BRPI1003751B1 - SUGAR CANE PROMOTERS, EXPRESSION CASSETTES, VECTORS AND THEIR USE IN TRANSGEN EXPRESSION - Google Patents

Abstract

sugarcane promoters, expression cassettes, vectors and their use in transgene expression the present invention provides nucleic acid sequences isolated from sugarcane gene promoters (seq id nº 1 to seq id nº 10) , its expression cassettes containing at least one of said sequences and at least one gene of interest heterologous and/or native to the plant and its expression vectors. additionally, the present application deals with the use of these structures in the scope of functional genomics, molecular biology, genetic engineering, particularly in the regulation of gene expression in plants, preferably grasses, more particularly, sugarcane.

Description

FIELD OF THE INVENTION

[01] The present invention provides nucleic acid sequences isolated from sugarcane gene promoters (SEQ ID N°1 to SEQ ID N°10), their expression cassettes containing at least one of said sequences and at least a gene of interest heterologous and/or native to the plant and its expression vectors. Additionally, the present application deals with the use of these structures in the scope of functional genomics, molecular biology, genetic engineering, particularly in the regulation of gene expression in plants, preferably grasses, more particularly, sugarcane.

BACKGROUND OF THE INVENTION

[02] Sugarcane belongs to the genus Saccharum L., which is part of the Poaceae (Graminea) family. Still, there is knowledge of a domesticated species called Saccharum officinarum (noble sugarcane). S. officinarum refers to a group of thick, succulent stalks cultivated in Southeast Asia and the Pacific Islands before expanding across the tropics between 1500 and 1000 BC (Daniels J, Roach BT (1987) Taxonomy and evolution in sugarcane, p 7-84. In: Heinz DJ (ed), Sugarcane improvement through breeding, Elsevier Press, Amsterdam).

[03] In China and India, S. officinarum crossed with wild relatives forming natural hybrids (S. sinense and S. barberi) which were then selected and cultivated. Sugar extraction probably developed in India and China from these hybrids (Daniels J, Daniels C (1975) Geographical, historical and cultural aspect of the origin of the Indian and Chinese sugarcanes S. barberi and S. sinense. Sugarcane Breeding newsletter 36:4-23).

[04] In the late nineteenth century, S. spontaneum, a wild species that did not produce sugar and with thin stems, was developed through breeding programs seeking to obtain resistant varieties to diseases that intensely attack the susceptible genus S. officinarum .

[05] Interspecific hybridization represented a milestone in modern breeding, as it not only solved the problem for a large number of diseases, but also allowed for increased productivity, sugar content, ratoon sprouting and resistance to stress (Roach BT (1972) Nobilization of sugarcane. Proc Int Soc Sugar Cane Technol 14:206-216). All modern cultivars are essentially derived from some interbreeding steps of these interspecific hybrids (Arceneaux G (1967) Cultivated sugarcanes of the world and their botanical derivation. Proc Int Soc Sugar Cane Technol 12:844-854; Price S (1965) Interspecific hybridization in sugarcane breeding. Proc Int Soc Sugar Cane Technol 12:1021-1026).

[06] While obtaining hybrids represents a milestone in traditional breeding programs, this technology has brought genomics a formidable challenge.

[07] The genus Saccharum includes six polyploid taxonomic groups: two wild species, S. spontaneum (2n=40 to 128) and S. robustum (2n=60, 80 and up to 200); three groups of ancestral cultivars, S. officinarum (2n=80), S. barberi (2n=81-124), and S. sinense (2n=116-120); and the sterile marginal group, S. edule (2n = 60 to 122) (Daniels J, Roach BT (1987) Taxonomy and evolution in sugarcane, p 7-84. In: Heinz DJ (ed), Sugarcane improvement through breeding, Elsevier Press, Amsterdam (review in Daniels and Roach 1987; Sreenivasan TV, Ahloowalia BS, Heinz DJ (1987) Cytogenetics. P. 211-253 In: Heinz DJ (ed), Sugarcane improvement through breeding. Elsevier, Amsterdam).

[08] Modern cultivars are highly polyploid and aneuploid, with about 120 chromosomes. GISH studies of chromosome preparations have determined that 15-25% of chromosomes are derived from S. spontaneum and that recombination of homologous chromosomes is possible (D'Hont A, Grivet L, Feldmann P, Rao PS, Berding, N, et al (1996) Characterization of the double genome structure of modern sugarcane cultivars (Saccharun spp) by molecular cytogenetics Mol Gen Genet 250:405-413; Piperidis G, D'Hont A (2001) Chromosome composition analysis of various Saccharum interspecific hybrids by genomic in situ hybridization (GISH) Proc Int Soc Sugar Cane Technol 11:565-566; Cuadrado A, Acevedo R, Moreno Dias de la Espina S, Jouve N, de la Torre C (2004) Genome remodeling in three modern S. officinarum x S. spontaneum sugarcane cultivars. J Exp Bot 55:847-854). It is estimated that the genome size of S. officinarum (2n=8x=80) is 7.68 pg, which corresponds to 7440 mega base pairs (Mbp) for a somatic cell and to 926 Mbp for the monoploid genome (x =10) (D'Hont A, Glaszmann JC (2001) Sugarcane genome analysis with molecular markers, the first decade of research. Proc Int Soc Sugar Cane Technol 24:556-559). For S. spontaneum with 2n=8x=64, the genome size was estimated at 6.30 pg, which corresponds to 6.080 Mbp for a somatic cell and 760 Mbp for the monoploid genome (x=8) (D'Hont A, Glaszmann JC (2001) Sugarcane genome analysis with molecular markers, the first decade of research. Proc Int Soc Sugar Cane Technol 24:556-559). Also, at about 760 to 926 Mbp, the size of the basic monoploid genome of Saccharum is about twice the size of rice (389 Mbp), similar to that of Sorghum bicolor Moench (760 Mbp), and significantly smaller than that of maize. (2500 Mbp). The genome size of a somatic cell (2C) of the modern cultivar R570 (2n=about 115) was estimated to be 10,000 Mb (D'Hont A (2005) Unraveling the genome structure of polyploids using FISH and GISH; examples of sugarcane and banana Cytogenet Genome Res 109 (1-3): 27-33).

[09] The analysis of the sugarcane genome is considered a great challenge, as an average gene is represented by 10 alleles. Collections of ESTs represented, until a few years ago, the fastest alternative for an initial characterization of the expressed sequences, which could significantly contribute to the identification of genes associated with agronomic tracts of interest (such as tolerance to biotic and abiotic stresses, mineral nutrition, sugar content, among others ), and also to obtain functional and evolutionary information. Several collections of ESTs have been developed (Carson DL, Botha FC (2000) Preliminary analysis of expressed sequence tags for sugarcane. Crop Sci 40:17691779; Carson DL, Botha FC (2002) Genes expressed in sugarcane maturing internadal tissue. Plant Cell Rep 20 :1075-1081; Casu R, Dimmock C, Thomas M, Bower N, Knight D, et al (2001) Genetic and expression profiling in sugarcane. Proc Int Soc Sugar Cane Technol 24:542-546; Casu RE, Grof CP , Rae AL, McIntyre CL, Dimmock CM, et al (2003) Identification of a novel sugar transporter homologue strongly expressed in maturing vascular stem tissues of sugarcane by expressed sequence tag and microarray analysis Plant Mol Biol 52:371-86; HM, Schulze S, Lee S, Yang M, Mirkov E, et al. (2004) An EST survey of the sugarcane transcriptome. Theor Appl Genet 108:851-863), the largest of which, called SUCEST, was a Brazilian initiative (Vettore AL, da Silva FR, Kemper EL, Souza GM, da Silva AM, et al. (2003) Analysis and functional annotation of an expressed sequence tag collection for tropical crop sugarcane. Genome Res 13:2725-35).

[10] All public ESTs were assembled into contigs or putative consensus sequences (virtual transcripts), singletons, and mature transcripts in the so-called Sugarcane Gene Index (SGI; http://compbio.dfci.harvard.edu/tgi/cgi-bin/ tgi/gimain.pl?gudb=s officinarum).

