KR20160023588A - Transgenic plants for increasing biomass production and method for producing the same - Google Patents

Abstract

The present invention relates to a transgenic plant into which a DX15 promoter and a Pinus densiflora GA20-oxidase (PdGA20ox) gene connected to the promoter to be operable are introduced; and a transgenic plant into which a PtrMYB003 gene connected to the promoter to be operable is additionally introduced. In addition, the present invention relates to a method for producing biomass comprising a step of producing the transgenic plant. The transgenic plant in accordance with the present invention showed qualitative changes in: inhibition of side effects such as reduction of the width of a leaf or the fresh weight of roots due to the consistent overexpression of a Gibberellin 20-oxidase (GA20ox) gene; an increase in biomass production up to 300% or more compared with a non-transgenic control group; and an increase in saccharides used as biosmass fuels due to a marked increase in the glucose and xylose content in the composition of monosaccharides of a cell wall. Additionally, the length of fiber cells of woody tissue is nearly doubled. Therefore, the transgenic plant of the present invention is expected to ultimately increase a raw material for the pulp and paper industry, a raw material for the timber industry, and a raw material of bioethanol.

Description

TECHNICAL FIELD The present invention relates to a transgenic plant having an increased biomass and a method for producing the same,

The present invention provides a transgenic plant into which a DX15 promoter and a PdGA20ox ( Pinus densiflora GA20-oxidase) gene operably linked to the promoter are introduced; And additionally a transgenic plant into which the PtrMYB003 gene operably linked to said promoter has been introduced. The present invention also relates to a method for producing biomass comprising the step of producing the transgenic plant.

Since the industrialization, worldwide use of fossil fuels has led to serious crises such as high oil prices, pollution of atmospheric pollution, increase of carbon dioxide concentration, and global climate change. Therefore, the importance of environmentally friendly and renewable biomass energy has been highlighted. The production of first-generation biofuels, which are obtained by decomposing and fermenting starch or sugar in grains, are causing side effects such as rising crop prices, food shortages and economic difficulties. Recently, research on second generation biomass energy using woody cellulose present in non-edible crops such as itch and poplar has been actively researched. In order to produce efficient biomass energy to replace fossil fuels, it is urgently required to dramatically increase the productivity of biomass and the content of cellulose.

Unlike the herbaceous plants which are the primary growth plants, which are long growing plants, the woody plants grow through the secondary growth. The secondary growth is the accumulation of the thorns (secondary conduits) It is the growth process that the circumference of the root increases. Thickening of the woody plants through this secondary growth produces over 90% of the total land biomass produced annually. The neck consists of secondary cell walls composed of cellulose, hemicellulose and lignin, which are used as raw materials for biomass energy production.

Various plant growth hormones are involved in throat formation. One of them, gibberellin, is known to play an important role in various developmental mechanisms of plants, including stem growth of plants. Gibberellin 20-oxidase (GA20ox) protein is known to catalyze the last reaction to synthesize active gibberellin in the process of gibberellin biosynthesis (Plant J., 17, 547-556, 1999). Experiments have also shown that gibberellin plays a decisive role in relation to biomass accumulation. Increased gibberellin content by overexpression of the gibberellin biosynthesis gene was able to induce a high level of lignification in tobacco and a change in lignin S / G ratio in poplar (Plant Mol. Biol. 52, 893-903, 2003 Plant Physiol., 135, 254-265, 2004). Also, overexpression of GA20ox in hybrid aspen resulted in an increase in biomass amount and a growth rate of length of neck fiber and growth rate.

To increase the productivity of plant biomass using the quantitative increase of gibberellin, we have developed transformants that overexpress mainly Arabidopsis or Poplar GA20ox gene in all tissues of plants using CaMV 35S promoter. However, in all reported cases, the stem elongation of the plant was promoted, but side effects such as remarkable inhibition of leaf and root development appeared.

To overcome this problem, the present inventors have isolated from the woody plants poplar, the neck tissue-specific promoter (DX15) and pine (Pinus Identification Transgenic plants prepared by using GA20ox (PdGA20ox) gene isolated and isolated from densiflora ; And the PtrMYB003 gene were further inhibited by the side effects of the reduction of leaf or root growth due to overexpression of PdGA20ox, and the biomass was significantly increased compared to the wild type, thus completing the present invention.

It is an object of the present invention to provide a transgenic plant in which a DX15 promoter and a PdGA20ox ( Pinus densiflora GA20-oxidase) gene operably linked to the promoter are introduced; Wherein the PtrMYB003 gene operably linked to the promoter is additionally introduced.

It is another object of the present invention to provide a method for producing a transgenic plant comprising introducing into the plant a DX15 promoter and a PdGA20ox gene operably linked to the promoter.

Another object of the present invention is to provide a DX15 promoter; A PdGA20ox gene operably linked to the promoter; And introducing the PtrMYB003 gene operably linked to the promoter into the plant.

Yet another object of the present invention is to provide a method for producing a biomass comprising introducing a DX15 promoter and a PdGA20ox gene operably linked to the promoter into a plant to produce a transformed plant.

Another object of the present invention is to provide a DX15 promoter; A PdGA20ox gene operably linked to the promoter; And introducing the PtrMYB003 gene operably linked to the promoter into a plant to produce a transgenic plant.

In some embodiments for achieving the above object, the present invention DX15 promoter and operably linked thereto PdGA20ox (Pinus densiflora Gibberellin 20-oxidase) gene. In addition, the transgenic plants of the present invention may further introduce the PtrMYB003 gene operably linked to the DX15 promoter.

The transgenic plant of the present invention suppresses the adverse effects of reduction of leaf or root growth due to overexpression of Gibberellin 20-oxidase (GA20ox) due to the introduced thrombus-specific promoters DX15 and PdGA20ox genes, Is significantly higher than that of the wild type. In addition, the transgenic plants of the present invention have a biomass that is significantly increased compared to the wild-type as a result of the simultaneous expression of PdGA20ox and PtrMYB003 as a result of the addition of the PtrMYB003 gene, The side effect of the root growth reduction does not occur or is remarkably suppressed.

The term " promoter " in the present invention is a gene region located upstream of an encoding site, which generally includes a site at which transcription is initiated, and is usually a TATA box, a CAAT box site, And an enhancer that promotes the expression of almost all genes regardless of the location and direction of the gene in response to the gene expression. In addition, among these promoters, there are a promoter that induces expression constantly in all tissues and a promoter that induces expression in a time or position-specific manner. Specifically, the promoter according to the present invention is a DX15 promoter to be.

The DX15 promoter is a promoter identified by the present inventor in Poplar, and is capable of significantly increasing the expression of the PdGA20ox gene while being specific to the tissues of the throat. Specifically, the DX15 promoter may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, and may contain not only the nucleotide sequence of SEQ ID NO: 1 but also 70% or more, specifically 80% or more, more specifically 90 , More specifically at least 95%, and most particularly at least 98%.

The term " Gibberellin " in the present invention refers to a plant hormone capable of enhancing a kidney, promoting seed germination, promoting flowering, fading, or promoting the growth of fruit in a plant. .

The term " Gibberellin 20-oxidase (GA20ox) " in the present invention is an oxidase enzyme involved in the biosynthetic step of gibberellin. When the expression of the enzyme is increased in plants, gibberellin biosynthesized may be increased . Specifically GA20ox according to the present invention, the inventors pine (Pinus densiflora) may be PdGA20ox (Pinus densiflora Gibberellin 20-oxidase ) was identified in.

Specifically, the PdGA20ox may be a protein consisting of the amino acid sequence of SEQ ID NO: 3, but the amino acid sequence having gibberellin activity may be included without limitation.

Accordingly, the PdGA20ox gene of the present invention may be a base sequence coding for the amino acid sequence of SEQ ID NO: 3 and variants thereof due to genetic code degeneracy of the genetic code, and more specifically, the base sequence of SEQ ID NO: 2 Lt; / RTI > polynucleotide.

The term "PtrMYB003" in the present invention means a transcription factor that promotes biosynthesis of cellulose, hemicellulose or lignin in plants. Specifically, the PtrMYB003 may be a protein consisting of the amino acid sequence of SEQ ID NO: 16, but an amino acid sequence having biosynthesis promoting activity such as cellulose, hemicellulose or lignin may be included without limitation.

Accordingly, the PtrMYB003 gene of the present invention may be a nucleotide sequence coding for the amino acid sequence of SEQ ID NO: 16 and mutants thereof, due to genetic code degeneracy of the genetic code, and more specifically, the nucleotide sequence of SEQ ID NO: Lt; / RTI > polynucleotide.

Each protein of the present invention has not only the amino acid sequence shown in each of the above SEQ ID NOs, but also a homology of not less than 80%, preferably not less than 90%, more preferably not less than 95%, particularly preferably not less than 97% ≪ / RTI > As the sequence having such homology, any amino acid sequence which represents a protein exhibiting substantially the same or equivalent activity as the respective proteins is included without limitation. It is also apparent that amino acid sequences in which some sequences are deleted, modified, substituted or added are also included within the scope of the present invention, provided that they have such homology.

In addition, the gene encoding each protein of the present invention may contain not only the nucleotide sequence coding for the amino acid sequence shown in the above SEQ ID NOs, but also the nucleotide sequence having 80% or more, preferably 90% or more, more preferably 95% or more , More preferably not less than 98%, and most preferably not less than 99% as long as it is a gene sequence encoding a protein that substantially or substantially has the same or equivalent efficacy as the respective proteins. Also, it is obvious that base sequences having deletion, modification, substitution or addition of some sequences are also included within the scope of the present invention if they are base sequences having such homology.

As used herein, the term "homology" means a base sequence or a similar degree of amino acid sequence of a gene encoding a protein. If the homology is sufficiently high, the expression product of the gene may have the same or similar activity have.

The term " transformation " in the present invention means that DNA is introduced into a host and DNA can be replicated as a chromosome factor or by chromosome integration completion. Specifically, in the present invention, the DX15 promoter and the PdGA20ox gene described above may be introduced into the host.

In the present invention, the term "transgenic plant" means a plant produced by the transformation using a plant as a host.

In the present invention, transformation into a host cell includes any method of introducing the nucleic acid into an organism, cell, tissue or organ, and can be carried out by selecting a standard technique suitable for the plant, as is known in the art. Such methods include electroporation, electroporation, protoplast fusion, calcium phosphate precipitation, calcium chloride precipitation, agitation with silicon carbide fibers, Agrobacterium-mediated transformation, PEG, dextran sulfate lipomethamine and drying / Mediated transformation methods, and the like, but the transformation method mediated by Agrobacterium is preferred.

More specifically, the transgenic plants according to the present invention can be applied to any herbaceous plants and herbaceous plants, if the plant is capable of increasing biomass by the introduction of the DX15 promoter and the PdGA20ox gene.

