WO1998046773A1 - Gene involved in basidiomycete fungi fruit body development and uses - Google Patents

Abstract

The invention concerns a gene involved in the fruit body development of the edible mushroom Agrocybe aegerita and the use thereof to induce said fruit body development in basidiomycete fungi. The invention concerns in particular a nucleotide sequence corresponding to: a) a sequence according to SEQ ID N°1 partially or wholly; b) a sequence hybridised with a); or c) a sequence with at least 80 % homology with a) or b).

Description

 GENE INVOLVED IN THE FRUCTIFICATION OF BASIDIOMYCET MUSHROOMS AND USES

The present invention relates to a gene involved in the fruiting of the edible fungus Agrocybe aegerita as well as the use of this gene. The basidiomycetes of the basidiomycetes are the most complex structures produced among fungi; in edible industrial mushrooms these structures, which constitute the commercial product, are of great economic added value. Consequently, knowledge of the genes which trigger the start of basidiocarp differentiation is of interest both for the elucidation of the regulation of eukaryotic differentiation and for the industrial culture of fungi because the yield of an industrial culture depends on the regulation fruiting. In fungi, most studies have been carried out on the influence of environmental factors on fruiting (Raudaskoski and Viitanen, 1982; Sietsma et al., 1977), but the regulation of genes that play a specific role in little is known about this phenomenon.

Indeed, one of the major problems in the industrial culture of edible mushrooms is to obtain the initiation or the initiation of fruiting. Under industrial culture conditions, fruiting is obtained after the culture substrate has been invaded by the dicaryotic mycelium, under the action of external factors (temperature, humidity, CO2, casing, etc.) and complex nutrients. These factors, which are often difficult to control, activate specific differentiation genes.

In basidiomycetes, the genes which intervene at various stages in the development of basidiocarps have been isolated from the fungus Schizophyllum commune and from the fungi Agaricus bisporus and

Lentinula edodes. The Sc7 gene (Shuren et al., 1993) and the mfbAc gene (Kondoh et al., 1995) are preferentially expressed during the later stages of basidiocarp differentiation in S. commune and L. edodes, respectively. The hypA (De Groot et al., 1996) and ABH1 (Lugones et al., 1996) genes, both isolated from A. bisporus, and the Sc4 gene (Shuren and Wessels, 1990) from

S. commune are expressed from initiation to maturation of the basidiocarp, which suggests that these genes have a role in the expansion and / or maintenance of the basidiocarp rather than in its differentiation. Three genes are characterized as being specific for the initiation of fruiting, the priA (Kajiwara et al., 1992) and priBc (Endo et al., 1994) genes from L.edodes and the Salt gene (Dons et al., 1984) of S. commune. A different case is that of the FRT1 gene isolated from S. commune, which is expressed in mycelial growth as well as in fruiting, however the ectopic genomic integration of the FRT1 gene makes it possible to induce fruiting (Horton and Râper, 1995 ).

The study of new morphogenesis genes intervening specifically in the early stages of the development of primordia seems to be of particular interest for understanding the genetic determinism of fruiting. Eight cDNA clones (EMAa-1 to EMAa-8) preferentially expressed during the differentiation of basidiocarp primordia have been isolated from the edible fungus Agrocybe aegerita (Salvado and Labadère, 1991). This fungus is a powerful model for development studies, because it can differentiate the basidiocarps on a synthetic medium, and transformation is mastered in this organism (Noël and Labarère, 1994), also it provides a good tool to study the function and regulation of cloned morphogenetic genes.

A genomic bank άA. aegerita was screened using the previously cloned EMAa-4 cDNA as a probe, resulting in the isolation of the corresponding genomic sequence called Aa-Pril. The nucleotide sequences of the cDNA and the cloned genomic DNA were determined and compared in order to determine the structure of the Aa-Pril gene and the possible presence of introns. The regions of the neighboring gene at 5 'and 3' have been sequenced and studied, looking for the regulatory signals involved in transcription. Transcription start points (tsp) were identified by primer extension. Gene expression

Aa-Pril during fruiting was confirmed using EMAa-4 cDNA to probe a Northern blot of total RNA from vegetative mycelia and three stages of basidiocarp development (aggregates / pre-primordial, primordial primordia, basidiocarps). The amino acid sequence deduced from the Aa-Pril gene was analyzed and compared with protein databases. The Aa-Pril gene is specifically transcribed at the immature primordia / basidiocarp stage, suggesting that it plays a role during the start of fruiting, but not for maintaining the structure of the basidiocarp. The complete nucleotide sequence of the gene (492 bp) and the regions upstream and downstream has been determined.

