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SI20215B - Uravnavanje ekspresije kinolat fosforibozil transferaze - Google Patents

Uravnavanje ekspresije kinolat fosforibozil transferaze Download PDF

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SI20215B
SI20215B SI9820033A SI9820033A SI20215B SI 20215 B SI20215 B SI 20215B SI 9820033 A SI9820033 A SI 9820033A SI 9820033 A SI9820033 A SI 9820033A SI 20215 B SI20215 B SI 20215B
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dna
quinolate
phosphoribosyl transferase
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Mark A Conkling
Nandini Mendu
Wen Song
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Univ North Carolina State
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Abstract

Izum zagotavlja DNA, ki kodira encim rastlinske kinolat fosforibozil transferaze (QPRTaze) in konstrukte, ki vsebujejo tako DNA. Izum zagotavlja tudi metode za uravnavanje ekspresije kinolat fosforibozil transferaze.

Claims (40)

  1. λ PATENTNI ZAHTEVKI 1. Izorirana molekula DNA, značilna po tem, da vsebuje zaporedje izbrano iz skupine, ki obsega: (a) Zaporedje št.l (b) DNA zaporedja ki kodirajo encim, ki vsebuje zaporedje št. 2 (c) DNA zaporedja, ki imajo vsaj 65% homolognost do izolirane DNA (a) ali (b) iz tega zahtevka in ki kodirajo encim kinolat fosforibozil transferazo;· in (d) DNA zaporedja ki se razlikujejo od DNA po (a), (b) ali (c) iz tega zahtevka zaradi degeneracije genetske kode.
  2. 2. DNA konstrukt, značilen po tem, da obsega ekspresijsko kaseto, katere konstrukt obsega v smeri od 5' do 3' promoter, ki je operativen v rastlinski celici in DNA segment, ki kodira zaporedje kinolat fosforibozil transferaze po zahtevku 1, ki je nameščen pod promoter in je z njim operativno povezan.
  3. 3. DNA konstrukt, značilen po tem, da obsega ekspresijsko kaseto, katere konstrukt obsega v smeri od 5' do 3' rastlinski promoter in DNA segment ki kodira zaporedje kinolat fosforibozil transferaze po zahtevku 1, ki je nameščen pod promoter in je z njim operativno povezan, omenjeni DNA segment pa je nekodogeno orientiran.
  4. 4. DNA konstrukt, značilen po tem, da obsega v smeri 5' do 3' promoter, ki je operativen v rastlinskih celicah in DNA, ki kodira rastlinsko kinolat fosforibozil transferazo, kjer je omenjeni DNA operativno povezan z omenjenim promoterjem. 2,
  5. 5. DNA konstrukt, značilen po tem, da obsega v smeri 5' do 3’ promoter, ki je operativen v rastlinskih celicah in DNA, ki kodira rastlinsko kinolat fosforibozil transferazo, kjer je omenjeni DNA nekodogeno orientiran in operativno povezan z omenjenim promoterjem.
  6. 6. DNA konstrukt po zahtevkih 2, 3, 4 ali 5, značilen po tem, da je promoter bistveno aktiven v rastlinskih celicah.
  7. 7. DNA konstrukt po zahtevkih 2, 3, 4 ali 5, značilen po tem, da je omenjeni promoter selektivno aktiven v celicah tkiva rastlinskih korenin ali v celicah tkiva skorje rastlinskih korenin..
  8. 8. DNA konstrukt po kateremkoli od zahtevkov od 2 do 7, značilen po tem, da nadalje obsega plazmid.
  9. 9. DNA konstrukt po kateremkoli od zahtevkov od 2 do 8, značilen po tem, da ga nosi vektor transformacije rastlin.
  10. 10. DNA konstrukt po zahtevku 9, značilen po tem, da je vektor transformacije rastlin Agrobacterium tumefaciens vektor.
  11. 11. Rastlinska celica značilna po tem, da vsebuje DNA konstrukt po kateremkoli od zahtevkov od 2 do 10.
  12. 12. Transgena rastlina značilna po tem, da vsebuje rastlinske celice po zahtevku 11.