Specifically, the herbaceous plant is Oryza sativa ; Rice, Zea mays ; Corn), Miscanthus spp. Or Pennisetum spp. purpureum and the like. The woody plants may be Eucalyptus species (for example, E. alba , E. albens , E. amygdalina, E. aromaphloia , E. baileyana , E. balladoniensis , E. bicostata , E. botryoides , E. brachyandra , E. brassiana , E. brevistylis, E. brockwayi E. camaldulensis , E. ceracea , E. cloeziana , E. coccifera, E. cordata , E. cornuta , E. corticosa , E. crebra , E. croajingoleisis , E. curtisii, E. dalrympleana, E. deglupta, E. delegatensis, E. delicata, E. diversicolor, E. diversifolia, E. dives, E. dolichocarpa, E. dundasii, E. dunnii, E. elata, E . erythrocoiys, E. erythrophloia, E. eudesmoides , E. falcata, E. gamophylla, E. glaucina, E. globulus, E. globulus subsp. bicostata, E. globulus subsp . globulus, E. gongylocarpa, E. grandis, E. grandis × urophylla, E. guilfoylei, E. gunnii, E. hallii, E. houseana, E. jacksonii, E. lansdowneana, E. latisinensis, E. leucophloia, E. leucoxylon, E. lockyeri, E. lucasii, E. maidenii, E. marginata, E. megacarpa, E. melliodora, E. michaeliana, E. microcorys, E. microtheca, E. muelleriana, E. nitens, E. nitida, E. obliqua , E. obtusiflora , E. occidentalis , E. optima , E. ovata, E. pachyphylla , E. pauciflora , E. pellita , E. perriniana , E. petiolaris, E. pilularis , E. piperita , E. platyphylla, E. polyanthemos, E. populnea, E. preissiana, E. pseudoglobulus, E. pulchella, E. radiata, E. radiata subsp. radiata , E. regnans , E. risdoni , E. robertsonii E. rodwayi , E. rubida, E. rubiginosa , E. saligna , E. salmonophloia , E. scoparia , E. sieberi , E. spathulata , E. staeri E. stoatei, E. tenuipes, E. tenuiramis, E. tereticornis, E. tetragona, E. tetrodonta, E. tindaliae, E. torquata, E. umbra, E. urophylla, E. vernicosa, E. viminalis, E. wandoo, E. wetarensis , E. willisii, E. willisii subsp . falciformis , E. willisii subsp . willisii , E. woodwardii ) , populus ( Populus ) Species (e.g., P. alba, P. alba × P . Grandidentata, P. alba × P. Tremula, P. alba × P. Tremula there is . glandulosa , P. alba x P. tremuloides , P. balsamifera, P. balsamifera subsp . trichocarpa , P. balsamifera subsp . trichocarpa × P. deltoides, P. ciliata, P. deltoides, P. euphratica, P. euramericana, P. kitakamiensis, P. lasiocarpa, P. laurifolia, P. maximowiczii, P. maximowiczii × P. balsamfera subsp . trichocarpa , P. nigra , P. sieboldii × P. grandideiztata , P. suaveolens , P. szechuanica , P. tomentosa , P. tremula, P. tremula x P. tremuloides, P. tremuloides, P. wilsonii, P. canadensis, P. yunnanensis), conifers (e.g., loblolly pine (Pinus taeda ) , slash pine ( Pinus elliotii ) , ponderosa pine ( Pinus ponderosa ) , lodgepole pine ( Pinus contorta ), Monterey pine ( Pinus radiata ); Douglas-fir ( Pseudotsuga menziesii ); Western hemlock ( Tsuga canadensis ); Sitka spruce ( Picea glauca ) ; redwood ( Sequoia sempervirens ); silver oven ( Abies amabilis ); balsam oven ( Abies balsamea )) And cedar (for example, Western red cedar ( Thuja plicata , Alaska yellow-cedar ( Chamecyparis nootkatensis )), and the like.

In particular, it may be a herbaceous plant among herbaceous plants, and may be, but is not limited to, poplar in woody plants.

The term in the invention, "Arabidopsis thaliana (Arabidopsis thaliana is an important plant for genetics and molecular biology, which is a flowering plant belonging to the family Brassicaceae, which has no agricultural significance but is widely used as a model plant in modern botany. The Arabidopsis thaliana according to the present invention can be used as a representative plant of herbal plants, and specifically Arabidopsis thaliana , ecotype Columbia (Col-0).

The term " poplar " in the present invention is a generic term for plants belonging to the broad-leaved willow tree willow tree and the squash tree. Specifically, in the present invention, poplar can be used as a representative plant of woody plants, and Populus alba x P. tremula there is. glandulosa . < / RTI >

In an embodiment of the present invention, in order to design a binary vector capable of inducing the expression of a transgene in a thrombus-specific manner, the 2x35S promoter in the pMDC32 vector is restricted to HindIII and KpnI The enzyme was used to replace the DX15 promoter (SEQ ID NO: 1) to produce a DX15-pMDC32 vector. In addition, pine trees ( Pinus PdGA20ox amplified using cDNAs of densiflora stems and Gateway attBails (Invitrogen, Carlsbad, Calif.) primers were introduced into the DX15-pMDC32 vector following the DX15 promoter using Gateway cloning, followed by the DX15 :: PdGA20ox construct Respectively. The vector was then transformed into Agrobacterium tumefaciens strain C58 and the strains were transfected with Arabidopsis thaliana , ecotype Columbia (Corynebacterium glutamicum) according to the floral-dip method (Clough and Bent, 1998) and the agrobacterium mediated gene transformation method (Choi et al. Col-0) and Populus alba x P. tremula there is. glandulosa ).

In order to produce a transformed plant (DX15 :: PdGA20ox-2A-PtrMYB003) into which the DX15 promoter, PdGA20ox gene and PtrMYB003 gene have been introduced, a virus-derived 2A peptide sequence (QLLNFDLLKLAGDVESNPGP, SEQ ID NO: 13), the nucleotide sequence (SEQ ID NO: 14) was made so as to be optimized for codon usage of poplar. Although a single transcript is produced by linking two genes with this sequence, a very inefficient peptide bond of G (glycine) and P (proline) shown in red in FIG. 18 causes two proteins do. A fusion gene in which the PdGA20ox gene with the stop codon removed and the PtrMYB003 whole gene (SEQ ID NO: 15) were linked using the base sequence of the 2A peptide was prepared and linked to the DX15 promoter according to Example 2 to obtain DX15 :: PdGA20ox- 2A-PtrMYB003 construct (Fig. 17). The resulting recombinant gene was cloned into pMDC32 vector to transform Arabidopsis and poplar.

Specifically, the transgenic plant according to the present invention may be introduced with a gene coding for the DX15 promoter and PdGA20ox such that a decrease in leaf area due to overexpression of GA20ox or a side effect of reducing root weight loss is suppressed and the biomass is increased as compared with the wild type have.

In one embodiment of the present invention, 35S :: PdGA20ox poplar was observed to have adverse effects such as remarkable inhibition of leaf and root development caused by overexpression (35S :: PdGA20ox poplar) in all tissues of PdGA20ox. As a result, The leaf area decreased by 33 ~ 41% (Fig. 14a), and the leaf color showed pale green (Figs. 9a and 14a). However, the four lines of DX15 :: PdGA20ox poplar showed a darker green color than the 35S :: PdGA20ox poplar, especially line 4 showing the darkest green (Fig. 14a). In addition, DX15 :: PdGA20ox leaves showed a reduction of at least 21% compared to wild type, and overall leaf area was wider than 35S :: PdGA20ox (FIG. 14b). Thus, it was found that the DX15 :: PdGA20ox poplar of the present invention suppresses the side effect of leaf area reduction unlike the 35S :: PdGA20ox poplar even when PdGA20ox is overexpressed.

As a result of the analysis of the roots, the 35S :: PdGA20ox line showed a reduction of 73-78% in root weight compared to the wild type, while the DX15 :: PdGA20ox line showed a reduction of 40-67% (FIG. 15). The root weight of DX15 :: PdGA20ox poplar 3 corresponds to 170% of the root weight of 35S :: PdGA20ox Poplar 22 (FIG. 15b). Thus, it was found that the DX15 :: PdGA20ox poplar of the present invention suppresses side effects of root weight loss unlike 35S :: PdGA20ox poplar even when PdGA20ox is overexpressed.

In another embodiment, the stem diameter and stem weight were measured in order to confirm the biomass increase of DX15 :: PdGA20ox poplar. As a result, the transgenic poplar of the present invention showed remarkable increase of biomass (200% 300%) (Fig. 10B). From the above results, the DX15 :: PdGA20ox poplar of the present invention suppresses the side effects of reduced leaf area or root weight loss due to the overexpression of PdGA20ox (35S :: PdGA20ox poplar), and at the same time, the biomass is greatly increased And it can be used effectively in biomass related industries.

In another embodiment of the present invention, stem diameter and stem fresh weight were measured in order to confirm biomass increase of DX15 :: PdGA20ox Arabidopsis. As a result, all transgenic Arabidopsis spp. (Fig. 5A and 5B), which was a result consistent with the expression pattern of the transcription step (Fig. 2B). Lines 1-4 showed the largest increase in biomass by about 200% and lines 2-1, 3-3, and 4-7 showed 118%, 140%, and 97% biomass increases, respectively, and 35S :: PdGA20ox Arabidopsis thaliana (OX) also showed a similar increase (Figure 5b). As a result, it was confirmed that the overexpression of PdGA20ox of the present invention resulted in an increase in biomass. This increase was observed not only in the use of a general promoter but also in the case of using the DX15 promoter of the present invention as a thrombotic specific promoter.

Further, in one embodiment of the present invention, in comparison with the known neck specific promoter of the DX15 promoter of the present invention, the known thrombotic specific promoter was found in the woody development (DX) of the neck, , Expression was decreased in the mature woody part (MX), and even when expression was increased in the mature woody part, the total expression amount was much smaller than that of the DX15 promoter (Fig. 16), indicating that the DX15 promoter of the present invention Specific and at the same time the highest overall expression level.

In conclusion, the transgenic plants overexpressing PdGA20ox using the DX15 promoter express PdGA20ox at the same time as specific for the thymus. Therefore, plants that are known to suppress the adverse effects of leaf area reduction or root weight reduction and increase biomass It was found that it had a more remarkable effect.

Specifically, the transgenic plant according to the present invention is characterized in that the DX15 promoter, the gene encoding PdGA20ox and the PtrMYB003 are encoded so that the side effect of reducing the leaf area or reducing the root weight due to overexpression of GA20ox is suppressed and the biomass is increased as compared with the wild type, Can be introduced.

In one embodiment of the present invention, stem length, diameter, and biomass were measured to confirm the effect of overexpression of PdGA20ox and PtrMYB003 in Arabidopsis thaliana (DX15 :: PdGA20ox-2A-PtrMYB003) DX15 :: PdGA20ox-2A-PtrMYB003 The Arabidopsis thaliana was longer than the wild type (Fig. 19) and the stem diameter was larger (Fig. 20A). It was also confirmed that the stem weight of DX15 :: PdGA20ox-2A-PtrMYB003 Arabidopsis was increased by about 200% or more as compared with the wild type (Fig. 20B).

In addition, the expression patterns of PdGA20ox-2A-PtrMYB003 transcripts in the wild type (WT) and stem tissues of each transgenic Arabidopsis thaliana showed that expression of PdGA20ox-2A-PtrMYB003 transcript in DX15 :: PdGA20ox-2A-PtrMYB003 Arabidopsis And the stem fresh weight was remarkably increased as the expression amount of the transcript was increased (Figs. 20b and 20c).

In another embodiment of the present invention, in order to confirm the effect of overexpression of PdGA20ox and PtrMYB003 on overexpression of PdGA20ox and PtrMYB003 in the transformation (DX15 :: PdGA20ox-2A-PtrMYB003) poplar, the length, diameter and fresh weight of the stem As a result, it was confirmed that DX15 :: PdGA20ox-2A-PtrMYB003 poplar had a longer stem (FIG. 21) and a larger stem diameter than the wild type (FIG. 22A). In addition, it was confirmed that DX15 :: PdGA20ox-2A-PtrMYB003 poplar significantly increased stem fresh weight compared to wild type and 35S :: PdGA20ox poplar (Fig. 22B).

