BRPI0705917B1 - COMPOSITIONS AND METHODS TO DIRECT GENE EXPRESSION USING THE COFFEE ISOFLAVONES FAMILY FAMILY GENE PROMOTER - Google Patents

Abstract

compositions and methods for directing gene expression using the coffee plant isoflavone family gene promoter. The present invention comprises the description of a novel regulatory sequence provided for directing expression of a nucleotide sequence, such as structural genes, in plant leaves, including monocotyledons and dicotyledons. More specifically the invention relates to regulatory sequences of polynucleotides isolated from coffee plants that are capable of initiating and triggering transcription of polynucleotides, and the use of these regulatory sequences in directing endogenous and / or heterologous polynucleotide transcription to leaf tissues. and polypeptide production. The invention further describes DNA constructs containing the coffee plant isoflavone family gene promoter that is operably linked to a heterologous and / or endogenous gene. furthermore, the invention relates to the use of these constructs in the form of expression vectors, recombinant vectors and transgenic plants, plant cells or protoplasts. The invention further discloses a method utilizing such constructs containing the coffee plant isoflavone family gene promoter for producing transgenic plants, plant cells or protoplasts.

Description

COMPOSITIONS AND METHODS TO DIRECT EXPRESSION 1) AND GENES

ISANDO THE PROMOTER OF THE GENE OF THE FAMILY OF THE ISOFI AVOXES OF

PIAM AS DE (AEF (ΛΜΡ (/ 1) _AJ IN L \ ÇÀ O

The present invention relates to a \ promoter for specific gene expression- cm

sheet ¹ -; plantar More specifically the invention relates to regulatory sequences of polynuclotides isolated from catechin plants that are capable of initiating and triggering transcription of polynuclotides. c the use of these regulatory sequences in the transcription modification of endogenous and or heterologous polynuclotides and production of

polypeptides in leaf tissue. The invention further describes DNA constructs that contain the promoter of a gene that encodes a protein from the coffee plant isoflavone family that is operably linked to a heterologous and / or endogenous gene. In addition, the invention relates to the use of these constructs in the form of expression vectors, recombinant vectors and in plants, plant cells or transgenic protoplasts. The invention further describes a method using such constructs containing the promoter of a gene that encodes a protein from the coffee plant isoflavone family for plant production.

plant cells or transgenic protoplasts.

BACKGROUND OF THE INVENTION

Λ coffee production currently plays an important role in the national economy, generating foreign exchange in the order of US $ 2 billion per year with 26 million bags exported per year (Companhia Nacional do Abastecimento - Conab). With a market still expanding, the caie agribusiness generates, for everyone, resources in the order of 91 billion dollars by marketing an average of 115 million bags. Brazil occupies a dominant position in the international market, standing out as the largest producer (contribution of

30 ₍ > of world production in the last harvests) and world coffee exporter, with an average share of 25 ° o. The agribusiness of coffee represents more than 2.5% of the total value of Brazilian exports, in addition to generating eight million direct and indirect jobs, standing out as the sector that most employs in Brazil. Despite the excellent performance, the expansion of coffee production in Brazil is limited, among other factors, by pests and diseases that attack the crop. Among the main diseases of coffee. stands out. aletanil the leaves, the fungus // umi / ciu vox / í / fr / .v that causes coffee rust.

\ growing worldwide demand for better quality food. principally lix] <> of agricultural pesticides, has promoted a significant advance in several arks of plant production. Among these, plant breeding stands out.

The genetic improvement of plants aiming at agronomic improvements has been carried out by man for thousands of years using classical procedures based on

in controlled crossings between species, however. with the advent of molecular biology in recent years, coupled with the development of advanced biotechnological techniques, plant breeding via genetic engineering has become an important tool

in obtaining superior gnotypically superior plants in a short time, becoming of great interest to the agricultural sector.

Advanced methods of plant genetic improvement have stood out mainly due to the possibility of obtaining uniform and targeted results 15 for desirable characteristics. Among the countless biotechnological methods used worldwide for breeding, it stands out as one of the most important, the production of genetically modified plants. Transgenics have allowed the improvement of genotypes previously selected by conventional methods through the introduction of one or a few genes that. in many cases, they are found in distant species that could not be transferred via recombination. what necessarily happens between scxualmentc compatible individuals. On the other hand, if they were transferred in this way,

years to obtain the desired genotype. When it comes to breeding perennial species, more precisely the species CoJJea ara bica. a minimum of 25 years can be expected between the first hybridizations until new cultivars are obtained.

Improvement of perennial species by obtaining genetically modified plants has been suggested as an alternative to classical breeding, in this context, the introduction via transgenics of desirable characteristics such as resistance to biotic and abiotic factors tends to be facilitated, since a a large number of genes induced by such factors in different species have been identified and characterized.

Many of these genes have been identified in projects where entire genonies have been screened. as in the case of Arabidopsis ihaliaiaia and rice, or in projects in which thousands of cDNAs originating from libraries built from tissue, with different stress conditions, have been created. also known as ESI s projects (express scquunce tags). As an example of the latter case, in the plant area, the project stands out (ieiioma (afc.

The aforementioned research projects are of paramount importance, however, it is known that the simple identification of a gene is not the only guarantee for its use in obtaining transgenics. The efficient production of a heterologous protein in a plant depends, for example, on obtaining high levels of transcription of the introduced gene for

that highly active promoters are needed.

The expression of a gene is regulated. in part, skipping cellular processes involved in transcription. During transcription, a single-stranded RN A, complementary to the DNA sequence to be transcribed, is formed by the action of RN A polymerases and transcription factors. The initiation of transcription in karyotic cells is regulated by complex interactions between cis-acting DNA motifs, located within the gene to be transcribed, and //yzh.s-acting protein factors. Among the rv.s-active regulatory regions are the DNA sequences. called promoters, to which the polymerase RNA is first linked, directly or directly. For the purposes of the present invention, the term “promoter refers to specific DNA sequences. usually located upstream (5 ¹ ) of the coding region of a structural gene, and which controls its expression due to its ability to provide a recognition site for RNA polymerase and / or other factors 20 necessary for the initiation of transcription, and so that it starts at the correct location of the gene.

Typically, a promoter has an “ΤΑΤΛ box” with an activation region located upstream. This “TATA Box is responsible for the location of the transcription start site, and is located approximately 25 base pairs from the beginning of the gene coding sequence (in the? 'Direction).

There are two basic types of promoters, the inducible and the non-inductive, also called constitutive. The constitutive promoters are capable of directing the expression of DNA sequences throughout the development of a plant, in addition to not having a specificity regarding the expression site of this sequence. this being then expressed in a wide variety of plant cells and tissues. Despite this, the term “constitutive does not imply that the sequence is expressed at the same levels in all plant cells. The inducible promoter. in turn, it is a capable promoter that activates (directly or indirectly) the transcription of one or more DNA sequences. or genes in response to a particular

35 nidumi. In the absence of inducing dowry. a>sequence> dc DNA. or gcnu>. will not be transcribed The inducer can be a chemical component (described, for example, in the patent document \\ () <> 5 1 ^l > 443), a stress of physiological origin (as in the case of injuries, which is described, for example, in patent document l'S66775l) 5). or an endogenous compound generated in

response to changes in plant development.

Promoters are therefore a key tool in biotechnological processes to ensure that the expression of a gene of interest is effective and

spatially and temporally regulated. Among the promoters usually employed in the production of genetically modified plants, the promoter 35S of the

Mosaic of ('hears flower (CaMV 3? S). The promoters of the nopaline and octopine genes

Agrobíuteiiuni tumejacicns synthetase and the promoter of the gene encoding ubiquitin in corn. Despite the great advances obtained with the employment of these promoters. the expression patterns of transgenes subjected to their regulation are varied and low in some cases, with no guarantee of expression in the appropriate tissue organ. The use of 15 ubiquitous promoters such as those mentioned, determines the expression of the gene product in all organs of the plant, which is not always desirable. Such a feature, which is unnecessary and undesirable in genetically modified plants intended for human consumption, tends to decrease the acceptance of the final product, or its derivatives, by consumers resulting in resistance to genetically modified products. In this case, attorneys

specific measures that guarantee spatial regulation of the transgene would be of great importance and several studies are being undertaken with this purpose.

There are numerous tidicotypic promoters described for plants, such as the specific expression in seed (\ V () 8903887). tuber (as mentioned in patent application S2 <) () 3 () 175783. Kcil et al .. 1989 EMBO J. 8: 1323-1330), leaves (as mentioned in patent application ES2OO3O1 75783. Hudspcth et al. 1989 Plant Mol Biol

12: 579-589), fruit (Edvvards and Coruzzi, 1990 Annu.Rcv .Gcnct. 24: 275-303 and

17S5753475). stem (as mentioned in patent application 17S200301 75783. Kellcr et al .. 1988 EMBO J. 7: 3625-3633), vascular tissues (as mentioned in patent application l iS20030175783. Pelcman et al., 1989 Gene 84: 359- 369 and Schmülling et al., 1989 Plant Cell 30 1: 665-670). root (l. 4820060143735 c as mentioned in the patent application

I S2OO3O175783. Kcller et al .. 1989 Genes Devei. 3: 1639-1646). stamens (WO8910396.

\\ οο2 | 3450). specific promoters <ki dciscence zone (W () 47 1 3805) and mcrisleme (Ito et al .. 1444 Pkmt Molecular Biologv. 24: 863-878).

Other examples of promoters that control the expression of cspcci genes remain in a given organ tissue and are important for resistance to biological and abiotic stresses may be \ isuali / ai in patent documents: \\ () 2007044442 (specific cat promoters for expression of genes in seeds): W02007005480 (promoter of the gene encoding coffee hydrochlorine): W () 20077 () 44751 (promoters encoding emokido genes

in the biosynthesis of chlorogenic acids in coffee plants): 1, 'Sóó 10840 (potato promoter specific for mcsõtllo): 1.8 () 800744 (promoter of alfalfa caused by biotic stress): US7153453 (specific coffee promoter) .

Although there are a large number of specific promoters for different tissues \ egetais. only the promoter of the patent document L’S7153953 is specific for coffee leaves. Although the promoter of the present invention is also specific for the leaf tissues of coffee, it differs from the patent LJS7153953 in that it is an isolated promoter of a different gene 15 (the promoter of the L S7153453 patent comes from the rbeS gene. Whereas the promoter of the present one. invention belongs to the gene that encodes a protein of the isoflavone family) and because it has different specificity (while the promoter of the present invention controls expression in response to mechanical damage, the promoter of the rbeS gene is of expression

constitutive). Furthermore, the promoter of the present invention has no similarity with the promoters already described in the prior art (see Example 1). These facts demonstrate the need to identify and characterize new promoters for dicotyledonous plants. cspccialnicnte to those specific to leaf tissues of coffee plants, and the importance of the promoter of the present invention for the production of gen et i c a m c n t c m od i 11 ca d a s plants.

l 'cause for concern report the use of genetically modified plants refers to the expression of toxic proteins such as those used to control certain insects and pests. Many studies report the efficient use of Bt protein (from Bete iilus ihiiringie / isis) in transgenic plants aiming at the control of pest insects (Shu. Q.Y .. Ye.