[11] The SUCEST collection, derived from transcripts obtained from various Brazilian cultivars, was grouped into 43,141 possible unique transcripts, also known as sugarcane assembled sequences (SAS) http://sucest-fun.org (Vettore AL, da Silva FR, Kemper EL, Souza GM, da Silva AM, et al. (2003) Analysis and functional annotation of an expressed sequence tag collection for tropical crop sugarcane. Genome Res 13:272535).

[12] Several studies have described and discussed possible functions of the obtained sequences, in particular for genes involved in general metabolism, signaling, growth, development and stress response (Menossi, M., Silva-Filho, MC, Vincentz, M. Van- Sluys, MA and Souza, GM (2008) Sugarcane Functional Genomics: discovery gene for agronomic trait development Int. J. Plant Genomics (eprint 2008:458732) About 30% of the identified transcripts do not have similarity to genes found in the banks of public data.

[13] The expression profile of the sugarcane stalk has been extensively studied. Sugarcane partitions carbon into sucrose, which accumulates in the stalks, as opposed to seeds, which is most common in most cultivated grasses. This intriguing and unique capacity of sugarcane, selected by man, who initially chewed the sweet stalks, and which was later incorporated into improvement programs, has been the subject of numerous studies. Accumulated sucrose reaches about 0.7 M in mature internodes (Moore PH: Temporal and spatial regulation of sucrose accumulation in the sugarcane stem. Austr J Plant Physiol 1995, 22:661-679), which corresponds to approximately 50 % of dry weight. This physiological specialization makes sugarcane an important model for studying the processes of synthesis, transport and accumulation of sugars.

[14] The regulation of gene expression represents one of the mechanisms used by plants to respond to internal and external stimuli. The regulatory regions of genes, the promoters, are capable of detecting these signals and activating or repressing gene expression. The promoter is the central processor of the regulation of a gene since it contains the binding sites for RNA polymerases, responsible for gene transcription.

[15] The identification of promoter regions of genes of interest is essential for the assembly of regulatory networks as they represent a hardwired entity in the recognition process of TFs and their respective target genes.

[16] Traditionally, promoter regions are cloned by Genome Walking using nested primers and primers of a gene of interest for the amplification of regions 5' from this one from genomic libraries (Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA , Lukyanov SA: An improved PCR method for walking in uncloned genomic DNA. Nucleic acids research 1995, 23(6):1087-1088; Devic M, Albert, S., Delseny, M., Roscoe, TJ: Efficient PCR walking on plant genomic DNA, Plant Physiology and Biochemistry 1997, 35(4):331-339). Alternatively, promoter regions are estimated from complete genome sequencing data and cloned using specific primers from genomic DNA. In all cases, cloned sequences must be tested in vivo for expression of a reporter gene. Promoter sequences can commonly extend from 500 bp to 5kb, and not infrequently up to 20 kb, which makes it difficult to clone promoters on a large scale.

[17] The availability of tissue-specific and constitutive promoters is critical for the development of transgenic plants. Currently, the most used promoters in genetically modified plants are the 35S promoter of the cauliflower mosaic virus (CaMV35S) and the promoters of the nopaline and octopine genes of Agrobacterium tumefaciens. Despite the great advances that have been made with these promoters, the expression patterns of transgenes are varied and low in some cases (Zheng Z, Kawagoe Y, Xiao S, Li Z, Okita T, Hau TL, Lin A, Murai N: 5' distal and proximal cis-acting regulator elements are required for developmental control of the rice seed storage protein glutelin gene. Plant J 1993, 4(2):357-366; Green J, Vain, P., Fearnehough, MT, Worland , B., Snape, JW, Atkinson, HJ: Analysis of the expression patterns of Arabidopsis thaliana tubulin-1 and Zea mays ubiquitin-1 promoters in rice plants in association with nematode infection. -205) with no guarantee of expression in adequate tissue (Neuteboom LW, Kunimitsu, WY, Webb, D., Christopher, DA: Characterization and tissue-regulated expression of genes involved in pineapple (Ananas comosus L.) root development. Plant Sci 2002, 163:1021-1035.)

[18] The Ubil promoter of the ubiquitin gene in maize is very active in monocotyledons (Christensen AH, Quail PH: Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous. Transgenic Res 1996, 5() 3):213-218), producing significantly higher expression than other promoters such as CaMV35S, the synthetic Emu promoter (Last KI, Brettell, RIS, Chamberlain, DA, Chaudhury, AM, Larkin, PJ, Marsch, EL, Peacock, WJ, Dennis, ES: pEmu: an improved promoter for gene expression in cereal cells. Theor Appl Genet 1991, 81:581-588) and the rice actin promoter (Act1) (McElroy D, Blowers AD, Jenes B, Wu R: Construction of expression vectors based on rice actin 1 (Act1) 5' region for use in monocot transformation. Mol Gen Genet 1991, 231(1):150-160). The Ubi-1 promoter is still the most used in current research for constitutive expression of transgenes in sugarcane (Lakshmanan P, Geijskes, RJ, Aitken, KS, Grof, CLP, Bonnett, GD, Smith: Sugarcane Biotechnology: The Challenges and Opportunities, In Vitro Cell Dev Biol Plant 2005, 41:345-363).

[19] In recent studies, there is great interest in developing more suitable promoters for sugarcane (Liu DW, Oard, SV, Oard, JH: High transgene expression levels in sugarcane (Saccharum officinarum L.) driven by the rice ubiquitin promoter RUBQ2. Plant Sci 2003, 165:743-750; Braithwaite KS, Geijskes, RJ, Smith, GR: The region variable of the SCBV genome can be used to generate a range of promoters for transgene expression in sugarcane. Cell Rep 2004, 23:319-326). Although a relatively constant pattern is maintained, such promoters can drive different expression models when introduced in different species, cell types or culture conditions, with no guarantee of expression in the desired tissue (Neuteboom LW, Kunimitsu, WY, Webb, D., Christopher, DA: Characterization and tissue-regulated expression of genes involved in pineapple (Ananas comosus L.) root development. Plant Sci 2002, 163:1021-1035; Benfey PN, Chua, NH: Regulated genes in transgenic plants. Science (New York, NY 1989, 244:174-181).

[20] The use of constitutive promoters represents one of the causes of great concern regarding the effects of transgenic organisms on the environment. Promoters such as CaMV35S, for example, when linked to genes used for selection of transformants (antibiotic resistance genes) determine, in general, the expression of the gene product in all plant tissues. Such feature, unnecessary and undesirable in genetically modified vegetables, tends to reduce the acceptance of derived products by consumers.

[21] Furthermore, the constitutive expression of a target gene in plants can lead to phenotypic problems, such as dwarfism and developmental and growth deficiency. For example, the expression of DREB1A under the control of the CaMV35S promoter improves the performance of transgenic plants compared to wild type during water stress, however, transgenic plants showed poor growth and development (Liu Q, Kasuga M, Sakuma Y, Abe H , Miura S, Yamaguchi-Shinozaki K, Shinozaki K: Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis The Plant Cell 1998, 10(8):1391-1406).

[22] The isolation of specific promoters is essential for adaptive responses in order to minimize the undesirable effects common to genetically modified plants, such as morphological alterations, silencing, altered temporal and spatial expression.

[23] In sugarcane, little is known about the specificity of promoters in a given tissue. The handling of products that accumulate on the stalk or the successful control of sugarcane pests that develop on stalks and roots through a transgenic approach depends very much on the availability of promoters that are specific or highly active on these targets (Lakshmanan P, Geijskes, RJ, Aitken, KS, Grof, CLP, Bonnett, GD, Smith: Sugarcane Biotechnology: The Challenges and Opportunities, In Vitro Cell Dev Biol Plant 2005, 41:345-363). Stem specific promoters have recently been described (Hansom S, Bower, R., Zhang, L., Potier, B., Elliot, A., Basnayake, S., Lamb, G., Hogarth, DM, Cox, M., Berding, N., Birch, RG: Regulation of transgene expression in sugarcane. Proc Int Soc Sugar Cane Technol 1999, 23:278-289).

