LAB93770- Application of Molecular Biology to Rice: Oryzacystatin Expression

Robert Carver  |  11/21/2008 3:14:25 AM

ACCESSION NO: 0205599 SUBFILE: CRIS
PROJ NO: LAB93770 AGENCY: CSREES LA.B
PROJ TYPE: HATCH PROJ STATUS: NEW
START: 01 OCT 2005 TERM: 30 SEP 2010 FY: 2007

INVESTIGATOR: Murai, N.

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

APPLICATION OF MOLECULAR BIOLOGY TO RICE: ORYZACYSTATIN EXPRESSION

CLASSIFICATION
KA Subject Science Pct
201 1530 1040 100

CLASSIFICATION HEADINGS: R201 . Plant Genome, Genetics, and Genetic Mechanisms; S1530 . Rice; F1040 . Molecular biology

BASIC 70% APPLIED 20% DEVELOPMENTAL 10%

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.

KEYWORDS: molecular biology; gene transfer; agrobacterium tumefaciens; binary vector; rice genomics; rice chromosome 1 and 5; oryzacystatin; arabidopsis thaliana; arabidopsis genomics; arabidopsis chromosome 5; functional genomics; protein purificaiton

PROGRESS: 2007/01 TO 2007/12
OUTPUTS: 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 this basic understanding to genetic improvement in rice cultivars. Toward this end we proposed to enhance the level of expression and recovery of oryzacystatin or cystatin (cysteine protease inhibitor) from the rice grain. A first objective of this proposal is to construct new binary vectors of the Ti plasmid of Agrobacterium tumefaciens. This objective is a necessary first step since we are required to obtain licensing agreements to use the existing vectors for commercial applications of project findings. We have the 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. The oryzacystatin genes will be constructed using polymerase chain reaction (PCR) 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. Up to this date we have made a reasonable progress toward achieving the first objective of this research project. However, we will not discuss the details of our construction process and final products until the results are successful. PARTICIPANTS: The project has provided opportunities for training and professional development of two graduate students in my laboratory. Seokhyun Lee is at a final stage of Ph.D. degree, and Guiying Su is a first year Ph.D. student. Professor Jack N. Losso of Food Science Department is one of our main collaborators at LSU. TARGET AUDIENCES: Plant biology, biotechnology and agricultural science communities. PROJECT MODIFICATIONS: No major changes in approach for the current project.

IMPACT: 2007/01 TO 2007/12
The seed storage protein phaseolin accumulates in native common bean (Phaseolus vulgaris L.) as a trimeric glycoprotein in the vacuolar protein bodies of developing cotyledon. Here we characterized the post-translational modifications of phaseolin, glycosylation and trimer formation, after expression in Sf9 cells of fall armyworm (Spodoptera frugiperda) infected by baculovirus. When a cDNA for the mature phaseolin protein (without its own signal peptide) was placed under control of the signal peptide of viral protein GP67 in baculovirus transfer vectors pAcGP67A, phaseolin accumulated within cells at a high level (40 microg/mL). To facilitate the protein purification, six histidines were added to the carboxyl terminal of phaseolin coding sequence as a metal-chelating affinity tag. Phaseolin was extracted from Sf9 cells by 6.0 M guanidinium chloride or 4.0 M urea as a protein solubilizing agent not as a denaturant, and purified by step-wise elution from a nickel column. Phaseolin was modified with a high-manose glycan at two potential glycosylation-sties in insect Sf9 cells as demonstrated by digestion with endoglycosidare H and peptide N-glycosidase F. Asn228 and Asn317 of two potential glycosylation-sites were converted either singly or simultaneously to Glu by site-directed mutagenesis of the cDNA. Similar amounts of wild-type and glycosylation-minus mutants were purified from Sf9 cells. Analytical equilibrium centrifugation analysis demonstrated trimer formation of both wild-type and glycosylation-minus phaseolin. The results indicate that glycosylation is not required for the protein stability or trimer formation of phaseolin. When phaseolin was expressed under control of its own signal peptide in a second transfection vector pAcSG2, phaseolin was accumulated within cells similarly to the first constructs. However, elimination of two but not one glycosilation-sites resulted in the endoproteolytic cleavage (s) of the mature protein. Circular dichroism analysis indicated the proper secondary structure formation of phaseolin in insect Sf9 cells. Taken together, phaseolin was glycosylated with a high-manose glycan, folded into the proper tertiary structure, and assembled into a trimer in insect Sf9 cells, providing a useful source of a large quantity of homogeneous protein to be used for further structural analysis.