SEQ ID NO: 1 represents a nucleotide DNA genomic sequence of Agrocybe aegerita comprising the coding part of the Aa-Pril gene (comprising an intron) framed by the 5 'and 3' non-coding portions. This sequence is also shown in Figure 1B. The present invention therefore relates to a nucleotide sequence corresponding to: a) a sequence according to SEQ LD No. 1 in whole or in part; b) a sequence hybridizing with a); or c) a sequence having at least 80% homology with a) or b). More particularly, it relates to a nucleotide sequence corresponding to a gene or a gene fragment comprising: a) a part of the sequence according to SEQ LD No. 1; b) a sequence hybridizing with a); c) a sequence having at least 80% homology with a) or b); d) a sequence coding for a protein coded by a gene or a gene fragment according to a), b) or c), or for an equivalent protein.

The nucleotide sequences can be either DNA or RNA (including mRNA) or sequences in which some of the nucleotides are modified in any way (including substituted by unnatural nucleotides) , either to improve any of their properties, or to allow their identification.

The sequences mentioned in (b) are essentially the total or partial complementary sequences (in particular for the cases mentioned above). In addition, it should be recalled that the present invention covers both the nucleotide sequence corresponding to the whole gene and fragments of this gene, in particular when they code for an equivalent protein as will be described below.

The invention also relates to the nucleotide sequences having a strong homology with the nucleotide sequence or the gene mentioned above, preferably a homology greater than 80% on the essential parts of said gene, ie in general at least 50% of the sequence; more preferably the homology on the essential parts will be greater than 90% (determined in particular by the software Clustal. W, version 1.4 for Macintosh).

Finally, when said gene or gene fragment codes for a protein, the present invention also relates to the sequences coding for the same protein, taking into account the degeneration of the genetic code, but also for equivalent proteins, that is to say producing the same effects, in particular proteins deleted and / or having undergone point mutations.

Figure 1 shows the structure of the Aa-Pri gene from Agrocybe aegerita. FIG. 1A is a schematic representation of the EcoRI- genomic fragment

2.4 Kb EcoRI containing Aa-Pri. The gray box represents the coding region for Aa-Pri with the intron (dark gray segment) and the 5 'and 3' positions not translated. FIG. 1B represents the nucleotide sequence and the predicted amino acid sequence of the Aa-Pri gene. An asterisk (*) marks the stop codon (TTA). The CCAAT and TATAAAT boxes are grayed out. The starting points for transcription determined by primer extension and by S1 nuclease mapping are indicated by arrows and by a black circle respectively. The intronic sequences possibly involved in the splicing phenomenon and the formation of a lasso are identified in bold and are underlined. The sequences resembling the polyadenilation signal are framed and the beginning of the poly (A) tail is represented by a triangle.

Figure 2 shows the structural characteristics of the deduced Aa-Pri protein. The AMP cyclic-dependent protein kinase phosphorylation site is represented by a gray box, the casein kinase II phosphorylation sites are represented by gray boxes and the protein kinase C phosphorylation is shown below. Putative glycosylation sites (NAS S and NKTI) are indicated by arrows.

For the translation of the gene, only 49 of 61 sense codons are used, which shows a bias in the use of codons. However, the degree of bias of the Aa-Pril gene is less marked than in the highly expressed fungal genes in which barely 28 codons can be used (Gurr et al., 1987). The Aa- Pril gene follows the basic patterns in the use of the third base described for the constitutive or highly expressed genes originating from yeasts and filamentous fungi (Gurr et al, 1987): when there is a choice between purines and pyrimidines, there is a preference for codons which end in pyrimidines, in particular C which is used in 66% of the codons, and similarly, if a purine is required in the floating position, 66% of the codons use G .

The comparison of cDNA and genomic sequences shows a short intron 54 bp long at nucleotide positions +125 to +179. Short introns located in the 5 ′ part of the fungal genes are described in the pyrG ά'Aspergillus nidulans and cutA genes of Fusarium solani, which have an intron of 59 bp at nucleotide +53 and 52 bp at the level of nucleotide +66, (Gurr et al., 1987). The intron of the Aa-Pril gene is a class II intron according to the consensus sequences at the intron-exon limits (5'GT / AG 3 ') (Michel et al., 1989). At the 5 'limit, instead of the classic GT, the intron of the Aa- gene

Pril has the GC nucleotide pair. Other exceptions to the GT limit rule have been found in fungi in the des-1 and qa-IS genes from Neurospora crassa (Gurr et al., 1987) and in the pgal and ras genes spAspergillus niger and L .edodes, respectively (Unkless, 1992). Analysis of the 5 'non-coding region reveals the presence of a motif

