KR101568342B1 - A complex trafficking through plasmodesmata and method of transporting targeted material through plasmodesmata - Google Patents

Abstract

The present invention relates to a complex capable of moving through plant cell intergenic plasmids and a method for moving a target substance through a plant cell intergenic plasmatic liaison using the complex. A target substance which can not be migrated through the plasmatic liaison can be moved through the plasmatic liaison by binding with a peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention.

Description

[0001] The present invention relates to a complex capable of moving through a plasmatic liaison, and a method for transferring a target substance between plant cells using the same,

The present invention relates to a complex capable of moving through plant cell intergenic plasmids and a method for moving a target substance through a plant cell intergenic plasmatic liaison using the complex.

The intercellular transport of regulatory proteins and mRNAs in the plant system is a symplasm established by plasmodesmata (PD), which enables non-cell-autonomous (NCA) control of plant development. (1-7). A transcription factor (TF), which acts as an important regulator of the major developmental program, is a major subject of the present invention. The first reported internal NCA-TF is maize KNOTTED1 (KN1) (8-10). KN1 is known to be expressed in the sub-L1 layer of the shoot fissure tissue, but the protein is detected in the L1 layer, which regulates it to determine cell fate (8, 11).

Antirrhinum flower proteins such as DEFICIENS and GLOBOSA (12), transcription factors related to root development such as Arabidopsis GRAS family members, SHORT ROOT (SHR) (13), and CAPRICE, Myb-like DNA- , 15) shows an example of another well-known NCA-TF. In the case of SHR, it is expressed in the central stele, but its active site is deviated from these tissues, which regulates unbalanced cellular imbalance and endodermal cell fate in the early stages of the cortex / endoderm. Interestingly, conserved GRAS domains in SHR and nuclear localization have been found to be necessary for migration (16).

Such intercellular migration of NCA-TF may include selective (10, 14-17) or non-selective (18-21) pathways. Some proteins can migrate non-selectively by simple diffusion through PD. LFY and cytoplasmic GFP (green fluorescent protein) also follow this pathway (18, 21, 22). These proteins do not seem to require specific motifs to interact with each other and to intercellularly transfer through PD. Conversely, selective migration of NCA-TF is associated with specific peptides or motifs mediating their targeting and migration through PD (10, 15, 22-25).

The present invention relates to the NCA capability of the Arabidopsis Dof family of TF. A detailed analysis was performed on INTERCELLULAR TRAFFICKING DOF 1 (ITD1) to reveal its intercellular transport pattern. The mutation analysis revealed that motifs within ITD1 are necessary and sufficient to travel through the PD.

It is an object of the present invention to provide a complex for moving a target substance through a plant cell interplanar communicator and a method for moving a target substance through the plasmatic liaison using the complex.

One aspect of the present invention provides a complex capable of moving through a plasmatic liaison comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 and a target substance.

"Plasma contactor" means a cytoplasmic channel formed in a cell wall adjacent to a multi-celled plant, and the cytoplasm extends toward the outside of the cell. Substances of sufficiently small size (less than 800 Da) can diffuse nonselectively through the plenum liaison, but substances with a size above a certain level are selectively transmitted through the plasma liaison using energy.

"Target substance" means a substance which is involved in the regulation of physiological activity by transferring it to another cell through the plasma-cytoplasmic receptor or exhibits a pharmacological effect or a substance having a biological activity in the cell. For example, nucleic acids, carbohydrates, lipids, glycolipids, proteins, and the like. The target material itself can be a substance that can not move through the plasmatic liaison. Examples of such materials include substances that are large in size and can not diffuse through the plasma liaison, cell-autonomous proteins, and the like.

The amino acid sequence of SEQ ID NO: 1 is the transcription factor protein of Arabidopsis thaliana, which means the amino acid sequence from the 61st amino acid residue to the 110th amino acid residue of AtDof4.1, one of the Dof family members. The amino acid sequence portion has been demonstrated by the experiments of the present invention to be sufficient for the N-terminal variable region of AtDof4.1 and the zinc finger domain region to be sufficient for the said moiety to undergo selective migration through the plasmatic liaison. Therefore, when the target substance and the peptide comprising the amino acid sequence of SEQ ID NO: 1 are linked, the target substance can move through the plasmatic liaison in the plant cell.

The one or more target substances may be linked physically / chemically, covalently or non-covalently with the peptide comprising the amino acid sequence of SEQ ID NO: 1, or may be incorporated into a fused form using a mediator . The target substance may be linked to the N-terminal or C-terminal of the peptide comprising the amino acid sequence of SEQ ID NO: 1. When the target substance is a protein, the cDNA of the protein can be obtained by PCR, followed by obtaining a fusion protein by a cloning technique using a vector, or a chemical fusion method. For example, a target substance having activity may be ligated to a plurality of linkers, and then the peptide may be bound to a peptide comprising the amino acid sequence of SEQ ID NO: 1.

According to one embodiment of the present invention, the peptide may have the amino acid sequence of SEQ ID NO: 2.

The amino acid sequence of SEQ ID NO: 2 means the amino acid sequence of AtDof4.1, the transcription factor protein of Arabidopsis thaliana. Since the amino acid sequence of SEQ ID NO: 2 includes an N-terminal variable region and a zinc finger domain (ZFM) region of the AtDof4.1 protein, the target substance linked to a peptide having the amino acid sequence of SEQ ID NO: Lt; / RTI >

According to one embodiment of the present invention, the target substance may be selected from the group consisting of nucleic acids, carbohydrates, lipids, glycolipids, proteins, and combinations thereof.

According to one embodiment of the present invention, the target substance is a protein, and the protein may be a cell-autonomous protein. "Cell-autonomous protein" refers to a protein that does not migrate to another cell and plays its role in the expressed cell. In the case of the cell-autonomous protein, it is not necessary to move to another cell. Therefore, when expressed in a plant cell, the cell-autonomous protein can not migrate to another plant cell through a plasmatic liaison, but when it is combined with a peptide comprising the amino acid sequence of SEQ ID NO: 1, Can move through. Examples of such cell-autonomous proteins include, but are not limited to, the GLABROUS1 protein of Arabidopsis thaliana and the AtDof5.4 protein of Arabidopsis thaliana.

According to one embodiment of the present invention, when the target substance is a protein, the size of the protein may be 1 kDa to 80 kDa.

When the size of the protein to be transferred through the plasmatic liaison is larger than a certain size, it is difficult to move through the plasmatic liaison. However, the protein can be selectively moved through the plasmatic liaison by connecting with the peptide containing the amino acid sequence of SEQ ID NO: have.

According to one embodiment of the present invention, the target substance is a protein, and the complex may be a peptide comprising the amino acid sequence of SEQ ID NO: 1 and a fusion protein in which the target substance is bound.

The fusion protein can be obtained by expressing in a plant a vector comprising a nucleotide sequence encoding a peptide comprising the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding the target protein. The vector is prepared by amplifying a nucleotide sequence encoding the target substance and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 using PCR and inserting it into an appropriate vector capable of transforming into a plant using a restriction enzyme . Here, the nucleotide sequence encoding the target substance may be connected to the N-terminal region of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 or may be connected to the C-terminal region. Such methods of making vectors are well known in the art (33).

One aspect of the present invention provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

Further, one aspect of the present invention provides a composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a nucleic acid encoding the same. The composition may be for use in transferring a target material through a plasmatic liaison. In addition, the composition may be for use in fusing the peptide or nucleic acid so that the target substance can be transferred through the plasmatic liaison.

One aspect of the present invention provides a method for producing a fusion protein comprising the steps of: preparing a vector comprising a nucleotide sequence encoding the fusion protein;

Transducing said vector into Arabidopsis thaliana; And

And expressing the fusion protein in an Arabidopsis thaliana cell. The present invention also provides a method for moving the target substance through a plasmatic liaison.

The step of preparing the vector means a step of preparing a vector containing the amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence encoding the protein to be transferred through the plasmatic liaison. Methods for producing general recombinant vectors are well known in the art (33). Such vectors may include expression promoters, terminators, selectable markers, reporter genes, tag sequences, enzyme recognition sequences, multiple cloning sites, and the like that are conventional in the art.

The step of transducing the vector into Arabidopsis thaliana may be carried out by the Agrobacterium method well-known in the art as a method of transforming plants, and the Agrobacterium method may be a deeping method, a vacuum infiltration method or a tissue culture method 34, 35). Arabidopsis thaliana transformed with this vector can be selected by germination in an MS plate containing the appropriate antibiotic.

The step of expressing the fusion protein in the Aribbicycytalliana cell can be carried out by growing the Arabidopsis thaliana transformed with the vector in MS plate or soil.

A target substance which can not be migrated through the plasmatic liaison can be moved through the plasmatic liaison by binding with a peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention.

Fig. 1 shows the transfer of various proteins between plant cells according to Example 1 and Example 2. Fig.
Fig. 2 shows the results of a trichome rescue according to Example 2 and Example 3. Fig.
Fig. 3 shows the sequence comparison between the zinc finger motifs of the respective transcription factors according to Examples 4, 5 and 6, and whether or not the structure is a ridge structure.
Figure 4 shows the GFPer expression pattern observed in the J0571 enhancer traps.
Figure 5 It indicates that ITD1-mCherry is able to migrate the leaf tissue-epidermal layer boundary in young leaves.
Figure 6 shows the alignment of sequences in the ZFM range region from 35 Arabidopsis Dof family members. Only ZFM exhibits a high level of sequence conservation. ZFM; CX ₂ CX ₂₁ CX ₂ C. The red box represents ITD1 (AtDof4.1).
Figure 7 shows the neighboring joining tree of the Arabidopsis Dof family based on the full-length protein sequence. The white box represents the Dof member of the ZFM tested for intercellular mobility in the scaly structure analysis of Example 4. Rod = 0.05% sequence difference. Cell-autonomous Dof: red letter, non-cell-autonomous Dof: blue letter.

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. In the following examples, the following experimental materials and methods were used.

(1) plant material, growth conditions and plant transduction

Enhancer Trap strain J0571 was obtained from the Arabidopsis Biology Research Center and the gll-I mutant (36) was used for the screening of the trichome rescue of this example. N. benthamiana, O. sativa cv. Leaf and Physcomitrella patens cultures from Japonica, Glycine max and Chlamydomonas reinhardtii strain CC-503 cw92 mt + were used to isolate genomic DNA. Seeds were germinated directly on MS plates or soil. Transgenic plants were selected on MS plates containing the appropriate antibiotic (50 mg / L kanamycin or hygromycin). Two main transgenic plants were transplanted into the soil. The plants were grown under the conditions of 22 ° C and 60% RH, 16: 8 h light / dark. The light intensity is 150 μmoles m ^-2 sec ^-1 .

(2) Score in statistical structure analysis

The analysis of the structure was carried out in the same way as in the conventional method (17). The number of scars was calculated from the roots of 4-week-old plants grown in soil.

(3) laser  Scanning and Electron Microscope

One week old Arabidopsis seedlings were used to investigate the cell distribution of GFP in roots and leaves. Fluorescence images were obtained using Olympus (Model FV1000, Tokyo, Japan) co-focal laser scanning microscope, excitation at 488 and 543 nm, emission at 510-540 and 587-625 nm for GFP and mCherry, respectively. With respect to the scanning electron microscope (SEM), a second pair of leaves of one-week-old seedlings was prepared as previously described (37) and observed on a JSM-6380LV (JEOL Ltd., Japan) SEM.

(4) software and web resources

The CNLS Mapper software 31 was used to find the predicted nuclear localization signal. Sequence alignment was performed using ClustalW2 software (38). Construction of vector maps and in-silico cloning were performed in Vector NTI version 9 (Invitrogen). The phylogenetic tree was created using MEGA4 software (39).

(5) UAS  Production of fusion systems and other vectors

The GFPer sequence was removed by restriction enzyme treatment with pZY375 (obtained from David Jackson Lab, CSHL) vector (SEQ ID NO: 18) with SalI / SacI and then the mCherry sequence between SalI / SacI was inserted into the removed region to obtain pCCL276 UAS :: mCherry). The histone-2B (H2B) fragment coding region was amplified with primers H2B-Avr-d1 and H2B-HpaK-r1 and inserted into the AvrII / KpnI site of pCCL276 to obtain pCCL284 (UAS :: mCherry-H2B). GW fragment (ITD1 N-var and ZFM-encoding nucleotide sequence (SEQ ID NO: 3), ITD1-encoding nucleotide sequence (SEQ ID NO: 4)) introduced to examine whether or not they migrate through the plasmatic liaisons in the following examples, (SEQ ID NO: 5) encoding AtDof2.2, a nucleotide sequence encoding AtDof2.2 ZFM (SEQ ID NO: 6), a nucleotide sequence encoding AtDof5.6 ZFM (SEQ ID NO: 7), AtDof4.5 ZFM Nucleotide sequence (SEQ ID NO: 8) encoding AtDof3.7 ZFM (SEQ ID NO: 9), nucleotide sequence encoding AtDof5.4 ZFM (SEQ ID NO: 10), nucleotide sequence encoding CrDof ZFM ), A nucleotide sequence (SEQ ID NO: 12) encoding PpDof15 ZFM, a nucleotide sequence (SEQ ID NO: 13) encoding PpDof09 ZFM, a nucleotide sequence (SEQ ID NO: 14) encoding PpDof19 ZFM, a GmDof (SEQ ID NO: 15) encoding a ZFM, a nucleotide sequence encoding OsDof ZFM (SEQ ID NO: 16), and a nucleotide sequence (SEQ ID NO: 17) encoding NtDof ZFM) was obtained from pEarlyGate 103 as an XhoI fragment , and inserted into the SalI site in pCCL276 to obtain pCCL292 (UAS :: GWmCherry). pCCL292 was treated with AvrII / KpnI restriction enzyme and H2B fragment was inserted to obtain pCCL306 (UAS :: GW-mCherry-H2B). GW fragment was amplified from pDest22 (Invitrogen) with primer attR-Bsgl-d1 / attR-BKpn-r1 to clone the pCCL702 (pRbcS :: GL1-GW) gateway destination vector and pK1401 (obtained from David Jackson Lab, CSHL) (SEQ ID NO: 19) at the BglII / KpnI site.

