JP2001517450A - Method for creating a plant with petals-specific promoter and petal-free flowers - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method for obtaining plants having petal-specific promoters and petal-free flowers.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention particularly relates to a method for producing a plant having a petal-specific promoter and a petal-free flower.

[0002] The advantage of creating plants that lack petals is based on the finding that senescent petals can fall on leaves to provide a favorable bed of infection for certain pathogenic spores. For rape, for example, the mode of infection of Sclerotio rum sclerotioum mainly follows this route. This fungus is indeed responsible for significant damage in rape cultivation (Lamarque, 1983), and no genetic resistance is known for this fungus in either rape or closely related species. Therefore, at present, only preventive chemical treatment can be used.

[0003] The control of sclerotinia sclerothiolam by plants that have no petals in the flower
The use of fungicides is reduced, thus making it possible to limit the subsequent contamination of the soil.

[0004] It therefore produces plants with petal-free flowers, thus controlling the aforementioned fungal control on the basis of "physical" resistance rather than on the basis of the use of resistance genes in the traditional sense. Including testing strategies.

[0005] It is therefore an object of the present invention to create plants whose flowers lack petals. that is,
Specifically in petals is to use a promoter region that controls the expression of a sequence (orf) encoding a molecule that can modify the natural properties of the petals or inhibit their formation.

[0006] As described above, altering the structure, shape, color and / or structure of petals of a flower can be achieved by adding a gene involved in the biosynthesis of a pigment or a regulatory gene such as MYB protein downstream of the promoter region. Can be intended by placing (Noda et al. 1994)
. This type of experiment has already been performed (Elomma et al., 1996; Gutterson, 1995).
. However, the promoter used is rather of a constitutive type, such as 35S of CaMV, which would be advantageous in limiting the expression of the transgene into the target organ. Thus, the present invention contemplates the creation of an original ornamental plant.

The subject of the present invention is thus a nucleotide sequence for which the corresponding gene has been demonstrated to be specifically expressed in petals, this nucleotide sequence corresponding to SEQ ID NO: 5.

Accordingly, the subject of the present invention is: a) a sequence of SEQ ID NO: 5, or b) a sequence hybridizing with the sequence of a), or c) a sequence having at least 80% homology with a) or b) Is a nucleotide sequence corresponding to all or a part of

According to the present invention, the most effective part of this nucleotide sequence is the promoter region, which is defined as the sequence before (5 ′) the translation initiation codon (ATG).
Strictly, this promoter region extends from nucleotide 1 to nucleotide 3265 of SEQ ID NO: 5 (i.e., the last nucleotide immediately before the ATG codon), but considering the restriction sites, this region is from nucleotide 1 to nucleotide 3
233 (corresponding to the AvaI site), preferably at nucleotide 2
More preferably, it extends from position 911 to nucleotide 3233.

[0010] Accordingly, this promoter region naturally precedes an orf that is specifically expressed in petals, and when this orf is replaced with another orf (by genetic manipulation), the product becomes a cytotoxic molecule. , It can only destroy petals. This substitution may also be made by a portion of the gene capable of altering its initial properties upon specific expression in the petals.

Thus, the subject of the present invention also comprises a promoter region as described above, located upstream of a DNA sequence coding for a product capable of altering the structure, shape, color and / or petal tissue of a flower. A method for producing an ornamental plant comprising inserting a cell expression vector and one of these vectors into a plant. The present invention also provides such a D
This includes the case where the NA sequence encodes a cytotoxic product.

Advantageously, the cytotoxic substance of interest is a ribonuclease. In particular, when this RNase is specifically expressed in the petals, all of its RNA is destroyed, resulting in the petals being unable to survive. Preferably, the RN-ase is barnase, its corresponding orf are those isolated from Bacillus amyloliquefaciens (B acillus amyloliquefasciens) (Hartley RW , 1988).

[0013] Thus it comprises introducing a vector of the present invention, the thin strain capable of performing transformation of plant cells as Agrobacterium tumefaciens (A grobacterium tumefaciens). In particular, this may be done by the method of infiltrating Arabidopsis thaliana plants described by Bechtold et al., 1993. This technique consists of introducing bacteria into the cells of the flower stem by infiltration under vacuum. The plant is then planted under glass and the seeds are collected. From about one in 1,000 seeds, plants were obtained in which all cells had the transgene. Transformation of other plants, particularly rapeseed, is accomplished by a variety of current techniques (leaf discs) that combine the steps of co-culturing bacteria and plant tissue, followed by selection of transformed cells and regeneration into complete plants. Agrobacterium tumefaciens and / or Agrobacterium rhizogenes
( Agrobacterium rhizogenes ). Other transformation techniques do not use this bacterium, but the cloned gene can be introduced directly into cells or tissues (electroporation, particle gun, etc.) and selected to obtain transformed plants (Siemens and Schieder) Technology outlined by).