TATAAAT at nucleotide -83 and a CCAAT motif at nucleotide -156 (73 nucleotides upstream of the predicted TATA box) (Fig. 1). These two sequences involved in the transcription of genes coding for eukaryotic proteins (McKnight and Kingsbury, 1982), have been identified in numerous fungal genes respectively around 30-350 bp and 83-500 bp from the site of initiation of translation (Gurr et al., 1987 and Unkless, 1992). By way of examples, TATA and CAAT boxes have been located in positions -96 and - 188 respectively in the cbh2 promoter from Trichoderma reesei (Unkless, 1992) and in positions -111 and -167 in the priA promoter from L.edodes (Kajiwara et al., 1992). Out of three potential transcription starting points (pdt) identified by primer extension (nt -49, -85 and -91), the pdt located at -49 seems more likely, and the two other pdt are both located in upstream of the putative TATAA box. In addition, this pdt begins with an A preceded by a C, which is the most common dinucleotide pair found in eukaryotic pdt (Breathnach and Chambon, 1981) and is surrounded by a strong inducing sequence (CCA ⁺ * TTCC) corresponding to the consensus initiation sequence YYA ⁺ ^ NWYY

(W = A or T; Y = C or T) (Javahery et al., 1984) in which a T at position +3 and an environment rich in pyrimidine seem to be important for a strong initiation (Javahery et al., 1984) .

At the 3 'end of the Aa-Pril gene, the poly (A) tail begins 145 nucleotides downstream from the predicted translation stop codon (TAA). The polyadenylation sequence AATAAA which is frequently found in eukaryotic genes (Gurr et al., 1987) is not found in the non-coding Aa-Pril region at 3 ', but similar sequences: [T (A) 5TA] and [T (A) 3T (A) 4T (A) J were identified at 44 nucleotides and 29 nucleotides respectively upstream of the polyadenylation site.

The Aa-Pril gene potentially codes for a polypeptide at 145 aa of 16093 Da (calculated according to the DNA Strider 1.2 software). Although its homology with Asp-hemolysin to A. fiimigatus (Ebina et al., 1994) is significant, it does not provide concrete elements on the function to! Aa-Pril in the differentiation of primordia. Cytolytic agents are synthesized by various microorganisms, plants and animals. Fungal hemolysins have been reported in the fungi Gyromitra esculenta (Gray, 1973), Tricholoma populinum (Lindequist et al., 1989) and Laetiporus sulfureus (Konska et al., 1994); a cytolytic protein has been purified (pleurotolysin) from the fungus Pleurotus ostreatus (Bemheimer and Avigad, 1979), and the cytolytic activity of rubescenslysine and phallolysin from manAmanita has been reported rubescens and Amanita phalloides, respectively (Seeger and Wachter, 1981); however, the in vivo biological functions in fungal cells are unclear. In any case, from the ability of Asp-hemolysin, classified as cytolysin, to interact with specific receptors in the membranes (Thelestam and Môlby, 1979), we can predict a hypothetical role for the protein.

Aa-Pril, in the binding to specific receptors located on the surface of the vegetative hyphae, which could allow their agglutination and their packing to form the primordia. The binding property to a cell and / or nuclear membrane has been reported for the priA protein from L.edodes which seems to play one or more roles during the beginning of fruiting (Kajiwara et al., 1992), but the mechanism of the putative bond seems to be different for the two proteins since no Cys-Aaa-Aaa-Xaa C-terminal box (Aaa = aliphatic acid, Xaa = any aa) was found in the protein Aa-Pril.

It seems particularly interesting that the protein Aa-Pril contains several residues (lysine, serine and threonine) which can act as substrates for phosphorylation by different types of protein kinases. A putative site of protein kinase dependent phosphorylation of cAMP (KRNT) begins at residue lysine 106. τj _nr 5i _e possible to cyclic AMP in the formation of basidiocarps Coprinus macrorhiΣus was assumed based on available protein for phosphorylation by cyclic AMP-dependent protein kinases (Ishika a and Uno, 1977). In addition, it is known that high levels of cyclic AMP are closely related to the onset of fruiting and / or the formation of primordia in the fungus L.edodes (Takagi et al., 1987) and that a putative AMP-dependent protein kinase phosphorylation site (RRNS) has been found in the priBc protein which is preferentially synthesized at the primordial stage (Endo et al., 1994).

Two casein kinase II phosphorylation sites, SNKD and SGTE were located at the serine residues and

respectively (Fig. 2). The SNKD site overlaps with a protein kinase C (SNK) phosphorylation site; two other consensus sites for protein kinase C phosphorylation, TIR and

SKR, are located at the tailings

respectively. Activation of the protein Aa-Pril by phosphorylation can lead to reactions involved in the differentiation of basidiocarps from primordia by analogy with other growth phenomena such as cell division or the development of a tumor in which functions protein kinase regulators seem to be involved (Hanks et al., 1988). In basidiomycetes, various genes intervening at different stages of basidiocarp development have been isolated. Studies on the regulation of their transcription have shown that only three genes are specific for the primordial stage / immature fruiting body: the PriA and PriBc genes of the fungus L.edodes, the corresponding proteins of which presumably control the expression of the gene or genes which are in relationship with the appearance of fruiting bodies (Kajiwara et al., 1992; Endo et al., 1994) and of the salt gene of the common fungus, coding for a hydrophobin protein which develops an insoluble complex in the cell walls of the emerging hyphae (Dons et al. 1984; Wessels et al. 1991).

