KR101247355B1 - Transgenic plant for regulating for plant root growth and method thereof - Google Patents

Abstract

The present invention relates to a transgenic plant for controlling plant root growth and a method thereof.

Description

Transgenic plant for regulating plant root growth and method thereof

The present invention relates to a transgenic plant for controlling plant root growth and a method thereof.

Root growth proceeds by cell proliferation and cell expansion and is very cooperative through interactions between hormones and genetic programs [Benkova, E., and Hejatko, J. (2009). Plant Mol. Biol. 69 , 383-396.

Recent studies have shown that gibberellin (hereinafter referred to as 'GA') signaling regulates cell proliferation [Achard, P., et al. (2009). Curr. Biol. 19 , 1188-1193; Ubeda-Tomas, S., et al. (2009). Curr. Biol. 19 , 1194-1199], endodermis-specific disruption of the GA pathway leads to nonintegrative cell expansion in Arabidopsis roots (Ubeda-Tomas, S., et al. (2008). Nat Cell Biol. 10 , 625-628].

GA signaling, together with SHORT ROOT (SHR) and SCARECROW (SCR), also regulates the time and amount of formal division in ground tissue maturation [Cui, H., and Benfey, PN (2009). ). Plant J. 58 , 1016-1027; Paquette, AJ, and Benfey, PN (2005). Plant Physiol. 138 , 636-640].

However, it is not known how a comprehensive GA pathway is involved in the root endothelium to regulate cell elongation and cell division.

The present invention has been made by the necessity of the above, an object of the present invention to provide a transgenic plant that regulates cell elongation and cell division.

Another object of the present invention to provide a plant cell growth method.

In order to achieve the above object, the present invention provides a transgenic plant for controlling plant root growth, which is transformed with a gene encoding a SCARECROW-LIKE'SCL 3 polypeptide.

In one embodiment of the present invention, the SCL3 polypeptide is preferably the amino acid sequence of SEQ ID NO: 1, but one or more substitutions, deletions, additions, etc. in the amino acid sequence of SEQ ID NO: 1 to control plant root growth All variants having an effect are included in the scope of the present invention.

In one embodiment of the present invention, the gene encoding the SCL 3 polypeptide is preferably the nucleotide sequence set forth in SEQ ID NO: 2, but the degenerate and the mutant having the effect of the present invention as described above In consideration of this, genes having a homology of SEQ ID NO: 2 and at least 80% are also included in the protection scope of the present invention.

The present invention also provides a method of increasing plant root growth compared to wild type by overexpressing the SCARECROW-LIKE'SCL 3 gene under the control of the Cauliflower Mosaic Virus 35S promoter.

In one embodiment of the present invention, the 35S promoter preferably has a nucleotide sequence set forth in SEQ ID NO: 3, but is not limited thereto.

The present invention also provides a recombinant vector comprising the gene according to the present invention. The recombinant vector is preferably a recombinant plant expression vector.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes have been modified and reintroduced into cells by artificial means.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. Vectors can replicate DNA and be reproduced independently in host cells. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host are viral vectors, such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc. For example, it may be selected from an incomplete plant viral vector. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. It is not limited to this.

In the plant expression vector according to an embodiment of the present invention, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter. The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

 The terminator may be a conventional terminator, and examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agrobacterium tumefaciens (ocrobacterium tumefaciens) Terminator of the Fine (Octopine) gene, etc., but is not limited thereto. With regard to the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

The present invention also provides a plant transformed with the recombinant vector according to the present invention. Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. Method is calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplasts Electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185 ), (DNA or RNA-coated) particle bombardment of various plant elements (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumulopasis by plant infiltration or transformation of mature pollen or vesicles And infection with (incomplete) virus (EP 0 301 316) in en mediated gene transfer. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Particularly preferred is the use of so-called binary vector techniques as described in EP A 120 516 and U.S. Pat. No. 4,940,838.

"Plant cell" used for transformation of a plant may be any plant cell. The plant cells may be cultured cells, cultured tissues, cultured organs or whole plants, preferably cultured cells, cultured tissues or cultured organs and more preferably any form of cultured cells.

"Plant tissue" refers to tissues of differentiated or undifferentiated plants, such as but not limited to roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, i.e., single cells, protoplasts. (protoplast), shoots and callus tissue. The plant tissue may be in planta or in an organ culture, tissue culture or cell culture.

Plants that can control the growth of plants by the method of the present invention include food crops, including rice, wheat, barley, corn, beans, potatoes, red beans, oats, sorghum; Vegetable crops including Arabidopsis, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, carrot; Special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, leeks, grapes, citrus fruits, persimmons, plums, apricots, bananas; Flowers, including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And feed crops including lygras, redclover, orchardgrass, alphaalpha, tolskew, perennial licegrass and the like.

The present invention also provides a transgenic plant in which the growth of the plant produced by the method is adjusted.

Plants promoted development or differentiation of the plant according to the present invention can be obtained through the sexual propagation method or asexual propagation method which is a conventional method in the art. More specifically, the plant of the present invention can be obtained through the oily breeding process of producing seeds through the pollination process of flowers and breeding from the seeds. In addition, after transforming a plant with a recombinant vector comprising the gene according to the present invention can be obtained through the asexual propagation method which is a process of induction, rooting and soil purification of the callus according to a conventional method. That is, the fragment of the plant transformed with the recombinant vector containing the gene of the present invention is inoculated in a suitable medium known in the art and then cultured under appropriate conditions to induce the formation of callus, and when the shoot is formed, a hormone-free medium Transfer to culture. After about 2 weeks, the shoots are transferred to rooting medium to induce roots. Plants with accelerated development or differentiation can be obtained by inducing roots and then transplanting them into the soil to purify them. In the present invention, the transformed plant may include not only the whole plant, but also tissues, cells, or seeds obtainable therefrom.

Hereinafter, the present invention will be described.

We found that SCARECROW-LIKE 3 (SCL3) acts directly on the DELLA protein inhibitor (REPRESSOR) downstream of ga1-3 (RGA) to participate in and maintain the functional GA pathway in the endothelium and to regulate the organization of cell proliferation. Confirmed.

In addition, acting downstream of the SHR / SCR pathway, SCL3 regulates the timing and amount of constitutive root basal tissue divisions.

These findings of the inventors provide a molecular framework for cell- / tissue-specific integration of the comprehensive GA pathway to developmental or physiological responses during plant growth and development.

In the GA signaling pathway, Arabidopsis DELLA proteins (hereinafter referred to as 'DELLAs') consisting of five members in thaliana (GAI, RGA, RGL1, RGL2 and RGL3) are major inhibitors of plant growth [Harberd, NP, Belfield, E., and Yasumura , Y. (2009). Plant Cell 21 , 1328-1339; Sun, T.-p. (2008). Gibberellin metabolism, perception and signaling pathways in Arabidopsis In The Arabidopsis Book., Volume doi: 10.1199 / tab. 0103. (American Society of Plant Biologists. Rockville, MD).

