their variations are different. The change of the RCA gene expression patterns indicate that RCA gene may play an important role in drought and salt stress. This has great value for the following-up research such as the relationship between the gene's functional and plants stress tolerance improvement.