The function of the protein Aa-Pril, specifically expressed during the formation of primordia, remains unknown. Although the homology with Asp-hemolysin dA.fumigatus is significant, it does not provide clear evidence of the putative function of the protein Aa-Pril because the functional regions of Asp hemolysin have not been identified. It is however interesting to note that the two proteins could interact with the membrane. In addition, like the pri Bc protein of L. edodes, the Aa-Pril protein has a cAMP-dependent phosphorylation site, which suggests that the corresponding structural gene could be regulated by the amount of AMP in cells. . The present invention also relates to the use of a sequence mentioned above to induce fruiting not only in Agrocybe aegerita but also in any other species of basidiomycete fungus. This use includes the insertion by transformation of said sequence into a cell, a hyphe or a sexual or asexual spore of basidiomycete, said transformation possibly being carried out by means of a vector. In fact, the aim of the present invention is in particular to better control the fruiting of edible basidiomycete fungi. As indicated above, the "natural" fruiting of mushrooms is only triggered by the activation of key genes themselves under the influence of more or less complex and difficult to control external factors. As will be shown in the example below, the Aa-Pril gene is a good candidate to be one of these key genes. Consequently, its use, or rather that of SEQ LD N ° 1 or N ° 2, in whole or in part, modified if necessary (for example by mutation) should allow, by its insertion within hyphae of basidiomycetes by a means transforming any, for example a vector, then possibly by its integration within the genome, to overcome the uncertainty of the expression of the natural gene. For example, the introduced sequence could be sensitive to lower thresholds of “stimulation” by external factors or only to some of them or could be spontaneously functional as soon as the basidiocarp has reached a certain stage of maturation.

The subject of the invention is therefore the cells, hyphae and sexual or asexual spores of basidiomycete fungus transformed by a nucleotide sequence according to the invention.

Another subject of the invention relates to the use of a nucleotide sequence according to the invention comprising at least one coding part for producing a protein or protein fragment. This use includes the insertion of the sequence in question into an expression vector capable of transforming prokaryotic or eukaryotic cells and the transformation of said cells with said expression vector. In addition to the transforming vector mentioned above, the invention also relates to expression vectors capable of transforming prokaryotic or encaryotic cells. These vectors in question express the protein sequence corresponding to the nucleotide sequence which they contain in the transformed cell, said protein sequence which can then be recovered by conventional techniques. Another subject of the invention relates to prokaryotic or eukaryotic cells transformed by an expression vector according to the invention or by any means whatsoever.

Finally, the invention relates to a protein or a protein fragment capable of being obtained by culture of the above-mentioned transformed cells.

The present invention is not limited to the description above. It will be better understood in the light of the example below which is only mentioned for illustrative purposes. Example: Strains and growing conditions

The dicaryotic strain of A. aegerita comes from subcultures of basidiocarp fragments of the wild type strain SM51 (A1B1 / A2B2). To obtain basidiocarps at various stages of development, 5 mm dicaryotic mycelium fragments are inoculated into petri dishes containing a solid complete grating medium (Râper et al., 1972), and cultured for 10 days in the dark at 25 ° C (vegetative growth) then they are transferred under fruiting conditions (room temperature under natural conditions of light / dark). The stages of development are defined previously (Salvado and Labarère, 1991). The vegetative mycelium is cultivated on complete liquid medium (Râper et al., 1972) in Roux vials without shaking in the dark at 25 ° C.

The JM83 strain of Escherichia coli (Yanish-Perron et al., 1985) used for the propagation of the plasmids is cultivated at 37 ° C. in a liquid or solid LB medium containing 50 μg / ml of ampicillin. DNA isolation and analysis

The total DNA is isolated according to the process of N-cethyl-NNN-trimethyl ammonium bromide (Noël and Labarère, 1989). The digested DNA is separated in a 0.8% agarose gel and then transferred to a nylon membrane (Hybond-N ⁺ , Amersham Corp.) according to the manufacturer's method and using a vacuum blotting system. (Appligene). Hybridization of the total digested DNA is carried out with the labeled cDNAs (see preparation of the radioactive probes) in a reference hybridization buffer containing 6 x SSC (1 x SSC in 0.15 M NaCl and 0.015 M sodium citrate, pH 7.6), 5 x Denhardt solution, 0.5% SDS (w / v) , DNA from herring sperm denatured at 100 μg / ml. The filters are washed successively twice (2 x 5 minutes) in 6 x 0.1% SSC / SDS, twice (2 x 10 minutes) in 2 x 0.1% SSC / SDS then twice (2 x 15 minutes) in 0.2 x

0.1% SSC / SDS.