G.Y .. Cui. H.R .. Cheng. X .. Xiang. λ ’., Wu. D., et al. 2000. Transgenie rice plants with a 30 synthctic herb lAb gene from Bacillus thuringiensis were highly resistant to eight lepidopteran rice pest spccies. Molecular Breed. 6: 433-434: Lcroy, Henry .. Henry. A.M .. Roycr, M .. Altosaar.

I .. Fruits. R .. D uris. D .. Philippe. R. 2000. Genctically modified coffec plants expressing the

35 ιί / α> ιhίπ'ΐfigir / ?. s / s crv 1 Ac gene ior rcsistcnce lo leaf mincr. Molecular Plant RcporK. ! 0352-tSO; Ncrtcr. F.W .. Fhomashow. E.S .. Matthcvv. M. and Gordon. \1. 2002. 100 Ycaix of

liiiii / hfs ihin /// y / c / oâ: j scicntific a> ^ cssmcnl. American Acadumv ol Microbiologv. 1 ~ 52 \ Strect. \\ V Washington. OF. 10 pp). However, such methodology can cause 5 target insects to acquire resistance when kept under constant selection pressure (Mcng. F .. Shcn. J .. /.liou. W .. and Ccn. H .. 2004. Eong-tcrm sclcction for resisting the transgenic cotton expressing Bacillus thurmgiensis inxm in Hchcoverpa urmigiiti íllubiic!) (I.cpidoptcra: Noctuidac). Pest Managc Sci. 60: 167-1 ⁷ 2). what normally happens when similar promoters are used to express the proteins of interest.

These unwanted effects could be minimized through the use of teeido-specific organ promoters that. if possible. were responsive to certain stimuli, such as a certain biotic stress. In this case, the expression of the transgene would be limited to the duration of the stimulus and is mainly directed to the locus! where the stimulus was produced, emphasizing once again the importance of the promoter of the present invention.

The obtaining of genetically modified plants with genes of agronomic interest (resistance to pests, diseases, drought, frost, etc.) isolated from wild species of Cogea represents a significant advance, as it allows to dramatically accelerate the process of obtaining new cultivars. In this context, directing the expression of genes of interest to certain specific organs of the plant, using specific teeido-specific promoters from the species itself, constitutes an important biotechnological tool. Such innovation makes possible not only the construction of expression cassettes composed of subunits (promoter and gene) belonging to the Coffca genus, but also the achievement of a highly desirable tissue-specific expression pattern from the point of view of biosafety and public opinion. . The search for promoters with such characteristics has been a priority in biotechnological programs that aim at the production and commercial release of genetically modified organisms.

SUMMARY OF THE INVENTION

The present invention comprises the description of a new regulatory sequence provided to direct the expression of a nucleotide sequence in leaf tissues, such as structural genes, in plants, including monocots and dicots. In accordance with the pressing invention, a tissue-spacial promoter isolated from catechin plants (Co / A'u iribibicí} called CalsoR is described together with a method for using this polynuclotide regulatory region to target the expression of endogenous polynucleotides or holograms on leaf tissues of transgenic plants.

Imi a first aspect, the present invention provides a sequence of isolated polinuclcotídco of coffee plants (CoJJcci arabica} having 40 ° C. Prefcrcncialmentc 60 ° _(,. More prcferencialmcnte ^ 5 ° _tJ. More prefcrcncialmentc even where identity SI. Q ID NO1: S1 reverse sequence: Q ID Ν.Ό1; probes and primers corresponding to SLQ ID XOI.

In another aspect, the present invention provides chimeric genes comprising the polynucleotide of the present invention, either alone, or in combination with one or more known polynucleotides, together with cells and organisms comprising these chimeric genes.

In a related aspect, the present invention provides recombinant vectors 15 comprising, in the 5'-3 'direction. a polynucleotide promoter sequence of the present invention, a polynucleotide to be transcribed, and a transcription termination sequence. The polynuclotide to be transcribed may comprise an open reading region of a polynuclotide that encodes a polypeptide of interest, or a region of non-coding, or untranslated, of a polynucleotide of interest. The open reading matrix can be oriented both in the direction of ^tv sense ”and“ antisense ”. Preferably, the transcription termination sequence is functional in a host plant. More preferably, this termination sequence comes from the gene of interest, but others can be described in the prior art (Grifftths. AJF. Gelbart. WM. Miller, JH. Lcwontin. RC 2000. Rcgulation of gene transcríption In: Modern Genctic Analysis 3rd edition. WH Frceman 25 and Companv. Xevv York. ΧΎ.) As the terminator of the nopaline sintetasc dc .1 gene. iiunctascicns. The recombinant vectors may further include a marker for identifying transformed cells.

In another aspect, cells from transgenic plants comprising the rccombinant vector of the present invention are provided, together with organisms, as plants.

comprising these transgenic cells. and fruits, seeds and other products, derivatives, or progeny of these plants. Propagules of inventive transgenic plants are included in the present invention.

S 35

Another aspect of the present invention is a method for modifying genes expression in an organism, such as a plant. including stable incorporation into the genome of the organism containing the recombinant ctor of the present invention.

In another aspect of the present invention, a method is provided for producing a transformed organism, such as a plant, having the expression of a polypeptide directed to leaf tissues. This method comprises transforming a plant cell with the recombinant vector of the present invention to provide a transgenic cell with conditions that lead to regeneration and growth of the mature plant.

In yet another aspect of the present invention, a method is provided for identifying a gene responsible for a desired function or phenotype. The method comprises: I) transforming a plant cell containing a recombinant vector comprising a polynucleotide promoter sequence of the present invention operably linked to a polynuclotide to be tested, 2) culturing the plant cell under conditions that lead to regeneration and growth of the mature plant in order to provide a transgenic plant, and 3) compare the phenotype of the transgenic plant with the phenotype of untransformed or wild-type plants.

The above mentioned and additional aspects of the present invention and how to obtain them will become evident, and the invention will be better understood by reference in the "Detailed Description of the Invention".

BRIEF DESCRIPTION OF THE FIGURES

Figure I Schematic representation of the protocol adopted for amplification (by genome n (i / king) and cloning of the CalsoR promoter from the genomic DNA of coffee.

Figure 2 - Scheme of the location of regulatory elements found in the promoter

CalsoR by comparative analysis with Pl.ACE database (Higo et al. Plant cis-acting regulatory DNA clements (Pl.ACE) databasc: 1Ó99. Nuclcic Acids Res. 27: 297-300, 1999).

Figure 3 - Schematic representation of the vector pR H03-Gus G5 Kb) that presents the reporter gene tiid. 1 (GUS) inserted between the BamHI and Acol sites under the control of the constitutive promoter CaM \ '35S.

Figure 4 - Schematic representation of the chimeric gene developed for the functional analysis of the promoter region amplified via the walkiiig genome (the promoter is not found

35 cm scale). Ο promoter (alsoR has been fused to the reporter gene md.i that encodes the cu / iin (Uglucoronidasc (G (S) of Escbcrichiuíoli.

Figure 5 - Schematic representation of the pC AX1BI \ -l 3 * 1 (Cambia) plaque used in the construction of the chiineric gene \ is the subsequent transformation of tobacco using. Igrobacíuriuin litniu / acic / i ^.

Dl S (RK / ÂO Dl I Al IIADA OF THE INVENTION \ the context of »the description.

inhuman> iurm>> used skull> c by is> o> more detailed skull to be used:

A “chimeric gene is a coder of different oriens.

gene comprising a promoter and a region In the case of the present invention, the chimeric gene comprises the polynucleotide of the present invention linked to regions encoding endogenous and / or exogenous genes.

A “consensus sequence is an artificial sequence in which the base in each position represents the base most often found in the current sequence when comparing different alleles, genes or organisms.

A “promoter” is the portion of the DNA located upstream of the coding region of a gene c that contains the binding sites for transcription factors and polymeric RNA. which are necessary to enable the beginning of DNA transcription into RNA.

“Expression is the transcription or translation of a structural, endogenous or heterologous gene.

“GC box is a common regulatory element in the promoter that can increase his activity.

Ί ATA box is an element of the promoter, located approximately 30 bases above the transcription start site. TATA box is associated with transcription factors in general, including the RNA polymerase enzyme.

The term “gene means a physical and functional unit of heredity.

represented by a segment of DNA that encodes a functional protein or an RNA molecule.

An “endogenous gene is a gene specific to the cell or organism.

I 'm “heterologous gene” is a gene isolated from a donor organism and combined in the transformed recipient organism. It is a gene that is not naturally present in the cell or organism.

1 (1 35 l ιη “reporter gene is a coding unit whose product is easily tested, for example, CA I. GUS. GAE. I.UC and GI P. genes. The expression of a reporter gene can be used to test function nm promoter linked to cs.sc reporter gene.

The term “1'ST includes labels for express strings (from English Expresscd Scquencc 1'ags). that is, they are short strings at the ends of a dc string

transcribed nucleotide (encoding a protein or not). These strings. directly from the attached genes. can be used in the large-scale identification of genes and in determining the pattern of gene expression.

The term “contig as used in the invention refers to an x 1 vertical assembly of a collection of ESTs corresponding to the same gene.

The term "propagule" as used in the present invention means any part of a plant that can be used for reproduction or propagation, sexual or asexual, including seedlings.

“Sense means that the polynucleotide sequence is in the same orientation 5'-3 'in relation to the orientation of the promoter that regulates it.

"Anti sense" means that the polynucleotide sequence is in the opposite direction with respect to the 5'-3 'orientation of the promoter that regulates it.

As used here, the term “x-mero”. as a reference to a specific value of “x”. refers to a sequence comprising at least a specific number (“x) of 20 residues of the polynucleotide identified as SEQ ID NO1. According to preferred embodiments, the value of x is preferably at least 20. more preferably at least 40. most preferably at least 60 and more preferably at least 80. Thus, polynucleotides of the present invention comprise a 20 mer polynucleotide. 40 mere. 60 mere. <80 mere. 100 mere. 120 mere. 150 mere. 180 mere.

