Validation of putative candidate gene function in vivo can be achieved by developing transgenic for down regulation or over expression of gene/s of interest. In this regards, the study was taken up with the isolation of full length cDNA clones for DREB1B and Metallothionein-like protein from rice EST resources. The plant expression vectors under 35S promoter for Agrobacterium were constructed and genetic transformation was done in indica rice.

DREB protein in as stress responsive transcription factor. This protein binds to DRE/LTRE/CRT elements on promoter region and regulates the expression of stress responsive genes. Metallothioneins (MTs) are a group of proteins with low molecular mass and high cystein content, which are organized into two clusters and bind to transition metals with strong affinity.

In the lab, EST database was generated by partial sequence of cDNA clones from drought stress rice seedlings. Two cDNA clones represented the DREB family while six different cDNA clones for Metallothionein-like protein were found in the database. The cDNA clones were individually sequenced from both ends MegaBace 500 automated capillary-based sequencer. The longest sequence length obtained for DREB1B was 862bp (AccNo: BU673228) and for metallothionein-like protein was 525bp (AccNo: CB199137). These genes were found to encode for DREB1B and metallothionein-like protein through in-silico analysis.

DREB1B and metallothionein like protein sequence were individually aligned against public database using BLASTN algorithm. DREB1B showed 98 per cent sequence identity while metallothionein-like protein revealed 90 per cent identity. The amino acid sequence of DREB1B and metallothionein-like protein were independently aligned against protein database using BLASTP. DERB1B showed 61 per cent sequence identity while metallothionein-like protein revealed 66 per cent similarity. The comparative analysis of amino acid sequence deduced from the gene encoding DREB1B and metallothionein-like protein using CLUSTALW showed high degree of conservation among very diverse plant species.

Full length DREB1B and metallothionein like protein were individually cloned into 35S promoter with a poly ‘A’ signal in sense orientation using pRT100 vector and sub cloned in to PstI site of binary vector pCAMBIA1301. These recombinant plasmids were mobilized into Agrobacterium tumefaciens by electroporation for transformation in rice. The transformation of rice and tobacco using these constructs for analysis of the gene function is under way.