RESUMO
The activity of a green tissue-specific promoter of the Rubisco small subunit gene from Arabidopsis (AraSSU) was studied using transgenic chickpea lines. We generated transgenic chickpea lines expressing an AraSSU promoter-driven cry2Aa gene through the Agrobacterium-mediated transformation method. Lines with AraSSU expressed the gene in all green tissues at high levels (> 90 ng/mg of fresh weight tissue) compared to lines generated using CaMV35S (< 10 ng/mg FW). We used vertical cross sections of various tissues of homozygous progeny using microtome for immunolocalization. The immunolocalization showed the expression of the cry2Aa gene in the green mesophyll cells of the leaves of both AraSSU and CaMV35 chickpea lines. Moreover, the accumulation of AraSSU-regulated Cry2Aa protein was also observed in vascular tissues, including enucleate sieve elements and their companion cells. However, no expression was observed in the roots of AraSSU lines. In the case of CaMV35 lines, the transgene expression was observed in all the tissues. Since our data indicated that the AraSSU promoter is active in non-green tissues such as vascular bundles. Therefore, we validated this by RT-PCR. We found Cry2Aa RNA transcripts in leaves, stems without epidermis (for vascular tissues), and roots with and without epidermis. Thus, the AraSSU promoter is active in all above-ground tissues of the chickpea plant.
