Whitefly ecdysone receptor nucleic acids, polypeptides, and uses thereof

a technology of ecdysone receptor and whitefly, applied in the field of biotechnology, can solve the problems of significant effect on other animals, significant effect on insect populations, and ineffective regulation of reporter gene expression

Inactive Publication Date: 2008-10-30
PRECIGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ecological side effects of these pesticides, due in part to their broad activity and lack of specificity, and in part, to the fact that some of these pesticides are not easily biodegradable, significantly affect populations of both insect and other species of animals.
Furthermore, as the insect populations evolve in directions to minimize the effects of the applied pesticides, the amounts of pesticides applied are often elevated so high as to cause significant effects on other animals, including humans, which are affected directly or indirectly by the application of the pesticides.
Unfortunately, these USP-based systems are constitutive in animal cells and therefore, are not effective for regulating reporter gene expression.
Drawbacks of the above described EcR-based gene regulation systems include a considerable background activity in the absence of ligands and non-applicability of these systems for use in both plants and animals (see U.S. Pat. No. 5,880,333).

Method used

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  • Whitefly ecdysone receptor nucleic acids, polypeptides, and uses thereof
  • Whitefly ecdysone receptor nucleic acids, polypeptides, and uses thereof
  • Whitefly ecdysone receptor nucleic acids, polypeptides, and uses thereof

Examples

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example 1

[0284] This Example describes the cloning of full-length cDNA encoding whitefly Bamecia argentifoli ecdysone receptor polypeptide. To isolate the full length coding sequence of this previously unknown whitefly ecdysone receptor isoform (herein named “BaEcR”), a whitefly cDNA library prepared from total RNA obtained from mixed stage whitefly nymphs (first to fourth instars) and pupa was used. Briefly, the cDNA library was constructed in the UNI-Zap XR™ vector using Zap Express cDNA Gigapack II Gold™ cloning kit (Stratagene, La Jolla, Calif.) following the manufacturer's instructions. Applicants used degenerate oligonucleotides (see Table 2; SEQ ID NOs: 3 and 4) designed based on conserved regions of ecdysone receptor C (KKCLSVGM; SEQ ID NO: 5) and E (KLIREDQI; SEQ ID NO: 6) domains to amplify and obtain a 441 base pair (bp) cDNA fragment (SEQ ID NO: 7) from whitefly total RNA using RT-PCR.

Primer NucleicPrimer and SEQ ID NO:Acid SequencePrimer 1 (SEQ ID NO: 3)5′-aa(a / g)aa(a / g)tg(t / c...

example 2

[0288] This Example describes the construction of whitefly ecdysone receptor gene expression cassettes and their use in an ecdysone receptor-based gene expression modulation system. The results presented herein demonstrate that a whitefly ecdysone receptor is functional in an ecdysone receptor-based gene expression modulation system in both insect and mammalian cells.

A) Insect Cells:

[0289] Briefly, the BaEcR CDE domains (amino acids 53416 of SEQ ID NO: 2) were fused to a VP16 transactivation domain (SEQ ID NO: 8) as follows. A construct was prepared by fusing a polynucleotide (nucleotides 259-1349 of SEQ ID NO: 1) encoding a BaEcR-CDE polypeptide to a polynucleotide (SEQ ID NO: 9) encoding a VP16 activation domain at the NH2 terminal end. This VP16BaEcR fusion was then cloned under the control of baculovirus IE1 promoter (SEQ ID NO: 10). The VP16BaEcR gene expression cassette was transfected into L57 cell line (Drosophila cell line that lacks endogenous EcR) and a cotton boll wee...

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Abstract

The present invention relates to a novel isolated whitefly ecdysone receptor polypeptide. The invention also relates to an isolated nucleic acid encoding the whitefly ecdysone receptor polypeptide, to vectors comprising them and to their uses, in particular in methods for modulating gene expression in an ecdysone receptor-based gene expression modulation system and methods for identifying molecules that modulate whitefly ecdysone receptor activity.

Description

FIELD OF THE INVENTION [0001] This invention relates to the field of biotechnology. Specifically, this invention relates to isolated nucleic acids, vectors comprising them, and polypeptides encoded by them, and to their use in the field of gene expression and insecticide discovery. More specifically, this invention relates to a novel nucleic acid encoding an ecdysone receptor polypeptide from the homopteran whitefly (Bamecia argentifoli, “BaEcR”) and its use in methods of modulating the expression of a gene within a host cell using BaEcR, and in methods of identifying molecules that modulate the activity of the BaEcR BACKGROUND OF THE INVENTION [0002] Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties. However, the citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application. [0003] Cultivated agriculture has greatly increased efficien...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01N37/18C07H21/04C07K14/72C12N15/09A61K31/7105A61K35/76A61K38/00A61K39/395A61K48/00A61P33/14A61P43/00C07K14/435C07K14/705C07K16/28C12N1/15C12N1/19C12N1/21C12N5/02C12N5/10C12P21/02C12P21/08C12Q1/02
CPCC07K14/43577G01N2333/723G01N33/566A61P33/14A61P43/00
Inventor ZHANG, JIANZHONGCRESS, DEAN ERVINPALLI, SUBBA REDDYDHADIALLA, TARLOCHART SINGH
Owner PRECIGEN INC
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