HRP980455A2 - Gene coding for androctonine, vector containing it and disease-resistant transformed plants obtained - Google Patents
Gene coding for androctonine, vector containing it and disease-resistant transformed plants obtainedInfo
- Publication number
- HRP980455A2 HRP980455A2 HR9710632A HRP980455A HRP980455A2 HR P980455 A2 HRP980455 A2 HR P980455A2 HR 9710632 A HR9710632 A HR 9710632A HR P980455 A HRP980455 A HR P980455A HR P980455 A2 HRP980455 A2 HR P980455A2
- Authority
- HR
- Croatia
- Prior art keywords
- peptide
- sequence
- androctonin
- nucleic acid
- plants
- Prior art date
Links
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
- C07K14/43522—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from scorpions
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
Sadašnji izum odnosi se na sekvenciju DNA šifriranu za androktonin, vektor za transformaciju organizma domaćina koji ju sadrži, te postupak transformacije rečenog organizma. The present invention relates to a DNA sequence coded for androctonin, a vector for the transformation of a host organism that contains it, and a process for transforming said organism.
Izum se posebice odnosi na transformaciju biljnih stanica i biljaka, te na androktonin koji proizvode tranformirane biljke, pri čemu nastaje otpornost prema bolestima, ponajprije bolestima gljivičnog podrijetla. The invention particularly relates to the transformation of plant cells and plants, and to androctonin produced by transformed plants, whereby resistance to diseases, primarily diseases of fungal origin, is created.
Danas postoji sve veća potreba za dobivanjem biljaka otpornih na bolesti, osobito gljivične bolesti, da bi se time smanjile, štoviše potpuno izbjegle obradbe proizvodima za protugljivičnu zaštitu, a sa ciljem zaštite okoliša. Jedan od načina povećanja te otpornosti na bolesti sastoji se u transformaciji biljaka, tako da one proizvode tvari koje same po sebi omogućuju njihovu obranu od bolesti. Today, there is an increasing need to obtain disease-resistant plants, especially fungal diseases, in order to reduce, even completely avoid treatments with anti-fungal protection products, with the aim of protecting the environment. One of the ways to increase this resistance to diseases consists in transforming plants, so that they produce substances that by themselves enable their defense against diseases.
Poznate su različite tvari prirodnog podrijetla, ponajprije peptidi koji pokazuju baktericidna ili fungicidna svojstva, posebice protiv gljiva odgovornih za biljne bolesti. Međutim, problem je u pronalaženju onih tvari koje nisu samo proizvedene od transformiranih biljaka, nego i zadržavaju svoja baktericidna ili fungicidna svojstva, te ih daju rečenim biljkama. U smislu sadašnjeg izuma pojmovi baktericidan ili fungicidan odnose se kako na stvarna baktericidna ili fungicidna svojstva, tako i na bakteriostatička i fungistatička svojstva. Different substances of natural origin are known, primarily peptides that show bactericidal or fungicidal properties, especially against fungi responsible for plant diseases. However, the problem is finding those substances that are not only produced by transformed plants, but also retain their bactericidal or fungicidal properties, and give them to said plants. In the sense of the present invention, the terms bactericidal or fungicidal refer both to actual bactericidal or fungicidal properties, as well as to bacteriostatic and fungistatic properties.
Androktonini su peptidi koje proizvode škorpioni, ponajprije oni vrste Androctonus Australis. Jedan androktonin i njegovu pripravu kemijskom sintezom opisali su Ehret-Sabatier et al., kao i njegova in vitro protugljivična i protubakterijska svojstva. Androctonins are peptides produced by scorpions, primarily those of the species Androctonus Australis. One androctonin and its preparation by chemical synthesis were described by Ehret-Sabatier et al., as well as its in vitro antifungal and antibacterial properties.
Androktoninski geni sada su identificirani, a pronađeno je također da mogu biti umetnuti u organizam domaćina, ponajprije u biljku, sa ciljem ekspresije androktonina kako za pripravu i izolaciju tog androktonina, tako i za pridjeljivanje rečenom organizmu domaćinu svojstva otpornosti prema gljivičnim bolestima i bolestima bakterijskog podrijetla, čime se priskrbljuje posebice prikladno rješenje gore istaknutog problema. Androctonin genes have now been identified, and it has also been found that they can be inserted into a host organism, primarily into a plant, with the aim of expressing androctonin both for the preparation and isolation of that androctonin, and for conferring on said host organism the property of resistance to fungal diseases and diseases of bacterial origin , which provides a particularly suitable solution to the above-mentioned problem.
Stoga je predmet izuma ponajprije fragment nukleinske kiseline šifriran za neki androktonin, kimerni gen koji sadrži rečeni fragment šifriran za neki androktonin, potom heterologne elemente regulacije na položajima 5’ i 3’ koji mogu funkcionirati u organizmu domaćinu, ponajprije u biljkama, te vektor transformacije organizama domaćina koji sadrži taj kimerni gen kao i transformirani organizam domaćina. Izum se također odnosi na transformiranu biljnu stanicu koja sadrži barem jedan fragment nukleinske kiseline šifriran za androktonin, te biljku otpornu na bolesti, koja sadrži spomenutu stanicu. Izum se, na kraju, odnosi i na postupak uzgoja transformiranih biljaka prema izumu. Therefore, the subject of the invention is primarily a nucleic acid fragment encoded for some androctonin, a chimeric gene containing said fragment encoded for some androctonin, then heterologous regulatory elements at the 5' and 3' positions that can function in the host organism, primarily in plants, and a vector for the transformation of organisms the host containing that chimeric gene as well as the transformed host organism. The invention also relates to a transformed plant cell containing at least one nucleic acid fragment encoded for androctonin, and a disease-resistant plant containing said cell. Finally, the invention also relates to the process of growing transformed plants according to the invention.
Prema izumu, izraz androktonin odnosi se na bilo koji peptid koji mogu proizvesti škorpijoni ili se iz njih može izolirati, a ponajprije iz vrste Androctonus australis, pri čemu ti peptidi sadrže najmanje 20 aminokiselina, ponajprije najmanje 25, te 4 cisteinska ostatka koji među sobom tvore disulfidne mostove. According to the invention, the term androctonin refers to any peptide that can be produced by scorpions or that can be isolated from them, preferably from the species Androctonus australis, wherein these peptides contain at least 20 amino acids, preferably at least 25, and 4 cysteine residues that together form disulfide bridges.
U prednosti je da androktonin u suštini sadrži peptidnu sekvenciju donje općenite formule (I): It is advantageous that androctonin essentially contains a peptide sequence of the following general formula (I):
Xaa-Cys-Xab-Cys-Xac-Cys-Xad-Cys-Xae (I) Xaa-Cys-Xab-Cys-Xac-Cys-Xad-Cys-Xae (I)
u kojoj where
Xaa predstavlja peptidni ostatak koji sadrži najmanje 1 aminokiselinu, Xaa represents a peptide residue containing at least 1 amino acid,
Xab predstavlja peptidni ostatak od 5 aminokiselina, Xab represents a peptide residue of 5 amino acids,
Xac predstavlja peptidni ostatak od 5 aminokiselina, Xac represents a peptide residue of 5 amino acids,
Xad predstavlja peptidni ostatak od 3 aminokiseline, a Xad represents a peptide residue of 3 amino acids, a
Xae predstavlja peptidni ostatak koji sadrži najmanje 1 aminokiselinu. Xae represents a peptide residue containing at least 1 amino acid.
U prednosti Xab i/ili Xad i/ili Xae sadrže najmanje jednu bazičnu aminokiselinu, ponajprije 1. Prema izumu se izrazom bazična aminokiselina podrazumijeva aminokiselina odabrana između lizina, asparagina i homoasparagina. Advantageously, Xab and/or Xad and/or Xae contain at least one basic amino acid, preferably 1. According to the invention, the term basic amino acid means an amino acid selected from lysine, asparagine and homoasparagine.
Ponajprije, First of all,
Xaa predstavlja peptidnu sekvenciju Xaa’-Val, u kojoj Xaa represents the peptide sequence Xaa'-Val, in which
Xaa’ predstavlja NH2 ili peptidni ostatak koji sadrži najmanje 1 aminokiselinu, i/ili Xaa' represents NH2 or a peptide residue containing at least 1 amino acid, and/or
Xab predstavlja peptidnu sekvenciju -Arg-Xab’-Ile, u kojoj Xab represents the peptide sequence -Arg-Xab'-Ile, in which
Xab’ predstavlja peptidni ostatak od 3 aminokiseline, i/ili Xab' represents a peptide residue of 3 amino acids, and/or
Xac predstavlja peptidnu sekvenciju -Arg-Xac’-Gly-, u kojoj Xac represents the peptide sequence -Arg-Xac'-Gly-, in which
Xac’ predstavlja peptidni ostatak od 3 aminokiseline, i/ili Xac' represents a peptide residue of 3 amino acids, and/or
Xad predstavlja peptidnu sekvenciju -Tyr-Xad’-Lys, u kojoj Xad represents the peptide sequence -Tyr-Xad'-Lys, in which
Xad’ predstavlja peptidni ostatak od 1 aminokiseline, i/ili Xad' represents a peptide residue of 1 amino acid, and/or
Xae predstavlja peptidnu sekvenciju -Thr-Xae’, u kojoj Xae represents the peptide sequence -Thr-Xae', in which
Xae’ predstavlja COOH ili peptidni ostatak koji sadrži najmanje 1 aminokiselinu. Xae' represents COOH or a peptide residue containing at least 1 amino acid.
Ponajprije, First of all,
Xaa’ predstavlja peptidnu sekvenciju Arg-Ser-, i/ili Xaa' represents the peptide sequence Arg-Ser-, and/or
Xab’ predstavlja peptidnu sekvenciju -Gln-Ile-Lys-, i/ili Xab' represents the peptide sequence -Gln-Ile-Lys-, and/or
Xac’ predstavlja peptidnu sekvenciju -Arg-Arg-Gly-, i/ili Xac' represents the peptide sequence -Arg-Arg-Gly-, and/or
Xad’ predstavlja peptidni ostatak -Tyr-, i/ili Xad' represents the peptide residue -Tyr-, and/or
Xae’ predstavlja peptidnu sekvenciju -Asn-Arg-Pro-Tyr. Xae' represents the peptide sequence -Asn-Arg-Pro-Tyr.
