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Subject: Biotechnology and molecular Biology × Author: Redhu, Neeru Singh × End date: 2023 × Start date: 2023 × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    In silico characterization and transcriptome analysis of transgenic pigeon pea [Cajanus cajan (L.) Millsp.] with OsRuvB gene for salt tolerance
    (CCSHAU, Hisar, 2023-01) Redhu, Neeru Singh; Kharb, Pushpa
    Soil salinity reduces the productivity of many agricultural crops, including legumes, which are highly sensitive. In this study, OsRuvBL1a, a DNA helicase, was computationally characterized and analyzed through transcriptomics to elucidate its potential pathway for imparting salt tolerance in transgenic pigeon pea. The sequence with locus ID Os01g0837500 was retrieved. DNA binding (from amino acid residues 1-15, 63-67, and 218-224), RNA binding (from amino acid residues 2-13 and 219-223), and Protein binding (from amino acid residues 9-14, 295-297 and 427-429) sites were predicted. NCBI-CDD predicted it as a member of AAA+ ATPase family and TIP49 (TBP interacting) superfamily. SOMPA used self-optimized prediction method for predicting 49.89% of alpha helix, 14.95% of beta strand, 29.67% of coils and 5.49% of beta turn. RuvB-like helicase (PDB-Id: 2C9O) with 99% query coverage and 73% sequence identity was selected as a template. MODELLER, SWISS-MODEL, Phrye, I- TASSER, Robetta, and IntFOLD predicted model with -15187.475, -19170.473, -12729.712, -18859.584, -21191.418, and -15212.321 kJ/mol energy, respectively. Molecular dynamic simulation was carried out using GROMACS-2019 software for representative structure of all models. RMSD between pre-simulated and simulated models of MODELLER, SWISS-MODEL, Phrye, I- TASSER, Robetta, and IntFOLD were observed to be 1.207, 1.148, 1.201, 1.274, 1.063, and 1.101 (Å), respectively. Even though little change in RMSD was observed with 2C9O template against simulated and pre-simulated models, still the simulated models gave a good understanding of its tertiary protein structure. A total of 19,59,13,490 (~190 million) raw reads were generated by Illumina HiSeq sequencer from transgenic and non-transgenic samples. The 100% of reads were mapped to the pigeon pea genome. The reads were mapped to a total of 21676 transcripts out of which 9407 genes were differentially expressed. The gene ontology of annotation uniquely identified 78 biological processes, 51 cellular component and 128 molecular functions in differentially expressed genes. A total to 378 genes with significant differential expression were identified with >= 3.0 for –log10(p adjusted value). Downstream analysis revealed 32 key genes with potential role in acquiring salt tolerance. 21 genes were found upregulated and 11 were found downregulated belonging to pathways like ABC transporters, flavonoid biosynthesis, fatty acid elongation, tropane, piperidine and pyridine alkaloid biosynthesis and circadian rhythm – plant among others. Although, the present study identifies key genes and pathways involved in the OsRuvB mediated salt stress tolerance in transgenic pigeon pea, more research is required to identify the exact molecular mechanism and the underlying signaling pathways of OsRuvB.