Application of Molecular Biology to Rice: Oryzacystatin Expression

INVESTIGATOR: Murai, N.

PERFORMING INSTITUTION:
PLANT PATHOLOGY & CROP PHYSIOL
LOUISIANA STATE UNIVERSITY
BATON ROUGE, LOUISIANA 70893

NON-TECHNICAL SUMMARY: After successful completion of this project it is possible to enter commercial application of the project outcomes and to apply for patents covering new binary vectors, oryzacystatin genes and transgenic plants. The patent rules essentially prevent us from disclosing full details of procedures even in the case of confidential research proposal to be reviewed by the peers. Thus, we would discuss the procedure in general terms and add as much as details allowed within the patent restriction. The principal and co-principal investigators are familiar with the all procedures to be described in this proposal, and enlist the refereed publications demonstrating our competence in applying the procedures to achieve the proposal objectives fully. A long-term goal of our research is to understand the molecular biology of growth and development processes of rice (Oryza sativum L.), and to apply the basic understanding to genetic improvement in rice cultivars. Toward this end we propose here to enhance the level of expression and recovery of oryzacystatin or cystatin (cysteine protease inhibitor) from rice grain. After successful completion of this project it is possible to enter commercial application of the project outcomes and to apply for patents covering new binary vectors, oryzacystatin genes and transgenic plants.

OBJECTIVES: A first objective of this proposal is to construct new binary vectors of Ti plasmid of Agrobacterium tumefaciens. This objective is the necessary first step because otherwise we are required for a licensing agreement to use the existing vectors for commercial application of this project. We have on-hand resources and past experiences to develop binary vectors superior to any commercially available binary vectors. A second objective is to construct genomic and cDNA clones for oryzacystatin. The sequences of cDNA and genomic clones for oryzacystatin are available in GenBank and described in detail in the Procedure Section below. The oryzacystatin genes will be constructed using polymerase chain reaction and designed synthetic oligonucleotides. Specific promoter and terminator sequences will be used to express the oryzacystatins in seeds. A third objective of this proposal is to express in and to purify the oryzacystatin proteins from seeds of Arabidopsis thaliana and rice. A model organism Arabidopsis thaliana is used to test oryzacystatin expression in plant seeds because of its many advantages as a transgenic host. After confirmation of optimal expression of the oryzacystatins in Arabidopsis thaliana, we will express the genes in rice seeds.

APPROACH: A new second-version of binary vector derived from Ti-plasmid of Agrobacterium tumefaciens will be the smallest binary vector in size to our knowledge to enhance the efficiency of cloning and other DNA manipulation in vitro and in E.coli. We will obtain from GenBank the DNA sequences of replication origins of E. coli plasmids and broad-host range plasmids, and of antibiotic resistance genes. We will define an essentially minimum region required for the promoter and terminator functions of the genes based on published results from functional analyses, known sequence motifs, secondary structures of RNA products. We will design primer sequences for polymerase chain reaction (PCR) based on the melting temperature (Tm), intra- and inter-molecular hybridization potentials and other pertinent considerations. We will order the primers to Sigma-Genosis with the cost of 30 cents per base. Added to this backbone are the T-DNA right and left boarder, a selectable marker gene and multi-cloning sites. We will test the validity of our reconstruction of the oryzastatin I and II genes in this project. We will express the oryzacystatin I gene in seeds of Arabidopsis thaliana and will purify the mature oryzacystatin protein. Amino acid sequence analysis of the amino terminal region of the mature protein should test the presence or absence of the signal peptide in the reconstructed gene for oryzacystatin I and II. We will design primer sequences for polymerase chain reaction (PCR) based on the melting temperature (Tm), intra- and inter-molecular hybridization potentials and other pertinent considerations. We will order the primers to Sigma-Genosis with the cost of 30 cents per base. We will introduce the genomic or cDNA clone of oryzacystatin under specific promoter and terminator sequences.

PROJECT CONTACT:

Name: Murai, N.
Phone: 225-578-1380
Fax: 225-578-1415
Email: nmurai@agcenter.lsu.edu  

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