DD288618A5 - METHOD FOR SELECTIVELY REDUCING THE PRODUCTION OF AN ISOENZYME OF GLUTAMINE SYNTHASE FROM MEDICAGO SATIVA L. - Google Patents
METHOD FOR SELECTIVELY REDUCING THE PRODUCTION OF AN ISOENZYME OF GLUTAMINE SYNTHASE FROM MEDICAGO SATIVA L. Download PDFInfo
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- DD288618A5 DD288618A5 DD33378989A DD33378989A DD288618A5 DD 288618 A5 DD288618 A5 DD 288618A5 DD 33378989 A DD33378989 A DD 33378989A DD 33378989 A DD33378989 A DD 33378989A DD 288618 A5 DD288618 A5 DD 288618A5
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Abstract
Die Erfindung betrifft ein Verfahren zur selektiven Verringerung der Produktion eines Isoenzyms der Glutamin-Synthetase von Medicago sativa L. Ziel der Erfindung ist ein Verfahren zur Erhoehung der biologischen Stickstoffixierung und damit zur Steigerung der Trockenmasse von Medicago sativa L. Erfindungsgemaesz wird dies dadurch erreicht, dasz eine "Anti-sense"-DNA, die auf der Konstruktion eines Gens mit kodierenden und nichtkodierenden DNA-Sequenzen beruht, dessen Transkription zu einer RNA-Sequenz fuehrt, mittels eines an sich bekannten Binaervektors stabil in das Genom von Luzerne integriert wird. Diese RNA-Sequenz ist der mRNA des zu eliminierenden Genes komplementaer. Die Erfindung kann in der Landwirtschaft zur Ertragssteigerung von Medicago sativa L. eingesetzt werden.{Verfahren; Hemmung; Isoenzym; RNA; Transkription; Glutamin - Synthetase; Binaervektor; DNA; Sequenz; Gen}The invention relates to a process for the selective reduction of the production of an isoenzyme of the glutamine synthetase of Medicago sativa L. The aim of the invention is a process for increasing the biological nitrogen fixation and thus for increasing the dry mass of Medicago sativa L. According to the invention, this is achieved by :. an "antisense" DNA, based on the construction of a gene with coding and non-coding DNA sequences whose transcription leads to an RNA sequence, is stably integrated into the alfalfa genome by means of a per se known Bina vector. This RNA sequence is complementary to the mRNA of the gene to be eliminated. The invention can be used in agriculture for increasing the yield of Medicago sativa L. {method; Inhibition; isoenzyme; RNA; Transcription; Glutamine synthetase; binary vector; DNA; Sequence; Gene}
Description
In einer Veröffentlichung von KNIGHT und LANGSTON-UNKEFER (1988) wird bewiesen, daß durch selektive, partielle Hemmung eines Isoenzyms der Glutamin-Synthetase Anzahl und Größe der Wurzelknöllchen bei Luzerne nach Beimpfen mit Rhizobium meliloti soweit erhöht wurden, daß assimilierter Stickstoff für den Trockenmassezuwachs nicht mehr begrenzend war. Bekannt ist die Hemmung durch ein mikrobiell permanent produziertes Toxin, dessen Wirkung aus biochemischen und physiologischen Gründen auf die in den Wurzeln gebildete Glutamin-Synthetase beschränkt bleibt.In a publication by KNIGHT and LANGSTON-UNKEFER (1988) it is proved that by selective, partial inhibition of an isoenzyme of glutamine synthetase number and size of root nodules in alfalfa after inoculation with Rhizobium meliloti were increased so much that the assimilated nitrogen for the dry mass gain not was more limiting. The inhibition is known by a microbially permanently produced toxin, the effect of biochemical and physiological reasons remains limited to the glutamine synthetase formed in the roots.
Zu experimentellen Zwecken Ist auch die gezielte oder zufällige Mutagenese entwickelt worden. Beide Methoden sind jedoch nicht praktikabel. Außerdem hat die letztere Methode den Nachteil, daß entweder noch andere physiologische Funktionen betroffen werden und/oder daß die entsprechende Enzymproduktion vollständig verloren geht.For experimental purposes, targeted or random mutagenesis has also been developed. Both methods are not practical. In addition, the latter method has the disadvantage that either other physiological functions are affected and / or that the corresponding enzyme production is completely lost.
Ziel der Erfindung ist ein Verfahren zur Erhöhung der biologischen Stickstoff bindung und damit zur Erhöhung der Trockenmasse von Medicsgo sativa L.The aim of the invention is a method for increasing the biological nitrogen bond and thus to increase the dry matter of Medicsgo sativa L.
Die Erfindung hat ein Verfahren zum Gegenstand, mit dem selektiv ein bestimmtes Isoenzym der Glutamin-Synthetase von Medicago sativa L. verringert produziert wird.The invention has a method for selectively producing a specific isoenzyme of Medicago sativa L. glutamine synthetase.