220 mere. 250 mere. 300 mere, 400 mere. 500 mere or 600 mere identified as

SEQ ID NO1 c xariants thereof.

The term "polynucleotide (s)" as used herein, means a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases and includes corresponding RNA and DNA molecules. including HnRNA and niRNA molecules. of filaments 30 both “sense and“ antisense. c comprises cDNA, genomic DNA, and recombinant DNA. as well as fully or partially synthesized polynucleotides. A HnRNA molecule contains introns and corresponds to a DNA molecule

35 considering one-to-one complementarity. One R \ Am molecule corresponds to a DNA and HnRNA molecule from which the introns have been excised. A polynucleotide may consist of a complete gene, or any portion thereof. The "antisense operable polynucleotides can comprise a fragment of the corresponding polynucleotide, and the definition of" polynucleotide "thus includes all of these

operable 'antisense ¹ ' fragments. The "antisense ¹¹ polynucleotides and techniques involving the use of" antisense ¹¹ polynucleotides are well known in the art (Sambrook. .1 .:

tld

E.F.Fritsh and T. Maniatis - Molecular cloning. A laboratory manual. 2 ed .. Cold Spring

Harbor Laboratory Prcss. 1989).

The polynucleotides described in the present invention are preferably about

80% pure, more preferably at least about 90% pure, and most preferably at least about 99% pure.

The term "oligonucleotide" refers to a relatively short segment of a polynucleotide sequence, generally comprising between 6 to 60 nucleotides. These 15 oligonucleotides can be used as probes or primers. where the probes can be used in hybridization tests and as primers in DNA amplification reactions through the polymerase chain reaction (PCR).

The term "probe" used in the present invention refers to an oligonucleotide. polynucleotide or a nucleic acid. it may be R.NA or DNA, naturally occurring, or 20 resulting from digestion with a restriction enzyme and purified, or produced synthetically, which is capable of annealing to or specifically hybridizing to a nucleic acid containing sequences complementary to it. A probe can also be single-stranded or double-stranded. The exact length of the probe will depend on many factors, including temperature, source and use of the method. For example, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. The probes here are selected to be complementary to differentiate strands from a sequence of a particular nucleic acid. This means that the probe can be sufficiently complementary to be able to "hybridize specifically" or ring with its respective target strands 30 under a number of predetermined conditions. Consequently, the probe sequence does not need to accurately reflect the target's complementary sequence. For example, a non-complementary nucleotide fragment may be attached to the 5 'or 3' end of the probe, with the remainder of the 5 'probe sequence being shared by the target strand. Alternalivente. basc> non-complementary or long sequences may be interspersed within the probe if it has sufficient complementarity with the target nucleic acid sequence to specifically anneal to it.

(.) primer term as used herein refers to an oligonucleotide. being R \ \ or

DNA. single-stranded or double-stranded, derived from a biological system, generated through digestion with restriction enzymes, or produced synthetically. when placed in its own environment, it is capable of acting functionally as a primer for the synthesis of a mold-dependent nucleic acid. When presented with a suitable nucleic acid template, nucleotide suitable triphosphate nucleic acid precursors. an enzyme

polymerase. suitable cofactors and conditions such as suitable temperature and pH, the primer can be extended to its 3 'end by the addition of nucleotides by the action of a polymerase enzyme. or with similar activity, to produce a first extension of the product. The ‘primer’ can vary in length depending on particular conditions and requirements for your application. For example, in diagnostic applications, the oligonucleotide primer is typically 15-25 or more nucleotides in length. () ‘Primer’ must have sufficient complementarity with the desired mold to initiate the synthesis of the desired product extension. This does not mean that the 'primer' sequence must represent an exact complement to the desired template. For example, a non-complementary nucleotide sequence can be linked to the 5 'end of a complementary primer.

Alternatively, non-complementary bases may be interspersed within the sequence

primer oligonucleotide. provided that it has sufficient complementarity with the sequence of the desired template chain to provide a functional template-primer in the product extension.

The term “specifically hybridizing” refers to the association between two single-stranded nucleic acid molecules that have sufficiently complementary sequences to allow such hybridization under predetermined conditions generally described in the prior art (Anderson. MUM Nucleic Acid Hybridization. Springer, Bios Seientifie Publishers. Nevv York. 1999. 240 p: Innis. Michael A .; Gelfand. David H .: Sninsky. John .1 .;

White. Thomas .1. PCR protocols. A guide to methods and applications. Academic Press. San Diego. HERE. USA. 1090,482 p).

35

l.m particular. the term refers to the hybridization / action of an oligonucleotide with a substantially complementary sequence containing a molecule of DNA or RN The simple chain of the present invention. Appropriate conditions necessary to perform specific hybridization between single-stranded nucleic acid molecules of varying complementarity are well described in the prior art (Anderson. M 1 \ 1. Nucleic Acid Hybridi / ation. Springer. Bios Seientifie Publishers. Neu York. 1999. 240 p;

Innis. Michaei A .: Geltand. David H .: Sninskx. John J.: White. Thomas .1. Protected PCR. \ guidc to mcthods and applications. Acadcmic Prcss. San Diego. HERE. USA. 1990. 482 p). A common formula for calculating the stringency conditions required to have a hybridization between nucleic acid molecules is shown below (Sambrook et al .. Molecular

Cloning. A Laboratory Manual. 2 ^ml ed. (1989). Cold Spring Harbor Laboratory Prcss):

Tm 8l, 5C 16.6 Log / Na ·] - 0.4 / (>, G (') 0.63 (<> fonnamida) -600 ph in the daplex (probe)

As an illustration of the above formula, using [Na—] - [0.368] and 50% formamide. with a GC content of 42% and an average probe size of 200 bases, the Tm will be 57 ^Ü C.

Probes or primers are described as corresponding to the polynucleotide of the present invention identified as SEQ ID NO1 or a variant thereof, if the oligonucleotide probe or primer. or its complement, is contained within the sequence specified as SEQ ID ΝΌ1. or a variant of this.

The term "oligonucleotide" is referred to herein as 'primers' and 'probes' in this

invention, and is defined as a nucleic acid molecule comprising two or more ribo or deoxyribonucleotides. preferably more than three. The exact size of the oligonucleotides will depend on several factors and their particular application and use. Preferred oligonucleotides comprise 15-50 base pairs of consccutixes complementary to SEQ ID NO 1. Probes can be easily selected using

2s procedures well described in the prior art (Sambrook et al "Molecular Cloning. A laboratory manual", C SHL Prcss, Cold Spring Harbor, NY. 1989). taking into account the stringency of DNA-DNA hybridization. recombination and melting temperatures, and the potential for bonding and other factors, which are known in the art.

The definition of the terms complement, rexerse complement ”and“ rexersed sequence ”.

as used herein, is illustrated by the following example. For the sequence 5'AGTGAAGT3 '. the complement is 3'TCACTTCA5 \ the re-inverse complement is 3'ACTTC'ACT5 'and the reverse sequence is 5'TGAAG'I GA3'.

Τ>

As used herein, the term "or substantially similar" variant comprises amino acid sequences or nucleotides other than specifically identified sequences, in which one or more nucleotides or amino acid residues is substituted, substituted or added. The variants may be alkaline, naturally occurring variants, or non-naturally occurring variants. The striking or substantially similar sequences concern fragments of nucleic acids that can be characterized by the percentage of similarity of their nucleotide sequences to the nucleotide sequence described here (SEQ II) XO 1). as determined by common algorithms

employed in the state of the art. The preferred nucleic acid fragments are those whose nucleotide sequences have at least about 40 or 45 ”sequence identity. preferably about 50% or 55% of sequence identity. more preferably about 60% or 65% sequence identity. more preferably about 70 ° or 75% of sequence identity. more preferably about 80% or 85% sequence identity. more preferably still about 90%. 91%.

92%. 93%. 94%. 95%. 96%. 97%. 98% or 99% sequence identity when compared to the reference sequence. The percent identity is determined by aligning two sequences to be compared, determining the number of identical residues in the aligned portion, dividing this number by the total number of residues in the searched sequence and multiplying the result by 100. This alignment can be done using softvvares existing on the internet, one of them is BLASTN. which is available on the Xalional Ccnier for Bioíec / mology hifonnation ^ CBi page (vvvvvv.ncbi.nlm.nih.gov).

The term "vector" refers to a replicon. such as a plasmid. cosmid. baemid, phage or virus, in which other genetic sequences or elements (be it DNA or RNA) can be linked to be replicated when associated with it. Preferably the virus-derived vector is selected from bacteriophages. vaccinias. retrovirus or bovine papilloma virus. The recombinant vector ”is the result of combining a commercial vector with chimeric genes. or the polynuclotide of the present invention optionally linked to an endogenous and heterologous polynuclotide of interest which in turn is operably linked to a transcription termination signal. Such vectors can be obtained commercially, including Clontech Laboratorics. Inc. (Paio Alto, Calif), Stratagene (La Jolla. Calif). Invitrogyn (Carlsbad. Calif.). Xcvv England Biolabs (Beverly. Mass.) And Promega (Madison. Wis.). Some examples of vectors used in the present invention, but

35 not limited to. are the \ chlorine pljEM κ -1 l.as \ (Promcga). pR 1 10? c p <A \ 1B1. \ - 1 3SI (Cambia).

() the term clone as used in the present term corresponds to fragments of DN \ inserted inside a bacterium and propagated asexually. These DNA fragments are normally attached to a cloning vector as described above.

The term “expression enhancing sequences” corresponds to amplifiers (enhancers) that enhance the rate of transcription and that can be located at great distances from the promoter (before or after, upstrcam or “doxvnstrcam”). These amplifiers are not specific and enhance the transcription of any promoter that is in your vicinity. The efficiency of the expression of a gene in a specific tissue depends on the

appropriate combination and integration of amplifiers, promoters and adjacent sequences.

The first enhancer discovered as a stimulator for the transcription of eukaryotic genes was SV40 (present in the Simian Virus 40 genome). After the discovery of this 15 intensifier. others have been identified in different viral genomes as HSV-1. AMV.

TMV. HPV-16.

The term "operationally linked" means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in appropriate positions relative to the coding sequence for the purpose of its expression.