Prema najpovoljnijem obličju izuma, androktonin je predstavljen peptidnom sekvencijom od 25 aminokiselina, opisanom sekvencijskim identifikatorom br. 1 (SEQ ID No 1) i homolognim peptidnim sekvencijama. According to the most favorable form of the invention, androctonin is represented by a peptide sequence of 25 amino acids, described by sequence identifier no. 1 (SEQ ID No 1) and homologous peptide sequences.
Pod homolognom peptidnom sekvencijom podrazumijevaju se sve ekvivalentne sekvencije koje su barem 65% homologne sa sekvencijom predstavljenom sekvencijskim identifikatorom br. 1, pri čemu se podrazumijeva da 4 cisteinska ostatka i niz aminokiselina koje ih razdvajaju ostaju identični, dok se neke aminokiseline zamjenjuju drugačijima ali ekvivalentnima, na položajima koji ne induciraju bitne promjene glede protugljivične ili protubakterijske aktivnosti spomenute homologne sekvencije. Ponajprije homologne sekvencije sadrže barem 75% homolognosti, posebice barem 85% homolognosti, a prije svega 90% homolognosti. Homologous peptide sequence means all equivalent sequences that are at least 65% homologous with the sequence represented by sequence identifier no. 1, whereby it is understood that the 4 cysteine residues and the series of amino acids that separate them remain identical, while some amino acids are replaced by different but equivalent ones, in positions that do not induce significant changes regarding the antifungal or antibacterial activity of the mentioned homologous sequence. Primarily homologous sequences contain at least 75% homology, especially at least 85% homology, and above all 90% homology.
NH2-terminalni ostatak androktonina može pokazati poslijetranslacijsku modifikaciju, primjerice acetiliranje, dok C-terminalni ostatak može pokazati poslijetranslacijsku modifikaciju, primjerice amidiranje. The NH2-terminal residue of androctonin can show post-translational modification, for example acetylation, while the C-terminal residue can show post-translational modification, for example amidation.
Pod ekspresijskom peptidnom sekvencijom koja u biti sadrži peptidnu sekvenciju općenite formule (I) podrazumijevaju se ne samo gore navedene sekvencije, nego i sve one sekvencije koje sadrže na jednom ili drugom svom kraju, ili na oba kraja, peptidne ostatke, osobito one potrebne za njihovo izražavanje i ciljanje u organizama domaćina, ponajprije u biljnih stanica ili biljaka. An expression peptide sequence that essentially contains a peptide sequence of the general formula (I) means not only the above-mentioned sequences, but also all those sequences that contain at one or the other end, or at both ends, peptide residues, especially those necessary for their expression and targeting in host organisms, primarily in plant cells or plants.
To se ponajprije odnosi na “peptid-androktonin” ili “androktonin-peptid”, ponajprije “peptid-androktonin”, cijepanje kojega u enzimskim sustavima biljnih stanica, dozvoljava dolje definirano oslobađanje androktonina. Peptid spojen s androktoninom može biti signalni peptid ili tranzitni peptid koji dopušta kontrolu i usmjeravanje proizvodnje androktonina na specifičan način u dijelu biljne stanice ili biljke, kao što je primjerice citoplazma ili stanična membrana, ili u slučaju biljaka u specifičnom tipu staničnih odjeljaka ili tkiva ili u izvanstaničnoj matrici. This primarily refers to "peptide-androctonin" or "androctonin-peptide", primarily "peptide-androctonin", the cleavage of which in the enzymatic systems of plant cells allows the release of androctonin as defined below. The peptide linked to androctonin can be a signal peptide or a transit peptide that allows the control and direction of androctonin production in a specific way in a part of the plant cell or plant, such as for example the cytoplasm or the cell membrane, or in the case of plants in a specific type of cell compartment or tissue or in extracellular matrix.
Prema jednom obličju, tranzitni peptid može biti signal kloroplastnog ili mitohondrijskog adresiranja, koji se potom cijepa u kloroplastu ili mitohondriju. In one embodiment, the transit peptide can be a chloroplast or mitochondrial targeting signal, which is then cleaved in the chloroplast or mitochondrion.
Prema drugom obličju izuma, signalni peptid može biti N-terminalni signal ili “prepeptid”, opcijski povezan sa signalom odgovornim za zadržavanje proteina u endoplazmičkom retikulumu, ili peptid vakuolnog adresiranja ili “propeptid”. Endoplazmički retikulum je mjesto na kojemu “stanična strojarnica” preuzima operacije sazrijevanja proizvedenog proteina, kao na primjer cijepanje signalnog peptida. According to another aspect of the invention, the signal peptide can be an N-terminal signal or "prepeptide", optionally linked to a signal responsible for the retention of the protein in the endoplasmic reticulum, or a vacuolar targeting peptide or "propeptide". The endoplasmic reticulum is the place where the "cellular machine shop" takes over the maturation operations of the produced protein, such as cleavage of the signal peptide.
Tranzitni peptidi mogu biti bilo jednostruki, bilo dvostruki, a u tome slučaju opcijski odvojeni intermedijernom sekvencijom, to jest onu koja sadrži u smjeru transkripcije sekvenciju šifriranu za tranzitni peptid biljnog gena koji je šifriran za plastidni lokalizacijski enzim, dio sekvencije zrelog N-terminalnog dijela biljnog gena šifriranog za plastidni lokalizacijski enzim, te sekvenciju šifriranu za drugi tranzitni peptid biljnoga gena koji je šifriran za plastidni lokalizacijski enzim, kako je opisano u patentnoj prijavi EP 0 508 909. Transit peptides can be either single or double, in which case they are optionally separated by an intermediate sequence, that is, one that contains in the direction of transcription the sequence coded for the transit peptide of the plant gene that is coded for the plastid localization enzyme, part of the sequence of the mature N-terminal part of the plant gene coded for a plastid localization enzyme, and a sequence coded for another transit peptide of a plant gene that is coded for a plastid localization enzyme, as described in patent application EP 0 508 909.
Kao tranzitni peptid koristan prema izumu može se navesti ponajprije signalni peptid duhanovoga PR-1α gena (WO 95/19443), predstavljen svojom šifriranom sekvencijom pomoću sekvencijskog identifikatora br. 2 (SEQ ID NO.2), a fuzioniran s androktoninom pomoću sekvencijskog identifikatora br. 3 (SEQ ID NO. 3), ponajprije u odgovarajući fuzijskom peptidu koji odgovara bazama 12 do 176 te sekvencije, a posebice kada je androktonin proizveden u biljnim stanicama ili biljkama, ili prekursor Mat α1 faktora kada je androktonin proizveden u kvascima. As a transit peptide useful according to the invention, the signal peptide of the tobacco PR-1α gene (WO 95/19443), represented by its coded sequence using the sequence identifier no. 2 (SEQ ID NO.2), and fused with androctonin using sequence identifier no. 3 (SEQ ID NO. 3), preferably in the corresponding fusion peptide corresponding to bases 12 to 176 of that sequence, and especially when androctonin is produced in plant cells or plants, or the precursor of the Mat α1 factor when androctonin is produced in yeasts.
Stoga se sadašnji izum odnosi u prvom redu na fragment nukleinske kiseline, ponajprije fragment DNA, šifriran za gore definiran androktonin. Prema izumu, to može biti fragment izoliran iz Androctonus australis, ili alternativno izvedeni fragment, prilagođen za ekspresiju androktonina u organizmu domaćinu u kojemu će peptid biti izražen. Fragment nukleinske kiseline može se dobiti prema standardnim metodama za izolaciju i čišćenje, ili alternativno sintezom prema uobičajenim tehnikama uzastopne hibridizacije sintetičkih oligonukleotida. Te tehnike opisali su ponajprije Ausubel et al. Therefore, the present invention relates primarily to a nucleic acid fragment, primarily a DNA fragment, coded for androctonin as defined above. According to the invention, it can be a fragment isolated from Androctonus australis, or an alternatively derived fragment adapted for the expression of androctonin in the host organism in which the peptide will be expressed. The nucleic acid fragment can be obtained according to standard methods for isolation and purification, or alternatively by synthesis according to conventional techniques of sequential hybridization of synthetic oligonucleotides. These techniques were first described by Ausubel et al.
Prema sadašnjem izumu izraz “fragment nukleinske kiseline” odnosi se na nukleotidnu sekvenciju koja može biti bilo DNA tipa ili RNA tipa, ponajprije DNA tipa, a prije svega cDNA, ponajprije dvolančanog tipa. According to the present invention, the term "nucleic acid fragment" refers to a nucleotide sequence that can be either of the DNA type or the RNA type, preferably of the DNA type, and primarily of the cDNA, preferably of the double-stranded type.
Prema obličju izuma, fragment nuleinske kiseline šifriran za androktonin je DNA sekvencija opisana sekvencijskim identifikatorom br. 1 (SEQ ID NO. 1), homologna sekvencija ili sekvencija komplementarna rečenoj sekvenciji, ponajprije šifrirajući dio toga SEQ ID NO.1, koji odgovara bazama 1 do 75. According to the invention, the nuleic acid fragment coded for androctonin is the DNA sequence described by the sequence identifier no. 1 (SEQ ID NO. 1), a homologous sequence or a sequence complementary to said sequence, preferably encoding a part of it SEQ ID NO. 1, which corresponds to bases 1 to 75.