Open reading frames (ORFs) of full length and protein fragments were amplified by PCR using sequence specific primers (Table 1) and Pfu DNA polymerase (Solgent, Korea) according to the manufacturer's instructions. The PCR product with the attB site next to it was cloned into pDonr207 using the BP reaction kit (Invitrogen, USA) according to the manufacturer's instructions. All PCR primers used in the present invention are shown in Table 1. To obtain the required expression vector, the target fragment cloned into the above described entry vector was transferred to the desired destination vector according to the manufacturer's instructions using the LR reaction kit (Invitrogen, USA).

( 6) The primer used in the present invention

Name of the primer SEQ ID NO: Name of the primer SEQ ID NO: UAS_ApaI_F 20 Dof-Cr-GW-d1 72 NOS_ApaI_R 21 Dof-Cr-GW-r1 73 Dof4.1_F 22 attB1-ad-F * 74 Dof4.1_R 23 attB2-ad-R * 75 Dof4.1_ZFM_F 24 mCherry-S-d1 76 Dof4.1_ZFM_R 25 mCherry-AvrKSac-r3 77 Dof4.1-ΔZFM_F 26 H2B-Avr-d1 78 Dof4.1-ΔZFM_R 27 H2B-HpaK-r1 79 Dof5.4_F 28 attR-Bsgl-dl 80 Dof5.4_R 29 attR-BKpn-r1 81 Dof5.4-ZFM_F 30 Dof4.1-del1_F1Bg 82 Dof5.4-ZFM_R 31 Dof4.1-del1_R1k 83 CrDof-ZFM_F 32 Dof4.1-del2_F2Bg 84 CrDof-ZFM_R 33 Dof4.1-del2_R2k 85 PpDof19-ZFM_F 34 Dof4.1-del3_F3Bg 86 PpDof19-ZFM_R 35 Dof4.1-del3_R3k 87 Dof4.1_GW_d1 36 Dof4.1-del4_F4Bg 88 Dof4.1_GW_d2 37 Dof4.1-del4_R4k 89 Dof4.1_GW_d3 38 Dof4.1-del5_F5Bg 90 Dof4.1_GW_d4 39 Dof4.1-del5_R5k 91 Dof4.1_GW_d5 40 Dof2.2-ZFM_F3Bg 92 Dof4.1_GW_r1 41 Dof2.2-ZFM_R3k 93 Dof4.1_GW_r2 42 Dof5.4-ZFM_F3Bg 94 Dof4.1_GW_r3 43 Dof5.4-ZFM_R3k 95 Dof4.1_GW_r4 44 2Myc_BamHI_F1 96 Dof4.1_GW_r5 45 2Myc_BglII_R1 97 Dof4.1_GW_r6 46 2Myc_BglII_F2 98 Dof4.1_GW_r7 47 2Myc_KpnI_R2 99 Dof2.2_GW_d1 48 GL1_AscI_F 100 Dof2.2_GW_r1 49 Dof2.2-ZFM_BamHI_R 101 Dof2.2_GW_d2 50 Dof5.4-ZFM_BamHI_R 102 Dof2.2_GW_r2 51 PpMKN1-3_BglII_F 103 Dof3.7_GW_d2 52 PpMKN1-3_KpnI_R 104 Dof3.7_GW_r2 53 PpMKN2_BglII_F 105 Dof4.5_GW_d1 54 PpMKN2_KpnI_R 106 Dof4.5_GW_r1 55 PpMKN4_BamHI_F 107 Dof5.6_GW_d1 56 PpMKN4_KpnI_R 108 Dof5.6_GW_r1 57 PpMKN6_BamHI_F 109 Dof5.4_GW_d1 58 PpMKN6_KpnI_R 110 Dof5.4_GW_r1 59 PpHD6_BglII_F 111 Dof-Os04-GW-d1 60 PpHD6_KpnI_R 112 Dof-Os04-GW-r1 61 CrGSM1_BglII_F 113 Dof-Nt-GW-d1 62 CrGSM1_KpnI_R 114 Dof-Nt-GW-r1 63 CrHDG1_BglII_F 115 Dof-Gm-GW-d1 64 CrHDG1_KpnI_R 116 Dof-Gm-GW-r1 65 CrGSP1_BglII_F 117 Dof-Pp09-GW-d1 66 CrGSP1_KpnI_R 118 Dof-Pp09-GW-r1 67 CrCO_BglII_F 119 Dof-Pp15-GW-d1 68 CrCO_KpnI_R 120 Dof-Pp15-GW-r1 69 CrYABBY_BglII_F 121 Dof-Pp19-GW-d1 70 CrYABBY_KpnI_R 122 Dof-Pp19-GW-r1 71

Example  One: Arabidopsis  In root and leaf tissue ITD1 Intercellular migration

To investigate the evolutionary relationship between cell-automonous and non-cell-autonomous (NCA) TF, genome-wide screening of Arabidopsis TF was performed. Through this screening, we isolated the putative NCA-TF, AtDof4.1, from the Dof family and named it INTERCELLULAR TRAFFICKING DOF 1: ITD1. To investigate the migration capability of this NCA-TF, various ITD1-mCherry fusions were expressed under the control of the Upstream Activation Sequence (UAS) in the Haseloff J0571 enhancer trap background (26, http: //www.arexdb. org / db ).

Fig. 1 shows the transfer of various proteins between plant cells according to Example 1 and Example 2. Fig. (AC) mCherry has a wide range of synthesis sites in the cortex (C) and endothelium (E) to cells spanning the entire root tip, including the lateral root cap (L), epithelium (Ep) It seems to move across. (D-F) Myb88-mCherry failed to move beyond the expression site. (G-I) ITD1-mCherry shows multiple cell layer movements (arrowheads) inside and outside of one cell layer. Inner box: Enlarge the box area. (J-L) mCherry-H2B is restricted to the nuclei of the cortex and endothelial cells. (M-O) ITD1-mCherry-H2B exhibits mCherry fluorescence outside of its expression domain, indicating its ability to migrate through PD. Inner box: Brighten mCherry signal to contrast. Rod = 20 ㎛

This J0571 strain expresses the UAS :: GFPer reporter in the fascia and endoderm of the root (Figs. A, D and Fig. 4). As a control for this experiment, the cell pattern of the fluorescence was examined for the UAS :: mCherry (27 kDa) reporter. As shown in Figure 1B, the free form of mCherry can migrate from its synthetic site (shown in Figure 1 A) to most cell types throughout the root tip (B and C in Figure 1). This indicates that the PD connecting most of the root cells expands sufficiently to allow diffusion of the non-selective intercellular movement of the 27 kDa mCherry. Conversely, the larger Myb88-mCherry fusion (81 kDa) did not migrate beyond its synthetic site (D-F in Figure 1). Red fluorescence from the Myb88-mCherry plant is restricted to the nuclei of crusted cells and endoderm cells. This means that larger size and / or nucleated mCherry fusion proteins were prevented from diffusing through the PD.

The UAS :: ITD1-mCherry (61.7 kDa) transgenic JO571 enhancer trap analysis showed the presence of fluorescent signals on both sides of the periphery, although weak, but with GFPer (GI in FIG. 1). The inward movement from the endoderm to the central body tissue was over a wider range than the outward movement from the cortex to the epidermis. No migration to the lateral and peripheral root cap cells was observed. Significant differences in the mobility patterns between mCherry and ITDl-mCherry indicate distinct forms of PD size exclusion limitation or selectivity in ITDl movement.

These two possibilities were tested by conducting experiments with AtDof2.2, a distant relative of ITD1 (27), previously known as cell-autonomous TF. This protein is 36.7 kDa Dof TF, similar in size to ITD1 (33.4 kDa). As expected, the analysis of nine independent T1 AtDof2.2-mCherry (64.9 kDa) indicates that the fluorescent signal is restricted to the expression period indicated by GFPer. These results support the hypothesis that ITD1, not AtDof2.2, has selective mobility over PD.

In the J0571 background, the UAS :: GFPer reporter was also expressed in the leaf tissue of the leaves and mature cervical cells (FIGS. 4 and 5A). Since mature guinea pigs have been reported to be separated symplasmically by PD cleavage during the final stages of maturation (28, 29), the present inventors have tested whether ITD1 can migrate between the mesophyll tissue and epithelial layers. ITDl-mCherry was detected in leaf epithelial cells and demonstrates its migration from the sub-epithelial layer (Fig. 5, B and C). Similarly, mCherry's cell-autonomous fusion protein was restricted to the expression site (G-I in Figure 5), whereas free mCherry was detected in epithelial cells (Figure D-F).

Figure 4 shows the GFPer expression pattern observed in the J0571 enhancer traps. The J0571 plant has a chimeric TF with a GAL4 DNA binding domain and a VP16 activation domain driven by an arabicids intrinsic enhancer controlling expression in cortex, endodermis, leaf mesophyll and guard cells . G4V16 activates the expression of the ER-targeted mGFP construct (GFPer), which is controlled by an upstream activation sequence (UAS) component. (A) A young leaf-surface confocal microscope photograph of Arabidopsis shows the presence of GFP in the epithelial layer. (B) Higher magnification of the surface image indicates that GFP expression is restricted to the guard cells. (C) The leaf section in the brightfield represents the boundary of the epithelium and the leaf tissue. (D) The same focal image of the leaf section indicates that GFP was expressed in the leaf tissue. (E) The same focal image of root cross-section indicates that GFP was expressed in the cortex and endothelial layer. Abbreviation: C, cortex; E, endothelial layer; Ep, epithelial layer; L, lateral cap cells; Me, leaf tissue cells; St, central state. Ratio bars: 100 탆 (A), 5 탆 (B), 20 탆 (C, D), 10 탆 (E).

Figure 5 shows that ITD1-mCherry is able to migrate through the leaf tissue-epidermal layer boundary in young leaves. Transgenic Arabidopsis plants expressing UAS :: GFPer and UAS :: ITD1-mCherry in the (AC) J0571 enhancer trap state. (A) The same focal image on the surface of the leaves indicates the expression of GFPer in the guard cells. (B) ITD1-mCherry fluorescence signals were detected in both the guard cells and the pavement cells. (C) Composition of images (A) and (B). (DF) < / RTI > J0571 enhancer trap strain expressing UAS :: GFPer and UAS :: mCherry. (D) A focal image of a leaf surface that expresses GFPer in a guard cell. (E) mCherry fluorescence signal is detected in both cervical and epithelial cells. (F) (D) and (E) Overlapping of images. (GI) mCherry fused with the cell - autonomous transcription factor failed to pass through the follicular tissue - epithelial cell boundary. Arrows indicate guanosine cells. Arrowheads indicate mCherry signals around the cytoplasm of epithelial cells with large vacuoles. Rod = 20 탆.

Example  2: Optional NCA  By path ITD1  carrying

Non-selective protein transfer through PD can be blocked or significantly reduced by increasing the size of the protein or through isolation in the cytoplasm. Histone 2B (H2B, 16kDa) is a nuclear chromatin binding protein with a strong nuclear localization signal (NLS) that can mediate the nuclear sequestration of the fusion protein (30). Expression of the mCherry-H2B fusion protein (44.2 kDa) was confined to the nucleus in the GFPer labeled expression region at the root of the fluorescent signal (JL in FIG. 1). Thus, the activity of H2B NLS prevented mCherry from diffusing into surrounding cells. Conversely, plant ITD1-mCherry-H2B (77.5 kDa) expression analysis revealed that the fusion protein could still migrate from the cell to the cell, although it is much larger than mCherry-H2B (M-O in FIG. This implies that ITD1 is not moved by simple diffusion, which supports the hypothesis that it travels through a selective path of PD.

The inventors then used a trichome rescue assay as an additional test for the selective transport capability of ITD1. The present inventors wondered if ITD1 could provide the migration function to other cell-autonomous proteins, GLABROUS1 (GL1). For this, the GL1-ITD1 fusion protein was specifically expressed in the glial mutant plant livers.

