PLANTS

Timid of thm Invention

[ 1 ] This invention belongs to the field of biotechnology, in particular, as regards to the plant development _/ and describes a method for accelerating plant growth through gene silencing of the gene that encodes DELLA protein -

[2] The invention has particular utility in connection with accelerating growth of sugar cane and to transgenic sugar cane plants obtained from the method, and will be described in connection with such utility, although other utilities are contemplated. In particular, the invention may be used for accelerating growth of other plants and to transgenic plants obtained for the method* including but not. limited to sorghum, corn., eucalyptus and miscanthus sinensis.

[3] Said gene that encodes a DELLA protein, that is responsible for. the control of plant development, and the sugar cane that comprises this gene silenced or with reduced expression feature a faster development without compromised sucrose levels.

Background of thm invention:

[4] In the evolutionary process, plants have developed intrinsic mechanisms to adjust their metabolic status and their growth to harsh environmental conditions _* In this scenario, the regulatory hormone-dependent pathways are the main targets for sugar signaling in the adaptation to stress conditions.

[5] Sugarcane {Saccharvim spp. ) is an extremely efficient culture in the conversion of solar energy into carbohydrates. The photosynthesis and. carbon assimilation occurs in the chloroplasts of the leaf mesophyll and in the cells of the vascular bundle sheath, where the same is fixed and converted into sugar or sugar derivatives _* Sucrose is then distributed to the distal sink cells, by the phloem for two destinations in the high internodes: consumption or storage.

[6] During the first stages of the sugarcane cycle, environmental factors (such as high temperature, water availability and nutrient rich soil) promote the growth and the thatch elongation. Under unfavorable environmental Conditions (such as low temperature and moderate water deficit during long-term period) _/ the growth phase is replaced by the maturation stage and the cell elongation is halted in the apical internodes,. with sucrose being directed to accumulation in the whole plant.

[7] The maturation in the sugarcane development is particularly interesting, since it is directly related to the time of the crop harvest and to the sucrose content. Although the sugarcane life cycle is reasonably understood under an ecophysi.ological. point of view, the molecular mechanisms that coordinate the regulation remain poorly understood.

[8] Nowadays, there is an increasing demand for greater agricultural productivity.. The acceleration of the plant life cycle to reach maturity earlier is highly desirable.

[9]. The DELIA protein is a candidate for the regulation of plant growth, since the genes encoding this protein are widely known as the genes of the green revolution, being responsible for the semi-dwarf character in cereals, providing the increase in the world production during the decades of 60 and 70.

[10] DELLA protein is a transcriptional nuclear regulator that suppresses the gibberellin hormone Signaling (GA) restricting the plant growth. DELLA protein is characterized by presenting a specific domain in the N- terminal region, which, contains the DELLA amino acids (aspartic acid, D, glutamic acid, E, leucine, L, Leucine, L and Alanine, A) , from which its name is derived. Although this domain can present slight variations, it is highly conserved among the species. Therefore, DELLA proteins refer to proteins, containing the DELLA domain or related domains with small changes in the amino acid composition. For example, in sugarcane and sorghum, the first leucine is replaced by methionine (M) .

[11] It is worth mentioning the LHR regions in the GRAS domain, which are essential for protein-protein interactions (through which the DELLA protein performs its transcriptional regulatory activity) .

[12] It was observed in this invention that the silencing of ScRGA gene (SEQ ID NO: 1), which encodes the DELLA growth repressor from sugarcane, named ScRGA (SEQ ID NO: 7) triggered an early development in transgenic plants of this species (herein defined as HpScRGA plants ), particularly of the internodes, without causing a reduction in sugar levels.

State of the art

[13] Some ^¾ state of the art documents describe other molecular strategies used in plants. [14] Document US 2009/0144847 Al relates to over- expression of a plant gene from Arabidopsis plant in order to assess the effect on stress tolerance for cold and heat, without analyzing the development acceleration in the transgenic Arabidopsis plants.

[15] In document US 2015/0052634 Al, the rice DELLA gene was silenced, with a modification being observed in the cell wall of the transgenic plants. It should be noted that DELLA protein from rice has only 84% identity with DELLA protein from sugarcane, and therefore, a person skilled in the art would not find obvious promoting the same event on sugarcane. Moreover, this document does not report any changes in the development rate of rice plants- tie] Document US 7268272 B2 discloses the use of proteins that contain a region similar to the DELLA domain found in the sugarcane ScRGA protein, but the genetic modification aimed to inhibit plant growth..