In another embodiment of the present invention, DX15 :: PdGA20ox-2A-PtrMYB003 poplar of the present invention was different from 35S :: PdGA20ox poplar in that PdGA20ox overexpression was stalk specific (FIG. 23).

In another embodiment of the present invention, the DX15 :: PdGA20ox-2A-PtrMYB003 poplar of the present invention has no side effect of leaf area reduction unlike the 35S :: PdGA20ox poplar even when PdGA20ox is overexpressed, And no side effects were observed (Figs. 25 and 26).

In another embodiment of the present invention, DX15 :: PdGA20ox-2A-PtrMYB003 poplar grown in the LMO test foreshape has a longer stem than the wild type (Figs. 27a and 28a) like the poplar grown in a flowerpot (Figs. 27B and 28B).

In another embodiment of the present invention, it has been found that the DX15 :: PdGA20ox-2A-PtrMYB003 poplar of the present invention has no side effects of inhibiting leaf growth due to overexpression of PdGA20ox even when cultivated in LMO test soil (FIGS. 30 and 31 ).

In another aspect, the present invention provides a method for producing a transgenic plant, comprising introducing a DX15 promoter and a PdGA20ox gene operably linked thereto into a plant.

The DX15 promoter, PdGA20ox gene and transgenic plants are as described above, and the transgenic plants of the present invention can be produced by the above method.

The transgenic plants produced by the above method can suppress the leaf area reduction due to the overexpression of GA20ox or the side effect of reducing the root biomass, and the biomass can be increased as compared with the wild type.

Specifically, the method may further comprise growing the transgenic plant in a soil or a medium. Cultivation of the transgenic plants in the present invention can be carried out according to well-known methods, and conditions such as cultivation temperature, cultivation time and pH of the medium can be appropriately adjusted.

The medium used should suitably meet the requirements of a particular transgenic plant. The medium may be a solid medium, a liquid medium or a double layer medium. Examples of the medium include water, colloid materials, agar, agarose, gelite, etc. may be used.

As inorganic nutrients, there are 15 elements, namely C, H, O, N, P, K, S, Ca, Mg, Fe, Mn, Cu, Zn, B and Mo, Nutrients include carbohydrates, plant growth regulators, vitamins and the like, and amino acids include myo-inositol, glycine, L-glutamine, and the like.

Examples of the carbon source include glucose, fructose, mannose, ribose, xylose, sucrose, melibiose, cellobiose, lactose, amylose, carbohydrate, raffinose, sorbitol, mannitol and glycerol l.

In one embodiment of the invention, Arabidopsis thaliana , ecotype Columbia (Col-0)) were grown in soil at 25 ° C in a growth chamber (14 hours light / 10 hours darkness) or in half-strength MS medium containing appropriate antibiotic and 2% sucrose for screening and Skoog, Sigma). The hybrid poplar ( Populus < RTI ID = 0.0 > There is alba x P. tremula . glandulosa ) were grown under the same conditions as the Arabidopsis thaliana.

In another embodiment, the present invention provides a DX15 promoter comprising the nucleotide sequence of SEQ ID NO: 1; A PdGA20ox gene encoding the amino acid sequence of SEQ ID NO: 3 operably linked to the promoter; And introducing into the plant a PtrMYB003 gene encoding the amino acid sequence of SEQ ID NO: 16, operably linked to the promoter.

The DX15 promoter, the PdGA20ox gene, the PtrMYB003 gene and the transgenic plant are as described above, and the transgenic plants of the present invention can be produced by the above method.

The transgenic plants produced by the above method can suppress the leaf area reduction due to the overexpression of GA20ox or the side effect of reducing the root biomass, and the biomass can be increased as compared with the wild type.

In another aspect, the present invention provides a method of producing a biomass comprising introducing a DX15 promoter and a PdGA20ox gene operably linked thereto into a plant to produce a transgenic plant. In addition, the production method of the present invention can further introduce the PtrMYB003 gene operably linked to the DX15 promoter in the step of producing the transgenic plant.

Specifically, the biomass may have an increased amount of glucose or xylose as compared to the wild type. As described above, the transgenic plants are inhibited from reducing the leaf area due to overexpression of GA20ox or reducing the root weight loss, Biomass can be increased compared to wild type.

In addition, since quantitative increase of biomass in the biomass-related industry and qualitative aspect such as sugar content are also important in the embodiment of the present invention, the composition of the cell wall monosaccharide in the transgenic poplar was measured. As a result, It was found that the total content of glucose and xylose in the PdGA20ox Arabidopsis and DX15 :: PdGA20ox poplar was greatly increased compared to the wild type (FIGS. 8 and 13). These results show that DX15 :: PdGA20ox plants have an increase in biomass biomass quantitatively as compared with wild type plants, and also have an increase in sugar components used as biomass fuel qualitatively. This is useful for biomass-related industries .

In addition, in one embodiment of the present invention, properties such as the length of the fibroblasts, which are most important in the pulp and paper-related industries, were investigated. As a result of measuring the length of the fibroblasts in the transgenic plants, it was confirmed that the length of the fibroblasts in the DX15 :: PdGA20ox Arabidopsis of the present invention was greatly increased as compared with that of wild-type plants (FIGS. 7A and 7B). This suggests that DX15 :: PdGA20ox plants have significantly increased fiber cell lengths compared to wild-type plants, which may be useful in pulp and paper-related industries.

In addition, although the content of wild type, cellulose, hemicellulose, and lignin was not significantly different in DX15 :: PdGA20ox-2A-PtrMYB003 poplar, the amount of cellulose was slightly increased in one embodiment of the present invention (FIG.

The transgenic plants according to the present invention inhibited the adverse effects of reduction of leaf area or root weight loss due to the overexpression of Gibberellin 20-oxidase (GA20ox) in all tissues, , The biomass increased by up to 300%, and the content of glucose and xylose in the monosaccharide composition of the cell wall was significantly increased, resulting in a qualitative change in the amount of sugar used as the biomass fuel. The length of the fibroblast was about 2 times longer than that of the fibroblast. Thus, the transgenic plants of the present invention are expected to ultimately increase the raw materials of the pulp and paper industry, raw materials for the wood industry, and bioethanol.

