FR2689905A1 - Method for cloning a DNA sequence coding for a NADPH-cytochrome P450 reductase and DNA sequences coding for a NADPH-cytochrome P450 reductase, in particular for plants. - Google Patents

Abstract

Method of coding a DNA sequence coding for an NADPH-cytochrome P450 reductase of an organism, especially a plant. The process is characterized in that (1) a strain of yeast transformed with plasmids from a total cDNA bank of the organism, especially a plant, and having a deficiency phenotype of endogenous NADPH-cytochrome P450 reductase activity, is subjected to treatment with a P450 cytochrome inhibitor at a dose which makes lethal the absence of endogenous NADPH-cytochrome P450 reductase activity; (2) the surviving clones are collected and plasmids for the expression of an NADPH-cytochrome P450 reductase activity corresponding to the NADPH-cytochrome P450 of said organism, especially of said plant, are extracted.

Description

 The present invention relates to a method for cloning a NADPH Cytochrome P450 Reductase gene from a plant or from another organism by a screen based on functional complementation in a yeast strain where the endogenous NADPH cytochrome P450 Reductase is either deficient or made of modular level.

The present invention relates more particularly to the application of this method to the cloning of NADPH Cytochrome genes
P450 Reductases from plants.

The present invention further relates to the use of the cDNA of a plant NADPH Cytochrome P450 Reductase obtained by said method, for cloning NADPH Cytochrome P450 genes
Reductases from another plant by hybridization under the condition of reduced stringency.

 The present invention also relates to DNA sequences coding for a plant NADPH Cytochrome P450 Reductase obtained by the cloning methods according to the invention.

Technical field of the invention
NADPH Cytochrome P450 Reductases (hereinafter abbreviated as "P450Réd") are essential for the enzymatic activity of Cytochromes
P450 (hereinafter abbreviated as "P450") (1). In the functional membrane complex P450-P450 Red, they supply electrons to P450 with an electrochemical potential sufficiently negative to allow them to carry out reactions of the monooxygenase type. These reactions are most often characterized by the introduction of an oxygen atom into carbon-carbon or carbon-hydrogen bonds or by the oxidation of a heteroatom (2). Until now, P450 Reds were considered to be unique in a given species where they interacted with all of the P450s present. The present invention demonstrates the erroneous nature of this concept and that different classes of Reductase can control the activity of different families of P450.

Although the state of knowledge regarding the intervention of
P450 of plants in the different biosynthesis or detoxification pathways is not as advanced as in the field of mammalian P450, the implication of P450 in many reactions of the secondary metabolism of different plants, is described today in the literature (3). It is also known from the literature that plant P450s are involved in the metabolism of certain pesticides such as diclofop or chlortoluron (4, 5). The activity of P450 Réd as an essential electronic donor for the function of P450 is likely to be used in a wide range of fields of application.
- the bioconversion of atoms or natural pigments from precursors endogenous to the plant. Indeed, it has been shown that a number of molecules of very varied chemical natures such as phenyl propanoids, terpenoids, fatty acids and sterols are substrates of
Plant P450 (3). These molecules are also intermediaries in the biosynthesis of molecules of industrial interest such as aromas, pigments or phytoalexins (defense substances synthesized by the plant against pathogenic infections). To this end, the isolation of genes from
P450 from plants and associated P450 Reductases, their cloning and their coordinated overexpressions in a suitable microorganism in order to reconstruct a portion of biosynthesis pathway is of certain interest. We can cite as applications
- Replacement of the stereospecific hydroxylation stages during the chemical synthesis of molecules of industrial interest by biotechnological means. The introduction of hydroxyl groups at very precise positions by the means of synthetic chemistry is often tedious and expensive. This step can advantageously be replaced by the use of a P450 / P450 Red complex capable of carrying out hydroxylation reactions at the desired position. By way of example, there may be mentioned the conversion of digitoxin to digoxin by hydroxylation in position 12 beta (6). The enzyme which performs this reaction in cells in culture of Digitalis lanata is a hydroxylase with cytochrome P450
- In vivo modulation of plant biosynthesis pathways. The discovery of the existence of at least two P450 Reductases with non-interchangeable function is a strong indication that at least the two P450
Isolated reductases could intervene differentially in the biosynthetic pathways in plants. The modulation in transgenic plants of the relative activity of P450 Reductases could be a powerful means of global control of function such as flowering, growth, symbiosis, resistance to the parasite, fruiting, conservation and ripening of fruits , etc ...;
- An in vitro diagnosis of environmental pollution. A certain number of industrial pollutants are either inhibitors or activators of P450 from plants
- Oxidation of Xenobiotics. It has been shown that certain P450s constitute factors of resistance and selectivity with respect to pesticides. It is clearly advantageous to transfer these genes to plants of agronomic interest so as to make them resistant to a herbicide, fungicide, insecticide, etc. The availability of the corresponding Reductases in the same localization as the imbricated P450s is a critical factor. for this use.

State of the art
Although P450 and P450 Reductases were detected in plants in the early 1970s (7), genomic or cDNA clones of membrane P450 Reductases come from higher eukaryotes (man, rabbit, pig, rat or trout), or yeast, excluding the plant kingdom. The low content of P450 Reductase in plant tissues, as well as the poor performance of the purification processes described, resulted in a serious delay in the development of cloning tools in plants.

The search for the enzymatic activity of P450 Reductases requires the reconstitution of a multienzymatic membrane complex, a process which has been very poorly mastered until now in the case of plant enzymes. Recently, the homogeneous purification of a P450 protein
Red sweet potato (8) and Jerusalem artichoke (9) has been described. Their identity has been demonstrated by the reconstitution of a very low level of P450 activity from partially purified preparations of plant P450. A polyclonal antibody against Topinambour's P450 Red completely inhibits the hydroxylation of cinnamate by cinnamate 4-hydroxylase (P450) in Jerusalem artichoke microsomes (9).

This reaction is common in the plant kingdom since cinnamic acid is the common core of different biosynthetic pathways leading to lignin, many plant pigments, defense compounds and aromas. The same anti-P450 Red antibody inhibits dependent P450 reactions in a large number of plant species, on the other hand it does not show cross-reactivity with the P450 Reds from yeast, insects, fish or mammals (10) . Conversely, antibodies against P450 Red from yeast or mammals do not inhibit P450
Red vegetable.

 Further studies have shown that P450 Red of Jerusalem artichoke (and several other species) does not appear to be a unique form based on immunoblotting and affinity chromatography experiments (presence of three bands of molecular weight 80 , 82 and 84 kDa) (11). However, these studies did not allow to conclude in the existence of several genes insofar as it had been demonstrated in at least one of the cases that one of the bands corresponded to a glycoprotein.

