CLONING AND CHARACTERIZATION OF DROUGHT INDUCIBLE PM19 PROMOTER FROM WHEAT
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Date
2018
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DIVISION OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY ICAR-INDIAN AGRICULTURAL RESEARCH INSTITUTE NEW DELHI
Abstract
Roots are the primary organs to sense water availability in soil. However, the
mechanism of how roots sense water availability, perceive water deficit and respond
to the change in water potential; still remains largely unexplored. Identification and
characterization of genes and promoters defining the root traits under drought will
help in increasing the fundamental knowledge as well as in designing crops with
better water use efficiency and drought tolerance.
PM19 gene was identified as one of the differentially expressed genes in root
transcriptome study of two wheat genotypes Raj3765 (inductive root growth) and
HD2329 (less or no inductive root growth) under drought stress. PM19 is a highly
conserved plasma membrane protein and it gets induced by ABA, osmotic stress,
cold, heat and salt stress. In the present study, cloning and characterization of PM19
promoter from wheat was carried out. Drought inducibility of PM19 gene was
checked by expression analysis after 4, 6 and 8 days of moisture deficit stress in
wheat genotype Raj3765. The analysis revealed 20 and 28 fold increase in expression
of PM19 gene in shoot and root, respectively, after 4 days of stress. The maximum
expression was recorded after 6 days of stress, with 45 fold and 236 fold increase in
PM19 expression in shoot and root, respectively. PM19 expressed in both root and
shoot under drought stress, yet level of induction was higher in root than that of shoot.
The promoter sequences of PM19 gene from Raj3765 and HD2329 were cloned in
binary GUS reporter construct and sequenced. Comparative sequence analysis
revealed that except three single nucleotide polymorphisms (SNP), the promoter
sequences were highly conserved. One of the SNPs led to generation of an E-Box
element in HD2329. Functionality of PM19 promoter from Raj3765 (PM19pRaj) was
analysed by transient expression. To elucidate its transcriptional regulation,
Arabidopsis transgenic harbouring PM19pRaj:GUS were developed. The expression
of PM19pRaj was confirmed in Arabidopsis T1 transgenic leaves under control
condition by GUS histochemical assay. The study revealed that PM19 promoter is
highly conserved among the two genotypes analysed. It is a constitutively expressed
gene with early and high level of upregulation under drought stress. The transgenic
plants developed in the study will help in understanding transcriptional regulation and
spatio-temporal expression of PM19 gene in wheat.
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t-9991
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