We have begun to address the action of SCL3 downstream of both the GA and SHR / SCR pathways, which serve as a convergent point. In Arabidopsis roots, mRNA in situ hybridization analysis showed that SCL3 transcripts were located primarily in the endothelium (FIG. 5A).

In addition, SCL3 Transcriptional fusion between the promoter and beta-glucuronidase (GUS) ( pSCL3 :: GUS ) is basically that mRNA in The situ hybridization pattern was repeated (FIGS. 1B and 1C).

In addition, the green fluorescent protein (GFP) plus scl3 - translation fusion in (pSCL3 :: SCL3 GFP) is a division delay heart; and given the location in the cell's nucleus (quiescent center QC), cortex / endothelial cool (CEI) and endothelial Tuesday , It indicates that SCL3 acts as a transcriptional regulator (FIGS. 1A and 1D). We found that SCL3 at the root It was confirmed whether expression is regulated by the GA pathway. GA ₃ When treated with ( bio GA), SCL3 expression was substantially reduced (FIGS. 1E and 1F). In contrast, its expression was upregulated by GA biosynthesis inhibitor paclobutrazol (PAC) (FIGS. 1G and 1H). GA- defect -3 ga1 mutants was the ent -copalyl diphosphate synthase deficiency in the first step in GA biosynthesis, indicates a smaller size than the normal phenotype [Harberd, NP, et al. (2009). Plant Cell 21 , 1328-1339; Sun, T.-p. (2008). Gibberellin metabolism, perception and signaling pathways in Arabidopsis In The Arabidopsis Book., Volume doi: 10.1199 / tab. 0103. (American Society of Plant Biologists.Rockville, MD); Sun, TP, and Kamiya, Y. (1994). Plant Cell 6 , 1509-1518. In ga1 -3 SCL3 expression level was adjusted by more bioactive GA levels were higher than in wild-type decreased by GA ₃ increased by PAC (Fig. 5B). These data indicate that regulation of SCL3 expression in the roots is dependent on DELLA accumulation regulated by bioactive GA levels.

We therefore found that the loss of mutants of DELLAs - gai , rga , gai rga and della ( gai rga rgl1 rgl2 rgl3 ) SCL3 transcript levels were examined by reverse transcription based quantitative PCR (qRT-PCR). rga In single mutants, SCL3 levels were reduced by about 70% compared to wild type while gai showed a slight decrease (FIG. 5). The application of bioactive GA Wild type, ga1 -3 , gai , rga , and gai In rga roots SCL3 Expression levels were downregulated. SCL3 induction by PAC is a gai known as PAC-resistance It was not observed in the rga double mutant (FIG. 5B).

Also SCL3 Expression was further reduced in the della quintuple mutant, indicating that DELLA proteins were not found in SCL3. It means having a redundant function in the regulation of transcription (FIG. 5B). These data are consistent with SCL3 expression regulated by the amount of bioactive GA.

SCL3 in QC, CEI and endothelial mRNA and protein localization overlaps the SHR protein as well as the spatial domains of SCR mRNA and protein, which imply molecular interactions at the cellular level.

Thus, we found that scr , pSCL3 :: GUS transcriptional fusion and qRT-PCR shr and scr shr . SCL3 expression patterns were examined in mutants. SCL3 expression is scr , shr and scr decreased in shr root, scr It showed the lowest expression in shr double mutants (FIGS. 1I-1M), which further demonstrates that SCL3 transcription is regulated by the SHR / SCR regulatory module. In particular, shr and scr In shr roots, the decrease in SCL3 expression was more pronounced in the elongation / differentiation zone (EDZ) than in the meristem zone (FIGS. 1K and 1L; FIGS. 5C-5F).

Interestingly, we found that foreign GA Presence of SCR or SCR And Only a slight decrease in SCL3 expression was observed in all SHR mutations. We therefore deduced that SCL3 transcription is regulated by both the GA and SHR / SCR pathways. In order to test this, we have rga with loss-of-function mutants in two direct upstream genes. scr double mutants were generated and SCL3 Expression was examined.

SCL3 mRNA expression levels were significantly reduced to only 30% of wild type (FIG. 1M), indicating that SCL3 is the point of convergence of both pathways. These results show that SCL3 acts as a cell / tissue specific integrator that acts downstream of both the GA and SHR / SCR pathways.

Since SCL3 expression is actively regulated by bioactive GA through DELLAs, we hypothesized the role of SCL3 as a negative regulator in the GA response pathway. To test our hypothesis, the inventors were ga1 -3 characterize the functional loss scl3 mutants in the background Surprisingly scl3 ga1 -3 double mutants showed a shorter root phenotype compared with hayeoseo ga1 -3 in root growth analysis (FIGS. 2A and 2C). Like ga1 -3 anti Foreign GA ₃ In the presence, the scl3 ga1-3 phenotype was restored, although somewhat shorter than the wild type in both ga1-3 and scl3 ga1-3 roots.

Also SCL3 overexpressed under the control of the Cauliflower Mosaic Virus 35S promoter (35S :: SCL3) in ga1 -3 shows a slightly longer roots (Fig. 2b and 2c). Root growth of scl3 is more sensitive to PAC treatment, whereas 35S :: SCL3 Seedlings impart resistance to the PAC (FIGS. 2D and 2E). In the presence of PAC, both rga and scl3 rga are indistinguishable in root growth (FIGS. 2D and 2E), which means that RGA is epistatic to SCL3. It is interesting to note that RGA, an inhibitor of GA neosynthesis , positively regulates SCL3 transcription. However, the present invention suggests that SCL3 seems to be a positive regulator in the GA signaling pathway.

DELLAs are thought to be transcription regulators that act as inhibitors in the GA pathway. Like DELLAs, SCL3 also suggests that it acts as a transcriptional regulator due to nuclear localization and phylogenetic relationships with GRAS membrane (FIG. 1D). Maintenance of the functional GA pathway is known to be achieved at least in part through the transcriptional regulation of biosynthetic genes encoding GA 20-oxidases ( GA20ox ) and GA 3-oxidases ( GA3ox ) and catabolic genes encoding GA 2-oxidases ( GA2ox ). Sun, T.-p. (2008). Gibberellin metabolism, perception and signaling pathways in Arabidopsis In The Arabidopsis Book., Volume doi: 10.1199 / tab. 0103. (American Society of Plant Biologists. Rockville, MD); Hedden, P., and Phillips, AL (2000). Trends Plant Sci. 5 , 523-530. Therefore, to determine whether SCL3 is involved in the maintenance of GA pathway in the roots, we examined the expression profile of these GA metabolic genes with qRT-PCR in the presence or absence of foreign GA or PAC. Among the GA metabolic genes examined, GA20ox1 , GA20ox2 , And Transcription level was GA20ox3 (PAC or under ga1 -3 present) GA- deficiency Up-regulated in scl3 under conditions (FIGS. 2F and 2G). These results indicate that GA20ox1 , under GA deficiency conditions GA20ox2 And especially GA20ox3 transcription is regulated by the GRAS transcription factor SCL3. This suggests that SCL3 is a biosynthetic gene ( GA20ox1 , GA20ox2 And GA20ox3 ) strongly suggests that it is a positive regulator that maintains a functional GA pathway in the roots by tuning transcription.