That same definition is sometimes applied to the arrangement of coding sequences and elements that control transcription (for example, promoters, assistants or

“Enliancers” and terminating elements or strings) in an expression vector. An exogenous coding region is typically franchised by operationally linked regulatory regions that regulate the expression of that region in a transformed cell (which may be a microorganism, plant or animal). A typical regulatory region operationally linked to an exogenous coding region includes a promoter, i.e. c. a nucleic acid fragment that can modulate the transcription of exogenous coding regions and which is positioned in the 5 'region thereof. In the case of the present invention, the regulatory region concerns regions substantially similar to SEQ ID ΝΌ 1. To assist in increasing the transcription of a given polynucleotide. the promoter sequence of the present invention may be linked to other regulatory sequences already described, such as: ATATT (element of strong expression in the root). AACAAAG c

35

GCCACCK Al (scientifically applicable to specific spores from seeds). CA (. (Il (ic (CI \ CC (both sequences can be stimulated by a factor of otrcs ^ c). Among others [Ai-Min Wu et al .. 2006 Isolaiiou of a uotton rcvcrsiblv glycosxlatcd polypcptidc (GhRGPI) promoler and its expression aclivitx in transgenic tobacco. Journal of Plant Physiology 163:

426-435],

A termination sequence is a DNA sequence that signals the end of the transcription. Examples of termination strings are, but are not limited to. the SV40 dc termination signal. fate! adcnylation of HSV ΓΚ. nopaline synthase gene termination signal from Agrobcu lenimi /itnte/íLscien.x (NOS). octopine synthase gene termination signal. CaMV 19S and 35S gene termination signal. termination signal

corn alcohol dehydrogenase gene, signal for termination of the mannopine synthase gene. beta-phaseolin gene termination signal. termination signal from the ssRUBISCO gene. termination signal from the sucrose synthase gene. signal of termination of the virus that attacks the

Irijoliuni subieminuan (SCSV). termination signal of the trpC gene of Aspergilbis niduhins and 15 similar ones. The present invention provides a regulatory region for isolated polynuclotides that can be employed in the manipulation of plant phenotypes, together with isolated polynuclotides comprising these regulatory regions. More specifically

The present invention is related to the promoter or regulatory sequence that occurs naturally in coffee plants (Coffea arabica), responsible for the expression of a protein of the isollaxone family in response to mechanical damage in this plant species. The coffee promoter was isolated from the gene encoding isoflavone reductase and was named in the present invention by CalsoR (SEQ ID NO1).

The polypeptide of specific interest can be expressed specifically in leaf tissues of plants by incorporating genes, or coding sequences, encoding a polypeptide. operably linked to the promoter sequence of the present invention (SEQ ID NO 1). in the genome of an organism, like a plant. I. A decrease in the amount of the polypeptide can be obtained by transforming the plant with antisense copies ”of these genes.

The polynuclotide of the present invention was isolated from coffee plants, more specifically from Cotjca arabica. but it can be alternatively synthesized using conventional synthesis techniques. Specifically, the isolated polynuclotide of the present invention includes the sequence identified as SEQ ID NO1; the sequence complement

U 35 idcnl 111 falls as S1'Q II) NOi: reverse complement of the sequence identified as SI O l) \ () 1.

F> all of the activity of the promoter of the present invention are detailed in the examples.

of this report.

(.) polynuclotide of the present invention can be identified in plant genomic D \ λ sequences for which genomic information is available to the public, or isolated from various polynucleotide libraries. or it can be synthesized using techniques that are well known in the state of the art (Sambrook et al '' Molecular Cloiiing. a laboratory manual ”. CSII1. Press. Cold Spring Harbor. NM 1989). The polynucleotide can be synthesized, for example, using automated oligonucleotide synthesizers (eg, OL1GO 1000M Bcckman DNA synthesizer) to obtain polynucleotide segments of up to 50 or more nucleic acids. Several of these polynucleotide segments can then be linked using standard DNA manipulation techniques that are well known in the art (Sambrook et al "Molecular Cloning. A laboratory manual". CSHL Press. Cold Spring Harbor. NY. 1989). A conventional and exemplary polynucleotide synthesis technique involves the synthesis of a single-stranded polynucleotide segment, having, for example. 80 base pairs, and hybridizing this segment to a complementary segment of 85 base pairs, synthesized, to produce an overhang of 5 nucleotides. The next segment can then be synthesized in a similar way, as an overhang of 5 nucleotides in the opposite strand. The “sticky” or cocsive ends ensure proper connection when the two portions are hybridized. In this way, the polynuclotide of this invention can be synthesized completely in vitro.

As noted above, the promoter sequence of the present invention can be employed in recombinant c- or expression vectors to trigger transcription and expression of a polynuclotide of interest in leaf tissues. The polynuclotide of interest can be endogenous or heterologous to an organism, for example, a plant, to be transformed. The recombinant and / or expression vectors of the present invention can thus be used to target the transcription and or expression of a polynuclotide, for example, a gene that is present in the wild type plant, or can be used to provide transcription and / or expression of a DNA sequence that is not found in the

I S 3 5 wild type plant, medium. for example, a gene that eodites a reporter gene, eumo <d S.

In some embodiment of the present invention, the merose pohnucleotide comprises an open reading matrix encoding a polypeptide of interest. The open reading matrix and inserted into the vector in a "sense" orientation and transformation with that recombinant vector genetic construct will generally result in an expression of the selected polypeptide of the plant leaf. The polypeptide of interest, which will be regulated by the promoter of the present invention, can be inserted into the vector 11a sense orientation ”. antisense or in both directions. Transformation with a recombinant c or expression vector containing the promoter of the invention regulating the expression of the polynucleotide of interest in the antisense orientation ”or in both orientations (sense and antisense) will generally result in reduced expression of the selected polypeptide in leaf tissues.

The polynucleotide of interest, containing a coding region, is operatively linked to a sequence of the polynucleotide promoter of the present invention so that the host cell is capable of transcribing an A RNon triggered by the promoter sequence linked to the polynucleotide of interest. The polynuclotide promoter sequence is generally positioned at the 5 'end of the polynuclotide to be transcribed. () use of tissue-specific promoter. as the promoter sequence of the gene encoding Cojfea arabica isoflavone reductasc identified as SEQ ID NO1. will direct the transcription of the polynuclotide of interest only in leaf tissues of the transformed plant.

The recombinant vector or expression vector of the present invention can also contain a selection marker that is effective in cells of the target organism, such as a plant, to allow detection of transformed cells containing the inventive recombinant vector. These markers, which are well known, typically confer resistance to one or more toxins. An example of this marker is the nptll gene. whose expression results in resistance to kanamycin or neomycin. antibiotics that are generally toxic to plant cells in moderate concentration. The transformed cells can thus be identified by their ability to grow in medium containing the antibiotic in question. Other markers that can be used to construct recombinant and / or expression vectors containing the polynucleotide of the present invention can be. but are not limited to: gene ///? / confers resistance to the antibiotic hygromycin. The maiiA gene is the bar gene.

35 the system that uses the manA gene (which codes for the enzyme PM1 - phosphomannose isomerase) from Escberichia co / i (Miles c Guest. 1984. Complete nucleotide sequence of the lumarasc uene tumA. Ot / · .. coli. Xuclcic Acids Res. 25: 12 '3631 3642). having mannose as a selective agent, is one of the new systems suggested as alternatives to the first two described above (Joersbo et al .. 1998 Parameters intcracting with mannosc sclcction emploved for the production of transgenic sugar bcct. Pbysiologia Plantarum 105: 109 doi: 10.1034 j.] 399-3054.1999.1051 I ”.x). Plant species that do not metabolize manosu have severe growth inhibition when it is offered as the sole carbon source

in culture medium. The adverse and inhibitory effects of the use of mannose are consequences of the accumulation of mannose-6-phosphate, a product of the phosphorylation of mannose by a hexokinase. PMI promotes the interconversion of mannose-6-phosphate and fructose-6-phosphate, thus allowing the former to be eatolized in the glycolytic pathway (Souza Júnior et al. 2001. Analysis of alternative marker agent gene systems for positive embryo selection somatic transgenic papaya. Rev. strips. Eisiol. Feg., 13: 365-372; Joerbos. 2001, Advances in the selection of transgenic plants using non-antibiotic marker genes, Pbysiologia Plantarum 111: 269-272). The bar gene (encoding the PAT enzyme - phosphinothricin-Nacetyltransferase) from Sireptomyces hygroscopicus (Murakani et al., 1986 The bialaphos biosvnthetic genes cií Streptomyccs hygroscopicus'. Molecular eloning and characterization of the cluster gene. Molecular and General Genetics 205: 42- 50). with anionium glufosinate (PPT) as a selective agent, it is, among the herbicide tolerance gene type systems, one of the most widely used by genetic engineering in the development of plant GMOs. PAT inactive herbicides that present PPT as an active compound by detoxifying it. Detoxification, which results from the ethylation of the free amino group present in the PPT. makes it unable to compete in an inhibitory way with glutamine synthetase (GS). thus enabling the removal of toxic ammonia from the plant cell by converting glutamate to glutamine, a reaction catalyzed by GS (Lindsev, 1992. Molecular eloning of ICAM-3. a third ligand for Lf'A-1. constitutively expressed on resting leukocvtes. Xature 366: 481 - 484).

Alternatively, the presence of the kinetic gene in transformed cells can be determined using other techniques known in the art (Sambrook et al. “Molecular Cloning. A laboratory manual”. CSHL Press. Gol d Spring Harbor. NY. 1989), such as Southern blot and PCR.

35

Operationally linked components of the inventive recombinant or expression vectors are well known in the art and include the use of synthetic linkers containing one or more recognition sites for restriction cells, as described, for example, in Sambrook ct al. (Molecular Cloning. A manual laboraton. (SHL Press. Cold Spring Harbor. NM 1984). Chimeric genes of the present invention can be linked to a vector having at least one replication system, such as that of L. coli. after each manipulation, the resulting constructions can be cloned and sequenced.

Recombinant and / or expression of the present invention can be used to transform a variety of organisms including, but not limited to, plants. Plants that can be transformed using recombinant and / or expression vectors of the present invention include monocotyledonous angiosperms (for example grasses, corn, grains, oats, wheat and barley ...), dicotyledonous angiosperms (for example Coffea arabica. Arabidopsis, tobacco , vegetables, alfalfa, oats, eucalyptus, maple ...), and gymnosperms (for example pine, white spruce, larch ...). The protocols for plant transformation are already well known in the state of the art (Manual of genetic transformation of plants. Brasília: EMBRAPA-SPIEMBRAPA-CENARGEM. Chapters 3 and 7, 1998). In a form of

preferred embodiment, the recombinant and / or expression vectors of the present invention are used to transform dicotyledonous plants. Preferably, the plant is selected from the Rubiaceae family. more preferably of the species Coffea arabica. Other plants can be usefully transformed with the recombinant and / or expression vector of the present invention include, but are not limited to: Anacardiuni, Anona. Arachis, Artocarpus, Asparagus. Atropa, Avcna, Braxsica, Ccirica, Cilrus, Citrullus, Capsicum, Carthanuts. Coconuts, Coffea, Cucumis. Cucurhita. Daucus, Efacis, Pragaria, Cdvcine. Gossvpium, Hdianihits. llctcrocallis, lordcmn. Hyoscyamns, Laciuca, Limim, Lolium,

Lu pi nus, Lycopcrsicon, Ma / us, Manihot. Maj prays at. I was a shit, Ni cotia na. Olea. Orvza. Panicum, Panncsetuni, Passiflora, Persca, Phascolus, Pistac / üa, Pisam, Pvnts. Cousins. Psidiiun, Raphamis, Ricinus, Secalc. Senate. Sinapis, Solanutn, Sorghum, 1'hcobromus. Trigondla, Triticum. Supper, Pier, Pigna, eZea.