1 is a diagram illustrating a design of a gateway destination vector for neck-specific expression.
1A is a schematic diagram showing the replacement of the 2x35S promoter with the DX15 promoter using HindIII (5 ') and KpnI (3') .
1B is a diagram showing the result of verifying a novel vector by treating HindIII and KpnI with pMDC32 vector. The sizes of the digested fragments of pMDC32 and DX15-pMDC32 were 785 bp and 944 bp (white arrows), respectively. (Lane 1: DNA size marker, lane 2: pMDC32 (intact), lane 3: treatment of HindIII and KpnI with pMDC32 (H + K ), Lane 4: DX15-pMDC32 (intact), lane 5: DX15-pMDC32 treated with HindIII and KpnI (H + K).
FIG. 1C shows the result of analysis of the nucleotide sequence of the DX15 promoter in the novel vector. FIG.
FIG. 2 is a diagram showing the results of expressing PdGA20ox using the DX15 promoter (DX15 :: PdGA20ox plants: 1-4, 2-1, 3-3, 4-7, 6-5, 35S :: PdGA20ox plants: OX).
Figure 2a shows the expression of the PdGA20ox gene using semi-quantitative RT-PCR on the stem and rosette leaves of 35-year-old transgenic Arabidopsis thaliana (DX15 :: PdGA20ox plant: 3-3, 35S :: PdGA20ox plant: OX) Stem-specific expression.
FIG. 2b shows the expression pattern of PdGA20ox in a 7-year-old Arabidopsis plant to the transcription stage (Cycle = 26).
FIG. 3 shows the phenotype associated with the growth of DX15 :: PdGA20ox Arabidopsis plants.
FIG. 3A shows a 7-day-old T3 islet seedlings, wild type and OX grown on a half-strength MS medium (rod = 2 cm).
FIG. 3B is a graph showing the hypocotyl length of a seven-year-old plant grown under light conditions. The error bars mean the standard deviation, n = 15.
FIG. 3c is a diagram illustrating the hypocotyl length of a seven-year-old plant grown under dark room conditions. Error bars mean standard deviation and n = 15.
Fig. 4 shows a phenotype related to stem elongation of DX15 :: PdGA20ox Arabidopsis thaliana. A 35-year-old mature plant was used to analyze the error bars, which means standard deviation and n = 14.
Figure 4a compares the growth of DX15 :: PdGA20ox Arabidopsis with wild type and OX (rod = 10 cm).
FIG. 4B is a view comparing lengths of basic stems. FIG.
4C is a view showing stem elongation with time.
Figure 5 shows the increase in stem diameter (a) and stem fresh weight (b) of transgenic Arabidopsis thaliana (a: measured at 1 mm above the ground stem; b: Weight, 35-year-old plants, error bars mean standard deviation, n = 14)
6 is a diagram showing the results of histological analysis of thickened cell walls in transgenic Arabidopsis thaliana.
FIG. 6A is a view showing the result of using the bottom of the basic stem for hand cross-sectioning and staining it with toluidine blue O. FIG.
FIG. 6B is a view showing the result of using the base of the basic stem for hand cross-sectioning and staining it with fluoroglucinol-HCl.
6C is a graph showing the results of measurement of cell wall thickness using cells of a secondary layer of a tubular fiber. 35-year-old plants were used, the error bars were standard deviation, and n = 30.
FIG. 7 is a diagram showing elongation of fibroblasts in the transgenic Arabidopsis thaliana.
FIG. 7A is a graph showing the results of stem leaching of fibroblasts. FIG. 35 The stem base 0.5 mm was used and the rod was 200 μm in size.
FIG. 7B is a diagram showing the result of measuring the length of a fibrous cell. FIG. The error bars mean the standard deviation, n = 50.
Fig. 8 shows a monosaccharide composition of the cell wall in the transgenic Arabidopsis thaliana (Rha: Rhamos, Ara: Arabinose, Xyl: xylose, Man: mannose, Gal: galactose, Glu: glucose). The error bars indicate the standard deviation of the triplicate experiments.
Figure 9 shows the stem elongation of DX15 :: PdGA20ox in transgenic poplar. 60-year-old plants were used.
Figure 9a compares the growth of the DX15 :: PdGA20ox line and the 35S :: PdGA20ox line with the wild type (bar = 10 cm) in the transgenic poplar.
Figure 9b shows the expression pattern and stem specificity of the PdGA20ox gene at the enterprise level in poplar using semi-quantitative RT-PCR (30 cycles).
Figure 9c is a view showing the length of the base stem. The error bars indicate the standard deviation of the triplicate experiments.
FIG. 9D is a diagram showing the elongation of the stem according to time; FIG. The error bars indicate the standard deviation of the triplicate experiments.
10 is a graph showing changes in the stem diameter and biomass increase of DX15 :: PdGA20ox in the transgenic poplar. 60-year-old plants were used, and the error bars indicate the standard deviation of the triplicate experiments.
Figure 10a shows the increase in stem diameter as compared to the wild type in transgenic poplar.
Fig. 10B shows the increase in stem biomass in the transgenic poplar. Fig.
Fig. 11 shows the increase of the woody part of DX15 :: PdGA20ox poplar. Histological analysis (rod = 400 μm) was performed by staining the eleventh section of the 60-day-old seedlings with 0.5% toluidine blue O and phloroglucinol-HCl for 5 minutes.
Fig. 12 is a diagram showing woody characteristics in a transgenic poplar. Fig. The eleventh section of the 60-day-old plant was used, and the error bars mean the standard deviation, n> 8.
Figure 12a shows an increase in the thickness of the woody part in a transgenic poplar.
12B is a graph showing that the number of cell piles increased as well as the thickness of the woody part.
Figure 12c shows that the cell wall thickness of the woody part is reduced compared to the wild type.
Fig. 13 shows changes in the monosaccharide composition of the cell wall in the transgenic poplar (Rha: Rhamos, Ara: arabinose, Xyl: xylose, Man: mannose, Gal: galactose, Glu: glucose).
14 is a graph showing an effect of inhibiting the side effect of leaf area reduction caused by overexpression of PdGA20ox of DX15 :: PdGA20ox. 60-year-old plants were used, and the error bars indicate the standard deviation of the triplicate experiments.
FIG. 14A is a graph showing the results of analysis of the leaves of the tenth, eleventh and twelfth nodes.
FIG. 14B shows that the leaf area of DX15 :: PdGA20ox is wider than that of 35S :: PdGA20ox.
Fig. 15 is a graph showing an effect of suppressing side effects of root weight reduction due to overexpression of PdGA20ox of DX15 :: PdGA20ox. 60-year-old plants were used, and the error bars indicate the standard deviation of the triplicate experiments.
15A is a diagram showing that the root of DX15 :: PdGA20ox has a morphology similar to wild type as compared to 35S :: PdGA20ox.
15B shows that the root fresh weight of DX15 :: PdGA20ox is much higher than that of 35S :: PdGA20ox.
FIG. 16 is a graph comparing gene expression levels (a) and gene expression standardization (b) for various promoter activities in tissues in the transgenic poplar (6 dL: 6 day old female, SL: Mature leaf, BP: bark, DP: stem, Ca: cambium, DX: woody development, MX: mature woody part).
FIG. 17 is a schematic diagram showing a recombinant gene structure used in the production of a transgenic plant (DX15 :: PdGA20ox-2A-PtrMYB003).
18 is a diagram showing the amino acid sequence and base sequence of the 2A peptide. G (glycine) and P (proline) in red indicate amino acids that make ineffective peptide bonds.
19 is a diagram showing a phenotype related to stem elongation of DX15 :: PdGA20ox-2A-PtrMYB003 Arabidopsis thaliana. The numbers represent each independent T3 homozygous line number.
Fig. 19A is a photograph showing the appearance of the Arabidopsis thaliana grown in pots for 50 days. Fig.
FIG. 19B is a graph quantifying the growth of the stem. The error bars represent the standard deviation, and n = 14.
FIG. 20 shows growth of DX15 :: PdGA20ox-2A-PtrMYB003 Arabidopsis. The numbers represent each independent T3 homozygous line number.
20A is a graph quantifying the stem diameter of the Arabidopsis thaliana grown in pots for 50 days. The error bars represent the standard deviation, and n = 14.
20B is a graph quantifying the stem fresh weight (biomass) of Arabidopsis thaliana. The error bars represent the standard deviation, and n = 14.
20C is a diagram showing the change in expression of the PdGA20ox-2A-PtrMYB003 transcript in Arabidopsis thaliana.
21 is a view showing a phenotype related to stem elongation of DX15 :: PdGA20ox-2A-PtrMYB003 poplar. The numbers represent each independent T3 homozygous line number. WT is a wild type poplar, and the numbers represent each independent line number.
21A is a photograph showing the appearance of a poplar grown in pots for 60 days.
21B is a graph quantifying the stem growth of poplar. The error bars represent the standard deviation, and n = 3.
FIG. 22 is a graph showing an increase in the growth of DX15 :: PdGA20ox-2A-PtrMYB003 poplar. The error bars mean the standard deviation, n = 3
FIG. 22A is a graph quantifying the stem diameter of a poplar grown for 60 days in a flower pot. FIG.
22B is a graph quantifying the stem weight (biomass) of the poplar.
23 is an analysis of expression levels of PdGA20ox gene in the stem and leaf of transgenic poplar.
FIG. 23A is a diagram showing PdGA20ox gene expression patterns in the stem and leaves of wild type (WT) and the respective transgenic poplar (35S :: PdGA20ox, DX15 :: PdGA20ox-2A-PtrMYB003).
FIG. 23B is a graph comparing the expression of PdGA20ox gene in the stem of wild-type (WT) and the respective transgenic poplar (35S :: PdGA20ox, DX15 :: PdGA20ox-2A-PtrMYB003). The error bars represent the standard deviation, and n = 3.
24 is a graph showing the results of analyzing the woody component of DX15 :: PdGA20ox-2A-PtrMYB003 poplar.
25 is an analysis of leaf growth of DX15 :: PdGA20ox-2A-PtrMYB003 poplar.
25A is a photograph showing leaves of 10th, 11th and 12th nodes of poplar grown for 60 days in pots.
25B is a graph showing an average value of the leaf areas of the tenth, eleventh and twelfth nodes of the poplar. The error bars represent the standard deviation, n = 9
26 is a graph showing the root weight of the root of DX15 :: PdGA20ox-2A-PtrMYB003 poplar grown for 60 days in pots. The error bars represent the standard deviation, and n = 3.
FIG. 27 is an analysis of stem growth of DX15 :: PdGA20ox-2A-PtrMYB003 poplar grown in an outdoor LMO test forage for 3 months from spring to late summer. The error bars refer to the standard deviation. # 3, n = 6; # 4, n = 6), WT (n = 8), 35S :: PdGA20ox poplar (# 22, n = 6; # 32, n = 6), DX15 :: PdGA20ox- 4; # 7, n = 5)
27A is a graph quantifying the stem length of the poplar.
27B is a graph quantifying the stem diameter of the poplar.
FIG. 28 is an analysis of the stem growth of DX15 :: PdGA20ox-2A-PtrMYB003 poplar grown in an outdoor LMO test forage for 3 months from spring to late summer. # 3, n = 6; # 4, n = 6), WT (n = 8), 35S :: PdGA20ox poplar (# 22, n = 6; # 32, n = 6), DX15 :: PdGA20ox- 4; # 7, n = 5)
FIG. 28A is a graph showing the result of measuring the length growth of the stem by date. FIG.
FIG. 28B is a graph showing a result of measuring the diameter (volume) growth of the stem by date. FIG.
FIG. 29 is an analysis of leaf growth (leaf area) of DX15 :: PdGA20ox-2A-PtrMYB003 poplar grown in outdoor LMO test forage for 3 months from spring to late summer.
29A is a photograph showing the leaf of the 10th to 18th node of the poplar.
FIG. 29B is a graph showing an average area (3 repetitions) of the largest leaf among the 10th to 18th leaves of poplar. The error bars mean the standard deviation, n = 9.
FIG. 30 is an analysis of leaf growth (chlorophyll content) of DX15 :: PdGA20ox-2A-PtrMYB003 poplar grown in an outdoor LMO test forage for 3 months from spring to late summer.
30A is a photograph showing leaves of the 10th to 12th nodes of the poplar.
30B is a graph showing the chlorophyll content of leaves of the 10th to 12th nodes of poplar. The error bars represent the standard deviation, and n = 3.
FIG. 31 is a graph showing the results of measurement of winter bud formation of DX15 :: PdGA20ox-2A-PtrMYB003 poplar from LMO test forage from early autumn (September 21) to October 20. (# 3, n = 6; # 4, n = 5), WT (n = 8), 35S :: PdGA20ox poplar (# 22, n = 5; # 32, n = 5), DX15 :: PdGA20ox-2A-PtrMYB003 Poplar 6)

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1. Plant and growth conditions used in the present invention

Arabidopsis thaliana, ecotype Columbia (Col-0) used in the present invention is cultivated in soil at 25 ° C in a growth chamber (14 hours light / 10 hours darkness) or grown in soil with appropriate antibiotic and 2% sucrose for screening (MS) medium (Murashige and Skoog, Sigma). The hybrid poplar ( Populus < RTI ID = 0.0 > There is alba x P. tremula . glandulosa ) were also cultivated under the same conditions as the Arabidopsis thaliana.

Example  2. Vector design and plant transformation

The pMDC32 vector (Plant Physoiol., 133, 462-469, 2003) was modified as follows to design a binary vector capable of inducing the expression of the transgene in a throat-specific manner. 1A, the DX15-pMDC32 vector was prepared by replacing the 2x35S promoter with the DX15 promoter (SEQ ID NO: 1) using restriction enzymes HindIII and KpnI in the pMDC32 vector, and the new vector was verified by treating HindIII and KpnI (Fig. 1B). In addition, the nucleotide sequence of the DX15 promoter was analyzed in the DX15-pMDC32 vector to confirm that the 2x35S promoter was replaced with the DX15 promoter (FIG.

As a substrate pine ( Pinus (SEQ ID NO: 2) of PdGA20ox was amplified by PCR using cDNAs of densiflora stems and Gateway attBails (Invitrogen, Carlsbad, Calif.) primers. The amplified PdGA20ox gene was introduced into the DX15-pMDC32 vector following the DX15 promoter using a gateway cloning method to construct a DX15 :: PdGA20ox construct.

In order to overexpress PdGA20ox in all tissues of plants, a 35S :: PdGA20ox construct was prepared by introducing the PdGA20ox gene after the 35S promoter in the pBI121 vector (EMBO J., 6, 3901-3907, 1987) (Comparative Example).

The vector was then transformed into Agrobacterium tumefaciens strain C58 and transformed with Arabidopsis thaliana and poplar according to the floral-dip method (Clough and Bent, 1998) and the agrobacterium mediated gene transformation method (Choi et al., 2005). The construct used in the present invention was confirmed by DNA sequencing, and the primers used are shown in Table 1 below.

Gene Name Gene I.D. Primers Sequence (5 '- > 3') Restriction site DX15 promoter
Potri.009G012200
FW GGG AAGCTT TTGTATCCGGAGGACATATTTGTT (SEQ ID NO: 4) HindIII RV TTT GGTACC AGCTAGGAGTGCTGTTTTGTTGCA (SEQ ID NO: 5) KpnI PdGA20ox KC461180.1 ^a FW aaaagcaggctATGGGTACTTCGACTGTGAGT (SEQ ID NO: 6) - RV agaaagctgggtTTATGGCTGGTTTCTTGAGG (SEQ ID NO: 7) - RV-p2A GACGTCACCTGCAAGCTTAAGAAGG TCGAAGTT AAGAAGCTG TGGCTGGTTTCTTGAGGTGAA (SEQ ID NO: 8) - AtActin8
AT1G49240
FW ATGAAGATTAAGGTCGTGGCA (SEQ ID NO: 9) - RV TCCGAGTTTGAAGAGGCTAC (SEQ ID NO: 10) - PagActin2 FW GCCATCTCTCATCGGAATGGAA (SEQ ID NO: 11) - RV AGGGCAGTGATTTCCTTGCTCA (SEQ ID NO: 12) -

In Table 1, restriction enzymes, Gateway AttB1 and AttB2 sites are indicated by under- and lower-case letters, and partial 2A nucleotide sequences for forward and reverse primers are shown in italics. a is GenBank (http://www.ncbi.nlm.nih.gov/genbank/) accession number.

Example  3. Analysis method

Example  3-1. Histological analysis

Arabidopsis and poplar stem sections were stained with 2% phloroglucinol / HCl or 0.05% toluidine blue O.