Prema izumu, pod izrazom “homologno” podrazumijevaju se fragmenti nukleinskih kiselina koji uključuju jednu ili više modifikacija u usporedbi s nukleotidnom sekvencijom opisanom sekvencijskim identifikatorom br. 1 šifriranim za androktonin. Ove modifikacije mogu se dobiti uobičajenim tehnikama mutacije ili alternativno odabirom sintetičkih oligonukleotida uporabljenih u pripravi rečene sekvencije hibridizacijom. S obzirom na višestruke kombinacije nukleinskih kiselina koje mogu dovesti do ekspresije iste aminokiseline, razlike između referentne sekvencije opisane sekvencijskim identifikatorom br. 1 i homologne sekvencije mogu biti znatne, tim više ako se radi o fragmentu DNA čija je veličina manja od 100 nukleinskih kiselina, a koji se mogu pripraviti kemijskom sintezom. Ponajprije iznosi stupanj homolognosti barem 70% prema referentnoj sekvenciji, povoljnije barem 80%, a najpovoljnije barem 90%. Ove su modifikacije općenito neutralne, to jest one ne utječu na primarnu sekvenciju rezultirajućeg androktonina. According to the invention, the term "homologous" refers to nucleic acid fragments that include one or more modifications compared to the nucleotide sequence described by sequence identifier no. 1 coded for androctonin. These modifications can be obtained by conventional mutation techniques or alternatively by selecting synthetic oligonucleotides used in the preparation of said sequence by hybridization. Given the multiple combinations of nucleic acids that can lead to the expression of the same amino acid, the differences between the reference sequence described by sequence identifier no. 1 and homologous sequences can be substantial, especially if it is a DNA fragment whose size is less than 100 nucleic acids, and which can be prepared by chemical synthesis. Preferably, the degree of homology to the reference sequence is at least 70%, preferably at least 80%, and most preferably at least 90%. These modifications are generally neutral, that is, they do not affect the primary sequence of the resulting androctonin.
Sadašnji izum također se odnosi na kimerni gen (ili ekspresijsku kasetu) koji sadrži šifrirajuću sekvenciju i heterologne regulacijske elemente u položajima 5’ i 3’ koji mogu funkcionirati u organizmu domaćinu, ponajprije u biljnim stanicama ili biljkama, pri čemu su ti elementi funkcionalno spojeni s rečenom šifrirajućom sekvencijom, a šifrirajuća sekvencija sadrži najmanje jedan DNA fragment šifriran za androktonin kao što je gore definirano (uključujući fuzijski peptid “peptid-androktonin” ili androktonin-peptid”). The present invention also relates to a chimeric gene (or expression cassette) containing a coding sequence and heterologous regulatory elements in the 5' and 3' positions that can function in a host organism, preferably in plant cells or plants, wherein these elements are functionally linked to said coding sequence, and the coding sequence comprises at least one DNA fragment coding for androctonin as defined above (including a “peptide-androctonin” or androctonin-peptide fusion peptide”).
Podrazumijeva se da se izraz organizam domaćin odnosi na bilo koji jednostanični ili višestanični organizam nižeg reda ili višeg reda, u koji se može uvesti kimerni gen prema izumu, sa ciljem proizvodnje androktonina. Takvi organizmi su ponajprije E. coli, kvasci, prije svega kvasci roda Saccharomyces ili Kluyveromyces, Pichia, gljive, prije svega Aspergillus, bakulovirus ili ponajprije biljne stanice i biljke. It is understood that the term host organism refers to any unicellular or multicellular organism of lower order or higher order, into which the chimeric gene according to the invention can be introduced, with the aim of producing androctonin. Such organisms are primarily E. coli, yeasts, primarily yeasts of the genus Saccharomyces or Kluyveromyces, Pichia, fungi, primarily Aspergillus, baculovirus or primarily plant cells and plants.
Pod “biljnom stanicom” podrazumijevaju se prema izumu sve stanice nastale iz jedne biljke koje mogu sačinjavati nediferencirana tkiva kao što su žuljevi, diferencirana tkiva kao što su zametci, dijelovi biljaka, biljke ili klice. According to the invention, "plant cell" means all cells originating from a single plant that can form undifferentiated tissues such as calluses, differentiated tissues such as embryos, plant parts, plants or sprouts.
Pod “biljkom” se prema izumu podrazumijevaju svi diferencirani višestanični organizmi sposobni za provođenje fotosinteze, ponajprije monokotiledoni ili dikotiledoni, a posebice biljne kulture namijenjene hranjenju životinja ili ljudi, ili bez takve namjene, kao što su kukuruz, žito, repica, soja, riža, šećerna trska, šećerna repa, duhan, pamuk, itd. According to the invention, the term "plant" means all differentiated multicellular organisms capable of carrying out photosynthesis, primarily monocotyledons or dicotyledons, and especially plant crops intended for feeding animals or humans, or without such a purpose, such as corn, grain, rapeseed, soy, rice, sugar cane, sugar beet, tobacco, cotton, etc.
Regulacijski elementi potrebni za izražavanje fragmenta DNA šifriranog za androktonin dobro su poznati stručnjaku kao funkcija organizma domaćina. Oni uključuju ponajprije promotorske sekvencije, transkripcijske aktivatore, tranzitne peptide i terminacijske sekvencije, te kodone start i stop. Sredstva i metode identifikacije i odabira regulacijskih elemenata dobro su poznati stručnjaku iz tog područja. The regulatory elements required for the expression of the DNA fragment coding for androctonin are well known to those skilled in the art as a function of the host organism. They primarily include promoter sequences, transcriptional activators, transit peptides and termination sequences, and start and stop codons. Means and methods of identification and selection of regulatory elements are well known to a person skilled in the art.
Za transformaciju mikroorganizama kao što su kvasci ili bakterije, regulacijski elementi dobro su poznati stručnjacima u tome području, te uključuju ponajprije promotorske sekvencije, transkripcijske aktivatore, tranzitne peptide, terminacijske sekvencije i kodone start i stop. For the transformation of microorganisms such as yeasts or bacteria, regulatory elements are well known to those skilled in the art, and primarily include promoter sequences, transcriptional activators, transit peptides, termination sequences and start and stop codons.
Sa ciljem usmjerenja ekspresije i sekrecije peptida u kvaščevoj uzgojnoj sredini, DNA fragment šifriran za heliomicin ugrađen je u prijenosni vektor koji sadrži sljedeće elemente: In order to direct the expression and secretion of the peptide in the yeast culture medium, the DNA fragment coded for heliomycin was incorporated into a transfer vector containing the following elements:
• markere koji omogućuju odabir tranformanata, • markers that enable the selection of transformers,
• sekvenciju nukleinske kiseline koja omogućuje replikaciju (ishodište replikacije) plazmida u kvascu, • a nucleic acid sequence that enables the replication (origin of replication) of a plasmid in yeast,
• sekvenciju nukleinske kiseline koja omogućuje replikaciju (ishodište replikacije) plazmida u E. coli, • the nucleic acid sequence that enables the replication (origin of replication) of the plasmid in E. coli,
• ekspresijsku kasetu koja se sastoji od • an expression cassette consisting of
1. promotorske regulacijske sekvencije, 1. promoter regulatory sequences,
1. sekvencije šifrirane za signalni peptid (ili prepeptid) kombinirane s nekim adresirajućim peptidom (ili propeptidom), 1. sequences coded for a signal peptide (or prepeptide) combined with an addressing peptide (or propeptide),
1. poliadenilacijske ili terminatorske regulacijske sekvencije. 1. polyadenylation or terminator regulatory sequences.
Ovi su elementi opisani u nekoliko publikacija, uključujući Reichhart et al., Invert. Reprod. Dev., 21 (1992) 15-24, te Michaut et al., FEBS Letters, 395 (1996) 6-10. These elements are described in several publications, including Reichhart et al., Invert. Reprod. Dev., 21 (1992) 15-24, and Michaut et al., FEBS Letters, 395 (1996) 6-10.
Ponajprije, kvasci specija S. cerevisiiae transformiraju se pomoću ekspresijskih plazmida metodom litijevog acetata (Ito et al., J. Bacteriol. 153 (1993) 163-168). In particular, yeasts of the S. cerevisiae species are transformed using expression plasmids by the lithium acetate method (Ito et al., J. Bacteriol. 153 (1993) 163-168).
Izum se odnosi posebice na transformaciju biljaka. Kao promotorsku regulacijsku sekvenciju u biljaka moguće je uporabiti bilo koju promotorsku sekvenciju gena koja je prirodno izražena u biljkama, ponajprije promotor bakterijskog, virusnog ili biljnog podrijetla, kao što je primjerice onaj gena za malu podjedinicu ribuloza biskarboksilaza/oksigenaza (RuBisCO), ili onaj biljnog virusnog gena kao što je primjerice onaj virusa mozaika cvjetače (CAMV 19S ili 35S), ili promotor koji može biti induciran patogenima kao što je duhanski PR-1α, pri čemu se mogu uporabiti svi poznati prikladni promotori. Ponajprije se rabi promotorska regulacijska sekvencija koja favorizira nadekspresiju šifrirane sekvencije na konstitutivan način ili induciranu napadajem patogenih, kao što je primjerice ona koja sadrži najmanje jedan histonski promotor kao što je opisano u patentnoj prijavi EP 0 507 698. The invention relates in particular to the transformation of plants. As a promoter regulatory sequence in plants, it is possible to use any gene promoter sequence that is naturally expressed in plants, primarily a promoter of bacterial, viral or plant origin, such as that of the gene for the small subunit of ribulose biscarboxylase/oxygenase (RuBisCO), or that of plant a viral gene such as for example that of cauliflower mosaic virus (CAMV 19S or 35S), or a pathogen inducible promoter such as tobacco PR-1α, where all known suitable promoters can be used. Preferably, a promoter regulatory sequence is used which favors the overexpression of the encoded sequence in a constitutive manner or induced by pathogen attack, such as for example one containing at least one histone promoter as described in patent application EP 0 507 698.
Prema izumu se također može primijeniti, zajedno sa promotorskom regulacijskom sekvencijom i druge regulacijske sekvencije, koje su smještene između promotorske i šifrirane sekvencije, kao što su transkripcijski aktivatori (“enhancers”), primjerice translacijski aktivator virusa mozaika duhana (TMV) opisan u patentnoj prijavi WO 87/07644, ili translacijski aktivator duhanovog virusa etch (TEV) koji su opisali Carrington & Freed. According to the invention, it is also possible to apply, together with the promoter regulatory sequence, other regulatory sequences, which are located between the promoter and the coding sequence, such as transcriptional activators ("enhancers"), for example the translational activator of the tobacco mosaic virus (TMV) described in the patent application WO 87/07644, or the tobacco etch virus (TEV) translational activator described by Carrington & Freed.
