Expression of active beta-glucuronidases enzyme in tobacco plant seeds
Volume 8, Issue 3, Winter 2018, Pages 231-241
https://doi.org/10.52547/JCT.8.3.231
Kh Bagheri, B Maleki Zanjani, M Mekanik
Abstract -
Aim: The purpose of this research was to design and prepare a suitable gene construct and transfer it to the tobacco plant and analysis of transgenic plants.
Material and methods: Seed-specific construct that containing Napin promoter, Ω sequence, GUS gene, and SAR sequence was prepared in pBI121 plasmid and proliferated in E.coli.. Then tobacco leaf explants were inoculated with LBA4404 agrobacterium strain by standard protocol. Selection of regenerated shoots also were performed in a selection media (co-culture medias + 25 mg/L Kan + 200 mg/L Cef). Transgenic plants were analyzed by PCR, RT-PCR and histochemical assay.
Results: Analysis of regenerated plantlets by using PCR and specific primers of nptII and GUS indicated that transfer of these genes to plantlets was successful. RT-PCR reaction results showed that nptII is transcribed in both tissues while GUS is transcribed only in the seed tissue. This finding was expected because Nos promoter (which controls the nptII transcription) is a constitutive and Napin is a seed specific promoter.. Expression and activity of Beta-glucuronidase enzyme in seeds of selected plants was confirmed by SDS-PAGE and histochemical assay.
Conclusion: The result of this research showed that designed gene construct was appropriate, because Napin promotes the expression of GUS gene in the seeds and Omega sequences have also been effective in increasing of transgene expression. In the fallowing, this construct can be used for the production of recombinant proteins by replacing valuable genes with GUS..