Fig. 2 shows the results of a trichome rescue according to Example 2 and Example 3. Fig. (A) gl1 transgenic strain expressing GL1-ITD1 exhibited a scar structure. (B) gl1 plants develop hairless leaves. (C) a wild-type leaf that usually represents a gouge pattern. Inner box: Enlarge the box area. Rod = 1 mm. (D-F) Scanning electron micrographs of the leaves of the plants indicated above (A-C, respectively). Rod = 200 탆. (G) Missing mutations tested in predicted domain structure of ITD1 and statistical structure analysis. The numbers correspond to amino acid residues at specific motif boundaries and deletion sites. ZFM, zinc finger motif; NLS, nuclear localization sequence; SR, serine rich region. Low bar: five defective mutations; (-) No gouge structure, (+) Gouge structure. (H) Scintillation structure analysis was performed with the indicated GL1 fusion protein. Statistical structure statistics are provided in Table 2. The KN1 constellation data come from Kim et al (24).

Analysis of the transgenic strain (T1) indicated that GL1-ITD1 could indeed rescue squamish development in the gl1 genetic background (Fig. 2A). Controls in this experiment were the gl1 mutation (Figure 2B) and the wild-type Columbia-0 (Figure 2C) plants. Scanning electron microscopy analysis of leaves from the same plant states confirmed the presence of squamous cells in the GL1-ITD1 (Fig. 2D) and wild-type (Fig. 2F) plants and the absence of squamous in the gl1 mutant strain Of E).

We observed the structure of the strand at 75 transgenic plants, 27% of the 271 tested, ie, 28% structure (H in Figure 2 and Table 2). As a control, we used AtDof 2.2, a cell-autonomous-TF. Only 9 out of 207 independent transgenic plants had a goblet in their leaves (H in FIG. 2). This result indicates that AtDof2.2-mCherry can not move through the PD in the root. The results of the ITD1-mCherry-H2B fusion protein experiments demonstrate that ITD1 contains a selective migration signal capable of providing migration functions to cell-autonomous proteins such as GL1. This is not a general feature of the Arabidopsis Dof family member, as AtDof2.2 lacks the structure of the ashes.

Table 2: Statistical structure analysis of ITD1 deficit experiment

Protein / peptide Total number of independent transgenic Number of weeks Structure Rate (%) AtDof2.2 207 9 4.3 ITD1 _{1 -294} 271 75 27.7 ITD1 _{1 -60} 176 7 4.0 ITD1 _{1 -207} 181 80 44.2 ITD1 _{61 -207} 122 36 29.5 ITD1 _{61 -294} 110 26 23.6 ITD1 _{61 -158} 274 68 24.8 ITD1 _{61 -110} 233 82 35.2 ITD1 _{111 -158} 299 12 4.0 ITD1 _{111 -207} 259 10 3.9 ITD1 _{159 -207} 297 9 3.0 ITD1 _{208 -294} 96 10 10.4

Example  3: ITD1 of Zinc Finger  Motifs provide intercellular transport capability.

The domain structure analysis was then performed on ITD1 to identify potential motifs that could be associated with providing NCA functionality to ITD1. Like the other members of the Dof TF family, ITD1 has a conserved CX ₂ CX ₂₁ CX ₂ C zinc finger motif (ZFM) in its N-terminal region (G in FIG. 2; amino acid residues 70-98). NLS consisting of 35 residues (amino acid residues 92-127) was also predicted using CNLS MApper software (31); NLS has been shown to partially overlap with ZFM. The motif scan program ( http://myhits.isb-sib.ch/cgi-bin/motif_scan ) predicted the serine richness (SR) region located close to the NLS.

We analyzed the structure of the ropes to test the role of the motives that mediate the movement of ITD1 through the PD. A variety of deletions were made to provide a series of GL1-ITD1 constructs and transfected into the gl1 mutant strain. Initially, the ITD1 ORF was divided into five fragments (G in Fig. 2).

Interestingly, the ITD11-207, ITD161-207, and ITD161-294 fragments, including both the ZFM, NLS, and SR regions, have significantly higher mosaic structures (44% compared to ITD11-60 (4%) or ITD1208-294 , 30 and 24%) (Figure 2 H and Table 2). The present inventors have further searched for functional motifs necessary for PD movement by cutting ITD161-207 region. These studies confirmed that ITD161-110 and ITD161-158 (ie residues in the ZFM range) provided 35% and 25% structural ratios, respectively. In contrast, ITD1111-158, ITD1111-207 and ITD1159-207 all failed to mediate the geometry structure (4, 4 and 3%, respectively). Taken together, these results confirm that the region within ITD161-110 containing DNA-bound ZFM is necessary and sufficient for PD movement of ITD1.

Example  4: Arabidopsis Dof family  Preserve movement motif between members

Sequence alignment of the ZFM range region of the Arabidopsis Dof family shows a high degree of conservation (Figure 6). Therefore, the present inventors have tested whether an area equivalent to ITD161-110 of other family members can provide intercellular translocation capability to GL1. An additional five Dof members from various clusters (FIG. 7) were selected for the statistical structure analysis. All five Dof members are expressed in both leaf and root organ (Genevesgator). As predicted from the phylogenetic analysis and the sequence homology relationship level of ZFM (Fig. 3 A), AtDof 3.7 and AtDof 4.5 belonging to the same branching group as ITD1 mediated significant strand conformation Table 3), and the more distant AtDof2.2 and AtDof5.4 were ineffective in rescuing tectonic development. Based on the phylogenetic analysis of this ZFM, it is noteworthy that the more distant AtDof 5.6 also facilitated the scaly structure. Similar conclusions are supported by the phylogenetic tree combined using the entire open reading frame of this Dof TF (Fig. 7). Therefore, the selective movement capability through PD is an early-derived characteristic.

Fig. 3 shows the sequence comparison between the zinc finger motifs of the respective transcription factors according to Examples 4, 5 and 6, and whether or not the structure is a ridge structure. (A) Arranged the sequences of the zinc finger motif (ZFM) range region of the selected AtDof member. There is a high degree of sequence conservation between these regions. The phylogenetic tree (left) shows the relationship between these proteins. Rod = 0.1% sequence difference. The numbers represent ITD1 residues. N-var, N-terminal variable region; ZFM, CX ₂ CX ₂₁ CX ₂ C. (B) The sequences of the ZFM range region of selected members of the Dof family of TFs were aligned (species are provided in Table 4). Phylogeny trees appear on the left. Rod = 0.1% sequence difference. (C) Scaly structure analysis was performed with the indicated ZFM fused to GL1. Statistical structure statistics are provided in Table 2. (D) Both ITD1 N-var and ZFM regions are required to convert AtDof5.4 to NCA-TF. The structures used for the analysis of the structure of the scratches involved the exchange of indicated AtDof5.4 (yellow) and ITD1 (blue) N-var and ZFM. The number of plants showing the structure of the ridges / the total number of plants tested (in%) are shown on the right of each structure.

Table 3: Analysis of the structure of filaments carried out with Dof ZFM of Arabidopsis and other species.

Protein / peptide Total number of independent transgenic lines Number of weeks Structure Rate (%) AtDof5.6 129 28 21.7 AtDof4.5 159 52 32.7 AtDof3.7 139 25 18.0 ITD1 233 82 35.2 AtDof2.2 163 15 9.2 AtDof5.4 173 6 3.5 CrDof 210 48 22.9 PpDof15 277 50 18.1 PpDof09 240 47 19.6 PpDof19 105 27 25.7 GmDof 212 37 17.5 OsDof 162 12 7.4 NtDof 181 3 1.7

Example  5: Dof TF family  Evolution of Intercellular Protein Transfer Capacity in

The Dof family of TF is plant-specific with members present in unicellular green algae, lower and upper plant species (32). A single Dof gene has been reported in Chlamydomonas (32), which appears to represent the ancient ancestor of the Dof family. Since Dof TF has already existed in a single cell organism that does not yet develop PD, the present inventors hypothesized that a particular Dof TF acquired NCA migration capability through subfunctionalization after gene cloning.

Phylogenetic analysis based on the ZFMs of the six different species shows a high degree of conservation between them (Figure 3, B and Table 4). In order to test whether the variable domain sequence associated with algae and moss (non-vascular tracheophyte) is ineffective in mediating intercellular GL1 migration in the higher plant Arabidopsis, the region corresponding to ITD161-110 of these species Were used for statistical structure analysis.

Surprisingly, Dof ZFM (CrDof) from Chlamydomonas , Dof ZFM (PpDof) from Physcomitrella and Glycine max of ZFM (GmDof) and the scaly structure (Fig. 3C and Table 3). On the contrary Nicotiana tabacum and Oryza Sativa 's Dof ZFM (NtDof, OsDof, respectively) was ineffective in mediating the constellation structure like AtDof5.4's ZFM. Taken together with our AtDof ZFM test results and these results, it is speculated that this motif, which provides members of the Dof TF family with NCA function, may precede the evolution of the PD selective pathway.

Table 4: Species used in Dof ZFM transfer experiments

Specification Accession Number / AGI Code / other name Arabidopsis thaliana ) AtDof2.2 (At2g28810), AtDof3.7 (At3g61850) AtDof4.1 (At4g00940), AtDof, 4.5 (At4g21080) AtDof5.4 (At5g60850), AtDof5.6 (At5g62940) Nicotiana tabacum (Nt ) CAA66601.1 Oryza sativa japonica (Os ) Os04g0567800 Glycine max (Gm ) ABI16018.1 Physcomitrella patens (Pp ) PpDof09 (gi | 22491945 |), PpDof15 (gi | 18326732 |), PpDof19 (gi | 7207220 |). Chlamydomonas reinhardtii (Cr ) gi | 10126755 |

Example  6: ITD1  And AtDof5 .4 < / RTI > n- ZFM  Functional analysis of the area.

In order to investigate the role of the N-terminal variable region (N-var) and ZFM in the intercellular translocation capacity of the Arabidopsis Dof TF family, the present inventors have identified a series of exchanged ITD1 and AtDof5.4 domains (Fig. 3, D). When the N-var region of ITD1 was replaced with AtDof5.4, the structure of the ridges could not be observed. AtDof5.4 Interchanging the N-var domain with ITD1 also showed loss of its mobility (Fig. 3D). This experiment demonstrates that the ITD1 N-var domain is required but is not sufficient to deliver NCA functionality to AtDof5.4. Equivalent experiments in which the N-var and ZFM regions were exchanged between ITD1 and AtD5.4 indicated that the modified AtDof5.4, rather than ITD1, could rescue the ridge development (Fig. 3D). This study demonstrates that both the ITD1 N-var and ZFM domains contain signature motifs that are both necessary and sufficient to mediate movement through the PD.

