Isolation, Cloning and Characterization of Promoter of Rubisco Small Subunit 2B (rbcS2B) Gene of Arabidopsis thaliana
Neetu Singh Kushwah*
National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi–1100012, India
Present Address: Indian Institute of Pulses Research, Kanpur–842002, India
DOI: NIL
Keywords: Arabidopsis, GUS assay, rbcS2B gene, tissue specific promoter, light regulated elements
Abstract
Cloning and characterization of tissue-specific or developmentally-regulated promoters is of paramount importance for the development of transgenics and for basic studies requiring ectopic gene expression such as activation tagging. In this study, we characterized the expression patterns conferred by promoters of rbcS2B genes of Arabidopsis thaliana. Transgenic approach was followed to characterize the promoter of rbcS2B gene. In silico analysis of the 5′ upstream intergenic region of rbcS2B gene showed mainly light-regulated elements. A 1.8 kb fragment upstream to the coding sequence of rbcS2B gene was PCR amplified and cloned into the pORE-R2 vector upstream to the β-glucuronidase (GUS) reporter gene. Histochemical assay of stably transformed Arabidopsis plants for GUS expression indicated that AtrbcS2B promoter has wider expression pattern including in roots. qRT-PCR showed the presence of higher level of GUS transcripts than the endogenous AtrbcS2B in transgenic Arabidopsis harbouring pAtrbcS2B::GUS, indicating cloned upstream fragment (1.8 kb) of rbcS2B gene has stronger promoter activity than the native promoter. This strong promoter of AtrbcS2B will find application in activation tagging as well as in development of GM crops.
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