The transcription termination signal and the polyadenylation region of the present invention includes, but is not limited to. SV40 termination signal, HSV TK adenylation signal, A. tnmefasciens (nos) nopaline synthase gene termination signal, dc signal

35 termination of the Àf \ gene. 1,355 of the CaMV. signal of termination of the virus that attacks ο Ίrüolumi whicmmcíin (S ('SV). smal dc termination of the gene y / y; C d. hpcr ^ i / lu · ^ niclii / un ^. c other-,' • cmclliantes. the transcription terminator used in the present invention is the terminator of the gene encoding the isoylavone protein reduced by the cell.

The recombinant vectors of expression or of the invention can be introduced into the genome of the desired host plant through a variety of techniques

conventional. For example, introduction mediated by .1. tunicfusc íc / iv. cloportionation: protoplast fusion: injection into reproductive organs: injection into immature embryos: microinjection of plant cell protoplasts: using ballistic methods, such as bombardment of particles coated with DNA and others. The choice of technique will depend on the plant to be transformed. For example, dicotyledonous plants and some monocotyledons and gymnosperms can be transformed using the technology based on the Ti dc Agrohycierium plasmid. The recombinant c or dc vectors can be combined with appropriate T-DNA flanking regions and introduced into a conventional host vector .4. lumejàsciens. The host's virulence function .4. lumefasciens will direct the insertion of gene constructs and adjacent marker into the DNA of the plant cell when it is infected by the bacterium. Transformation techniques mediated by.4. uimejascicns. including disarmament and the use of binary vectors, are well described in the scientific literature (as mentioned in patent application IS 20 (120152501. Horsch et al. Science 233: 406-498. 1984: and Fraley et al. Proc. Natl. Acad I know, USA 80: 4803, 1983).

Microinjection techniques are known in the prior art and well described in the scientific and patent literature. The introduction of recombinant and expression vectors using polyethylene glycol precipitations is described in Paszkowski et al. (Embo J.

3: 2717-2722. 1984) and as mentioned in patent application US20020152501. Electroporation techniques are described in From et al. (Proc. Natl. Acad. Sci. USA 82: 5824. 1985) and as mentioned in patent application US20020152501. Ballistic transformation techniques are described in Klein et al. (Nature 327: 70-73. 1987) and as mentioned in patent application US20020152501. The introduction of recombinant vectors c or tlc 30 expressing the present invention can be done in tissues, such as leaf tissue, dissociated cells, protoplasts. seeds, embryos, meristematic regions. cotyledons. hypocotyledons. and others. Preferably the present invention uses the transformation

35 through the introduction mediated by. í. fiime / asciens using .1. thaliana with model plant (Clough at al. 1998. Floral dip: a simplified method for / fgro /? uc / m'm? / - mcdiatcd transformation of .1. í / iíiIííhkí. Plant J. 16: 735-743). However, other transformation methods can be used to insert recombinant and / or expression vectors of the present invention, such as biobalistics. which consists of a transformation technique

DXA that uses microprojects driven at high speed to carry the

DXA into cells [Rcch. F.L: Aragon. F..LI Biobalistics. In: Manual of Genetic Transformation of Plants (Brasileiro. A.C.M. & Carneiro. V.T.C7 eds.).

EMBRAPA Information Production Service -SPI. 1998. Opop]. and via pollen tube. The pollen tube transformation method was first disclosed by Zhou et al.

(Zhou. G., Wang, J „Zeng, Y .. Huang. J., Qian. S., and Liu, G. Introduction of exogenous DNA into cotton cmbryos. Meth. In Enzymol. 101: 43.3-448, 1983 ) and consists of the application of a DNA solution to the top of the young apple from the cotton tree after pollination. Using this technique, exogenous DNA can reach the ovary through the passage left by the pollen tube and integrate the zygotic cells already fertilized, but not divided.

Once the cells have been transformed by any of the techniques mentioned above, cells having the recombinant and / or expression vector of the present invention incorporated into their genome can be selected using a marker, such as the hygromycin resistance marker or kanamycin. The transformed plant cells 20 can then be grown to regenerate an entire plant that has the transformed genotype e. finally, the desired phenotype. Such regeneration techniques

they rely on the manipulation of certain phytohormones in tissue culture growth medium, typically containing a biocidal marker and / or herbicide, which must be introduced together with the desired nucleotide sequence. Plant regeneration from protoplast culture is described in Evans et al. (Evans et al. Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company. Nevv York. 1983; and Binding. Regcneration of Plants. Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. as mentioned in patent application US20020152501). Regeneration can also be achieved through plant calluses, explants. organs, or part of it. Such regeneration techniques are well described in the prior art, such as in Leelavathi et al. [Leelavathi et al. 2004. A simple and rapid Agrobcictcriiim-mcdiaicd transformation protocol for cotton (G. hirsutiun L.Y Embryogenic calli as a source to generate large numbers

35 ol transgenic planls. The cat iranslorination is effected efficiently using the microparticle bombardment technique. as described in Ribas cl al. (Ribas. Al .. Kobavashi. AK. Pereira. I IP. Vieira. I Gl. 20 ()> Gcnctic translbnnation of Co / Zec cancphora by particlc bombardmcnt. Biol. Plantarum 49: 493-49 ^ ⁷ ).

The production of RN A in cells can be controlled by choosing the promoter sequence, by selecting the number of functional copies, or by using the integration site of polynuclotides incorporated into the host genome. l’m organism can be transformed

using a recombinant c or expression vector of the present invention containing more than one open reading frame encoding a polypeptide of interest.

The isolated polynuclotide of the present invention is also useful in

genome mapping. physical mapping and positional gene cloning. The sequence identified as SEQ ID ΝΌ1 and its variants can be used to design probes and oligonucleotide primers. Oligonucleotide probes designed using the polynucleotide of the present invention can be used to detect the presence of the promoter of the gene encoding the isoflavone reductase protein in any organism having sufficiently similar DNA sequences in its cells using techniques well

known in the art, such as DNA hybridization and doi hlot techniques (Sambrook. .1 .. Fritsch. EF. Maniatis. T. Molecular cloning. a laboratory manual. 2 ^lki edition. Nevv York: Cold Spring Harbor Laboratory Press 1989: Andcrson, MLM Nucleic Acid 20 Hybridization, Springcr, Bios Scientific Publishers, Nevv York, 1999, 240 p).

Oligonucleotide primers designed using the polynucleotide of the present invention can be used for PCR amplifications. The polynucleotide of the present invention can also be used to tag or identify an organism or reproductive material therein. This tag can be obtained, for example, by the stable introduction of a heterologous polynucleotide identifier. non-functional, not disruptive. in an organism, under the control of the polynucleotide of the present invention.

EXAMPLES

The present invention is further defined in the following examples. It should be understood that these Examples, while indicating part of the invention, are provided as a form of illustration only, and therefore have no limiting feature on the scope of the present inventions.

35 usual molecular biology techniques such as transformation of bacteria and cycophoresis from nucleic acids into agaroscopic gel. they are referred to through common terms to describe them. Details of the practice of these techniques, well known in the prior art, are described in Sambrook. et al. (Molecular Cloning. A Laboratory Manual, 2 ^nJ ed. Cold Spring Harbor Laboratory Press. 1980). Various solutions used in experimental manipulations are referred to by their common names such as "agarosc". "TBE," miniprep ". etc. The compositions of these solutions can be found in the reference

Sambrook. et al. (above).

Example 1

Comparison of the promoter sequence of the present invention with the promoter sequences described in the prior art

The promoter of the present invention has been compared with promoter sequences described in patent documents, such as: W02007044992 (specific coffee promoters for expression of genes in seeds); W02007005980 (promoter of the gene that encodes a coffee dehydrin): WO20077044751 (promoters that encode genes involved in the biosynthetic pathway of chlorogenic acids in coffee plants); US6610840 (potato promoter specific for mesophile); US6800794 (promoter of alfalfa induced by biotic stress): US7153953 (promoter of coffee with specific leaf expression). The sequences were analyzed using an alignment generated by the Clustal X 1.83 application using the “default” parameters. The alignment result was analyzed in the Genedoc program, to calculate the% identity between the strings. Table 1 and the alignment below show the results obtained and show that the promoter of the present invention is significantly different from the promoters described in the prior art.

ί abcla i. Percentage of identity between the promoters described in the prior art and the ptomolor of the present invention.