As a result, it was not known whether the different bands corresponded to different post-translational modification states of the same protein or to proteins of distinct sequences. Digestion fragments in the form of 80 kDa were microsequenced, and the peptide sequences obtained could be aligned with the known sequences of trout, rabbit, rat, pig, and yeast. Over a distance ranging from 10 to 15 amino acids, l the homology found in the best of cases is around 60%. Very recently, a monoclonal antibody made from a mixture of the three forms of P450 Réd from Topinambour, in the hope that it will be able to recognize a common structural motif between the three isoforms, has been found to recognize only a structure called "dinucleotide fold", a common motif present in a very large number of NAD (P) H dependent enzymes (12).

 Conventional approaches for cloning a plant reductase gene either by screening libraries with a specific antibody, or by hybridization with heterologous probes or deduced from the amino acid sequences obtained by microsequencing of plant reductases, have proved ineffective for the reasons following - the expression level of P450 Red at the mRNA level of plants is very low, compared to the expression levels measured in mammals.

Induction in the plant by injury or other means is modest and, moreover, a transient phenomenon. Even under optimal induction conditions, P450 Red therefore remains a minor protein in plant tissues (12). Screening for sparse cDNA in a total plant mRNA library is a tedious and slow procedure. It should also be added that, in general, it is more difficult to obtain a good quality mRNA library in plants than from mammalian tissues.

- No nucleic acid sequence of plant P450 Red was known, hybridization screening had to be carried out either with a mammalian or yeast probe, or with oligonucleotides derived from the amino acid micro sequences of P450 Red Jerusalem artichoke. In both cases, the use of low stringency conditions was required, which greatly increased the possibility of obtaining false positives. If there exist in the literature a certain number of examples which have described the successful application of specific antibodies for the screening of expression libraries, the success of this procedure, which entirely depends on the specificity of the antibody , is somewhat random.

Summary of the invention
The present invention provides a method for cloning genes from all NADPH-Cytochrome P450 Reductases regardless of their origins, which method consists in using a screen making it possible to detect the very activity of a Cytochrome P450.

 Compared to the conventional cloning methods cited above, which are based on criteria independent of the function, such as the degree of homology or the recognition of structural patterns, the cloning method according to the present invention comprises a considerably higher degree of reliability since based on function.

 The absence of endogenous NADPH-cytochrome P450 Reductase activity is not lethal in yeast if cytochrome P450 activity is expressed.

In particular, in yeast, and more particularly Saccharomyces cerevisiae, a P450, nanosterol 14-demethylase (C14DM), constitutes an essential step in the biosynthesis of ergosterol, a major constituent of the cell membrane. The absence of Cl4DM activity results in the accumulation of methyl intermediates in 14 of the biosynthesis of ergosterol in the membrane and in a defect in ergosterol, which is lethal for the cell (14). The P450 deletion
Réd, on the other hand, is curiously not lethal while that of the
C14DM is, because an unknown NADH-dependent microsomal reductase (based on its activity to reconstitute an activity
Ethoxyroresorufin O-deethylase in the presence of cytochrome P450 (mouse AI and NADH), can alternatively provide reducing equivalents to C14DM so as to ensure the rate of synthesis of ergosterol required for the cell to survive.

 Subjected to high doses of cytochrome P450 inhibitors, the yeast does not survive. However, a yeast strain which no longer expresses P450 Reductase activity does not survive doses of P450 inhibitor such as ketoconazole, normally insufficient to be lethal, i.e. insufficient to be lethal when P450 Red activity is expressed (13).

The method according to the invention is based on the discovery that this phenotype of hypersensitivity to a P450 inhibitor, in particular to the antifungal ketoconazole of a yeast strain deficient in activity
Endogenous P450 Reductase is Suppressed by Expression of a P450
Exogenous reductase (regardless of the electron donor).

The subject of the present invention is in fact a method for cloning a DNA sequence coding for a Cytochrome P450 NADPH
Reductase, in particular from plants, or from another organism, characterized in that the following steps are carried out
1- We submit a yeast strain transformed with plasmids
a bank of total cDNA of said organism, in particular of said
plant, and having an activity deficiency phenotype
Endogenous NADPH-Cytochrome P450 Reductase, for treatment
with a cytochrome P450 inhibitor at a dose that makes
lethal lack of activity NADPH Cytochrome P450 Reductase
endogenous then
2- The surviving clones are recovered and the plasmids are extracted therefrom
allowing the expression of NADPH Cytochrome P450 activity
Reductase which corresponds to the NADPH Cytochrome P450 of said
organism, in particular of said plant.

The expression “dose which renders lethal” is therefore understood to mean a dose which -normally, that is to say when the endogenous P450 Red activity is expressed, is not lethal for the yeast and only becomes so in l absence of endogenous P450 Red activity. The dose of P450 inhibitor should therefore not exceed the lethal dose, even in the presence of P450
Endogenous reductase.

 The transformation of Step 1 is done according to techniques well known to those skilled in the art; for example, from a strain of yeast URA- and plasmids carrying the URA 3 gene as selection marker, which makes it possible to exert a selection pressure with uracyl.

 In the present invention, yeasts which are totally or partially deficient in endogenous P450 Reductase can be used to test a P450 Red activity.

The obtaining and description of the strains of S. cerevisiae, in which the endogenous NADPH-Cytochrome P450 Reductase gene has been either deleted or put under the control of an inducible promoter enabling it to be activated, are described in the Patent application No. 91 08884 (strains PES1-3D and PES1-3U). In the strain PES1-3U, the endogenous gene for NADPH-Cytochrome P450 Reductase was put under the control of the promoter GAL10-CYC1, which gives to the strain a wild phenotype in galactose medium. In the strain PES1-3D, the coding region of the
NADPH-Cytochrome P450 Reductase has been replaced by the marker gene TRP1, which gives it a permanent deficient phenotype in
NADPH-Cytochrome P450 Reductase.

 The term “Cytochrome P450 inhibitor” is understood to mean a compound which interacts with said Cytochrome P450 in particular by complexation, so that said Cytochrome P450 is inactivated.

A large number of azole compounds, and more particularly ketoconazole, have been developed as potent fungicides in the medical and agricultural fields and can be used according to the invention (15).
The lethal dose is variable according to the agents and according to the strain, in general a dose of 5 to 100 ug / ml, preferably from 10 to 50 ug / ml, may be sufficient in the process according to the invention.

