Reproductive ablation constructs
View Patent ↗The present invention relates to the regulation of reproductive development, particularly to the genetic ablation of reproductive tissues in angiosperm and gymnosperm species. Reproductive-preferred promoters, regulatory elements, and cytotoxic nucleotide sequences are disclosed herein, as are constructs and methods for genetic ablation.
1. A method for producing a transgenic plant, comprising:
(a) transforming a plant cell with a construct that comprises (i) a functional Pinus radiata male cone (PrMC) promoter operably linked to (ii) a polynucleotide that comprises a sequence encoding an E73G barnase mutant; and
(b) culturing the transformed plant cell under conditions that promote growth of a plant, wherein said plant is a transgenic plant that exhibits a phenotype that is different from a plant of the same species that has not been transformed with the construct.
2. The method of claim 1 , wherein the sequence that encodes the E73G barnase mutant comprises the sequence of SEQ ID NO. 5.
3. The method of claim 1 , wherein the polynucleotide comprises a sequence that encodes the amino acid sequence of SEQ ID NO. 9.
4. The method of claim 1 , wherein the PrMC promoter comprises the sequence of SEQ ID NO. 4 or 16.
5. The method of claim 4 , wherein the sequence encoding the E73G barnase mutant comprises either (i) the sequence of SEQ ID NO. 5, or (ii) a polynucleotide that comprises a sequence that encodes the amino acid sequence of SEQ ID NO. 9.
6. The method of claim 1 , wherein the PrMC promoter/E73G barnase mutant construct comprises the sequence of SEQ ID NO. 15.
7. The method of claim 1 , further comprising obtaining wood pulp from the transgenic plant.
8. The method of claim 7 , wherein the PrMC promoter comprises the sequence of SEQ ID NO. 4 or 16.
9. The method of claim 8 , wherein the sequence encoding the E73G barnase mutant comprises either (i) the sequence of SEQ ID NO. 5, or (ii) a polynucleotide that comprises a sequence that encodes the amino acid sequence of SEQ ID NO. 9.
10. A construct, comprising a Pinus radiata male cone (PrMC) promoter operably linked to a polynucleotide encoding an E73G barnase mutant, wherein expression of the E73G barnase mutant disrupts reproductive development of at least one of a male reproductive structure in a plant, which expresses the construct.
11. The construct of claim 10 , wherein the sequence encoding the E73G barnase mutant comprises either (i) the sequence of SEQ ID NO. 5, or (ii) a polynucleotide that comprises a sequence that encodes SEQ ID NO. 9.
12. The construct of claim 11 , wherein the PrMC promoter comprises the sequence of SEQ ID NO. 4 or 16.
13. The construct of claim 10 , wherein the construct comprises the sequence of SEQ ID NO. 15.
14. A transgenic plant, comprising the construct of claim 10 .
15. The transgenic plant of claim 14 , wherein the sequence encoding the E73G barnase mutant comprises either (i) the sequence of SEQ ID NO. 5, or (ii) a polynucleotide that comprises a sequence that encodes SEQ ID NO. 9.
16. The transgenic plant of claim 15 , wherein the PrMC promoter comprises the sequence of SEQ ID NO. 4 or 16.
17. The transgenic plant of claim 14 , wherein the construct comprises the sequence of SEQ ID NO. 15.
18. A transgenic progeny plant of the transgenic plant of claim 14 , wherein the progeny plant either (i) has a disrupted male reproductive structure, or (ii) expresses the construct.
19. The progeny plant of claim 18 , wherein the progeny plant is obtained from the cross of pitch pine Pinus rigida with loblolly pine P. taeda.