[17] None of these documents provides for DELLA gene silencing for accelerating plant growth without harming the plant sugar levels.

Brief description of the invention:

{18] The present invention relates to a method for accelerating plant growth comprising the steps of;

a) producing constructs for silencing genes among genes encoding DELLA proteins containing the DELLA domain or its variations, in particular the genes disclosed on SEQ ID NO: 1 to 5; b) genetic plant transformation with a silencing construct; and

c) Phenotypic analysis of transgenic plants. wherein, in step (a) , the final construct produced is pUbi:hpRGAi and has at least 77% of DNA sequence identity and, in step (c), the plants phenotypically evaluated contain in its genome the pUbi:hpRGAi construct.

[19] More specifically, the plant is selected from the group comprising sugar cane, sorghum, corn, eucalyptus and miscanthus sinensis.

[ 20] The invention further relates to a transgenic plant comprising the gene that encodes silenced DELLA protein, wherein the plant is selected from sugar cane, sorghum, corn,, eucalyptus and miscanthus sinensis,

Brief dmsczripti&n of the figures:

[21] In order to achieve a. full and complete view of the object of this invention, referenced figures are presented, as follows.

[22.3 Figure 1 contains a ScRGA protein scheme, showing the different domains (A) and the alignment of the DELIA domain of the ScRGA protein with DELIA proteins from other species (BJ - In (A) , the DELLA and GRAS domains are indicated. The DEMLA sequence, similar to. the DELLA domain sequence, is indicated, as well as the TVHYNP sequence, the LH conserved domains, the nuclear · localization signal (NLS), and the VHIID, PFYRE and SAW regions that comprise the GRAS domain are found interlinked by the variable region poly S/T/V. (B) shows, in gray background, identical amino acids in all. proteins analyzed. I and II indicate the two conserved regions of the DELLA domain, and the underlined amino acids DEMLA and TVHYNP in the protein alignment .

[23] Figure 2 is a schematic drawing of the gene silencing construct of ScRGA mediated by double-stranded RNA (A) and the hairpin sequence hpRSAi/ used for silencing the ScRGA gene (B) . In (A) , the hpRGAi hairpin is shown in detail, inserted into the expression cassette comprised by the ubiquitin promoter (maize Ubi ^w l gene) , the untranslated 5' region, the Ubi-1 gene intron and the gene transcription terminator of nopaline synthase (NOS) . This complete vector was named pDbirhpRGAi. SEQ ID NO: 6 shows the gene sequence of the hpRGAi hairpin, with the anti- sense gene of the ScRGA gene indicated in the nucleotides

1 to 500, followed by the intron II sequence of ScRlMYBl gene (nucleotides 501 to 595} and the sense sequence of the ScRGA gene (nucleotides 596 to 1095) .

[24] E'igure 3 corresponds to the integration analysis of the construct pUbi:hpRGAx in the sugarcane genome. (A) shows the expression cassette pUBI:hpScRGAi. The site of the pair of primers used in the analysis of the polymerase chain reaction (PCR) is indicated by arrows. This pair of primers amplifies a fragment of 392-base pairs (BP) in transgenic plants. (B) shows an agarose gel with the PCR products with the expected size of 392 bp. M - molecular weight marker "100 bp ladder" (Promega, Brazil); 1-water;

2 - non-transformed plant (negative control) ; 3 - hpScRGAi vector (positive control); 4-HRA; 5- HRB; 6-HR.C; 7-HR1; 8- HR11; 9-HR12; 10-HR13; 11-HR15; 12-HR16; 13-HR19; 14-HR21; 15- HR22; 16- HR23; 17- HR24; 18- HR25; 19- HR27; 20- HR28? 21- HR29; 22- HR30; 23- HR31; 24- HR32; 25- HR34; 26- HR35; 27- HR37; 28-HR38. The site of the 392 bp fragment is indicated on the left of the agarose gel figure. Three biological replicates and techniques were used. 125] Figure 4 corresponds to the graphic that indicates the levels .of expression of the ScRGA gene in wild (WT, black bar) and silenced transgenic plants due to the expression of the hairpin hpRGAi (white bars) obtained by real-time PCR.