Kao poliadenilacijska ili teminacijska regulacijska sekvencija može se primijeniti bilo koja odgovarajuća sekvencija bakterijskog podrijetla, kao primjerice terminator Agrobacterium tumefaciens, ili alternativno biljnog podrijetla kao primjerice histonski terminator opisan u patentnoj prijavi EP 0 633 317. Any appropriate sequence of bacterial origin, such as the Agrobacterium tumefaciens terminator, or alternatively of plant origin, such as the histone terminator described in patent application EP 0 633 317, can be used as a polyadenylation or termination regulatory sequence.
Prema sadašnjem izumu kimerni gen može se kombinirati i sa selekcijskim markerom adaptiranim u odnosu na transformirani organizam domaćina. Takvi selekcijski markeri dobro su poznati stručnjacima u tom području. Takvi markeri mogu biti antibiotički rezistencijski gen ili herbicidni tolerancijski gen za biljke. According to the present invention, the chimeric gene can be combined with a selection marker adapted to the transformed host organism. Such selectable markers are well known to those skilled in the art. Such markers can be an antibiotic resistance gene or a herbicide tolerance gene for plants.
Sadašnji izum također se odnosi na klonski ili ekspresijski vektor za transformaciju organizma domaćina koji sadrži najmanje jedan kimerni gen kao što je gore definirano. Osim gornjeg kimernoga gena, taj vektor sadrži najmanje jedno replikacijsko ishodište, te prema potrebi i prikladan selekcijski marker. Taj se vektor može sastojati od plazmida, kozmida, bakteriofaga ili virusa, koji su transformirani uvođenjem kimernoga gena prema izumu. Ovisno o organizmu domaćinu koji treba transformirati, takvi transformacijski vektori dobro su poznati stručnjacima u tome području i potanko su opisani u literaturi. The present invention also relates to a clonal or expression vector for transformation of a host organism containing at least one chimeric gene as defined above. In addition to the above chimeric gene, this vector contains at least one origin of replication and, if necessary, a suitable selection marker. This vector can consist of a plasmid, cosmid, bacteriophage or virus, which have been transformed by introducing a chimeric gene according to the invention. Depending on the host organism to be transformed, such transformation vectors are well known to those skilled in the art and are described in detail in the literature.
Za transformaciju biljnih stanica ili biljaka takav je vektor ponajprije virus koji se može primijeniti za transformaciju razvijenih biljaka, a koje sadrže i vlastite replikacijske i ekspresijske elemente. Ponajprije, vektor za transformaciju biljnih stanica ili biljaka prema izumu je plazmid. For the transformation of plant cells or plants, such a vector is primarily a virus that can be used for the transformation of developed plants, which also contain their own replication and expression elements. First of all, the vector for the transformation of plant cells or plants according to the invention is a plasmid.
Predmet izuma također je postupak transformacije organizma domaćina, ponajprije biljnih stanica, inkorporiranjem najmanje jednog fragmenta nukleinske kiseline ili jednog kimernog gena prema gornjoj definiciji, pri čemu je moguće postići tu transformaciju bilo kojim od poznatih prikladnih načina koji su dostatno opisani u specijaliziranoj literaturi, a posebice u navodima citiranim u sadašnjoj patentnoj prijavi, prije svega pomoću vektora prema izumu. The subject of the invention is also the process of transformation of the host organism, primarily plant cells, by incorporating at least one nucleic acid fragment or one chimeric gene according to the above definition, whereby it is possible to achieve this transformation by any of the known suitable methods that are sufficiently described in the specialized literature, and in particular in the statements cited in the present patent application, primarily by means of the vector according to the invention.
Niz metoda sastoji se u bombardiranju stanica, protoplasta ili tkiva česticama na koje su povezane sekvencije DNA. Drugi niz metoda sastoji se u primjeni kimernog gena umetnutog u Ti plazmid iz Agrobacterium tumefaciens ili Ri plazmid iz Agrobacterium rhizogenes kao transfernog sredstva u biljku. A number of methods consist of bombarding cells, protoplasts or tissues with particles to which DNA sequences are attached. Another series of methods consists in the application of a chimeric gene inserted into the Ti plasmid from Agrobacterium tumefaciens or the Ri plasmid from Agrobacterium rhizogenes as a means of transfer into the plant.
Od ostalih se metoda mogu primijeniti mikroinjekcijska ili elektroporna metoda, ili alternativno izravno taloženje primjenom PEG. Among other methods, microinjection or electroporation methods can be used, or alternatively, direct deposition using PEG.
Stručnjak u tom području odabrati će prikladnu metodu prema prirodi organizma domaćina, ponajprije biljne stanice ili biljke. An expert in the field will select a suitable method according to the nature of the host organism, preferably a plant cell or plant.
Predmet sadašnjeg izuma također je transformirani organizmi domaćini, ponajprije biljne stanice ili biljke, koji sadrže djelotvornu količinu kimernog gena koji uključuje sekvenciju šifriranu za gore definirani androktonin. The subject of the present invention is also transformed host organisms, in particular plant cells or plants, which contain an effective amount of a chimeric gene including the sequence coding for androctonin as defined above.
Također su predmet sadašnjeg izuma biljke koje sadrže transformirane stanice, ponajprije biljke regenerirane iz transformiranih stanica. Regeneracija se postiže bilo kojim prikladnim postupkom koji ovisi o prirodi vrste, kao što je opisano primjerice u gornjim navodima. Also subject to the present invention are plants containing transformed cells, especially plants regenerated from transformed cells. Regeneration is achieved by any suitable process depending on the nature of the species, as described for example in the above references.
Za postupke transformacije biljnih stanica i regeneracije biljaka treba ponajprije citirati sljedeće patente i patentne prijave: US 4,459,355, US 4,536,475, US 5,464,763, US 5,177,010, US 5,187,073, EP 267 159, EP 604 662, EP 672 752, US 4,945,050, US 5,036,006, US 5,100,792, US 5,371,014, US 5,478,744, US 5,179,022, US 5,565,346, US 5,484,956, US 5,508,468, US 5,538,877, US 5,554,798, US 5,489,520, US 5,510,318, US 5,204,253, US 5,405,765, EP 442 174, EP 486 233, EP 486 234, EP 539 563, EP 674 725, WO 91/02071 i WO 95/06128. For plant cell transformation and plant regeneration procedures, the following patents and patent applications should first be cited: US 4,459,355, US 4,536,475, US 5,464,763, US 5,177,010, US 5,187,073, EP 267 159, EP 604 662, EP 672 752, US 4,945,050 US 5,100,792, US 5,371,014, US 5,478,744, US 5,179,022, US 5,565,346, US 5,484,956, US 5,508,468, US 5,538,877, US 5,554,798, US 5,489,520, US 5,510,318, US 5,204,253, US 5,405,765, EP 442 174, EP 486 233, EP 486 234 , EP 539 563, EP 674 725, WO 91/02071 and WO 95/06128.
Predmet sadašnjeg izuma također su transformirane biljke dobivene uzgojem i/ili križanjem regeneriranih biljaka, kao i sjeme transformiranih biljaka. The subject of the present invention are also transformed plants obtained by growing and/or crossing regenerated plants, as well as seeds of transformed plants.
Tako transformirane biljke otporne su prema nekim bolestima, ponajprije nekim gljivičnim ili bakterijskim bolestima. Posljedično tome, sekvencija DNA šifrirana za androktonin može se umetnuti s glavnim sredstvom za proizvodnju biljaka otpornih na rečene bolesti, budući da je androktonin djelotvoran protiv gljivičnih bolesti kao što su one prouzročenih vrstama Cerospora, ponajprije Cerospora beticola, Cladosporium, ponajprije Cladosporium herbarum, Fusarium, ponajprije Fusarium culmorum ili Fusarium graminearum, ili Phytophtora, ponajprije Phytophtora cinnamoni. Plants transformed in this way are resistant to some diseases, especially some fungal or bacterial diseases. Consequently, the DNA sequence coding for androctonin can be inserted with the main means of producing plants resistant to said diseases, since androctonin is effective against fungal diseases such as those caused by Cerospora species, in particular Cerospora beticola, Cladosporium, in particular Cladosporium herbarum, Fusarium, primarily Fusarium culmorum or Fusarium graminearum, or Phytophtora, primarily Phytophtora cinnamoni.
Kimerni gen može se ponajprije kombinirati s najmanje jednim selekcijskim markerom, kao što je jedan ili više herbicidnih tolerancijskih gena. The chimeric gene can preferably be combined with at least one selection marker, such as one or more herbicide tolerance genes.
Sekvencija DNA šifrirana za androktonin može se također umetnuti kao selekcijski marker za vrijeme transformacije biljaka drugim sekvencijama šifriranim za druge peptide ili proteine od interesa, kao što su primjerice herbicidni tolerancijski geni. The DNA sequence coding for androctonin can also be inserted as a selection marker during transformation of plants with other sequences coding for other peptides or proteins of interest, such as for example herbicide tolerance genes.
Takvi herbicidni tolerancijski geni dobro su poznati stručnjacima u tom području i ponajprije su opisani u patentnim prijavama EP 115 673, WO 87/04181, EP 337 899, WO 96/38567 ili WO 97/04103. Such herbicide tolerance genes are well known to those skilled in the art and are particularly described in patent applications EP 115 673, WO 87/04181, EP 337 899, WO 96/38567 or WO 97/04103.
Naravno, stanice i biljke transformirane prema izumu mogu također sadržavati sekvenciju šifriranu za androktonin, druge heterologne sekvencije šifrirane za proteine od interesa kao što su drugi komplementarni peptidi sposobni za pružanje otpornosti biljkama prema drugim bolestima bakterijskog podrijetla ili gljivičnog podrijetla, i/ili druge sekvencije šifrirane za herbicidne tolerancijske proteine, ponajprije one definirane ranije, i/ili druge sekvencije šifrirane za insektne rezistencijske proteine, kao što je ponajprije protein Bt. Of course, cells and plants transformed according to the invention may also contain a sequence coding for androctonin, other heterologous sequences coding for proteins of interest such as other complementary peptides capable of conferring resistance to plants against other diseases of bacterial origin or fungal origin, and/or other sequences coding for for herbicide tolerance proteins, preferably those defined earlier, and/or other sequences encoded for insect resistance proteins, such as preferably the Bt protein.
