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Chemically inducible cucumber mosaic virus protein expression system

a technology of chemical induction and plant protein, which is applied in the field of plant molecular biology and plant breeding, can solve the problems of low infection efficiency, risk of releasing competent recombinant virus vectors into the environment, and hindering the use of recombinant plant viruses, and achieves neither increased nor decreased raat production. , the effect of increasing the raat level

Active Publication Date: 2012-01-05
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is a new system for inducing the expression of genes in plants using a Cucumber mosaic virus (CMV) inducible viral amplicon (CMViva). This system allows for tightly regulated and chemically inducible expression of genes in plants. The CMViva expression system is controlled by a CMV replicase gene that is activated by the addition of a chemical inducer. The system can produce high levels of functional proteins in plants, with up to 30-fold more rAAT being produced compared to the levels obtained using a constitutive promoter. The CMViva expression system can be stably integrated into the plant genome or transiently expressed. The invention also provides a method for producing heterologous proteins in plants by contacting them with a bacterial cell containing the CMViva expression system. The heterologous gene can encode for other proteins, RNAs, or enzymes.

Problems solved by technology

Despite the ease and short turnaround time for transient production, use of recombinant plant viruses has been hindered by various shortcomings, including low infection efficiency, the risks associated with the release of the competent recombinant virus vectors into the environment (Manske and Schiemann, 2005), limitations on the size of the transgene, and transgene instability (Rabindran and Dawson, 2001; Shivprasad et al., 1999).
Production of heterologous proteins utilizing constitutively-expressed viral amplicons has also been investigated in stably transformed plants, however, low recombinant viral levels, and low product titers resulting from post-transcriptional gene silencing (PTGS) have been continuously problematic (Angell and Baulcombe, 1997; Mori et al., 1993).
However, low recombinant viral levels, and low product titers resulting from post-transcriptional gene silencing continue to be a problem.
Although there have been advances in improving the activity of enzymes involved in the degradation of lignocellulosic biomass, design of engineered cellulase mixtures, development of more productive host strains, and utilization of inexpensive medium components, the high cost of producing, recovering and formulating cellulase enzymes using traditional fungal or bacterial fermentation continues to impact the economics of ethanol production from lignocellulosic biomass.
Microbial fermentations require large energy inputs due to agitation, aeration, temperature control and in some cases cell disruption and enzyme recovery, as well as high capital equipment costs for fermentors and downstream processing unit operations.
The application of cellulases often requires pretreatment of lignocellulosic biomass to facilitate accessibility of the enzymes to the complex substrate, again requiring additional energy inputs and / or costly treatment of acid / base wastes.

Method used

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  • Chemically inducible cucumber mosaic virus protein expression system
  • Chemically inducible cucumber mosaic virus protein expression system
  • Chemically inducible cucumber mosaic virus protein expression system

Examples

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example 1

Construction of the CMViva Expression System 1

[0088]The following materials were used in this example:

[0089]The complete cDNA clones, pQCD1, pQCD2, and pQCD3, corresponding to genomic RNAs 1, 2 and 3, of CMV strain Q (Ding et al., 1995), respectively.

[0090]The binary plasmid vectors pER8 (Zuo et al., 2000) and pER10 containing the estradiol inducible expression system (XVE system); plasmid pER10 is similar to pER8 except it contains nptII as the selectable marker instead of the hygromycin resistance gene.

[0091]The binary vector p35S:p19 (Voinnet et al., 2003) containing the TBSV p19 gene

[0092]The binary vectors pDU97.1005 (Uratsu and Dandekar, unpublished), a modified version of pCGN1547 (McBride and Summerfelt, 1990)), pDU99.2215 (Escobar et al., 2001) and pART7 (Gleave, 1992).

[0093]A. tumefaciens strain EHA105:pCH32 (Hamilton, 1997) was used to carry all the binary vectors used in this study except for p35S:p19, where A. tumefaciens strain C58C1 was used.