  13. 13. Peptid značilen po tem, da vsebuje zaporedje št. 2. 3
  14. 14. Peptid, značilen po tem, da ga kodira DNA zaporedje izbrano iz skupine, ki obsega: (a) Zaporedje št. 1; (b) DNA zaporedja, ki imajo vsaj 65% homolognost do izolirane DNA (a) iz tega zahtevka in ki kodirajo encim kinolat fosforibozil transferazo; in (c) DNA zaporedja, ki se razlikujejo od DNA po točki (a) ali (b) tega zahtevka zaradi degeneracije genetske kode.
  15. 15. Metoda pridobivanja celice transgene rastline, ki ima zmanjšano ekpresijo kinolat fosforibozil transferaze (QPRTaze), značilna po tem, da obsega: zagotavljanje rastlinske celice takega tipa, za katerega je znano da izraža kinolat fosforibozil transferazo; zagotavljanje eksogenega DNA konstrukta, kjer konstrukt vsebuje v smeri 5' do 3' promoter, ki je operativen v rastlinskih celicah in DNA zaporedje, ki kodira kinolat fosforibozil transferazno mRNA in je izbrano iz DNA zaporedij po zahhtevku 1, omenjena DNA pa je operativno povezana z omenjenim promoterjem; in transformacijo omenjenih rastlinskih celic z omenjenim DNA konstruktom, da dobimo transformirane celice, kjer ima omenjena rastlinska celica zmanjšano ekpresijo QPRTaze v primerjavi z netransformirano celico.
  16. 16. Metoda po zahtevku 15, značilna po tem, da ima omenjena DNA, ki vsebuje delež zaporedja, ki kodira kinolat fosforibozil transferazno mRNA, kodogeno ali nekodogeno orientacijo.
  17. 17. Metoda po zahtevku 15 ali 16, značilna po tem, da je rastlinska celica Nicotiana tabacum.
  18. 18. Metoda po kateremkoli od zahtevkov od 15 do 17, značilna po tem, da nadalje obsega regeneracijo rastline iz omenjene transformirane rastlinske celice.
  19. 19. Metoda po kateremkoli od zahtevkov od 15 do 18, značilna po tem, da je omenjeni promoter bistveno aktiven.
  20. 20. Metoda po kateremkoli od zahtevkov od 15 do 18, značilna po tem, da je omenjeni promoter selektivno aktiven v celicah tkiva rastlinskih korenin ali v celicah tkiva skorje rastlinskih korenin.
  21. 21. Metoda po kateremkoli od zahtevkov od 15 do 20, značilna po tem, da je omenjeni korak transformacije izveden s pomočjo obstreljevanja omenjenih rastlinskih celic z mikrodelci, ki nosijo DNA konstrukt.
  22. 22. Metoda po kateremkoli od zahtevkov od 15 do 20, značilna po tem, da je omenjeni korak transformacije izveden z infekcijo omenjenih rastlinskih celic z Agrobacterium tumefaciens, ki vsebuje Ti plazmid, ki nosi omenjeni DNA konstrukt.
  23. 23. Metoda pridobivanja transgenih tobačnih semen, značilna po tem, da obsega zbiranje semen iz transgene tobačne rastline pridobljene po metodi po zahtevku 17. 5
  24. 24. Metoda po kateremkoli od zahtevkov od 15 do 23, značilna po tem, da zaporedje omenjene eksogene DNA vsebuje zaporedje, ki kodira kinolat fosforibozil transferazo in je izbrano iz DNA zaporedij po zahtevku 1 ter je komplementarno omenjeni kinolat fosforibozil transferaza mRNA (QPRT mRNA), ki je izražena v omenjeni rastlinski celici v področju, ki je izbran izmed: (a) 5'-neprevedenega zaporedja omenjene QPRT mRNA (b) 3'-neprevedenega zaporedja omenjene QPRT mRNA (c) prevedenem področju omenjene QPRT mRNA.
  25. 25. Metoda po kateremkoli od zahtevkov od 15 do 23, značilna po tem, da zaporedje omenjene eksogene DNA vsebuje zaporedje, ki kodira kinolat fosforibozil transferazo in je izbrano iz DNA zaporedij po zahtevku 1 ter da je omenjeno eksogeno zaporedje DNA komplementarno z vsaj 15 nukleotidi omenjene kinolat fosforibozil transferaze mRNA izražene v omenjeni rastlinski celici.
