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Team:Valencia UPV/Project/results/biosafety
From 2014.igem.org
Project > Results > Biosafety
Our goal was to develop a sterile and easily identifiable plant. In order to do this, we created a module in collaboration with NRP-UEA-Norwich which incorporated an RNAse (barnase) under the regulation of a tapetum specific promoter (TA29) and a chromoprotein
Male Sterility strategy was not possible to test in a transient approach, but both components are well documented (see Biosafety Module).
The NRP-UEA-Norwich provided us with blue and yellow chromoproteins transcriptional units (TU) and we agroinfiltrated both TUs in N. benthamiana along with a GFP control. Chromoprotein detection was impossible by the naked eye (Figure 1) even though GFP control was expressed (Figure 2). Plants agroinfiltration was correct since GFP was expressed in the leaves.
Figure 1. Plants agroinfiltrated with NRP-UEA-Norwich chromoproteins. TUs containing Blue (left) and Yellow (Right) chromoproteins were agroinfiltrated in N.benthamiana. None of the chromoproteins could be detected by the naked eye.
Figure 2. Agroinfiltration GFP control. Agroinfiltration control shows GFP expression, indicating that non-detection of chromoproteins is not due to a failure in agroinfiltration.
Nevertheless, identity preservation is an important part of the biosafety module, and it must be happen efficiently. NRP-UEA-Norwich team suggested leaf degreening in order to observe the chromoproteins, but we don’t consider this strategy convenient for our purpose since identity should be easily recognisable in plants without any kind of treatment. As an alternative to chromoproteins, we propose the use of two transcriptional factors to enhance anthocyanins production.
Our identity preservation construct consists of two transcriptional units carrying the tomato (Solanum lycopersicum) transcriptional factors (SlANT1, SlJAF13) which are regulated by the 35S constitutive promoter. Both transcriptional factors are involved in flavonoids biosynthetic pathway regulation; it is that they enhance anthocyanin accumulation.
SlANT1 and SlJAF13 are S. lycopersicum orthologous of the Antirrhinum majus Rosea1 and Delila genes. In previous researchs, ectopic over-expression of these transcription factors under the control of the E8 fruit-specific promoter increases the transcript levels of most of the anthocyanin biosynthetic genes in tomato fruit leading to high levels of anthocyanins [1,2]. In N. benthamiana, transient expression of SIANT1 and SIJAF3 activates the expression of several flavonoid biosynthetic genes leading to a change of the colour on the leaf due to accumulation of anthocyanins.
We transiently transformed this new identity preservation module (containing SIANT1 and SIJAF13 transcriptional units) by agroinfiltration into N. benthamiana. As result, the anthocyanin accumulation drives our plant to a violet colour change that can be observed by the naked eye (Figure 3).
Figure 3. Anthocyanins accumulation in N.benthamiana. SlANT1 and SlJAF13 expression produces accumulation of anthocyanins in agroinfiltrated plant (Left), showing a purple colour in contrast with a green wild type leaf (Right, held by hand).