[24] Records of promoters that are capable of promoting stable expression in sugarcane are scarce. Detailed evaluations of the activity of these promoters are lacking in all stages of sugarcane development, from the seedling stage to the adult plant (Lakshmanan P, Geijskes, RJ, Aitken, KS, Grof, CLP, Bonnett, GD, Smith : Sugarcane Biotechnology: The Challenges and Opportunities. In Vitro Cell Dev Biol Plant 2005, 41:345-363) and an assessment of the potential for expression of transgenes.

[25] Despite the good knowledge of sugarcane biology and the efficient practice of its cultivation, the development of this plant's technology is still at the beginning of a detailed biochemical and genetic characterization (Hotta, CT, Lembke , CG, Ochoa, EA, Cruz, GMQ, Domingues, DS, Hoshino, AA, Santos, WD, Souza, AP, Crivellari, A., Marconi, TG, Santos, MO, Melotto-Passarin, DM, Mollinari, M. , Margarido, GRA, Carrer, H., Souza, AP, Garcia, AAF, Buckeridge, MS, Menossi, M., Van Sluys, MA. and Souza, GM .

[26] There is a pressing need to obtain information that will enable biotechnological development and enable the long-term success of this industry.

[27] Large-scale sequencing projects such as SUCEST not only identify genes, but also allow the development of tools to search for promoters. Although the individual role of many genes has been revealed, the larger structure of the system is still unknown.

[28] In 50 years of sugarcane research, the processes and enzymes involved in sucrose accumulation have been identified and used to produce transgenics, but the results are still not encouraging, in part due to complex regulation, lack of suitable promoters and feedback processes (Moore G: Cereal genome evolution: pastoral pursuits with 'Lego' genomes. Current opinion in genetics & development 1995, 5(6):717-724).

[29] The first transgenic sugarcane was obtained in 1992, but so far there is no commercial genetically modified sugarcane due to the lack of stability in the expression of the transgene.

[30] Biotechnology in agriculture has become an important tool for genetic improvement and increased production. Given the importance of sugarcane, there is great effort in all sectors related to the sugar-alcohol industry to improve the cultivation of this crop, prioritizing the search for improved varieties, better adapted to adverse environmental conditions and resistant to pathogens and pests.

[31] In general, the focus of biotechnology companies has been the search for genes and promoters that can serve for the production of transgenic plants resistant to herbicides and insects and, more recently, the search for tissue-specific promoters that can be noted direct gene expression to increase biomass, increase sucrose, drought tolerance and fiber content. In the latter case, the aim is to produce sugarcane with an altered cell wall so that the sugarcane bagasse is less recalcitrant to the action of hydrolase enzymes that release the glucose residues from cellulose and hemicellulose and allow them to be fermented increasing the amount of ethanol produced per hectare of plant.

[32] The prospect of ethanol production from bagasse (cellulosic ethanol), in addition to the production of ethanol from sucrose, a technology that Brazil already dominates, has brought investments in R&D in the sector, which may increase the productivity of ethanol production by 40-50%. It is noteworthy that one third of the carbon in sugarcane is in sucrose, one third in bagasse and one third in straw. Currently, bagasse and straw are burned in boilers to co-generate electricity, but the availability of transgenic sugarcane with greater saccharification potential allows the creation of new alternatives for the sector and adds value.

[33] The development of new technologies involving the manipulation of the expression of specific genes can increase several characteristics of agronomic interest. The regulation of gene expression represents one of the mechanisms used by plants to respond to internal and external stimuli. The regulatory regions of genes, the promoters, are capable of detecting these signals and activating or repressing gene expression (ROOK, F. and BEVAN, MW 2003. Genetic approaches to understanding sugar-response pathways. Journal of Experimental Botany. 54(382) ): 495-501).

[34] The identification and characterization of sugarcane promoter elements are important tools for the genetic manipulation of this species. The availability of tissue-specific and constitutive promoters is critical for the development of transgenic plants, as it is necessary to express the transgenes in the places and in the adequate amounts to obtain better agronomic responses.

[35] As described above, there is an interest in sugarcane development in obtaining transgenic plants with high sucrose content and better adapted to water stress conditions. However, few sugarcane promoter sequences are available for use in the transformation process.

[36] Although not from sugarcane, the Ubi-1 promoter of the corn ubiquitin gene is very active in monocots (CHRISTENSEN AH and QUAIL PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Research 5, 213-218), producing significantly higher expression than other promoters, including CaMV35S and the rice actin promoter (Act1) (MCELROY, D., BLOWERS , AD, JENES, B., and WU, R. (1991). Construction of expression vectors based on rice actin 1 (Act1) 50 region for use in monocot transformation. Mol. Gen. Genet. 231:150-160) . The Ubi-1 promoter is still the most used in current research for the constitutive expression of transgenes in sugarcane (LAKSHMANAN, P., GEIJSKES, RJ, AITKEN, KS, GROF, CLP, BONNETT, GD, and SMITH. ( 2005). Sugarcane Biotechnology: The Challenges and Opportunities. In Vitro Cell. Dev. Biol. Plant. 41:345-363).

[37] Furthermore, it is known from the isolation of two polyubiquitin gene promoters from sugarcane, which showed constitutive expression in rice (WEI, H., ALBERT, HH, MOORE, PH (1999) Differential expression of sugarcane polyubiquitin genes and isolation of promoters from two highly expressed members of the family gene, J. Plant Physiol. 155:513-519; WEI, H., WANG, ML, Moore PH, ALBERT, HH (2003). two sugarcane polyubiquitin promoters and flanking sequences in transgenic plants. J Plant Physiol. 160(10):1241-51).

[38] Constitutive gene expression is essential when one wants to express a protein in high amounts and in all tissues. However, this strategy does not apply when it is necessary to express a gene in a certain tissue and in a smaller amount, in order to avoid a possible deleterious effect of agronomically toxic concentrations of the protein encoded by the gene.

[39] Some tissue-specific promoters active in monocots are isolated and present specific expression during barley germination (WOF, N. (1992) Structure of the encoding genes Hordeum vulgare (1,3-1,4)-beta-glucanase isoenzymes I and II and functional analysis of their promoters in barley aleurone protoplasts Mol. Gen. Genet. 234(1), 33-42; JENSEN, LG, OLSEN, O., KOPS, O., WOLF, N., THOMSEN , KK, VON WETTSTEIN D. (1996) Transgenic barley expressing a protein-engineered, thermostable (1,3-1,4)-beta-glucanase during germination. Proc. Natl. Acad. Sci. 93(8), 3487 -3491; MIKKONEN, A., PORALI, I, CERCOS, M., HO, TH (1996). A major cystein proteinase, EPB, in germinating barley seeds: structure to two introless genes and regulation of expression. Plant Molecular Biology, 31, 239-254; JENSEN, LG, POLITZ, O., OLSEN, O., THOMSEN, KK, VON WETTSTEIN, D. (1998) Inheritance of a Codon-Optimized Transgene Expressing Heat Stable (1,3-1, 4)-β-Glucanase in Scutellu m and Aleurone of Germinating Barley. Hereditary. 129, 215-225;) and corn (MUHITCH, MJ, LIANG, H., RASTOGI, R., SOLLENBERGER, KG (2002) Isolation of a promoter sequence from the glutanine synthetase gene capable of conferring tisue-specific gene expression in transgenic maize. Plant Science, 163, 865-872) or specific for phloem and xylem tissues in rice (YIN, Y., CHEN, L., BEACHY, R. (1997) Promoter elements required for phloem-specific gene expression from the RTBV promoter in rice. Plant Journal, 12, 1179-1188; ITO, H., HIRAGA, S., TSUGAWA, H., MATSUI, H., HONMA, M., OTSUKI, Y., MURAKAMI, T., OHASHI, Y. (2000) Xylem-specific expression of woundinducible rice peroxidase genes in transgeneic plants. Plant Science, 155, 85-100). However, the activities of these promoters specifically for sugarcane are not proven.

