Pradeep K. Burma

Exploring transgene expression in plants
 
The production of transgenic plants with expression of the transgene at the right place, time and level is important for the success of crop improvement programs based on genetic engineering. The expression of the transgene also needs to be stable over generations. Thus a transgenic developer makes hundreds of independent lines of which only few or may be even one is useful for field applications. The current research focus of the lab revolves around promoters, 5’ Untranslated Regions (5’UTR) and codon usage that are some of the key components in regulation of transgene expression. Analysis of the architecture of tapetum specific promoters like A9 from Arabidopsis and TA29 from tobacco is currently being carried out. A novel promoter from cotton expressing in tapetum as well as in roots has been identified. Attempts are on to redesign this promoter to make it tapetum specific. We have earlier developed strategies to design synthetic constitutively expressing promoters using CaMV35S as a model. This was attempted to circumvent the problem of promoter-homology based transgene silencing.
 
Investigation in the role of 5’UTR in influencing transgene expression led to the concept of ‘Upstream Regulatory Modules’ (URM) and the importance of 5’UTR in realizing the full potential of a promoter. A serendipitous observation led to the identification of a synthetic 5’UTR (synJ) that enhances transgene expression by several folds.
  
The lab has been a part of the efforts (along with Prof. Deepak Pental) in the improvement of mustard and cotton by biotechnological approaches.  In mustard, the technology for producing hybrid seeds through use of transgenics containing barnase (male sterile) and barstar (restorer) genes expressed in tapetum specific manner, as a pollination control mechanism has been developed. These transgenics are currently undergoing toxicological and other tests before they can be commercialized. Maintaining tapetum specific expression of barnase protein  (a cytotoxic protein) was a challenge during development of these transgenics.  The concept of ‘Spacer DNA Fragment’ to insulate tissue-specific expression was thus developed which was later patented.
 
In collaboration with Prof. Pental we have also developed an insect resistant transgenic cotton line expressing the Bt. endotoxin, Cry1Ac. The transgenic line in the cultivar Coker 310FR has been transferred to Punjab Agricultural University and Central Institute of Cotton Research for introducing the gene into commercial varieties. Our efforts to develop and identify a cotton line showing proper insect resistance without affecting other features of the plant was an uphill task. We showed that expression of Cry1Ac effects growth and development of tobacco and cotton transgenics. We further demonstrated that the problem could be circumvented by targeting the Cry1Ac protein to the chloroplast. Reasons for the negative effects of Cry1Ac protein are currently being investigated.
 

Selected papers and patents

  • Agarwal P, Garg V, Gautam T, Pillai B, Kanoria S and Burma PK (2014) A study on the influence of different promoter and 5’UTR (URM) cassettes from Arabidopsis thaliana on the expression level of the reporter gene β-glucuronidase in tobacco and cotton Transgenic Research 23:351-363
  • Kanoria S and Burma PK (2012) A 28nt long synthetic 5¢UTR (synJ) as an enhancer of transgene expression in dicotyledonous plants BMC Biotechnology 12:85
  • Rawat P, Singh AK, Ray K, Chaudhary B, Kumar S, Gautam T, Kanoria S, Kaur G, Kumar P, Pental D and Burma PK (2011) Detrimental effect of expression of Bt endotoxin Cry1Ac on in vitro regeneration, in vivo growth and development of tobacco and cotton transgenics Journal of Bioscience 36(2): 363-376.
  • Bhullar S, Datta S and Burma PK (2011) Delayed trans-inactivation of synthetic domain A 35S promoters by “T obacco 271 locus” due to reduced sequence homology Plant Molecular Biology Reporter 29: 1-11.
  • Bhullar S, S Datta, S Advani, S Chakravarthy, T Gautam, D Pental and P K Burma. 2007. Functional analysis of Cauliflower Mosaic Virus 35S promoter: re-evaluation of the role of subdomains B5, B4 and B5 in promoter activity. Plant Biotechnology Journal. 5: 696-708.
  • Bhullar S, S Chakravarthy, S Advani, S Datta, D Pental and P K Burma. 2003. Strategies for development of functionally equivalent promoters with minimum sequence homology for transgne expression in plants :cis-elements in a novel DNA context versus domain swapping. Plant Physiology. 132: 988-998.
  • Regulation of lethal gene expression in plants. US Patent No.: US6,833,494. (2004).
 

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