molecular and functional characterization of putative sigma factor i

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MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF PUTATIVE SIGMA FACTOR IN FINGER MILLET FOR DESICCATION RESPONSE PRAKASH V., RAMEGOWDA HV., GEETHA GOVIND AND UDAYAKUMAR M. Department of Crop Physiology, University of Agricultural Sciences , GKVK, Bangalore – 560065 E-mail: prakashvs23@yahoo.com Introduction: Finger millet ( Eleusine coracana ( L)Gaertn. ) is one of the major food crop in southern parts of India. It is said to be drought tolerant crop expressing stress responsive genes which are not normally expressed in susceptible crop species. We have isolated a putative sigma factor ( Fdr-75 ) from subtracted stress cDNA library showed homology to sigma factors of Maize ( Zm SIG2), Rice (OsSIG2) and Arabidopsis ( At SIGB). In higher plants plastid encoded RNA polymerase (PEP) in chloroplast has nuclear encoded subunit called sigma factor. PEP is associated with sigma factor to bring in activation under abiotic stress conditions for transcription of photosynthetic genes in turn protecting damage occurring to photosymtem. Hypothesis: Isolating the genes expressed in stress tolerant crops will perform better under abitotic stresses than the genes expressed in susceptible crops. The heterologous genes having more than 90% homology with host gene will be better to assess its functional relevance through VIGS ( V irus I nduced G ene S ilencing) approach. Experimental Results: Phenotype of Finger millet subjected for different moisture stress levels. Total RNA isolated from 20%,40% 60% FC and control leaf samples and quantified later .Subtraction was carried out. C 20% 40% 60% Assessment of stress response in Finger millet by RT-PCR showing expression of known stress responsive gene NCED ( 9-cis-epoxycarotenoid dioxygenase ) at different moisture stress levels Note: RT-PCR was carried out using groundnut NCED gene specific primers. C 20% 40% 60% RT-PCR showing the expression of stress responsiveness of putative sigma factor under different moisture stress levels in finger millet. Note: Reaction was done using Fdr-75 gene specific primers C 20% 40% 60% Northern expression pattern of finger millet putative sigma factor in groundnut under different moisture stresses. Note: Fdr-75 was labelled with  32 PdCTP (20Ci)by using PCR labelling method with gene specific primers. PCR amplification of putative sigma factor genomic DNA in crop plants of cereals and dicot species using gene specific primers of Fdr-75 M=1 kb Marker, Cereals: 1=Finger millet,2= Rice,3= Wheat,4= Maize,5= Oat,6= Bajra,7= Barn yard millet and Dicots: 8= Soybean,9= Red gram,10= Horse gram,11= Field bean,12= Bengal gram,13= Cowpea,14= Beans C(T) values 18sRNA 20% 19.155 18sRNA control 22.237 FM20% 28.359 FM 40% 26.521 FM 60% 26.006 FM control 29.883 Quantitative RT - PCR in Finger millet putative sigma factor in different stress levels, 18s RNA was run as a positive control in severe stress (20% FC) and control. 850 bp M C M I RT-PCR analysis showing the confirmation of virus pTRV1and pTRV2 infection upon infiltration in to Nicotiana benthamiana plants after 14 DPI. M=Marker, C=Control, M=Mock, I= Infiltrated Sigma factor Control pTRV1::pTRV2 Control pTRV1::pTRV2 pTRV2::SF Pale green leaf,leaf curling, yellowing along the margin and midrib are the phenotypic changes due to silencing of Fdr-75 (putative sigma factor) in Nicotiana banthamiana (NB) under control and moisture stress condition. Upper panel: Control, Lower Panel: Stress Conclusions: Results showed that putative sigma factor (Fdr-75) transcripts increased under drought stress. Putative sigma factor is fairly conserved across cereals and dicot species It has more than 90% homology and hence proved that heterologous genes will work for VIGS approach to know its functional relevance. M 1 2 3 Amplification of putative Sigma factor (Fd r-75) from FM and NB genomic DNA with gene specific primers 1&2 = Finger millet genomic DNA 3= NB genomic DNA GenBank accession number for Fdr–75 as a putative sigma factor is CV478067