[40] In a recent study, the GUS gene driven by a sugarcane UDP glucose dehydrogenase promoter showed developmental stage-regulated transgene expression in young leaves and developing internodes (VAN DER MERWE, MJ , GROENEWALD, JH, BOTHA, FC (2003). Isolation and evaluation of a developmentally regulated sugarcane promoter. Proc. South African Sugar Cane Technol. 77:146-169). Such promoter alone was ineffective in directing transgene expression in sugarcane, but showed high GUS activity when fused to an intron (980 bp) derived from the 5' UTR of the same gene. Furthermore, other factors such as post-transcriptional changes are not foreseen in the promoters study system, therefore, it is not possible to compare the gene transcript accumulation with the activity of its promoter.

[41] Additionally, as already mentioned, sugarcane is an extremely complex genome organism, so there is no guarantee that the cloned promoter region is actually the promoter region of the functional allele. Another relevant fact is that biolistic transformation is an event with little reproducible results, in which there is no control of the region of the genome in which the exogenous DNA will integrate, in addition to being associated with a high level of post-transcriptional silencing.

[42] Two sugarcane Ubi promoters, Ubi-4 and Ubi-9, capable of producing high transient expression levels in calli of some species, such as sugarcane and rice, have been recently identified (WEI, H ., ALBERT, HH, MOORE, PH (1999) Differential expression of sugarcane polyubiquitin genes and isolation of promoters from two highly expressed members of the family gene. J. Plant Physiol. 155:513-519; WEI, H., WANG, ML, Moore PH, ALBERT, HH (2003). Comparative expression analysis of two sugarcane polyubiquitin promoters and flanking sequences in transgenic plants. J Plant Physiol. 160(10):1241-51). Future characterization of these promoters showed that Ubi-9 was able to drive high transgene expression in stable rice transformants, but not in sugarcane due to post-transcriptional silencing (WEI, H., WANG, ML, Moore PH , ALBERT, HH (2003). Comparative expression analysis of two sugarcane polyubiquitin promoters and flanking sequences in transgenic plants. J Plant Physiol. 160(10):1241-51).

[43] Biolistic transformation is still the main method used in sugarcane to introduce transgenes. The transformants produced by this method do show great variation in transgene expression (LAKSHMANAN, P., GEIJSKES, RJ, AITKEN, KS, GROF, CLP, BONNETT, GD, and SMITH. (2005). Sugarcane Biotechnology: The Challenges and Opportunities. In Vitro Cell. Dev. Biol. Plant. 41:345-363). The integration and rearrangement mechanism seems to be the same in both systems, Agrobacterium and biolistics (SOMERS, DA; MAKAREVICH, I. (2004). Transgene integration in plants: poking or patching holes in promiscuous genomes? Curr. Opin. Biotechnol. 15:126-131). However, biolistic transformations are different from those mediated by Agrobacterium. The biolistic method often results in plants with multiple and fragmented copies of the transgenes, which is attributed to the cause of gene silencing.

[44] Although the exact mechanism is not known in sugarcane, transgene silencing occurs at both the transcriptional and post-transcriptional levels, by promoter methylation and RNA degradation, respectively (Ingelbrecht et al., 1999). These observations indicate that there are records of promoters that are capable of promoting a high level of stable expression in sugarcane, however, there is still a lack of detailed evaluation of the activity of these potentially useful promoters in the direction of transgene expression in all phases of sugarcane development, from the seedling stage to the adult plant (LAKSHMANAN, P., GEIJSKES, RJ, AITKEN, KS, GROF, CLP, BONNETT, GD, and SMITH. (2005). Sugarcane Biotechnology: The Challenges and Opportunities. In Vitro Cell. Dev. Biol. Plant. 41:345-363).

[45] US patent US 6,359,196 deals with tissue-specific regulatory sequences, including barley gene promoters (Hordeum vulgare) that encode alpha-glucosidase and cystatin-1 (cysteine protease inhibitor). Furthermore, the patent refers to methods of producing transgenic plants using chimeric genes, cassettes, vectors, among others, comprising the promoter and the signal sequence coding region of the gene.

[46] The US patent US 2006195919A1 provides Arabidopsis taliana promoters, tissue-specific or constitutive, with strong or weak expression. Genes, promoters and tissues are unrelated to the present application for protection of sugarcane promoter sequences. Arabidopsis does not produce high levels of sucrose like sugarcane and its tissues are not specialized to accumulate sucrose like sugarcane tissues. Therefore, the expression pattern of sugarcane genes are different from those of Arabidopsis. It is worth remembering that sugarcane carbohydrate metabolism and photosynthetic processes are C4 type while Arabidopsis are C3 type, which makes this monocot even more different from the Arabidopsis dicotyledon.

[47] Given the above, the Depositor developed constitutively and/or tissue-specific active promoters in sugarcane, expression cassettes and their vectors.

DESCRIPTION OF THE FIGURES

corresponde à Raíz,

corresponde à Folha,

corresponde à Flor,

diz respeito à Gema

corresponde ao Entrenó 1 e, finalmente,

diz respeito ao Entrenó 4.[48] Figure 1 shows the expression profile analyzed by real-time PCR of the six target genes used to obtain 5' regulatory sequences, the normalizing gene being polyubiquitin (PUB) and (1A) SCRFLR1012F12.g/ COMT. (1B) SCQGLR1085F11.g/ Dehydrin; (1C) SCCCLR1066D10.g/ GTPase; (1D) SCCLR1126E09.g/ no match; (1E) SCCQRT2100B02.g/aquaporin and (1F) SCCCLR1048F12.g/14-3-3, where

corresponds to the Root,

corresponds to Folha,

corresponds to the Flower,

it concerns the gem

corresponds to Entrenode 1 and finally

it concerns Entrenod 4.

[49] Figure 2 depicts the expression pattern of GUS under the control of the COMT2 promoter of the SAS SCRFLR1012F12.g. Histochemical assay performed on seedlings cultivated for three months in vitro (A and B) and plants acclimated in a greenhouse for three months (C: sheath; D: root; and E: internode), where F represents a cross section of the internode showing in detail the vascular bundle and CN refers to the negative control.

[50] Figure 3 shows the histochemical assay of sugarcane plants transformed with the Dehydrin promoter (DEID1:GUS) controlling the expression of the uidA reporter gene. A and B refer to sugarcane seedlings after three months of in vitro cultivation, C and D represent the sheath and leaf cross-section, respectively, of sugarcane plants after three months of cultivation in vegetation House.

[51] Figure 4 shows the expression pattern of the GTP1 promoter (A and B) and transcript accumulation (C and D) of a GTPase (SCCCLR1066D10.g) in sugarcane internodes. Hybridization signal present in the reserve parenchyma (pr) and weakly expressed in the phloem (f). The expression concentration is in the bundle parenchyma and in the sclerenchyma, mainly in the adaxial (xylem sclerenchyma) and abaxial (phloem sclerenchyma) regions.

[52] Figure 5 demonstrates the localization of SCACLR1126E09.g transcripts on sugarcane leaves detected by in situ hybridization using antisense probe. The expression of these genes in cells of the leaf cross section is visible as a pink precipitate (arrow). The hybridization signal is observed in the cells of the leaf vascular bundle sheath, where f refers to the phloem and x to the xylem.

[53] Figure 6 shows the expression pattern of GUS under the control of the AQUA1 promoter (826bp) (SCEQRT2100B02.g). Histochemical assay performed on explants cultivated for three months in vitro (A and B) and plants acclimated in a greenhouse for three months (C: sheath; D: internode). CN: negative control.

[54] Figure 7 depicts the expression pattern of GUS under the control of the AQUA2 promoter (2169bp) (SCEQRT2100B02.g). Histochemical assay performed on explants cultivated for three months in vitro (A, B and C) and plants acclimated in a greenhouse for three months (D: sheath and; E: leaf).

[55] Figure 8 reveals the in situ location of aquaporin transcripts (SCEQRT2100B02.g) in sugarcane root. The expression signal is visualized in the root epidermis (ep).