Antifungal agents of the ketoconazole type in particular inhibit the demethylation of lanosterol at the level of C14DM by forming a 1: 1 complex with this P450. Due to the very high affinity of ketoconazole, lanosterol as a physiological substrate cannot displace the inhibitor, which leads to cell death. A wild strain of yeast is resistant up to a concentration of 50 μm or 80 μg / ml of ketoconazole. The observation according to the present invention that a strain deficient in P450 Red is more sensitive to ketoconazole up to 10 times, and perhaps lethal at 5, ug / ml (because of the poor coupling between the
Non-specific Réd and C14DM), was used to develop a mode of selection for NADPH P450-Red activity. Indeed, after transformation of a deficient yeast strain into NADPH P450 Red with an exogenous DNA library , can only grow in the presence of 50 ug / ml of ketoconazole transformed cells in which the exogenous DNA received has introduced sufficient P450 Red activity. The essential point of this screening method by functional complementation is its universality, since one can take into account the differences in coupling efficiency between the yeast C14DM and the exogenous P450 Red by varying the inhibitor concentration.

In one embodiment, the method according to the present invention was applied to the cloning of plant P450 Reds, in particular of the species Arabidopsis thaliana. In the absence of knowledge as to the maximum conditions for induction of expression of P450 Red in
Arabidopsis and tissues where P450 Red is expressed, the total mRNA extracted from young whole seedlings in the growth phase requires intense synthetic activity, it can therefore be assumed that all the factors necessary for the growth of the seedling are expressed at this point.

The use of whole seedlings dilutes the population of tissue-specific mRNA expressed in the total population of mRNA, but has the enormous advantage of not taking into account specific conditions for induction of P450 Red, and of facilitate the preparation of starting materials.

 An expression library in S. cerevisiae can be constructed in a known manner by insertion of a double-stranded cDNA in a vector between a promoter and a yeast transcription terminator.

The well known promoter of the PGK type (16) can be used, it being understood that the nature of the promoter is not critical for the method according to the invention. Preferably, however, a strong promoter will be used in the yeast. The multicopy nature of the vector used, although desirable, is also not critical.

The library thus obtained is fractionated before transformation in yeast according to the size of the cDNAs synthesized. As the size of the coding sequence of P 450 Red of eukaryote described in the literature is of the order of 2 kbp apart from the flanking 5 'and 3' sequences, the mRNA coding for a P450 Red in a plant and in particular in
Arabidopsis must reasonably be present in the fraction having a molecular weight greater than 2 kbp.

According to the invention, in Step 1, the cDNA library is preferably enriched in the gene of interest (NADPH-Cytochrome P450 gene
Reductase of said plant or other organism) by fractionation according to size, keeping only fragments larger than 2 kbp. Such a fractionation leads in fact to an enrichment of the library for the gene of interest.

Advantageously according to the invention, a so-called "in flight" transformation method is used of a yeast strain carrying a gene made artificially conditional for the endogenous P450 Reductase. Indeed, the deficiency in P450 Reductase induces a significant loss of transformability which would make it difficult, if not impossible in certain cases, the constitution of a representative bank in a strain deleted for P450
Reductase unconditionally, that is, permanently.

 Preferably therefore, a yeast strain is used in which the NADPH Cytochrome P450 Reductase gene is placed under the control of an inducible promoter such as the PES1-3U strain. In this case, in Step 1, a P450 Reductase deficiency is induced by inactivating said promoter in the strain already transformed.

The present invention therefore also relates to a process characterized in that the endogenous yeast NADPH Cytochrome P450 Reductase gene is placed under the control of an inducible promoter and in step 1, the following substeps are carried out successively
1-a) A yeast strain is transformed with plasmids of a
bank of total cDNA of said organism, in particular of said
plant then
lb) NADPH-Cytochrome P450 activity deficiency is induced
Endogenous reductase by inactivating said promoter, and
lc) Subjecting said strain to treatment with said dose
cytochrome P450 inhibitor.

In one embodiment, the strain PES1-3U is, firstly, transformed by a cDNA library of said organism, in particular of the plant and the transformants selected solely on the basis of the selection marker URA3 of the plasmid. Secondly, the transformants are subjected to the P450 inhibitor, in particular to ketoconazole, to render lethal the absence of NADPH Cytochrome P450 activity.
Reductase. The surviving clones then contain a plasmid allowing the expression of an exogenous P450 Reductase activity (approximately 0.1% in the case of Arabidopsis).

The presence of cloning artifacts is possible by the selection of an inducible promoter regulatory factor such as GAL 10-CYC1 in a glucose medium. This is why a series of annexed control steps makes it possible to ensure, in particular after extraction of the plasmid and retransformation of a yeast strain totally deficient in P450.
Reductase, that complementation is due to the expression of a
NADPH Cytochrome P450 Exogenous reductase. To do this, a yeast strain is used in which the endogenous P450 Reductase gene is entirely deleted, such as PES 1-3D. The plant origin of this reductase can then be checked by hydridation of the clone with the genomic DNA of the plant of origin. The biochemical nature of the cloned gene is then verified by sequence.

 The present invention therefore also relates to a method characterized in that after the transformation "in flight" and using the plasmids recovered in step 2), a yeast strain is transformed in which the NADPH gene Endogenous cytochrome P450 Reductase has been deleted and treatment with a cytochrome P450 inhibitor is repeated at a dose which makes the absence of endogenous cytochrome P450 cytochrome P450 activity lethal. The surviving clones are then recovered and the plasmids allowing it are extracted. expression of a NADPH-cytochrome P450 activity which corresponds to the cytochrome NADPH, in particular of said plant.

The present invention further provides the demonstration of the existence, in certain organisms, of several genes independent of
NADPH cytochrome P450 Reductase coding for proteins of very distinct predictable physicochemical characters capable of interacting with different classes of cytochromes, P450.

 The method according to the invention made it possible to clone two DNA sequences coding for NADPH Cytochrome P450 Reductase from Arabidopsis ara-B and ara-C as shown in Figures 9 and 10 respectively.

 The present invention therefore also relates to cDNA sequences coding for NADPH Cytochrome P450 Reductase of Arabidopsis ara-B or ara-C, or of Jerusalem artichoke heliantus tuberosus, as shown in Figures 9 to 1 1 respectively.

 Indeed, according to the present invention, an entire coding sequence of arabidopsis (ara-C), including the flanking 5 'and 3' sequences, was then used as a probe to screen a total cDNA library of Jerusalem artichoke heliantus tuberosus under conditions of reduced stringency.

This library was obtained from Jerusalem artichoke nodules which were induced for the synthesis of P450 Red by injury, but was not enriched by fractionation according to the size of the cDNAs. The use of a monospecific antibody directed against a mixture of the three isoforms present in Jerusalem artichoke did not prove fruitful, neither the screening using a heterologous probe (yeast and human reductase), or derived from the sequence protein, had not allowed cloning. Screening of the same library with a cDNA probe of the ara-C clone immediately revealed positives having an insert of more than 2 kbp with a frequency of 1 per 10,000 after three rounds of screening.