Druge sekvencije mogu se umetnuti pomoću istog vektora koji sadrži kimerni gen prema izumu, a koji sadrži sekvenciju šifriranu za androktonin i najmanje jedan drugi gen koji uključuje drugu sekvenciju šifriranu za neki drugi peptid ili protein od interesa. Other sequences can be inserted using the same vector containing the chimeric gene according to the invention, which contains the sequence coding for androctonin and at least one other gene which includes another sequence coding for some other peptide or protein of interest.
One mogu također biti umetnute pomoću nekog drugog vektora koji sadrži najmanje jednu od rečenih drugih sekvencija, prema uobičajenim gore navedenim tehnikama. They may also be inserted by means of another vector containing at least one of said other sequences, according to the usual techniques mentioned above.
Biljke prema izumu mogu također biti dobivene križanjem roditelja, pri čemu jedan od njih donosi gen prema izumu šifriran za androktonin, a drugi donosi gen šifriran za najmanje jedan drugi peptid ili protein od interesa. Plants according to the invention can also be obtained by crossing parents, one of which carries a gene according to the invention encoding androctonin, and the other carries a gene encoding at least one other peptide or protein of interest.
Među sekvencijama šifriranim za druge protugljivične peptide može se spomenuti onu šifriranu za drosomicin, opisanu u patentnoj prijavi FR 2,725,992 i koju su opisali Fehlbaum et al. (1994), te u neobjavljenoj patentnoj prijavi FR 97/09115 zaprimljenoj 24. srpnja 1997. Among the sequences encoding other antifungal peptides, one can mention that encoding drosomycin, described in patent application FR 2,725,992 and described by Fehlbaum et al. (1994), and in unpublished patent application FR 97/09115 received July 24, 1997.
Sadašnji izum odnosi se konačno na postupak uzgoja biljaka transformiranih prema izumu, postupak koji se sastoji od sijanja sjemena rečenih transformiranih biljaka u površinu uzgojnog okoliša, ponajprije polja prikladnog za uzgoj rečene biljke, primjene agrokemijskog pripravka na rečenu površinu, bez bitnog utjecaja na rečeno transformirano sjeme ili biljke, potom pobiranje kultiviranih biljaka koje su postigle željenu zrelost i opcijski odvajanje sjemena od pobranih biljaka. The present invention finally relates to the process of growing plants transformed according to the invention, a process that consists of sowing the seeds of said transformed plants in the surface of the growing environment, primarily a field suitable for growing said plant, applying an agrochemical preparation to said surface, without significant impact on said transformed seeds or plants, then harvesting the cultivated plants that have reached the desired maturity and optionally separating the seeds from the harvested plants.
Prema izumu, izrazom agrokemijski pripravci podrazumijevaju se svi agrokemijski pripravci koji sadrže najmanje jedan djelotvoran produkt koji posjeduje bilo herbicidnu, fungicidnu, baktericidnu, virucidnu ili insekticidnu aktivnost. According to the invention, the term agrochemical preparations means all agrochemical preparations that contain at least one effective product that has either herbicidal, fungicidal, bactericidal, virucidal or insecticidal activity.
Prema načinu realizacije postupka uzgoja koji je u prednosti prema izumu, agrokemijski pripravak sadrži najmanje jedan djelotvoran produkt koji pokazuje najmanje fungicidnu i/ili baktericidnu aktivnost, te uz to ponajprije posjeduje aktivnost komplementarnu onoj androktonina proizvedenog u biljci transformiranoj prema izumu. According to the method of realization of the cultivation process which is advantageous according to the invention, the agrochemical preparation contains at least one effective product which shows at least fungicidal and/or bactericidal activity, and in addition, it primarily possesses an activity complementary to that of androctonin produced in the plant transformed according to the invention.
Prema izumu, kao ekspresijski produkt koji ima aktivnost komplementarnu onoj androktonina podrazumijeva se produkt koji posjeduje spektar komplemantarnih aktivnosti, što znači produkt koji će biti aktivan protiv napadaja kontaminanata neosjetljivih na androktonin (gljive, bakterije ili virusi), ili alternativno produkt čiji spektar aktivnosti potpuno ili djelomice prekriva onaj androktonina, te čija je aplikacijska doza znatno smanjena zbog prisutnosti androktonina proizvedenog u transformiranoj biljci. According to the invention, an expression product that has an activity complementary to that of androctonin means a product that possesses a spectrum of complementary activities, which means a product that will be active against the attack of contaminants insensitive to androctonin (fungi, bacteria or viruses), or alternatively a product whose spectrum of activity completely or it partially covers that of androctonin, and whose application dose is significantly reduced due to the presence of androctonin produced in the transformed plant.
Konačno, uzgoj transformiranih organizama domaćina omogućuje široku ljestvicu proizvodnje androktonina. Predmet sadašnjeg izuma je stoga također postupak priprave androktonina, koji uključuje korake uzgoja transformiranih organizama domaćina koji sadrže gen šifriran za androktonin prema gornjoj definiciji u prikladnom uzgojnom okruženju, te potom ekstrakciju i potpuno ili djelomično čišćenje dobivenog androktonina. Finally, cultivation of transformed host organisms allows large-scale production of androctonin. The subject of the present invention is therefore also a process for the preparation of androctonin, which includes the steps of growing transformed host organisms containing the gene encoded for androctonin as defined above in a suitable culture environment, and then extracting and completely or partially purifying the obtained androctonin.
Primjerima navedeni dalje omogućena je ilustracija izuma, priprave sekvencije šifrirane za androktonin, kimernog gena, integracijskog vektora i transformiranih biljaka. Priložene slike 1 do 5 ocrtavaju shematske strukture nekih plazmida pripravljenih za konstrukciju kimernih gena. U tim slikama su različiti restrikcijski položaji označeni kurzivom. The following examples illustrate the invention, the preparation of the sequence coded for androctonin, the chimeric gene, the integration vector and the transformed plants. Attached figures 1 to 5 outline the schematic structures of some plasmids prepared for the construction of chimeric genes. In these figures, different restriction sites are marked in italics.
Primjer 1 Example 1
Konstrukcija kimernih gena Construction of chimeric genes
Sve dalje primijenjene tehnike standardne su laboratorijske tehnike. Detaljni postupci tih tehnika posebice su opisali Ausubel et al. All further applied techniques are standard laboratory techniques. The detailed procedures of these techniques were particularly described by Ausubel et al.
pRPA-MD-P: Priprava plazmida koji sadrži signalni peptid za duhanov PR-1α gen. pRPA-MD-P: Preparation of a plasmid containing a signal peptide for the tobacco PR-1α gene.
Dva komplementarna sintetička oligonukleotida Oligo 1 i Oligo 2 navedena dalje, hibridzirana su pri 65 °C kroz 5 minuta, a potom uz polagano sniženje temperature na 30 °C kroz 30 minuta. Two complementary synthetic oligonucleotides, Oligo 1 and Oligo 2, listed below, were hybridized at 65 °C for 5 minutes, and then slowly lowering the temperature to 30 °C for 30 minutes.
[image] [image]
Nakon hibridizacije između Oligo 1 i Oligo 2, preostala jednolančana DNA služi kao matrica za klenow fragment iz E. coli polimeraze 1 (pod standardnim uvjetima preporučenima od proizvođača (New England Biolabs)) za pripravu dvolančanog oligonukleotida počevši od 3’ kraja svakog oligo. Dobiveni dvolančani oligonukleotid potom je digeriran s restrikcijskim encimima SacII i NaeI i kloniran u plazmid pBS II SK(-) (Stratagene) digeriran s istim restrikcijskim encimima. Dobiven je time klon koji je sadržavao regiju šifriranu za signalni peptid duhanovog PR-1α gena (SEQ ID NO. 2). After hybridization between Oligo 1 and Oligo 2, the remaining single-stranded DNA serves as a template for the klenow fragment from E. coli polymerase 1 (under standard conditions recommended by the manufacturer (New England Biolabs)) to prepare a double-stranded oligonucleotide starting from the 3' end of each oligo. The resulting double-stranded oligonucleotide was then digested with restriction enzymes SacII and NaeI and cloned into plasmid pBS II SK(-) (Stratagene) digested with the same restriction enzymes. A clone was obtained that contained the region coded for the signal peptide of the tobacco PR-1α gene (SEQ ID NO. 2).
pRPA-PS-PR1a-andro: Priprava sekvencije šifrirane za androktonin spojen na PR-1α signalni peptid bez netranskribiranog 3’ područja. pRPA-PS-PR1a-andro: Preparation of the sequence coding for androctonin fused to the PR-1α signal peptide without the non-transcribed 3' region.
Dva komplementarna sintetička oligonukleotida Oligo 3 i Oligo 4 hibridzirana su prema radnim uvjetima navedenima za pRPA-MD-P. Two complementary synthetic oligonucleotides Oligo 3 and Oligo 4 were hybridized according to the operating conditions specified for pRPA-MD-P.
[image] [image]
Nakon hibridizacije između Oligo 3 i Oligo 4, preostala jednolančana DNA služi kao matrica za klenow fragment iz E. coli polimeraze 1 (pod standardnim uvjetima preporučenima od proizvođača (New England Biolabs)) za pripravu dvolančanog oligonukleotida počevši od 3’ kraja svakog oligo. Dobiveni dvolančani oligonukleotid koji sadrži dio šifriran za androktonin (SEQ ID NO. 1) potom je kloniran izravno u plazmid pRPA-MD-P, koji je digeriran s restrikcijskim enzimom NaeI. Pravilna orijentacija dobivenog klona provjerena je sekvenciranjem. Dobiven je time klon koji je sadržavao regiju šifriranu za fuzijski protein PR-1α-androktonin, smještenu između restrikcijskog položaja na N-terminalnom kraju te restrikcijskim položajima ScaI, ScaII i BamHI C-terminalnog kraja (SEQ ID NO. 3). After hybridization between Oligo 3 and Oligo 4, the remaining single-stranded DNA serves as a template for the klenow fragment from E. coli polymerase 1 (under standard conditions recommended by the manufacturer (New England Biolabs)) to prepare a double-stranded oligonucleotide starting from the 3' end of each oligo. The obtained double-stranded oligonucleotide containing the part encoded for androctonin (SEQ ID NO. 1) was then cloned directly into the plasmid pRPA-MD-P, which was digested with the restriction enzyme NaeI. The correct orientation of the obtained clone was verified by sequencing. This resulted in a clone containing the region coded for the fusion protein PR-1α-androctonin, located between the restriction site at the N-terminal end and the restriction sites ScaI, ScaII and BamHI at the C-terminal end (SEQ ID NO. 3).
pRPA-RD-238: Priprava ekspresijskog vektora u biljaka koji sadrži sekvenciju šifriranu za fuzijski protein PR-1α androktonin. pRPA-RD-238: Preparation of an expression vector in plants containing the sequence coded for the fusion protein PR-1α androctonin.
