Patent
Plant viral expression vectors and use of same for generating genotypic variations in plant genomes
| العنوان: | Plant viral expression vectors and use of same for generating genotypic variations in plant genomes |
|---|---|
| Patent Number: | 8,791,324 |
| تاريخ النشر: | July 29, 2014 |
| Appl. No: | 12/988636 |
| Application Filed: | April 21, 2009 |
| مستخلص: | A method of generating genotypic variation in a genome of a plant is disclosed. The method comprising introducing into the plant at least one viral expression vector encoding at least one chimeric nuclease which comprises a DNA binding domain, a nuclease and a localization signal to a DNA-containing organelle, wherein the DNA binding domain mediates specific targeting of the nuclease to the genome of the plant, thereby generating genotypic variation in the genome of the plant. |
| Inventors: | Vainstein, Alexander (Rechovot, IL); Zuker, Amir (Nes Ziona, IL) |
| Assignees: | Danziger Innovations Ltd. (Moshav Mishmar HaShiva, IL), Yissum Research Development Company of the Hebrew University of Jerusalem Ltd. (Jerusalem, IL) |
| Claim: | 1. A method of generating genotypic variation in a genome of a plant, the method comprising introducing into the plant at least one Tobacco Rattle Virus (TRV) expression vector encoding at least one chimeric nuclease which comprises a zinc finger binding domain, a nuclease, and a localization signal to a DNA-containing organelle, wherein said zinc finger binding domain mediates specific targeting of said nuclease to the genome of the plant, and wherein a nucleic acid sequence of said TRV expression vector is devoid of the 2b sequence as set forth in SEQ ID NO: 43, thereby generating genotypic variation in the genome of the plant. |
| Claim: | 2. A plant viral expression vector comprising a pTRV backbone and comprising a nucleic acid sequence encoding at least one chimeric nuclease which comprises a zinc finger binding domain, a nuclease, and a localization signal to a DNA-containing organelle, said nucleic acid sequence is devoid of the 2b sequence as set forth in SEQ ID NO: 43. |
| Claim: | 3. An expression vector comprising a pTRV backbone and comprising a nucleic acid sequence encoding at least two heterologous polypeptides, said nucleic acid sequence is devoid of the 2b sequence as set forth in SEQ ID NO: 43. |
| Claim: | 4. A transgenic plant comprising the plant viral expression vector of claim 2 . |
| Claim: | 5. The method of claim 1 , wherein said generating genotypic variation is transient. |
| Claim: | 6. The method of claim 1 , wherein said at least one expression vector encodes for two chimeric nucleases. |
| Claim: | 7. The method of claim 1 , wherein said at least one chimeric nuclease comprises two chimeric nucleases. |
| Claim: | 8. The plant viral expression vector of claim 2 , further comprising a second nucleic acid sequence encoding a heterologous polypeptide. |
| Claim: | 9. The plant viral expression vector of claim 2 , wherein said nucleic acid sequence comprises a Ω enhancer as set forth in SEQ ID NO: 44. |
| Claim: | 10. The expression vector of claim 3 , wherein said nucleic acid sequence comprises two separate sub genomic promoters (sgPs) for regulating transcription of said at least two heterologous polypeptides. |
| Claim: | 11. The expression vector of claim 2 , wherein the sequences of said at least two heterologous polypeptides are separated by a nucleic acid sequence encoding a cleavage domain. |
| Claim: | 12. The expression vector of claim 11 , wherein said cleavage domain comprises a T2A-like protein sequence as set forth in SEQ ID NO: 52. |
| Claim: | 13. The expression vector of claim 3 , wherein said amino acid sequence of at least two heterologous polypeptide sequences comprise SEQ ID NOs: 85, 87 or 89. |
| Claim: | 14. The expression vector of claim 3 , wherein said at least two heterologous polypeptides comprise chimeric proteins, wherein each of said chimeric proteins comprise a zinc finger binding domain, a nuclease, and a localization signal to a DNA-containing organelle. |
| Claim: | 15. The method of claim 1 , wherein said zinc finger binding domain binds a 9 nucleotide sequence. |
| Claim: | 16. The method of claim 1 , wherein said zinc finger binding domain comprises three zinc finger domains. |
| Claim: | 17. The method of claim 1 , wherein the plant comprises a Petunia hybrida or a Nicotiana tabacum. |
| Claim: | 18. The method of claim 17 , wherein said specific targeting of said nuclease to the genome of said Petunia hybrida is to a phytoene desaturase (PDS) or a flavanone 3 beta-hydroxylase (FHT) of said Petunia hybrida. |
| Claim: | 19. The method of claim 1 , wherein said nuclease comprises a type II restriction endonuclease. |
| Claim: | 20. The method of claim 1 , wherein said nuclease is selected from the group consisting of FokI, I-SceI, I-CeuI, artificial meganuclease, modified meganuclease, homing nuclease, DNA gyrase, eukaryotic topoisomerase II, bacterial topoisomerase W, topoisomerase VI, Cre recombinase, Hin recombinase, Rad51/RecA, deoxyribonuclease I, deoxyribonuclease II and micrococcal nuclease. |
| Claim: | 21. The method of claim 1 , further comprising introducing into the plant a DNA vector. |
| Claim: | 22. The method of claim 21 , wherein said DNA vector is based on an Abutilon mosaic virus (AbMV) or a Tomato yellow leaf curl virus. |
| Claim: | 23. The method of claim 1 , wherein a nucleic acid sequence of said TRV expression vector further comprises two separate sub genomic promoters (sgPs) for regulating transcription of at least two heterologous polypeptides. |
| Claim: | 24. The method of claim 23 , wherein said sub genomic promoters (sgPs) are as set forth in SEQ ID NOs: 45. |
| Claim: | 25. The method of claim 23 , wherein said at least two heterologous polypeptide sequences are separated by nucleic acid sequence encoding a cleavage domain. |
| Claim: | 26. The method of claim 25 , wherein said cleavage domain comprises a T2A-like protein sequence as set forth in SEQ ID NOs: 52 . |
| Claim: | 27. The method of claim 10 , wherein said sub genomic promoters (sgPs) are as set forth in SEQ ID NOs: 45. |
| Current U.S. Class: | 800/278 |
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| Primary Examiner: | Ibrahim, Medina A |
| رقم الانضمام: | edspgr.08791324 |
| قاعدة البيانات: | USPTO Patent Grants |
| الوصف غير متاح. |