Thus, by the method of the present invention, it was for the first time isolated and identified three cDNAs from plant sources coding for NADPH Cytochrome P450 Reductases in two different species (Arabidopsis and Jerusalem artichoke heliantus tuberosus). The microsomes prepared from these strains transformed by yeast expression vectors are capable of reducing a non-physiological substrate of P450 Red, the
Cytochrome c. The alignment of the amino acid sequences with the known sequences of P450 Red from other eukaryotic sources makes it possible to identify them as being P450 Red of Arabidopsis on the basis of a very high conservation in the functional domains of the
P450 Réd described in the literature. A direct comparison at amino acid level between the two Arabidopsis P450 Red shows long blocks of homology almost perfect for the same fields while, outside these fields in particular, the distribution of charged amino acids varies significantly. In southern genomics with Arabidopsis DNA digested by different restriction enzymes, the two cDNA clones hybridize to different genomic loci clearly indicating the presence of two genes. A more detailed analysis of the sequences shows that (Figure I) - the amino acid divergence between the two Arabidopsis Reductases (ara-B and ara-C) is 36%, while that between the genes of the
Ara-B and topi-A reductase is 31% and that between ara-C and topi-A is 27%. This is a strong indication that the two genes in Arabidopsis have evolved independently for a time at least comparable to that of the divergence between these distant plant species.

 Furthermore, the analysis of nucleotide conservation on the third base of codons (Figure 1) after alignment between the amino acid sequences of the Reductases, demonstrates a very low evolutionary conservation residue confirming the previous data. This very low conservation at the nucleotide level, on the unconstrained bases compared to the relatively high homology of the plant reductases between them at the amino acid level, demonstrates the existence of a strong evolutionary constraint on the function of the two genes of Arabidopsis. This indicates that the activity of these two genes is evolutionarily critical for the plant and suggests that these two genes are not interchangeable. They therefore seem to play individual roles in the life of the likely plant due to their association with specific biosynthetic pathways.

In addition to cDNA sequences encoding a NADPH P450
Plant reductase obtained by the cloning methods according to the invention, the present invention also relates to DNA sequences hybridizing under weakly stringent conditions at 50 "C and / or having at least 60% homology with one of the sequences of arabidopsis or Jerusalem artichoke heliantus tuberosus shown in FIGS. 9 to 11, or any other sequence of NADPH Cytochrome P450 Reductase, in particular of plants, obtained by the cloning methods according to the invention.

 Obviously, the present invention also relates to all equivalent DNA sequences, that is to say which differs from the cDNA sequences mentioned above, only by one or more neutral mutation (s), that is to say, the change or substitution of the nucleotides in question, does not affect the primary sequence of the resulting protein.

 Other characteristics and advantages of the present invention will appear in the light of the detailed description of the following embodiment.

 Figure 1 is a schematic representation of the constituent elements of the vector pFL 61, the unique restriction sites and their physical location as well as the cloning cassette inserted in the site N "1.

 FIG. 2 represents the nucleotide sequence of the region of insertion of the cloning cassette into the Not I site of the vector pFL 61 with the restriction sites. The two Not I sites at the edge of the cassette and the two BstX I sites carried by the cassette are indicated by the arrows.

 Figure 3 is a schematic representation of the principle of cloning using synthetic adapters with incompatible ends.

 FIG. 4 represents the scheme for obtaining a mutant strain of yeast in which the endogenous promoter of P450 Red is replaced by the modular promoter pGK after homologous recombination with a linear Eco RI fragment of the vector pGPlPC.

 FIG. 5 represents the diagram for obtaining a mutant strain of yeast in which the gene for the marker TRP1 has been introduced at the endogenous locus of P450 Red to give the strain PES 1-3D.

 FIG. 6 represents the classification of the clones capable of conferring a ketoconazole R phenotype on the PES 1-3U strain after transformation according to their restriction profile and the frequency of appearance.

Figure 7 shows the measurement of NADPH CYT activity
P450 Reductase in microsomes isolated from the mutant strain PES 1-3D after transformation with plasmid DNA from classes A, B,
C, E. As controls were included the untransformed PES 1-3D strain (absence of P450 Red activity) and the wild strain W303.1B (endogenous P450 Red activity).

Figure 8 shows the homology between NADPH-P450
Plant reductase and comparison to known P450 reductases, human and yeast. Column 1 shows the calculation of the overall conservation rate at amino acid level between the two Arabidopsis P450 Red, columns 2 to 4 show the conservation rate between amino acid sequences of the two Arabidopsis P450 Red with P450
Human red (HRed) and the yeast P450 Red (YRed) known from the literature. Column 5 shows the evolutionary distance between the yeast P450 Red and the human P450 Red. Columns 6 and 7 show the conservation rate between the two Arabidopsis P450 Red and an eP450 Red from another plant species, isolated by cross-hybridization with AraB as probe.

 FIGS. 9 to 11 represent the nucleotide sequences and the deduced amino acid sequences of the two cDNAs of Arabidopsis P450 Red (Ara B -Figure 9- and Ara C -Figure 10-) isolated by functional complementation and of P450 Red of Jerusalem artichoke (Figure 11) isolated by crosshybridization (TopA). The functional areas of these reductrases have been defined by alignment with the known P450 Reds and are indicated by square brackets.

 FIG. 12 represents the alignment of the amino acid sequences deduced from the cDNAs of the three plant P450 Reds. The strict storage positions between the plant reductases are indicated by asterisks in line 4. The cysteine conserved among all the known plant and animal P450 reductases and which is important for the binding of NADPH is highlighted by an asterisk. The sequences necessary for fixing the FMN are indicated by the blocks At and A2.

The sequences necessary for fixing the FAD are represented by blocks B1 and B2. The sequences necessary for the attachment of NADPH are represented by block C and the conserved cysteine highlighted with an asterisk.

 The strain of Saccharomyces cerevisiae PES1-3U was deposited with the National Collection of Cultures and Microorganisms on March 17, 1992, under the deposit number I-1187.

1) Construction of the expression vector in S. cerevisiae
The vector pFL61, used here for the cloning of a cDNA library of Arabidopsis thaliana, was obtained from the vector pFL60, described in the literature (17). It is characterized by the following modifications: In place of the ClaI-BglII polylinker present in pFL60, a single Note site has been created. In this unique site was then introduced a cassette containing inside two unique BstXI sites and lined with 5 ′ outgoing ends compatible with NotI. Digestion with BstXI generates a linearized vector with two incompatible ends (18). Recirculation of the vector will only be possible in the presence of a DNA fragment having ends compatible with those created on the vector by BstXI digestion. The insertion cassette is placed between the inducible PGK promoter and the PGK terminator, which will allow expression of the inserted heterologous DNA sequences. In addition, this vector contains the URA3 auxotrophy marker gene for S. cer., A small part of the 2u plasmid comprising the origin of replication of S. cer. and sequences from the vector of E. coli pUCl9 which are unchanged compared to the original vector pFL 60.