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<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> A complex trafficking through plasmodesmata and method of          transporting targeted material through plasmodesmata <130> PN092500 <160> 122 <170> Kopatentin 2.0 <210> 1 <211> 50 <212> PRT <213> Artificial Sequence <220> ITD1 N-var + ZFM peptide seq [ITDN'-5.4_ZfM] <400> 1 Ser Thr Arg Pro Gln Glu Pro Arg Asn Cys Pro Arg Cys Asn Ser Leu   1 5 10 15 Asn Thr Lys Phe Cys Tyr Tyr Asn Asn Tyr Asn Leu Ser Gln Pro Arg              20 25 30 His Phe Cys Lys Asn Cys Arg Arg Tyr Trp Thr Lys Gly Gly Val Leu          35 40 45 Arg Asn      50 <210> 2 <211> 294 <212> PRT <213> Artificial Sequence <220> <223> ITD1 protein seq <400> 2 Met Asp His His Gln Tyr His His His Asp Gln Tyr Gln His Gln Met   1 5 10 15 Met Thr Ser Thr Asn Asn Asn Ser Tyr Asn Thr Ile Val Thr Thr Gln              20 25 30 Pro Pro Thr Thr Thr Thr Met Thr Thr Thr Thr Thr Met          35 40 45 Ile Met Asp Asp Glu Lys Lys Leu Met Thr Thr Met Ser Thr Arg Pro      50 55 60 Gln Glu Pro Arg Asn Cys Pro Arg Cys Asn Ser Ser Asn Thr Lys Phe  65 70 75 80 Cys Tyr Tyr Asn Asn Tyr Ser Leu Ala Gln Pro Arg Tyr Leu Cys Lys                  85 90 95 Ser Cys Arg Arg Tyr Trp Thr Glu Gly Gly Ser Leu Arg Asn Val Pro             100 105 110 Val Gly Gly Gly Ser Arg Lys Asn Lys Lys Leu Pro Phe Pro Asn Ser         115 120 125 Ser Thr Ser Ser Thr Lys Asn Leu Pro Asp Leu Asn Pro Pro Phe     130 135 140 Val Phe Thr Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Thys 145 150 155 160 Asn Asn Asn Asp Leu Ser Leu Ser Phe Ser Ser Pro Met Gln Asp Lys                 165 170 175 Arg Ala Gln Gly His Tyr Gly His Phe Ser Glu Gln Val Val Thr Gly             180 185 190 Gly Gln Asn Cys Leu Phe Gln Ala Pro Met Gly Met Ile Gln Phe Arg         195 200 205 Gln Glu Tyr Asp His Glu His Pro Lys Lys Asn Leu Gly Phe Ser Leu     210 215 220 Asp Arg Asn Glu Glu Glu Ile Gly Asn His Asp Asn Phe Val Val Asn 225 230 235 240 Glu Glu Gly Ser Lys Met Met Tyr Pro Tyr Gly Asp His Glu Asp Arg                 245 250 255 Gln Gln His His His Val Arg His Asp Asp Gly Asn Lys Lys Arg Glu             260 265 270 Gly Gly Ser Ser Asn Glu Leu Trp Ser Gly Ile Ile Leu Gly Gly Asp         275 280 285 Ser Gly Gly Pro Thr Trp     290 <210> 3 <211> 150 <212> DNA <213> Artificial Sequence <220> ITD1 N-var + ZFM DNA seq [ITDN'-5.4_ZfM] <400> 3 agcactaggc cgcaagaacc aagaaactgt cctcgttgta attctcttaa cacaaagttc 60 tgttactaca acaattacaa tctttctcag cctcgtcact tttgcaagaa ctgtcgtcgt 120 tactggacta aaggtggtgt tctccgtaac 150 <210> 4 <211> 885 <212> DNA <213> Artificial Sequence <220> <223> ITD1 CDS sequence <400> 4 atggaccatc atcagtatca tcatcatgat caataccaac atcagatgat gactagtact 60 aacaataatt cctataacac catcgtcaca acacaaccac caccaacaac aacaacaatg 120 gattcaacaa cagcaacaac tatgataatg gatgacgaga agaagttgat gacgacaatg 180 agcactaggc cgcaagaacc aagaaactgt ccaagatgca actcaagcaa caccaagttt 240 tgttattaca acaactacag cttagcacag cctaggtact tgtgtaagtc ttgtcggaga 300 tattggactg aaggtggctc tctccgtaac gtccccgtag gcggaggttc tagaaagaac 360 aagaagcttc catttcctaa ttcctctact tcttcttcca ccaagaacct cccggatctc 420 aaccctcctt tcgtcttcac atcatcagct tcatcatcaa accctagcaa gacgcatcaa 480 aacaataatg acctcagcct atccttctcc tcccctatgc aagacaagcg agctcaaggg 540 cattacggtc atttcagtga gcaagttgtg acaggagggc agaactgtct tttccaagct 600 cctatgggaa tgattcagtt tcgtcaagag tatgatcatg agcaccccaa aaagaatctt 660 gggttttcat tagacaggaa cgaggaagag attggtaatc atgataactt cgttgttaat 720 gaggaaggaa gtaagatgat gtatccttat ggagatcatg aagaccgtca acaacatcac 780 catgtgagac acgatgatgg taataagaag agagaaggtg gttcaagcaa tgagctatgg 840 agcggaatca tcctaggtgg tgatagtggt ggaccaacat ggtga 885 <210> 5 <211> 1023 <212> DNA <213> Artificial Sequence <220> <223> AtDof2.2 DNA seq <400> 5 atggttttct catccgtctc aagcttttta gatccaccaa ttaattggcc acagtctgcg 60 aatccaaata accatcctca tcatcatcag ctacaagaaa atggaagttt agttagtggc 120 caccaccaag tactctctca ccacttccca caaaacccta accctaacca ccaccatgtt 180 gagacagcag ccgccaccac cgttgatccg agcagtctca atggccaggc ggctgagaga 240 gcgaggctag ctaagaactc tcagccgcca gagggagccc taaagtgtcc tcgatgtgac 300 tcagccaata ccaagttctg ttacttcaac aactacaacc tcacgcagcc acgccacttc 360 tgcaaagctt gccgtcgcta ctggacacgt ggcggtgcct tgaggaacgt acctgtcggt 420 ggtggctgcc ggaggaataa gaagggtaaa tccggaaatt caaagtcttc ctcttcctct 480 cagaacaagc agtcaacgtc tatggtcaac gctacaagcc ctactaatac tagtaatgtc 540 cagctccaaa caaatagcca attcccattt ttgcccactc tacaaaacct cactcaactt 600 ggtggtattg gtttaaactt agccgccatt aatggaaata atggtggaaa tggtaacact 660 agctcaagtt tcttgaatga cttagggttt tttcatggtg gtaacacttc aggtccggtc 720 atgggtaaca acaacgagaa taacctaatg acttctcttg gatcatccag ccactttgct 780 ttgttcgatc gaaccatggg attatataat ttccctaacg aggtaaatat gggattatct 840 tctattggtg ctactagggt ttctcaaact gctcaggtga aaatggagga caaccatttg 900 ggtaatataa gccgcccggt ttcggggttg acatctccag ggaatcaatc caatcaatat 960 tggaccggtc aaggtctccc cggttcttca tctaacgatc atcatcacca gcatcttatg 1020 tga 1023 <210> 6 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> AtDof2.2 ZFM <400> 6 tctcagccgc cagagggagc cctaaagtgt cctcgatgtg actcagccaa taccaagttc 60 tgttacttca acaactacaa cctcacgcag ccacgccact tctgcaaagc ttgccgtcgc 120 tactggacac gtggcggtgc cttgaggaac 150 <210> 7 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> AtDof5.6 ZFM <400> 7 cgtccacctc acgaccatcc tcaaaagtgt cctcgttgcg agtcaacaca tactaagttc 60 tgttactaca ataactacag cctctctcag cctcgttact tctgcaagac ttgtcgccgt 120 tactggacaa aaggcggaac tctaaggaat 150 <210> 8 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> AtDof4.5 ZFM <400> 8 aagcccccac caccaccacc tcgagtgtgt gcaaggtgtg attctgataa tactaaattt 60 tgttattaca acaactactg tgagtttcag ccacgatact tctgcaagaa ctgtcgtaga 120 tactggactc atggtggggc tttaagaaac 150 <210> 9 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> AtDof3.7 ZFM <400> 9 aaggcgaggc cacaagagaa agtaaattgt ccaagatgca actcaacaaa cacaaagttc 60 tgttattaca acaactacag tctcacgcaa ccaagatact tctgcaaagg ttgtcgaagg 120 tattggaccg aaggtggctc tcttcgtaac 150 <210> 10 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> AtDof5.4 ZFM <400> 10 atacttaacc atcatcaatc tctcaagtgt cctcgttgta attctcttaa cacaaagttc 60 tgttactaca acaattacaa tctttctcag cctcgtcact tttgcaagaa ctgtcgtcgt 120 tactggacta aaggtggtgt tctccgtaac 150 <210> 11 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> CrDof ZFM <400> 11 cttccacgcc ccgacaagaa ggaggcatgc cctcgctgca acagcatgga caccaaattc 60 tgctactaca acaattacaa catcaagcag ccccgctttt actgcaagac gtgtcagcgg 120 tactggactg ccggcggcac gttgaggaac 150 <210> 12 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> PpDof15 ZFM <400> 12 ggcactacca agcccaagga ccttccttgc ccgcgctgcc agtccatgaa caccaaattc 60 tgctactaca acaactatag tgtcaaccag ccccgccact tctgccgcaa ttgccaacgc 120 tactggaccg tgggtggtac tctccgcaac 150 <210> 13 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> PpDof09 ZFM <400> 13 cgcgagaagc cagacaaggt gctgccgtgt cctcgctgcg agtccatgaa caccaagttc 60 tgctactaca acaattacag tgtaacacag ccccgccact tctgcaggca gtgtcagcgc 120 tactggaccg cgggagggac gcttcgcaat 150 <210> 14 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> PpDof19 ZFM <400> 14 ctgaagcctc cagatcaagt tattgcgtgc cctcgctgcc aatcccttaa caccaagttt 60 tgttactaca acaactacag ccttacgcag ccccgacatt tttgtaaaag ttgtcgccgc 120 tactggactg caggtgggac tttaagaaat 150 <210> 15 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> GmDof ZFM <400> 15 aaaacaaggc cacaagagca actgaattgt ccaaggtgca attcaaccaa cacaaagttc 60 tgttattaca acaactacag cctcacacag ccaagatact tctgcaagac ttgtagaagg 120 tattggacag aaggagggtc tctgagaaac 150 <210> 16 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> OsDof ZFM <400> 16 cggccgcaga aggagaaggc gctcaactgc ccgcggtgca actcgacgaa caccaagttc 60 tgctactaca acaactacag cctccagcag ccgcgctact tctgcaagac gtgccggcgc 120 tactggacgg agggcggctc gctccgcaac 150 <210> 17 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> NtDof ZFM <400> 17 aggccacaaa aagaacaagc aataaattgt ccaagatgca attcaacaaa cacaaaattc 60 tgttattata acaattatag cctttctcag ccaaggtatt tttgcaaaac ttgtagaagg 120 tattggactg atggtggttc tttaagaaat 150 <210> 18 <211> 13394 <212> DNA <213> Artificial Sequence <220> <223> pZY375 vector sequence <400> 18 cctgtggttg gcatgcacat acaaatggac gaacggataa accttttcac gcccttttaa 60 atatccgatt attctaataa acgctctttt ctcttaggtt tacccgccaa tatatcctgt 120 caaacactga tagtttaaac tgaaggcggg aaacgacaat ctggatccaa gcttcgtatt 180 ggctagagca gcttgccaac atggtggagc acgacactct cgtctactcc aagaatatca 240 aagatacagt ctcagaagac caaagggcta ttgagacttt tcaacaaagg gtaatatcgg 300 gaaacctcct cggattccat tgcccagcta tctgtcactt catcaaaagg acagtagaaa 360 aggaaggtgg cacctacaaa tgccatcatt gcgataaagg aaaggctatc gttcaagatg 420 cctctgccga cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag 480 aagacgttcc aaccacgtct tcaaagcaag tggattgatg tgataacatg gtggagcacg 540 acactctcgt ctactccaag aatatcaaag atacagtctc agaagaccaa agggctattg 600 agacttttca acaaagggta atatcgggaa acctcctcgg attccattgc ccagctatct 660 gtcacttcat caaaaggaca gtagaaaagg aaggtggcac ctacaaatgc catcattgcg 720 ataaaggaaa ggctatcgtt caagatgcct ctgccgacag tggtcccaaa gatggacccc 780 cacccacgag gagcatcgtg gaaaaagaag acgttccaac cacgtcttca aagcaagtgg 840 attgatgtga tatctccact gacgtaaggg atgacgcaca atcccactat ccttcgcaag 900 accttcctct atataaggaa gttcatttca tttggagagg acacgctgaa atcaccagtc 960 tctctctaca aatctatctc tctcgagctt tcgcagatcc gggggggcaa tgagatatga 1020 aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg 1080 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag 1140 gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt 1200 atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg 1260 agtttagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag 1320 acctgcctga aaccgaactg cccgctgttc tacaaccggt cgcggaggct atggatgcga 1380 tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg 1440 gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact 1500 ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga 1560 tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca 1620 acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt 1680 tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta 1740 tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccacgac 1800 tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca 1860 atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg 1920 ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg 1980 tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaagaaat 2040 agagtagatg ccgaccggat ctgtcgatcg acaagctcga gtttctccat aataatgtgt 2100 gagtagttcc cagataaggg aattagggtt cctatagggt ttcgctcatg