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Subject: Molecular Biology and Biotechnology × Author: Singh, Uma Maheshwar × End date: 2016 × Start date: 2010 × Type: Thesis ×
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    ThesisItemOpen Access
    Transcriptomic approach for understanding the role of calcium signaling and transport genes in relation to grain calcium content and calcium responsiveness in finger millet
    (G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2013-08) Singh, Uma Maheshwar; Anil Kumar
    Finger millet is a plant with exceptionally high seed Ca content. Little is known about the genetic and epigenetic basis of calcium accumulation in seed. In order to understand the mechanisms of high seed Ca accumulation, two genotypes of finger millet differing in seed Ca content i.e. GPHCPB-1 (low Ca) and GPHCPB-45 (high Ca) were selected in present study. Through PCR based approach a total of 8 genes of Calcium regulated protein kinases were isolated, but these were quite few to be able to explain the exact mechanism of high seed Ca accumulation. Therefore, to cover all Ca signalling and transport genes expressed during seed development, the transcriptome of developing spike of both GPHCPB-1 and GPHCPB-45 genotypes were sequenced. A total of 55 and 52 Ca signaling and transport genes were identified in GPHCPB-1 and GPHCPB-45 genotype respectively. On the basis of phylogeny, per cent sequence identity, and conserved sequence analysis all the genes were classified into Ca sensor and transporter gene families. Differential expression analysis showed that 15 genes were highly expressed in GPHCPB-45, 7 were highly expressed in GPHCPB-1 and remaining 32 had very less (FPKM<1) expression value. Structural and functional analysis of these genes were also done and high level of sequence similarity among Ca sensor genes and low level of sequence similarity among Ca transporter genes of finger millet with their rice orthologues was found. Simultaneously effect of different levels of exogenously supplied Ca on both GPHCPB-1 and GPHCPB-45 genotypes were also studied. The responsiveness of both the genotypes towards increasing exogenous application of Ca was determined in terms of changes in Ca accumulation in different tissues and different morpho-physio-agro-biochemical parameters. During the spike development (S1, S2, S3 and S4) the effect of exogenous Ca (Ca deficient-0.1mM, Ca sufficient-5.0 mM, Ca excess-10 mM and Ca toxic- 20 mM) on Ca accumulation in root, stem, leaf and spike and also on root length, root dry matter accumulation, plant height, RWC, chlorophyll content, SPAD value, oxalate and phytate content was recorded. During Ca deficient conditions, genotype GPHCPB-45 performed better than GPHCPB-1; while at toxic level of Ca genotype GPHCPB-1 withstand that condition better than GPHCPB-45 in terms of Ca accumulation as well as different agro-morphophysio-biochemical parameters. Based on such studies it was revealed that threshold potential of Ca accumulation was high for GPHCPB-1 in even Ca toxic conditions, however GPHCPB-45 was efficient accumulator under Ca deficient conditions. The results of present study clearly suggest the differential responsiveness of genotypes towards exogenous application of Ca in different tissues, which is suggestive of genetic and epigenetic basis of seed Ca accumulation trait and induction of differential calcium signaling and transport machinery. The responsiveness of differentially expressed genes (highly expressed in GPHCPB-45; FPKM>4) were studied towards exogenous application of Ca in both GPHCPB-1 and GPHCPB-45 genotypes. The overall high expression of most of the Ca sensor genes i.e., EcCDPK16, EcCDPK19, EcCIPK2, EcCIPK9, and EcCIPK11 were observed in GPHCPB-45 genotype in later stages of spike development, in a concentration dependent manner. Similarly, the overall high expression of Ca transporter genes i.e., EcPM3ATPase2, EcPM4ATPase8, EcPM12ATPase4, EcER3ATPase3 and EcCaX3 were observed in GPHCPB-45 in later stages of spike development, in a concentration dependent manner. The expression of two genes EcCIPK19 and EcPM8ATPase was comparatively higher in GPHCPB-1 as compared to GPHCPB-45 genotype. The exceptionally higher expression of EcCaX3 in response to exogenous Ca and at later stages of spike development in high Ca containing genotype establishes this gene as a strong candidate for higher seed Ca accumulation and its further use for Ca biofortification. However, exploration of its spatial distribution within seed, over expression and knockout studies will help in understanding the exact role of this gene in high seed calcium accumulation. Finally, transcriptome based functional annotation provides a greater insight into the mechanisms related to seed calcium accumulation and would help to accelerate future research in this very important area of seed biology.