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Isolation and characterization of stress inducible promoters from Pennisetum glaucum and their role in abiotic stress adaptation
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Isolation and characterization of stress inducible promoters from Pennisetum glaucum and their role in abiotic stress adaptation

  1. Feb 2017 Isolation and characterization of stress inducible promoters from Pennisetum glaucum and their role in abiotic stress adaptation About ICRISAT: www.icrisat.org ICRISAT’s scientific information: http://EXPLOREit.icrisat.org Divya K1 , Reddy PS1 *, Bhatnagar-Mathur P1 , Reddy MK2 , Vadez V1 , Sharma KK 1 *E-mail: p.sudhakarreddy@cgiar.org Introduction • Environmental stresses are serious threat to agriculture and the primary cause of crop loss worldwide and become a major challenge in our quest to achieve sustainable food production. • Genetic engineering thus provides alternatives to combat this problem addressed. • Transgenic plants developed with a strong constitutive expression of functional genes and/or transcription factors often suffer from undesirable phenotypes. • It is therefore desirable to generate transgenic plants that accumulate transgenic products only under stress conditions. • Use of stress inducible promoters has proven to be an alternative strategy for elimination of such phenotypes induced by constitutive transgene expression. Objective: Development of the abiotic stress tolerant crops The relative expression of five abiotic stress inducible genes under different abiotic stress conditions. The fold change between 2 to 5 fold are given a single (+) or (-) sign for up regulation and down regulation respectively. Similarly (++) or (- -) denotes 5 to 10 fold change, (+++) or (- - -) denotes 10 to 15 and (++++) or (- - - -) denotes 15 fold or above change in the expression values. Heat (45 o C) Cold (4 o C) Drought (withholding the water for 48 h) Salt (250 mM) Hsp10 ++++ - + + Hsc70 ++++ - ++ +/- Lea +++ ++++ + ++++ Dhn + - + ++++ APX ++++ ++ + + Isolation and cis-motif analysis of promoter regions of five abiotic stress inducible genes. Schematic diagram of pMDC164 vector Cloning in to pMDC164 vector Hsp10S Dhn Hsc70 Apx Lea M 1 2 3 4 5 6 7 8 TC TC 1 2 3 4 5 6 7 8 M TC TC P N Cloning, transformation and molecular confirmation of the tobacco transgenic plants. Conclusion • PgHsc70 pro and PgLea pro were active in the heat, PgDhn pro in heat, cold and drought and PgApx pro in drought stress conditions. • PgHsc70 pro and PgDhn pro can be used to drive the expression of selected target gene to develop abiotic stress tolerant crops. References • Reddy PS, Mahanty S, Kaul T, Nair S, Sopory SK, Reddy MK (2008) A high- throughput genome-walking method and its use for cloning unknown flanking sequences. Anal Biochem 381:248-253. Acknowledgments Innovation in Science Pursuit for Inspired Research (INSPIRE), Department of Science and Technology, Govt. of India for the research grant through INSPIRE Faculty Program (Award No. IFA-LSPA-06). This work has been undertaken as part of the CGIAR Research Program on Dryland Cereals. GUS Histochemical assay. Symbols, + (mild expression), ++ (strong expression) and - (no expression) represents the intensity of GUS activity. 2 International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi-110 067, India 1 International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, 502 324, Telangana, India Results Hsp10 Hsc70 Apx LeaRab Walker Primers Phi29 DNA polymerase Genomic DNA Whole genome is copied into large number of overlapping fragments with unique sequence Cis-elements of stress inducible promoters ABREHSE LTR GC-motif CircadianSkn-1_motif ARE CGTCA TGACGTGABox-W1MBS CCGTCC-box GCN4_motif -100 -300-200 -400 -600-500 -700 -800-1 APX Hsc70 Dhn Lea Hsp10 -900 Reddy et al., 2008 1. Expression profiling of five abiotic stress inducible genes. 2. Isolation and analysis of promoter regions of five abiotic stress inducible genes. 3. Construction of plant expression vector with five different promoters. Relative GUS mRNA level in abiotic stressed transgenic tobacco plants. 4. Functional validation in tobacco using histochemical assay and molecular approaches. Drought tolerant crop Pearl millet Crop plants Model plants Rice Legumes Sorghum MaizeWheat Evaluation in model plants Stress tolerant crops Agriculturally important crops
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