The construction and isolation of the plasmid DNAs is carried out according to Maniatis et al. (1982). Isolation and Analysis of RNA Total RNA is isolated using the hot phenol method (De Vries et al., 1980). The RNAs are fractionated in 1.4% formaldehyde agarose gel according to Maniatis et al. (1982) then it is blotted on a nylon membrane by capillary transfer using 1.5 M NaCl / 0.5 M NaOH as transfer buffer. Hybridizations are carried out with the EMAa-4 cDNA or the 18 S rDNA of Pleurotus comucopiae used as probes, at 42 ° C. in the reference hybridization buffer in the presence of 50% formamide. The nylon membranes are washed for 20 minutes with 1 x 0.1% SSC / SDS at 25 ° C and then for 10 minutes with 0.2 x 0.1% SSC / SDS at 55 ° C.

Poly (A) RNAs are prepared using the polyAtract mRNA system (Promega), according to the manufacturer's instructions. The poly (A) isolated by this method represents 0.5% of the total RNA. Preparation of radioactive probes

The EMA inserts released after the digestion of the pEMA plasmids with BamHI are purified, from an electrophoresis on agarose gel by the "Gene Clean" system (BIO 101). The linearized pEMA inserts and plasmids are labeled with BamYΩ. by means of a random priming with 25 μCi of a-32p. dCTP (3000 Ci / mmole, Amersham Corp.) up to a specific activity of 10 ^ - 10 ^ cpm / μg, using the Primer-a-Gene labeling system (Promega).

The 18S rDNA of Pleurotus comucopiae used in the Northern blot analysis is obtained by digestion with Sali-Eco RI of the insert pPcRl containing a part of the rDNA unit of P.cornucopiae (Iraçabal and Labardère, 1994) and mark as described above.

Construction of the genomic library and screening

The genomic DNA isolated from the vegetative mycelium of A. is digested with Pst 1. aegerita and separated on 0.8% agarose gels. The gel segment containing DNA fragments of between 2.9 and 3.3 kb is cut and the DNA is purified from an agarose gel using the "Gene clean" system (Bio 101). The purified DNA is ligated to the vector pUC18 previously digested with Pst 1 and dephosphorylated by treatment with bacterial alkaline phosphatase. The ligation mixture is used to transform E. coli JM83 cells, 3000 transforming clones are screened by hybridization on colonies using a labeled EMAa-4 cDNA insert (Salvado and Labarère, 1991) as probe. Sequencing and sequence analysis

The nucleotide sequences of the 2.4 kb EcoRI genomic fragment and the corresponding dΕMAa-4 cDNA insert on the two strands are determined using a series of overlapping deleted subclones produced by the method d 'Ffenikoff (1984) with the Erase-A kit (Promega). Ambiguities in the sequence are removed using specific oligonucleotides as the sequencing primers. Sequencing is carried out with the Sequenase version 2.0 kit (United States Biochemical) using the deoxyribonucleotide method (Sanger et al., 1977).

The predicted amino acid sequence is determined using the universal genetic code and compared with proteins from GenBank and EMBL databases using the search algorithm (Altschul et al., 1990). To compare DNA sequences, we use the Genetics program

Computer Group (BESTFIT - GCG - Wisconsin group). A hydrophobicity curve is produced with the DNA Strider program (1.2) based on the Kyte-Doolittle algorithm. Extension of the primer The transcription starting points are determined by extension of the primer.

We follow the method of Kajiwara et al. (1992) with minor modifications: (i) 2.2 μg are fixed on an 18-mer oligonucleotide (5'-GAG CAT AGG CCC TTT CAT) corresponding to the antisense strand going from nucleotide 17 to 34, the poly (A) RNAs (4.4 μg) for 5 minutes at 70 ° C, (ii) a reverse transcriptase (67 units of AMV reverse transcriptase, Promega) and 25 μCi of α-32p-dCTP (3000 Ci / mmol, Amersham Corp.) are added and incubation at 42 ° C for 60 minutes.

After extraction with chloroform phenol and precipitation, the extension products are air dried, they are separated in a gel on electrophoresis of 7M urea / 6% polyacrylamide and they are visualized by autoradiography. The size of the extension products is determined by comparison with the sequencing scales (GATC) which are derived from the control DNA (bacteriophage M13mpl 8) included in the Sequenase 2.0 kit using a universal 17-mer primer. Cloning of a fragment of genomic DNA ά ^} Agrocybe aegerita hybridizing with the cDNA insert of EMAa-4 We digest the total DNA at! A. aegerita with the endonucleases EcoRl, Hindïïl and