2) Preparation of a total cDNA library of sorted Arabidopsis thaliana
by molecular weight of cDNA
Young whole seedlings, including leaves, stems and roots, are collected at the two-leaf stage, frozen in liquid nitrogen and then ground in a mortar containing frozen homogenization medium (10 mM Tris-Hcl pH8.0, I mM EDTA pH8, 0.5% sodium Dodecyl
Sulfate). The powder thus obtained is then thawed and extracted twice with an equal volume of a 1: 1 phenol / chloroform mixture. After adding 1/10 volume of a 4M NaCl solution, the aqueous solution is again extracted twice with a 1: 1 phenol / chloroform mixture, and the nucleic acids are precipitated by the addition of two volumes of ethanol.

The total nucleic acids obtained are immediately enriched in poly (A +) RNA by chromatography on oligo dT sepharose (19). After elution and precipitation, the poly (A +) RNAs are resuspended in water, their amount is determined by absorption at 260 nm, then they are frozen immediately until use. A total cDNA library is synthesized from an aliquot of 5ug poly RNA (A +), using the "cDNA Synthesis System Plus" supplied by Amersham. The ANDc are then made blunt end before ligation with adapters having 5 'CACA outgoing ends which are compatible with the GTGT ends produced on the cloning vector by digestion with BstX I (see FIG. 2). The cDNAs are then prepared according to their size by electrophoresis on agarose gel "low gelling temperature" (Sigma, Cat. NO. A 9539), which also removes the excess of annoying adapters during ligation. When eluting the agarose gel, the cDNAs are divided into two classes according to their size: one ranging from 0.2 to approximately 2 kb, and one representing the cDNAs larger than 2 kb. After quantification, the ligation was carried out in the presence of stolchiometric amounts of vector pFL61 digested with Bst XI (after removing the small internal Bst XI fragment) and cDNA of size greater than 2 kb. After transformation by electroporation in E. coli (strain MR 32), 5 2.0 × 10 clones were obtained. All the clones were combined for a plasmid DNA preparation on cesium color gradient, after removing a aliquot for future amplifications.

3) Description of the mutant strains of yeast used and conditions for
culture.

The strains described below were described in French patent application No. 91 08884. They were constructed from a strain which has the name W303.1B. This strain is haploid (mating type alpha) and carries as known mutations Ura3, Ade 2, His 1,
Trp 1, Leu 2; she breathes normally and is able to use galactose as a carbon source.

 The construction of the PES 1-3U and PES 1-3D strains is repeated here.

PES 1-3U strain
This haploid strain (mating type alpha) is completely isogenic with strain W303.1B, except the promoter region preceding the coding phase of the endogenous P450 Red, which is modified by homologous recombination so as to replace the natural promoter of P450 Red by the inducible promoter GALlO-CYCI. The procedure used to obtain this recombinant strain is shown schematically in Figure 3.

a) The strain W303.1B is transformed (using the standard lithium chloride method) with a mixture of four linear fragments of DNA obtained after exhaustive digestion of the vector pGPl-PC with
Eco RI. The only fragment capable of entering into recombination with the sequences naturally present in the strain, carries, in addition to the recombination bounds - the 5 'non-coding region of P450 Red and the 5' region coding of P450 Red - the marker gene Ura3 linked to the promoter GAL10-CYC1. Firstly, the Ura + markers were selected, to test the integration of the marker in the yeast genome.

b) The second screen consists of the selection of the Ura transformants obtained according to their slow growth rate relative to
W303 lB in complete glucose environment and growth identical to W303 1.B in complete galactose environment. Indeed, a very low level of P450 Red -which is under the control of the promoter GAL10-CYC1 after recombination leads to the accumulation of intermediates for synthesis of ergosterol in the cell membrane, which results in a considerable lowering of the cell growth rate. Conversely, a high level expression of P450 Red in a galactose complete medium will ensure correct biosynthesis of ergosterol, and must have the same growth characteristics as the wild strain. One of the clones meeting these criteria was chosen and stored under the name of PES 1-3.

 c) A spontaneous revertant of the Ura + phenotype to Ura 3 was selected by a replica made on a medium containing uracil and 5-fluoro-orotic acid. A stable mutant URA- (reversion rate less than 10-7), indistinguishable by any other criterion of PES 1-3, is stored under the name of PES 1-3U.

d) The characteristics relating to the recombinant phenotype of the locus of P450 Red are then tested
i) verification of the Ura 3 phenotype of the PES1-3U strain
ii) PCR amplification of a 2.2 kb band using a
first primer located at the 3 'end of the GAL10-CYC1 promoter
and a second primer located at the 5 'end of the non-coding strand of
the coding phase.

iii) testing of the ketoconazole R phetotype in a galactose complete medium,
linked to overexpression of P450 Réd, compared to the phetotype
ketoconazole S in a glucose medium.

PES 1-3D strain
This strain is completely isogenic with the strain PES1-2, with the exception of the sexual locus which has been changed to mating type a by the technique of the plasmid HO according to the published method (Methods in
Enzymology, vol 194, pp 132-146, and the locus of P450 Red. The entire coding phase as well as part of the 5 'and 3' flanking sequences are eliminated and the gene for the TRP1 marker is introduced at the level of the deletion. A schematic representation of the recombinant locus of P450
Red endogenous is given in Figure 4.

Culture Conditions
The different strains are cultured at 28 "C with moderate stirring (rotary shaker at 100 rpm) in a semisynthetic medium, composed as follows
- SWA6 medium: D-glucose 20 g / l (w / v), Yeast nitrogen base (Difco)
0.7% (w / v), 0.1% casein acid hydrolyzate (w / v), adenine 40
mg / l, tryptophan 20 mg / l.

- SWAPS medium: D-Galactose 20 g / l (w / v), yeast nitrogen base (Difco)
0.7% (w / v), 0.1% casein acid hydrolyzate (w / v), adenine 40
mg / l, tryptophan 20 mg / l.