PUBLICATIONS (not previously reported): 2007/01 TO 2007/12
The above results have been written in a refereed article format to be submitted in 2008 to either Plant Physiology, Canadian Journal of Botany or Open Plant Science Journal.

PROGRESS: 2006/01/01 TO 2006/12/31
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 this basic understanding to genetic improvement in rice cultivars. Toward this end we proposed to enhance the level of expression and recovery of oryzacystatin or cystatin (cysteine protease inhibitor) from the rice grain. A first objective of this proposal is to construct new binary vectors of the Ti plasmid of Agrobacterium tumefaciens. This objective is a necessary first step since we are required to obtain licensing agreements to use the existing vectors for commercial applications of project findings. 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. The oryzacystatin genes will be constructed using polymerase chain reaction (PCR) 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. Up to this date we have made a reasonable progress toward achieving the first objective of this research project. However, we will not discuss the details of our construction process and final products due to patent considerations. Implicit with the first objective is an improved efficiency for A. tumefaciens-mediated transformation of rice. Initially we have tested a number of experimental parameters using leaf disks of tobacco (Nicotiana tabacum L. cv. Xanthi) for the ease of culture. The test results from tobacco should be extended to rice with minor modifications. We found that co-cultivation temperature at 20 C is one the most critical factor to obtain the reproducible enhancement of GUS activity. pCAMBIA 1305.01 resulted in higher GUS activity than the other two pCAMBIA vectors 1301 and 1305.02. The highest GUS activity and transformation efficiency were achieved under the following experimental conditions: A. tumefaciens strain LBA4404 containing pCAMBIA1305.01 was grown overnight at 28 C in liquid Agrobacterium media, and the concentration was adjusted to 3x107 cells/mL (0.3 A600 units/mL). Tobacco leaf disks were inoculated with bacteria under 50 mm Hg vacuum infiltration for 20 min in the presence of 0.001% (w/v) Silwet L-77. Leaf disks were co-cultivated for four days under constant light at 20 C in MS shoot media containing 200 mM acetosyringone without antibiotics. Leaf disks were then transferred to MS shoot selection media containing 50 mg/L hygromycin and 500 mg/L carbenicillin, and grown for an additional 14 days under constant light at 25 C. b-Glucuronidase (GUS) activity was measured at the end of the growth period by quantitative GUS assay and GUS histochemical staining.

IMPACT: 2006/01/01 TO 2006/12/31
A. tumefaciens-mediated transformation has been generally used for genetic transformation of higher plants since 1983. The transformation procedures appear to be routine for some model plants, but not for the most of crop plants. We have learned a hard lesson in 1989 when we attempted to generate a large number of transgenic tobacco plants to study the effect of 5'-deletion mutation on the promoter activity of the bean seed storage protein phaseolin gene. We aimed to gererate a minimum of ten independent transgenic plants for seven deletion constructs. We repeated eight tobacco transformation experiments and found out only two worked successfully to generate sufficient number of transgenic plants. At the moment we had no clue as to why we had to waste so much time and effort. Based on the results from our experiments we now knew why. We found that a co-cultivation temperature at 20 C was one of the most critical factors to achieve reproducibility and consistency of GUS activity expression after A. tumefaciens-mediated transformation of tobacco leaf disks. We believe the controlled temperature envirnment at 20 C for co-cultivation is one of the critical conditions for efficient transformation of other crop plants, including the Louisiana's major corps rice, sugarcane, soybean and cotton. Among the five temperatures tested from 15, 18, 20, 22 to 25 C, co-cultivation at 18, 20 and 20 C resulted in statistically higher GUS activity than that at 25 C. Co-cultivation at 15 C resulted in a statistically intermediate value between the highest and lowest GUS activities.

PUBLICATIONS: 2006/01/01 TO 2006/12/31
Park, S. 2006. Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L.) leaf disks: Evaluation of the co-cultivation conditions to increase b-glucuronidase activity. M. S. Thesis. Louisiana State University etd-07052006-173930, Baton Rouge, LA.

PROJECT CONTACT:

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