Pst I and are hybridized with the plasmid probe pEMAa-4 according to Southern (1975) under highly stringent hybridization conditions. The plasmid pEMAa-4 carries a cDNA corresponding to an mRNA preferentially transcribed during the differentiation of the basidiocarp primordia (Salvado and Labarère, 1991). In all cases, a single hybridization band is detected having an estimated size of 2.4, 2.7 and 3.1 kb respectively (results not shown). To clone the genomic fragment carrying the corresponding gene, the total DNA of A. is digested. aegerita with the endonuclease Pst I and it is separated by electrophoresis. The DNA fragments ranging from 2.9 to 3.1 kb are purified from the agarose gel and ligated to the vector pUC18 linearized with Pst I. The recombinant plasmids are used to transform the dΕ cells. .coli. When 3000 recombinant clones were subjected to hybridization in colonies with the EMAa-4 insert labeled as probe, 12 positive clones were identified carrying a Pst I insert at 3.1 kb. One of them is chosen randomly. Analysis of the restriction map of the Pst I insert of 3.1 kb from A. aegerita by 10 restriction endonucleases (Ava I, BamH I, EcoR I, Hind III, Kpn I, Sal I, Sma I, Sph I, Xba I and Sma I), these restriction sites being located in the multisite linker (linker) pUC18, does not make it possible to find a combination of a sensitive site and a site resistant to digestion by exonuclease III, that is to say that is, two types of sites located on the same side of the insert. Therefore, it was decided to subclone the 2.4 kb internal EcoR I fragment. In fact, it was previously established by Southern blot analysis of the total DNA of A. agerita with the insert EMAa-4 as probe, that this fragment carries the sequence homologous to the cDNA. In addition, this allows the use of the Xba I and Sph I sites for cloning. The 2.4 kb fragment is released by digestion with EcoR I of the 3.1 kb Pst I fragment, it is separated by agarose gel electrophoresis then it is purified and it is introduced into the vector pUC18 following the method described for the 3.1 kb Pst I fragment. The EcoR I fragment is subcloned in the two orientations as shown from the analysis of the BamH I digestion schemes of the recombinant plasmids obtained (two BamH I fragments of 2.9 and 2.2 kb for orientation and two BamH I fragments of 0.2 and 4.9 kb for the other). The recombinant plasmids representing each orientation of the EcoR I fragment are used as bases for DNA sequencing after construction by treatment with exonuclease III of the deleted overlapping subclones. Nucleotide sequence analysis

The complete nucleotide sequence of the 2.4 kb EcoR I fragment and of the hybridizing cDNA (insert ΕMAa-4a) is determined on the two strands. Analysis of the 2.4 kb EcoR I sequence (1.2 DNA Strider program) reveals an open reading frame of 492 nt, homologous to the cDNA sequence, starting with the methionine codon ATG (+1) and ending with the TAA translation stop codon (+492) (Figure 1A). The nucleotide sequences of the entire gene, of the open reading frame (ORF) called the Aa-Pril gene (492 bp) and of a part of its neighboring regions in 5 '(400 bp) and 3' (186 bp) are reported in Figure 1B.

The ATACCATGGG sequence around the start of tracuction site (ATG) of 4 nucleotides out of 7 (A in position -3, C in positions -1 and -2 and G in position +1) corresponds to the consensus sequence CC (A / G ) CCATGGC de Kozac (1984) in which the nucleotide -3 is always a purine, and generally an A. On supposes that this sequence is involved in the recognition of the correct AUG codon by the ribosome (Ballance, 1991).

The alignment of the genomic sequences and of the corresponding cDNA (insert EMAa-4) shows that the genomic sequence contains a 54 bp intron located at nucleotides + 125 to +179. The 3 'intronic splicing site (CAG) carries the typical consensus sequences for splicing (YAG; Y = C or T) (Gurr et al., 1987). However, at the 5 'limit, the intron has the GC nucleotide pair instead of the classic GT. The putative internal sequence required for lasso formation (TACTTAC) resembles the consensus NNCTRAY sequence (N = A, C, G or T; R = A or G; Y = C or T) described for filamentous fungi ( Unkless, 1992), and it is only the T at position 5 of the element which does not correspond to the consensus sequence.

In the neighboring region 5 ′ of the genomic sequence, a TATA box (TATAAAT) at nucleotide -83 and a CAAT box (CCAAT) at nucleotide -156 is identified. In the neighboring region in 3 ′, two sequences similar to the consensus polyadenylation sequence AATAAA are located in the nucleotide positions +593 [T (A) ₅ TA] and +608 [T (A) ₃ T (A) ₄ T ( A) ₆ ].