Plazmid pRTL-2 GUS izveden iz plazmida pUC-19 dobiven je od Dr. Jim Carringtona (Texas A&M University, neopisan). Taj plazmid čija je shematska struktura prikazana na slici 3, sadrži dvostruki promotor CaMV 35S izoliran iz virusa mozaika cvjetače (promotor CaMV 2x35S; Odell et al., 1985) koji usmjerava ekspresiju RNA, koja sadrži netranslatiranu sekvenciju na 5’ duhanovog virusa etch (TEV 5’ UTR; Carrington i Freed, 1990), β-glukuronidazni gen iz E. coli (GUS; Jefferson et al., 1987), potom CaMV RNA 35S poliadenilacijski položaj (CaMV polyA; Odell et al., 1985). Plasmid pRTL-2 GUS derived from plasmid pUC-19 was obtained from Dr. Jim Carrington (Texas A&M University, undescribed). This plasmid, the schematic structure of which is shown in Figure 3, contains a CaMV 35S double promoter isolated from cauliflower mosaic virus (CaMV 2x35S promoter; Odell et al., 1985) that directs the expression of an RNA containing the 5' untranslated sequence of tobacco etch virus (TEV). 5' UTR; Carrington and Freed, 1990), the β-glucuronidase gene from E. coli (GUS; Jefferson et al., 1987), then the CaMV RNA 35S polyadenylation site (CaMV polyA; Odell et al., 1985).
Plazmid pRTL-2 GUS digeriran je s restrikcijskim enzimima NcoI i BamHI i glavni fragment DNA je očišćen. Plazmid pRPA-PS-Pr1a-andro digeriran je s restrikcijskim enzimima NcoI i BamHI i mali fragment DNA koji je sadržavao regiju šifriranu za fuzijski protein PR-1a-androktonin također je očišćen. Dva očišćena fragmenta DNA potom su zajedno spojena u ekspresijskoj kaseti u biljkama koje sintetiziraju fuzijski protein PR-1a-androktonin. Shematska struktura tih ekspresijskih kaseta prikazana je na Slici 2. “PR-1a-androktonin” predstavlja područje šifrirano za fuzijski protein PR-1a-androktonin iz pRPA-RD-230. Androktonin je transportiran u ekstracelularnu matricu biljke djelovanjem peptidnog signala PR-1a. Plasmid pRTL-2 GUS was digested with restriction enzymes NcoI and BamHI and the main DNA fragment was purified. Plasmid pRPA-PS-Pr1a-andro was digested with restriction enzymes NcoI and BamHI and a small DNA fragment containing the region coding for the fusion protein PR-1a-androctonin was also purified. The two purified DNA fragments were then joined together in an expression cassette in plants that synthesize the PR-1a-androctonin fusion protein. The schematic structure of these expression cassettes is shown in Figure 2. “PR-1a-androctonin” represents the region encoded for the PR-1a-androctonin fusion protein from pRPA-RD-230. Androctonin is transported into the extracellular matrix of the plant by the action of the signal peptide PR-1a.
pRPA-RD-195: Priprava plazmida koji sadrži modificiran višestruki položaj kloniranja. pRPA-RD-195: Preparation of a plasmid containing a modified multiple cloning site.
Plazmid pRPA-RD-195 je plazmid deriviran iz pUC-19 koji sadrži modificiran višestruki položaj kloniranja. Komplementarni sintetički oligonukleotidi Oligo 5 i Oligo 6 prikazani dolje, hibridizirani su i imaju dvostruki lanac prema postupku opisanom za pRPA-MD-P. Plasmid pRPA-RD-195 is a plasmid derived from pUC-19 containing a modified multiple cloning site. The complementary synthetic oligonucleotides Oligo 5 and Oligo 6 shown below were hybridized and double-stranded according to the procedure described for pRPA-MD-P.
[image] [image]
Dobiveni dvolančani oligonukleotid potom je umetnut u pUC-19 koji je prethodno digeriran s restrikcijskim enzimima EcoRI i HindIII i otupljen na vrhovima primjenom fragmenta klenow DNA polimeraze 1 iz E. coli. Dobiven je vektor koji je sadržavao višestruke klonske položaje za omogućavanje uvođenja ekspresijskih kaseta u plazmidni vektor iz Agrobacterium tumefaciens. Shematska struktura višestrukog klonskog položaja prikazana je na slici 3. The resulting double-stranded oligonucleotide was then inserted into pUC-19, which was previously digested with the restriction enzymes EcoRI and HindIII and blunted at the ends using a fragment of klenow DNA polymerase 1 from E. coli. A vector containing multiple cloning sites was obtained to enable the introduction of expression cassettes into a plasmid vector from Agrobacterium tumefaciens. The schematic structure of the multiple clone position is shown in Figure 3.
pRPA-RD-233: Uvođenje ekspresijske kasete PR-1a-androktonina iz pRPA-RD-230 u pRPA-RD-195. pRPA-RD-233: Introduction of the PR-1a-androctonin expression cassette from pRPA-RD-230 into pRPA-RD-195.
Plazmid pRPA-RD-230 digeriran je s restrikcijskim enzimom HindIII. Fragment DNA koji je sadržavao ekspresijsku kasetu PR-1a-androktonina očišćen je. Očišćeni fragment potom je umetnut u pRPA-RD-195 koji je prethodno digeriran s restrikcijskim enzimom HindIII i defosforiliziran s telećom intestinalnom fosfatazom. Plasmid pRPA-RD-230 was digested with the restriction enzyme HindIII. The DNA fragment containing the PR-1a-androctonin expression cassette was purified. The purified fragment was then inserted into pRPA-RD-195, which was previously digested with the restriction enzyme HindIII and dephosphorylated with calf intestinal phosphatase.
pRPA-RD-174: Plazmid izveden iz pRPA-BL-150A (EP 0 508 909) koji je sadržavao tolerancijski gen na bromoksinil iz pRPA-BL-237 (EP 0 508 909). pRPA-RD-174: Plasmid derived from pRPA-BL-150A (EP 0 508 909) which contained the bromoxynil tolerance gene from pRPA-BL-237 (EP 0 508 909).
Tolerancijski gen na bromoksinil izoliran je iz pRPA-BL-237 pomoću PCR genske amplifikacije. Dobiveni fragment ima tupe vrške i kloniran je u položaju EcoRI iz pRPA-BL-150A koji je dobio tupe vrške djelovanjem polimeraze klenow pod standardnim uvjetima. Dobiven je vektor Agrobacterium tumefaciens koji je sadržavao tolerancijski gen na bromoksinil u blizini svojega desnog kraja, tolerancijski gen na kanamicin u blizini svojeg lijevog kraja i višestruki klonski položaj između ta dva gena. The bromoxynil tolerance gene was isolated from pRPA-BL-237 by PCR gene amplification. The resulting fragment has blunt ends and was cloned in the EcoRI position from pRPA-BL-150A, which was blunted by Klenow polymerase under standard conditions. An Agrobacterium tumefaciens vector was obtained that contained a bromoxynil tolerance gene near its right end, a kanamycin tolerance gene near its left end, and a multiple clonal position between these two genes.
Shematska struktura pRPA-RD-174 predstavljena je na slici 4. Na toj slici “nos” predstavlja poliadenilacijski položaj nopalin sintaze iz Agrobacterium tumefaciens (Bevan et al., 1983), “NOS pro” predstavlja promotor nopalin sintaze iz Agrobacterium tumefaciens, “NPT II” predstavlja neomicin fosfotransferazni gen transposona Tn5 iz E. coli (Rothstein et al., 1981), “35S pro” predstavlja promotor 35S izoliran iz virusa mozaika cvjetače (Odell et al., 1985), “BRX” predstavlja nitrilazni gen izoliran iz K. ozaenae (Stalker et al., 1988), “RB” i “LB” predstavljaju desni, odnosno lijevi kraj sekvencije Ti plazmida iz Agrobacterium tumefaciens. The schematic structure of pRPA-RD-174 is presented in Figure 4. In this figure, "nos" represents the polyadenylation site of nopaline synthase from Agrobacterium tumefaciens (Bevan et al., 1983), "NOS pro" represents the promoter of nopaline synthase from Agrobacterium tumefaciens, "NPT II" represents the neomycin phosphotransferase gene of transposon Tn5 from E. coli (Rothstein et al., 1981), "35S pro" represents the 35S promoter isolated from cauliflower mosaic virus (Odell et al., 1985), "BRX" represents the nitrilase gene isolated from K. ozaenae (Stalker et al., 1988), “RB” and “LB” represent the right and left ends of the Ti plasmid sequence from Agrobacterium tumefaciens, respectively.
pRPA-RD-184: Dodatak novog, jedinstvenog restrikcijskog položaja u pRPA-RD-174. pRPA-RD-184: Addition of a new, unique restriction site in pRPA-RD-174.
Komplementarni sintetički dolje prikazani oligonukleotidi Oligo 7 i Oligo 8 hibridizirani su i pripravljen im je dvostruki lanac prema postupku opisanom za pRPA-RD-183. Complementary synthetic oligonucleotides Oligo 7 and Oligo 8 shown below were hybridized and their double chain was prepared according to the procedure described for pRPA-RD-183.