A subject of the present invention is also a plant cell transformed with a vector according to the invention, and a plant comprising such a cell. The subject of the present invention is also a plant in which the flowers have no petals.

As indicated above, the present invention is thus able to create plants without petals in the flower, and the method of the present invention comprises a DNA encoding a cytotoxic product, as set forth above. Inserting the vector comprising the sequence into a plant.

The present invention may also contemplate the production of a hybrid plant by crossing two lines for which a combination of their agronomic qualities is required. However, for optimal pollination, the target hybrid parent must have petals. Therefore, such crossing is only possible with the activation of the two-component toxic gene. The principle of such a system consists of having two lines each having a component without cytotoxic activity.
Thus, specific toxic activity is restored to these two hybrids by a combination of the two components.

A possible example of such a system is to insert at least one stop codon at the beginning of the corresponding coding sequence and then to recognize the stop codon in the system and retain it instead of terminating translation. T called “suppressor” that supplies amino acids
The addition of RNA consists of inactivating the expression product whose regulation is desired. Thus, this protein is capable of full-length translation and its activity is restored. Such a system has already been attempted for sequences encoding the GUS gene with an amber stop codon inserted, and the suppressor tRNA used is a leucine carrier. In addition, Arabidopsis Sariana and Nicotiana Tabacam (Nic
otiana tabacum), the functionality of such a transactivation system using the tRNA ^Leu suppressor varied from plant to plant. This model was then applied in the case of barnase. Mutant genes encoding barnase and dependent on the expression of the tRNA ^Leu gene (ie, genes with a stop codon inserted) were obtained and tested for transient expression in tobacco protoplasts (Choisne Nathalie, 199).
7).

Accordingly, the present invention also provides a method for producing a hybrid plant having no petals in a flower, the method comprising: a) encoding a cytotoxic sequence obtained by modifying a plant of line A by inserting at least one stop codon; B) crossing the plant of line A thus obtained with a plant of line B expressing the tRNA suppressor gene, c) petals Selecting a hybrid plant having a flower without flowers.

In the present invention, plants of line A are transformed with a construct similar to pIB352 shown in FIG.

Advantageously, the plant of the invention belongs to the Brassicaceae, preferably the plant is rape.

The present invention is not limited only to the above description, but is better understood by the following examples. However, this is only given as an example.

Example 1 Demonstration of a Petal-Specific Promoter The first step is to obtain a complementary DNA (cDNA) clone that is specifically expressed in petals. For this purpose, messenger RNA (m
CDNA was synthesized from RNA). At the same time, cDNA was synthesized from mRNA from leaves, petals from which petals were removed, and stamens.

[0023] cDNAs from these organs or tissues were subtracted from cDNAs derived from mRNA expressed in rape petals. The molecule obtained from this subtraction is called Atanasso
A technique similar to that proposed by v et al., 1996 was used for differential hybridization experiments on petal cDNA libraries.

As a result of this experiment, several rapeseed DNA clones were isolated. Their expression profiles were studied by Northern molecular hybridization. In the absence of a clone strictly specific to petals (at the limit of detection of this technique), the best candidate for the rest of the experiment was reserved, which is clone 9.2. This clone was strongly expressed in young petals (about 3 mm flower buds) but very weakly in stamen (FIG. 1).

[0025] Sequence homology searches in the databank indicate proteins deduced from the open reading frame (orf) of clone 9.2 and Arabidopsis thaliana encoding a putative wall protein whose expression is regulated by gibberellin. Strong similarity between the coding sequence of the gene (X74360) was shown (Phi
llips and Huttly, 1994) (Figure 2). The degree of homology indicated by the corresponding individual cDNA sequences is higher than 80% in the first 500 bases and the remaining 22
All disappeared over 0 bases (FIG. 3).

A rape genomic library was screened using the rape cDNA clone 9.2 as a probe. Seven genomic clones were isolated. Based on the restriction map and sequence, these seven clones were divided into two groups, suggesting the presence of rape of at least two gene families, referred to herein below as 4.1.1 and 8.1.1. (FIG. 4). cDNA 9.2 is derived from the gene corresponding to genomic clone 4.1.1.

