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Methods and means for obtaining modified phenotypes

phenotype technology, applied in the field of reducing the phenotypic expression of a nucleic acid sequence of interest in eucaryotic cells, can solve the problems of difficult detection of the same plant and easy virus transmission

Inactive Publication Date: 2008-05-01
COMMONWEALTH SCI & IND RES ORG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides methods for reducing the expression of a nucleic acid of interest in a eucaryotic cell, particularly a gene or a foreign transgene. The methods involve introducing a chimeric DNA molecule into the cell, which contains a promoter and a DNA region that can be transcribed into a sense RNA molecule with a nucleotide sequence that matches at least a portion of the nucleotide sequence of the nucleic acid of interest. The chimeric DNA molecule also contains a DNA region that can be transcribed into an antisense RNA molecule with a nucleotide sequence that matches the complement of the sense RNA molecule. The resulting RNA molecules can form a double stranded RNA structure that reduces the expression of the nucleic acid of interest. The invention also provides methods for identifying the phenotype associated with the expression of a nucleic acid of interest in a eucaryotic cell.

Problems solved by technology

However, the same plants were susceptible to the virus if they contained no GUS sequences.
This hypothetical complementary RNA (cRNA) has not been detected (Baulcombe 1996) but it is expected that the cRNAs will be small and heterodisperse RNAs rather than full complements (Schiebel 1993ab, Baulcombe 1996) and therefore difficult to detect.

Method used

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  • Methods and means for obtaining modified phenotypes
  • Methods and means for obtaining modified phenotypes
  • Methods and means for obtaining modified phenotypes

Examples

Experimental program
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Effect test

example 1

Comparison of Chimeric Genes Comprising Only Antisense, Only Sense, or Both Sense and Antisense (Complimentary Pair (CoP)) Sequence for Reduction in Phenotypic Expression of an Integrated β-Glucuronidase Gene

[0174] Transgenic rice tissue expressing β-glucuronidase (GUS) from a single transgene (and hygromycin resistance from a hph gene) (lines V10-28 and V10-67) was supertransformed using vectors that contained the bar gene conferring phosphinothricin resistance and various sense, antisense and CoP constructs (see FIG. 1A) derived from a crippled GUS (GUSd) gene. The supertransformed tissue was maintained on hygromycin and bialaphos selection media for 3 weeks then analyzed for GUS activity. A crippled GUS gene was used so that expression from this gene would not be superimposed on the endogenous GUS activity.

[0175] The figures in Table 2 represent the rate of MU production measured by absorption at 455 nm, with excitation at 365 nm of 1.5 μg of total protein in a reaction volume ...

example 2

Comparison of the Efficiency of Using Chimeric Genes Comprising Only Antisense Genes, Only Sense Genes, or Both Genes Simultaneously for Obtaining Virus Resistance in Transgenic Plants

[0178] Gene constructs were made using the PVY protease encoding sequence SEQ ID No 1) in a sense orientation, an antisense orientation and in a complimentary pair (CoP) orientation, where the T-DNA comprised both the sense and antisense chimeric genes each under control of their own promoter. In all three arrangements the CaMV35S promoter was used. Five different versions of CoP constructs were made in which the second promoter was either the CaMV35S promoter, the S4 promoter, the double S4 promoter, the S7 enhanced S4 promoter, or the vascular specific rolC promoter (see FIG. 1B).

[0179] These constructs were transformed into tobacco (via Agrobacterium mediated DNA transfer) and approximately 25 independently transformed plants were recovered per chimeric gene construct. The transgenic plants were t...

example 3

Inheritance of Extreme Resistance in Plants from Example 2

[0182] Plants from Example 2 were allowed to self-fertilize and their seeds were collected. Seeds originating from plants showing extreme resistance and low transgene copy number for CoP constructs 35S-Nia / S4-AntisenseNia and 35S-Nia / 35S-AntisenseNia, and seeds from the sense and the antisense plants showing extreme resistance, were germinated and grown in the glasshouse. Plants were also grown from seed collected from two susceptible CoP lines, two susceptible sense gene only lines and two susceptible antisense gene only lines. Twenty plants from each line were selected for overall uniformity of size and development stage, put into individual pots, allowed to recover for one week, then inoculated with PVY. The plants were scored for virus symptoms 2, 4, and 7 weeks after inoculation. The results (Table 5) showed that all eight plant lines of 35S-Nia / S4-antisenseNia and 35S-Nia / 35S-antisenseNia containing one or two gene cop...

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Abstract

Methods and means are provided for reducing the phenotypic expression of a nucleic acid of interest in eucaryotic cells, particularly in plant cells, by introducing chimeric genes encoding sense and antisense RNA molecules directed towards the target nucleic acid, which are capable of forming a double stranded RNA region by base-pairing between the regions with sense and antisense nucleotide sequence or by introducing the RNA molecules themselves. Preferably, the RNA molecules comprises simultaneously both sense and antisense nucleotide sequence.

Description

1. FIELD OF THE INVENTION [0001] The invention relates to methods for reducing the phenotypic expression of a nucleic acid sequence of interest in eucaryotic cells, particularly plant cells, by simultaneously providing the cells with chimeric genes encoding sense and anti sense RNA molecules comprising nucleotide sequences respectively homologous and complementary to at least part of the nucleotide sequence of the nucleic acid of interest. The sense and antisense RNA molecules may be provided as one RNA molecule, wherein the sense and antisense RNA may be linked by a spacer nucleotide sequence and are capable of forming a double stranded RNA molecule. In one aspect of the invention, the methods are directed towards reducing viral infection, resulting in extreme virus resistance. In another embodiment the methods are directed towards reducing the phenotypic expression of an endogenous gene in a plant cell. The invention further relates to high throughput screening methods for identif...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01H5/00C12N15/82C12N15/87C12N5/04C12N5/06C12N7/00C12Q1/68
CPCC12N15/8247C12N15/8218
Inventor WATERHOUSE, PETER MICHAELWANG, MING-BOGRAHAM, MICHAEL WAYNESMITH, NEIL A.
Owner COMMONWEALTH SCI & IND RES ORG
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