Example  3-2. RNA  Extraction and RT - PCR

Total RNA was extracted according to the instructions using Trizol reagent. The plant tissue was ground with a fine powder using LN ₂ and mixed with the triazole reagent to treat the chloroform. The mixture was treated with isopropanol to separate the RNA. 1 μg of total RNA isolated from the 20 μl reaction mixture using Superscript III reverse transcriptase (Invitrogen) was reverse-transcribed and diluted 2-fold. RT-PCR was then performed with 1 [mu] l of the reaction as substrate.

Example  3-3. Fiber length analysis

For fiber length measurements, plant stems were named according to Franklin's method (Franklin, 1945). Trimmed pieces of plant stems were incubated in 30% hydrogen peroxide and glacial acetic acid solution at room temperature overnight. The next day, the sample was washed three times with distilled water, neutralized with sodium hydrogencarbonate, and washed with distilled water. Vortexing and shaking were used to separate the fibrous tissue and observed with a microscope.

Example  3-4. Alcohol insoluble cell wall Residue ( AIR ) Extraction and analysis of monosaccharide composition of cell wall

35 The 10 cm of the upper part of the stem of the Arabidopsis thaliana was made into fine flour using LN ₂ and a mortar bowl. The samples were treated with 70% ethanol and incubated at 70 ° C for 20 minutes with vortexing periodically. The insoluble cell wall was separated by a centrifuge, and then washed three times or more. Two of them were washed with 100% ethanol. Finally, the cell walls were again washed with 100% acetone and vacuum dried at 70 < 0 > C.

5 mg of AIRs were hydrolyzed with 72% sulfuric acid at room temperature for 30 minutes. An inositol solution of 0.4 mg / ml was used as internal standards. The sulfuric acid was diluted to 6%, the standard was added, and the sample was incubated at 105 ° C for 2 hours. After cooling at room temperature, 0.6 ml of a 25% ammonia solution was added, and 1.5 ml of the mixture was taken out with a glass pipette. After spun down at 12,000 rpm for 3 minutes, 300 μl of the supernatant was transferred to a new vial. The mixture of the solution and 2 ml of sodium borohydride was incubated at 42 캜 for 90 minutes while periodically turning every 30 minutes. Then 4 ml of acetic anhydride and 0.4 ml of methylimidazole were added. After incubation at room temperature for 30 minutes, the solution was transferred to a separatory funnel, the anhydrous acetic acid was dissolved in 20 ml of demineralized water, and 8 ml of dichloromethane was added. The lower layer of the solution was washed three times with water, and the supernatant was collected. The solution was then completely dried at 50 < 0 > C for 16 hours. The dried lumps were dissolved in 50 mu l of dichloromethane, and then 1 mu l was injected into gas chromatography (HP 4890).

Example  4. Analysis results of transgenic Arabidopsis thaliana

Example  4-1. In Transgenic Arabic Pole DX15  Promoter PdGA20ox Of stem-specific overexpression

The expression of PdGA20ox in the stems and leaves was confirmed by semi-quantitative RT-PCR (Fig. 2) in order to confirm that stem-specific overexpression of PdGA20ox was caused by the DX15 promoter in the DX15 :: PdGA20ox Arabidopsis of the present invention.

First, two kinds of Arabidopsis thaliana transformed to overexpress PdGA20ox (35S :: PdGA20ox or DX15 :: PdGA20ox) were prepared. Among the 10 DX15 :: PdGA20ox transformed Arabidopsis thaliana lines 1-4, 2-1, 3-3 and 4-7 were selected for this experiment and one representative line (OX) line for 35S :: PdGA20ox Arabidopsis was selected. Were used.

Semi-quantitative RT-PCR of the transgenic Arabidopsis thaliana showed that PdGA20ox was not expressed in the leaves of DX15 :: PdGA20ox Arabidopsis thaliana (3-3) unlike the stem, whereas 35S :: PdGA20ox Arabidopsis In the case of the pole (OX), it was confirmed that the PdGA20ox gene was overexpressed strongly in both the stem and leaf (Fig. 2a).

In addition, most DX15 :: PdGA20ox Arabidopsis PdGA20ox genes similar to 35S :: PdGA20ox Arabidopsis (Fig. 2B).

As a result, the DX15 :: PdGA20ox Arabidopsis of the present invention overexpresses PdGA20ox similar to 35S :: PdGA20ox Arabidopsis, and at the same time, the overexpression was specific to the stem.

Example  4-2. Analysis of transgenic Arabidopsis plants

Gibberellin is known to be an important positive regulator of stem and hypocotyl cell growth, and hypocotyl length was identified in 7 - day - old plants in the transgenic Arabidopsis thaliana overexpressing PdGA20ox.

As a result, it was confirmed that all seven life plants of DX15 :: PdGA20ox and OX Arabidopsis have longer hypocotyl length than the wild type (WT) (FIGS. 3A and 3B). Also, in the light conditions, the hypocotyl of all transgenic Arabidopsis lines was about 27% longer than the wild type (Fig. 3b), while there was no change in the dark room conditions (Fig. 3c).

In other words, it was confirmed that PdGA20ox overexpression of PdGA20ox overexpresses wild-type transgenic Arabidopsis thaliana regardless of the promoter, resulting in increased hypocotyl length biosynthesis and hypocotyl extension.

Example  4-3. Stem height analysis of transgenic Arabidopsis thaliana

Stem length, stem elongation rate and bolting time were measured to confirm the effect of PdGA20ox overexpression in transgenic Arabidopsis thaliana.

As a result of measuring stem length, it was confirmed that all the DX15 :: PdGA20ox Arabidopsis poles had longer stem than the wild type, just as the hypocotyl length was confirmed in the young plant stage (FIG. 4B). Also, by confirming that most DX15 :: PdGA20ox Arabidopsis thaliana has a stem length similar to that of 35S :: PdGA20ox Arabidopsis, PdGA20ox overexpressed transgenic Arabidopsis overexpresses PdGA20ox over wild type regardless of promoter, I could see the lengthening.

As a result of measuring the stem elongation rate, the elongation rate (18.2 mm / day) of the present invention having the longest stem was faster than OX (17.7 mm / day), and most of the transgenic Arabidopsis thaliana was wild type Lt; / RTI > mm / day (Figure 4c). Thus, it can be seen that the DX15 :: PdGA20ox Arabidopsis of the present invention can shorten the time required to produce biomass due to the faster growth rate than the wild type and 35S :: PdGA20ox Arabidopsis.

As can be seen in Table 2 below, DX15 :: PdGA20ox Arabidopsis 1-4 and 35S :: PdGA20ox Arabidopsis OX started to harvest when they had 8 and 7.8 rosette leaves (RL), respectively, In comparison with the wild type, the petioles started to grow when they had 11 rosette leaves, and the length of petiole showed a similar tendency to stem length (Table 2). As a result, it was found that the phenotype as shown in FIG. 4A was caused not only by the elongation rate but also by the faster chasing time.

As a result, PdGA20ox overexpression of PdGA20ox overexpressed Arabidopsis thaliana was overexpressed in PdGA20ox over wild type, regardless of the promoter, resulting in longer stem length, faster stem extension rate, and faster chimerism. Further, it was found that the DX15 :: PdGA20ox Arabidopsis of the present invention can shorten the time required to produce biomass due to the faster growth rate than the wild type and 35S :: PdGA20ox Arabidopsis.

n = 14

Example  4-4. Transgenic Arabian Pole Biomass  Analysis

Stem diameter and stem fresh weight were measured to confirm the biomass increase of DX15 :: PdGA20ox Arabidopsis of the present invention (FIG. 5).

All transgenic Arabidopsis thalamuses increased stem diameter and stem viability (Figs. 5a and 5b), consistent with the expression pattern of the transcriptional stage. 1-4 showed the greatest increase in biomass of about 200%, 2-1, 3-3 and 4-7 showed 118%, 140% and 97% biomass increase respectively, and OX showed similar increase (Fig. 5B).

As a result, it was confirmed that the overexpression of PdGA20ox of the present invention resulted in an increase in biomass. This increase was observed not only in the use of a general promoter but also in the case of using the DX15 promoter of the present invention as a thrombotic specific promoter.

Example  4-5. Secondary cell wall thickness and fibroblast kidney analysis of transformed Arabidopsis thaliana

The number or length of the thymus tissue fibers during tree formation is known to be affected by gibberellin (Ann. Bot., 30, 539-548., 1966), through histological analysis of stem sections in the transformed Arabidopsis thaliana Fiber cell elongation was measured by secondary cell wall thickness and stem leaching (maceration).

As a result, it was confirmed that all of the transformed Arabidopsis thaliana cells had increased secondary cell wall thickness (Fig. 6). Although the cell wall thickness results do not exactly correspond to the stem diameter results (Fig. 5A), it was found that the PdGA20ox overexpressed transgenic Arabidopsis thaliana increases the secondary cell wall thickness by overexpressing PdGA20ox compared to the wild type regardless of the promoter.

In addition, it was confirmed that the fibroblasts of the transgenic Arabidopsis thaliana were greatly elongated through stem leaching (Fig. 7A). The fiber length of DX15 :: PdGA20ox Arabidopsis thaliana (1-4), which has the greatest stem elongation, was about 93% higher than that of wild type and was similar to 35S :: PdGA20ox Arabidopsis thaliana (OX) (FIG. These trends were consistent with gene expression pattern, stem elongation and hypocotyl extension at the transcriptional stage. As a result, it was found that PdGA20ox overexpressed transgenic Arabidopsis thaliana overexpresses PdGA20ox over the wild type, irrespective of the promoter.

Example  4-6. Analysis of cell wall monosaccharide composition in transgenic Arabidopsis thaliana

The quantitative increase of biomass in the biomass-related industry, as well as the qualitative aspect such as sugar content, are important, so the composition of cell wall monosaccharide was measured in the transformed Arabidopsis thaliana (Fig. 8).

As a result, it was confirmed that the total content of glucose and xylose was greatly increased. In particular, the glucose ratio of the whole composition of the cell wall was increased in agreement with the expression pattern of the PdGA20ox gene. The xylose content was also increased compared to the wild type, but not to the glucose (Fig. 8).

As a result, PdGA20ox overexpression of PdGA20ox overexpressed PdGA20ox over the wild type, regardless of the promoter, resulting in an increase in the amount of sugar used as biomass fuel in addition to a quantitative increase in biomass, And can be usefully used in related industries.

Example  5. Analysis of transgenic poplar

Example  5-1. From Transgenic Poplar DX15  Promoter PdGA20ox Of stem-specific overexpression

The expression of PdGA20ox in the stems and leaves was confirmed by semi-quantitative RT-PCR (Fig. 9B) in order to confirm that stem specific-overexpression of PdGA20ox was caused by the DX15 promoter in DX15 :: PdGA20ox poplar of the present invention.

First, four DX15 :: PdGA20ox lines (3, 4, 7 and 13) and two 35S :: PdGA20ox lines (22 and 32) were prepared. Semi-quantitative RT-PCR of the transgenic poplar revealed that DX15 :: PdGA20ox poplar did not express PdGA20ox in the leaves, unlike the stem, whereas 35S :: PdGA20ox poplar showed no expression in both the stem and leaf It was confirmed that PdGA20ox was strongly overexpressed (FIG. 9B).

Thus, the DX15 :: PdGA20ox poplar of the present invention overexpresses PdGA20ox in a stem-specific manner, unlike the 35S :: PdGA20ox poplar.

