Isolation, Cloning and Sequence Characterization of the Salinity-Responsive Transcription Factor Gene OsbHLH024 from Rice (Oryza sativa L.) Cv. Ratnagiri-8

Abstract

Oryza sativa L. (rice) is a staple food crop whose productivity is increasingly constrained by soil salinity, a stress that disrupts ionic balance and limits growth and yield. Basic helix–loop–helix (bHLH) transcription factors are known regulators of plant abiotic stress responses, and OsbHLH024 has been reported to act as a negative regulator of salinity tolerance in rice, making it a promising candidate gene for functional and genome-editing studies. As a foundational step towards such studies, the present investigation reports the isolation, cloning and sequence-level validation of OsbHLH024 from the salt-tolerant indica cultivar Ratnagiri-8. The gene sequence (locus Os01g0575200) was retrieved from the Rice Annotation Project Database (RAP-DB), and five overlapping primer pairs were manually designed and evaluated in silico to amplify the ~4.35 kb gene as five fragments (P1–P5; 1001, 1001, 1001, 1051 and 701 bp). High-molecular-weight genomic DNA extracted from Ratnagiri-8 leaves by the CTAB method served as a template, and all five fragments were amplified as single, specific bands. The fragments were purified, ligated into the pJET1.2/blunt vector and transformed into Escherichia coli DH5α; colony PCR and plasmid-based PCR identified positive recombinant clones for all five fragments, which were subsequently confirmed by Sanger sequencing. Alignment of the assembled consensus sequence against the RAP-DB reference using BLAST and Clustal Omega showed 99.10% sequence identity, confirming successful isolation and cloning of OsbHLH024 from Ratnagiri-8. This sequence-validated clone constitutes a genomic resource for future comparative and functional characterisation of OsbHLH024 in salinity tolerance.