  26. 26. Metoda po kateremkoli od zahtevkov od 15 do 23, značilna po tem, da zaporedje omenjene eksogene DNA vsebuje zaporedje, ki kodira kinolat fosforibozil transferazo in je izbrano iz DNA zaporedij po zahtevku 1 ter da je omenjeno eksogeno zaporedje DNA komplementarno z vsaj 200 nukleotidi omenjene kinolat fosforibozil transferaze mRNA izražene v omenjeni rastlinski celici.
  27. 27. Metoda po kateremkoli od zahtevkov od 15 do 23, značilna po tem, da je omenjeno eksogeno zaporedje DNA vsebuje kinolat fosforibozil transferazo, ki kodira zaporedje izbrano izmed DNA zaporedij po zahtevku 1.
  28. 28. Transgena rastlina vrste Nicotiana, značilna po tem, da ima zmanjšano ekpresijo kinolat fosforibozil tranferaze (QPRTaze) v primerjavi z netransformiranimi kontrolnimi rastlinami, kjer transgena rastlina obsega transgene rastlinske celice, ki vsebujejo: - eksogeni DNA konstrukt, ki vsebuje v smeri od 5' proti 3' promoter, ki je v omenjeni rastlinski celici operativen in DNA, ki vsebuje segment DNA zaporedja, ki kodira rastlinsko kinolat fosforibozil transferazno mRNA in je izbrano iz zaporedij po zahtevku 1, kjer je omenjena DNA operativno povezana z omenjenim promoterj em; kjer omenjena rastlina kaže znamjšano ekpresijo QPRTaze v primerjavi z netransformiranimi kontrolnimi rastlinami.
  29. 29. Metoda po zahtevku 28, značilna po tem, da je segment DNA, ki vsebuje segment DNA zaporedja, ki kodira kinolat fosforibozil transferazno mRNA izbran iz DNA zaporedij po zahtevku 1 in ima kodogeno ali nekodogeno orientacij i.
  30. 30. Transgena rastlina vrste Nicotiana, značilna po tem, da ima zmanjšano ekpresijo kinolat fosforibozil transferaze (QPRT-aze) v primerjavi z netransformirano kontrolno rastlino, kjer je omenjena transgena rastlina potomec rastline po zahtevku 28 in vsebuje transgen po zahtevku 1.
  31. 31.Semena transgenih rastlin vrste Nicotiana, značilna po tem, da imajo zmanjšano ekpresijo kinolat fosforibozil transferaze (QPRT-aze) v primerjavi z netransformirano kontrolno rastlino, kjer je omenjena transgena rastlina rastlina po zahtevku 28 ali njen potomec in katere semena vsebujejo transgen po zahtevku 1.
  32. 32. Pridelek, značilen po tem, da obsega množico rastlin po zahtevku 28, ki so posajene skupaj na poljedeljskem polju.
  33. 33. Metoda zmanjševanja ekspresije gena kinolat fosforibozil transferaze v rastlinski celici, značilna po tem, da obsega: vzgajanje rastlinske celice, ki je transformirana tako, da vsebuje eksogeno DNA, ki je izbrana iz DNA zaporedja po zahtevku 1 in kjer je prepisana veriga eksogene DNA komplementarna s kinolat fosforibozil transferazne mRNA, ki je endogena v omenjeni celici, pri čemer prepisovanje omenjene komplementarne verige zmanjša ekspresijo omenjenega kinolat fosforibozil gena.