[56] Figure 9 shows the expression pattern of GUS under the control of the UB1 (A) and UB2 promoter in a sugarcane sheath grown in a greenhouse.

[57] Figure 10 shows examples of constructions containing promoters (sequences of the present invention) for controlling the expression of the gene of interest (target gene "X"), where, in A, the promoter controls the expression of gene fragments from interest in sense and antisense, flanking an intron for the production of a hairpin of the gene of interest that acts in its silencing by RNAi; in B the promoter directs the target gene “X” in the sense sense for its overexpression; in C the promoter directs the expression of the target gene “X” in the antisense direction leading to the silencing of the endogenous gene and in D, E and F the promoters are used in two copies to increase the expression effect of the A, B and C. Buildings are not presented to scale.

DESCRIPTION OF THE INVENTION

[58] The present invention provides nucleic acid sequences isolated from sugarcane gene promoters (SEQ ID N°1 to SEQ ID N°10), their expression cassettes containing at least one of said sequences and at least a gene of interest heterologous and/or native to the plant and its expression vectors. Additionally, the present application deals with the use of these structures in the scope of functional genomics, molecular biology, genetic engineering, particularly in the regulation of gene expression in plants, preferably grasses, and more particularly sugarcane.

[59] In a first embodiment, the present application is directed to isolated nucleic acid sequences, such as, SEQ ID NO. 1 to SEQ ID NO. 10.

[60] SEQ. ID AT THE. 1 refers to the regulatory sequence of the SCCCLR1066D10.g gene, which encodes a GTPase, 800 bp cloned fragment.

[61] SEQ. ID AT THE. 2 represents the regulatory sequence of the SCQGLR1085F11.g gene, which codes for Dehydrin, a cloned fragment of 936bp.

[62] SEQ. ID AT THE. 3 corresponds to the regulatory sequence of the SCRFLR1012F12.g gene that codes for COMT, a cloned 1920bp fragment (COMT1).

[63] SEQ. ID AT THE. 4 refers to the regulatory sequence of the SCRFLR1012F12.g gene, which codes for COMT, a cloned fragment of 1523bp (COMT2).

[64] SEQ. ID AT THE. 5 identifies the regulatory sequence of the SCCCLR1048F12.g gene, which codes for the 14-3-3 protein, a cloned 1298bp fragment (Ub1).

[65] SEQ. ID AT THE. 6 refers to the regulatory sequence of the SCCCLR1048F12.g gene, which codes for the 14-3-3 protein, cloned fragment of 2010bp (Ub2).

[66] SEQ. ID AT THE. 7 represents the regulatory sequence of the SCEQRT2100B02.g gene, which codes for Aquaporin, a cloned 826bp fragment (Aqua1).

[67] SEQ. ID AT THE. 8 corresponds to the regulatory sequence of the SCEQRT2100B02.g gene, which codes for Aquaporin, a cloned 2169bp fragment (Aqua2).

[68] SEQ. ID AT THE. 9 refers to the regulatory sequence of the SCACLR1126E09.g gene, which codes for a “no match” sequence, cloned fragment of 619bp (Nm1).

[69] SEQ. ID AT THE. 10 corresponds to the regulatory sequence of the SCACLR1126E09.g gene, which codes for a “no match” sequence, cloned fragment of 386bp (Nm2).

[70] In a second embodiment, the present invention provides expression cassettes containing at least one sequence (SEQ. ID No. 01 to SEQ. ID No. 10), at least one gene of interest and a 3' terminator sequence.

[71] The sequences of the present invention can be cloned into single or multiple copy expression vectors, followed by the gene of interest to be regulated by it. The gene of interest can be in the sense sense (for overexpression) or antisense sense (for silencing).

[72] The expression cassettes of the present application can be constructed, particularly, with the structures COMT1, COMT2, DEID1, GTP1, GTP2, NM1, NM2, AQUA1, AQUA2, UB1 and/or UB2 combined with a gene of interest (gene - target "X"), preferably COMT1: target gene; COMT2: target gene; DEID1: target gene; GTP1: target gene; GTP2: target gene; NM1: target gene; NM2: target gene; AQUA1: target gene; AQUA2: target gene; UB1:target gene and/or UB2:target gene.

[73] In addition, the present patent application provides constructs that include genomic fragments of COMT, DEID, GTP, NM1, NM2, AQUA and UB alleles with a similarity range of at least 80% sequence identity, including also alleles from other cultivars and/or genotype derived from the genus Saccharum.

[74] Optionally, this application allows the inclusion in its constructions of genes associated with sucrose content, genes associated with cell wall metabolism, genes associated with productivity (biomass production) and genes associated with the response to sugarcane drought -of sugar.

[75] In a third embodiment, the present application also refers to expression vectors containing said expression cassettes of the present invention, inserted in vectors that can be chosen among pAHC17, pAHC15, pAHC27, pAHC18, pAHC20, pAHC25 , pGPro 1, porE Series, pSTARLING, pSTARGATE, pCAMBIA0380, pCAMBIA0390, pCAMBIA1281Z, pCAMBIA1291Z, pCAMBIA1381Z and pCAMBIA1391Z, but not limited to these.

[76] In a fourth embodiment, the present application is intended for use of these structures, as expression cassettes and vectors comprising the sequences of the present invention, in the scope of functional genomics, molecular biology, genetic engineering, particularly in the regulation of gene expression in plants , preferably, grasses, more particularly, sugar cane.

[77] Particularly, the structures of the present invention are capable of directing constitutive expression in calluses, leaves, internodes and sugarcane roots.

EXAMPLES

[78] To obtain regions with promoter activity, PCR amplifications were performed on unknown 5' genomic regions located upstream of the coding sequences of the genes of interest, using the Universal GenomeWalker™ Kit (CLONTECH, USA). Initially, the sugarcane genomic DNA was digested separately with four different restriction endonucleases (DraI, EcoRV, Stu I and Pvu II), which leave the blunt ends. The digestion products were purified and linked to adapters provided in the kit, creating four GenomeWalker “libraries”. In an attempt to obtain the 5' region of the genes of interest, two specific oligonucleotide primers for each gene (GSP - gene specific primer) were synthesized (Table 1), which looped in the 5' region of the gene's coding sequence.

[79] For each “library”, two consecutive PCR reactions were performed. In the first PCR, a primer provided by the kit (API: adapter primer, 10 μM) and a gene-specific primer of interest (GSP1, “ gene-specific primer 1 ”, 10 μM; Table 1) were used. The solution for the primary PCR reaction also consisted of the following components: BD Advantage 2 PCR Buffer (BD Biosciences), 0.2 mM dNTP mix, Advantage Genomic Polymerase Mix (BD Biosciences), 1 μL of each "library" GenomeWalker and enough milli-Q water to complete the reaction volume to 50 µL. The reaction followed a routine of seven initial cycles of 94°C for 2 seconds for denaturation, 72°C for 3 minutes for annealing and extension, followed by 32 cycles of denaturation at 94°C for 2 seconds and annealing and extension at 67°C for 3 minutes. After the cycles, the reaction was subjected to an additional polymerization period of 4 minutes at 67°C. The product from the first PCR reaction was diluted 50 times and 1 μL used as a template for a second PCR. A primer (nested type) also provided by the kit (AP2 nested) at 0.2 μM, and a gene-specific nested primer (GSP2 nested) also at 0.2 μM were used. The solution for this secondary PCR still consisted of the reagents described above for the primary PCR, with the exception of the oligonucleotides. The reaction followed a program of 5 cycles of denaturation at 94°C for 2 seconds, annealing and extension at 72°C for 3 minutes, followed by 20 cycles of denaturation at 94°C for 2 seconds and annealing and extension at 67°C for 3 minutes, finished by incubation at 67°C for 4 minutes. Both reactions were carried out in a PTC-200 DNA Engine Thermocycler (MJ Research). Subsequently, the products of the two PCR reactions were resolved in 2% (w/v) agarose gel electrophoresis. The desired fragments were purified using the Perfect Gel Cleanup kit (Eppendorf).

