Biocidal protein

Details Biocidal protein Biocidal protein

2000-10-11

2003-11-25

Bugaisky, Gabriele (Department: 1653)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C536S023500, C536S023700, C435S069100, C435S320100

Reexamination Certificate

active

06653463

ABSTRACT:

BACKGROUND
Advances in biotechnology have enabled the generation of plants which express recombinant proteins. Thus, plants can be engineered to overproduce a variety of polypeptides with desirable qualities. Such polypeptide can include enzymes which produce secondary metabolites, proteins with medicinal or pharmaceutical properties, and proteins which endow the plants with new traits, for example, resistance to diseases, pathogens, and environmental conditions.
Given the vulnerability of agricultural crops to damage by insects, and other pests and pathogens, the ability to provide additional protective means and agents is of considerable importance. Moreover, traditional breeding techniques have identified plant lines with Mendelian traits endowing resistance to pests and pathogens. Modern molecular biological techniques can now be applied to isolate the critical nucleic acids and proteins with these properties in order to enhance the resistance of more sensitive plants or to antagonize the pests and pathogens in a variety of scenarios.
SUMMARY
The invention is based, in part, on the discovery of a novel mung bean nucleic acid which is expressed in a mung bean plant line that is resistant to insect attack, but not expressed in sensitive plant lines. The nucleic acid encodes a polypeptide which has insecticidal activity and which has similarity to thionin proteins. The sequence of the mung bean thionin nucleic acid (SEQ ID NO:1), designated as “VrCRP”, is shown below:
5′-ACCTCAACAATTCATCACTC
ATG
GAGAGAAAAACTTTCAGCTTCTTGTT CTCGCTCCTTCTCGTCTTAGCCTCTGATGTGGCCGTAGAGAGAGGAGAGGCTAGA ACTTGTATGATAAAGAAAGAAGGGTGGGGAAAATGCTTAATTGACACCACCTGT GCACATTCGTGCAAGAACCGCGGTTACATAGGTGGAGATTGCAAAGGCATGACG CGCACCTGCTATTGCCTCGTCAACTGT
TGA
ACCCTTTTCGAATATCATATCATCTT ATCACAAATAAATATAGCAGCATCACTGCTACTAGTACCGCCCTCCGCACCACG CCCT-3′
The initiator and terminator codons are underlined and in boldface. The sequence of the mung bean thionin polypeptide sequence (SEQ ID NO:2), designated as “VrCRP”, is shown below:
MERKTFSFLFSLLLVLASDVAVERGEARTCMIKKEGWGKCLIDTTCAHSCKNR GYIGGDCKGMTRTCYCLVNC
The invention is also based on the discovery the a polypeptide derived from VrCRP which has the VrCRP signal sequence removed is biologically active as an insecticide and fungicide. This polypeptide is encoded by the nucleic acid sequence (SEQ ID NO:3) below:
GAGAGAGGAGAGGCTAGAACTTGTATGATAAAGAAAGAAGGGTGGGGAA AATGCTTAATTGACACCACCTGTGCACATTCGTGCAAGAACCGCGGTTACATAGGT GGAGATTGCAAAGGCATGACGCGCACCTGCTATTGCCTCGTCAACTGTTGA
The polypeptide sequence (SEQ ID NO:4) of this form of VrCRP lacking the signal sequence is shown below:
ERGEARTCMIKKEGWGKCLIDTTCAHSCKNRGYIGGDCKGMTRTCYCLVNC
Accordingly, in one aspect, the invention features isolated nucleic acid sequences which include a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO:1. The nucleic acids of the invention can further include nucleic acids which hybridize under stringent conditions to SEQ ID NO:1, as well as nucleic acids which are at least 50% identical, e.g., at least 60%, 70%, 80%, 90%, or 95% identical, to SEQ ID NO:1. Such nucleic acid sequences can encode a polypeptide which inhibits translation of messenger RNAs in a wheat germ extract, a polypeptide which has insecticidal activity, e.g., insecticidal activity against bruchids such as
Callosobruchus chinensis, Callosobruchus maculates
, and
Zabrotes subfasciatu
, or a polypeptide which has anti-fungal activity, e.g., against
Rhizoctonia solani.
In another aspect, the invention features polypeptides comprising the amino acid sequence of SEQ ID NO:2. Featured polypeptides also include polypeptides which are at least 50% identical, e.g., at least 60%, 70%, 80%, 90%, or 95% identical, to SEQ ID NO:2. Such polypeptides can have at least one, two, three, four, five, eight, ten, twelve, or twenty conservative amino acids substitutions. The polypeptides can inhibit translation of messenger RNAs in a wheat germ extract, can have insecticidal activity, e.g., insecticidal activity against bruchids such as
Callosobruchus chinensis, Callosobruchus maculates
, and
Zabrotes subfasciatus
, or can have anti-fungal activity, e.g., against
Rhizoctonia solani
. Also encompassed by the invention are nucleic acid sequences encoding such polypeptides.
The featured polypeptides can be recombinant and/or purified. For example, they can be overexpressed in a variety of host cells, such as
E. coli
, Sf9 insect cells, plant cells and mammalian tissue culture cells using overexpression vectors known in the art. Lysates are made from the host cells, e.g., after overexpression is induced if induction is required. The polypeptides are purified from the lysate. Alternatively, the polypeptides are secreted, by the inclusion nucleic acid sequences encoding the signal peptide or a heterologous signal peptide. In another example, the featured polypeptides are encoded by a transgene and overproduced in a plant or a plant tissue. The plant is harvested and the polypeptides purified from the plant.
The purified and/or recombinant polypeptides can be formulated a composition. The composition can include an agriculturally acceptable carrier, e.g., one described below. The composition can contain the polypeptide at a concentration of about 0.005% to 10%, or about 0.01% to 1%, or about 0.1% to 0.5% by weight of composition. The composition can further include a cyclopeptide alkaloid, e.g., vignatic acid A and vignatic acid B, e.g., a cyclopeptide alkaloid with insecticidal properties. The composition can also include other desirable compounds, e.g., protease inhibitors, endotoxins, and the like. The contemplated compositions can have insecticidal activity, e.g., against bruchids such as
Callosobruchus chinensis, Callosobruchus maculates
, and
Zabrotes subfasciatu
, and/or anti-fungal activity, e.g., against
Rhizoctonia solani
. The compositions can be applied to plants and their environs by methods described below.
The nucleic acids of the invention can also include a heterologous promoter such that the promoter is operably linked to a coding genomic nucleic acid or a cDNA. The promoter can direct transcription of the nucleic acid in wounded or pathogen infected cells. The promoter can be induced by a signalling molecule, e.g., methyl jasmonate, salicylic acid, ethylene, absiscic acid, gibberillins, HgCl
2
, and H
2
O
2
. The invention also features transformed cells which contain such nucleic acids, i.e., an aforementioned nucleic acid operably linked to a heterologous promoter. Also included are transgenic plants whose genomic DNA includes such nucleic acids, as are transgenic seeds from such plants.
A “purified polypeptide”, as used herein, refers to a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated. The polypeptide can constitutes at least 10, 20, 50 70, 80 or 95% by dry weight of the purified preparation.
An “isolated nucleic acid” is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid, or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of different (i) DNA molecules, (ii) transfected cells, or (iii) cell clones: e.g., as thes

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