US 2006/0143737 A1 · Method of controlling gene silencing using site specific recombination

Abstract

This invention relates to methods of controlling gene silencing using site-specific recombination. A variety of constructs are provided which are useful for conditional or regulated gene silencing in plants, comprising a suite of constitutive, inducible, tissue-specific or developmental stage-specific promoters operably linked to target sequences (TS). Recombinase inversion or excision yields double-stranded TS RNA, which thereby functions to trigger endogenous gene silencing mechanisms. By matching promoters, responsive to various inducers, plant tissues or plant developmental states with the recombinase systems, transcriptional stop fragments or introns and target sequences, gene silencing of virtually any target sequence may be modulated at any plant development stage or in any plant generation. This is especially useful, when genes responsible for gene silencing are down-regulated to permit expression of particular transgenes at levels greater than permitted when gene silencing is activated.

Claims38

Dependent claims

A gene silencing site-specific recombination system comprising:

a) a first recombinase element having the general structure P1-R, wherein P1 is a first promoter and R is a recombinase coding sequence and 3′ region;
and
b) a second gene silencing-recombinase element having the general structure:
RS-X-RS*-Y wherein:
i) RS and RS* are opposingly oriented recombinase sites responsive to the recombinase;
ii) X is a nucleic acid fragment comprising at least one second promoter in a 3′ to 5′ orientation;
and
iii) Y is a nucleic acid fragment comprising at least one target sequence;
wherein expression of the recombinase results in inversion of the element contained between RS and RS* and transcription of the second gene silencing-recombinase element resulting in the production of double-stranded RNA and silencing of the target gene.

6 more elements

A gene silencing site-specific recombination system comprising:

a) a first recombinase element having the general structure P1-R, wherein P1 is a first promoter and R is a recombinase coding sequence and 3′ region;
and
b) a second gene silencing-recombinase element having the general structure:
RS-TSINV-P2INV-RS*-TSINV-polyA, wherein:
i) RS and RS* are opposingly oriented recombinase sites responsive to the recombinase;
ii) TSINV is an inverted target sequence whose orientation is from 3′-5′;
iii) P2INV is an inverted second promoter whose orientation is from 3′-5′;
and
iv) polyA is the 3′ region of a gene;
wherein P1 and P2 are operably linked to their down stream elements and wherein expression of the recombinase results in inversion of the element contained between RS and RS* and transcription of the gene silencing-recombinase element resulting in production of double-stranded RNA and silencing of the target gene.

7 more elements

A gene silencing site-specific recombination system comprising:

a) a first recombinase element having the general structure P1-R, wherein P1 is a first promoter and R is a recombinase coding sequence and 3′ region;
and
b) a second gene silencing-recombinase element having the general structure RS-5′ Intron-TSINV-P2INV-RS*-3′ Intron-TSINV-polyA, wherein:
i) RS and RS* are opposingly oriented recombinase sites responsive to the recombinase;
ii) 5′ Intron is the N-terminal portion of an intron;
iii) TSINV is an inverted target sequence and whose orientation is from 3′-5′;
iv) P2INV is an inverted second promoter whose orientation is from 3-5′;
v) 3′ Intron is the C-terminal portion of an intron;
and
vi) polyA is the 3′ region of a gene;
wherein P1 and P2 are operably linked to their down stream elements and wherein expression of the recombinase results in inversion of the element contained between RS and RS* and transcription of the gene silencing-recombinase element, resulting in excision of the intron by mRNA splicing and production of double-stranded RNA and silencing of the target gene.

8 more elements

A gene silencing site-specific recombination system comprising:

a) A recombinase element having the general structure P1-R, wherein P1 is a first promoter and R is a recombinase coding sequence and 3′ region;
and
b) A gene silencing-recombinase element having the general structure P2-TS-5′ Intron-RS-STP-RS-3′ Intron-TSINV-polyA, wherein:
i) P2 is a second promoter;
ii) TS is a target sequence;
iii) 5′Intron is the N-terminal portion of an intron;
iv) RS is a recombinase site responsive to the recombinase;
v) STP is a Transcriptional ‘STOP’ fragment;
vi) 3′Intron is the C-terminal portion of an intron;
vii) TSINV is an inverted target sequence whose orientation is from 3′-5′;
and
viii) polyA is the 3′ region of a gene;
wherein P1 and P2 are operably linked to their down stream elements and wherein expression of the recombinase results in excision of the element contained between RS and transcription of the gene silencing-recombinase element resulting in excision of the intron by mRNA splicing and production of double-stranded RNA silencing the target gene.

10 more elements

A method for effecting systemic gene silencing in a plant comprising:

A) providing constructs comprising:
i) a first recombinase element having the general structure P1-R;
ii) a second gene silencing-recombinase element having a general structure selected from the group consisting
1) RS-P2INV-RS-TS-P3INV;
2) RS-TSINV-P2INV-RS-TSINV-polyA;
3) RS-5′ Intron-TSINV-P2INV-RS-3′ Intron-TSINV-polyA;
4) P2-TS-RS-STP-RS-TSINV-polyA;
or
5) P2-TS-5′ Intron-RS-STP-RS-3′ Intron-TSINV-polyA;
iii) a third gene-silencing-mobility element having the general structure P4-TSR;
wherein:
a) P1 is a first promoter that is chemically inducible;
b) R is a recombinase;
c) RS is a recombinase site responsive to the recombinase;
d) P2 and P3 are second and third promoters that have overlapping expression profiles and are either the same or different;
e) P2INV is an inverted second promoter whose orientation is from 3′-5′;
f) TS is a target sequence optionally having an operably-linked poly A region at the 3′ end of each complementary strand;
g) TSINV is an inverted target sequence whose orientation is from 3′-5′;
h) 5′Intron is the N-terminal portion of an intron;
i) 3′Intron is the C-terminal portion of an intron;
j) STP is a Transcriptional ‘STOP’ fragment;
and
k) polyA is the 3′ region of a gene;
l) P4 is a strong constitutive promoter;
and
m) TSR is redundant target sequence comprising at least a target sequence selected from the group consisting of TS and TSINV;
wherein P1, P2, P2INV, optionally P3, and P4 are operably linked to their down stream elements;
B) providing a plant having a target gene and comprising the first recombinase element, the second gene silencing-recombinase element, and the third gene silencing-mobility element;
and
C) chemically inducing the P1 promoter to cause inversion or excision of the elements between the recombinase sites on the second gene silencing-recombinase element permitting the formation of double-stranded RNA encoding the target sequence to silence the target gene and creation of a mobile gene-silencing signal in the plant for systemic silencing.

27 more elements

Dependencies are inferred from the claim text.

Method of controlling gene silencing using site specific recombination

Life of this patent

Method of controlling gene silencing using site specific recombination

Life of this patent

Abstract

Claims38