tgttgagcat 2160 ataagaaacc cttagtatgt atttgtattt gtaaaatact tctatcaata aaatttctaa 2220 ttcctaaaac caaaatccag tactaaaatc cagatccccc gaattaattc ggcgttaatt 2280 cagtacattg ctctggtggt ggttctggtg gcggctctga gggtggtggc tctgagggtg 2340 gcggttctga gggtggcggc tctgagggag gcggttccgg tggtggctct ggttccggtg 2400 attttgatta tgaaaagatg gcaaacgcta ataagggggc tatgaccgaa aatgccgatg 2460 aaaacgcgct acagtctgac gctaaaggca aacttgattc tgtcgctact gattacggtg 2520 ctgctatcga tggtttcatt ggtgacgttt ccggccttgc taatggtaat ggtgctactg 2580 gtgattttgc tggctctaat tcccaaatgg ctcaagtcgg tgacggtgat aattcacctt 2640 taatgaataa tttccgtcaa tatttacctt ccctccctca atcggttgaa tgtcgccctt 2700 ttgtctttgg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca 2760 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga 2820 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt 2880 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca 2940 agcttgcatg cctgcaggtc ggagtactgt cctccgagcg gagtactgtc ctccgagcgg 3000 agtactgtcc tccgagcgga gtactgtcct ccgagcggag tactgtcctc cgagcggaga 3060 ctctagaagc tactccacgt ccataaggga cacatcacaa tcccactatc cttcgcaaga 3120 cccttcctct atataaggaa gttcatttca tttggagagg acgacctgca ggtcgacgga 3180 tccaaggaga tataacaatg aagactaatc tttttctctt tctcatcttt tcacttctcc 3240 tatcattatc ctcggccgaa ttcagtaaag gagaagaact tttcactgga gttgtcccaa 3300 ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg 3360 aaggtgatgc aacaggaaaa cttaccctta aatttatttg cactactgga aaactacctg 3420 ttccatggcc aacacttgtc actactttct cttatggtgt tcaatgcttt tcaagatacc 3480 cagatcatat gaagcggcac gacttcttca agagcgccat gcctgaggga tacgtgcagg 3540 agaggaccat cttcttcaag gacgacggga actacaagac acgtgctgaa gtcaagtttg 3600 agggagacac cctcgtcaac aggatcgagc ttaagggaat cgatttcaag gaggacggaa 3660 acatcctcgg ccacaagttg gaatacaact acaactccca caacgtatac atcatggccg 3720 acaagcaaaa gaacggcatc aaagccaact tcaagacccg ccacaacatc gaagacggcg 3780 gcgtgcaact cgctgatcat tatcaacaaa atactccaat tggcgatggc cctgtccttt 3840 taccagacaa ccattacctg tccacacaat ctgccctttc gaaagatccc aacgaaaaga 3900 gagaccacat ggtccttctt gagtttgtaa cagctgctgg gattacacat ggcatggatg 3960 aactatacaa acatgatgag ctttaagagc tcgaatttcc ccgatcgttc aaacattggc 4020 aataaagttt cttaaagatt gaatcctgtt gccggtctgc gatgattatc atataatttt 4080 ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga 4140 tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata 4200 tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ntatgttact agatcgggaa 4260 ttaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac 4320 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca 4380 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcgc ccgctccttt cgctttcttc 4440 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 4500 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat 4560 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 4620 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc 4680 tattcttttg atttataagg gattttgccg atttcggaac caccatcaaa caggattttc 4740 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga 4800 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccacccca gtacattaaa 4860 aacgtccgca atgtgttatt aagttgtcta agcgtcaatt tgtttacacc acaatatatc 4920 ctgccaccag ccagccaaca gctccccgac cggcagctcg gcacaaaatc accactcgat 4980 acaggcagcc catcagtccg ggacggcgtc agcgggagag ccgttgtaag gcggcagact 5040 ttgctcatgt taccgatgct attcggaaga acggcaacta agctgccggg tttgaaacac 5100 ggatgatctc gcggagggta gcatgttgat tgtaacgatg acagagcgtt gctgcctgtg 5160 atcaaatatc atctccctcg cagagatccg aattatcagc cttcttattc atttctcgct 5220 taaccgtgac aggctgtcga tcttgagaac tatgccgaca taataggaaa tcgctggata 5280 aagccgctga ggaagctgag tggcgctatt tctttagaag tgaacgttga cgatatcaac 5340 tcccctatcc attgctcacc gaatggtaca ggtcggggac ccgaagttcc gactgtcggc 5400 ctgatgcatc cccggctgat cgaccccaga tctggggctg agaaagccca gtaaggaaac 5460 aactgtaggt tcgagtcgcg agatcccccg gaaccaaagg aagtaggtta aacccgctcc 5520 gatcaggccg agccacgcca ggccgagaac attggttcct gtaggcatcg ggattggcgg 5580 atcaaacact aaagctactg gaacgagcag aagtcctccg gccgccagtt gccaggcggt 5640 aaaggtgagc agaggcacgg gaggttgcca cttgcgggtc agcacggttc cgaacgccat 5700 ggaaaccgcc cccgccaggc ccgctgcgac gccgacagga tctagcgctg cgtttggtgt 5760 caacaccaac agcgccacgc ccgcagttcc gcaaatagcc cccaggaccg ccatcaatcg 5820 tatcgggcta cctagcagag cggcagagat gaacacgacc atcagcggct gcacagcgcc 5880 taccgtcgcc gcgaccccgc ccggcaggcg gtagaccgaa ataaacaaca agctccagaa 5940 tagcgaaata ttaagtgcgc cgaggatgaa gatgcgcatc caccagattc ccgttggaat 6000 ctgtcggacg atcatcacga gcaataaacc cgccggcaac gcccgcagca gcataccggc 6060 gacccctcgg cctcgctgtt cgggctccac gaaaacgccg gacagatgcg ccttgtgagc 6120 gtccttgggg ccgtcctcct gtttgaagac cgacagccca atgatctcgc cgtcgatgta 6180 ggcgccgaat gccacggcat ctcgcaaccg ttcagcgaac gcctccatgg gctttttctc 6240 ctcgtgctcg taaacggacc cgaacatctc tggagctttc ttcagggccg acaatcggat 6300 ctcgcggaaa tcctgcacgt cggccgctcc aagccgtcga atctgagcct taatcacaat 6360 tgtcaatttt aatcctctgt ttatcggcag ttcgtagagc gcgccgtgcg tcccgagcga 6420 tactgagcga agcaagtgcg tcgagcagtg cccgcttgtt cctgaaatgc cagtaaagcg 6480 ctggctgctg aacccccagc cggaactgac cccacaaggc cctagcgttt gcaatgcacc 6540 aggtcatcat tgacccaggc gtgttccacc aggccgctgc ctcgcaactc ttcgcaggct 6600 tcgccgacct gctcgcgcca cttcttcacg cgggtggaat ccgatccgca catgaggcgg 6660 aaggtttcca gcttgagcgg gtacggctcc cggtgcgagc tgaaatagtc gaacatccgt 6720 cgggccgtcg gcgacagctt gcggtacttc tcccatatga atttcgtgta gtggtcgcca 6780 gcaaacagca cgacgatttc ctcgtcgatc aggacctggc aacgggacgt tttcttgcca 6840 cggtccagga cgcggaagcg gtgcagcagc gacaccgatt ccaggtgccc aacgcggtcg 6900 gcgtgaagc ccatcgccgt cgcctgtagg cgcgacaggc attcctcggc cttcgtgtaa 6960 taccggccat tgatcgacca gcccaggtcc tggcaaagct cgtagaacgt gaaggtgatc 7020 ggctcgccga taggggtgcg cttcgcgtac tccaacacct gctgccacac cagttcgtca 7080 tcgtcggccc gcagctcgac gccggtgtag gtgatcttca cgtccttgtt gacgtggaaa 7140 atgaccttgt tttgcagcgc ctcgcgcggg attttcttgt tgcgcgtggt gaacagggca 7200 gagcgggccg tgtcgtttgg catcgctcgc atcgtgtccg gccacggcgc aatatcgaac 7260 aaggaaagct gcatttcctt gatctgctgc ttcgtgtgtt tcagcaacgc ggcctgcttg 7320 gcctcgctga cctgttttgc caggtcctcg ccggcggttt ttcgcttctt ggtcgtcata 7380 gttcctcgcg tgtcgatggt catcgacttc gccaaacctg ccgcctcctg ttcgagacga 7440 cgcgaacgct ccacggcggc cgatggcgcg ggcagggcag ggggagccag ttgcacgctg 7500 tcgcgctcga tcttggccgt agcttgctgg accatcgagc cgacggactg gaaggtttcg 7560 cggggcgcac gcatgacggt gcggcttgcg atggtttcgg catcctcggc ggaaaacccc 7620 gcgtcgatca gttcttgcct gtatgccttc cggtcaaacg tccgattcat tcaccctcct 7680 tgcgggattg ccccgactca cgccggggca atgtgccctt attcctgatt tgacccgcct 7740 ggtgccttgg tgtccagata atccacctta tcggcaatga agtcggtccc gtagaccgtc 7800 tggccgtcct tctcgtactt ggtattccga atcttgccct gcacgaatac cagcgacccc 7860 ttgcccaaat acttgccgtg ggcctcggcc tgagagccaa aacacttgat gcggaagaag 7920 tcggtgcgct cctgcttgtc gccggcatcg ttgcgccaca tctaggtact aaaacaattc 7980 atccattaaa atataatatt ttattttctc ccaatcaggc ttgatcccca gtaagtcaaa 8040 aaatagctcg acatactgtt cttccccgat atcctccctg atcgaccgga cgcagaaggc 8100 aatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc cacttacttt 8160 gccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga caagttcctc 8220 ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa atggagtgtc 8280 ttcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt aatccaattc 8340 ggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga tggagtgaaa 8400 gagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt catcttcata 8460 ctcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc agccatcatg 8520 ccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata gcatcatgtc 8580 cttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca tttttaaata 8640 taggttttca ttttctccca ccagcttata taccttagca ggagacattc cttccgtatc 8700 ttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc tcattttagc 8760 catttattat ttccttcctc ttttctacag tatttaaaga taccccaaga agctaattat 8820 aacaagacga actccaattc actgttcctt gcattctaaa accttaaata ccagaaaaca 8880 gctttttcaa agttgttttc aaagttggcg tataacatag tatcgacgga gccgattttg 8940 aaaccacaat tatgggtgat gctgccaact tactgattta gtgtatgatg gtgtttttga 9000 ggtgctccag tggcttctgt gtctatcagc tgtccctcct gttcagctac tgacggggtg 9060 gtgcgtaacg gcaaaagcac cgccggacat cagcgctatc tctgctctca ctgccgtaaa 9120 acatggcaac tgcagttcac ttacaccgct tctcaacccg gtacgcacca gaaaatcatt 9180 gatatggcca tgaatggcgt tggatgccgg gcaacagccc gcattatggg cgttggcctc 9240 aacacgattt tacgtcactt aaaaaactca ggccgcagtc ggtaacctcg cgcatacagc 9300 cgggcagtga cgtcatcgtc tgcgcggaaa tggacgaaca gtggggctat gtcggggcta 9360 aatcgcgcca gcgctggctg ttttacgcgt atgacagtct ccggaagacg gttgttgcgc 9420 acgtattcgg tgaacgcact atggcgacgc tggggcgtct tatgagcctg ctgtcaccct 9480 ttgacgtggt gatatggatg acggatggct ggccgctgta tgaatcccgc ctgaagggaa 9540 agctgcacgt aatcagcaag cgatatacgc agcgaattga gcggcataac ctgaatctga 9600 ggcagcacct ggcacggctg ggacggaagt cgctgtcgtt ctcaaaatcg gtggagctgc 9660 atgacaaagt catcgggcat tatctgaaca taaaacacta tcaataagtt ggagtcatta 9720 cccaattatg atagaattta caagctataa ggttattgtc ctgggtttca agcattagtc 9780 catgcaagtt tttatgcttt gcccattcta tagatatatt gataagcgcg ctgcctatgc 9840 cttgccccct gaaatcctta catacggcga tatcttctat ataaaagata tattatctta 9900 tcagtattgt caatatattc aaggcaatct gcctcctcat cctcttcatc ctcttcgtct 9960 tggtagcttt ttaaatatgg cgcttcatag agtaattctg taaaggtcca attctcgttt 10020 tcatacctcg gtataatctt acctatcacc tcaaatggtt cgctgggttt atcgcacccc 10080 cgaacacgag cacggcaccc gcgaccacta tgccaagaat gcccaaggta aaaattgccg 10140 gccccgccat gaagtccgtg aatgccccga cggccgaagt gaagggcagg ccgccaccca 10200 ggccgccgcc ctcactgccc ggcacctggt cgctgaatgt cgatgccagc acctgcggca 10260 cgtcaatgct tccgggcgtc gcgctcgggc tgatcgccca tcccgttact gccccgatcc 10320 cggcaatggc aaggactgcc agcgctgcca tttttggggt gaggccgttc gcggccgagg 10380 ggcgcagccc ctggggggat gggaggcccg cgttagcggg ccgggagggt tcgagaaggg 10440 ggggcacccc ccttcggcgt gcgcggtcac gcgcacaggg cgcagccctg gttaaaaaca 10500 aggtttataa atattggttt aaaagcaggt taaaagacag gttagcggtg gccgaaaaac 10560 gggcggaaac ccttgcaaat gctggatttt ctgcctgtgg acagcccctc aaatgtcaat 10620 aggtgcgccc ctcatctgtc agcactctgc ccctcaagtg tcaaggatcg cgcccctcat 10680 ctgtcagtag tcgcgcccct caagtgtcaa taccgcaggg cacttatccc caggcttgtc 10740 cacatcatct gtgggaaact cgcgtaaaat caggcgtttt cgccgatttg cgaggctggc 10800 cagctccacg tcgccggccg aaatcgagcc tgcccctcat ctgtcaacgc cgcgccgggt 10860 gagtcggccc ctcaagtgtc aacgtccgcc cctcatctgt cagtgagggc caagttttcc 10920 gcgaggtatc cacaacgccg gcggccgcgg tgtctcgcac acggcttcga cggcgtttct 10980 ggcgcgtttg cagggccata gacggccgcc agcccagcgg cgagggcaac cagcccggtg 11040 agcgtcgcaa aggcgctcgg tcttgccttg ctcgtcggtg atgtacttca ccagctccgc 11100 gaagtcgctc ttcttgatgg agcgcatggg gacgtgcttg