FROM RICE 'EST' RESOURCES: CONSTRUCTION OF PLANT EXPRESSION VECTORS FOR FUNCTIONAL TESTS
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Date
2004
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Professor Jayashankar Telangana State Agricultural University
Abstract
loping transgenics for down regulation or over expression of genels of interest. In this regard, the study was taken up with the isolation of full length cDNA clones for DREBlB and Metallothionein-like protein from rice EST resources. The plant expression vectors under 35s promoter for Agi-obacterium were constructed and genetic transformation was done in indica rice. DREB protein is a 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 afiimty. In the lab, EST database was generated by partial sequence of cDNA clones fiom drought stress rice seedlings. Two cDNA clones represented the DREB fdy while six dfierent cDNA clones for metallothionein-like protein were in the database. The cDNA clones were individually sequenced from both sequence length obtained for DREBlB was 862bp (AccNo: BU673228) and for metallothionein-like protein was 525 bp (AccNo : CB 1 99 1 3 7 j . These genes were found to encode for DREB lB and metallothionein-like protein through in-silico analysis. DREB 1 B and metallothlonein lke protein sequence were individually aligned against public database using BLASTN algorithm. DREB 1 B showed 98 per cent sequence identity while metallotbonein-like protein revealed 90 per cent identity. The amino acid sequence of DREB 1 B and metallothionein-lke protein were independently aligned against protein database using BLASTP. DERBlB 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 DREB 1 B and metallothionein-like protein using CLUSTALW showed high degree of conservation among very diverse plant species. Full length DREBlB and metallothionein like protein were individually cloned into 35s promoter with a poly 'A' signal in sense orientation using pRTl00 vector and sub cloned in to PstI site of binq vector pCAMBL41301. These recombinant plasmids were mobilized into Agrobacterium tzrinefacie~zs by electroporation for transformation in rice. The transformation of rice and tobacco using these constructs for analysis of the gene function is under way.
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D7081