4) Transformation "in flight" of the recombinant strain PES 1-3U
A necessary condition during the design of cloning by functional complementation is the obtaining of a sufficiently large number of transformants in yeast so as to statistically find a representative of rare cDNAs from the library previously enriched according to the size of the cDNAs. however, the level of endogenous P450 Red present in wild yeast seems to be strictly linked to the good transformability of this yeast. Overexpression of endogenous P450 Red (PES 1-3U culture in galactose medium) as well as very low levels or absence of P450
Réd (PES 1-3U culture in glucose medium or PES 1-3D in glocose / galactose medium) considerably reduce the transformability of these mutant strains. We describe here a protocol which solves the transformation problems linked to the concentration of P450 Red : After an overnight culture (2 × 107 cells / ml of the PES 1-3U strain) in galactose medium allowing the overexpression of P450 Red, the cells are collected by centrifugation, washed and 106 cells / ml are replaced in glocose medium where the expression of the endogenous P450 Red gene under the control of the gal 10-cycl promoter is suppressed. The culture is then continued for 5 h (approximately 107 cells / ml), the time necessary for the initial level of P450 Red to fall to a residual level corresponding to that of the wild strain. At this time, the cells are collected by centrifugation, washed and used for transformation according to the standard lithium chloride method, described in the literature.

 After transformation, the cells are maintained in a glucose medium, which brings the level of endogenous P450 Reductase present in the cells at the time of transformation to zero. Then, the functional complementing described below is carried out.

5) Functional complementation in the PES l-3U and PES 1-3D strains
After transformation of the PES l-3U strain with 10 μg of the cDNA library in the vector pFL61 according to the procedure described above, the cells are spread on 20 Ura dishes, in order to initially select for the presence of Plasmid DNA in transformed yeasts, the Ura3 gene present on the cloning vector making it possible to complement the Ura phenotype of the recipient cell. Approximately 2 x initial transformants are thus obtained, distributed in two pools and then spread out in two independent series in double selection on uraet dishes comprising 0, 5, 10, 20 or 50 ug / ml of ketoconazole to test for the presence of P450 Red activity in the initial transformants. 52 clones of independent secondary transformants, which can complement the Ura phenotype and which can grow in the presence of 10 or 20 μg / ml of ketoconazole were isolated and reselected on SWA-6 / ketoconazole medium 10 μg / ml. 24 clones were randomly selected for mini-preparations of plasmid DNA according to standard methods, which are then used to transform a strain of E. coli (DH5-1). The clones resistant to ampicillin (bla gene carried by the vector pFL61) were selected and their plasmid DNA analyzed by double Bam digestion.
HI / Hind III after amplification. According to their restriction profile (schematic representation in Figure 5), the 24 clones were divided into 5 different classes. The size of the inserts has been estimated to be between 2 and 3 kb, which corresponds to the size expected after the pre-fractionation.

In order to study the hereditary character of the phenotype conferred by the transformed plant DNA, 4 members of each class established on the basis of the restriction profile were randomly chosen for the transformation of the PES1-3D strain, in which the gene for the P450 Red has been disrupted. After transformation of 10 μg of plasmid DNA from the four different members of classes A, B, C, D and E into the strain
PES1-3D, the cells are spread on a series of 4 SWA6 / agarose dishes containing 0, 20, 40 or 80 ug / ml of ketoconazole. The transformants from classes A, B, C and E showed resistance to ketoconazole up to 40 ug / ml, a level comparable to that of the strain PES1-3 cultivated in SW A5 medium (endogenous reductase expressed). from class D were unable to grow in the presence of ketoconazole and were abandoned.

6) Enzymatic determination of the P450 Red activity in the microso fraction
male from the 4 classes of PES 1-3D transformants resistant to
ketoconazole
Cultures (250 ml) of PS1-3U, transformed by classes
A, B, C and E are stopped when the cell density is 5 × 10 7 cells / ml. After centrifugation for 5 min at 2500 g, the cell pellet is suspended in 35 ml of 50 mM Tris-HCl buffer pH 7.4.5 mM EDTA, 100 mM KC1, 20 mM beta-mercaptoethanol, incubated at 20 "C for 10 min , then recentrifuged at 10,000 g for 3 min. After washing in 35 ml of breakage buffer (50 mM Tris-CHl ph7.4.1 m EDTA, 0.6 M Sorbitol), the pellet is resuspended in one volume of buffer breaks equal to that of the pellet, then glass beads (Braun, diameter 0.45-0.5 mm) are added in an amount just sufficient to level the surface of the cell suspension. The tubes are tightly closed and violently shaken on horizontal agitator at a speed of 3 strokes per second for the time necessary for more than 70% of the cells to be broken. 3 ml of breakage buffer are then added to the mixture, the tubes are briefly vortexed then the liquid present between the beads is transferred to another tube. The extraction is repeated twice with 3 ml of case buffer, and the supernatants are transferred to the same tube. All the extracts are centrifuged for 15 min at 10,000 g. The supernatant is transferred to another tube and the volume is adjusted to 10 ml with case buffer.

Three hundred microliters of a 4M NaCl solution are added, then 3.5 ml of a 40% solution (w / v) polyethylene glycol-4000. After incubation for 15 min at 0 C and centrifugation at 10 min at 10,000 g, the supernatant is carefully discarded without disturbing the pellet (which contains the microsomes), then the tube and pellet are rinsed with a small volume of recovery buffer (50 mMTris -HCl pH 7.4 1 mM EDTA, 20% glycerol) in order to remove traces of polyethylene glycol. The pellet thus obtained is resuspended and homogenized in 1 ml of recovery buffer, then the suspension is aliquoted by 200 μl for storage at -80 C.

The activity of P450 Red contained in the microsomal fraction is measured by reduction kinetics of a non-physiological substrate, horse heart cytochrome c (Sigma type vr). The reaction mixture contains in 1 ml of final volume: 2 to 10 μl of a 30-fold diluted solution of microsomes, 10 μM cytochrome c, 100 μg / ml
NADPH dissolved in 50 mM Tris-HCl pH 7.4, 1 mM EDTA. The activity is calculated using a differential coefficient of 21 mM 1 at 550 nm. One P450 Red activity unit is defined as the quantity of enzyme necessary for the reduction of 1 nmole of cytochrome c / min to 200C.

The activity is related to the total amount of microsomal proteins present in the reaction which has been previously determined by the test
BCA of PIERCE.

 The activities obtained are shown in FIG. 6: all the clones obtained by functional complementation express an activity of P450 Red type at least 3 times greater than the background noise measured in the strain deficient in P450 Red, transformed with an empty vector. Clone B having shown the same phenotype of resistance to ketoconazole as clones A, C and E, it must be concluded that approximately one third of the P450 Red activity measured in the wild strain is sufficient to establish this phetotype in the strain deficient after transformation.

7) Determination of the nucleotide sequence of the cDNA inserts in the
vector pFL61 isolated from the 4 classes of PES transformants
1-3D resistant to ketoconazole.