Extension of the primer reveals three transcription start points (pdt) at nucleotides -49, -85 and -91. One of them (pdt at nt -49) is surrounded by a CCA ⁺ ^ TTCC sequence corresponding to the consensus induction sequence YYA ⁺¹ NWYY (W = A or T; Y = C or T) (Javahery et al., 1984). Amino acid sequence deduced from the protein Aa-Pri

The translation of the open reading frame stripped of its intron, using the universal genetic code, shows that it codes for an acidic protein (pi = 5.8 calculated with the Isoelectric-GCG-WISCONSLN Package program, v 9.0) of 145 amino acids with a calculated molecular weight of 16093 Da (calculated from DNA Srider 1.2 software). The protein deduced is rich in aspartic acid (7.5%), asparagine (8.9%), glycine (11-1%), lysine (7.5%) and serine (9.6%). The hydrophobicity curve of Kyte and Doolittle reveals that the Aa-Pril protein is mainly hydrophilic. A hydrophobic region of 20 aa

(from aal2 to aa32) is predicted in the NH2-terminal sequence, determining a putative α helix (TopPredlI program), suggesting the possibility that the Aa-Pril protein could be a membrane-associated protein.

One can highlight potentially significant motifs for glycosylation or phosphorylation in the amino acid sequence (Figure 2). Two putative N-glycosylation sites (NXS / TX, X'P) namely NASS and NKTI are located at positions 73 and 91, respectively. We highlight six putative phosphorylation sites: (i) one for a protein kinase p dependent on cyclic AMP (R / KR KXS / T) starting with lysine ^^, (ii) two for a casein kinase protein II (S / TXXD / E) starting at

and (iii) three for a protein kinase C (S / TXR K) starting at the

threonine ^ and serine ^

Comparison of the amino acid sequence Aa-Pril with the GenBank and EMBL databases shows that it exhibits homologies with Asphemolysin originating from the amphomycete Aspergillus fumigatus (GenBank access number D 16501).

Expression of the Aa-Pril gene during the life cycle

In order to study the expression of the Aa-Pril gene, the total cellular RNAs were isolated from four stages of development: vegetative mycelia, aggregates, primordia / immature fruit bodies and mature fruit bodies. For each stage, the total RNAs were subjected to Northern blot analysis using the labeled EMAa-4 cDNA insert as a probe. The same amount of total RNA (30 μg) is loaded into each lane, which is verified by staining the gel with ethidium bromide before transfer. Northern analysis with the EMAa-4 cDNA probe revealed a strong signal corresponding to a 600 b RNA band in the primordium extract. Vegetative mycelia, aggregates and mature fruiting bodies do not contain detectable amounts of the Aa-Pril transcript (data not shown).

As a control, after hybridization of the Northern blot with the probe, the stoichiometric transfer of the four RNA preparations is examined by washing and new hybridization of the band with the 18S rDNA of Pleurotus comucopiae (see above) differential expression during the life cycle. 18S rDNA signals were detected in all RNA bands with similar intensities (data not mentioned). Since the RNA concentrations in each sample are similar, it is evident that the Aa-Pril gene is only actively transcribed at the stage of primordia / immature fruiting bodies.

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(1) GENERAL INFORMATION:

(i) DEPOSITOR:

(A) NAME: NATIONAL INSTITUTE OF AGRONOMIC RESEARCH

(INRA)

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(E) COUNTRY: FRANCE

(F) POSTAL CODE: 75007

(i) DEPOSITOR:

(A) NAME: VICTOR SEGALEN BORDEAUX 2 UNIVERSITY

(B) STREET: 146 RUE LEO SAIGNAT

(C) CITY: BORDEAUX

(E) COUNTRY: FRANCE

(F) POSTAL CODE: 33000

(ii) TITLE OF THE INVENTION: GENE INVOLVED IN THE FRUCTIFICATION OF BASIDIOMYCET MUSHROOMS AND USES it) NUMBER OF SEQUENCES: 1

(iv) COMPUTER-DETACHABLE FORM:

(A) TYPE OF SUPPORT: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 1079 base pairs (3) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: cDNA

(ix) CHARACTERISTIC: (A) NAME / KEY: CDS (3) LOCATION: 401..523

(ix) CHARACTERISTIC:

(A) NAME / KEY: intron

(B) LOCATION: 525..578

(ix) CHARACTERISTIC: (A) NAME / KEY: CDS! 3) LOCATION: 581..889

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:

TGTTTAAAAA CTCGCGAGTT ATGCGTGGGT CCGCGCGAAG GATGCTTTCT CGATTGAATA 60

GAGAATGGAG GAGAGGTTCG TTTGGCAAGT GATACTTTCA TCAACGCCGA GAATGGAAGT 120

CGAGCTGCTG TCTCCGATTT CCGTAGCCTA CAAGTTGCTC TTTGAATGGA TCATCCATGG 180

GGCTCGTGGC GATGTGGCGT GGTCGGGTGG TACTGACCAA CCTTCTGGCA TATTCCTCAG 240 AGCTCCAATT AGACGTAAAG AGAACATCGT CCGGCTCGAC GAAGAAAAAA AGGGAAATTG 300

