Analysis of activity driven by upstream regulatory modules (URM) of tapetum specific genes TA29 and A9 at ectopic locations in tobacco transgenics
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TA29 and A9 are genes from Nicotiana tabacum and Arabidopsis thaliana respectively, which express in a tapetum specific manner. The upstream regulatory modules (URMs; i.e. the promoter and the 5′UTR) of these genes have been used in development of male sterile and restorer lines expressing the barnase and barstar genes for hybrid seed production. While initial studies show that these URMs drive the expression in a tapetum specific manner, there are no recordings of unintended (leaky) expression driven by these URMs at ectopic locations due to position effect in developed transgenic lines. The information on leaky expression driven by tissue specific URMs is important for their use in developing transgenic plants. The present study records the leaky activity of both these URMs in transgenic tobacco lines using β-glucuronidase as a reporter gene. Leaky activity was observed in about one-fourth of the lines developed with TA29. Most interestingly in these lines, the leaky expression of the reporter gene was observed to be restricted to the meristematic tip region of the roots and at the leaf gap from where leaf trace diverges from stem bundles. Such a restricted and unique pattern of leaky activity of a tissue specific promoter or a URM has never been reported before, including the URM of the A9 gene analyzed in the present study. This observation suggests the presence of cryptic cis-elements within the URM of TA29 gene that can possibly activate it in meristematic tissue when integrated at certain ectopic locations. The URM of the A9 gene was also observed to show leaky activity. However, there was no unique pattern as observed with that of TA29. Further, in the study we also show that while the smaller (290 bp) length of TA29 URM can be used to drive the expression of barnase gene to develop male sterile lines, it adversely affects the regeneration of transgenic tobacco lines due to leaky expression. This adverse effect is significantly reduced when the full length (1.5 kb) URM of the TA29 gene is used.
KeywordsTA29 promoter A9 promoter Tissue specific expression Tapetum Leaky expression Reporter gene Barnase gene
Intron containing β-glucuronidase gene
Intron containing barnase gene
Murashige and Skoog’s medium
1-Naphthalene acetic acid
A part of this work was supported by grant-in-aids from Council of Scientific and Industrial Research (CSIR#38(1368)/13/EMR-II, University of Delhi (R&D Grant) and DU-DST PURSE grants. The authors acknowledge CSIR for research fellowships and thank Dr. Surijit Sarkar and Prof. J P Khurana for use of Olympus Fluorescent microscope and Leica Stereo zoom microscope, respectively.
PAS and NV contributed equally to the work. PAS, NV and PKB designed the experiments, analyzed the data and wrote the manuscript. PAS and NV carried out the experiments.
Compliance with ethical standards
Conflict of interest
The authors declare that no conflict of interest exists.
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