Plant gene construct encoding a protein capable of disrupting the biogenesis of viable pollen

• US 6,172,279 B1
• Filed: 11/07/1995
• Issued: 01/09/2001
• Est. Priority Date: 01/26/1989
• Status: Expired due to Fees

• Current Assignee: Zeneca Incorporated (Astrazeneca PLC)
• Original Assignee: Zeneca Incorporated (Astrazeneca PLC)
• Inventors: Bright, Simon William Jonathan, Bridges, Ian George, Schuch, Wolfgang Walter, Greenland, Andrew James
• Primary Examiner(s): Benzion, Gary
• Application Number:
  US08/554,811
• Time in Patent Office: 1,890 Days
• Field of Search: 435/172.3, 435/172.1, 536/24.1, 536/23.5, 536/23.6, 536/23.7, 47/58, 800/205, 800/320.1, 800/268, 800/271, 800/274, 800/303
• US Class Current: 800/274
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/415 from plants
• CPC Class Codes: C07K 14/4703 Inhibitors; Suppressors
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/73 Expression systems using phage (lambda) regulatory sequences
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8217 Gene switch
• CPC Class Codes: C12N 15/8222 Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
• CPC Class Codes: C12N 15/8231 Male-specific, e.g. anther, tapetum, pollen
• CPC Class Codes: C12N 15/8237 Externally regulated expression systems
• CPC Class Codes: C12N 15/8238 chemically inducible, e.g. tetracycline
• CPC Class Codes: C12N 15/8289 Male sterility
• CPC Class Codes: C12N 2795/10022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 9/1088 Glutathione transferase (2.5.1.18)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: Y10S 47/01 Methods of plant-breeding and including chromosome multiplication

Abstract

Male sterility is imparted to a plant by a cascade of gene sequences which expresses a protein which disrupts the biosynthesis of viable pollen. Expression of the disrupter protein is restricted to male parts of the plant by an upstream promoter sequence which is specific to male flowers, the male specific promoter being under control of an operator sequence. The cascade also includes a gene encoding a repressor protein specific for that operator. Expression of the repressor protein is under control of a chemically inducible promoter which is inducible by the application to the plant by, spraying or like process, of an exogenous chemical. In the absence of the exogenous chemical inducer, no repressor protein is expressed, resulting in expression of the disrupter protein and, consequently, male sterility. Fertility may be restored to the plant, when required for maintenance of the line, by spraying with the inducer, resulting in expression of the repressor which binds the operator and inhibits expression of the disrupter protein.

Claims

1. An expression system comprising:

(a) a first promoter sequence responsive to the presence or absence of an exogenous chemical inducer;

(b) a first structural gene encoding a repressor protein operably linked to and under the control of said first promoter sequence;

(c) an operator sequence responsive to the repressor protein expressed by said first structural gene sequence;

(d) a male flower specific second promoter sequence operably linked to and under the control of said operator sequence; and

(e) a second structural gene sequence encoding a protein which when expressed disrupts pollen biogenesis operably linked to said second promoter sequence.

2. The expression system of claim 1 in which said first promoter sequence is a promoter of a 27 kd subunit isolated from the maize glutathione-S-transferase (GSTII) gene.

3. The expression system of claim 2 in which the exogenous chemical inducer for the first promoter is selected from the group consisting of N,N,dially1-2,2-dichloro-acetamide and benzyl -2-chloro-4-(trifluoro-methyl)-5-thiazole-carboxylate.

4. The expression system of claim 3 in which the exogenous chemical inducer is N,N-diallyl-2,2-diochloro-acetamide.

5. The expression system of claim 1 in which said first structural gene encodes a DNA-binding protein which upon expression inhibits expression of said second structural gene sequence.

6. The expression system of claim 5 in which said DNA-binding protein and said operator sequence are of non-plant origin.

7. The expression system of claim 6 in which said DNA-binding protein gene and said operator sequence are of bacterial origin.

8. The expression system of claim 7 in which said DNA-binding protein and said operator sequence are derived from the LacI gene of Escherichia coli, isolated from the plasmid p35SlacI, and deposited in an Escherichia coli, strain TG-2, host under NCIB Accession Number 40092.

9. An expression system according to claim 1 wherein said first promoter sequence and said first structural gene are on a first vector and said operator sequence, said male flower specific second promoter and said second structural gene sequence are on a second vector.

10. An expression system according to claim 1 which comprises a single vector.

11. A method of producing a reversibly sterile male plant, said method comprising transforming a plant with an expression system which comprises:

(a) a first promoter sequence responsive to the presence or absence of an exogenous chemical inducer;

(b) a first structural gene encoding a repressor protein operably linked to and under the control of said first promoter sequence;

(c) an operator sequence responsive to the repressor protein expressed by said first structural gene sequence;

(d) a male flower specific second promoter sequence operably linked to and under the control of said operator sequence; and

(e) a second structural gene sequence encoding a protein which when expressed disrupts pollen biogenesis operably linked to said second promoter sequence,

wherein external application of said exogenous chemical inducer induces expression of said repressor protein, whereby said repressor protein represses expression of said protein which when expressed disrupts pollen biogenesis.