One clone of each class (A, B, C, E), which previously was resistant to ketoconazole and also showed significant activity of
P450 Réd, was chosen at random and the plasmid DNA was prepared from a miniculture by standard methods. The fragments corresponding to the inserted plant cDNAs were purified by elution of agarose gel after digestion of the vector with Not I. The fragments thus obtained are then subcloned into the vector pUC19 / NotI, linearized by
Note. This vector is derived from the standard vector pUCl9 and characterized by the following modifications: A unique NotI site is created by ligation of NotI linkers to the vector pUCl9 linearized in Hindi in the polylinker, followed by recirculation. The vector thus obtained is then linearized in EcoRI, the outgoing 5 ′ ends are made blunt for the Klenow polymerase, then subsequently religated, which restores the open phase in the LacZ gene. Thus, this vector allows a double screening after transformation in a host cell of the white colonies growing on a complemented medium.
Ampicillin X-Gal and IPTG contain the vector (the ampicillin resistance gene is carried by the plasmid) whose open phase of the gene
LacZ was destroyed by inserting a DNA fragment at the polylinker (eg NotI site). After transformation, the transformants obtained for each class are tested for the presence of cDNA inserted by enzymatic restriction, then are then used for sequencing.

The nucleotide sequence of the entire insert of a representative of each class (A, B, C, E) was determined by sequencing on double-stranded DNA according to a modified Sanger method using the T7 polymerase supplied by BRL, exactly under the conditions described by the supplier. Starting from the universal primers located 5 'and 3' from the insertion site on the two strands of the vector, the complete sequence was obtained by gradual progression using sequence-specific oligonucleotides, derived from the 5 'part of the newly read sequences. on both strands. The coding region of clones A, C and E being identical, the sequencing of the complete insert was continued with the clones
B and C. The nucleotide sequence comprising the 5 'and 3' flanking regions and the entire coding region from the first ATG followed by the open reading phase for the clones B and C is given in the
Figures 9 and 10. The numbering of the nucleic acids is given in the right column, the numbering of the amino acids is indicated below the amino acids Concerning the sequences described in the
Figures 9 and 10, several remarks can be made
a) Despite a relatively low homology of the order of 36% with P450 Red sequences known from yeast (21) or from man (22) at the deduced amino acid level, the identification of the cDNA sequences isolated from functionality criteria such as P450 Red has been helped by the existence of high conservation blocks in all the functional areas previously described for higher eukaryotes except plants.

 These domains have been identified on the basis of comparison of known P450 Red sequences with those of other flavoproteins as well as on the basis of chemical modifications of P450 Red.

(24) Haniu, M., Iyanagi, T., Legesse, K., and Shively, J.E. 1984 J. Biol. Chem.

259, pp 13703-13711 (25) Porter, TD, and Kasper, CB 1986 Biochemistry 25, pp 1682-1687 (26) Porter, CD and Kasper, CB 1985 Proc. Natl. Acad. Sci. USA 82, pp
973-977 (27) Katagiri, M., Murakami, H., Yabusaki, Y., Sugiyama, T., Okamoto, M.,
Yanano, T. and Ohkawa, H. 1986, J. Biochemistry 100, pp 945-954 (28) Vogel, F. and Lumper, L. 1986, Biochem. J. 236, pp 871-878
The locations of the binding sites of the FMN (A1 / A2) of the FAD (B1 / B2) and of the NADPH (C) for the plant P450 Reds are obtained by comparison with the alignment according to Yabusaki et al, 1988 (21) and are indicated by square brackets in Figure 12.

b) The amino acid sequences deduced from the AraB clones and
AraC show only 64% of strict conservation between themselves although having been screened on an identical function and coming from the same organism. In addition, the two cloned cDNAs show a different hybridization pattern in a southern genomic, suggesting that they are transcripts of two very distinct genes, present in simple copy at different locations in the Arabidopsis genome.

 This is a very strong indication that the two cDNAs come from independent genes, and that there is selective pressure to maintain two proteins with identical function. This observation is reinforced by the difference of the third base of the codon on identical amino acids (Figure 8): the choice of the third base is very close to the random distribution and much less than in the case of genes which diverged there are 300 million years (Figure 8, columns 8 and 9).

 c) On the N-terminal side, clone B contains a significant extension of 25 amino acids (ie 75 nucleotides), comprising in particular a block of 7 serines which immediately follows the first ATG.

d) Using the algorithm of Kyte and Doolittle for the calculation of the hydropathy index, one can detect in the two sequences ARaB and
AraC, a very hydrophobic segment in the NH2-terminal region of the two proteins, composed of a sequence of at least twenty hydrophobic amino acids.

This is a typical signal from a protein located in the endoplasmic reticulum and has also been described in all P450s known to date.

e) Using the ISOELECTRIC program provided by
Wisconsin GCG Software Package, the calculated net charge of putative proteins AraB and AraC deduced from nucleotide sequences, shows a very clear difference at physiological pH (7.5): AraB is very close to the
P450 Red human in terms of net charge, AraC being much more acidic. This is a strong indication for an interaction of the two P450s.
Réd of Arabidopsis with distinct P450 classes and / or a different subcellular localization, which explains well the selective pressure on the maintenance of the two P450 Réd in Arabidopsis.

8) Use of Arabidopsis P450 Red cDNAs for the Cloning of Others
P450 Reduced plants by cross-species hybridization.

Taking into account the low degree of conservation between the P450s
Reduced known eukaryotes (yeast, rat, rabbit, pig, trout, man), it is easily understandable that all attempts to use probes
P450 Réd known to isolate a P450 Réd by crosshybridization were doomed to failure. The first plant P450 Red cDNAs were used for the screening of a Jerusalem artichoke cDNA library under conditions of reduced stringency to take account of inter-species variations.

 Pieces of Jerusalem artichoke nodules which were kept in water at 4 "C for 24 hours to induce the synthesis of P450 Red mRNA (12), were used as plant material to prepare total Jerusalem artichoke mRNA according to standard procedures. A total Jerusalem artichoke cDNA library in phage lambda gt 10 was obtained according to the method of OKAYAMA and BERK (13). About 20,000 pfu are seeded per petri dish containing solid LB medium and then transferred on nitrocellulose filter (Schleicher and Schuell, BA85). 400,000 pfu from the Jerusalem artichoke cDNA library are screened with 500 ng of purified AraB cDNA, marked with high specificity by random priming, according to standard methods, but at 50 " C instead of 65 "C to take account of the divergences in the sequence for an identical protein in different plant species. The filters are / then washed under conditions of increasing stringency until disappearance of the hybridization signal not specific.