[80] The fragments of desired size obtained in the second PCR reaction were purified from a 2% agarose gel (w/v) and ligated into pCRII vector (Invitrogen) using the TA Cloning® Kit (Invitrogen). For the ligation reaction, 4 U of T4 DNA ligase enzyme (New England Biolabs), 0.5 μL of T4 DNA Ligase Reaction Buffer 10X”, 25 ng of pCRII vector, the purified fragment from the PCR reaction, were used in an insert:vector ratio of 3:1 and MilliQ H2O for a total volume of 5 µL. The binding reaction was kept overnight at 16°C.

[81] Subsequently, 1 μL of the binding reaction was added to 50 μL of electrocompetent DH5 bacteria and the mixture transferred to an electroporation cuvette previously cooled on ice. The bacterial solution was subjected to electroporation under conditions of 2.55 KV, 50 mF and 129 W. Then, 150 μL of LB medium (1% Bactotryptone, 0.5% yeast extract, 0 NaCl) were added. 17 M), which were spread on Petri dishes containing LBC medium solidified with X-Gal (LB containing 1.5% agar, 50 μg/mL carbenicillin and 1.2mg X-Gal (5-bromo-4-chloro -3-indolyl-beta-Dgalactopyranoside) Plates were incubated at 37°C for 16 hours. White colonies positive for the presence of the insert were isolated and subcultured at 37°C for 16 hours in 100 μL of LB liquid medium containing carbenicillin (50 μg/ml) Two μl of each culture were then used as a template in a PCR using T7 and SP6 primers in order to confirm the presence of the insert.

[82] Positive clones were subjected to plasmid DNA extraction for further sequencing. Plasmid mini-preparation was performed using the Fast Plasmid Mini kit (Eppendorf) according to the manufacturer's instructions. Inserts were sequencing using the Big Dye Terminator 3.1 kit (Applied Biosystems) and the ABI 3100 automatic sequencer. The primers used in the sequencing reactions were T7 and SP6.

[83] Table 1 below lists, by way of example, target genes to be expressed in transgenic sugarcane under control of the promoter sequences of the present invention, not limited to them. The genes were identified in transcriptome experiments using DNA chips as associated with agronomic tracts of interest in sugarcane.TABLE 1

[83] To analyze the spatial expression of these sequences, gene constructs involving the uidA reporter gene, which encodes the enzyme β-glucuronidase (GUS), were performed. For this, the vector pCambia1281z was used, which was properly designed for cloning and characterization of plant promoter regions. To carry out the gene constructions in the pCambia1281z vector, primers were designed based on the sequences obtained, comprising the entire fragment, without, however, covering the region of the initial ATG (Table 2). At the ends of the primers, restriction enzyme sites were added for further cloning into the pCambia1281z vector. The primers (Table 2) were used to amplify the fragments using the pCRII vector containing the desired inserts as a template.

PCR

[84] Amplification reactions were performed using BD Advantage 2 PCR Buffer (BD Biosciences), 0.2 mM dNTP mix, 0.2 μM each of the primers (Integrated DNA Technologies), Advantage Genomic Polymerase Mix (BD Biosciences), 0.5 μL of the pCRII vector miniprep product, and enough milli-Q water to make up the reaction volume to 25 μL. The PCR was subjected to specific cycling for each fragment and pair of primers used. The entire PCR volume was subjected to electrophoresis on a 0.7% agarose gel and the amplified fragment purified from the gel using the PerfectPrep Gel Cleanup kit (Eppendorf). The eluted product was quantified via absorbance at 260 nm. The eluted fragments were again ligated into the pCRII vector and transformed into electrocompetent E. coli DH5, as described above. Confirmation of insertion of the fragment into the vector and extraction of plasmid DNA occurred as described above. For the cloning of the fragments of interest in the pCambia1281z vector, digestion reactions with appropriate restriction enzymes were carried out (Table 2) as per standard procedure. Subsequently, the entire volume of the reaction was subjected to 1% agarose gel electrophoresis (w/v) and the band referring to the insert was isolated using the PerfectPrep Gel Cleanup kit (Eppendorf). For the purification of the digested pCambia1281z, the paper DNA extraction protocol was used. The gel was cut below the band of interest to allow for the introduction of a Whatman DE81 piece of paper. Electrophoresis was continued until the DNA was adsorbed to the paper. The DNA fragment transferred to the paper was placed in a tube with 500 µL of TE (10 mM Tris-HCl, 1 mM EDTA), 1 N NaCl, and 10 µg of tRNA and incubated at room temperature for 1 hour. Then, it was centrifuged for 10 minutes at 14000 rpm. The supernatant is transferred to another tube containing the same volume of phenol/chloroform (1:1).

[85] After shaking and centrifugation at 14000 rpm for 2 minutes, the aqueous phase was transferred again to another tube and two volumes of ethanol were added to pellet the precipitated DNA, followed by centrifugation for 15 minutes at 14000 rpm. The precipitate was washed with 500 µL of 70% ethanol (v/v) and then dried at 37°C and resuspended with 20 µL of TE (10 mM Tris-HCl, 1 mM EDTA).

[86] For the ligation reactions 4 U of T4 DNA ligase (New England Biolabs), 0.5 μL of 10x T4 DNA Ligase Reaction Buffer (New England Biolabs), 70 ng of the vector prepared as described above and digested insert were used. and purified at the insert:vector ratio of 5:1 and MilliQ H2O for a total volume of 5 μL. The binding reaction was maintained overnight at 16°C. The products of the binding reactions were used for electroporation of E. coli DH5 , as described above, and the selection antibiotic chloramphenicol was used. Selection of positive colonies was performed by PCR using 2 μL of culture as a template, as described above. Positive clones were then used for large scale plasmid DNA extraction using the Perfectprep Plasmid Maxi kit (Eppendorf).

CALLUSES

[87] The process of transforming embryogenic calluses from sugarcane was carried out via the biolistic method. DNA was precipitated onto tungsten particles (50 μL of solution at 60 ng/μL) through the mixture containing calcium chloride (50 μL of 2.5 M solution), 10 μg of DNA and spermidine free base (20 μL of solution 0.1M). This mixture was centrifuged for 10 seconds in a microcentrifuge and the pellet washed twice in 95% ethanol and resuspended in 25 µL of 95% ethanol. Tungsten particles containing plasmid DNA were bombarded at a pressure of 7kgf/cm2.

[88] After bombardment, calli were transferred to Cl-3 culture medium without 2,4-D and kept in a growth room, in the dark, at 25 ± 2°C for 10 days. After this period, the calluses were transferred to a culture medium with 2,4-D and 35 mg/mL of the antibiotic geneticin for regeneration and selection of transformed plants. The plants were kept in a growth room with a 14 h photoperiod and a temperature of 25 ± 2°C, with regular changes of culture medium every two weeks. Subsequently, the rooted seedlings were transferred to pots and acclimated in a greenhouse, together with wild plants, which served as control.

[89] Plants regenerated from the transformation events were subjected to a verification of transgene integration by PCR using specific primers. The extraction of DNA from the plants was carried out according to standard protocol. The amplification reaction consisted of 35 cycles each of a compound of denaturation (95°C for 45 seconds), annealing (55°C for 45 seconds) and elongation (72°C for 150 seconds).

HISTOCHEMICAL TESTS

[90] Two histochemical assays were performed for GUS activity at different periods of plant growth. In the first, in vitro seedlings were used with approximately three months of cultivation in selective medium, while in the second trial, plant tissues were used three months after acclimation. In the histochemical assays, the transformed plants were immersed in a reaction buffer composed of 100 mM NaH2PO4.H2O, 0.5 mM K4Fe(CN)6.3H2O, 10 mM Na2EDTA.2H2O, 0.1% Triton X- 100 and 1 mM X-Gluc and incubated in the dark at 37°C for 16 hours. After this period, the reaction buffer was replaced by 70% ethanol whose objective is to stop the reaction. Tissues were observed under a stereoscopic magnifying glass and photographed. As a positive control, the vector pAHC27 was used, which contains ubi1:GUS:NOS as an expression cassette in plants. For the negative control, non-transformed plants were used, which underwent the tissue culture process.

