Peroxidase gene sequences

Details Peroxidase gene sequences Peroxidase gene sequences

1999-07-30

2001-08-21

Bui, Phuong T. (Department: 1649)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide confers pathogen or pest resistance

C800S278000, C800S301000, C435S069100, C435S410000, C435S418000, C435S419000, C435S252300, C435S320100, C435S183000, C530S370000, C536S023100, C536S023600, C536S024100

Reexamination Certificate

active

06278041

ABSTRACT:

FIELD OF THE INVENTION
The present invention generally relates to novel peroxidase gene sequences and more particularly to synthetic peroxidase gene sequences optimized for expression in plants. The present invention also relateds methods of making and methods of using the novel peroxidase gene sequences to control insects.
BACKGROUND OF THE INVENTION
Insect pests are a major factor in the loss of the world's commercially important agricultural crops. Broad spectrum chemical pesticides have been used extensively to control or eradicate pests of agricultural importance. Although insecticides have been effective in controlling most harmful insects, there are considerable problems associated with the use of these compounds. Insecticides are expensive and costly to apply. Often repeated applications are necessary for effective control. There is also concern that insects have or will become resistant to many of the chemicals used in controlling them. Insecticides often kill beneficial insects which are pollinators or prey on the herbivorous insects. Additionally, there are environmental hazards associated with the long term use of chemical insecticides.
Programs of pest management are being introduced which lower the use of chemical insecticides. These programs include the improvement of crops by selection, the employment of biological control agents and insect predators, and the incorporation of insect resistant genes through breeding programs and genetic engineering. The most widely utilized genes for genetic engineering are the crystal protein genes from
Bacillus thuringiensis
. See, for example, Rice et al., EP-A-292 435 and Koziel et al., WO 93/07278. The majority of the crystal proteins made by Bacillus are toxic to larvae of insects in the orders Lepidoptera, Diptera and Coleoptera. In general, when an insecticidal crystal protein is ingested by a susceptible insect, the crystal is solubilized and acts as a toxic moiety. To avoid the development of insects which are resistant to these toxins, additional toxins are needed which have additive or synergistic affects.
Peroxidases are a subclass of oxido-reductases that use a peroxide such as H
2
O
2
as substrate. Peroxidases are heme-containing monomeric glycoproteins able to bind divalent cations (mainly Ca
2+
, but also Mn
2+
) (Maranon and Van Huystee,
Phytochemistry
37: 1217-1225 (1994)). The prosthetic groups for peroxidase have different roles. While the heme group is involved in catalysis, the divalent cations stabilize the heme moiety, and the glycosyl groups may help to stabilize the peroxidase by decreasing its turnover rate (Maranon and Van Huystee,
Phytochemistry
37: 1217-1225 (1994)).
Peroxidases are often grouped into anionic, cationic, and neutral forms according to their migration on isoelectric focusing gels. Although as enzymes they are considered to have wide substrate specificity, they do appear to have some substrate “preferences” for different isoenzymes (Van Huystee,
Ann. Rev. Plant Physiol
., 205-219 (1987)). There are several types of peroxidases and related enzymes including guaiacol peroxidase, NADH peroxidase, cytochrome-C peroxidase, catalase, glutathione peroxidase, L-ascorbate peroxidase, and manganese peroxidase.
In plants, peroxidases are monomeric proteins which are highly complex enzymes whose activities are closely regulated by the plant. Peroxidases are critical in the biosynthesis of plant cell walls. Peroxidases promote the peroxidative polymerization of the monolignols coniferyl, r-coumaryl, and sinapyl alcohol into lignin (Greisbach, In: The Biochemistry of Plants, Ed. Conn, Academic, New York pp. 457-480 (1991)). Different plant species have varying ratios of the monolignol species assembled in a semi-random fashion (Hwang et al.,
Carbohydrate Polymers
14:77-88 (1991)). Lignification serves to strengthen and reinforce cell walls. The overall result is a toughening of the plant tissue.
A tobacco anionic peroxidase has been utilized to transform
N. tabacum
and
N. sylvestris
(Lagrimini,
Plant Cell
2:7-18 (1990); Lagrimini,
Plant Physiology
96:577-583 (1991); both of which are incorporated herein by reference). These transgenic plants constitutively overexpressed a tobacco anionic peroxidase from a CaMV 35S promoter. The same construct has also been utilized to transform tomato plants (Lagrimini et al.,
J. Am. Soc. Hort. Sci
. 117:1012-1016 (1992); Lagrimini et al.,
Hortscience
28:218-221(1993); both of which are incorporated herein by reference). Some tissues of these transgenic dicotyledonous plants expressing a tobacco anionic peroxidase gene were resistant to some insects (Dowd et al., presentation at the National Meeting of the Entomological Society of America, Indianapolis, December 1993).
In addition, the tobacco anionic peroxidase has been utilized to transform
Zea maize
(WO 98/27218). These transgenic plants constitutively overexpressed a tobacco anionic peroxidase from a CaMV 35S promoter. Maize leaves expressing the tobacco anionic peroxidase enzyme conferred 100% mortality against
Ostrinia nubilalis
(European corn borer) and
Heliothis zea
(Corn earworm), and also had a strong antifeeding effect against
Spodoptera frugiperda
(Fall armyworm).
Despite the previous successes realized by incorporation of insect resistant genes through breeding programs and genetic engineering, there remains a long-felt but unfulfilled need to discover new and effective insect control agents. Particularly needed are control agents that are targeted to economically important insect pests and that efficiently control insect strains resistant to existing insect control agents. Furthermore, agents whose application minimizes the burden on the environment are desirable.
SUMMARY OF THE INVENTION
The present invention addresses the aforementioned needs by providing novel peroxidase gene sequences, including synthetic peroxidase gene sequences optimized for expression in plants. The peroxidase enzymes encoded by the novel gene sequences are highly active against economically important insect pests such as
Ostrinia nubilalis
(European corn borer),
Heliothis zea
(Corn earworm), and
Spodoptera frugiperda
(Fall armyworm). The peroxidase enzymes can be used in multiple insect control strategies, resulting in maximal efficiency with minimal impact on the environment.
Hence, in one embodiment, the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a
Nicotiana tomentisiformis
anionic peroxidase. In another embodiment, the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence that encodes an anionic peroxidase that comprises the amino acid sequence set forth in SEQ ID NO:2. In a particularly preferred embodiment, the present invention is directed to an isolated nucleic acid molecule comprising a nucleotide sequence that encodes an anionic peroxidase, wherein the nucleotide sequence is SEQ ID NO:1.
The present invention is further directed to a nucleic acid molecule comprising a synthetic nucleotide sequence that encodes an anionic peroxidase, wherein the synthetic nucleotide sequence has been optimized for expression in plants. In a preferred embodiment, the synthetic nucleotide sequence is SEQ ID NO:3 or SEQ ID NO:4.
The present invention is further directed to an isolated nucleic acid molecule comprising a nucleotide sequence that encodes an anionic peroxidase, wherein said nucleotide sequence comprises a 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion of SEQ ID NO:3 or SEQ ID NO:4.
The present invention also provides a chimeric gene comprising a heterologous promoter sequence operatively linked to a nucleic acid molecule of the invention. Further, the present invention provides a recombinant vector comprising such a chimeric gene. Still further, the present invention provides a host cell comprising such a chim

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