More specifically, the hybridization conditions making it possible to detect cDNA sequences coding for an identical protein in different plant species are as follows: A batch of 10 nitrocellulose filters is prehybridized at 50 "C in a hybridization oven in a tupperware box hermetically sealed containing 100 ml of prehybridization solution on a rotary shaker. The prehybridization solution which was previously kept at 50 "C contains per 100 ml: 10 ml 50x
Denhardt's, 25 ml 20x SSPE, 5 ml 10% Sarcosyl, 0.5 ml DNA of sperm of salmon at 10 mg / ml denatured, 59.5 ml water. The composition of the ingredients is completely standard and described in reference books (24). The prehybridization time is 2 hours minimum. The prehybridization solution is then eliminated and replaced by the same volume of hybridization solution also preheated to 50 "C. This solution is identical to the prehybridization solution with the exception of the salmon sperm DNA which has been replaced with 2.5 µg of radioactive specific probe actively randomized, then denatured by heating just before adding it to the hybridization solution. The hybridization time is 16 hours at 50 "C with stirring, then Washes: The hybridization buffer is eliminated and replaced with 200 ml of a washing solution containing lxSSPE, 0.1% SDS, 0.2% pyrophosphate and the mixture is left to stir at room temperature for 20 minutes. solution, it is replaced by 200 ml of the same solution preheated to 50 "C and left to stir at 50" C for 20 minutes. This last step is repeated at least once, then the filters are drained, the remaining liquid is removed on sheets of Whatman paper and then the wet but not dry filters are sealed in a plastic bag for exposure.

Colonies that had specifically hybridized with the probe
AraB, have been highlighted by exposure in the presence of a "Chronex Lightening Plus" screen (Dupont), then are subjected to 2 additional rounds of hybridization. The positive clones after 3 rounds of hybridization were used for DNA mini-preparations and identified by analysis of the restriction profile. Selected restriction fragments were purified by LMT (low melting temperature) agarose electrophoresis, then ligated into the bacteriophage M13mpl8. The single-stranded phage DNA was isolated from cultures of infected bacteria, by precipitation with polyhylene glycol, digestion with proteinase K, phenolic extraction then precipitation with ethanol. The nucleotide sequence of single-stranded DNA was determined by Sanger's method (20) using T7 polymerase supplied by
BRL under the conditions indicated by the supplier. Starting from the universal primer, the complete sequence was obtained by gradual progression using sequence-specific oligonucleotides, derived from the 5 'part of the newly read sequences on the two strands. The sequence of a randomly selected clone which was positive after 3 rounds of hybridization with a P450 Red probe from Arabidopsis in the conditions described above, is shown in Figure 11. Several comments can be made
a) Although the sequenced clone is not complete in the NH2 terminal end of the protein, the Jerusalem artichoke cDNA isolated by cross-hybridization with a cDNA of Arabidopsis P450 Red is irrevocably identified as that of a P450 Red of Jerusalem artichoke, since it presents 69 respectively i3% homology in terms of amino acids with the 2 vegetable Red P450 previously identified (Figures 6 and 10).

 b) Alignment of the amino acid sequence of the putative Jerusalem artichoke P450 with the Arabidopsis P450 Red shows a particular conservation in the functional areas previously described for higher eukaryotes with the exception of plants (Yabusaki et al, 1988) .

 c) The P450 Red cDNA from Topinambour shows a significantly higher homology with the AraC form (73%) than with the AraB form (69%), which suggests the existence of several independent genes in the Jerusalem artichoke, unlike what has been reported in the literature for mammalian P450 Red.

 d) In view of the poor homology of Jerusalem artichoke P450 Red with the P450 Reds known from the literature, it is not possible to isolate this cDNA using cDNA sequences of P450 Red already known from the literature.

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Claims (12)

 1. Method for cloning a DNA sequence coding for a NADPH Cytochrome P450 Reductase of an organism, especially of plants, characterized in that  1- We submit a yeast strain transformed with plasmids  a bank of total cDNA of said organism, in particular of said  plant, and having an activity deficiency phenotype  Endogenous NADPH-Cytochrome P450 Reductase, for treatment  with a cytochrome P450 inhibitor at a dose that makes  lethal lack of activity NADPH Cytochrome P450 Reductase  endogenous then  2- The surviving clones are recovered and the plasmids are extracted therefrom  allowing the expression of a NADPHCytochrome P450 activity  Reductase which corresponds to NADPHCytochrome P450  Reductase of said organism, in particular of said plant.  2. Method according to claim 1 characterized in that the endogenous yeast NADPH Cytochrome P450 Reductase gene is placed under the control of an inducible promoter and in step 1, the following substeps are carried out successively  l-a) A yeast strain is transformed with plasmids of a  bank of total cDNA of said organism, in particular of said  plant then  l-b) NADPH-Cytochrome P450 activity deficiency is induced  Endogenous reductase by inactivating said promoter, and  l-c) Subjecting said strain to treatment with said dose  cytochrome P450 inhibitor.  3. Method according to claim 2 characterized in that, using the plasmids recovered in step 2, a yeast strain is transformed in which the endogenous NADPH cytochrome P450 Reductase gene has been deleted and a treatment is repeated with a cytochrome P450 inhibitor at a dose which renders the absence of endogenous NADPH cytochrome P450 activity lethal, then the surviving clones are recovered and the plasmids are extracted allowing the expression of a NADPH cytochrome P450 activity which corresponds to the NADPH cytochrome P450 Reductase of said organism, in particular of said plant.  4. Method according to claim 1 to 3 characterized in that the strain is a strain of saccharomyces cerevisiae.  5. Method according to one of claims 1 to 4 characterized in that the activity inhibitor P450 is an inhibitor of lanosterol 14-demethylase such as an antifungal azole compound, in particular ketoconazole.  6. Method according to claim 5 characterized in that the activity inhibitor P450 such as ketoconazole is used at a dose of between 5 and 100, ug / ml of culture medium, preferably between 10 and 50 ug / ml .  7. Method according to one of claims 1 to 6 characterized in that in step 1; the cDNA bank is enriched with NADPH-Cytochrome P450 Reductase of interest, by fractionation according to size, keeping only fragments larger than 2 kbp.  8. Method according to one of claims 1 to 7 characterized in that the cDNA sequence coding for a Cytochrome NADPH is cloned P450 Arabidopsis Reductase from a total and enriched ANDc bank of said plant.  9. Method for cloning a NADPH Cytochrome P450 gene Plant reductase by hybridization under low stringency condition with a cDNA of a NADPH Cytochrome P450 of another plant obtained by a method according to one of claims 1 to 8 from a total or or enriched cDNA library in fragments greater than 2 kbp of said plant.  10. CDNA sequences encoding a Cytochrome NADPH P450 Plant reductase obtained by a process according to one of claims 1 to 9.  11. CDNA sequences encoding Cytochrome NADPHs P450 Reductase of arabidopsis ara-B or ara-C, or of Jerusalem artichoke heliantus tuberosus, as shown in Figures 9 to 11 respectively.  12. DNA sequences coding for Cytochrome NADPHs P450 Reductase from plants hybridizing under weakly stringent conditions at 50 "C and / or having at least 60% homology with one of the sequences according to claims 10 or 11.