Development of dual reporter vector system for estimating translational activity of regulatory elements

Abstract

Background: For the needs of modern biotechnology, a quantitative approach to the control of regulatory elements at all stages of gene expression has long become indispensable. Such a control regime is impossible without a quantitative analysis of the role of each regulatory element or pattern used. Therefore, it seems important to modify and develop the accuracy, reproducibility, and availability of methods for quantifying the contribution of each regulatory code to the implementation of genetic information.

Results: A new vector system for transient expression in plants is described; this system is intended for quantitative analysis of the contribution of regulatory elements to transcription and translation efficiencies. The proposed vector comprises two expression cassettes carrying reporter genes (of the Clostridium thermocellum thermostable lichenase and E. coli β-glucuronidase) under the control of different promoters. Herewith we also propose a new method for quantification of the effect of tested regulatory elements on expression, which relies on assessment of the enzyme activities of reporter proteins taking into account the transcription of their genes.

Conclusions: In our view, this approach makes it possible to precisely determine the amounts of reporter proteins and their transcripts at all stages of expression. The efficiency of the proposed system has been validated by the analysis of the roles of known translation enhancers at the stages of transcription and translation.

Keywords: High throughput screening; Lichenase; Regulatory elements; Transient expression; Vector; β-glucuronidase.

Fig. 1

Scheme of the experiment. The experiment consists of three main stages: construction of plant expression vectors bearing tested enhancers; transient expression in plants and analysis of the expression levels of the reporter genes with linnear regression

Fig. 2

Scheme of the pLAUMe vector. p19 – Silencing supressor from tombusviruses. TCTP – arabidopsis translationally controlled tumor protein promoter. en35SCaMV –enhanced 35S CaMV promoter. LicB – lichenase gene. pAct – arabidopsis actin promoter. uidA – gene of beta-glucuronidase

Fig. 3

Map of the pLAUMe vector. OCS – octopin synthase terminator. p19 – Silencing supressor from tombusviruses. TCTP – arabidopsis translationally controlled tumor protein promoter. en35SCaMV –enhanced 35S CaMV promoter. LicB – lichenase gene. pAct – arabidopsis actin promoter. uidA – gene of β-glucuronidase. T-Nos – nopaline synthase terminator. CBCI – castor bean catalase intron. Data are based on [9]

Fig. 4

Standart curve for β-glucuronidase. Along the abscissa – concentration of 4-Methylumbelliferyl- β-D-glucuronide, nM; along the ordinata – fluorescence intensity

Fig. 5

Standart curve for lichenase by enzyme. Along the abscissa – total amount of lichenase in the sample, ng; along the ordinata – fluorescence intensity

Fig. 6

Left pane: comparison of reporter gene expression under the control of tested enhancers at the transcription stage (absolute expression is indicated for both lichenase and glucuronidase); Right pane : at the translation stage. For the reporter proteins, relative expression is shown. Lichenase activity is indicated in ng/ μl of lichenase, β-glucuronidase activity – in nmol/ μl of 4-Methylumbelliferyl- β-D-glucuronide