The inventors have ga1 -3 or in the presence or absence of PAC was analyzed wild type, root growth is mediated by GA scl3 and 35S :: SCL3 seedlings, the inventors of the primary cell from QC to the parameters for the meristem size and length can Was measured (FIG. 3A). Under standard conditions, the root meristem size of scl3 and 35S :: SCL3 was similar to that of the wild type (FIGS. 3B and 3C). However, the root meristem size of 35S :: SCL3 seedlings appeared slightly but significantly larger than that of scl3 in the presence of wild-type and PAC (FIGS. 3B and 3C), indicating that SCL3 overexpression confers resistance to PAC. it means. We also monitored CYCB1 :: GUS mitotic markers that have been shown to correlate with mitotic potential in root meristem [Achard, P., Gusti, A., Cheminant, S., Alioua, M., Dhondt, S., Coppens, F., Beemster, GT, and Genschik, P. (2009). Curr. Biol. 19 , 1188-1193; Ubeda-Tomas, S., Federici, F., Casimiro, I., Beemster, GT, Bhalerao, R., Swarup, R., Doerner, P., Haseloff, J., and Bennett, MJ (2009). Curr. Biol. 19 , 1194-1199.

The number of dividing cells in the presence of PAC is similar to that of wild type scl3 Decreased in the root meristem (FIG. 6). Also scl3 ga1 -3 root meristem size of the double mutant ga1 - were not separated as in 3 (6), which indicates that the GA- mediated cell proliferation in the root meristem that is independent of the SCL3 function.

In fact, reduction of root cell elongation is observed in scl3 in the presence of PAC treatment, while root elongation of 35S :: SCL3 is PAC-resistant (FIG. 3D). The inventors in GA- deficiency ga1 -3 background we observed the opposite effect of the loss and obtaining the function from the root SCL3 kidney cells (Figure 6). Also F scl3 The rga double mutant exhibits a PAC resistant phenotype in cell elongation that is indistinguishable from the rga single mutant and confirms the synergistic relationship of RGA to SCL3 (FIG. 3D). Overall, scl3 , 35S :: SCL3 , rga and scl3 The root cell elongation of rga correlates well with the EDZ length of each individual (FIG. 3E), which indicates that the sum of each cell elongation mainly contributes to the EDZ length and ultimately the overall root length (FIG. 2E).

In order to further define the features of the GA- SCL3 in cell-mediated kidney, the inventors pSCR each scl3 and wild-type plants :: gai - GR - YFP Constructs were imported. In the absence of dexamethasone (-DEX), we found no difference in root cell elongation in both wild type and scl3 seedlings (FIGS. 3F and 3G). Upon induction of (+ DEX), pSCR : gai - GR - YFP seedlings in the wild-type background showed reduced root growth with deviant cell elongation in EDZ (FIGS. 3F and 3G).

In particular, in the background scl3 pSCR: gai - GR - YFP seedlings exhibited a severe inhibition of root growth. The surface of the pSCR : gai - GR - YFP root in scl3 is protruding because the direction of cortical cell extension is shifted vertically to that of the untreated root (FIG. 3g). In the presence of DEX, due to the shift in cell extension direction, the average length of individual cells in EDZ is significantly reduced in scl3 compared to that of wild type (FIG. 3H). The present invention thus indicates that endothelial-limited GA signaling by SCL3 is essential for the regulation of linked cell elongation and thus for the progression of root growth.

Our evidence SCL3 acts as a convergence point of SHR / SCR path in GA and endothelium takes place the questions of whether the primary role in the organization of mature marketing SCL3 the formative (formative) coordinating the amount and timing of the split. To test this, the timing and frequency of constitutive division in wild type and scl3 with tissue specific markers were investigated. Unlike the scr and shr mutants, scl3 mutant cells in contact with the pattern did not show defects in root Thus's telephone (stele) are shown the properties of the endothelium, such as those seen in wild-type (Fig. 7). In wild type, incipient development of MC was observed 6 days after germination, and half of the seedlings in a given group showed MC formation 12 days after germination under this experimental condition (FIG. 8). At 7 days after germination, only 12% of the wild type showed constitutive basic tissue division but 42% of scl3 seedlings had already undergone periclinal division (FIGS. 4A-4E; FIG. 8).

In contrast, 35S :: SCL3 seedlings showed a delay in the development of constitutive division than in wild-type 7 days after germination (2% versus 12%), indicating that SCL3 was associated with the timing of constitutive basic tissue division for MC formation. Implying to control the frequency (FIG. 4E; FIG. 8). In addition, the scr mutants often perform additional constitutive divisions in later stages resulting in sporadic production of two underlying tissue layers (FIG. 8). Specifically, scl3 In the scr double mutant, constitutive cleavage occurred much earlier than the scr mutant; 55% scl3 at 4 days after germination scr splits to create already two basic tissue layers: an inner layer (fused as a mutant layer) with fused properties of the endothelium and cortex and an outer layer with cortical properties (FIGS. 4H-4K; FIG. 8). However, at 4 days after germination in scr :: 35S SCL3 postponed the initial occurrence of the formation of cleavage on the scr (6% vs. 24%) . These findings are consistent with the regulation of the timing and amount of constitutive basic tissue division by regulation of SCL3 activity. Also scl3 shr In double mutants we observed no additional constitutive division in the underlying tissue, indicating that SHR is required for constitutive basic tissue division.

In order to define the function of the tissue in the main SCL3 GA- parameters, the inventors have investigated the timing and amount of divisive form in the presence of GA, PAC or ga1 -3. In the presence of foreign GA, we found that only 18% of the wild type roots showed MC formation 12 days after germination, which is about three times inhibition of untreated roots (FIG. 4G). In addition, foreign GA treatment also increased the inhibition of premature periclinal cleavage in scl3 and 35S :: SCL3 (FIG. 4G). In contrast, under the GA- deficient conditions caused by foreign PAC treatment or -3 ga1 mutants, the inventors have increased similarly to the occurrence of immature formative cleavage; highest in scl3, the lowest in the 35S :: SCL3 (Fig. 4e and 4f). scl3 Asymmetrical divisions in ga1 -3 double mutants occurred more frequently in ga1 -3 on day 6 after germination, whereas 35S :: SCL3 seedlings showed somewhat less divisions in in ga1 -3 (FIG. 4F). This fact indicates that inhibition of GA biosynthesis or foreign GA has the opposite effect on the regulation of the timing and amount of constitutive lesion division.