[image] [image]
Hibridizirani dvolančani oligonukleotidi (96 parova baza) očišćen je nakon separacije na agarnom gelu (3 % Nusieve, FMC). Plazmid pRPA-RD-174 digeriran je s restrikcijskim enzimom XmaI i glavni fragment DNA je očišćen. Dva dobivena fragmenta DNA potom su međusobno spojena. Hybridized double-stranded oligonucleotides (96 base pairs) were purified after separation on agar gel (3% Nusieve, FMC). Plasmid pRPA-RD-174 was digested with the restriction enzyme XmaI and the main DNA fragment was purified. The two obtained DNA fragments were then connected to each other.
Dobiven je plazmid izveden iz pRPA-RD-174 koji je sadržavao druge restrikcijske položaje između tolerancijskog gena za bromoksinil i selekcijskog markera kanamicin gena. A plasmid derived from pRPA-RD-174 was obtained which contained other restriction sites between the tolerance gene for bromoxynil and the selection marker of the kanamycin gene.
Shematska struktura plazmida pRPA-RD-184 prikazana je na slici 5, u kojoj izrazi “nos”, “NPT II”, “NOS pro”, “35S pro”, “BRX gen”, “RB” i “LB” imaju isto značenje kao i na slici 4. The schematic structure of plasmid pRPA-RD-184 is shown in Figure 5, in which the terms “nos”, “NPT II”, “NOS pro”, “35S pro”, “BRX gene”, “RB” and “LB” have the same meaning as in Figure 4.
pRPA-RD-236: Priprava vektora Agrobacterium tumefaciens koji sadrži gensku konstrukciju šifriranu za androktonin usmjeren prema ekstracelularnoj matrici. pRPA-RD-236: Preparation of an Agrobacterium tumefaciens vector containing a gene construct encoding androctonin targeting the extracellular matrix.
Plazmid pRPA-RD-233 digeriran je s restrikcijskim enzimima PmeI i AscI, a DNA fragment koji sadrži gen PR-1a-androktonin je očišćen. Plazmid pRPA-RD-184 digeriran je s istim restrikcijskim enzimima. DNA fragment koji sadrži PR-1a-androktonin ekspresijsku kasetu potom je umetnut u pRPA-RD-184. Tako je dobiven vektor iz Agrobacterium tumefaciens koji je sadržavao sekvenciju šifriranu za fuzijski protein PR-1a-androktonin koji je doveo do ekspresije androktonina u ekstracelularnoj biljnoj matrici. Plasmid pRPA-RD-233 was digested with restriction enzymes PmeI and AscI, and the DNA fragment containing the PR-1a-androctonin gene was purified. Plasmid pRPA-RD-184 was digested with the same restriction enzymes. The DNA fragment containing the PR-1a-androctonin expression cassette was then inserted into pRPA-RD-184. Thus, a vector was obtained from Agrobacterium tumefaciens that contained a sequence coded for the fusion protein PR-1a-androctonin, which led to the expression of androctonin in the extracellular plant matrix.
Primjer 2 Example 2
Tolerancija na herbicide u transformiranih biljaka duhana Tolerance to herbicides in transformed tobacco plants
2.1 - Transformacija 2.1 - Transformation
Vektor pRPA-RD-236 uveden je u lanac Agrobacterium tumefaciens EHA101 (Hood et al., 1987) koji je u sebi nosio kozmid pTVK291 (Komari et al., 1986). Tehnika transformacije temeljena je na postupku koji su opisali Horsh et al. (1985). The pRPA-RD-236 vector was introduced into the Agrobacterium tumefaciens EHA101 strain (Hood et al., 1987) carrying the pTVK291 cosmid (Komari et al., 1986). The transformation technique is based on the procedure described by Horsh et al. (1985).
2.2 - Regeneracija 2.2 - Regeneration
Regeneracija duhanskih biljaka PBD6 (podrijetlo SEITA Francuska) počevši od folijarnih eksplanata provedena je u bazičnoj sredini Murashige-Skoog (MS) uključujući 30 g/l sukroze i 200 μg/ml kanamicina. Folijarni eksplanti su unaprijed uzeti s kultiviranih biljaka u stakleniku ili in vitro i regenerirani tehnikom folijarnih diskova (Horsh et al., 1985) u tri uzastopna koraka: prvi korak uključuje indukciju mladica u sredinu u koju je dodano 30 g/l saharoze koja sadrži 0.05 mg/l naftiloctene kiseline (NAA) i 2 mg/l benzilaminopurina (BAP) kroz 2 tjedna. Mladice formirane tijekom tog koraka potom su uzgajane kroz 10 dana kultiviranjem u sredini MS kojoj je dodano 30 g/l saharoze ali bez hormona. Potom, razvijene mladice izvađene su i kultivirane u sredini MS za ukorjenjivanje sa polovicom sadržaja soli, vitamina i šećera, a bez sadržaja hormona. Nakon približno 2 tjedna ukorijenjene mladice su premještene u staklenik. Regeneration of tobacco plants PBD6 (origin SEITA France) starting from foliar explants was carried out in basic Murashige-Skoog (MS) medium including 30 g/l sucrose and 200 μg/ml kanamycin. Foliar explants were taken in advance from cultivated plants in the greenhouse or in vitro and regenerated by the foliar disk technique (Horsh et al., 1985) in three successive steps: the first step involved the induction of seedlings in a medium supplemented with 30 g/l sucrose containing 0.05 mg/l naphthylacetic acid (NAA) and 2 mg/l benzylaminopurine (BAP) for 2 weeks. The saplings formed during this step were then grown for 10 days by culturing in MS medium to which 30 g/l of sucrose was added but without hormones. Then, the developed saplings were taken out and cultured in MS medium for rooting with half the content of salt, vitamins and sugar, and without the content of hormones. After approximately 2 weeks, the rooted saplings were moved to the greenhouse.
2.3 - Tolerancija na bromoksinil 2.3 - Tolerance to bromoxynil
Dvadeset transformiranih biljaka regenerirano je i stavljeno u staklenik, s ciljem konstrukcije pRPA-RD-236. Te su biljke u stakleniku obrađene vodenom suspenzijom po Pardner-u koja odgovara 0.2 kg bromoksinilne aktivne tvari po hektaru do stadija od 5 listova. Twenty transformed plants were regenerated and placed in the greenhouse, with the aim of constructing pRPA-RD-236. These plants were treated in the greenhouse with an aqueous suspension according to Pardner, which corresponds to 0.2 kg of bromoxynil active substance per hectare until the stage of 5 leaves.
Sve biljke koje su pokazale potpunu toleranciju prema bromoksinilu potom su uporabljene za različite pokuse koji pokazuju da ekspresija androktonina u transformiranih biljaka rezultira njihovom otpornošću prema gljivičnom napadaju. All plants that showed complete tolerance to bromoxynil were then used for various experiments showing that expression of androctonin in transformed plants results in their resistance to fungal attack.
LITERATURNI IZVORI LITERARY SOURCES
Ausubel, F.A. et al. (eds. Greene), Current Protocol in Molecular Biology, Publ. Wiley & Sons. Ausubel, F.A. et al. (eds. Greene), Current Protocols in Molecular Biology, Publ. Wiley & Sons.
Bevan, M. et al., Nuc. Acids Res. 11 (1983) 369-385. Bevan, M. et al., Nuc. Acids Res. 11 (1983) 369-385.
Carington and Freed, J. Virol. 64 (1990) 1590-1597. Carrington and Freed, J. Virol. 64 (1990) 1590-1597.
Ehret-Sabatier et al., The Journal of Biological Chemistry, 271, 47 (1996) 29537-29544. Ehret-Sabatier et al., The Journal of Biological Chemistry, 271, 47 (1996) 29537-29544.
Horsch et al., Science 227 (1985) 1229-1231. Horsch et al., Science 227 (1985) 1229-1231.
Jefferson et al., EMBO J. 6 (1987) 3901-3907. Jefferson et al., EMBO J. 6 (1987) 3901-3907.
Komari et al., J. Bacteriol. 166 (1986) 88-94. Komari et al., J. Bacteriol. 166 (1986) 88-94.
Rothstein et al., Cold Spring Harb. Symp. Quant. Biol. 45 (1981) 99-105. Rothstein et al., Cold Spring Harb. Symp. Quant. Biol. 45 (1981) 99-105.
Stalker et al., J. Biol. Chem. 263 (1988) 6310-6314. Stalker et al., J. Biol. Chem. 263 (1988) 6310-6314.
Odell, J.T. et al., Nature 313 (1985) 810-812. Odell, J.T. et al., Nature 313 (1985) 810-812.