Example  5-2. Stem height analysis of transgenic poplar

To determine the effect of overexpression of PdGA20ox in transgenic poplar, stem length (FIG. 9c), stem elongation rate (FIG. 9d) and length of interstices and petiole lengths (Table 3) were measured.

9a and 9b, the stem elongation was greatly increased by PdGA20ox overexpression by the 35S and DX15 promoter in Poplar, similar to the transgenic Arabidopsis thaliana. Thus, the transgenic Poplar overexpressing PdGA20ox was found to be more potent than the wild type regardless of the promoter PdGA20ox was overexpressed.

As a result of measuring the elongation rate of the stem, the elongation rate (1.36 cm / day) of the DX15 :: PdGA20ox Poplar 13 of the present invention having the longest stem was similar to 35S :: PdGA20ox Poplar 32 (1.44 cm / day) The transgenic poplar was much faster than the wild type elongation rate 0.61 cm / day (Fig. 9d).

These results indicate that PdGA20ox overexpressed PdGA20ox overexpresses wild-type transgenic poplar regardless of the promoter, and thus can be usefully used in biomass related industries because it has a rapid stem growth rate.

In addition, as shown in Table 3, the length of intersex and petiole was elongated according to stem height and the number of total intersex sections also increased (Table 3). As a result, the transgenic Poplar overexpressing PdGA20ox overexpressed PdGA20ox over the wild type regardless of the promoter, indicating that the length and length of the petiole were extended.

a: the length between the tenth, eleventh and twelfth sections

b: Length of 10th, 11th and 12th petiole

n = 3

Example  5-3. Transgenic Poplar Biomass  And changes in woody parts

In order to confirm the increase of the biomass of DX15 :: PdGA20ox poplar of the present invention, stem diameter and stem weight were measured (FIG. 10).

First, the diameter of the stem was measured to confirm that the stem diameter of DX15 :: PdGA20ox poplar was larger than that of the wild type and 35S :: PdGA20ox poplar (FIG. 10A). It was found that the transgenic poplar of the present invention is characterized by the transgenic poplar, which is a woody plant, and that side effects due to the overexpression of PdGA20ox can be suppressed due to the relatively large stem.

In addition, as a result of the stem biomass measurement, DX15 :: PdGA20ox poplar increased stem biomass about twice to three times compared to the wild type for 60 days (FIG. 10B). As a result, it was found that the DX15 :: PdGA20ox poplar of the present invention showed a remarkable increase (200% to 300%) of biomass than the wild type poplar.

As a result, the DX15 :: PdGA20ox poplar showed a remarkable increase in biomass compared to the wild type poplar, and it was found that the side effect due to overexpression of PdGA20ox could be suppressed due to the thick stem.

Example  5-4. Analysis of changes in the neck of transgenic poplar

To determine the effect of PdGA20ox overexpression in transformed poplar, stem sections (Fig. 11) and neck changes (Fig. 12) were measured by histological analysis.

Stem cross sections of the transgenic poplar were checked, and the neck was increased in all transformed lines (Fig. 11) when the same growth stage (eleventh stage) was compared with the wild type.

In addition, the results of the neck changes showed that the neck area of 35S :: PdGA20ox 22 and 32 increased only 30% and 33%, respectively, while the DX15 :: PdGA20ox line increased up to 69% compared with the wild type (FIG. This increased neck was due to an increase in the number of xylem cell piles (Fig. 12b). These results suggest that GA20ox overexpression promotes cell division rather than cell size. In contrast to the Arabidopsis thaliana, the cell wall thickness of the woody tissue fibers was reduced compared to the wild type (Fig. 12C).

The results showed that DX15 :: PdGA20ox poplar increased the number of throat and thymus cell pals compared to wild type and 35S :: PdGA20ox poplar.

Example  5-5. Analysis of cell wall monosaccharide composition in transgenic poplar

In the biomass-related industry, the amount of monosaccharide of cell wall was measured in the transgenic poplar (Fig. 13), because quantitative increase of biomass and qualitative aspect such as sugar content are important.

As a result, it was confirmed that the content of xylose and glucose was greatly increased in the transgenic poplar, as well as in the Arabidopsis thaliana. These results indicate that DX15 :: PdGA20ox poplar has an increase in sugar content as a biomass fuel compared with wild type, as well as a quantitative increase in biomass compared with wild type and 35S :: PdGA20ox poplar, Indicating that it can be usefully used in industry.

Example  6. Trace phone of poplar PdGA20ox  Effect of over-expression of side effects

The method of increasing the plant biomass using the quantitative increase of gibberellin promotes the stem elongation of the plant, but the side effects such as the remarkable inhibition of the development of the leaf and the root are appeared. Therefore, the leaves and roots of DX15 :: PdGA20ox poplar of the present invention And the presence or absence of the side effect was measured (FIGS. 14 and 15).

First, 35S :: PdGA20ox poplar showed a leaf area reduced by 33 ~ 41% (Fig. 14a) compared with that of wild type, and leaf color showed pale green (Figs. 9a and 14a). However, the four lines of DX15 :: PdGA20ox poplar showed a darker green color than the 35S :: PdGA20ox poplar, especially line 4 showing the darkest green (Fig. 14a). In addition, DX15 :: PdGA20ox leaves showed a reduction of at least 21% compared to wild type, and overall leaf area was wider than 35S :: PdGA20ox (FIG. 14b).

Thus, it was found that the DX15 :: PdGA20ox poplar of the present invention suppresses the side effect of leaf area reduction unlike the 35S :: PdGA20ox poplar even when PdGA20ox is overexpressed.

As a result of root analysis, root growth in 35S :: PdGA20ox poplar was significantly affected by PdGA20ox overexpression. The 35S :: PdGA20ox line showed a 73-78% reduction in root biomass compared to the wild type, while the DX15 :: PdGA20ox line showed only a 40-67% reduction (Fig. 15). The root weight of DX15 :: PdGA20ox poplar 3 corresponds to 170% of the root weight of 35S :: PdGA20ox Poplar 22 (FIG. 15b).

Thus, it was found that the DX15 :: PdGA20ox poplar of the present invention suppresses side effects of root weight loss unlike 35S :: PdGA20ox poplar even when PdGA20ox is overexpressed.

From the above results, the DX15 :: PdGA20ox poplar of the present invention has the effect of suppressing the leaf area reduction due to overexpression of PdGA20ox or the side effect of reducing the root weight, and at the same time, the biomass is significantly increased compared to the wild type poplar, And can be usefully used in related industries.

Example  7. DX15  Comparison with other thrombotic specific promoters of promoters

Through the above examples, it was confirmed that the DX15 promoter of the present invention can suppress not only the effect of overexpression but also the side effect that may be caused by overexpression when overexpressing PdGA20ox due to thrombotic specificity, In the examples, the DX15 promoter of the present invention was compared with other known thrombotic specific promoters. Specifically, the neck-specific expression intensity of the DX15 promoter and the known thymus-specific promoter was compared (Fig. 16).

As a result, the known thrombotic specific promoter was generally expressed in the woody developmental part (DX) of the neck, but the expression was decreased in the mature woody part (MX). In addition, it was confirmed that even when the expression level is increased in the mature woody part, the total expression amount is much smaller than the DX15 promoter.

As a result, it was found that the DX15 promoter of the present invention was the most thrombotic specific and the highest total expression amount.

In conclusion, the transgenic plants overexpressing PdGA20ox using the DX15 promoter express PdGA20ox at the same time as specific for the thymus. Therefore, plants that are known to suppress the adverse effects of leaf area reduction or root weight reduction and increase biomass It was found that it had a more remarkable effect.

Example  8. DX15  Promoter, PdGA20ox  Gene and PtrMYB003  Transgenic transformation with gene DX15 :: PdGA20ox -2A- PtrMYB003 ) Production of plants

To prepare the transformed (DX15 :: PdGA20ox-2A-PtrMYB003) plants into which the DX15 promoter, PdGA20ox gene and PtrMYB003 gene were introduced, the virus 2A system was used. The nucleotide sequence (SEQ ID NO: 14) was made to optimize for the codon usage of the poplar while encoding the 2A peptide sequence (QLLNFDLLKLAGDVESNPGP, SEQ ID NO: 13) derived from the virus. Although a single transcript is produced by linking two genes with this sequence, a very inefficient peptide bond of G (glycine) and P (proline) shown in red in FIG. 18 causes two proteins do.

A fusion gene was constructed by linking the PdGA20OX gene and PtrMYB003 whole gene (SEQ ID NO: 15) from which the stop codon was removed by using the nucleotide sequence of the 2A peptide. The fusion gene was linked to the DX15 promoter according to Example 2 to obtain DX15 :: PdGA20ox- 2A-PtrMYB003 construct (Fig. 17). The resulting recombinant gene was cloned into pMDC32 vector to transform Arabidopsis and poplar.

The construct used in the present invention was confirmed by DNA sequencing, and the primers used are shown in Table 4 below.

Gene Name Gene I.D. Primers Sequence (5 '- > 3') PtrMYB003 Potri.001G267300 FW aaaaagcaggctATGAGGAAGCCGGATCTAATG
(SEQ ID NO: 17) FW-p2A CTTCTTAAGCTTGCAGGTGACGTCGAGTCAAACCCAGGTCCA ATGAGGAAGCCGGATCTAATG (SEQ ID NO: 18) RV agaaagctgggtTTATAAAACTTGGAAATCAAGGAAAGGAAAGG
(SEQ ID NO: 19)

In Table 4, Gateway AttB1 and AttB2 sites are shown in lower case, and partial 2A nucleotide sequences for forward and reverse primers are shown in italics.

Example  9. DX15 :: PdGA20ox -2A- PtrMYB003  Analysis of Arabidopsis

DX15 :: PdGA20ox-2A-PtrMYB003 To determine the effect of PdGA20ox and PtrMYB003 on the neck-specific overexpression in Arabidopsis, stem length, diameter and biomass were measured.

As a result, the stem of DX15 :: PdGA20ox-2A-PtrMYB003 Arabidopsis was longer than that of wild type (Fig. 19) and the stem diameter was larger (Fig. 20A). It was also confirmed that the stem weight of DX15 :: PdGA20ox-2A-PtrMYB003 Arabidopsis was increased by about 200% or more as compared with the wild type (Fig. 20B).

In addition, expression patterns of PdGA20ox-2A-PtrMYB003 transcripts in wild type (WT) and stem tissues of each transformed Arabidopsis thaliana were analyzed by semi-quantitative RT-PCR (26 cycles).

As a result, the PdGA20ox-2A-PtrMYB003 transcript was expressed in DX15 :: PdGA20ox-2A-PtrMYB003 Arabidopsis, and the stem fresh weight was remarkably increased as the expression amount of the transcript was increased (FIGS. 20b and 20c) .

Example  10. DX15 :: PdGA20ox -2A- PtrMYB003  Analysis of Poplar

Example  10-1. DX15 :: PdGA20ox -2A- PtrMYB003  Stem Growth Analysis of Poplar

Transformation (DX15 :: PdGA20ox-2A-PtrMYB003) To determine the effect of PdGA20ox and PtrMYB003 on the neck-specific overexpression in poplar, stem length, diameter and biomass were analyzed.

As a result, it was confirmed that the stem of DX15 :: PdGA20ox-2A-PtrMYB003 poplar was longer than the wild type (Fig. 21) and the stem diameter was larger (Fig. 22A). In addition, it was confirmed that DX15 :: PdGA20ox-2A-PtrMYB003 poplar significantly increased stem fresh weight compared to wild type and 35S :: PdGA20ox poplar (Fig. 22B).