We also scr In the background, GA-mediated regulation of the formation of basal tissue divisions in the loss or acquisition of SCL 3 function was investigated. In the presence of PAC, the delay of immature MC formation by 35S :: SCL3 is scr Disappearing from the background (FIG. 4K), this indicates that SCR plays a role in GA mediated basic tissue maturation. Interestingly, scl3 The rga scr triple mutant showed faster MC formation in the absence of PAC (FIG. 4L), which plays an important role in basic tissue maturation during the post-embryogenic development of regulatory networks of SHR / SCR, GA / DELLA and SCL3 in the endothelium. Indicates that

As can be seen from the present invention, SCL3, a member of the family of GRAS transcription factors, directly acts downstream of the DELLA inhibitor RGA to regulate the expression of the GA biosynthetic genes GA20ox1 , GA20ox2 and GA20ox3 , thereby regulating the functional GA pathway. It can be seen that it is maintained (Fig. 9).

GA deficiency by ga1-3 or PAC in scl3 also leads to further growth inhibition, indicating that SCL3 acts as a positive regulator in the GA pathway. In addition, disruption of GA signaling by pSCR :: gai-GR-YFP in the root endothelium increases uncontrolled cell expansion, which provides evidence that the endothelial restricted GA pathway by SCL3 contributes to the regulation of root cell growth. During development after embryonic formation, SCL3 cooperates with the SHR / SCR pathway to regulate constitutive lesion division for basic tissue maturation through at least partial maintenance of the functional GA pathway in the endothelium (FIG. 9).

Since cell migration does not occur during plant development, regulation of cell division (formal and proliferative) and regulation of cell elongation are important for specific tissue / organ formation, final shape and size. Plant hormones play a major role in integrating dynamic changes in external identities into a network of internal programs that regulate the amount and direction of cell elongation and the timing and amount of cell division throughout the life cycle. The present invention provides a molecular framework for cell- / tissue-specific integration of hormone signaling pathways in plant growth and development.