[26] Figure 5 demonstrates the phenotypic analysis of the transgenic plants silenced for the ScRGA gene (HpScRGAj . (A) On the left, 3-month-old plants are represented, with WT being a wild plant,, and on the right, a HpScRGA plant, silenced for the ScRGA gene. By way of comparison, it is also shown in the central position a ScRGAOE plant, which over-expresses the scRGA gene and features a phenotype opposite of the HpScRGA plants. To the right of figure A, height data., number of interno.des and plant tillers of the pl¾nts of 3 months of age are shown. (B) We draw the attention to the plants of 3 months of age, showing the early formation of internodes in HpScRGA silenced plants. Scale = 5 cm.. The graph columns show averages ± standard deviation from the mean (n = 8, P < 0.05; One way AHOVA followed by Bonferroni multi- comparative post-test); the arrows indicate the first visible auricle in plants. The internodes were counted from the ground.

Detailed demcziptton of the invention:

[27] This invention describes a method for accelerating plant growth through silencing of a DELLA gene, as well as the transgenic plants obtained from the method described, particularly sugarcane.

[29] It is known that gibberellin (GA.) signaling has an important role in the plant growth, in which the defense is prioritized in relation to growth by increased levels of DELLA, a growth repressor protein.

[291 In a preferred embodiment of this invention, a single copy of the ScRGA gene in sugarcane was identified, which encodes a protein homologous to the DELLA growth repressor of GA signaling (ScRGA is a protein with high similarity to other DELLA proteins) - see figures 1 and 2. Then, GA signaling was manipulated by the generation of transgenic strains of sugarcane with repressed expression of the ScBGA gene, which led to lower levels of DELLA protein encoded by them,

[30] The method for accelerating plant growth of this invention comprises the steps of:

a) Producing constructs for silencing ScRGA gene? b) Genetic plant transformation;

c) Analysis of the ScRGA gene expression levels; and d) Phenotypic analysis of transgenic plants.

(31] The ScRGA gene (SEQ ID NO: 1) has homologous genes with high similarity at the DNA sequence level, and can be selected from the group consisting of sorghum gene {SEQ ID NO: 2) with 97% identity, corn gene (SEQ ID NO: 3) with 94% identity, Miscanthus sinensis gene (SEQ ID NO: 4), with 97% identity and eucalyptus gene (SEQ ID NO: 5), with 77% identity.

Step a) Construct production for silencing ScRGA gene;

[32] In a preferred embodiment of this invention, the. ScRGA cDNA clone was obtained and its encoding sequence was amplified via polymerase chain reaction (PCR) using specific primers using genomic DNA from the commercial sugarcane variety SP80-3280. [3.3] For the silencing construct using the hpRGAi hairpin, the antisense and sense fragments of the ScRGA gene were amplified through specific primers and subcloned in the pGEMTEasy vector, generating the constructs pGEMTEasy:asDELLA and pGEMTEasy:sde11a. In parallel, the genomic DNA of the sugarcane variety RB92579 was used as a template for amplifying, the intron II of sugarcane ScRlMYBl gene. The intron II. fragment was cloned in pGEMTEasy, generating the construct pGEMTEasy: IntronlI.

[34} Once the three constructs were sequenced to confirm the cloning, the sDELIA inserts (corresponding to the sense fragment of ScRGA gene) and the intron II of gene ScRlMYBl were linked to pUBILN vector through the EcoRV/Kpnl enzymes, generating the construct pUbi : IntronlI: sDELLA. After confirming this vector by restriction ana-lysis, the insert asDELLA {corresponding to the antisense gene fragment ScRGA) was inserted through the EcoRV/BamHI enzymes, generating the final vector pUbirhpRGAi. This vector was confirmed by sequencing. The sequence of HpRGAi hairpin corresponds to SEQ ID NO: 6. Step b) Genetic plant transformation.

[3.53 The commercial variety of. sugarcane Q208 (Saccharum offlcinarum L. var. Q208A) was used as raw material. The leaf sheaths and culms were removed and the remaining tissues were cleaned with ethanol 70%.. The apex of the aerial part and approximately 3-4 rolled leaves linked were cut into thin discs that were placed in the initial medium "(MS medium (Murashige and Skoog, 1962) lx supplemented with synthetic 2,4D auxin -1 mg/L and adjusted to pH 5.7 using KOH before autoclaving) . [36] The best explants were selected and placed in fresh initial medium. After two days, the explants were placed, in osmotic medium (MS medium lx supplemented with sorbitol and mannitol to 3.6% and adjusted to pH 5.? using KOH prior to autoc.laving) during 3 hours before the shooting by biolistic process.