Preliminary experiments by PCR amplification were performed on clone 9.4.1 belonging to group 4.1.1. Especially due to the structure of genomic clones, amplification of large DNA fragments and P
Using the technique of progressive sequencing by CR, it became possible to amplify the 3233 bp upstream region.

This 3233 bp region extends from nucleotide 1 to nucleotide 3233 of the sequence shown in FIG. 5 and terminates at the level of the AvaI site, at which level cleavage and cloning occur to create a “blunt end”. Obtained.

The upstream region, possibly containing regulatory sequences, was then cloned into both genomic clones (4.1.
1 and 8.1.1) into a cloning vector.

To date, a sequence of more than 4 kb in the majority corresponding to the orf and upstream regions (FIG. 5) has been obtained for clone 4.1.1.

Example 2: Demonstration of promoter region specificity In transgenic plants derived from Arabidopsis thaliana and rapeseed, these chimeric genes (that is, the coding sequence of a known gene preceded by the promoter region of the present invention) are used. In order to study), various constructs comprising the GUS reporter gene placed under the control of expression of these specific sequences were generated.

These constructs belong to two categories as functions of orf placed under the control of regulatory sequences: GUS to study expression profiles and to demonstrate the specificity conferred by the promoter Reporter gene Wild-type or inactivated barnase gene to prevent petal formation by expression of this toxic gene in this organ (Figures 6 and 7 detail the composition of each construct).

The expression profile of the GUS reporter gene in the Arabidopsis transformants obtained in the case of pIB100 shows a constant variation among the plants as a whole (see Table 1 below, which shows the blue transformation). List of parts of plants). However, in nearly half of the plants with blue color (13/30), the reporter gene is expressed only in petals (the limit of detection of this technique). Some plants show stamenally weak expression, which is not surprising given the results of Northern hybridization, and sometimes expression in other organs of the flower, indicating that the transgene It is suggested that this is due to the effect of the positional result due to the small. However, the presence of a significant proportion of plants with the expected profile suggests that a 322 bp flanking fragment could confer specific expression on the petals. The stability of this expression was examined in the progeny of self-pollination of these plants. "Petal" specificity was indeed observed for most (data not shown).

[0034] Transgenic plants derived from Arabidopsis thaliana were also observed with constructs pIB102 and pIB105 using longer promoter sequences (Table 2).
Lists some plants transformed with pIB102 and has a blue color, and Table 3 lists some plants transformed with pIB105 and has a blue color). In almost all cases, the reporter gene is effectively expressed in the petals and also in other organs of the flower, so petal specificity is now not observed at the previously observed rates.

Similarly, the transformed rapeseed plant has 3233 of gene 4.1.1 as a regulatory sequence.
obtained using a construct comprising the bp upstream fragment, which was cloned after PCR amplification. In nine rapeseed plants that could already be observed, the reporter gene was expressed in petals and in other organs of flowers, as was seen in Arabidopsis using these large promoter regions. (Data not shown).

The results indicate that these fragments are too long, while the preceding (322 bp)
Is thought to be slightly shorter, suggesting that it amplifies potential positional effects. However, this has the most promising results. Similar to pIB100, but instead of the reporter gene coding sequence,
Promoters pIB351 and pBI352 (FIG. 7), each comprising the coding sequence of the wild-type barnase gene and the same sequence inactivated by insertion of a stop codon (hereinafter referred to as mutant barnase), were added to Arabidopsis thaliana. Introduced (no results yet).

[0037]

[Table 1]

[0038]

[Table 2]

[0039]

[Table 3]

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a diagram showing analysis of rapeseed poly A + RNA (2 μg) and total RNA (10 μg) by Northern hybridization. This membrane hybridized with all cDNAs 9.2 labeled with ³² P. -80 with screen
Detection is performed after 24 hours exposure to ° C. The identified mRNA is approximately 800 bp in size. Plant 1: 1 week old plant; Plant 2: 2 week old plant.

FIG. 2. cDNAs X74360 (SEQ ID NO: 1) and 9.2 (SEQ ID NO: 2), respectively.
FIG. 2 is a diagram showing a comparison of protein sequences derived from Arabidopsis thaliana (upper) and rapeseed (lower) deduced from the above. The protein from Arabidopsis thaliana has a length of 140 amino acids, while the protein from rapeseed has a length of 147 amino acids, with 74.6% homology between the two. The asterisk indicates an amino acid common to both sequences, and the dots in the cDNA from Arabidopsis thaliana only indicate that the sequence common to both plants can be placed in the opposite orientation to each other. The array is read continuously, ie irrespective of this dot.