) ( ^M Wool. ^{11 X} 0) S_DI P. \ 1 1 XI] Sn6 I í) S4f i s ~ 1 3X3;

\\ (p! i \\ (Ρι K) XH) 5QS

Ca tic:

C <> R

800794

6X84 0

Ca L \ oR

US6800794

US 6 G1C 8 4 0 US7153953W02 9770059: CCAA ·. GTAG: AAGG: GAAG

GG

T

T

T: GTGAGC

11)! X I IDADI

42%

36 °>

.> X ^{, J} o

.ATTCACAA.A ^ ' A TATGA A T T O! 7 T AAA2íG G A AA A GATT The; t -ATC I Air / X. CGTT AT lTACTTTGCT -G CAAAA GAAG --- AATATT GA-A AATT AT --C GATG TTT TAAA CT XTGCACATA CG AGTGA ACT7AACTGTTGA AC CA GATCC TC ATC TGTCGATAAC7TT AT 'TAAGTTTTA TG CTTCTT ’C CTCCTTTAATGTT CAA- - AGCT OK - - - - -GGT AGur TGTG G- 'CATTCTTCC GGAAGAGGG TTCTTCTGGG CAAAA CGAGAAAGGA TAAGTTC GTCT GAG TACAAAG * 0 0 * 120 140 ★ .16 0

TCATAGC

TTAC-GT TTCAGGC ATGG-AT

T G ---- C

OK----*

TCAGACATi

G

ACAGTCACGG

AAC-TTCAGTTGAT

Itaa-aaaaat

JCAC-CTCTAT

G ----- rtCGG-AGGAAC

TTTTATgAGGC-AATGGA

ITCT TT

CAA HATTATA

GAAC0TGG

ATT

GGA

*

TGG-GCA CC

TGGTGTGTCT AAAAATA AGACAATAACA CACG AGTGAAC

GTCC G GTTT A GTTT

- -CG

TGCA

200

220 *

240

Ca IsoR : AGC -A TTCTGACCT ACAAG AATAC THE- US683X794- : AAC THE GTTG GGTTTAT CTT GTTAC THE- US 6 610 8 4 0 - : GGCA Ç ACG ACTTC G TACT TAGGC GT US7153953- : TAT T CCT AACTA THE AGA AGGG- - «020070059 : TGG GG CTT GTACC THE AAC CGAAAA GT ’

260

CTAAAGAAACTCG

TCATCCGTTCTAT

X ----- The TT

1AGGAGGA CG '

TTCCTCATTC GgAATTAGTTGTT TAGGAACTAAC TACACAG

CATTAGGCTT CATTTAÀ

280

ATTATCTTAC

Ca IxoR

US6800794

US 6610840US7153953W029070059: G -------- ACC: AGTA GAATG: CACA --- CGGAAGACTT

AAAT: ---------- AC: TACATCCCGCAT

TAGAAGAM ÕA CT ----- TACTAG-TATTTTiy «T TT - - - - -tgtaaggagus cgaaattS cSaattgtgttcgtccgtct

AAAGAGAg | T GA ----- CACCACTTTTfi

TCTCCTCTTGATMC HG TGATTTCTGCAGATTC

TACC

GTTA

cc

TT

320 ac-c taatSctaattScta

AT-G

ACAC ‘CACTGCAGGl

CAGA GGGCgACGAAAÕGCA

TGCTrtGAAT ”TT

TTGGüGGTC TT iaaattRgag

* 340 * 360 * 380 * 400

Ca Ixafí: ACCTTAcMaG T TTATCT B-Bg-aRa T TC ------- TG cKtTACATGACCTTACAAGATATTGT AG A CA ·

US6 800794: TTTTTGTOGG T ATGTAT gBcSa-A || A A TTGGAAACACG CgCTGTCAAAGAGTACACGGTAAAGG GGTGGTAT

US661C 840-; ACTTCTCgAGGT AGCAAT WGgATAAG A GTCGC - TCTCCTgATATTTATCCGAACAATAAAAAAT AT T TGT

0873 ^^ 953- :→TACWTA C - - · gCWG GHCCC CCTTGACAATC A »GAGCCAAGCTCCGTGGTTTGTTGGAGG A AAWO2 0070059: TGCAGGGgGC GGCAATCAGC gTgAATgC ACCCTGTTCTGTGTGA

420

440: AG ATCG AAGTTG ATGTG CTTTTTTTGTTTTTTCCTTTCAA: AC AAAG GTGC; TTGCATATGCAT: TT TGAA ATAA: TG AGAT CACT

TCGCTCT TTCTTCAGGTCATTTGGTTTGCT

T

G c

GATCACACACCCCCCCCCCCCCCGCCCCT

AGGCA CGGCCATCA-CACCAGAGTCTAT *

AAGGG AGACCATACAGATGGGGAAATGA

Ca LxoR

US6800794 *

: CCATtBtTTTtBttAA A ----- AAAG: ACTGlJrAACGgCAAA A TGCTCGGTTA

500

520

* 460 * 480 TATT GATG C j MGT CAATTGCG --TGC TT CAGT TAGG A Htt GGAGGAA Gg AATAA- CA GATTCCCTCGAT ΉΤΆ ATTAAAT ATCAT-CTA ; tccg ccag a Kgtactgaaga ATGATGTA TTCA ΤΑΤΑ Τ gAG AAAAAGG GATGA-TA * 540 * 560

GGG GC CAGW Tg- T ------- CTCGACCATA

CAGGTGG AGAjjAAj- ΤΤΆΑ TTTCTTGCTTTTG

ATCT- GGAT

GGGTG GGC

35

Cffi-TTTCT

AACCACCCCCACC J-Λ n / VC 4 'cG' -

GCTGTTTACTGTTTA 'GGTGAA There: A CAG GG.- ·.

CAAGA0TTCCGHt iG 7777ΆΤ7 TU i G

CAAATWAAATaRcTTAAG AAACG TA.AG

GGGTCgGGGCGgCCGT C TCATG AACTAG

Ί A

TCT

CGAT

TTCATG ΑΛ7

C d / \ <> R in à - X-à s β D *

600 *

620

4 0

TGACCATA GTTG7TTAC GG T T THE THE- - --RcacctaccBL · l ACAGTTATTAT GA-- -TAGCAAA R GG- - ACATAGGCg AAAGTTT TTCTCTTCC TTCAA THE ç G G - -nGGCGGCTigC - -HGAACTTCAgz ; ccgtttaccat c TTGCTCT CACTGGCTC ATGTA GAC CAAA-- VCTGGGCAGGTGA ATGCT ^ TAG ^ -AGO 'M AAAGACC GACATTTCTG - - - - •HERE TG THE Ç--- - SG - -CTGCgC 5 ATGATTCTGGGC CGA-- AAACGAA g TAGTTTG ATATATGCGT GCGTT THE 2 .7 GATT tc8aaatc'gat @ i 7 CTATGGGCCGA acgcc; ttggagac y

* / u 0 '*

CATTGGG

ATTT

CCTC

Ca fsoR

US6H0C794

US 6 610 8 4 0 US 7: 5 ^ 953W023070059

TCG-- C

CAAGT C

AGAGA C

GGAAGTG

TTTCCATT

C AAm <hA G

TTTTTTGTGíTTTTGGGT

CCCATCTCT TCTAT GACGGATCGGG

T --- TOA CTGAACCAACTT TGTTGACA M G CGA

T TGT AATACTGTTTTG CTTTGGTG gC T CC7

AGCCAG AGTAATTGATGC ATCAAAAG «A G AGC

G AAAAAAGATATATCGACTGGACCCAAAA RaAC AAG ·

TCATGATGTTGA CCTTTCAACHT TGACT * 740 * 760

AAGAATCCATAGAgRv AGATTAA CC ------- TGGAGC agtacaccttgtgcRag AGGACTA tt GATTTGGTAATAT

GCAGGTGGAGTTAGKA to GCTTCAG AA T-TGGCTCAGGCTT

GTTACTTATCATCCHT TTCACGTCCT ------- CCATTC

AGATGGGATTTGTG0T CGTGTTGT AACTTGTTACTGACCG

78C * 800

TT ---- G AATTT AGAGGCCAATAGAGC TCA

TC TTTTA CCTCT AAAAAAAAATCAGGA AAG

CTT GATCA CATGT CCTCAGCTTAATTAA ATG

CG ---------- AAAACAGATGAGATAAG TGA

C CAGAAGAC GCGGA TCTGACTTCACCACGTGTC: AAAGG: AAAAAG; GAGGA: TTGAA: TCTTT (

820

CACCC CACgS (GGTCGGAAGlS ¹ AGATT

TCGCC

AATTT

0

ACGGCAGCG CAGGgCTATC-1 TA

CC

TC

GTG x ^ rtGTgTTACCTG TGTTgTAAAATC cgtgScagtgcg aaagRcatttttc

THE

G

G

CAAT ---- AT

T- CTATCCTAG

-------- _G * 860 CC CTAtS ATT

AAATTG

CTA CGG

AA GAT. AG TTA & ¹ g ATA

S CAATTT gGGCT g TGT

880

CTAAAA G TTgCTCCTA C AAgACCTTG g TT0TTAAAAGA

CAGCC- GgSc --C

TTT C- AAgT AG CAA T-CA TAffiATTT

CCT CA G GGHC -tcg T- ggtaJt TT

900 * 920 * 940 * 960 --- CACATTCÍ Be TCTCTT THE CTC CAA TAC --- G TG CBT ATTTGCAA AGATGAAAT | Bt attttc ç TT -AT TATGTA GT TT TTT TTTT TAT V ----- AGTT! g- AGTCCA G AT GTG CGTGGGT THE GAAG CCC TGCC AGC --- TAGGGA Ι Btggtgaac AC TA CCA GGCGGACG GG -CTA GATC GGA -*s ---- GATGG | gG TTGGATGG GGGATATC GATAT- T HERE CCC- -TTCC AAA

* 980 * 1000 * 1020 * 1040 Ca IsoR CAG A » ΓΓΤ ACCATCCTAGAGTTGGAAATGGCTGT A A GC® -G TT TGATC TTGGCG Ç CCGGATAC TTG-G USE 80 07 94 : ACG Ώ ΚΤΤ TGCTTTGAAATTTGAGTGGTCTTGGA G G GAg -THE CAAAAGAG AAAGA AA TTAATAGT GAT-G US661C840- : CAG G3 BGA ATAGAAACAAAACATGTAATAAAGAGAACA ATg TG GT TCGAA AGAAC THE GTTAGCAT AGG-A US 7: 53953- : TGA Gj gAT TTTGCCCGTTAGATACAGGAACCATG A G GCg GT GT GCATT TATAT THE AGGGTTCTGTAGAG WC) 2 0 070059 : TTCAGj gACCATGA - CGTATTTAATATCCCCCAGC G AGACgCGTGCC TGATGTCTTAT GTGGCAATAC CTT-CA

* 106 ' Ca IsoR : ATACGTAG GGAGGCAAC USE 8 0 07 94 : TAATTTTG T GTCCAAj US6610840- ; G GAATCACA T TCTTAGC US '/ 1 53 95 3 - : AGGCCATA G T ----- WO2 007 00 5 9 : GCTTCCTCTGCT ATACGTí

c c

THE

THE

1Acmp.fo Nplunicmofoa DNA sequence promoting the iso11 a_v <ma reductasc protein gene

In order to identify the promoter region of the present invention, express sequences or ESTs were selected that showed a pattern of expression in bubbles. These ESTs were selected /// si / iuo in the Genome Café database (http: wvvvv.lgc.ibi.unicamp.hr cafe). For this purpose, the tools of

Electronic honhcin dispomôci> on the website. the identity of the ES I are expressed exclusively in toliet toita teita by comparing the contigs present in the libraries of the towel with the contigs present in the other libraries. The contigs formed by sequences coming only from leaf libraries were considered tissue-specific. Such contigs were then analyzed for the presence of them in libraries from leaf tissues subjected to certain stresses (leaves infected with weed and rust: leaves of plants with water stress), all selected ESTs and considered as tissue-specific were submitted to Blast analyzes at the NCBI bank (http: vvvvvv.ncbi.nlm.nih.gov.br) to confirm their identity. In the case of EST (CA00-XX-LV5084-Al0-EZ.F) used in cloning the polynuclotide of the present invention, tblastx analyzes at NCBI indicated that its deduced amino acid sequence has <89% identity to a reducent isoflavone ( Isoflavonc Reductase Related Protein) from Xicoliana sy / ves / ris.

