Functional Validation of Novel Rice gene for its role in flower and Seed development through over expression and silencing approach

Abstract

Calcium dependent protein kinases (CDPKs) are Ca2+ sensor protein kinases that are prevalent in plants. They play diverse roles in plant signaling mechanism. Thirty one CDPK genes have been identified in rice, of which a few are involved during seed development newlineprocess and some are functionally haracterized. Expression profiling of this gene in indica genotype indicated that the gene expressed only in endosperm. The present work was undertaken to validate the newly newlineidentified CDPK gene, OsCPK31 through transgenic approach in BPT 5204 and Taipei 309 rice genotypes. Putative transgenic plants were developed in both genotypes with two gene constructs viz., overexpression and silencing vectors and they were confirmed by PCR and Southern analysis for transgene integration. Selected transgenic newlineplants were advanced up to T3 generation and all the transgenic plants were confirmed by PCR and Southern hybridization. Northern newlinedot-blot analysis of non-transformed control plant (BPT 5204) revealed that the endogenous gene expressed from milky stage (S3) to maturity stage (S5). Transgene expression analysis of overexpression plant newlinetissues (leaf and five stages of seed) revealed that the transgene OsCPK31 overexpressed in all the tissues examined whereas in silencing plants, the endogenous gene was suppressed 50% as compared to control plant. Phenotypic studies of overexpression transgenic homozygous lines showed that seeds matured early (19-20 newlinedays after pollination) and the average maturity days of the transgenic plants was reduced to about 10 days. In contrast, high rate of sterility and no difference in maturity days was observed in all the transgenic silencing lines. Thus, the present findings suggested that the CDPK gene OsCPK31 might be involved in signal transduction pathways for distribution of photosynthetic assimilates during grain filling stage of newlineseed development process where it might be associated with genes related to grain filling and seed development.