Die Aufgabe wird so gelöst, daß eine »Anti-sense"-DNA, die auf der Konstruktion eines Gens mit kodierenden und nichtkodierenden DNA-Sequenzen beruht, dessen Transkription zu einer RNA-Sequenz führt, mittels eines an sich bekannten Binärvektors stabil in das Genom von Luzerne integriert wird. Diese RNA-Sequenz ist der mRNA des zu eliminierenden Gens komplementär. Wird ein solches Genprodukt mindestens in der mit der Anzahl von mRNAKopien vergleichbaren Menge transkribiert, kommt es zu einem Vorgang, der in den Zellen sonst nicht vorkommt, zur Paarung von RNA-Molekülen zu einem Doppelstrang. Dieser „unphysiologische" Vorgang ist ausschließlich auf das „Zielgen" beschränkt und vermindert dessen Translation um ca. 80%, was zu der erforderlichen Verminderung der Glutamin-Synthetaseaktivität in der Wurzel führt. Bei der Konstruktion des Binärvektors muß beachtet werden, daß Neomycin als Selektionsmarker für Medicage sativa L. ungeeignet ist.The object is achieved in such a way that an anti-sense DNA, which is based on the construction of a gene with coding and non-coding DNA sequences whose transcription leads to an RNA sequence, stably into the genome by means of a known binary vector This RNA sequence is complementary to the mRNA of the gene to be eliminated, and if such a gene product is transcribed at least in the amount comparable to the number of mRNA copies, a process that does not otherwise occur in the cells results in mating This "unphysiological" process is restricted to the "target gene" and reduces its translation by about 80%, leading to the required reduction of glutamine synthetase activity in the root It should be noted that neomycin is unsuitable as a selection marker for Medicage sativa L.
Das reduzierte Niveau der Glutamin-Synthetaseaktivität in der Wurzel führt dazu, daß bei Medicage sativa L. nach Beimpfung mit Rhizobium meliloti eine Erhöhung der biologischen Stickstoffixierung und der Trockenmasse 'im mindestens 60% möglich sind.The reduced level of glutamine synthetase activity in the root means that in Medicage sativa L. after inoculation with Rhizobium meliloti an increase of the biological nitrogen fixation and the dry mass' in at least 60% are possible.
1. er enthält zwei je 25 bp große „Border"-Sequenzen (notwendig für Integration ins Pflanzengenom),1. it contains two 25 bp "Border" sequences (necessary for integration into the plant genome),
2. er weist einen pflanzenselektierbaren Marker auf, z. B. für Methotrexatresistenz (Dihydrofolatreduktase) oder für Hygromycinreslstenz (Hygromycinphosphotransferase) (notwendig, um transformierte Pflanzen ^u selektieren),2. he has a plant-selectable marker, z. For methotrexate resistance (dihydrofolate reductase) or for hygromycin resistance (hygromycin phosphotransferase) (necessary to select transformed plants),
3. er enthält ein DNA-Element, diis für die Organspenifität der Expression verantwortlich ist (Wurzelexpressionselement),3. it contains a DNA element which is responsible for the organ mobility of the expression (root expression element),
4. er besitzt den originalen Promoter des Luzerne-Glutamin-Synthetase-Genes für die zytosolische Untereinheit des Wurzelenzyms,4. he owns the original promoter of the alfalfa glutamine synthetase gene for the cytosolic subunit of the root enzyme,
5. er beginnt mit einem frühen Kodon der nativen mRNA, die die dieser mRNA analoge cDNA in umgekehrter Orientierung beinhaltet,5. it starts with an early codon of the native mRNA which contains the cDNA analogous to this mRNA in reverse orientation,
6. er enthält den natürlichen Terminatorbereich zur Beendigung der Transkription in richtiger Orientierung. Mittels dieses Binärvektors wird die „Anti-sense"-DNA in das Genom von Medicago sativa L. integriert.6. It contains the natural terminator region to complete the transcription in the correct orientation. This binary vector integrates the anti-sense DNA into the genome of Medicago sativa L.
Die dadurch eintretende Reduktion der Wurzel-Glutamin-Synthetaseaktivität führt dazu, daß nach Beimpfung mit Rhizobium meliloti eine Erhöhung der biologischen Stickstoffixierung und der Trockenmasse möglich Ist.The resulting reduction of the root glutamine synthetase activity means that after inoculation with Rhizobium meliloti an increase of the biological nitrogen fixation and the dry mass is possible.
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DD33378989A DD288618A5 (en) | 1989-10-23 | 1989-10-23 | METHOD FOR SELECTIVELY REDUCING THE PRODUCTION OF AN ISOENZYME OF GLUTAMINE SYNTHASE FROM MEDICAGO SATIVA L. |
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DD33378989A DD288618A5 (en) | 1989-10-23 | 1989-10-23 | METHOD FOR SELECTIVELY REDUCING THE PRODUCTION OF AN ISOENZYME OF GLUTAMINE SYNTHASE FROM MEDICAGO SATIVA L. |
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US10760086B2 (en) | 2011-09-13 | 2020-09-01 | Monsanto Technology Llc | Methods and compositions for weed control |
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US10968449B2 (en) | 2015-01-22 | 2021-04-06 | Monsanto Technology Llc | Compositions and methods for controlling Leptinotarsa |
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1989
- 1989-10-23 DD DD33378989A patent/DD288618A5/en not_active IP Right Cessation
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