After the selection phase in si / ico. specificity to a given tissue was confirmed using the Reverse Transcription (RT) -PCR technique using specific oligonucleotides and total RNA extracted from different organs. coffee fabrics. For such. pairs of specific oligonucleotides for each EST identified in the bank were synthesized and used in the amplification reactions. In this case, the nucleotide sequence of the EST (in the case CA0O-XX-LV5-084-A10-EZ.F) more representative of the selected contig was used to design the oligos.

In the case of the EST used to clone the polynuclotide of the present invention, the oligos designed for validation via PCR were: 5 '

TGAGCCTGCCTCAAGCTTAT 3 '(foward) and 5 ’GACAGACTGTTGGCAGGTGA

3 ’(rcverse).

The reactions were carried out as described below: 0.5 μΐ of cDNA. IX PCR

35

Biiffcr: 1.5 mM MgC12: 0.2 ni \ 1 dN IP mix: 1 μΜ of oligos lorxxard and re-inversed: c 5 u dc Ί aq polymcrasc DNA. The cycling conditions were: initial denaturation at 94 (for 5 minutes, followed by 30 cycles of ^l U '(' for 45 seconds. 52 C for 40 seconds polymerization at 72 '7' for 1 minute).

V

The region of the DNA located upstream (5) of the ESd xalidated by R7-P (R \\ as having a specific expression in leaf (C AOO-NX-EV5-O84-A1U-F / .. F). analysis of the gene under analysis, amplified using the dc gt name u alking (Dcx ic et al. Efficicnl PC R walking on plant genomie DNA. Plant Physiol Biochcm. 35: 1-9. 1997) _c genomic DNA extracted from leaves The putative promoter regions were amplified using the Universal kit (lenomelValker (Clontech; Cat. n ° Kl807-1). Following the protocol provided by the manufacturer as briefly outlined in Figure 1: A) DNA isolation coffee genomics: digestion of this with restriction enzymes / λγζΐ SmL / fcoRV c Cridl: connection of adapters B) Use of restriction fragments connected to the adapters as an amplification template x ia PCR. employing the AP and GSP oligonucleotides. C) Cloning of the amplified fragments in pGEM-Teasy vector and insertion in E. coli strain DH5 (x.

For such. two reverse genc-specific oligonucleotides (GSP1 5'CTCCCAAACTCTTGAAGCTCTCTAGGTTG-3 ' _and GPS2 5'-

i-Γ, Γ- I

GCACrTGCCTCCACTACG fATTTGCC-3 '). arranged in sequence. were synthesized based on the alpha sequence (EST deposited in the Genome Café CA00-XX-LV5-084-A10-EZ.F bank). These oligos (GSPs) were positioned as close to the 5 'region of the EST x alidate and whose sequence was available in the bank.

For the construction of the genome walking libraries, two aliquots of genomic DNA from coffee (20 pg each) were digested separately with the restriction enzymes Dra \ and S / nl, for 18 hours, with digestion being periodically monitored in agarosc gel. After digesting the genomic DNA with the described enzymes, adapters were then attached to the terminal portion of the fragments originated for the assembly of the libraries, exactly as recommended in the protocol described in the Universal CienonielValker Kit User Manual (Clontech: Protocol PT3O42-1) (step A of Figure 1). In order to amplify the promoter regions, DNA aliquots from the referred libraries (Iml) were submitted to PCR amplification sessions (from

35

according to the dcscrilo protocol in the GcnomcWaikcr Kit l scr Manual) using the GSP1 oligos (iPS2 described above and oligonucleotides complementary to the adapters provided by the kit (AP 1 -5 G1AA! A ('GA (IG \ (] ATAGGGC- 3 'c AP2-5 * -ACTATAGGGCACG (. G1GG-3') (step B in Figure I).

The single fragment of approximately 1.0 kb amplified in the gcnoHic iialking step (as shown in Figure 1) was removed from the agarosc gel with the aid of a disposable blade, and purified using the ΟΙ kit. ΙΛΑ II (ie! Exiraclion (Quiagcn) according to the specifications provided by the manufacturer. DNA was eluted from the column using ultrapure water at 70 ° C.

The purified amplification product was then inserted into the pGEMK-T Easy (Promcga) vector specifically used for insertion of PCR products having an adenine nucleotide pair at their ends (oxerhang) (step G of Figure I). Aliquots of approximately 150 ng of DNA were disposed in a 1 ml microtube along with 50% 2X Binding Buffer. 50 ng of vector and 5 u dc T4 DNA ligase. The bonding reaction remained for 16 hours at 14 ° C. After that time, an aliquot of the binding product was inserted into Escherichia coli strain DH5a by electroporation as described (Sambrook et al. Molecular Cloning. A laboratory manual. CSHL Press. Cold Spring Harbor. NY. 1989).

Immediately after the pulse (2,200-volts). the cletroporated cells were resuspended in 1 ml of liquid Luria-Bertani (LB) medium (10 g tryptone; 5 g lexedure extract and 10 g NaCl). transferred to a 1.5 ml microcentrifuge and kept under constant agitation at 300 rpm at 37 ° C for 1 hour. Then. 300 μΐ of the culture were plated on Pctri plates containing solid LB medium plus the appropriate antibiotic, in the case ampicillin (100 mg / l) plus IPTG (24 mg I) (isopropyl- (ID-thiogalactopyranoside) and X-gal (20 1 mg). the plates were kept in an incubator at 37 ^° C 'for 16 hours.

Three clones were selected from each transformation event for validation for the presence of the plasmid rccombinant. Proxáxeis rccombinantcs 30 colonies (white) were rcpicadas dc cm 3 ml liquid LB medium plus ampicillin (100 mg 1) and incubated under constant stirring at 300 rpm DC 37 ^J C for 16 hours. After the incubation period, the samples were centrifuged and successive mini-preparations ο 35

of plasmid DNA were performed ^. The OL ÍGA.V Kit Mifiiprcp (Quiagcn) was used. as described by the manufacturer.

The plasmids were analyzed via digestion pattern with / o/RI and visualization on 1% agarosc gel stained with acid bromide. Digestions were performed at 3 ^ (/ in the presence of reaction buffer IX. 30 ng of plasmid DNA and water. The presence of the expected size insert (-1.0 kb) was confirmed. The plasmid DNA (200 ng) was subjected to a sequencing reaction using the lugDvc 'mui lei / or I ervon 3. / Rcadv Reaclion (yc / c Sequcucing Kif (Applied Biosvstcms). 3 clones of each event were sequenced using an automatic equipment model ABI PRIS.VI 377 (Applied Biosvstems) .

The sequences were analyzed using the Chromas 2.30 program and a consensus was obtained from the three sequenced clones. A validation PC R was performed in order to confirm the overlap of the amplified regions in relation to their respective ESTs. For this purpose, a Jorward oligo (5'CAAGATATTG ΓΑ-3 ') was used, based on the putative promoter sequences obtained and the GSP1 and GSP2 gene-specific reverse oligos previously described.

The sequences obtained were subjected to comparative analysis with sequences deposited in the NCBI and Genoma Café database using the Blastn tool. These analyzes revealed the absence of any significant identity or similarity with the strings deposited in these banks, indicating that the sequence corresponding to the promoter Cal xo R identified in the present patent (SEQ ID NO 1) was hitherto unknown. At the same time searches were carried out for regulatory reasons normally present in promoter regions of plant genes using the P LACE database (http: vvvvvv.dna.affrc.go.jp htdocs PI.ACE) (Higo et al. Plant cis-actmg rcgulatory DNA elements (PLACE) database: 1999. Nuclcic Acids Research. 27: 297-300. 1999). The regulatory elements found throughout the nucleotide sequence of the promoter region of the present invention (TGTCA. W Box. TATA Box. GATA, GARE. MYC, ACE, MRE) are listed in Figure 2.

3 () Example 3 - Cloning of the promoter sequence of the gene encoding a coffee isotlavone reductase into an expression vector in plants

35

The amplified fragment ('1.0 kb) containing the promoter isolated by the technique of gc / zo / ac uo / Áò / e. as previously described. was inserted in the vector pRT103- (iu>

(Figure 3) (pR I 103 modified: I opíer cl al. \ Sul of plant expression vcctorx for transcriptional and translational fusions. Nucleic Acids Rc.c .. I>: o890. 1 0S7) \ isundo transient expression tests in coffee . For such. specific oligonucleotides (5 'CCCAAGCTTCACAAACAATTATG-3' c 5'- GATCCCATGGCCAACTCTAGG-

3 ') were used>> c used in the complete amplification of the regulatory sequence under study (CalsoR).

Recognition sites for the Hind \\\ restriction cn / imas in the 5 'and AGol region in the 3' region (underlined above) were added to the said oligos to make it possible to insert the amplified product into the equally digested pRT103-Gus vector (dc reaction) digestion: 1 pg of DNA, enzyme buffer diluted to IX.1 u of restriction enzyme and deionized water for a final reaction volume of 20 pl - the reaction was incubated at 37 ° C for 1h). In this case, a partial digestion of that vector with the Hind \\\ enzyme had to be performed since it has two restriction sites for that enzyme in its base sequence. The digestions were planned to allow the 35S promoter present in the pRT103-Gus vector to be replaced by the promoter sequence of the present invention, the same being inserted in transcriptional fusion with the nidA reporter gene (GLJS).

The ligations were carried out in a 3: 1 ratio (insert: vector) for IS hours at 16 ^° C. The ligation products were subsequently used for transformation of / 2 co / i electrocompelcnte. strain DH5a. The transformations were performed on an electroporator (BioRad) as described in example 2.

Five recombinant clones were then placed rcpicados and to grow in selective liquid LB medium at 37 ^J C for 16 hours under constant agitation (200 rpm). Mini-preparations of pkismidial DNA from the obtained clones and digestion tests with restriction enzymes were performed to confirm the correct insertion of the promoter fragment in the vector. The following construct was then obtained: chimeric gene containing the CalsoR promoter of the present invention transcriptionally fused to the nidA gene (Figure 4).

The chimeric gene described above was also transferred to the vector pCAMBIA-1381 (Cambia) (Figure 5) for the purposes of stable transformation into tobacco. For such. the gcnc reporter promoter set was released from the \ ctoi pR I 1 o> for digestion with the enzymes // mt / lll c Vcol (see reaction of digestion described above, inserted in the \ ctor dc plant expression p (. \\ 1 Bl \ -13x I also digested.