[0094]A series of binary p...

example 2

Construction of the CMViva Expression System 2

[0095]Whereas the CMViva expression system in Example 1 is a binary pCMV-SPAAT plasmid (FIG. 2B), CMViva expression system can also be two separate binary plasmids. The first binary plasmid is the pDUR22XLR1R (FIG. 27); the second binary plasmid is comprised of a series of ten plasmids containing the AscI fragment for pQA (FIG. 28) cloned into pDU97 giving plasmids pQA-2, pQA-4, pQA-6, pQA-7, pQA-8, pQA-9, pQA-10, pQA-11, pQA-12, and pQA-13, collectively called the pQA series hereafter. The pQA series is modified to have restriction sites including, but not limited to, Eco RI-Sac, I-Kpn, 1-Sma, I-BamHI-Xba, I-Acc, I-Sal, I-Pst, and I-SphI-Hind III. The heterologous gene is cloned into the multiple cloning site (MCS) of one or all of the pQA series of plasmids. The different restriction sites used for cloning the recombinant genes allow for easier and more efficient cloning and expression of the heterologous genes, as well as for the iden...

example 3

Agroinfiltration and Induction of Transactivation

[0098]A. tumefaciens EHA105:pCH32 cells containing the appropriate plasmids were grown for 24 to 48 hours in 2 ml LB broth. Approximately 0.5 ml was then transferred to 25 ml LB medium supplemented with 10 μl of 100 mM acetosyringone (3′,5′-dimethoxy-4′-hydroxyacetophenone) (Aldrich Chemicals, Milwaukee, Wis.) and 0.5 ml of MES buffer (pH 5.6) and grown overnight at 28° C. with shaking until cell density (0D600) reached 1.0 absorbance units. Cells were harvested by centrifuging at 2600 g, resuspended in 10 ml sterile de-ionized water, and cell density was adjusted to 1.0 absorbance units. Five ml of the A. tumefaciens cell suspension for each plasmid (p35S-SPAAT, pXVE-SPAAT, and pCMV-SPAAT) was separately mixed with either five ml of sterile water or A. tumefaciens cells containing the p35S:p19 plasmid, then supplemented with magnesium chloride to reach a final concentration of 10 mM and acetosyringone to 150 μM and incubated at room ...

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Abstract

The invention relates to a novel chemically inducible plant viral amplicon (CMViva) expression system that permits controllable, high level expression of foreign genes in plant hosts. This system employs agro-infiltration of plants to provide a transient production of a protein of interest, such as a human blood protein. This system provides a major advantage over existing plant expression systems because it allows for consistent expression of foreign or heterologous proteins in plant hosts.

Description

RELATED APPLICATION[0001]This application claims the benefit of U.S. Provisional Application No. 60 / 846,704, filed on Sep. 22, 2006, which is incorporated by reference herein in its entirety.GOVERNMENT INTEREST[0002]This invention was made with Government support under Grant (or Contract) No. BES-0214527 awarded by the National Science Foundation and NSF REU. The US Government has certain rights in the invention.FIELD OF THE INVENTION[0003]This invention is in the field of plant breeding and plant molecular biology. In particular, this invention relates to a novel chemically inducible plant expression system that allows controllable, high level expression of foreign genes in plant hosts.BACKGROUND OF THE INVENTION[0004]Plants offer enormous potential for production of recombinant forms of therapeutics and diagnostics for humans and animal applications, for example, human blood proteins, antibodies, vaccines, and the like. Plants have the ability to perform complex post-translational...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P21/00C12N9/00C12N5/04C12N15/83A01H5/00
CPCC07K14/8125C12N15/8203C12N15/8257C12N15/8217C12N15/8238C12N15/8216
Inventor MCDONALD, KAREN A.DANDEKAR, ABHAYAFALK, BRYCE W.SUDARSHANA, MYSORE R.URATSU, SANDRA L.PLESHA, MICHAEL A.HUANG, TING-KUO
Owner RGT UNIV OF CALIFORNIA
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