1 shows that SCL3 expression in the roots is regulated by the GA and SHR / SCR pathways. (A) Schematic of Arabidopsis roots. QC refers to the quenescent center, which is the tissue center of the root. (B) Transcriptional fusion ( pSCL3 :: GUS ) in the wild-type root section. Blue GUS Stain SCL3 in Similar to the situ hybridization pattern was observed. (C) pSCL3 :: GUS detection in wild type primary roots. (D) Translational fusions ( pSCL3 :: SCL3-GFP ) in roots were scl3 Has a mutation. GFP signals are observed in the endothelial line of roots and in the nucleus of QC. (E and F) wild-type in the absence (E) and presence (F) of exogenous 10 μM GA ₃ for 6 hours pSCL3 :: GUS . Expression is reduced by foreign GA ₃ . (G and H) in wild-type in the absence (G) and presence (H) of 10 μM PAC, a biosynthesis inhibitor for 6 hours pSCL3 :: GUS . Expression is upregulated by PAC treatment. (IL) Wild type (I), scr (J), shr (K) and scr shr (L) shows the expression of pSCL3 :: GUS . (M) wild type, scr , shr , scr shr, rga rga scr and QRT -PCR of SCL3 transcript in root. SCL3 transcript levels are substantially reduced in rga scr double mutants. Error bars indicate standard deviation.
Figure 2 shows that SCL3 acts as a positive regulator in the GA pathway (Figure 2a). In the absence of foreign GA ₃ , the scl3 mutant is indistinguishable from the wild type, but the scl3 ga1 -3 double mutant is less than the ga1 -3 mutant. Represents a smaller sized phenotype. (2b) Both scl3 and 35 :: SCL3 seedlings are indistinguishable from wild type in the presence of foreign 10 μM GA ₃ . (2c) outpatient 10 μM GA ₃ Determination of root growth in the absence or quasi-presence. (2d) In the presence of 1 μM PAC, root growth of scl3 was further inhibited while 35 :: SCL3 seedlings were more resistant to PAC compared to wild type. (2e) Determination of root growth in the absence or presence of 1 μM PAC. scl3 rga double mutants are PAC- resistant similar to rga . (2f and 2g) the presence of exogenous GA ₃ or PAC (2f), and ga1 -3 background shows the transcript levels of GA biosynthetic genes which are GA20ox1, GA20ox2 and GA20ox3. Under (2g). Under GA-deficient conditions, the expression level of GA20ox3 is Significantly upregulated in scl3 background. The statistical significance of the differences was determined by Student t-test (single asterisk; p <0.05, double asterisk; p <0.01). Scale bars in A, B and D represent 10 mm.
Figure 3 shows that the endothelial-specific GA pathway by SCL3 cooperates with cell elongation for root growth. (3a) Schematic zonation of the primary root along the longitudinal axis. EDZ is defined as the root-hypocotyl bifurcation from the border of MZ. (3b) The basal cell number is reduced in the presence of 1 μM PAC. SCL3 ( 35 :: SCL3 ) overexpression imparts resistance to PAC. (3c) The meristem length is indistinguishable from wild type and scl3 roots in the presence of 1 μM PAC. (3d) in the presence of 1 μM of PAC, while a root cell elongation of scl3 decreases, rga mutants suppress the inhibition of root elongation of the cells scl3. Red arrows indicate the boundaries of single cells in the EDZ. (3e) In the presence of 1 μM PAC, the side length of EDZ length is shown. (3f-3h) wild type and scl3 From seedlings Induction of pSCR :: gai - GR - YFP construct in the presence or absence of 10 μM of dexamethasone (-DEX) or (DEX). Under DEX, in wild-type background pSCR :: gai - GR - YFP seedlings exhibited the reduced root growth has a deviation of cell expansion in EDZ. scl3 In the background, the direction of cell elongation of pSCR :: gai - GR - YFP seedlings shifted vertically to that of untreated beak. Thus, mean cell length was significantly reduced in scl3 background compared to wild-type background (3h).
Scale bars represent 30 μm (D), 5 mm (F) and 100 μm (G), respectively. The statistical significance of the differences was determined by Student t-test (single asterisk; p <0.05, double asterisk; p <0.01).
4 is a diagram showing that SCL3 regulates constitutive division for basic tissue maturation. (4a-4d) scl3 mutants (B and D) exhibit precocious additional asymmetrical periclinal cleavage compared to wild type (a and c). The new layer (intermediate cortex) quickly lost the endothelial marked by pSCR :: GFP-SCR (a and b) and pSHR :: SHR-GFP (c and d). (4e and 4f) in the presence of a PAC absence (e) of 1 μM or presence and ga1 -3 (f), represents the generation of additional formative cleavage in wild type, and scl3 35 :: SCL3. (4g) in the presence of 10 μM in the absence or presence of GA ₃ and ga1 -3, indicates the occurrence of additional formative cleavage in wild type, and scl3 35 :: SCL3. (4h-4j) scl3 scr Indicative of the occurrence of additional formational splitting. scl3 scr double mutants show uncoordnated constitutive cleavage, which creates an inner layer with Casparian stripe (CS) and an additional cortex. Arrows h and i represent additional formational cleavage and arrowhead J represents Casparian stripe CS. (4k) in the absence or presence of 1 μM PAC (E), indicates the occurrence of additional constitutive cleavage in scr , scl3 scr and 35 :: SCL3 scr . Scr in the loss or acquisition of SCL3 features In the background, PAC treatment has a greater effect on the amount of constitutive cleavage. (4l) rga scr , with or without 1 μM PAC (E) And the occurrence of additional formational cleavage in scl3 rga scr . scl3 rga scr Roots exhibited an increased incidence of constitutive divisions despite PAC. Scale bars represent 30 μm.
5 GA / DELLA and SHR / SCR pathways regulate SCL3 expression. (A) in SCL3 at situ hybridization mRNAh. SCL3 expression was detected mainly in the endothelium. (B) GA biosynthesis ( ga1 -3 ) and GA signaling ( gai , rga , gai in the absence or presence of foreign GA ₃ or PAC rga and della) SCL3 in mutant roots 4 days after germination of qRT-PCR. (CF) of transcript, wild-type (C), scr (D), shr (E) and scr shr (F) pSCL3 at the first root :: GUS It represents the valuation of the transcriptional fusion. Expression of SCL3 has shr and scr It was significantly reduced in the EDZ of shr. Blue GUS staining is wild type, scr , shr and scr shr one is perfect (intact) from the meristem contributions (proximal) step by the (primary roots).
FIG. 6 shows that Arabidopsis root growth is regulated by GA in SCL3 and MZ in EDZ. (6a and 6b) in wild type and scl3 in the absence (a) or presence (1) of 1 μM PAC at 7 days after germination CYCB1 :: GUS expression. Black arrows markedly indicate zones of CYCB1 :: GUS expressing cells. (6c) Measurement of length of CYCB1 :: GUS expressing zones. (6d and 6e) GA- deficiency ga1 -3 background in the measurement of the root meristem of the basic cell can (d) and meristem (e). GA regulates proliferative division in Arabidopsis root meristem and it is independent of SCL3 function. (f and g) from GA- ga1 -3 deficient background, and obtaining loss of SCL3 function has the opposite effect on the root cell elongation. Red arrows indicate the boundaries of single cells in the EDZ. The statistical significance of the differences was determined by the Student t-test (double asterisk; p <0.01). The scale bar at f represents 30 μm.
Figure 7 shows that scl3 mutants show no visible damage in root pattern. (A) pSCR :: GFP-SCR in wild type and scl3 , (B) pSHR :: SHR-GFP in wild type and scl3 (C) QC25 and Lugol staining in wild type and scl3 . (DG) wild type (D), scl3 (E), scr (F) and shr Confocal image and Casparian stripe (CS) staining in (G). White arrows indicate Casparian stripe. In the picture co, cortex; en, endodermis; ep and epidermis are shown, respectively. Scale bars represent 30 μm.
FIG. 8 shows that SCL3, together with the SHR / SCR regulatory module, regulates GA-mediated asymmetric cleavage in Arabidopsis roots. (8a and 8b) pCO2 :: H ₂ B-YFP (cortical marker) in wild type on day 5 (a) and day 10 (b) after germination. Wild-type roots undergo additional morphological divisions that produce endothelial (en) and intermediate cortex (mc) in embryonic development. (8c) Development of constitutive division in wild type, scl3 and 35 :: SCL3 , which gradually increases as the roots mature. Loss or gain of SCL3 function regulates the timing and amount of formational basic tissue division. In the presence of (8d and 8e) PACs, formational cleavage at scr makes it more possible and it creates an inner mutation (mu) layer and an outer cortex (co). (8f) scr in loss or acquisition of SCL3 function Occurrence of formational divisions in the background. The loss of SCL3 function in scr has a profound effect on the timing and amount of formational disruption. Scale bars represent 30 μm.
9 shows an SCL3 functional model in the regulation of GA-mediated root growth during development after embryonic formation. SCL3, DELLA, SCR and SHR GRAS transcription factors interact in the root endothelium to regulate GA-mediated growth and development. SCL3 acts directly downstream of the DELLA inhibitor RGA and regulates the expression of GA20ox genes to maintain a functional GA pathway to achieve GA homeostasis. During post-embryogenesis, SCL3, along with the GA / DELLA and SHR / SCR pathways, regulates the formational dividing for cooperative root cell elongation and basic tissue maturation (purple arrows) in EDZ (orange arrows). Bars indicate negative adjustments and arrows indicate positive adjustments.

The present invention will now be described in more detail by way of non-limiting examples. However, the following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as limited by the following examples.

Example  1: plant material and growing condition

All plants are Arabidopsis thaliana Columbia (Col-0) background (except for della quintuple mutants in Landsberg erecta (Ler)). The origin of the mutants and transformation lines is as follows:

scl3 -1 ( SALK _002516), gai ( SALK _082622), and rga ( SALK _089146) , SALK T-DNA line ( http://signal.salk.edu ) [Alonso, J., et al. (2003). Science 301 , 653-657; Lee, MH, et al. (2008). Plant Mol. Biol. 67 , 659-670;

ga1 -3 [Tyler, L., et al. Plant Physiol. 135 , 1008-1019;

scr -5 and shr -6 (SALK _002744) [Lee , MH, et al. (2008). Plant Mol. Biol. 67 , 659-670; Paquette, AJ, and Benfey, PN (2005). Plant Physiol. 138 , 636-640;

QC25 [Sabatini, S., et al. (2003). Genes Dev. 17 , 354-358;

pSHR :: SHR - GFP [Nakajima, K., et al. (2001). Nature 413 , 307-311];

pSCR :: GFP - SCR Sabatini, S., et al. (2003). Genes Dev. 17 , 354-358; Gallagher, KL, et al. (2004). Curr. Biol. 14 , 1847-1851;

pCO2 :: H ₂ B-YFP Heidstra, R., Welch, D., and Scheres, B. (2004). Genes Dev. 18 , 1964-1969;

gai rga rgl1 rgl2 rgl3 ( della quintuple mutant) [Feng, S., et al. (2008). Nature 451 , 475-479;

pSCR :: gai-GR-YFP [Ubeda-Tomas, S., et al. (2008). Nat Cell Biol. 10 , 625-628;

pCYCB1 :: GUS [Donnelly, PM, et al. (1999). Dev. Biol. 215 , 407-419.