gcaatcacgc gcaccccccg 11160 gccgttttag cggctaaaaa agtcatggct ctgccctcgg gcggaccacg cccatcatga 11220 ccttgccaag ctcgtcctgc ttctcttcga tcttcgccag cagggcgagg atcgtggcat 11280 caccgaaccg cgccgtgcgc gggtcgtcgg tgagccagag tttcagcagg ccgcccaggc 11340 ggcccaggtc gccattgatg cgggccagct cgcggacgtg ctcatagtcc acgacgcccg 11400 tgattttgta gccctggccg acggccagca ggtaggccga caggctcatg ccggccgccg 11460 ccgccttttc ctcaatcgct cttcgttcgt ctggaaggca gtacaccttg ataggtgggc 11520 tgcccttcct ggttggcttg gtttcatcag ccatccgctt gccctcatct gttacgccgg 11580 cggtagccgg ccagcctcgc agagcaggat tcccgttgag caccgccagg tgcgaataag 11640 ggacagtgaa gaaggaacac ccgctcgcgg gtgggcctac ttcacctatc ctgcccggct 11700 gacgccgttg gatacaccaa ggaaagtcta cacgaaccct ttggcaaaat cctgtatatc 11760 gtgcgaaaaa ggatggatat accgaaaaaa tcgctataat gaccccgaag cagggttatg 11820 cagcggaaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 11880 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 11940 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 12000 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 12060 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 12120 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 12180 gtgagcgagg aagcggaaga gcgccagaag gccgccagag aggccgagcg cggccgtgag 12240 gcttggacgc tagggcaggg catgaaaaag cccgtagcgg gctgctacgg gcgtctgacg 12300 cggtggaaag ggggagggga tgttgtctac atggctctgc tgtagtgagt gggttgcgct 12360 ccggcagcgg tcctgatcaa tcgtcaccct ttctcggtcc ttcaacgttc ctgacaacga 12420 gcctcctttt cgccaatcca tcgacaatca ccgcgagtcc ctgctcgaac gctgcgtccg 12480 gaccggcttc gtcgaaggcg tctatcgcgg cccgcaacag cggcgagagc ggagcctgtt 12540 caacggtgcc gccgcgctcg ccggcatcgc tgtcgccggc ctgctcctca agcacggccc 12600 caacagtgaa gtagctgatt gtcatcagcg cattgacggc gtccccggcc gaaaaacccg 12660 cctcgcagag gaagcgaagc tgcgcgtcgg ccgtttccat ctgcggtgcg cccggtcgcg 12720 tgccggcatg gatgcgcgcg ccatcgcggt aggcgagcag cgcctgcctg aagctgcggg 12780 cattcccgat cagaaatgag cgccagtcgt cgtcggctct cggcaccgaa tgcgtatgat 12840 tctccgccag catggcttcg gccagtgcgt cgagcagcgc ccgcttgttc ctgaagtgcc 12900 agtaaagcgc cggctgctga acccccaacc gttccgccag tttgcgtgtc gtcagaccgt 12960 ctacgccgac ctcgttcaac aggtccaggg cggcacggat cactgtattc ggctgcaact 13020 ttgtcatgct tgacacttta tcactgataa acataatatg tccaccaact tatcagtgat 13080 aaagaatccg cgcgttcaat cggaccagcg gaggctggtc cggaggccag acgtgaaacc 13140 caacataccc ctgatcgtaa ttctgagcac tgtcgcgctc gacgctgtcg gcatcggcct 13200 gattatgccg gtgctgccgg gcctcctgcg cgatctggtt cactcgaacg acgtcaccgc 13260 ccactatggc attctgctgg cgctgtatgc gttggtgcaa tttgcctgcg cacctgtgct 13320 gggcgcgctg tcggatcgtt tcgggcggcg gccaatcttg ctcgtctcgc tggccggcgc 13380 cagatctggg gaac 13394 <210> 19 <211> 13201 <212> DNA <213> Artificial Sequence <220> <223> pK1401 vector sequence <400> 19 aattcgagct cactagcttc aaaaaacaga gttgccacgg gagacagaat gtttaaatgg 60 aatcaacaag tgaacacaaa ctgcatgttc tgtcacgaac ctatggaaac aagacaccat 120 cttttttttg ggtgctctta ctcgcagaag gtatgggaga agctgataag gggaatacta 180 cttgataagt actcgacata ttggagggaa gtctttaaaa ccatttgtga caaaaactat 240 gacaaaacaa aaatcttcat tctacggtat gtcttccaaa acacggttca ttcgatttgg 300 ggagaaagaa atgcttgaaa gcatggtgaa caaccatcac caatggagaa acttgtcaag 360 ttaattgaca aaaatgtttg gaaccggctg agcacaatac ggagtggggg gagctttgaa 420 gtatgaagga ggtcttcaag tttggtttgg tacaagacaa gcttaagacc aaatcctctg 480 ttttagattt gtttttcaaa aaagtatgca ttaagttgta aacacgttta tttctctttt 540 taaaatctaa tctaaaaatt ttctttttta aaatgacata gaaaaatcca actgtatatt 600 ttaaatccat aaacatgaat tctaaagccg caagtccaaa aatttagaga tgacatcata 660 gcaagcaagg acacgcaaga attaatgaaa tttataatgg agataacaaa gcatgaatat 720 ttataatttg gaaactgttt tacattaaat tagatagttt acgatacatt caaagctaaa 780 acacttattt acaatgaaaa gtaatcaatg tttcttgaat gggtttggct acttcttctc 840 atatggttgg accggggcat atatgagctt ttacgtggta ttcgtggacc catgaccata 900 tgagcttctt catgtggcct tagagtgtgg attcttccct cggatccaca ccatccattg 960 aagtgtgtta taaaatttcc ccatgatccg tcatggcttt tgtgcttaag ttgtcatggt 1020 gggaattatc ttgaggtgcc acattcgtgc ttaagtgggc cgtaagtgca attgcgttgt 1080 gctccaaaag gaacggtgcc gtgggttcct cttgttcatc agaaatatat taattagccg 1140 taaaacctga aaattcacaa gcatttggat tgttttctaa tttaatatcc attatgtgac 1200 taaaagttct agtgatcgta catactacat agaaaataat aacacaaaat actagtttac 1260 atttcccaat taaaaaccat tttgaatgaa ctctgtctga tttaattata cttttaaaat 1320 gtgggatgaa ttcaaagatt atacttatat tcttattatt taagattatc aagtggaaaa 1380 ataaaaatat gaatgtgtta atataaggta atagaaattt aatcattttt ttaatctata 1440 tgtaaaaagt atttaaccga tatctacaat ttgacgcctc ccaattgaaa ggagccaaaa 1500 gcaaccgatc aagtggagac cagtagccat acacattcac tcctaccctt acatgagaaa 1560 gataagatta tggagttttc tgccacgtga tcttatccta gtggtccaaa tcgataaggg 1620 tgtcaacacc tttccttaat cctgtggcaa ttaacgacgt tatcatgaat tatggcccct 1680 ttgatcatta gggctagttg cctctagcgg ttcccactat ataaagatga caaaaccaac 1740 agacaaacaa gtaagtaaga gaaaaaccaa aagaagaaga gaaacaacaa cccaaatcac 1800 tcttttcaaa gcaaatttaa ttctctgaaa attctcatta cttccaaaca atagcagctt 1860 cgaaggatcc agaatgagaa taaggagaag agatgaaaaa gagaatcaag aatacaagaa 1920 aggtttatgg acagttgaag aagacaacat ccttatggac tatgttctta atcatggcac 1980 tggccaatgg aaccgcatcg tcagaaaaac tgggctaaag agatgtggga aaagttgtag 2040 actgagatgg atgaattatt tgagccctaa tgtgaacaaa ggcaatttca ctgaacaaga 2100 agaagacctc attattcgtc tccacaagct cctcggcaat agatggtctt tgatagctaa 2160 aagagtaccg ggaagaacag ataaccaagt caagaactac tggaacactc atctcagcaa 2220 aaaactcgtc ggagattact cctccgccgt caaaaccacc ggagaagacg acgactctcc 2280 accgtcattg ttcatcactg ccgccacacc ttcttcttgt catcatcaac aagaaaatat 2340 ctacgagaat atagccaaga gctttaacgg cgtcgtatca gcttcgtacg aggataaacc 2400 aaaacaagaa ctggctcaaa aagatgtcct aatggcaact actaatgatc caagtcacta 2460 ttatggcaat aacgctttat gggttcatga cgacgatttt gagcttagtt cactcgtaat 2520 gatgaatttt gcttctggtg atgttgagta ctgccttgct gctgctgctg ctgctgctgc 2580 tgctgcagat cttgtcgaca tggaggagat cacccaacac tttggagttg gcgcaagcag 2640 ccacggccat ggccacggcc agcaccacca tcatcaccac caccaccacc cgtgggcatc 2700 ctccctcagc gccgtcgtag cgccgctgcc gccgcaaccg ccaagcgcag gcctgccgct 2760 gccctgaac acggtggcgg ccactgggaa cagcggcggt agcggcaacc cggtgctgca 2820 gcttgccaac ggtggcggcc tcctcgacgc atgcgtcaag gcgaaggagc cctcgtcgtc 2880 gtctccctac gcaggcgacg tcgaggccat caaggccaag atcatctcgc acccacacta 2940 ctactcgctc ctcactgcct acctcgagtg caacaaggtg ggggcaccac cggaggtgtc 3000 ggcgaggctg acggagatag cgcaggaggt ggaggcgcgg cagcgcacgg cgctcggcgg 3060 cctggccgct gcgacggagc cggagctgga ccagttcatg gaggcgtacc acgagatgct 3120 ggtgaagttc agggaggagc tgacgaggcc gctgcaggag gcgatggagt tcatgcgaag 3180 ggtggagtcg cagctgaact cgctttccat ctccggaagg tcgctgcgca acatcctttc 3240 atctggctct tctgaggagg atcaagaagg tagcggagga gagaccgagc tccctgaagt 3300 tgatgcacat ggtgtggacc aagagctgaa gcaccatctc ctgaagaaat acagtggcta 3360 tctaagctcg ctcaagcaag aactgtcaaa gaagaagaag aaagggaagc tccccaagga 3420 ggctcgccag cagctcctta gctggtggga tcagcactac aaatggcctt acccctcaga 3480 gactcagaag gtggcactgg ctgagtctac cgggcttgac ctgaagcaga tcaacaactg 3540 gttcatcaac cagcggaagc ggcactggaa gccatccgag gagatgcacc acctgatgat 3600 ggatgggtac cacaccacca atgccttcta catggacggc cacttcatca acgacggcgg 3660 gctgtaccgg ctcggctagc caccggtatc tcgcttccat ttcacacccc acggcctagc 3720 tataaagact aatggttcca ggtgtctgaa gtactgaaga caggggggct agctatctaa 3780 tgtttgtgcc gcacgcatga gctgtaagga ggccatgctt aattattctg ttgccgttgc 3840 tactctatct atatgcgcct atgcctccgt gcatgaacta tgctttaggt ggttgctgct 3900 ccacactgtg gtggtgtgct tttgcttttg tgtggtcgta ttgtatgcgt aacctgacag 3960 atggatccct gattgctaca tgtttgaata atttgcatga tctagctagt ttctgcctaa 4020 tcaaaaggaa ttctagatgc atcgcaaatc accagtctct ctctacaaat ctatctctct 4080 ctattttctc cagaataatg tgtgagtagt tcccagataa gggaattagg gttcttatag 4140 ggtttcgctc atgtgttgag catataagaa acccttagta tgtatttgta tttgtaaaat 4200 acttctatca ataaaatttc taattcctaa aaccaaaatc cagtgacctg cacgcgttaa 4260 ttaaggatcc tctagagtcg acctgcagca tgcaagcttg gcactggccg tcgttttaca 4320 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc 4380 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg 4440 cagcctgaat ggcgaatgct agagcagctt gagcttggat cagattgtcg tttcccgcct 4500 tcagtttaaa ctatcagtgt ttgacaggat atattggcgg gtaaacctaa gagaaaagag 4560 cgtttattag aataacggat atttaaaagg gcgtgaaaag gtttatccgt tcgtccattt 4620 gtatgtgcat gccaaccaca gggttcccct cgggatcaaa gtactttgat ccaacccctc 4680 cgctgctata gtgcagtcgg cttctgacgt tcagtgcagc cgtcttctga aaacgacatg 4740 tcgcacaagt cctaagttac gcgacaggct gccgccctgc ccttttcctg gcgttttctt 4800 gtcgcgtgtt ttagtcgcat aaagtagaat acttgcgact agaaccggag acattacgcc 4860 atgaacaaga gcgccgccgc tggcctgctg ggctatgccc gcgtcagcac cgacgaccag 4920 gacttgacca accaacgggc cgaactgcac gcggccggct gcaccaagct gttttccgag 4980 aagatcaccg gcaccaggcg cgaccgcccg gagctggcca ggatgcttga ccacctacgc 5040 cctggcgacg ttgtgacagt gaccaggcta gaccgcctgg cccgcagcac ccgcgaccta 5100 ctggacattg ccgagcgcat ccaggaggcc ggcgcgggcc tgcgtagcct ggcagagccg 5160 tgggccgaca ccaccacgcc ggccggccgc atggtgttga ccgtgttcgc cggcattgcc 5220 gagttcgagc gttccctaat catcgaccgc acccggagcg ggcgcgaggc cgccaaggcc 5280 cgaggcgtga agtttggccc ccgccctacc ctcaccccgg cacagatcgc gcacgcccgc 5340 gagctgatcg accaggaagg ccgcaccgtg aaagaggcgg ctgcactgct tggcgtgcat 5400 cgctcgaccc tgtaccgcgc acttgagcgc agcgaggaag tgacgcccac cgaggccagg 5460 cggcgcggtg ccttccgtga ggacgcattg accgaggccg acgccctggc ggccgccgag 5520 aatgaacgcc aagaggaaca agcatgaaac cgcaccagga cggccaggac gaaccgtttt 5580 tcattaccga agagatcgag gcggagatga tcgcggccgg gtacgtgttc gagccgcccg 5640 cgcacgtctc aaccgtgcgg ctgcatgaaa tcctggccgg tttgtctgat gccaagctgg 5700 cggcctggcc ggccagcttg gccgctgaag aaaccgagcg ccgccgtcta aaaaggtgat 5760 gtgtatttga gtaaaacagc ttgcgtcatg cggtcgctgc gtatatgatg cgatgagtaa 5820 ataaacaaat acgcaagggg aacgcatgaa ggttatcgct gtacttaacc agaaaggcgg 5880 gtcaggcaag acgaccatcg caacccatct agcccgcgcc ctgcaactcg ccggggccga 5940 tgttctgtta gtcgattccg atccccaggg cagtgcccgc gattgggcgg ccgtgcggga 6000 agatcaaccg ctaaccgttg tcggcatcga ccgcccgacg attgaccgcg acgtgaaggc 6060 catcggccgg cgcgacttcg tagtgatcga cggagcgccc caggcggcgg acttggctgt 6120 gtccgcgatc aaggcagccg acttcgtgct gattccggtg cagccaagcc cttacgacat 6180 atgggccacc gccgacctgg tggagctggt taagcagcgc attgaggtca cggatggaag 6240 gctacaagcg gcctttgtcg tgtcgcgggc gatcaaaggc acgcgcatcg gcggtgaggt 6300 tgccgaggcg ctggccgggt acgagctgcc cattcttgag tcccgtatca cgcagcgcgt 6360 gagctaccca ggcactgccg ccgccggcac aaccgttctt gaatcagaac ccgagggcga 6420 cgctgcccgc gaggtccagg cgctggccgc tgaaattaaa tcaaaactca tttgagttaa 6480 tgaggtaaag agaaaatgag caaaagcaca aacacgctaa gtgccggccg tccgagcgca 6540 cgcagcagca aggctgcaac gttggccagc ctggcagaca cgccagccat gaagcgggtc 6600 aactttcagt tgccggcgga ggatcacacc aagctgaaga tgtacgcggt acgccaaggc 