POPIS SEKVENCIJA LIST OF SEQUENCES
(1) OPĆENITE INFORMACIJE: (1) GENERAL INFORMATION:
(i) PRIJAVITELJ: (i) APPLICANT:
(A) NAZIV: RHONE-POULENC AGROCHIMIE (A) NAME: RHONE-POULENC AGROCHIMIE
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(ii) NASLOV IZUMA: Gen šifriran za androktonin, vektor koji ga sadrži, te dobivene transformirane biljke otporne na bolesti (ii) TITLE OF THE INVENTION: The gene coded for androctonin, the vector containing it, and the resulting disease-resistant transformed plants
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(B) TIP: nukleotid (B) TYPE: nucleotide
(C) LANČANOST: dvostruka (C) CHAINING: double
(D) TOPOLOGIJA: linearna (D) TOPOLOGY: linear
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(ix) DODATNE ZNAČAJKE: (ix) ADDITIONAL FEATURES:
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(B) POLOŽAJ: 1..75 (B) POSITION: 1..75
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(B) TIP: nukleotid (B) TYPE: nucleotide
(C) LANČANOST: dvostruka (C) CHAINING: double
(D) TOPOLOGIJA: linearna (D) TOPOLOGY: linear
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(ix) DODATNE ZNAČAJKE: (ix) ADDITIONAL FEATURES:
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(B) POLOŽAJ: 12..101 (B) POSITION: 12..101
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(A) DULJINA: 211 parova baza (A) LENGTH: 211 base pairs
(B) TIP: nukleotid (B) TYPE: nucleotide
(C) LANČANOST: dvostruka (C) CHAINING: double
(D) TOPOLOGIJA: linearna (D) TOPOLOGY: linear
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(ix) DODATNE ZNAČAJKE: (ix) ADDITIONAL FEATURES:
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(i) ZNAČAJKE SEKVENCIJE: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) DULJINA: 75 parova baza (A) LENGTH: 75 base pairs
(B) TIP: nukleotid (B) TYPE: nucleotide
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(D) TOPOLOGIJA: linearna (D) TOPOLOGY: linear
(ii) TIP MOLEKULE: druga nukleinska kiselina (ii) TYPE OF MOLECULE: other nucleic acid
(A) OPIS: /opis = “sintetički oligonukleotid 1” (A) DESCRIPTION: /opis = “synthetic oligonucleotide 1”
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(i) ZNAČAJKE SEKVENCIJE: (i) CHARACTERISTICS OF THE SEQUENCE:
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(D) TOPOLOGIJA: linearna (D) TOPOLOGY: linear
(ii) TIP MOLEKULE: druga nukleinska kiselina (ii) TYPE OF MOLECULE: other nucleic acid
(A) OPIS: /opis = “sintetički oligonukleotid 2” (A) DESCRIPTION: /opis = “synthetic oligonucleotide 2”
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(i) ZNAČAJKE SEKVENCIJE: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) DULJINA: 44 parova baza (A) LENGTH: 44 base pairs
(B) TIP: nukleotid (B) TYPE: nucleotide
(C) LANČANOST: jednostruka (C) CHAINING: single
(D) TOPOLOGIJA: linearna (D) TOPOLOGY: linear
(ii) TIP MOLEKULE: druga nukleinska kiselina (ii) TYPE OF MOLECULE: other nucleic acid
(A) OPIS: /opis = “sintetički oligonukleotid 3” (A) DESCRIPTION: /opis = “synthetic oligonucleotide 3”
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(i) ZNAČAJKE SEKVENCIJE: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) DULJINA: 97 parova baza (A) LENGTH: 97 base pairs
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(D) TOPOLOGIJA: linearna (D) TOPOLOGY: linear
(ii) TIP MOLEKULE: druga nukleinska kiselina (ii) TYPE OF MOLECULE: other nucleic acid
(A) OPIS: /opis = “sintetički oligonukleotid 4” (A) DESCRIPTION: /opis = “synthetic oligonucleotide 4”
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(i) ZNAČAJKE SEKVENCIJE: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) DULJINA: 85 parova baza (A) LENGTH: 85 base pairs
(B) TIP: nukleotid (B) TYPE: nucleotide
(C) LANČANOST: jednostruka (C) CHAINING: single
(D) TOPOLOGIJA: linearna (D) TOPOLOGY: linear
(ii) TIP MOLEKULE: druga nukleinska kiselina (ii) TYPE OF MOLECULE: other nucleic acid
(A) OPIS: /opis = “sintetički oligonukleotid 5” (A) DESCRIPTION: /opis = “synthetic oligonucleotide 5”
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(i) ZNAČAJKE SEKVENCIJE: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) DULJINA: 66 parova baza (A) LENGTH: 66 base pairs
(B) TIP: nukleotid (B) TYPE: nucleotide
(C) LANČANOST: jednostruka (C) CHAINING: single
(D) TOPOLOGIJA: linearna (D) TOPOLOGY: linear
(ii) TIP MOLEKULE: druga nukleinska kiselina (ii) TYPE OF MOLECULE: other nucleic acid
(A) OPIS: /opis = “sintetički oligonukleotid 6” (A) DESCRIPTION: /opis = “synthetic oligonucleotide 6”
(xi) OPIS SEKVENCIJE: SEQ ID NO. 9: (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 9:
[image] [image]
(2) INFORMACIJE ZA SEQ ID NO. 10: (2) INFORMATION FOR SEQ ID NO. 10:
(i) ZNAČAJKE SEKVENCIJE: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) DULJINA: 93 parova baza (A) LENGTH: 93 base pairs
(B) TIP: nukleotid (B) TYPE: nucleotide
(C) LANČANOST: jednostruka (C) CHAINING: single
(D) TOPOLOGIJA: linearna (D) TOPOLOGY: linear
(ii) TIP MOLEKULE: druga nukleinska kiselina (ii) TYPE OF MOLECULE: other nucleic acid
(A) OPIS: /opis = “sintetički oligonukleotid 7” (A) DESCRIPTION: /opis = “synthetic oligonucleotide 7”
(xi) OPIS SEKVENCIJE: SEQ ID NO. 10: (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 10:
[image] [image]
(2) INFORMACIJE ZA SEQ ID NO. 11: (2) INFORMATION FOR SEQ ID NO. 11:
(i) ZNAČAJKE SEKVENCIJE: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) DULJINA: 93 parova baza (A) LENGTH: 93 base pairs
(B) TIP: nukleotid (B) TYPE: nucleotide
(C) LANČANOST: jednostruka (C) CHAINING: single
(D) TOPOLOGIJA: linearna (D) TOPOLOGY: linear
(ii) TIP MOLEKULE: druga nukleinska kiselina (ii) TYPE OF MOLECULE: other nucleic acid
(A) OPIS: /opis = “sintetički oligonukleotid 8” (A) DESCRIPTION: /opis = “synthetic oligonucleotide 8”
(xi) OPIS SEKVENCIJE: SEQ ID NO. 11: (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 11:
[image] [image]
Claims (39)
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR9710632A FR2767537B1 (en) | 1997-08-20 | 1997-08-20 | GENE ENCODING ANDROCTONIN, VECTOR CONTAINING SAME AND OBTAINED TRANSFORMED PLANTS RESISTANT TO DISEASE |
Publications (1)
Publication Number | Publication Date |
---|---|
HRP980455A2 true HRP980455A2 (en) | 1999-04-30 |
Family
ID=9510484
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
HR9710632A HRP980455A2 (en) | 1997-08-20 | 1998-08-19 | Gene coding for androctonine, vector containing it and disease-resistant transformed plants obtained |
Country Status (14)
Country | Link |
---|---|
EP (1) | EP1007711A1 (en) |
JP (1) | JP2001514898A (en) |
AR (1) | AR016839A1 (en) |
AU (1) | AU751263B2 (en) |
CA (1) | CA2301978A1 (en) |
CO (1) | CO4790113A1 (en) |
FR (1) | FR2767537B1 (en) |
HR (1) | HRP980455A2 (en) |
HU (1) | HUP0003585A3 (en) |
IL (1) | IL134609A0 (en) |
NZ (1) | NZ502951A (en) |
PL (1) | PL338859A1 (en) |
WO (1) | WO1999009189A1 (en) |
ZA (1) | ZA987450B (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7414173B1 (en) | 2002-07-12 | 2008-08-19 | E.I. Du Pont De Nemours And Company | Isolated nucleic acid molecules encoding orally active androctonus amoreuxi pesticidal biopeptides |
WO2011095460A1 (en) | 2010-02-02 | 2011-08-11 | Bayer Cropscience Ag | Soybean transformation using hppd inhibitors as selection agents |
PL236444B1 (en) * | 2017-12-04 | 2021-01-11 | Politechnika Slaska Im Wincent | Method for obtaining degradable hydrogels based on derivatives of trehalose and their applications |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5614395A (en) * | 1988-03-08 | 1997-03-25 | Ciba-Geigy Corporation | Chemically regulatable and anti-pathogenic DNA sequences and uses thereof |
DE69034081T2 (en) * | 1989-03-24 | 2004-02-12 | Syngenta Participations Ag | Disease resistant transgenic plant |
FR2673643B1 (en) * | 1991-03-05 | 1993-05-21 | Rhone Poulenc Agrochimie | TRANSIT PEPTIDE FOR THE INSERTION OF A FOREIGN GENE INTO A PLANT GENE AND PLANTS TRANSFORMED USING THIS PEPTIDE. |
GB9321469D0 (en) * | 1993-10-18 | 1993-12-08 | Zeneca Ltd | Insecticidal proteins |
FR2745004B1 (en) * | 1996-02-16 | 1998-03-27 | Rhone Poulenc Agrochimie | ANTIBACTERIAL AND ANTIFUNGAL PEPTIDE |
-
1997
- 1997-08-20 FR FR9710632A patent/FR2767537B1/en not_active Expired - Fee Related
-
1998
- 1998-08-18 IL IL13460998A patent/IL134609A0/en unknown
- 1998-08-18 JP JP2000509852A patent/JP2001514898A/en active Pending
- 1998-08-18 ZA ZA987450A patent/ZA987450B/en unknown
- 1998-08-18 HU HU0003585A patent/HUP0003585A3/en unknown
- 1998-08-18 AU AU90766/98A patent/AU751263B2/en not_active Ceased
- 1998-08-18 PL PL98338859A patent/PL338859A1/en unknown
- 1998-08-18 NZ NZ502951A patent/NZ502951A/en unknown
- 1998-08-18 EP EP98942749A patent/EP1007711A1/en not_active Withdrawn
- 1998-08-18 WO PCT/FR1998/001814 patent/WO1999009189A1/en not_active Application Discontinuation
- 1998-08-18 CA CA002301978A patent/CA2301978A1/en not_active Abandoned
- 1998-08-19 HR HR9710632A patent/HRP980455A2/en not_active Application Discontinuation
- 1998-08-20 CO CO98047698A patent/CO4790113A1/en unknown
- 1998-08-20 AR ARP980104113A patent/AR016839A1/en unknown
Also Published As
Publication number | Publication date |
---|---|
PL338859A1 (en) | 2000-11-20 |
CO4790113A1 (en) | 1999-05-31 |
WO1999009189A1 (en) | 1999-02-25 |
AR016839A1 (en) | 2001-08-01 |
AU751263B2 (en) | 2002-08-08 |
IL134609A0 (en) | 2001-04-30 |
FR2767537B1 (en) | 2001-07-13 |
HUP0003585A3 (en) | 2002-10-28 |
EP1007711A1 (en) | 2000-06-14 |
FR2767537A1 (en) | 1999-02-26 |
NZ502951A (en) | 2001-11-30 |
ZA987450B (en) | 1999-02-22 |
CA2301978A1 (en) | 1999-02-25 |
AU9076698A (en) | 1999-03-08 |
JP2001514898A (en) | 2001-09-18 |
HUP0003585A2 (en) | 2001-02-28 |
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