Seeds were surface sterilized in 5% sodium hypochlorite and 0.15% Tween-20 for 3 minutes and rinsed with sterile water, then absorbed in sterile water for 4 days under dark conditions and mixed with 0.5x MS agar plates (0.5x Murashige-Skoog salt mixture; 0.5 Planted on mM 2- (N-morpholino) ethanesulfonic acid (MES), pH5.7-5.8; 1% sucrose; 1% agar). Svistoonoff, S., et al. (2003). Plates were grown vertically under continuous light at 22 ° C. as described in Plant Soil 254, 239-244. When using GA-deficient mutant backgrounds ( ga1 -3 , scl3 ga1 -3, and 35S :: SCL3 ga1-3), surface sterilized seeds are washed 10 times with distilled water before planting was absorbed in 5 days 100 μM GA ₃ (Duchefa). For mature plants, seedlings grown under continuous light were transferred to soil and grown under long daytime conditions (16 hr light / 8 hr dark cycle). GA- deficiency ga1 -3 background (ga1 -3, scl3 ga1 -3, and 35S :: SCL3 ga1 -3 ) were sprayed with 100 μM GA ₃ (Duchefa) three times a week.

Example  2: expression analysis

For qRT-PCR, seedlings were grown on filter paper strips (about 5 mm wide) in MS agar plates for 7 days and transferred to new MS agar plates supplemented with 1 μM PAC or 10 μM GA ₃ for 6 hours. Only roots were collected for RNA isolation and total RNA was isolated using the RNeasy Plant Mini kit according to manufacturer (Qiagen) instructions. After RNA extraction, we processed the sample with RQ1 RNase free-DNase (Promega) to remove potential contamination of the genetic DNA. The quality and quantity of the isolated RNA was examined by gel electrophoresis and spectroscopy [Lee, MH, et al. (2008). Plant Mol. Biol. 67 , 659-670;]. SYBR Premix ExTaq reagent (Takara) was used for qRT-PCR with Mx3000PQPCR system (Stratagene). For internal reference, primers specific for the GAPC gene (At3g04120) were used [Dill, A., et al. (2004). Plant Cell 16 , 1392-1405. Each experiment was performed at least three times independently. The mean value of three replicates was calculated and the standard deviation (± SE) was shown.

mRNA in For situ hybridization, 961-bp fragments of SCL3 were amplified and subcloned into the pCR4 Blunt-TOPO vector (Invitrogen). To generate antisense probes, the pCR4-SCL3 construct was linearized with restriction enzyme SpeI and the DIG-labeled SCL3 riboprobe was subjected to in vitro transcription according to the manufacturer's instructions using the DIG RNA Labeling Kit (Roche). Generated. Tissue immobilization and RNA hybridization were performed using wild type roots 4 to 5 days old.

Quantitative RT-PCR (qRT-PCR) involving whole RNA extraction and reverse transcription is Lee, M.-H., et al. (2008). Plant Mol. Biol. 67 , 659-670.

Sequence information of the primers used in the present invention are listed in Supplemental Table 1. mRNA in situ hybridization is Di Laurenzio, et al. (1996). It was performed as described in Cell 86 , 423-433.

Example  3: Treatment and Root Growth Analysis

For root growth analysis, approximately 36-40 hdg (time after germination) seeds were individually Cui, H., et al. (2007). As described in Science 316 , 421-425, GA ₃ Transfer to a new MS agar plate supplemented with (stock concentration 100 mM in ethanol, Duchefa), PAC (stock concentration in ethanol 100 mM, Duchefa), or DEX (stock concentration in ethanol 10 mM, Sigma). On the day of analysis, individually harvested seedling roots were washed with ethanol and GUS clearing method [Laplaze, L., et al. (2007). Plant Cell 19 , 3889-3900]. The number of cells in the root meristem was obtained by counting the cortical cells from the QC, showing no signs of rapid kidney, and the EDZ was Ubeda-Tomas, et al. (2009). Curr. Biol. 19 , 1194-1199, was specified from first cortical cells discharged from meristem. Root length was measured from digital images of plates using Image J software (http :: //rsb.info.nih.gov/ij). The experiments were repeated three times on their own and the data were analyzed using the Excel statistical package (Microsoft).

Example  4: Molecular Cloning and  Transgenic plant

To produce transcriptional and translational fusions and overexpressions, Gateway recombinant cloning technology (Invitrogen) was described by Lee, MH, et al. (2008). Plant Mol. Biol. The method described in 67 , 659-670 was partially improved.

For the pSCL3 :: GUS transcriptional fusion, approximately 2.5 kb upstream sites from the start codon of the SCL3 gene were transferred to the pMDC162 vector [Curtis, M., and Grossniklaus, U. (2003) by an LR recombination reaction according to the manufacturer's instructions (Invitrogen). ). Plant Physiol. 133 , 462-469. For pSCL3 :: SCL3 - GFP translational fusions, about 5 kb gene fragments (promoter and ORF) were subcloned into pDONR221 (Invitrogen) by BP recombination reaction and then for C-terminal GFP fusion [Curtis, M., and Grossniklaus, U. (2003). Plant Physiol. 133 , 462-469] to pMDC107. To overexpress the SCL3 gene under the control of the 35S promoter, the full-length SCL3 ORF was amplified from Col-0 genomic DNA using a pair of genes specific to the SCL3 gene and Phusion high-fidelity polymerase (Finnzymes). Sequence information of the primers used herein is listed in Supplemental Table S1. The PCR fragment was subcloned into a pENTR Directional TOPO vector and then LR recombination as directed by the manufacturer (Invitrogen) as a pEarleyGate100 gateway-comparable overexpression vector [Earley, KW, et al. (2006). Plant J. 45 , 616-629]. Delivered by reaction. floral dip method [Clough, S., and Bent, A. (1998). Plant J. 16 , 735-743, was performed using Col-0 or scl3 for the production of transgenic plants. After selecting the T ₁ plants, homozygous T ₂ plants were obtained through confirmation of T ₃ production.

Example  5: Histology and Microscopic method

To monitor the activity of GUS lipoto, Lee, M.-H., et al. (2008). Plant Mol. Biol. 67 , 659-670 soaked in GUS staining solution under 37 ℃ dark conditions to activate beta-glucrunidase activity. To limit the spread of blue staining, 5 mM K ₃ Fe (CN) ₆ and K ₄ Fe (CN) ₆ were added and the tissues were Laplaze, L., et al. (2007). Soak in 70% ethanol for 2 days as described in Plant Cell 19 , 3889-3900. The dehydration process for washing is described in Laplaze, L., et al. (2007). The method described in Plant Cell 19 , 3889-3900 was accompanied by some improved continuous ethanol treatment. At the end of dehydration, 10% (v / v) glycerol / 50% (v / v) ethanol and 30% after supplementing 0.7% (w / v) NaOH / 60% (v / v) ethanol for 10 minutes (v / v) glycerol / 30% (v / v) ethanol. Seedlings were then mounted in 0.05% (v / v) Triton X-100 / 50% (v / v) glycerol. Lugol staining Fukaki, H., et al. (1998). It was performed as described in Plant J. 14 , 425-430.