6660 aagaccatta ccgagctgct atctgaatac atcgcgcagc taccagagta aatgagcaaa 6720 tgaataaatg agtagatgaa ttttagcggc taaaggaggc ggcatggaaa atcaagaaca 6780 accaggcacc gacgccgtgg aatgccccat gtgtggagga acgggcggtt ggccaggcgt 6840 aagcggctgg gttgtctgcc ggccctgcaa tggcactgga acccccaagc ccgaggaatc 6900 ggcgtgacgg tcgcaaacca tccggcccgg tacaaatcgg cgcggcgctg ggtgatgacc 6960 tggtggagaa gttgaaggcc gcgcaggccg cccagcggca acgcatcgag gcagaagcac 7020 gccccggtga atcgtggcaa gcggccgctg atcgaatccg caaagaatcc cggcaaccgc 7080 cggcagccgg tgcgccgtcg attaggaagc cgcccaaggg cgacgagcaa ccagattttt 7140 tcgttccgat gctctatgac gtgggcaccc gcgatagtcg cagcatcatg gacgtggccg 7200 ttttccgtct gtcgaagcgt gaccgacgag ctggcgaggt gatccgctac gagcttccag 7260 acgggcacgt agaggtttcc gcagggccgg ccggcatggc cagtgtgtgg gattacgacc 7320 tggtactgat ggcggtttcc catctaaccg aatccatgaa ccgataccgg gaagggaagg 7380 gagacaagcc cggccgcgtg ttccgtccac acgttgcgga cgtactcaag ttctgccggc 7440 gagccgatgg cggaaagcag aaagacgacc tggtagaaac ctgcattcgg ttaaacacca 7500 cgcacgttgc catgcagcgt acgaagaagg ccaagaacgg ccgcctggtg acggtatccg 7560 agggtgaagc cttgattagc cgctacaaga tcgtaaagag cgaaaccggg cggccggagt 7620 acatcgagat cgagctagct gattggatgt accgcgagat cacagaaggc aagaacccgg 7680 acgtgctgac ggttcacccc gattactttt tgatcgatcc cggcatcggc cgttttctct 7740 accgcctggc acgccgcgcc gcaggcaagg cagaagccag atggttgttc aagacgatct 7800 acgaacgcag tggcagcgcc ggagagttca agaagttctg tttcaccgtg cgcaagctga 7860 tcgggtcaaa tgacctgccg gagtacgatt tgaaggagga ggcggggcag gctggcccga 7920 tcctagtcat gcgctaccgc aacctgatcg agggcgaagc atccgccggt tcctaatgta 7980 cggagcagat gctagggcaa attgccctag caggggaaaa aggtcgaaaa ggtctctttc 8040 ctgtggatag cacgtacatt gggaacccaa agccgtacat tgggaaccgg aacccgtaca 8100 ttgggaaccc aaagccgtac attgggaacc ggtcacacat gtaagtgact gatataaaag 8160 agaaaaaagg cgatttttcc gcctaaaact ctttaaaact tattaaaact cttaaaaccc 8220 gcctggcctg tgcataactg tctggccagc gcacagccga agagctgcaa aaagcgccta 8280 cccttcggtc gctgcgctcc ctacgccccg ccgcttcgcg tcggcctatc gcggccgctg 8340 gccgctcaaa aatggctggc ctacggccag gcaatctacc agggcgcgga caagccgcgc 8400 cgtcgccact cgaccgccgg cgcccacatc aaggcaccct gcctcgcgcg tttcggtgat 8460 gcggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg 8520 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc 8580 gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat 8640 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa 8700 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 8760 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 8820 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 8880 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 8940 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 9000 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 9060 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 9120 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 9180 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 9240 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 9300 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 9360 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 9420 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 9480 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 9540 aaaactcacg ttaagggatt ttggtcatgc attctaggta ctaaaacaat tcatccagta 9600 aaatataata ttttattttc tcccaatcag gcttgatccc cagtaagtca aaaaatagct 9660 cgacatactg ttcttccccg atatcctccc tgatcgaccg gacgcagaag gcaatgtcat 9720 accacttgtc cgccctgccg cttctcccaa gatcaataaa gccacttact ttgccatctt 9780 tcacaaagat gttgctgtct cccaggtcgc cgtgggaaaa gacaagttcc tcttcgggct 9840 tttccgtctt taaaaaatca tacagctcgc gcggatcttt aaatggagtg tcttcttccc 9900 agttttcgca atccacatcg gccagatcgt tattcagtaa gtaatccaat tcggctaagc 9960 ggctgtctaa gctattcgta tagggacaat ccgatatgtc gatggagtga aagagcctga 10020 tgcactccgc atacagctcg ataatctttt cagggctttg ttcatcttca tactcttccg 10080 agcaaaggac gccatcggcc tcactcatga gcagattgct ccagccatca tgccgttcaa 10140 agtgcaggac ctttggaaca ggcagctttc cttccagcca tagcatcatg tccttttccc 10200 gttccacatc ataggtggtc cctttatacc ggctgtccgt catttttaaa tataggtttt 10260 cattttctcc caccagctta tataccttag caggagacat tccttccgta tcttttacgc 10320 agcggtattt ttcgatcagt tttttcaatt ccggtgatat tctcatttta gccatttatt 10380 atttccttcc tcttttctac agtatttaaa gataccccaa gaagctaatt ataacaagac 10440 gaactccaat tcactgttcc ttgcattcta aaaccttaaa taccagaaaa cagctttttc 10500 aaagttgttt tcaaagttgg cgtataacat agtatcgacg gagccgattt tgaaaccgcg 10560 gtgatcacag gcagcaacgc tctgtcatcg ttacaatcaa catgctaccc tccgcgagat 10620 catccgtgtt tcaaacccgg cagcttagtt gccgttcttc cgaatagcat cggtaacatg 10680 agcaaagtct gccgccttac aacggctctc ccgctgacgc cgtcccggac tgatgggctg 10740 cctgtatcga gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg gctggctggt 10800 ggcaggatat attgtggtgt aaacaaattg acgcttagac aacttaataa cacattgcgg 10860 acgtttttaa tgtactgaat taacgccgaa ttaattcggg ggatctggat tttagtactg 10920 gattttggtt ttaggaatta gaaattttat tgatagaagt attttacaaa tacaaataca 10980 tactaagggt ttcttatatg ctcaacacat gagcgaaacc ctataggaac cctaattccc 11040 ttatctggga actactcaca cattattatg gagaaactcg agcttgtcga tcgacagatc 11100 cggtcggcat ctactctatt tctttgccct cggacgagtg ctggggcgtc ggtttccact 11160 atcggcgagt acttctacac agccatcggt ccagacggcc gcgcttctgc gggcgatttg 11220 tgtacgcccg acagtcccgg ctccggatcg gacgattgcg tcgcatcgac cctgcgccca 11280 agctgcatca tcgaaattgc cgtcaaccaa gctctgatag agttggtcaa gaccaatgcg 11340 gagcatatac gcccggagtc gtggcgatcc tgcaagctcc ggatgcctcc gctcgaagta 11400 gcgcgtctgc tgctccatac aagccaacca cggcctccag aagaagatgt tggcgacctc 11460 gtattgggaa tccccgaaca tcgcctcgct ccagtcaatg accgctgtta tgcggccatt 11520 gtccgtcagg acattgttgg agccgaaatc cgcgtgcacg aggtgccgga cttcggggca 11580 gtcctcggcc caaagcatca gctcatcgag agcctgcgcg acggacgcac tgacggtgtc 11640 gtccatcaca gtttgccagt gatacacatg gggatcagca atcgcgcata tgaaatcacg 11700 ccatgtagtg tattgaccga ttccttgcgg tccgaatggg ccgaacccgc tcgtctggct 11760 aagatcggcc gcagcgatcg catccatagc ctccgcgacc ggttgtagaa cagcgggcag 11820 ttcggtttca ggcaggtctt gcaacgtgac accctgtgca cggcgggaga tgcaataggt 11880 caggctctcg ctaaactccc caatgtcaag cacttccgga atcgggagcg cggccgatgc 11940 aaagtgccga taaacataac gatctttgta gaaaccatcg gcgcagctat ttacccgcag 12000 gacatatcca cgccctccta catcgaagct gaaagcacga gattcttcgc cctccgagag 12060 ctgcatcagg tcggagacgc tgtcgaactt ttcgatcaga aacttctcga cagacgtcgc 12120 ggtgagttca ggctttttca tatctcattg cccccccgga tctgcgaaag ctcgagagag 12180 atagatttgt agagagagac tggtgatttc agcgtgtcct ctccaaatga aatgaacttc 12240 cttatataga ggaaggtctt gcgaaggata gtgggattgt gcgtcatccc ttacgtcagt 12300 ggagatatca catcaatcca cttgctttga agacgtggtt ggaacgtctt ctttttccac 12360 gatgctcctc gtgggtgggg gtccatcttt gggaccactg tcggcagagg catcttgaac 12420 gatagccttt cctttatcgc aatgatggca tttgtaggtg ccaccttcct tttctactgt 12480 ccttttgatg aagtgacaga tagctgggca atggaatccg aggaggtttc ccgatattac 12540 cctttgttga aaagtctcaa tagccctttg gtcttctgag actgtatctt tgatattctt 12600 ggagtagacg agagtgtcgt gctccaccat gttatcacat caatccactt gctttgaaga 12660 cgtggttgga acgtcttctt tttccacgat gctcctcgtg ggtgggggtc catctttggg 12720 accactgtcg gcagaggcat cttgaacgat agcctttcct ttatcgcaat gatggcattt 12780 gtaggtgcca ccttcctttt ctactgtcct tttgatgaag tgacagatag ctgggcaatg 12840 gaatccgagg aggtttcccg atattaccct ttgttgaaaa gtctcaatag ccctttggtc 12900 ttctgagact gtatctttga tattcttgga gtagacgaga gtgtcgtgct ccaccatgtt 12960 ggcaagctgc tctagccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa 13020 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat 13080 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg 13140 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac 13200 g 13201 <210> 20 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> UAS_ApaI_F <400> 20 gagcgggccc acacaggaaa cagctatgac 30 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> NOS_ApaI_R <400> 21 gagcgggccc agtgagacgg gcaacagctg 30 <210> 22 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1_F <400> 22 caccatggac catcatcagt atcatcatc 29 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1_R <400> 23 gttaccatgt tggtccacca ctatca 26 <210> 24 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1_ZFM_F <400> 24 caccatggct agcactaggc cgcaagaacc 30 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1_ZFM_R <400> 25 gttacggaga gagccacctt c 21 <210> 26 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1-deltaZFM_F <400> 26 ttgatgacga caatggtccc cgtaggcgga ggttc 35 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1-deltaZFM_R <400> 27 cattgtcgtc atcaacttct tc 22 <210> 28 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Dof5.4_F <400> 28 caccatggct atgcaagata ttcatgattt ctc 33 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Dof5.4_R <400> 29 aggaaggtag agaccactct g 21 <210> 30 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Dof5.4-ZFM_F <400> 30 caccatggct atacttaacc atcatcaatc tc 32 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Dof5.4-ZFM_R <400> 31 gttacggaga acaccacctt 20 <210> 32 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CrDof-ZFM_F <400> 32 caccatggct cttccacgcc ccgacaagaa 30 <210> 33 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CrDof-ZFM_R <400> 33 gttcctcaac gtgccgccgg ca 22 <210> 34 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PpDof19-ZFM_F <400> 34 caccatggct ctgaagcctc cagatcaagt 30 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PpDof19-ZFM_R <400> 35 atttcttaaa gtcccacctg 20 <210> 36 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1_GW_d1 <400> 36 aaaaagcagg ctttatggac catcatcagt 30 <210> 37 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1_GW_d2 <400> 37 aaaaagcagg ctttagcact aggccgcaa 29 <210> 38 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1_GW_d3 <400> 38 aaaaagcagg ctttcgtcaa gagtatgatc 30 <210> 39 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1_GW_d4 <400> 39 aaaaagcagg ctttgtcccc gtaggcgga 29 <210> 40 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1_GW_d5 <400> 40 aaaaagcagg ctttcatcaa aacaataatg 30 <210> 41 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1_GW_r1 <400> 41 agaaagctgg gtttcacatt gtcgtcatca 30 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1_GW_r2 <400> 42 agaaagctgg gtttcaaaac tgaatcattc 30 <210> 43 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1_GW_r3 <400> 43 agaaagctgg gtttcaccat gttggtcca 29 <210> 44 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1_GW_r4 <400> 44 agaaagctgg gtttcagtta cggagagagc c 31 <210> 45 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1_GW_r5 <400> 45 agaaagctgg gtttcacgtc ttgctagggt t 31 <210> 46 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1_GW_r6 <400> 46 