Analysis of activity driven by upstream regulatory modules (URM) of tapetum specific genes TA29 and A9 at ectopic locations in tobacco transgenics
- 98 Downloads
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.
- Bhullar S, Chakravarthy S, Advani S, Datta S, Pental D, Burma PK (2003) Strategies for development of functionally equivalent promoters with minimum sequence homology for transgene expression in plants: cis-elements in a novel DNA context versus domain swapping. Plant Physiol 132(2):988–998CrossRefGoogle Scholar
- Jagannath A, Bandyopadhyay P, Arumugan N, Gupta V, Pradhan AK, Burma PK, Pental D (2001) The use of spacer DNA fragment insulates the tissue specific expression of a cytotoxic gene (barnase) and allows high frequency generation of transgenic male sterile lines in Brassica juncea L. Mol Breed 8:11–23CrossRefGoogle Scholar
- Jagannath A, Arumugan N, Gupta V, Pradhan AK, Burma PK, Pental D (2002) Development of transgenic barstar lines and identification of a male sterile (barnase)/restorer (barstar) combination for heterosis breeding in Indian oilseed mustard Brassica juncea. Curr Sci 82:46–52Google Scholar
- Ray K, Bisht NC, Pental D, Burma PK (2007) Development of transgenic barnase/barstartransgenics for hybrid seed production in Indian oilseed mustard (Brassic juncea) using a mutant acetolactate synthase gene conferring resistance to imidazole-based herbicide ‘Pursuit’. Curr Sci 93:1390–1396Google Scholar
- Sivaraman I, Arumugam N, Sodhi YS, Gupta V, Mukhopadhyay A, Pradhan AK, Burma PK, Pental D (2004) Development of high oleic and low linoleic acid transgenics in a zero erucic acid Brassica juncea L. (Indian mustard) line by antisense suppression of the fad2 gene. Mol Breed 13:365–375CrossRefGoogle Scholar
- Svab Z, Hajdukiewicz P, Maliga P (1995) Generation of transgenic tobacoo plants by cocultivation of leaf discs with Agrobacterium pPZP binary vectors. In: Maliga P, Klessig DF, Cashmore AR, Gruissem W, Varner JE (eds) Methods in plant molecular biology. Cold Spring Harbour Laboratory Press, New York, pp 55–77Google Scholar
- Vancanneyt G, Schmidt R, O’Connor-Sanchez A, Willmitzer L, Rocha Sosa M (1990) Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol Gen Genet 220(2):245–250CrossRefGoogle Scholar