[91] In the second histochemical assay, the same tissues used in the methodology described above were photographed in anatomical sections from fixed material. The material immersed in 70% ethanol (v/v) was dehydrated in an ethanol series (70, 80, 90 and 100% v/v) with intervals of 4 hours until the situation of absolute ethanol. Then there was treatment with a 3:1 mixture of ethanol:historesin (Leica) for 4 hours. This mixture was subsequently replaced by a 1:1 mixture (ethanol:historesin) for 4 hours and then a 1:3 mixture (ethanol:historesin) for a further 4 hours. The material was then kept in pure historesin for another 16 hours and then embedded in historesin. The included material was sectioned in a rotating microtome at five micrometers. The sections were mounted on microscope slides in Entellan medium and photographed on a Zeiss Axiovert 35 microscope.

PROMOTING ACTIVITY

[92] cDNA microarray experiments using samples from six sugarcane tissues (root, flower, lateral bud, leaf, and first and fourth internodes) identified genes with tissue-enriched expression as well as genes with ubiquitous expression. These results made it possible not only to characterize the expression of these genes, but also to select promoter regions that could direct the expression of proteins of interest only in the desired tissues or ubiquitously. The cloning of tissue-specific promoters represents a great advance for sugarcane research, as it enables functional studies of the expression of transgenes and the obtainment of biotechnologically improved varieties.

[93] Ten 5' sequences of six sugarcane genes were tested to verify the ability to activate transcription of a heterologous gene, the reporter gene uidA (GUS) was fused to them.

[94] The target genes were SCCCLR1048F12.g, SCQGLR1085F11.g, SCCCLR1066D10.g, SCACLR1126E09.g, SCEQRT2100B02.g and SCRFLR1012F12.g, which code, respectively, for the proteins caffeic acid 3-O-methylltransferase (COMT), Deidrine , GTPase, a straight in the match, for Aquaporin and 14-3-3. These genes were selected from expression analyzes performed using cDNA microarrays and real-time PCR (Figure 1) of samples from different sugarcane tissues, as they present preferential expression in internodes (COMT, dehydrin and GTPase ), leaf (no match) and root (aquaporin) or for presenting constitutive expression in all tissues evaluated (14-3-3).

[95] The 5' flanking sequence of the genes described above was obtained from sugarcane genomic DNA previously digested with restriction enzymes that produce blunt-ended fragments for subsequent ligation of adapters of known sequence. Such genomic DNA fragments were then used as a template for PCR amplifications, using a primer with a complementary sequence to the adapter and a complementary one to the target gene. Through this methodology, at least one fragment was amplified for each of the described genes. After cloning and sequencing, the fragments corresponding to the 5' region of the genes were identified (Table 3). Thus, two fragments for the COMT gene (SCRFLR1012F12.g), 1920bp (COMT1) and 1523bp (COMT2) were obtained; a fragment for the dehydrin gene (SCQGLR1085F11.g) of 936bp (DEID1); two fragments for GTPase (SCCCLR1066D10.g), 800bp (GTP1) and approximately 1200bp (GTP2); a fragment for the no match sequence (SCACLR1126E09.g), of 619bp, with which two constructions were obtained: of 619bp (NM1) and of 326bp (NM2); two fragments for the aquaporin gene (SCEQRT2100B02.g), 826bp (AQUA1) and 2169bp (AQUA2) and two fragments for gene 14-3-3 (SCCCLR1048F12.g), 1298bp (UB1) and 2010bp (UB2 ).

[96] The existence of multiple bands, even using stringent cycles, may be due to the complexity of the sugarcane octoploid genome, which is an interspecific hybrid. This complexity probably makes it difficult to obtain a single-band pattern when it comes to amplification from genomic DNA fragments. This fact involves the risk of isolating a promoter sequence of a non-functional allele of the gene of interest. Hence the need to analyze more than one fragment referring to the possible regulatory sequence of the same gene. Given the high level of polyploidy in sugarcane, it is possible, by this technique, to clone promoter regions of several alleles, depending on the level of similarity between them.

[97] Comparison of the COMT1 and COMT2 sequences shows that they are 98% identical to each other for a region of 1484bp. The COMT SAS in question contains a 218bp region of 5'-UTR region and is located from the site for which the AvrII site primer was designed. Comparison of this 218bp sequence with the sequences cloned in vector pCambia1281z shows 95% identity to COMT1 and 97% identity to COMT2.

[98] The sequences AQUA1 and AQUA2 are 99% identical to each other for a region of 755bp. The high similarity found between these sequences shows that they differ mainly in terms of size, as in one of them it was possible to advance further towards the 5’ region.

[99] To verify the gene expression activation capacity of the cloned fragments, they were fused to the uidA reporter gene (GUS), from the pCambia1281z vector. The primers used in the amplification of the promoter regions for cloning in the pCambia1281z vector were designed upstream of the initial ATG codon, to avoid the formation of a bicistronic messenger with the message encoded by the reporter gene. Thus, all constructions encompass the gene's 5' UTR sequence, without however including the gene's initial ATG region. The integration of exogenous DNA in plants grown in a greenhouse was confirmed via PCR, using an oligonucleotide primer that looped in the 3' region of the cloned fragment (promoter sequence) and another that looped in the 5' region of the sequence. intron of catalase, present in the pCambia1281z vector.

[100] Only plants whose presence of exogenous DNA was confirmed were used in this second histochemical assay. Thus, it was possible to analyze the expression of GUS in plants with the constructions referring to possible promoters of the genes of COMT (COMT1:GUS and COMT2:GUS), dehydrin (DEID1:GUS), GTPase (only GTP1:GUS), aquaporin (AQUA1 :GUS and AQUA2:GUS) and 14-3-3 (UB1:GUS and UB2:GUS), in different transformation events. The frequency of transformants (confirmed by PCR) guarantees that the biolistic transformation methodology used was efficient.

RESULTS

[101] The analysis of the expression of the uidA (GUS) gene under the control of these sequences in transgenic sugarcane plants and the analysis of the in situ expression of the tissue location of the proteins encoded by the described genes demonstrate the applicability of the promoter sequences in expression of genes of interest in sugarcane.

[102] From a phenotypic point of view, transgenic sugarcane plants with the promoter constructs were considered normal compared to non-transformed plants, with no phenotypic change being observed until the development stage reached at three months in vegetation House.

[103] To analyze the expression pattern of target genes and promoter activity of the cloned 5' regions, transgenic sugarcane plants were obtained (Table 3) containing the promoters fused to the uidA reporter gene with subsequent histochemical assays together with in situ hybridization analyzes using specific probes for each target gene.

[104] A sample of in vitro cultured explants, at least three months old, obtained after transformation and selection using the antibiotic gentamicin, were subjected to a preliminary histochemical assay to detect the functionality of the gene construct and the 5' sequence of the target gene as a promoter, that is, the ability to activate the expression of the reporter gene uidA (GUS). At this stage, the explants had leaf and root primordia, however, they still had a region with a disdifferentiated callus appearance. Afterwards, the seedlings originating from these explants were transferred to a greenhouse, where they were acclimated and cultivated for a period of three months, and then submitted to a second histochemical assay to determine tissue-specificity with greater precision.

[105] In the second trial, leaf, sheath, internode, and root tissues were analyzed separately. However, despite the integration of exogenous DNA into the sugarcane genome has been confirmed in many independent transformants, only a small number of plants showed expression of the GUS gene at this stage of development. For the vast majority of plants, the GUS gene remained inactive. As a positive control, petunia tissue transformed with vector pAHC27 was used, which contains the UBI1 promoter of corn ubiquitin fused to uidA (UBI1:GUS), a species that has a well-characterized uidA transformation and expression system.