  34. 34. Metoda pridobivanja tobačne rastline, ki ima zmanjšane stopnje nikotina v listih, značilna po tem, da obsega: vzgajanje tobačne rastline ali njenih potomcev, kjer omenjena rastlina vsebuje celice, ki vsebujejo DNA konstrukt, ki obsega transkripcijsko začetno področje, ki je v omenjeni rastlini funkcionalno in eksogeno DNA zaporedje, ki je izbrano izmed zaporedij po zahtevku 1 in ki je operativno združena z omenjenim transkripcijskim začetnim področjem, kjer je prepisana veriga omenjenega DNA zaporedja komplementarna z endogeno kinolat fosforibozil transferazo mRNA v omenjenih celicah. s
  35. 35. Metoda pridobivanja transgene rastlinske celice, ki ima povečano ekpresijo kinolat fosforibozil transferaze (QPRT-aze), značilna po tem, da obsega: zagotavljanje rastlinske celice takega tipa, za katerega je znano, da izraža kinolat fosforibozil transferazo; zagotavljanje eksogenega DNA konstrukta, ki vsebuje v smeri 5' do 3' promoter, ki je operativen v rastlinski celici in DNA zaporedje, ki kodira kinolat fosforibozil transferazo in ki je izbrano izmed zaporedij po zahtevku 1 ter operativno povezano z omenjenim promoterjem; in transformacijo omenjene rastlinske celice z omenjenim DNA konstruktom, da pridobimo transformirane celice, kjer imajo omenjene rastlinske celice, v primerjavi z netransformiranimi celicami, povečano ekspresijo QPRTaze.
  36. 36. Transgena rastlina vrste Nicotiana, ki ima v primerjavi z netransformirano kontrolno rastlino povečano ekpresijo kinolat fosforibozil transferaze (QPRT-aze), značilna po tem, da obsega transgene rastlinske celice, ki vsebuj ej o: eksogeni DNA konstrukt, ki vsebuje v smeri 5' do 3' promoter, ki je operativen v omenjeni rastlinski celici in DNA zaporedje, ki kodira rastlinsko kinolat fosforibozil transferazo in je izbrano iz zaporedij po zahtevku 1, kjer je omenjena DNA z omenjenim promoterjem operativno povezana; in da ima omenjena rastlina povečano ekpresijo QPRTaze v primerjavi z netransformirano kontrolno rastlino. 1
  37. 37. Transgena rastlina vrste Nicotiana, ki ima v primerjavi z netransformirano kontrolno rastlino povečano ekpresijo kinolat fosforibozil transferaze (QPRTaze), značilna po tem, da je potomec rastline po zahtevku 36 in vsebuje transgen po zahtevku 1.
  38. 38. Metoda za povečanje ekspresije gena kinolat fosforibozil transferaze v rastlinski celici, značilna po tem da obsega: vzgajanje rastlinske celice transformirane da vsebuje eksogeno DNA, ki je izbrano iz zaporedij po zahtevku 1, kjer omenjena eksogena DNA kodira kinolat fosforibozil transferazo.
  39. 39. Metoda po zahtevku 38, značilna po tem, da je transformirana rastlinska celica pridobljena s pomočjo metode, ki obsega: integracijo genoma rastlinske celice gostitelja v konstrukt, ki obsega v smeri prepisovanja promoter, ki je funkcionalen v rastlinskih celicah in DNA zaporedje, ki kodira kinolat fosforibozil transferazo, ki je v omenjeni celici funcionalna in ki je izbrano iz zaporedij po zahtevku 1, kjer je omenjeno DNA zaporedje operativno povezana z omenjenim promoterjem in transkripcijskim končnim področjem v omenjeni celici, s čimer pridobimo transformirano rastlinsko celico. 40
  40. 40.Metoda pridobivanja tobačne rastline, ki ima povečane stopnje nikotina v listih, značilna po tem, da obsega: vzgajanje tobačne rastline, ali njenih potomcev, kjer omenjena rastlina vsebuje celice, ki vsebujejo DNA konstrukt, ki obsega transkripcijsko začetno področje, ki j e v omenjeni rastlini funkcionalno in eksogeno DNA zaporedje, ki je izbrano iz zaporedij po zahtevku 1 in je operativno povezano z omenjenim transkripcijskim začetnim področjem, kjer omenjeno DNA zaporedje kodira kinolat fosforibozil tranferazo funcionalno v omenjenih celicah. Za NORTH CAROLINA STATE UNIVERSITY 103 Bynum Hall, Campus Box 7003 Raleigh, NC 27695 USA Zastopnik: Mark-Inventa
SI9820033A 1997-06-12 1998-06-10 Uravnavanje ekspresije kinolat fosforibozil transferaze SI20215B (sl)

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