My Work

My PhD thesis is on "Genetic transformation and DNA fingerprinting in Pigeonpea". In this I did the following three major objectives:

1. Developing a transgenic pigeonpea for drought tolerance using Mannitol-dehydrogenase gene through Agrobacterium mediated transformation.
2. DNA fingerprinting of cultivated pigeonpea and two of its wild relatives by using AFLP.
3. Isolation and characterization of a green tissue-specific promoter (rbcS) from pigeonpea.

I did my PhD work in Dr P.Ananda kumar's lab, NRCPB, IARI, New Delhi. I have registered my PhD at Ch.Ch.Singh University, Meerut, under Prof. P.C.Sharma.

In addition to my PhD work I have also worked on pigeonpea transgenic development for insect resistance, developing synthetic cry1F gene, cloning of many synthetic cry gene constructs and molecular markers in chickpea, maize and rice.

Currently I am working as Post-Doctoral fellow in Entomology Department, University of Kentucky, Lexington, USA. I am working with Dr S. R. Palli. My project work here is to refine the ecdysone based geneswitch system, testing the effects of ligands required for this geneswitch on mammalian cells and shuffling the nuclear receptors in the gene switch for high efficiency.

Ph.D work was carried out at National Research Centre on Plant Biotechnology (NRCPB), IARI, under co-chairmanship of Dr P. Ananda Kumar, Principal Scientist.
Thesis submitted to Division of Biotechnology, C.C.S.University, Meerut, under the chairmanship of Prof. P.C.Sharma.

Thesis: “Genetic engineering and genetic biodiversity in pigeonpea (Cajanus cajan L.)”.
Pigeonpea (Cajanus cajan (L.) Millsp.) is one of the major grain legume (pulse) crops of the tropics and subtropics, yielding nutritious protein rich seeds for human consumption and animal feed. Being highly recalcitrant to regeneration among all the plant species tropical grain legumes are more difficult to transform. Improvement of legumes by genetic engineering requires procedures to regenerate them in vitro. Studies on various regeneration protocols that are available to find the most efficient for Agrobactereium mediated transformation has been done. By using Mannitol dehydrogenase (MtlD) gene, drought resistant transgenic pigeonpea was developed through Agrobacterium mediated transformation. A green tissue specific promoter (rbcS3A) was isolated from pigeonpea and pea through PCR and their sequence analysis; tissue specificity, light indusibility, stage specificity and its strength were compared by using GUS gene in transgenic tobacco. Wild relatives of pigeonpea serve as a rich source of disease resistance genes that can be introgressed into the cultivars. However, development of improved types through hybridization and recombination of available variability in pigeonpea and other species of Cajanus have met with limited success. Further research efforts need to concentrate on developing a good understanding of genetic systems controlling qualitative and quantitative characters. Genetic diversity of various pigeonpea cultivars as well as two of its wild relatives has been studied by using various PCR based markers like AFLP, RAPD, URP’s, SSR’s and STMS; also analyzed to find the best marker system in pigeonpea, for further studies, like gene tagging and genome mapping.

Advisors: Prof P. C Sharma, Dean, School of Biotechnology, G.G.S. Indraprastha University, New Delhi.
Dr P. Ananda kumar, Principal Scientist, National Research Center on Plant Biotechnology, IARI, New Delhi, India.

Ecdysone based gene switch in mammalian system for medical use

Ecdysteroids (Ec) are signaling molecules widespread in the animal as well as in the plant kingdom (Lafont and Wilson, 1992). However, they do not occur naturally in vertebrates, a feature that makes them suitable as ligands in medical gene switch applications due to the reduced likelihood of pleiotropic effects.
Insect EcR can heterodimerize with retinoid X receptor (RxR) and transactivate genes that are placed under the control of Ecdysone response element (EcRE) in various cellular backgrounds including mammalian cells (Palli et al. 2003).
A chimeric protein composed of GAL4 DNA binding protein fused to an Ecdysone receptor protein and a VP16 activation domain fused with retinoid X receptor (RxR) along with a gene of interest under the control of a response element (here EcRE) not recognized by natural nuclear hormone receptors. Binding of specific inducer to the receptor dimer leads to its activation and consequently to the transcriptional up regulation of any gene of interest located downstream of the synthetic response element. An optimal gene switch should have low or no basal expression in the absence of ligand, high induced expression in the presence of ligand, rapid switch-off response after removal of the inducer and specific response to inducer with no pleiotropic effects.
The current version of EcR based gene switches does not have some of the desired characteristics of an optimal gene switch like high background in the absence of ligand, low sensitivity with the and pleiotropic effects of gene switch compounds/ ligands used.
To overcome these problems, the present investigation was carried out with the following objectives.
1. Studies on Two-hybrid gene switch and development of an efficient mono-receptor gene switch.
2. Studies on pleiotropic effects of gene switch compounds and ligands.