COMT

[106] The expression of the reporter gene driven by the COMT2:GUS construct (1523bp, SCRFLR1012F12.g) was observed in the histochemical assay with in vitro explants in leaf primordium tissues (Figure 2, A and B), indicating that the construct gene was functional and that the promoter was active. To verify tissue-specificity, a second histochemical assay was performed on sheath, leaf, internode and roots, with plants acclimated in a greenhouse. In this experiment, GUS expression was verified in the sheath, internode and root (Figure 2 C, D and E, respectively). These results corroborate those obtained by real-time PCR, which indicated a preferential expression in internode, but there was also the presence of transcripts in a root sample, while in leaf the expression signal was reduced (Figure 1 C).

[107] In the internode, blue staining was mainly detected in the vascular bundles, especially in the bundle parenchyma cells (Figure 2 F).

[108] The second cloned 1920bp SAS fragment SCRFLR1012F12.g, referring to COMT1:GUS did not show consistent GUS activity in the stages of plant development in which the histochemical assays were performed. Although the COMT sequence has a high similarity with the COMT2 sequence, as described above, the fact that this fragment does not present promoter activity may be related to the possible presence of repressor regulatory elements in the region upstream of this sequence.

DEIDRIN

[109] The activity of the SCQGLR1085F11.g dehydrin promoter was verified in the leaf primordia of explants grown in vitro from plants with the DEID1:GUS construct (Figure 3, A and B) and in the sheath and leaf of plants grown in a greenhouse (Figure 3, C and D, respectively). The pattern of expression on the leaf was generalized, as all cells had a blue color (Figure 3D). In fact, the presence of this gene transcript is observed in sugarcane leaves in real-time PCR analysis (Figure 1 B). However, despite the real-time PCR analysis showing that the dehydrin transcript accumulates in greater quantity in immature internodes, GUS activity was not verified in this tissue.

[110] The intense activity of the promoter in explants cultured in vitro and the fact that the dehydrin transcript accumulates in greater quantity in internode 1, indicates that this promoter can be regulated temporally. Therefore, the expression of the reporter gene has not been verified in the more developed internodes used in histochemical assays, since the real-time PCR analysis shows a marked reduction in the expression of this gene in internode 4 compared to internode 1 (Figure 1 B).

GTPASE

[111] Regarding the GTPase promoter SCCCLR1066D10.g, two fragments of 800 and 1200bp were obtained, named GTP1 and GTP2, respectively. However, the expression pattern was evaluated only in plants that contained the 800bp construction (GTP1:GUS), as those containing the 1200bp promoter (GTP2:GUS) are still in the process of transformation and selection.

[112] In in vitro cultured explants containing the GTP1:GUS construct, no expression of the reporter gene was observed. Expression analyzes performed by real-time PCR indicated the activity of this promoter in all tissues evaluated (Figure 1 C), however, in the histochemical assays performed with the material acclimated in a greenhouse for a period of three months, it was observed a weak marking in leaf tissue (data not shown) and internode (Figure 4), whereas in root there was no marking. Although the GTP1 sequence did not prove to be a strong promoter in the internode tissues (Figure 4), the preferential expression of this SAS in the tissues of this sugarcane organ was proven not only by microarray data and real-time PCR (Figure 1C), but also by in situ hybridization assays.

[113] Using an antisense probe of the SAS SCCCLR1066D10.g it was possible to detect an expression signal in the internode, specifically in the reserve parenchyma, weakly in the phloem and in higher concentration in the bundle parenchyma and in the sclerenchyma, mainly in the adaxial regions (sclerenchyma of the xylem) and abaxia (phloem sclerenchyma) (Figure 4, C and D).

NO MATCH

[114] Sugarcane plants were transformed with two 5' sequences of the SAS SCACLR1126E09.g, a no match sequence (no match) that showed preferential leaf expression in cDNA microarray and real-time PCR analysis (Figure 1). An antisense probe of SCACLR1126E09.g was used to analyze the accumulation of transcripts in sugarcane leaf, and it was verified that the expression of this gene was preferentially carried out by the parenchymal cells of the vascular bundle sheath (Figure 5).

AQUAPORIN

[115] Both 5' sequences of the SCEQRT2100B02.g aquaporin gene (AQUA1:GUS - 826bp; AQUA2:GUS - 2169bp) were able to activate the expression of the reporter gene in in vitro cultured explants, mainly in leaf primordia (Figure 6 A and B; Figure 7 A, B and C), indicating the functionality of the gene construct and the promoter sequence. Tissue-specificity was later analyzed in plants grown for three months in a greenhouse. In this analysis, the expression of GUS under the control of the AQUA1 promoter was restricted to sheath and internode samples (Figure 7 C and D), whereas in plants with the AQUA2 promoter the labeling was detected in the sheath and leaf tissues (Figure 7 D and E). The differences observed in the promoter activity between the AQUA1 and AQUA2 sequences are possibly related to the presence of regulatory elements located upstream, in the 5' region, of the AQUA2 fragment, as there was a great similarity between them, as discussed above.

[116] Although the cloned aquaporin promoter sequence did not show GUS activity in the root, this gene showed enriched expression in this tissue in microarray and real-time PCR assays (Figure 1 E).

[117] In fact, the presence of aquaporin gene transcripts (SCEQRT2100B02.g) in the root was confirmed in in situ hybridization assays, with the signal being specifically visualized in the epidermis of this tissue (Figure 8).

14-3-3

[118] The activity of GUS whose expression was controlled by the 5' UB1 (1298bp) and UB2 (2010bp) sequences of the 14-3-3 SCCCLR1048F12.g gene was not detected in the in vitro cultured explants. However, in the tests carried out with samples of plants grown in a greenhouse, there was a blue marking on the sheath, demonstrating the functionality of the gene constructs (Figure 9).

[119] Tissue-directed or ubiquitous (in the case of the 14-3-3 protein gene) expression data of selected genes were obtained from plant samples at different stages of development. The leaves and internodes samples, for example, were taken from plants aged 12 to 14 months for the experiments of “microarrays” - microarrays and real-time PCR. Root samples were obtained from adventitious roots developed after 12 days from explants taken from plants grown in the field for 12-14 months, and planted in moist white sand or on moist absorbent paper. Thus, further tests will be carried out with plants at a more advanced stage of development, where all tissues, including internodes, are visible. In principle, histochemical tests revealed that the 5' flanking region of these genes allowed the expression of the GUS protein in different tissues. Therefore, these regions have positive and negative regulatory elements capable of directing the expression of a gene.

[120] Table 2 shows the specific oligonucleotide primers used in the two PCRs used to obtain the promoter sequences of the genes of interest.

[121] Table 3 describes the primers used to amplify the 5' region of the genes of interest.

[122] Table 4 presents constructions of the sugarcane gene promoter regions cloned in the pCambia 1281z vector and results of histochemical assays for GUS activity.

Claims (8)

1. EXPRESSION CASSETTES, characterized by having the sequence SEQ. ID AT THE. 3 regulatory gene SCRFLR1012F12.g coding for COMT containing a cloned 1920bp fragment (COMT1) and operatively linked to at least one heterologous and/or native plant gene of interest. 2. EXPRESSION CASSETTES, characterized by having the sequence SEQ. ID AT THE. 4 regulatory gene SCRFLR1012F12.g, which codes for COMT containing a cloned fragment of 1523bp (COMT2). 3. EXPRESSION CASSETTES, according to claims 1 and 2, characterized in that the expression cassettes still consist of a 3' terminator sequence. 4. EXPRESSION CASSETTES, according to claims 1 and 2, characterized in that the cassettes also consist of constructs that include COMT allelic sequences. 5. VECTORS, according to claims 1 and 2, characterized in that the vectors consist of a single or multiple copy of the sequences followed by the gene of interest. 6. VECTORS, according to claim 5, characterized in that the gene of interest consists of the sense or antisense sense. 7. USE OF CASSETTES AND VECTORS, as defined in claims 1 to 6, characterized in that it is for applications in the scope of functional genomics, molecular biology, genetic engineering, particularly in the regulation of gene expression in plants, preferably grasses, more particularly still, sugar cane. 8. USE, according to claim 7, characterized in that the structures consist, particularly, the constitutive expression in calluses, leaves, internodes and sugarcane roots.

Images