For root sectioning, seedlings and GUS-stained seedlings were fixed overnight in FAA solution (10% (v / v) formaldehyde, 5% (v / v) acetic acid and 50% (v / v) ethanol), The fixed roots were filled into 1% agarose and dehydrated by ethanol series. After running through an ethanol series (50, 70, 80, and 100%, for 30 minutes each), the agarose gel blocks with root samples were transferred to a Peel-A-Way disposable embedding mold (Polysciences). Plastic resin blocks were made of Technovit 7100 according to the manufacturer's instructions (Heraeus Kulzer). Cereal sections (5 μm each) were generated with HM 355S microtome (Microm). To see the casparian stripe, the sections were stained and observed. All washed roots were observed with differential interference (DIC) optics, except for the root slices for Casparian stripe in FITC filters, and images were obtained using Axio Imager. The A1 microscope was equipped with an AxioCam MRc5 digital camera (Carl Zeiss). For confocal laser scanning microscopy, roots were placed in distilled water with 10 μM propidium iodide (Sigma) and images were obtained using Fluoview FV300 (Olympus).

<110> Konkuk University Industrial Cooperation Corp. <120> Transgenic plant for regulating for plant root growth and method          the <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 482 <212> PRT <213> SCL3 <400> 1 Met Val Ala Met Phe Gln Glu Asp Asn Gly Thr Ser Ser Val Ala Ser   1 5 10 15 Ser Pro Leu Gln Val Phe Ser Thr Met Ser Leu Asn Arg Pro Thr Leu              20 25 30 Leu Ala Ser Ser Ser Pro Phe His Cys Leu Lys Asp Leu Lys Pro Glu          35 40 45 Glu Arg Gly Leu Tyr Leu Ile His Leu Leu Leu Thr Cys Ala Asn His      50 55 60 Val Ala Ser Gly Ser Leu Gln Asn Ala Asn Ala Ala Leu Glu Gln Leu  65 70 75 80 Ser His Leu Ala Ser Pro Asp Gly Asp Thr Met Gln Arg Ile Ala Ala                  85 90 95 Tyr Phe Thr Glu Ala Leu Ala Asn Arg Ile Leu Lys Ser Trp Pro Gly             100 105 110 Leu Tyr Lys Ala Leu Asn Ala Thr Gln Thr Arg Thr Asn Asn Val Ser         115 120 125 Glu Glu Ile His Val Arg Arg Leu Phe Phe Glu Met Phe Pro Ile Leu     130 135 140 Lys Val Ser Tyr Leu Leu Thr Asn Arg Ala Ile Leu Glu Ala Met Glu 145 150 155 160 Gly Glu Lys Met Val His Val Ile Asp Leu Asp Ala Ser Glu Pro Ala                 165 170 175 Gln Trp Leu Ala Leu Leu Gln Ala Phe Asn Ser Arg Pro Glu Gly Pro             180 185 190 Pro His Leu Arg Ile Thr Gly Val His His Gln Lys Glu Val Leu Glu         195 200 205 Gln Met Ala His Arg Leu Ile Glu Glu Ala Glu Lys Leu Asp Ile Pro     210 215 220 Phe Gln Phe Asn Pro Val Val Ser Arg Leu Asp Cys Leu Asn Val Glu 225 230 235 240 Gln Leu Arg Val Lys Thr Gly Glu Ala Leu Ala Val Ser Ser Val Leu                 245 250 255 Gln Leu His Thr Phe Leu Ala Ser Asp Asp Asp Leu Met Arg Lys Asn             260 265 270 Cys Ala Leu Arg Phe Gln Asn Asn Pro Ser Gly Val Asp Leu Gln Arg         275 280 285 Val Leu Met Met Ser His Gly Ser Ala Ala Glu Ala Arg Glu Asn Asp     290 295 300 Met Ser Asn Asn Asn Gly Tyr Ser Pro Ser Gly Asp Ser Ala Ser Ser 305 310 315 320 Leu Pro Leu Pro Ser Ser Gly Arg Thr Asp Ser Phe Leu Asn Ala Ile                 325 330 335 Trp Gly Leu Ser Pro Lys Val Met Val Val Thr Glu Gln Asp Ser Asp             340 345 350 His Asn Gly Ser Thr Leu Met Glu Arg Leu Leu Glu Ser Leu Tyr Thr         355 360 365 Tyr Ala Ala Leu Phe Asp Cys Leu Glu Thr Lys Val Pro Arg Thr Ser     370 375 380 Gln Asp Arg Ile Lys Val Glu Lys Met Leu Phe Gly Glu Glu Ile Lys 385 390 395 400 Asn Ile Ile Ser Cys Glu Gly Phe Glu Arg Arg Glu Arg His Glu Lys                 405 410 415 Leu Glu Lys Trp Ser Gln Arg Ile Asp Leu Ala Gly Phe Gly Asn Val             420 425 430 Pro Leu Ser Tyr Tyr Ala Met Leu Gln Ala Arg Arg Leu Leu Gln Gly         435 440 445 Cys Gly Phe Asp Gly Tyr Arg Ile Lys Glu Glu Ser Gly Cys Ala Val     450 455 460 Ile Cys Trp Gln Asp Arg Pro Leu Tyr Ser Val Ser Ala Trp Arg Cys 465 470 475 480 Arg lys         <210> 2 <211> 1446 <212> DNA <213> SCL3 <400> 2 atggtggcta tgtttcaaga agataatgga acatcttctg tagcttcatc accacttcaa 60 gtcttctcaa ctatgtcact caacagaccg actctcctcg cttcttcatc tccgtttcat 120 tgtctcaaag atctcaaacc agaggagcgt ggtctctact taatccacct cttgctaact 180 tgtgccaacc acgtggcttc aggtagcctc caaaacgcta acgcagcgct cgagcagctc 240 tctcacctcg cttctcctga cggcgacacg atgcagcgaa tcgctgctta cttcaccgaa 300 gcgcttgcta acagaatcct taagtcctgg cctggtcttt acaaggctct taacgcaact 360 cagacaagaa ctaacaatgt ctctgaggag attcatgtta gaagactctt ctttgagatg 420 ttcccgatac tcaaagtctc ttacttgctc actaatcgag ctatactcga ggctatggaa 480 ggagagaaga tggttcatgt gattgatctc gatgcttctg