Biologia plantarum 50:352-358, 2006 | DOI: 10.1007/s10535-006-0049-6

Promoterless gus gene shows leaky β-glucuronidase activity during transformation of tomato with bspA gene for drought tolerance

R. Roy1, R. S. Purty1, V. Agrawal1, S. C. Gupta1,2,*
1 Department of Botany, University of Delhi, Delhi, India
2 Dr. B.R. Ambedkar Centre for Biomedical Research, University of Delhi, Delhi, India

Transformation of tomato (Lycopersicon esculentum Mill.) was carried out using disarmed Agrobacterium tumefaciens strain EHA 105 harboring a binary vector pBIG-HYG-bspA. The plasmid contains the bspA (boiling stable protein of aspen) gene under the control of a CaMV35S promoter and nopaline synthase (NOS) terminator, hygromycin phosphotransferase gene (hpt) driven by nopaline synthase promoter and polyadenylation signal of Agrobacterium gene7 as terminator and a promoterless gus gene. Very strong β-glucuronidase (GUS) expression was observed in transformed tomato plants but never in non-transformed (control). Since GUS expression was observed only in transformed plants, the possibility of the presence of endogenous GUS enzymes was ruled out. Possibility of false GUS positives was also ruled out because the GUS positive explants reacted positively to polymerase chain reaction (PCR) and PCR-Southern tests carried out for the presence of bspA gene, which indicated the integration of T-DNA in tomato genome. The promoterless GUS expression was hypothesized either due to leaky NOS termination signal of bspA gene or due to different cryptic promoters of plant origin. It was concluded that GUS expression was observed in the putative transgenics either due to the read through mechanism by the strong CaMV35S promoter or due to several cryptic promoters driving the gus gene in different transgenic lines.

Keywords: cryptic promoter; leaky GUS expression; Lycopersicon esculentum
Subjects: Agrobacterium tumefaciens; cryptic promoter; drought stress; β-glucuronidase; Lycopersicon esculentum; nutrient medium, Murashige and Skoog; polymerase chain reaction (PCR); Southern blot analysis

Received: November 12, 2004; Accepted: April 1, 2005; Published: September 1, 2006Show citation

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Roy, R., Purty, R.S., Agrawal, V., & Gupta, S.C. (2006). Promoterless gus gene shows leaky β-glucuronidase activity during transformation of tomato with bspA gene for drought tolerance. Biologia plantarum, 50(3), 352-358. doi: 10.1007/s10535-006-0049-6.
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