[37] For this procedure, the gold particles, were coated with a L:,l molar mixture of plasmids pUbi chpRGAi and pUKtS. The helper plasmid pUKN provides the NPTII gene, which produces the neomycin phosphotransferase II protein regu.ired for the selection of transformed plants. Spermidine and calcium chloride (CaCla); were used to precipitate DNA on gold.

[38] After shooting, the explants were placed in the initial medium for two weeks for recovery. Then, the explants were cut into small pieces and placed on plates containing MS medium containing 50 mg/L of geneticin and 1. mg/L of 2.4-D. These plates were kept during three weeks in the dark and replaced with new selective fresh medium for. three additional weeks. During this last interval., several independent events were selected and replicated in selective medium. For regeneration, the material was transferred to MS medium containing 50 mg/L of geneticin without 2.4-D and kept under a photoperiod of 16 hours of light and 8 hours of dark, with cell passages every three weeks. The plants obtained were transferred to MS medium containing 1.2 mg/L of 6-benzylaminopurine (BAP) for plant propagation. The seedlings were then transferred to pots containing 60% of soil and 40% of sand, and were kept in a greenhouse until the completion of the analysis described in the continuation.

(39] Leaves of the transgenic plants were collected for DMA extraction and to confirm the presence of the transgene by PCR, according to the protocol of kit GoTaq Green Master Mix (Promega) . The PCR genotyping result is shown in figure 3, and the plants that presented the amplification of a fragment comprised by 392 base pairs were considered genetically modified.

Step c) Analysis of the levels of expression of the ScRGA gene in transgenic sugarcane plants.

[4Ό] Leaf samples were collected from each independent event- and frozen in liquid nitrogen. The leaf tissue was macerated by using a mini-beater and the extraction of total RNA was performed according to the protocol of the kit Spectrum Plant total RNA (Sigma*- Aldrich) .

[41 ) For the cDNA synthesis, the total RNA was treated with RQl RNase-Free DNAse (Promega, USA), at 37° C during 30 minutes to remove the genomic DNA contamination. After that, cDNAs were synthesized according to the kit protocol of Improm-II reverse transcriptase enzyme II {Promega} . A PCR was performed to verify the absence of genomic DNA contamination.

[42] . ViiA ¹ *""? Real-Time PCR System (Life technologies, Australia) was used for the real-time PCR technique (qPCR) . All data generated was analyzed using DataAssist" ⁴ software (Life Technologies, Australia) . The level of expression was compared against a reference sample in accordance with the 2-AZ_cq method (Livak and Schmittgen, 2001), using the ADF gene as endogenous control (Actin depolymerizing factor) . The qPCR result confirming the ScRGA gene silencing in sugarcane plants is shown in Figure 4. It is worth mentioning that most of the transgenic events analyzed {white bar) showed a. reduction in the expression level of the endogenous gene ScRGA in comparison with WT plants (black bar) .

Step d) Phenotypic analysis of transgenic plants.

[43] For phenotypic analysis, 72 plants were randomly distributed in 8 blocks containing three independent events of each construct and unprocessed plants {WT; wild) . The entire experiment was conducted in a greenhouse with automatic irrigation. The planting was carried out with a 3:1 mixture of soil:vermiculite with periodic fertilization. The measurements of height and length of internodes. were carried out using a ruler.. Phenotypic differences are shown in Figure 5, highlighting the rapid growth and the early formation of internodes in plants with silenced ScRGA gene, while the overexpression of ScRGA gene results in reduced growth (dwarf plants) , highlighting the role of the ScRGA gene (DELIA) in the control of sugarcane plant growth.

[44] While the invention, has been described in detail of accelerating plant growth of sugar cane and to transgenic sugar cane plants, as mentioned above, the invention advantageously may be used with other plants including but not limited to sorghum, -corn, eucalyptus and miscanthus sinensis.

Rofarsncas

Livak KJ, Schmittgen TD (2001) Analysis of Relative Gene Expression Data Using Real-Time Quantitative PGR and the 2-ΔΔΟΤ Method. Methods 25: 402-408 Murashige T, S.koog F (1962) A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cult Physiol Plant 15: 473-49?