agccagctca atggcttgct 540 ttgcttcaag cttttaactc taggcctgaa ggtccacctc atttgagaat cactggtgtt 600 catcaccaga aggaagtgct tgaacaaatg gctcatagac tcattgagga agcagagaaa 660 ctcgatatcc cgtttcagtt taatcccgtt gtgagtaggt tagactgttt aaatgtagaa 720 cagttgcggg ttaaaacagg agaggcctta gccgttagct cggttcttca attgcatacc 780 ttcttggcct ctgatgatga tctcatgaga aagaactgcg ctttacggtt tcagaacaac 840 cctagtggag ttgacttgca gagagttcta atgatgagcc atggctctgc agctgaggca 900 cgtgagaatg atatgagtaa caacaatggg tatagcccta gcggtgactc ggcctcatct 960 ttgcctttac caagttcagg aaggactgat agcttcctca atgctatttg gggtttgtct 1020 ccaaaggtca tggtggtcac tgagcaagac tcagaccaca acggctccac actaatggag 1080 aggctattag aatcacttta cacctacgca gcattgtttg attgcttgga aacaaaagtt 1140 ccaagaacgt ctcaagatag gatcaaagtg gagaagatgc tcttcgggga ggagatcaag 1200 aacatcatat cctgcgaggg atttgagaga agagaaagac acgagaagct tgagaaatgg 1260 agccagagga tcgatttggc tggttttggg aatgttcctc ttagctatta tgcgatgttg 1320 caggctagga gattgcttca agggtgcggt tttgatgggt atagaatcaa ggaagagagc 1380 gggtgcgcag taatttgctg gcaagatcga cctctatact cggtatcagc ttggagatgc 1440 aggaag 1446 <210> 3 <211> 2549 <212> DNA <213> Artificial Sequence <220> <223> Promoter <400> 3 ttgtaacgaa gtctgttgtt ctcattttct acttattacg ttaattcgaa cgttggaatc 60 atcaaaagac cgtgccaaaa caaaaatgca aattgatgcg atagacattc ttttgctgat 120 ttgtatgcta taggttttca aatctctagc tacacttatg tatttcccta gattatcaaa 180 gttagcctgc cgttttctaa ttttattatt tgatattttg attatactgc ttcttaatgc 240 atcaaataag ttgattcatc catctaatat tttaactaca tcaaatgaag tcttttataa 300 gtacaggcac taattgaact tgaaccctat aaaatacatg aataatcaca aagaattgat 360 agtgttcttt cagtttttca atcacaaaga actcaccgac attatcaagt tgacttcact 420 tttagactta attggtgttc tggaggatcc acaaaactac tattacacag ttttcacaaa 480 attgtgattc tttatttttt attttctttg gtcaaactaa catttcattc aactttaaac 540 gagagttgcc tccacgtgga gagtatacac aaaaagctac gaggacaaat gctcaaacaa 600 aaagacataa gaaggggagc tcagaagctc aacccgagta gcagcatcca aagttgctaa 660 aacagaacca tcatttactt ctgaggcggt cttcgtacca agctccttca gcaaaattgt 720 gattctatgt ttggatatca tgagatctaa ctacaaagtt tttaataaca aaaacttacc 780 attctctaat ttgtagtcaa agctaaacaa atttcatact aaaacaattt tactgtgttg 840 ttatggatgc atatttttct cactttatag tactatttta ctattgataa gatgaaaaaa 900 aagttgaggt acaatagtag taaaatagca tcattctttt tctgtagaat ctctctggtc 960 aatggtcttt ttagtttaaa aactgaataa tgttctctct cacacagtaa aaaacaaaga 1020 gagagagaaa gaagagaaga agacaaaaat gtaggcatta aaaagaagaa aaagacaaag 1080 agaaaagtaa tgaaaagaca atgaacattt aaagaagaca gacaaatccc acacccaagc 1140 ctcagcctca tctcttttat tgttatttgt ttttctaaca aaacaacaaa aacaaaagag 1200 atggtttttg ggttttgttt tgtctttgtt agggcgtgtt tggacgtttc cttcttctca 1260 gaatcaatat tttaccaaaa cagctttctt tttttctctt taacaatttt tttgatgtag 1320 aaatatatag tcatgattcc aaatccacct catgccatga caaaaaccca taaaagtgga 1380 ggttccaatc tcgaatacag acatatatgt tgtattcgtg tatgtatcta ttataagata 1440 attccaattt gtataatgcc aaatgggttc attgaacttc ttcttccttc ataattaagt 1500 gttgcttttt cttcttagtt cctctaaatc aagaaaattc taggtgaaac ttctcttatc 1560 ttgactttat ataaaataat aactataaaa ataatcaata agactaattc ctacaagtct 1620 tacaaattta aaagaaaatg aaaatgcaac attagctaat acacataata gtataagatt 1680 gatggatgta ttcttattca gttttattta taaatgcaac ataagctaat acactctcat 1740 tcagttttat ttattaatgt tacaccactt taagagcact caaaatcaaa atacgtattc 1800 ttatatgata ttcattcatg tagtactagt acacataata gtataagata gatggatggg 1860 attggaaaat ttgacaacct tttgtttaat aattccttaa aaccaaggaa agaagaaaga 1920 ggaaacgaaa aaaacaaaaa ggaagaagac aaaaccgact atcacgcatt gtctggtacg 1980 tagagccaaa aggaacaatg cctctgtttt agtgttttct ttcttaaagt tttttcccaa 2040 aacacaaaca acatttcaca cacacgctat tactcacaag ataaccacca gagacgtgga 2100 tagagagata aagattgaga gaaccctctc actcccaaaa aaaaagcttt agacattttc 2160 tctaaatcca tgtaagtttt tttctttggt tattgttgtt tgttatcttc tttgatcatt 2220 tgctattatt ttgttttgtt tctttcttag tttatcaaat taggaaagtt ttaattcagc 2280 tctctgtttg tgtttcttcg tctatatcta tttcaaatag tttaactaga ttcaaaggtt 2340 tttaaaattc agtactttct ctgcaaaccc tagttcctct gttctttaac cccctttttt 2400 gttaaactgc agattctctg catctttcaa gaaattccga atcgtgggtt tggttcttct 2460 cgttacgagt ctctcaaagt ttaaatcttt ctttacaaaa aaggttactt ttggttctga 2520 gtaaatcaaa atcaagcttt tggccttca 2549

Claims (6)

delete delete delete SCARECROW-LIKE'SCL having the nucleotide sequence shown in SEQ ID NO: 2 under the control of the Cauliflower Mosaic Virus 35S promoter in the presence of PAC (paclobutrazole) 3 Method of overexpressing genes to increase plant root growth compared to wild type. delete delete

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