AGGGCCTGTA GAAATTGTAT AAATACTATA CCTGCTAAGA GCAGATTCCT CATTCCAAAG 360

CACCCACCGA GTACATCCAA CAGCCACCCG TTTGAATACC ATG GAT AGC AAC AAG 415

Met Asp Ser Asn Lys 1 5

GAT GAA AGG GCC TAT GCT CAA TGG GTG ATC ATC ATC CTC CAC AAC GTC 463 Asp Glu Arg Ala Tyr Ala Gin Trp Val Ile Ile Ile Leu His Asn Val 10 15 20

GGC TCG TCC CCT TTC AAG ATC GCG AAC CTC GGC CTC TCC TGG GGA AAG 511 Gly Ser Ser Pro Phe Lys Ile Ala Asn Leu Gly Leu Ser Trp Gly Lys 25 30 35

CTC TAC GCT GAC G GCAAGCCGC TTCATCAGTC CAAACGCATA TGGTACTTAC 563

Leu Tyr Ala Asp Gly 40

CATTGTCTTT AACAG GT AAC AAG GAC AAA GAA GTC TAT CCC AGT GAC TAC 613

Asn Lys Asp Lys Glu Val Tyr Pro Ser Asp Tyr 45 50

AAT GGA AAG ACG GTT GGG CCC GAC GAG AAG ATA CAA ATC AAT TCG TGC 661 Asn Gly Lys Thr Val Gly Pro Asp Glu Lys Ile Gin Ile Asn Ser Cys 55 60 65

GGC CGG GAG AAC GCC TCC TCT GGC ACC GAG GGG TCC TTC GAT ATC GTC 709 Gly Arg Glu Asn Ala Ser Ser Gly Thr Glu Gly Ser Phe Asp Ile Val 70 75 80 85

GAC CCC AAT GAT GGC AAT AAG ACC ATC CGT CAC TTC TAC TGG GAA TGC 757 Asp Pro Asn Asp Gly Asn Lys Thr Ile Arg His Phe Tyr Trp Glu Cys 90 95 100

CCC TGG GGG AGC AAA AGA AAC ACC TGG ACT CCT AGC GGC TCG AAT ACC 805 Pro Trp Gly Ser Lys Arg Asn Thr Trp Thr Pro Ser Gly Ser Asn Thr 105 110 115

AAG TGG ATG GTC GAG TGG AGC GGC CAG AAC TTG GAC AGC GGA GCA CTC 853 Lys Trp Met Val Glu Trp Ser Gly Gin Asn Leu Asp Ser Gly Ala Leu 120 125 130

GGC ACC ATC ACC GTC GAT GTC CTG CGC AAG GGC AAC TAAATACGTG 899

Gly Thr Ile Thr Val Asp Val Leu Arg Lys Gly Asn 135 140 145

AGT TACATA CGTACCTTAT CCAGTTCTCG CGAATGGTTA CTGAACCAGA AACCAAAGGT 959

GGCATCTGCT GCCAATTTCG TGAGGAAAAT CAGTAAAAAT AAGGATGGTA AATAAAATAA 1019

AAAATGATGA ACTTGTCTTG CTGTAGTTGT CGCCGAGGTC GTAGGGTTGA TTTGATGTAC 1079 REFERENCES

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Claims

1- Nucleotide sequence corresponding to: a) a sequence according to SEQ LD N ° 1 in whole or in part; b) a sequence hybridizing with a); or c) a sequence having at least 80% homology with a) or b).

2- Nucleotide sequence according to claim 1 corresponding to a gene or a gene fragment comprising: a) part of the sequence according to SEQ LD N ° 1; b) a sequence hybridizing with a); c) a sequence having at least 80% homology with a) or b); d) a sequence coding for a protein coded by a gene or a gene fragment according to a), b) or c), or for an equivalent protein.

3- Use of a sequence according to claim 1 or 2 to induce fruiting in a basidiomycete fungus.

4- Use according to claim 3 characterized in that the basiodiomycete fungus is Agrocybe aegerita.

5- Use according to claim 3 or 4 characterized in that it comprises the insertion by transformation of a sequence according to claim 1 or 2 in a cell, a hypha or a sexual or asexual spore of basidiomycete fungus.

6- Use according to claim 5 characterized in that the transformation is carried out by a vector.

7- Cell, hypha, sexual or asexual spore of basidiomycete fungus transformed by a sequence according to claim 1 or 2. 8- Use of a sequence according to claim 2 for producing a protein or a protein fragment characterized in that it comprises the insertion of a sequence according to claim 2 in an expression vector capable of transforming prokaryotic cells or eukaryotes and the transformation of said cells with said expression vector.

9- Vector for expression of a sequence according to claim 2.

10- Prokaryotic or eukaryotic cell transformed by a vector according to claim 9.

11 - Protein or protein fragment capable of being obtained by culturing a cell according to claim 10.