In addition, as shown in Table 5, the length and length of the stem and the petiole of DX15 :: PdGA20ox-2A-PtrMYB003 poplar were elongated according to the stem elongation, and the number of the whole stem was also increased. In addition, it was found that the length of the interspecies and petiole were also increased.

Example  10-2. DX15 :: PdGA20ox -2A- PtrMYB003  From poplar DX15  Promoter PdGA20ox Of stem-specific overexpression

The expression of PdGA20ox in the stem and leaf was confirmed by semi-quantitative RT-PCR in order to examine whether the DX15 :: PdGA20ox-2A-PtrMYB003 poplar of the present invention overexpressed PdGA20ox in the stem-specific overexpression by the DX15 promoter

As a result, in the case of 35S :: PdGA20ox poplar, the PdGA20ox gene was overexpressed strongly in both the stem and the leaf, but in the case of DX15 :: PdGA20ox-2A-PtrMYB003 poplar, PdGA20ox was not expressed in the leaves unlike the stem ). In addition, 35S :: PdGA20ox poplar showed higher expression level of PdGA20ox gene in stem than DX15 :: PdGA20ox-2A-PtrMYB003 poplar (FIG. 23b).

As a result, the DX15 :: PdGA20ox-2A-PtrMYB003 poplar of the present invention was found to be staphylococcal-specific, unlike the 35S :: PdGA20ox poplar.

Example  10-3. DX15 :: PdGA20ox -2A- PtrMYB003  Analysis of woody component of poplar

DX15 :: PdGA20ox-2A-PtrMYB003 The constituents of woody part of poplar were analyzed by Van Soest technique.

As a result, it was confirmed that the content of wild type, cellulose, hemicellulose, and lignin was not significantly different but the amount of cellulose slightly increased in DX15 :: PdGA20ox-2A-PtrMYB003 poplar (FIG. 24).

Example  10-4. DX15 :: PdGA20ox -2A- PtrMYB003  Poplar PdGA20ox  Effect of over-expression of side effects

The method of increasing the plant biomass using the quantitative increase of gibberellin promotes the stem elongation of the plant, but the side effects such as the remarkable inhibition of the development of the leaf and the root are appeared. Therefore, the leaves and roots of DX15 :: PdGA20ox poplar of the present invention And the adverse reaction was measured.

As a result, the leaf area of 35S :: PdGA20ox poplar was reduced compared with that of wild type, but DX15 :: PdGA20ox-2A-PtrMYB003 poplar had a leaf area similar to wild type and overall leaf area was smaller than that of 35S :: PdGA20ox poplar (Fig. 25).

Thus, the DX15 :: PdGA20ox-2A-PtrMYB003 poplar of the present invention was found to have no or less side effects of leaf area reduction unlike the 35S :: PdGA20ox poplar even when PdGA20ox was overexpressed.

As a result of root analysis, root growth in 35S :: PdGA20ox poplar was significantly affected by PdGA20ox overexpression. Although the 35S :: PdGA20ox poplar significantly reduced the root biomass compared to the wild type, the DX15 :: PdGA20ox-2A-PtrMYB003 poplar showed similar root-like or wild-type root weight (Fig. 26) .

Thus, the DX15 :: PdGA20ox-2A-PtrMYB003 poplar of the present invention was found to have no adverse effect on root weight loss unlike the 35S :: PdGA20ox poplar even when PdGA20ox was overexpressed.

The results showed that the DX15 :: PdGA20ox-2A-PtrMYB003 poplar of the present invention did not show a side effect of reduced leaf area or root weight loss due to the overexpression of PdGA20ox, and at the same time, the biomass of 35S :: PdGA20ox poplar And it can be used effectively in biomass related industries.

Example  11. LMO In Test Forage  Grown up DX15 :: PdGA20ox -2A- PtrMYB003  Analysis of Poplar

Example  11-1. LMO In Test Forage DX15 :: PdGA20ox -2A- PtrMYB003  Poplar cultivation

The LMO test forge was planted in accordance with the LMO law of the Ministry of Environment in the forest genetic resources department (Suwon city, Suwon city) and DX15 :: PdGA20ox-2A-PtrMYB003 poplar was cultivated in 30cm x 60cm intervals with wild type (WT) .

Example  11-2. LMO In Test Forage  Grown up DX15 :: PdGA20ox -2A- PtrMYB003  Poplar analysis result

The length, diameter and weight of the stem were analyzed in the DX15 :: PdGA20ox-2A-PtrMYB003 poplar grown in the LMO test fores.

As a result, the DX15 :: PdGA20ox-2A-PtrMYB003 poplar grown in the LMO test foreshadows a stem longer than the wild type, similar to the poplar grown in the pollen (Figs. 27A and 28A). In addition, it was confirmed that the stem diameter of DX15 :: PdGA20ox-2A-PtrMYB003 poplar was larger than that of wild type and 35S :: PdGA20ox poplar (FIGS. 27B and 28B).

Example  11-3. LMO In Test Forage  Grown up DX15 :: PdGA20ox -2A- PtrMYB003  Identification of the effect of poplar on PdGA20ox overexpression

Leaf area, chlorophyll content, and winter bud formation were measured to determine whether adverse effects of leaf growth were inhibited by the quantitative increase of gibberellin in DX15 :: PdGA20ox-2A-PtrMYB003 poplar grown in LMO test fores .

As a result, it was confirmed that Poplar grown in the LMO test fores showed similar tendency to poplar grown in the pollen. The 35S :: PdGA20ox poplar grown in the LMO test fores reduced leaf area compared to the wild type, whereas the DX15 :: PdGA20ox-2A-PtrMYB003 poplar grown in the LMO test fores had a leaf area similar to that of the wild type, : The leaf area was wider than that of PdGA20ox poplar (Fig. 29).

In addition, the 35S :: PdGA20ox poplar grown in the LMO test fores had a lighter green leaf color and lower chlorophyll content compared to the wild type, while DX15 :: PdGA20ox-2A-PtrMYB003 poplar showed similar wild type leaf color and chlorophyll content (Fig. 30).

In addition, 35S :: PdGA20ox poplar grown in the LMO test fores was found to be very late compared to the wild type, while DX15 :: PdGA20ox-2A-PtrMYB003 poplar was found to be in the same age as the wild type (Fig. 31).