Techniques used :
1. Cloning
2. Transfection in mammalian cells
3. Micro-array
4. Analysis of micro-array data with TIGR MeV software
5. Electro Mobility Shift Assay (EMSA)
6. Chromatin Immuno Precipitation Assay (ChIP-Assay)
7. Ligand Binding Assay
8. RNAi
9. Quantitative Real Time PCR (QRT-PCR)

Achievements:

1. Pleiotropic effects of various gene switch components and different ligands were analyzed.
2. An efficient monoreceptor gene switch (VGE), with less pleiotropic effects was developed and characterized.
3. An efficient two-hybrid gene switch, which is highly sensitive to ligand even at very low concentrations (which will help in reducing the pleiotropic effects of ligands on mammalian cells) was developed and characterized.
4. The mechanism of functioning of EcR based gene switch was studied.

References:
Lafont, R. D. and Wilson, I. D. (1992). The Ecdysone Handbook. Chromatographic society, Nottingham, UK.

Palli, S. R., Kapitskaya, M. Z., Kumar, M. B. and Cress, D. E. (2003). Improved Ecdysone receptor-based inducible gene regulation system. Eur. J. Biochem. (270): 1308-1315.

Anderson Paul, S. R. Sharma, T. V. S. Sresty, Suma Baisaria, P. S. Kumar, P. Parthasaradhi, Roger Frutos, I. Altosaar and P. Ananda Kumar* (2005). Transgenic cabbage (Brassica oleracea var. capitata) resistant to Dimondback moth (Plutella xylostella). Ind. J. Biotech. 4: 72-77.

S. K. Panguluri, K. Janaiah, J. N. Govil, P. A. Kumar and P. C. Sharma (2006). AFLP fingerprinting in pigeonpea (Cajanus cajan (L.) Millsp.) and its wild relatives. Genet. Res. Crop Evol.53:523-531.

Ritu Bhalla, Monika Dalal, Siva. K Panguluri, Borra Jagadish, Ajin. D. Mandoaker, A. K. Singh and Polumetla. A. Kumar* (2005). Isolation, characterization and expression of a novel vegetative insecticidal protein gene of Bacillus thuringiensis. FEMS Microbiol. Lett. 243:467-472.

S. K. Panguluri, J. Sridhar, B. Jagadish, P. C. Sharma and P. A. Kumar (2005). Isolation and characterization of a green tissue-specific promoter from pigeonpea [Cajanus cajan (L.) Millsp.]. Ind. J. Exp. Biol 43: 369-372.

Tazo Abraham, S. K. Panguluri, B. Jagadish, J. Sridhar, R. Mukesh and P. A. Kumar (2005). AFLP fingerprinting of some elite Indian cotton genotypes. Plant Cell Biology and Molecular Biology.

Srinivasa Rao Linga, Usha Rani papineni, Deshmukh P.S, Ananda Kumar Polumetla and Siva Kumar Panguluri*. RAPD and ISSR fingerprinting in cultivated chickpea [Cicer arietinum (L.)] and wild species (2006). Genetic Resources and Crop Evolution (in press).

S.K.Panguluri and S.R.Palli. Effects of ligands on ecdysone-inducible mammalian expression system in 293 and RKO cell lines. Manuscript under preparation.

S.K.Panguluri and S.R.Palli. Functional characterization of monopartate ecdysone based gene switches for mammalian system. Manuscript under preparation.

Books/Reviews:

Rajani Jaiswal, P. S. Kumar, M Z abdiin and P. Ananda Kumar* (2004). Chapter:21.Genetic Transformation of Grain Legumes. In; Plant biotechnology and its applications in tissue culture. I.K. International Private Ltd. New Delhi.pp:243-283.

P. S. Kumar, P. A. Kumar, K. Janaiah and P. C. Sharma (2003). AFLP fingerprinting to access genetic diversity in pigeonpea (Cajanus cajan (L.) Millsp.) and its wild relatives. Seventy third annual sessions on The National Academy of Sciences, India. Pp-15.

Ritu paruti, P. S. Kumar, P. A. Kumar and P. C. Sharma (2003). RAPD diversity in pigeonpea (Cajanus cajan (L.) Millsp.) and related species. Seventy third annual sessions on The National Academy of Sciences, India. Pp-05.

P. C. Sharma, P. S. Kumar, R. Paruthi, K. Janaiah, J. N. Govil and P. A. Kumar (2004). AFLP and RAPD fingerprinting in Pigeonpea and related species. Legumes for the benefit of Agriculture, Nutrition and the Environment: their genomics, their products and their improvement. Palais Des Congress, Dijon, France. pp-171.

Linga S. Rao, P. Usha Rani, P. S. Deshmukh, P. A. Kumar and S. K. Panguluri (2005). RAPD and ISSR fingerprinting in cultivated chickpea [Cicer arietinum (L.)] and wild species. First International Conference on Crop Wild Relative Conservation and Use, Italy.

For Full text of my publications, please see My work page in my other site:
http://www.geocities.com/skpanguluri/cv.html