The calls were made in proportion dc 3

hours at 16 ° C. the bonding products subsequently being used for transformation of the l '' .. with cloctrocompetentc. strain l) H5u (see example 2). The bacteria were then pkiqucadas selctiv cm LB medium containing the appropriate antibiotic (kanamycin 100pg ml) and incubated at 37 cm greenhouse ^':' C for 18 hours.

Three clones were seeded in selective liquid LB medium and placed to grow at 37 ° C for 16 hours under constant agitation (200 rpm). After obtaining plasmid DNA through miniprep using QIA (iE \ Kà Minipiep (Quiagen), these were sequenced in an automatic sequencer as previously described, in order to confirm the correct insertion of the promoter in the vector.

Example 4 - Transient expression in Co / fea arahica leaf via biobalistic

For the biobalistic assays, the chimeric gene obtained in example 3 was used and as a positive control the closed vector pRT103-Gus, that is. containing the uidA reporter gene under the control of the 35S promoter. The treatments used in the trial were:

Chimeric gene 1 - 35S promoter GUS reporter gene (Positive control: Figure 3)

Chimeric gene 2 - CalsoR promoter of the present invention has a GUS reporter gene (Figure 4)

The versions of the chimeric genes described above were bombarded both in the hypocotylcdonar region and in the hepicotyledonous seedlings of C. arahica using the procedures described in the protocol of the Genetic Transformation Manual for Plants, produced by Embrapa Milho e Sorgo - Sete Lagoas, with some modifications. In this case, 8 shots were taken per plant, four in the hypocotyledon region (pressure of 1100 psi), two shots on each side of the seedling and four in the hupiuotilecionar (prosàu of 6 (H) psi). the inversions being repeated.

After ik biobalistic tests. the plant material was maintained on germination paper moistened with Milh-Q water in a growth chamber at 30 ° C for 48 hours, after which time the enzymatic revision test for activating the activity was started

For such. the bombarded material was submerged in a? 0 mM solution

Potassium frrocyanate. 5 mM Potassium frrocyanide. 10 mM β-mercaptoctanol and 1 mM of the substrate X-Gluc. The samples were kept in the dark at 30'C for 16 hours. After that period, the material was washed with 70% ethanol. aiming to block complete GUSca activity removed from chlorophyll, and then fixed in 50 ° glycerol <>.

Appendix 1 shows the transient expression of the GL S reporter gene in a Coffea arabica leaf transformed via biobalance with the chimeric gene containing the CaísoR promoter. The detection of the activity of the GUS reporter gene (blue dots) in leaves transformed via biobalance reveals that the region isolated via gcnonie wa / kíug has specific transcriptional activity and restricted to this organ. Confirmation of the point transformation of epidermal cells by optical microscopy analysis can also be seen in Appendix 1 (A, B. C. D and E). More detailed analyzes also reveal the presence of a large number of transformed cstôinates in these leaves (B, C). In this test, BioRad 1.0 micron gold particles were used under firing pressure of 1,100 psi (F).

Example 5 - Obtaining transgenic tobacco plants containing the Ca IsoR promoter

Clones of Agrobacteriam fumejacieiis strain LBA4404 transformed with the chimeric gene containing the CalsoR promoter were incubated in 5 ml of selective liquid YEP medium (10 g Tryptone; 10 g Yeast Extract; 10 g NaCl containing 100 pg ml kanamycin c 100 pg rifampicin ) with agitation between 100-150 rpm at 28 ° C '. until they reach an absorbance (600 nm) between 0.5 and 1. Subsequently. 1 ml of this inoculum was transferred to a 50 ml centrifuge tube, which was

35 ml of 4 ml of Y1 P liquid medium is added to the amosia mixtures in the environment until use.

Tobacco plants (V / 7oòunu tnhaimn SR 1) grown in vino were used

in the tlc transformation process. 1 in foliar discs were removed with the aid of a dc spill (previously sterilized error, and kept in a Petri dish until the transformation process. For the transformation, five Petri dishes were used with 10 leaf discs each. The discs were immersed by 15 minutes in YEP medium containing the agrobacteria and then dried on sterile filter paper and then transferred to plates containing solid MS medium (Murashige. T. c Skoog. E. \ rev iscd medium for rapid growth and bio-assays vv ith tobacco tissue .... .. Cultures Plant Physiol 15: 473-497 1962) These plates were kept in the dark for 48 hours in a growth room at 28 ^C. After this period, the discs were transferred to Petri plates containing AIS solid added dc 100 pg ml of hygromycin. 500 pg ml of celotaxime. 1 pg'ml of 6-benzylaminopurine (BAP) and 100 pg / ml naphthalcnoacetic acid (ANA). The plates were kept for approximately 30 days in a growth room. then under a photoperiod of 16 hours of light until calluses appear. As a positive control, untransformed leaf discs maintained in the selective medium mentioned above were used. Subsequently, small seedlings formed from these calluses were individualized in glass pots containing rooting medium (MS medium described above, but lacking the hormone 6-benzylaminopurine) to induce the formation of roots.

17 plants transformed with a chimeric gene containing the specific leaf promoter fused to the GUS reporter gene were regenerated. In order to confirm the insertion of the chimeric gene. Genomic DNA from these plants was extracted from leaf tissue using the previously described protocol (Konieczny. A. and Ausubel. FM A proccdurc for mapping Arabidopsis mutations using co-dominant ccotypespecific PCR-bascd markers. Plant J .. 4: 403-410. 1993).

The insertion of the chimeric gene in the regenerated plants was continued in PCR reactions using 2 μΐ dc genomic DNA in reaction buffer IX. 1.5 mM MgCl2 is added. 0.2 mM decade dN ΓΡ. ΙμΜ dc each oligonuclotide [one promoter specific (5'-CAAGA IATTGTA-3 ') and another complementary to the GUS gene (5'-GTCTGC ( ^, AGTTCA (iTTCGTTGrfC-3 ^, j, and 5 u of DNA faq

35

pohmcrase (Invotrogen). The plants are said to be positive in the PCR reaction. qoc therefore had the fragment of expected size (-1.0 kb) corresponding to the promoter of the present invention plus the GCS gene. were subjected to GCS histochemical assay as described in example 4.

To carry out the reporter gene expression assays in response to abiotic stress (mechanical damage), five plants of the F l dc generation were randomly chosen, five different transformation events, all positive in the PCR analyzes and in the GCS histochemical tests (being therefore a total of vinyl and five plants were sampled). For such. seedlings of approximately 45 a.a. (days after germination) had part of their leaves injured. with the aid of a sterile forceps and 4 hours after treatment, they were subjected to a GUS histochemical test as described in the previous example.

Appendix 2 reveals the activity of B-glucuronidasc in transgenic tobacco leaves transformed with the chimeric gene containing the CalsoR promoter subjected to mechanical damage. Panel A shows the injured wild plant. where no reporter gene activity is detected. Panels B, C, D, E and F represent the GUS expression pattern observed in all transgenic plants subjected to stress due to mechanical damage. In that case it is possible to verify that the mechanical lesion is capable of activating a temporal and localized expression response of the reporter gene, continuing that the promoter of the present invention, in addition to presenting specific expression in leaf, is also induced by abiotic stress.

Claims (14)

1. A chimeric gene characterized by comprising: a) a polynucleotide according to the sequence described in SEQ ID NO 1, optionally linked to expression enhancing sequences or promoters of interest; operationally linked to b) a heterologous polynucleotide sequence of interest. A chimeric gene according to claim 1, characterized in that the polynucleotide sequence of interest may be a coding region or a non-coding region. A chimeric gene according to claim 1, characterized in that the polynucleotide sequence of interest may be in the sense or antisense orientation. 4. A chimeric gene according to claim 1 characterized by the fact that the expression enhancing sequences are selected from the group consisting of SV40, HSV-1, AMV, HPV-16, among others. A recombinant vector characterized in that it contains a chimeric gene according to claim 1. 6. A recombinant vector characterized by comprising: a) a polynucleotide according to the sequence described in SEQ ID NO1, optionally linked to expression enhancing sequences or promoters of interest; operationally linked to b) a heterologous polynucleotide sequence of interest; and c) a termination sequence. A recombinant vector according to claim 6, characterized in that the polynucleotide sequence of interest may be a coding region or a non-coding region. Petition 870180134006, of 09/25/2018, p. 7/13 2/3 8. A recombinant vector according to claim 6, characterized in that the termination sequence is selected from the group consisting of the SV40 termination signal, HSV TK adenylation signal, Agrobacterium tumefasciens nopaline synthase gene termination signal ( NOS), octopine synthase gene termination signal, CaMV 19S and 35S gene termination signal, corn alcohol dehydrogenase gene termination signal, manopine synthase gene termination signal, beta gene termination signal -phaseolin, ssRUBISCO gene termination signal, sucrose synthase gene termination signal, virus termination signal that attacks the subterranean Trifolium (SCSV), Aspergillus nidulans trpC gene termination signal and the like. A recombinant vector according to claim 6 characterized by the fact that the expression enhancing sequences are selected from the group consisting of SV40, HSV-1, AMV, HPV-16, among others. 10. A transformed microorganism cell characterized by the fact that it contains a recombinant vector according to any one of claims 8 to 13. 11. Method to produce a plant having the expression of a modified gene characterized by the fact that it comprises the following steps: a) transforming a plant, tissue, organ or embryo cell into a recombinant vector according to any one of claims 5 to 9 or a chimeric gene according to any one of claims 1 to 4; b) select transformed cells, cell calluses, embryos or seeds; c) regenerate mature transformed cell plants, cell calluses, embryos or seeds selected in step (b); d) select mature plants from step (c) having the gene expression modified when compared to an untransformed plant. 12. Method according to claim 11, characterized in that the transformation of the plant from (a) is carried out by means of a technique chosen from the group a Petition 870180134006, of 09/25/2018, p. 8/13 3/3 follow: biobalistics, Agrobacterium tumefasciens, electroporation, plant cell protoplast fusion. Method according to claim 12, characterized in that the selection of transformed cells from (b) is performed using a technique chosen from the following group: hygromycin antibiotic resistance, kanamycin antibiotic resistance, mannose selection (manA gene ), selection by ammonium glufosinate (bar gene). 14. Method according to claim 12, characterized in that the selection of mature plants defined in (d) is carried out using a technique chosen from the following group: polymerase chair reaction (PCR), agarose gel electrophoresis, DNA sequencing, endonuclease digestion, northem blot, western blot.