A study on the influence of different promoter and 5'UTR (URM) cassettes from Arabidopsis thaliana on the expression level of the reporter gene β glucuronidase in tobacco and cotton.
Several reports of promoters from plants, viral and artificial origin that confer high constitutive expression are known. Among these the CaMV 35S promoter is used extensively for transgene expression in plants. We identified candidate promoters from Arabidopsis based on their transcript levels (meta-analysis of available microarray control datasets) to test their activity in comparison to the CaMV 35S promoter. A set of 11 candidate genes were identified which showed high transcript levels in the aerial tissue (i.e. leaf, shoot, flower and stem). In the initial part of the study binary vectors were developed wherein the promoter and 5'UTR region of these candidate genes (Upstream Regulatory Module, URM) were cloned upstream to the reporter gene β glucuronidase (gus). The promoter strengths were tested in transformed callus of Nicotiana tabacum and Gossypium hirsutum. On the basis of the results obtained from the callus, the influence of the URM cassettes on transgene expression was tested in transgenic tobacco. The URM regions of the genes encoding a subunit of photosystem I (PHOTO) and geranyl geranyl reductase (GGR) in A. thaliana genome showed significantly high levels of GUS activity in comparison to the CaMV 35S promoter. Further, when the 5'UTRs of both the genes were placed downstream to the CaMV 35S promoter it led to a substantial increase in GUS activity in transgenic tobacco lines and cotton callus. The enhancement observed was even higher to that observed with the viral leader sequences like Ω and AMV, known translational enhancers. Our results indicate that the two URM cassettes or the 5'UTR regions of PHOTO and GGR when placed downstream to the CaMV 35S promoter can be used to drive high levels of transgene expression in dicotyledons.
Department of Genetics, University of Delhi, South Campus, Benito Juarez Road, New Delhi, 110021, India.
Parul
P
Agarwal
Varsha
V
Garg
Taru
T
Gautam
Beena
B
Pillai
Shaveta
S
Kanoria
Pradeep Kumar
PK
Burma
D020121:5' Untranslated Regions / Q000235:genetics*; D017360:Arabidopsis / Q000235:genetics*; D017796:Caulimovirus / Q000235:genetics; D003001:Cloning, Molecular; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism*; D003368:Gossypium / Q000201:enzymology* / Q000235:genetics; D046228:Microarray Analysis; D045331:Photosystem I Protein Complex / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D018709:Statistics, Nonparametric; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
D020121:5' Untranslated Regions; D045331:Photosystem I Protein Complex; D005966:Glucuronidase