FIG. 3 shows an alignment of the nucleotide sequences of rapeseed cDNA 9.2 (bottom) and Arabidopsis thaliana X74360 (top). Both sequences have a total homology of 83%.

FIG. 4 is a diagram showing a partial restriction map of a genomic clone (A: Aval, B: B
amH1, EI: EcoRI, EV: EcoRV, H: HindIII, Hc:
HincII, P: PstI, S: SacI, S1: Sal1, Xb: Xba1
, Xh: Xho1).

FIG. 5 shows the 5 ′ → 3 ′ sequence of genomic clone 4.1.1 (SEQ ID NO: 5). The palindromic sequence is shown as a double line and the coding sequence is shown as a single line. The following restriction sites are marked: BamHI (No. 1): GGATCC; S
alI (No. 2911): GTCGAC and AvaI (No. 3229): CCCG
AG.

FIG. 6 is a diagram showing a continuation of the array shown in FIG. 5;

FIG. 7 is a diagram showing a continuation of the array shown in FIG. 5;

FIG. 8 is a diagram showing a continuation of the array shown in FIG. 5;

FIG. 9 shows the construction performed using the promoters of the genomic clones 4.1.1 and 8.1.1. Distal promoter region of genomic clone 4.1.1 Palindromic sequence Proximal promoter region of genomic clone 4.1.1 322 bp promoter region of genomic clone 4.1.1 322 bp promoter of genomic clone 8.1.1 Region Terminator of nopaline synthase gene Coding sequence of gus reporter gene Coding sequence of gene 4.1.1 3 ′ untranslated region of gene 4.1.1

FIG. 10 is a view showing a continuation of 9;

FIG. 11 shows a construct prepared using the 322 bp promoter of genomic clone 4.1.1. 322 bp promoter region of genomic clone 4.1.1 coding sequence of gus reporter gene coding sequence of wild type barnase gene coding sequence of mutant barnase gene terminator of nopaline synthase gene terminator of CaMV 19S

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] March 23, 2000 (2000.3.23)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP , KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Philip, Gersh Bambu, France, Ryu, Marceau, 7F term (reference) 2B030 AA02 AB03 AD11 CA06 CA17 CA19 CB02 CD18 4B024 AA08 CA04 DA01 FA02 GA11 HA20 4B065 AA88X AA88Y AB01 CA53

Claims (12)

[Claims] 1. All or part of a) the sequence of SEQ ID NO: 5, or b) a sequence hybridizing with the sequence of a), or c) a sequence having at least 80% homology with a) or b) Nucleotide sequence corresponding to 2. a) a sequence spanning nucleotides 1 to 3233, preferably nucleotides 2911 to 3233 of SEQ ID NO: 5, or b) a sequence hybridizing with the sequence of a), or c) a) or 2. The nucleotide sequence according to claim 1, corresponding to all or part of a sequence having at least 80% homology with b). 3. A cell expression vector comprising a sequence according to claim 2, which is located upstream of a DNA sequence encoding a product capable of improving the structure, shape, color and / or petal organization of the flower. . 4. A cell expression vector comprising the sequence of claim 2, which is located upstream of a DNA sequence encoding a cytotoxic product. 5. The method according to claim 4, wherein the cytotoxic product is a ribonuclease, preferably barnase.
The described vector. 6. A plant cell transformed with the vector according to any one of claims 3 to 5. 7. A plant comprising the cell according to claim 6. 8. A plant having no petals in its flowers. 9. A method for producing an ornamental plant, comprising inserting the vector according to claim 3 into a plant. 10. A method for producing a plant having no petals in a flower, comprising inserting the vector according to claim 4 or 5 into the plant. 11. A method for producing a hybrid plant having no petals in a flower, the method comprising: a) modifying a plant of the A line by inserting at least one stop codon into a DNA coding sequence. B) crossing the plant of line A obtained in a) with the plant of line B expressing the tRNA suppressor gene, and c) selecting a hybrid plant having a flower without petals. A method comprising the steps of: 12. The rapeseed of claim 7 or 8, which belongs to the Brassicaceae family.
Or a plant obtained by using the method according to claim 10 or 11.