agaaagctgg gttccatgtt ggtccacc 28 <210> 47 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1_GW_r7 <400> 47 agaaagctgg gttccatgtt ggtccacca 29 <210> 48 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Dof2.2_GW_d1 <400> 48 aaaaagcagg ctttatggtt ttctcatcc 29 <210> 49 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Dof2.2_GW_r1 <400> 49 agaaagctgg gttcataaga tgctggtg 28 <210> 50 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Dof2.2_GW_d2 <400> 50 aaaaagcagg cttttctcag ccgccagagg 30 <210> 51 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Dof2.2_GW_r2 <400> 51 agaaagctgg gtttcagttc ctcaaggcac c 31 <210> 52 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Dof3.7_GW_d2 <400> 52 aaaaagcagg ctttaaggcg aggccacaag 30 <210> 53 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Dof3.7_GW_r2 <400> 53 agaaagctgg gtttcagtta cgaagagagc c 31 <210> 54 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Dof4.5_GW_d1 <400> 54 aaaaagcagg ctttaagccc ccaccaccac 30 <210> 55 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Dof4.5_GW_r1 <400> 55 agaaagctgg gtttcagttt cttaaagccc c 31 <210> 56 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Dof5.6_GW_d1 <400> 56 aaaaagcagg ctttcgtcca cctcacgacc 30 <210> 57 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Dof5.6_GW_r1 <400> 57 agaaagctgg gtttcaattc cttagagttc c 31 <210> 58 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Dof5.4_GW_d1 <400> 58 aaaaagcagg ctttatactt aaccatcatc 30 <210> 59 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Dof5.4_GW_r1 <400> 59 agaaagctgg gtttcagtta cggagaacac c 31 <210> 60 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Dof-Os04-GW-d1 <400> 60 aaaaagcagg ctttcggccg cagaaggaga ag 32 <210> 61 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Dof-Os04-GW-r1 <400> 61 agaaagctgg gtttcagttg cggagcgagc cgcc 34 <210> 62 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Dof-Nt-GW-d1 <400> 62 aaaaagcagg ctttaggcca caaaaagaac aa 32 <210> 63 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Dof-Nt-GW-r1 <400> 63 agaaagctgg gtttcaattt cttaaagaac cacc 34 <210> 64 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Dof-Gm-GW-d1 <400> 64 aaaaagcagg ctttaaaaca aggccacaag ag 32 <210> 65 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Dof-Gm-GW-r1 <400> 65 agaaagctgg gtttcagttt ctcagagacc ctcc 34 <210> 66 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Dof-Pp09-GW-d1 <400> 66 aaaaagcagg ctttcgcgag aagccagac 29 <210> 67 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Dof-Pp09-GW-r1 <400> 67 agaaagctgg gtttcaattg cgaagcgtcc c 31 <210> 68 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Dof-Pp15-GW-d1 <400> 68 aaaaagcagg ctttggcact accaagccc 29 <210> 69 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Dof-Pp15-GW-r1 <400> 69 agaaagctgg gtttcagttg cggagagtac c 31 <210> 70 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Dof-Pp19-GW-d1 <400> 70 aaaaagcagg ctttctgaag cctccagat 29 <210> 71 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Dof-Pp19-GW-r1 <400> 71 agaaagctgg gtttcaattt cttaaagtcc c 31 <210> 72 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Dof-Cr-GW-d1 <400> 72 aaaaagcagg ctttcttcca cgccccgaca ag 32 <210> 73 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Dof-Cr-GW-r1 <400> 73 agaaagctgg gtttcagttc ctcaacgtgc cgcc 34 <210> 74 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> attB1-ad-F * <400> 74 ggggacaagt ttgtacaaaa aagcaggct 29 <210> 75 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> attB2-ad-R * <400> 75 ggggaccact ttgtacaaga aagctgggt 29 <210> 76 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> mCherry-S-d1 <400> 76 gggcgtcgac atggtgagca agggcgagga 30 <210> 77 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> mCherry-AvrKSac-r3 <400> 77 gggcgagctc tccggtacct tacctaggct tgtacagctc gtccatg 47 <210> 78 <211> 30 <212> DNA <213> Artificial Sequence <220> H2B-Avr-d1 <400> 78 gggccctagg atggcgaagg cagataagaa 30 <210> 79 <211> 41 <212> DNA <213> Artificial Sequence <220> H2B-HpaK-r1 <400> 79 gggcggtacc tcagttaaca gaactcgtaa acttcgtaac c 41 <210> 80 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> attR-Bsgl-d1 <400> 80 ggcgttcgaa gatctcacaa gtttgtacaa aaaagc 36 <210> 81 <211> 42 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > attR-BKpn-r1 <400> 81 ggcgggtacc ttatccttcg aacaccactt tgtacaagaa ag 42 <210> 82 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1-del1_F1Bg <400> 82 ggcgagatct tatggaccat catcagtatc a 31 <210> 83 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1-del1_R1k <400> 83 ggcgggtacc tcacattgtc gtcatcaact tct 33 <210> 84 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1-del2_F2Bg <400> 84 ggcgagatct tagcactagg ccgcaagaac c 31 <210> 85 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1-del2_R2k <400> 85 ggcgggtacc tcagttacgg agagagccac ctt 33 <210> 86 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1-del3_F3Bg <400> 86 ggcgagatct tgtccccgta ggcggaggtt c 31 <210> 87 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1-del3_R3k <400> 87 ggcgggtacc tcacgtcttg ctagggtttg atg 33 <210> 88 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1-del4_F4Bg <400> 88 ggcgagatct tcatcaaaac aataatgacc t 31 <210> 89 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1-del4_R4k <400> 89 ggcgggtacc tcaaaactga atcattccca tag 33 <210> 90 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1-del5_F5Bg <400> 90 ggcgagatct tcgtcaagag tatgatcatg a 31 <210> 91 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Dof4.1-del5_R5k <400> 91 ggcgggtacc tcaccatgtt ggtccaccac 30 <210> 92 <211> 31 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Dof2.2-ZFM_F3Bg <400> 92 ggcgagatct ttctcagccg ccagagggag c 31 <210> 93 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Dof2.2-ZFM_R3k <400> 93 ggcgggtacc tcagttcctc aaggcaccgc cac 33 <210> 94 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Dof5.4-ZFM_F3Bg <400> 94 ggcgagatct tatacttaac catcatcaat c 31 <210> 95 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Dof5.4-ZFM_R3k <400> 95 ggcgggtacc tcagttacgg agaacaccac ctt 33 <210> 96 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> 2Myc_BamHI_F1 <400> 96 ggcggatcct aaagctaccc ctagcactag tatg 34 <210> 97 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 2Myc_BglII_R1 <400> 97 ggcagatctg cacgggttgg gcctttcaaa tcctc 35 <210> 98 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 2Myc_BglII_F2 <400> 98 ggcagatctt aaagctaccc ctagcactag tatgg 35 <210> 99 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 2Myc_KpnI_R2 <400> 99 ggcggtaccg cacgggttgg gcctttcaaa tcctc 35 <210> 100 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> GL1_AscI_F <400> 100 gcaggcgcgc ctccagaatg agaataagga gaag 34 <210> 101 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Dof2.2-ZFM_BamHI_R <400> 101 gcgggatcct ttcagttcct caaggcacc 29 <210> 102 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Dof5.4-ZFM_BamHI_R <400> 102 gcgggatcct ttcagttacg gagaacacca c 31 <210> 103 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> PpMKN1-3_BglII_F <400> 103 ggaagatctt gttgatgtgc gagaggagat cctac 35 <210> 104 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> PpMKN1-3_KpnI_R <400> 104 ggcgggtacc aaatggattg ctgtgccagt tc 32 <210> 105 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PpMKN2_BglII_F <400> 105 ggcgagatct ttatggtcgc cacatcggag 30 <210> 106 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> PpMKN2_KpnI_R <400> 106 gggcggtacc ttatttacct tcgcagctcc agtg 34 <210> 107 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> PpMKN4_BamHI_F <400> 107 ggaagatctt ggcagcaaga agggccgtga aaacc 35 <210> 108 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> PpMKN4_KpnI_R <400> 108 ggcgggtacc ttaaccctcg cagctccagt gtcgcttg 38 <210> 109 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> PpMKN6_BamHI_F <400> 109 ggaggatcct gaggacgttc gtgaggaaat tttgc 35 <210> 110 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> PpMKN6_KpnI_R <400> 110 ggcgggtacc ttagttgcta tgccagtttc gctttcgc 38 <210> 111 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PpHD6_BglII_F <400> 111 ggcgagatct tcttggaatg ctccaacagc 30 <210> 112 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PpHD6_KpnI_R <400> 112 ggcgggtacc ttataccatt ggcttccaca g 31 <210> 113 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> CrGSM1_BglII_F <400> 113 ggcgagatct tgtgcaggtg gctgccagcc t 31 <210> 114 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CrGSM1_KpnI_R <400> 114 gggcggtacc aatgcttgtg ccagtgcctc 30 <210> 115 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> CrHDG1_BglII_F <400> 115 ggcgagatct tagcaagaag ggccgtgaaa ac 32 <210> 116 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> CrHDG1_KpnI_R <400> 116 gggcggtacc tgatgagcgg cttccagatg c 31 <210> 117 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> CrGSP1_BglII_F <400> 117 ggcaagatct tcagctgtac ggcggcgtgg gggat 35 <210> 118 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> CrGSP1_KpnI_R <400> 118 gccgggtacc ttacatgatg gcgggccgcc agtgcct 37 <210> 119 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> CrCO_BglII_F <400> 119 ggcgagatct taacctgact cgcgagcagc g 31 <210> 120 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> CrCO_KpnI_R <400> 120 gggcggtacc tctcctcctt cttggcgaag c 31 <210> 121 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> CrYABBY_BglII_F <400> 121 ggcgagatct tattcggtct ttctcggaaa g 31 <210> 122 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CrYABBY_KpnI_R <400> 122 gggcggtacc aaatggaagc ccacttgtcg 30

Claims (8)

1. A complex capable of moving through a plasmid liaison of a target substance with a peptide consisting of the amino acid sequence of SEQ ID NO: 1, wherein the target substance is a protein having a molecular weight of 1 kDa to 80 kDa and the complex is a peptide composed of the amino acid sequence of SEQ ID NO: Wherein the fusion protein is a fusion protein in which the target substance is bound. 2. The complex of claim 1, wherein said peptide is comprised of the amino acid sequence of SEQ ID NO: 2. delete The complex according to claim 1, wherein the protein is a cell-autonomous protein. delete delete A peptide consisting of the amino acid sequence of SEQ ID NO: 1. Preparing a vector comprising a nucleotide sequence encoding said fusion protein of claim 1;
Transducing said vector into Arabidopsis thaliana; And
And expressing the fusion protein in an Arabidopsis thaliana cell, wherein the target substance is transferred through a plasmatic liaison.

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