Isolation and In Silico Analysis of OsGLP12-3 Gene’s Promoter from Rice Cultivar Dilrosh-97
Isolation and In Silico Analysis of OsGLP12-3 Gene’s Promoter from Rice Cultivar Dilrosh-97
Irfan Safdar Durrani*, Noreen Asim and Ammar Sohail
ABSTRACT
Germins and Germin-like proteins (GLPs) are the ubiquitous family of plant proteins that belong to the cupin superfamily and have been reported to play a major role in plant defense against pathogens attacks and different abiotic stresses. Current research deals with the study of cis-regulatory elements located in the promoter region of OsGLP12-3 gene of Nipponbare and newly sequenced Oryza sativa L. cultivar Dilrosh-97 using bioinformatics approach. The possible cis-acting regulatory elements were analyzed using PlantCARE and PlantPAN3.0. Phylogenetic tree was constructed using MEGA7 and docking of regulatory elements with the corresponding Transcription factors was performed using pyDockDNA. The OsGLP12-3 promoter region (1863bp) was PCR amplified using genomic DNA from Indica rice cultivar Dilrosh-97 and subsequently purified and sequenced. Sequence analysis using Plant Care online database revealed 32 different regulatory elements in Nipponbare while 31 different regulatory elements were found in Dilrosh-97. The cis-acting elements were found to be involved in plant hormonal inductions, developments and biotic and abiotic stress response. Notably, the OsGLP12-3 gene promoter from Dilrosh-97 was found to contain higher number of TATA-box and CAAT-box as compared to Nipponbare, reflecting it to be efficient in transcription initiation. Pairwise alignment using NCBI Nucleotide Blast tool showed 99% similarity between subject sequence (Nipponbare) and query sequence (Dilrosh-97). Localization of Transcription factors motif sequences indicate that the motifs including Homeodomain; TALE, NF-YB and GGAAAt in Nipponbare promoter and Alpha-amylase and B3 in Dilrosh-97 promoter were observed abundantly close to the transcriptional initiation site. Expression analysis using TENOR identified that OsGLP12-3 gene show high to minimal level of expression in response to different stress stimuli. Phylogenetic analysis of the Dilrosh-97 promoter sequence showed close relationship of this promoter with the Japonica rice cultivar Nipponbare. Three transcription factors including B3, GT-1 and NF-YB were subjected to docking with their corresponding cis-acting elements regulatory elements using pyDockDNA to evaluate the nature of interaction between the proteins and DNA sequences. The docking analysis revealed the hydrogen bonding interaction between cis-acting elements and their corresponding Transcription factors.
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