12. A method according to claim 11 wherein said first promoter sequence and said first structural gene of said expression system are on a first vector and said operator sequence, said male flower specific second promoter and said second structural gene sequence of said expression system are on a second vector.

13. A method according to claim 11 herein said expression system comprises a single vector.

14. An expression system for controlled disruption of pollen biogenesis comprising:

(a) a first promoter sequence responsive to the presence or absence of an exogenous chemical inducer;

(b) a first transcribed sequence encoding a first product operably linked to and under the control of said first promoter sequence;

(c) a male flower specific second promoter sequence; and

(d) a second transcribed sequence encoding a second product which when expressed disrupts pollen biogenesis, said second transcribed sequence being operably linked to and under the control of said male-flower specific second promoter sequence,

wherein expression of said first product from said first transcribed sequence inhibits disruption of pollen biogenesis by said second product.

15. An expression system of claim 14 wherein:

said first transcribed sequence is a structural gene encoding a first product which is a protein that inhibits disruption of pollen biogenesis by said second product.

16. An expression system of claim 14 wherein:

said first transcribed sequence is a structural gene encoding a first product which is a repressor protein;

said male flower specific second promoter sequence is operably linked to and under the control of an operator sequence, said operator sequence being responsive to said repressor protein; and

expression of said repressor protein from said first transcribed sequence represses transcription of said second transcribed sequence encoding said second product, thereby inhibiting disruption of pollen biogenesis by said second product.

17. An expression system of claim 14 wherein:

said first transcribed sequence encodes an antisense RNA molecule; and

expression of said antisense RNA molecule from said first transcribed sequence inhibits expression from said second transcribed sequence of said second product, thereby inhibiting disruption of pollen biogenesis by said second product.

18. A method of producing a reversibly sterile plant, said method comprising stably transforming a plant with an expression system for controlled disruption of pollen biogenesis, said expression system comprising:

(a) a first promoter sequence responsive to the presence or absence of an exogenous chemical inducer;

(b) a first transcribed sequence encoding a first product operably linked to and under the control of said first promoter sequence;

(c) a male flower specific second promoter sequence; and

(d) a second transcribed sequence encoding a second product which when expressed disrupts pollen biogenesis, said second transcribed sequence being operably linked to and under the control of said male-flower specific second promoter sequence,

wherein expression of said first product from said first transcribed sequence inhibits disruption of pollen biogenesis by said second product.

19. A method according to claim 18 wherein:

said first transcribed sequence is a structural gene encoding a first product which is a protein that inhibits disruption of pollen biogenesis by said second product.

20. A method according to claim 19 wherein:

said first transcribed sequence is a structural gene encoding a first product which is a repressor protein;

said male flower specific second promoter sequence is operably linked to and under the control of an operator sequence, said operator sequence being responsive to said repressor protein; and

expression of said repressor protein from said first transcribed sequence represses transcription of said second transcribed sequence encoding said second product, thereby inhibiting disruption of pollen biogenesis by said second product.

21. A method according to claim 18 wherein:

said first transcribed sequence encodes an antisense RNA molecule; and

expression of said antisense RNA molecule from said first transcribed sequence inhibits expression from said second transcribed sequence of said second product, thereby inhibiting disruption of pollen biogenesis by said second product.

22. A plant stably transformed with an expression system for controlled disruption of pollen biogenesis, said expression system comprising:

(a) a first promoter sequence responsive to the presence or absence of an exogenous chemical inducer;

(b) a first transcribed sequence encoding a first product operably linked to and under the control of said first promoter sequence;

(c) a male flower specific second promoter sequence; and

(d) a second transcribed sequence encoding a second product which when expressed disrupts pollen biogenesis, said second transcribed sequence being operably linked to and under the control of said male-flower specific second promoter sequence,

wherein expression of said first product from said first transcribed sequence inhibits disruption of pollen biogenesis by said second product.

23. A plant according to claim 22 wherein:

said first transcribed sequence is a structural gene encoding a first product which is a protein that inhibits disruption of pollen biogenesis by said second product.

24. A plant according to claim 23 wherein:

said first transcribed sequence is a structural gene encoding a first product which is a repressor protein;

said male flower specific second promoter sequence is operably linked to and under the control of an operator sequence, said operator sequence being responsive to said repressor protein; and

expression of said repressor protein from said first transcribed sequence represses transcription of said second transcribed sequence encoding said second product, thereby inhibiting disruption of pollen biogenesis by said second product.

25. A plant according to claim 22 wherein:

said first transcribed sequence encodes an antisense RNA molecule; and

expression of said antisense RNA molecule from said first transcribed sequence inhibits expression from said second transcribed sequence of said second product, thereby inhibiting disruption of pollen biogenesis by said second product.