Thus, it can be seen that the DX15 :: PdGA20ox-2A-PtrMYB003 poplar of the present invention has no side effects of inhibiting leaf growth due to overexpression of PdGA20ox even when cultivated in LMO test soil.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Transgenic plants for increasing biomass production and method          for producing the same <130> KPA140514-KR-P1 <150> KR 10-2014-0108553 <151> 2014-08-20 <160> 19 <170> Kopatentin 2.0 <210> 1 <211> 1025 <212> DNA <213> Populus maximowiczii x nigra <400> 1 ttcccccttt tggttcaatg ccttttattc ttccaaaatt atttcatatt ttgtatccgg 60 aggacatatt tgtttcaaaa ggtgtcagaa aatcaaagcc cattgaaaat atataaacat 120 atatagatat aaaaactcaa gggttcattc caaaatataa gaacaaactg attgaattaa 180 tttgttattt taagaacact gtctatatgt ttatatagtg ggaggtagtg ttttttaaat 240 catatactaa cttattataa aaataaatca taaaaaagga acctcaagca tcccctggta 300 agctcgtatg taggaatact cggagatcaa atgtccgaat gtcaaatgtt aaggcaagtg 360 aaatatccct gactttttag caagcaaatt gttgagtagc taaaatgaat tattttaata 420 tttttaaatc attttaatat attaatatta aaaaaaatta aatatttttt ttaatacatt 480 ttcaataaca aacactttaa aatataatct ttgtcacact cttaaacagt aacagcagaa 540 agcatatgtg agtgatatag ctatagttgc tgtttgacac ggacaatctc catctaaatt 600 catgaataat aaagttttgc ctacacaccc acttgaaatc tcctcctagt tttcctgatt 660 tgccatgcta actacaagaa caagatgcta gctagtatct tgttctgtct ctcgctctct 720 ctctatctct ccagttgata gttgatagtt gatagttgat agctgatacc ctcccacctt 780 tcccagaaag atgattgagg aactagtcac tgtgttcgtg taactaatac tgttcatggc 840 acctaacttg atcctctctt caccagacca ctataaaaac cctatctgtc ctcctcataa 900 tcatatcact acacccaaca cttctgcaag cacaactcca ttcaagaaca tcaagagtat 960 aggccgccgc tgcaacaaaa cagcactcct agctacttca agatgaggcc acaatctttc 1020 atctt 1025 <210> 2 <211> 1107 <212> DNA <213> Pinus densiflora <400> 2 atgggtactt cgactgtgag tgggccaaac gtgccagtgt tgttcgatgc tagtattctg 60 caacgagagg agaagatgcc tgaggtattt gtttggcccc aagtggatcg accctccaca 120 gatttggcgt tgaagggaag ggagacagag gtgccattgc cgattatcga tttgggcgga 180 ttctggaaaa atgatgaaaa ggcaaccaaa gaagcgtcag acgttatagg gaaagcctgt 240 gtggaacatg gtttctttca ggtcatcaac catcaagtct ctccagacct cctcactgct 300 gctcatcaac acatcagttt gttttttagt ttgagtttag tggaaaaacg aagggctcaa 360 aggaagcttg gcgagagctg tggatatgcc agtagtttta cggatcgctt tgcaaataaa 420 ctaccatgga aggagacact gtctttcgag cacagtccct cttcagacgt tcgtgactat 480 tttgtcaaag ctatgggtga agattttcaa gacgctggaa atgtattcaa agagtactgt 540 gaagcaatgg aatgcttggc tcttggattg atggagttgt tgggaatgag cctcggaata 600 gggcgtctgc atcttcggaa attttttgaa ggtggcaact caataatgag gttgaactat 660 tatcctcctt gtgcgcagcc aaatctgaca cttgggactg gtccccattg cgatccgaca 720 tcccttactc tccttcatct ggacgaagtg ggaggtctgg agattttcat aaataataag 780 tggcattctg tgcgacccaa cactgatgcc ttcgttgtca acatcggaga cacctttatg 840 gcactgtcca atggtaaata caagagctgc ctccacagag cagtggtgaa caagacaaca 900 cctcgcaaat cactggcatt tttcctgaac ccaccatatg acaaaattgt acgacctcca 960 gatgatcttt tggattctga tcatcctcga aaatatccag atttcacgtg gcctgtgttc 1020 ttggagttca ctcagaaaca ctacagatca gacatgaata ctcttgcagc ttttcagaaa 1080 tggttcacct caagaaacca gccataa 1107 <210> 3 <211> 368 <212> PRT <213> Pinus densiflora <400> 3 Met Gly Thr Ser Thr Val Ser Gly Pro Asn Val Pro Val Leu Phe Asp   1 5 10 15 Ala Ser Ile Leu Gln Arg Glu Glu Lys Met Pro Glu Val Phe Val Trp              20 25 30 Pro Gln Val Asp Arg Pro Ser Thr Asp Leu Ala Leu Lys Gly Arg Glu          35 40 45 Thr Glu Val Pro Leu Pro Ile Ile Asp Leu Gly Gly Phe Trp Lys Asn      50 55 60 Asp Glu Lys Ala Thr Lys Glu Ala Ser Asp Val Ile Gly Lys Ala Cys  65 70 75 80 Val Glu His Gly Phe Phe Gln Val Ile Asn His Gln Val Ser Pro Asp                  85 90 95 Leu Leu Thr Ala Ala His Gln His Ile Ser Leu Phe Phe Ser Leu Ser             100 105 110 Leu Val Glu Lys Arg Arg Ala Gln Arg Lys Leu Gly Glu Ser Cys Gly         115 120 125 Tyr Ala Ser Ser Phe Thr Asp Arg Phe Ala Asn Lys Leu Pro Trp Lys     130 135 140 Glu Thr Leu Ser Phe Glu His Ser Pro Ser Ser Asp Val Arg Asp Tyr 145 150 155 160 Phe Val Lys Ala Met Gly Glu Asp Phe Gln Asp Ala Gly Asn Val Phe                 165 170 175 Lys Glu Tyr Cys Glu Ala Met Glu Cys Leu Ala Leu Gly Leu Met Glu             180 185 190 Leu Leu Gly Met Ser Leu Gly Ile Gly Arg Leu His Leu Arg Lys Phe         195 200 205 Phe Glu Gly Gly Asn Ser Ile Met Arg Leu Asn Tyr Tyr Pro Pro Cys     210 215 220 Ala Gln Pro Asn Leu Thr Leu Gly Thr Gly Pro His Cys Asp Pro Thr 225 230 235 240 Ser Leu Thr Leu Leu His Leu Asp Glu Val Gly Gly Leu Glu Ile Phe                 245 250 255 Ile Asn Asn Lys Trp His Ser Val Arg Pro Asn Thr Asp Ala Phe Val             260 265 270 Val Asn Ile Gly Asp Thr Phe Met Ala Leu Ser Asn Gly Lys Tyr Lys         275 280 285 Ser Cys Leu His Arg Ala Val Val Asn Lys Thr Thr Pro Arg Lys Ser     290 295 300 Leu Ala Phe Phe Leu Asn Pro Pro Tyr Asp Lys Ile Val Arg Pro Pro 305 310 315 320 Asp Asp Leu Leu Asp Ser Asp His Pro Arg Lys Tyr Pro Asp Phe Thr                 325 330 335 Trp Pro Val Phe Leu Glu Phe Thr Gln Lys His Tyr Arg Ser Asp Met             340 345 350 Asn Thr Leu Ala Ala Phe Gln Lys Trp Phe Thr Ser Arg Asn Gln Pro         355 360 365 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> DX15 promoter forward primer <400> 4 gggaagcttt tgtatccgga ggacatattt gtt 33 <210> 5 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> DX15 promoter_reverse primer <400> 5 tttggtacca gctaggagtg ctgttttgtt gca 33 <210> 6 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> PdGA20ox forward primer <400> 6 aaaagcaggc tatgggtact tcgactgtga gt 32 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> PdGA20ox_reverse primer <400> 7 agaaagctgg gtttatggct ggtttcttga gg 32 <210> 8 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> PdGA20ox_RV-p2A <400> 8 gacgtcacct gcaagcttaa gaaggtcgaa gttaagaagc tgtggctggt ttcttgaggt 60 gaa 63 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AtActin8_forward primer <400> 9 atgaagatta aggtcgtggc a 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AtActin8_reverse primer <400> 10 tccgagtttg aagaggctac 20 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PagActin2_forward primer <400> 11 gccatctctc atcggaatgg aa 22 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PagActin2_reverse primer <400> 12 agggcagtga tttccttgct ca 22 <210> 13 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> 2A peptide <400> 13 Gln Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser   1 5 10 15 Asn Pro Gly Pro              20 <210> 14 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> 2A peptide <400> 14 cagcttctta acttcgacct tcttaagctt gcaggtgacg tcgagtcaaa cccaggtcca 60                                                                           60 <210> 15 <211> 984 <212> DNA <213> Populus trichocarpa <400> 15 atgaggaagc cggatctaat ggccagagat agagtgccaa tcaataataa tatgaacagg 60 gccaaactta gaaagggctt atggtcacca gaggaagatg agaagctcat caagtatatg 120 ttaactaatg gacaagggtg ttggagtgaa attgctagga atgctggctt gcaaaggtgt 180 ggcaagagtt gccgactgcg atggattaac tatttgagac ctgacctcaa gcgtggcgca 240 ttttctcctc aagaagaaga gcttatcatt catttacact ccattcttgg caacaggtgg 300 tctcaaattg cggcgcgtct tccaggaagg acagacaacg aaataaagaa tttttggaat 360 tctacattaa agaaaagatt caagatcaac agcacttcca catcctcacc aaacgatagt 420 tctgattcat cagaacctag agatcatgtc gtaggaaata tcatgcccat gcatgatcat 480 gacgttatga ctctgtgcaa ggactcatct tcctcaccat ccatatccat gcatggtgtg 540 gtcacaggca accaatttga tcctttcacc gtgctcagta accgctatga tgtgagtggt 600 gcagcaagtt tatttgacat gtccacatgc ttaacacagg tgggtatggg agatggattt 660 tatggtgatc attatgggat tttggagggt aataataaaa tagggctaga aagtgatctt 720 tctcttccgc cactcgagag tagaagcatt gaagagaata atgcagtgag taataacaga 780 attggcgtga aaagcagcag caacgacaac caccactttg atagtacctg cttcaataat 840 actgatcaga gatttaaagt agaggacatg ttggggcttg aaaatcattg gcaaggagaa 900 aacgtgagaa tgggagaatg ggatctggaa ggtttaatgg aaaacatatc ttcctttcct 960 ttccttgatt tccaagtttt ataa 984 <210> 16 <211> 327 <212> PRT <213> Populus trichocarpa <400> 16 Met Arg Lys Pro Asp Leu Met Ala Arg Asp Arg Val Pro Ile Asn Asn   1 5 10 15 Asn Met Asn Arg Ala Lys Leu Arg Lys Gly Leu Trp Ser Pro Glu Glu              20 25 30 Asp Glu Lys Leu Ile Lys Tyr Met Leu Thr Asn Gly Gln Gly Cys Trp          35 40 45 Ser Glu Ile Ala Arg Asn Ala Gly Leu Gln Arg Cys Gly Lys Ser Cys      50 55 60 Arg Leu Arg Trp Ile Asn Tyr Leu Arg Pro Asp Leu Lys Arg Gly Ala  65 70 75 80 Phe Ser Pro Gln Glu Glu Glu Leu Ile Ile His Leu His Ser Ile Leu                  85 90 95 Gly Asn Arg Trp Ser Gln Ile Ala Ala Arg Leu Pro Gly Arg Thr Asp             100 105 110 Asn Glu Ile Lys Asn Phe Trp Asn Ser Thr Leu Lys Lys Arg Phe Lys         115 120 125 Ile Asn Ser Thr Ser Thr Ser Ser Pro Ser As Ser Ser Ser As Ser Ser     130 135 140 Glu Pro Arg Asp His Val Val Gly Asn Ile Met Pro Met His Asp His 145 150 155 160 Asp Val Met Thr Leu Cys Lys Asp Ser Ser Ser Ser Pro Ser Ser Ser                 165 170 175 Met His Gly Val Val Thr Gly Asn Gln Phe Asp Pro Phe Thr Val Leu             180 185 190 Ser Asn Arg Tyr Asp Val Ser Gly Ala Ala Ser Leu Phe Asp Met Ser         195 200 205 Thr Cys Leu Thr Gln Val Gly Met Gly Asp Gly Phe Tyr Gly Asp His     210 215 220 Tyr Gly Ile Leu Glu Gly Asn Asn Lys Ile Gly Leu Glu Ser Asp Leu 225 230 235 240 Ser Leu Pro Pro Leu Glu Ser Arg Ser Ser Glu Glu Asn Asn Ala Val                 245 250 255 Ser Asn Asn Arg Ile Gly Val Lys Ser Ser Ser Asn Asp Asn His His             260 265 270 Phe Asp Ser Thr Cys Phe Asn Asn Thr Asp Gln Arg Phe Lys Val Glu         275 280 285 Asp Met Leu Gly Leu Glu Asn His Trp Gln Gly Glu Asn Val Arg Met     290 295 300 Gly Glu Trp Asp Leu Glu Gly Leu Met Glu Asn Ile Ser Ser Phe Pro 305 310 315 320 Phe Leu Asp Phe Gln Val Leu                 325 <210> 17 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> PtrMYB003_forward primer <400> 17 aaaaagcagg ctatgaggaa gccggatcta atg 33 <210> 18 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> PtrMYB003_FW-p2A primer <400> 18 cttcttaagc ttgcaggtga cgtcgagtca aacccaggtc caatgaggaa gccggatcta 60 atg 63 <210> 19 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> PtrMYB003_reverse primer <400> 19 agaaagctgg gtttataaaa cttggaaatc aaggaaagga aagg 44

Claims (15)

DX15 promoter consisting of the nucleotide sequence of SEQ ID NO: 1 and operably linked thereto, PdGA20ox (Pinus encoding the amino acid sequence of SEQ ID NO: 3 densiflora Gibberellin 20-oxidase) gene.
The method according to claim 1,
Wherein the PtrMYB003 gene encoding the amino acid sequence of SEQ ID NO: 16, operably linked to the promoter, is further introduced.
3. The method according to claim 1 or 2,
Wherein said transgenic plants are inhibited from reducing the leaf area due to overexpression of Gibberellin 20-oxidase or by reducing side effects of root weight loss, and the biomass is increased compared to the wild type.
The method according to claim 1,
Wherein the PdGA20ox gene comprises the nucleotide sequence of SEQ ID NO: 2.
3. The method of claim 2,
Wherein the PtrMYB003 gene consists of the nucleotide sequence of SEQ ID NO: 15.
3. The method according to claim 1 or 2,
Wherein said transgenic plants are Arabidopsis or Poplar.
DX15 promoter consisting of the nucleotide sequence of SEQ ID NO: 1 and operably linked thereto, PdGA20ox (Pinus encoding the amino acid sequence of SEQ ID NO: 3 densiflora Gibberellin 20-oxidase gene into a plant.
A DX15 promoter consisting of the nucleotide sequence of SEQ ID NO: 1; A PdGA20ox ( Pinus densiflora Gibberellin 20-oxidase) gene encoding the amino acid sequence of SEQ ID NO: 3 operably linked to the promoter; And introducing into the plant a PtrMYB003 gene encoding the amino acid sequence of SEQ ID NO: 16, operably linked to the promoter.
9. The method according to claim 7 or 8,
And culturing the transgenic plant in a soil or a medium.
9. The method according to claim 7 or 8,
Wherein said transgenic plants are inhibited by a decrease in leaf area or a reduction in root weight loss due to overexpression of Gibberellin 20-oxidase, and the biomass is increased compared with the wild-type plant .
9. The method according to claim 7 or 8,
Wherein the transgenic plants are Arabidopsis or Poplar.
DX15 promoter consisting of the nucleotide sequence of SEQ ID NO: 1 and operably linked thereto, PdGA20ox (Pinus encoding the amino acid sequence of SEQ ID NO: 3 densiflora Gibberellin 20-oxidase gene into a plant to produce a transgenic plant.
13. The method of claim 12,
Wherein the PtrMYB003 gene encoding the amino acid sequence of SEQ ID NO: 16, operably linked to the promoter, is further introduced into the plant to produce a transgenic plant.
The method according to claim 12 or 13,
Wherein the transgenic plant is inhibited from reducing the leaf area or reducing root weight loss due to overexpression of Gibberellin 20-oxidase, and the biomass is increased compared to the wild-type.
The method according to claim 12 or 13,
Wherein the biomass is increased in glucose or xylose content of the cell wall compared to the wild type.

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