KR100744760B1 - Lateral suppressor-likeDgLsL gene from Dendranthema grandiflorum Kitamura - Google Patents

Abstract

A lateral suppressor-like(DgLsL) gene isolated from Dendranthema grandiflorum Kitamura is provided to reduce the time and labor for removing lateral branching of Dendranthema grandiflorum Kitamura. The lateral suppressor-like(DgLsL) gene isolated from Dendranthema grandiflorum Kitamura 95B1-49 has the nucleotide sequence of SEQ ID NO:1 and is obtained by cloning cDNA(complementary DNA) of lateral suppressor-like gene from Dendranthema grandiflorum Kitamura 95B1-49 and analyzing the whole sequence of the cloned cDNA through RACE(Rapid amplification of cDNA ends). A protein encoded by the lateral suppressor-like(DgLsL) gene isolated from Dendranthema grandiflorum Kitamura 95B1-49 has the amino acid sequence of SEQ ID NO:2.

Description

Lateral suppressor-like (DgLsL) gene from Dendranthema grandiflorum Kitamura

Figure 1 shows the expected amino acid sequence analysis of the geodetic inhibitory gene derived from chrysanthemum. In addition, the sequence and the protein related to the initiation of aquatic meristems for blastocysts (At-Ls (GenBank accession no. AY196482; Arabidopsis thaliana), Le-Ls (GenBank accession no AF098674;. Lycopersicon esculentum ), Os-MOC1 (GenBank accession no. AY242058; Oryza sativa ), Dc-LSLP from GenBank accession no.AB189844; Daucus carota )). In this case, 1 represents a VHIID motif, 2 represents a leucine heptad repeat, 3 represents an LXXLL motif, and 4 represents a SAW motif.

Figure 2 shows the results of phylogeny analysis of the VHIID domain belonging to GRAS obtained as a result of running the TreeView software program using Clsustal W.

Figure 3 shows the Southern blot results of the geodetic suppressor genes in chrysanthemum 95B1-49. In this case, E represents EcoRV, H represents HamH1, and S represents SacI.

Figure 4 is a photograph showing the longitudinal section of the apical growth point after the etepon treatment to chrysanthemum 95B1-49. At this time, A is an apical bud, B is an axillary bud, T is apical meristem after etepon treatment, and C is an untreated control.

5 is a photograph showing the expression pattern of the geodetic inhibitory gene after the etepon treatment at the apical growth point of chrysanthemum 95B1-49. In this case, C represents a control before treatment.

Fig. 6 shows the accumulation degree of the geodetic suppressor gene transcript for each organ. In this case, A analyzes total RNA by tissue using RT-PCR, and PCR product is hybridized with DgLsL gene probe, and shows the result of PCR. B shows real-time PCR product on 2% agarose gel after PCR amplification. The results of the separation are shown and C is a graph showing the relative values for ribosomal RNA.

The present invention relates to a geodetic suppressor gene derived from chrysanthemum, and more particularly, the present invention relates to the development of a new non-geodetic varieties by cloning the geodetic inhibitory gene that controls the geodetic development in chrysanthemum.

During organogenesis, the main axes of growth are determined by two meristems, the SHOT Apical Meristem and the root meristem. SAM is a small group of mitotic tissues that started during the embryogenic period and is located at the end of the growing terrestrial. Cytoplasmic density and cell division rate are confined to three characteristic parts of SMA: peripheral, central and leaf veins. They will represent a functional subdivision of SAM, although there is no direct evidence. Geodetic organs are produced from cells in the periphery, while stem tissue is derived from cells in the leaf veins. The central part serves as a reservoir of stem cells, providing both peripheral and leaf veins while maintaining central integrity.

During embryonic development, plants begin to expand their axis of growth by forming new meristem called laterals. These germs develop flowers, leaves, and geodes (V. Grbic, AB Development). 122 (1996) 2395-2403). To maintain these plants, the germ and leaves share the same origin (IJ Furner et al. Development 115 (1992) 755-764; CJ Hung et al. J. Clin . Endocrinol . Metab . 88 (2003) 3694- 3699). As a result, the growth of the germ endows the geodetic properties of the plants. This geodetic starts with leaf stem cells derived from secondary meristem. This phenomenon is known as apical dominance and appears to be mediated by plant hormones. Basically, oxine suppresses the growth of the side, and cytokinin promotes the growth of the side. However, in geodetic chrysanthemums, ethylene has been reported to increase side growth.

Genetic mutants of GRAS family members showing different patterns of flanks have been reported in various plant species. For example, Arabidopsis with many geodesic auxin is a resistant1, supershoot, max1, max2 and maize teosinte branched. On the other hand, Maize barren with less geodetic inflorescence2 ( bif2 ) and barren stalk1 ( ba1 ), arbidopsis revoluta ( rev ), toamto lateral suppressor ( ls ), arabidopsis lateral suppressor ( las ), toamto blind ( bl ), rice lax panicle ( lax ) and monoculm1 ( moc1 ). Geodetic chrysanthemums 95B1-49 artificially generate geodesy when ethephons are treated during the geodetic period. In addition, these geodetic chrysanthemums are not always geodetic because they are affected by extreme summer temperatures and geodesic formations.

Therefore, the present inventors have tried to find a geodetic suppressor gene, which is a regulatory gene related to the geodetic in chrysanthemum, and apply it to foster an apologetic lineage. The geodetic inhibit gene transcription regulators (transcriptional regulators) tomato Ls (GenBank accession no AF098674 as;. Lycopersicon esculentum ) and rice MOC1 (GenBank accession no.AY242058; Oryza sativa ), carrot DcLSLP (GenBank accession no.AB189844; Daucus carota ), Arabidopsis LAS (GenBank accession no. AY196482; Arabidopsis thaliana ). The removal of geodetic from chrysanthemums is time-consuming and labor-intensive, and the development of perfect geodetic chrysanthemums is a high value-added business because geodetic chrysanthemums are not always geodetic by the extreme temperatures of ethylene and summer.

Therefore, an object of the present invention was to clone the geodetic inhibitory gene in chrysanthemum and to characterize the gene.

For this purpose of the present invention, the cDNA of the geodetic inhibitory gene in the geodetic chrysanthemum is cloned, the full length sequence is analyzed through RACE, the homology with other plants, and the phylogenetic analysis using the expected amino acid sequence is performed. Then, the composition and copy number of the gene in the chrysanthemum genome were examined, and the effect of etepon on the lateral organisms was investigated by treating the atheponic ephesians in the non-geodesic chrysanthemum, and through RT-PCR analysis This was achieved by investigating expression patterns and quantifying and comparing the expressions.

The present invention is a cDNA cloning step of the geodetic inhibitory gene in a geodetic chrysanthemum; Analyzing the gene sequence; And a step of examining the expression pattern of the gene.

The present invention provides a side-suppression gene derived from an azioid chrysanthemum having the nucleotide sequence set forth in SEQ ID NO: 1. The DgLsL gene derived from the geodetic chrysanthemum 95B1-49 has an effect of regulating the initiation of aquatic meristem for geodetic formation.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

EXAMPLE

Example  1: from chrysanthemum DgLsL  Of coding genes cDNA  Isolation and Sequencing of Clones

In order to isolate the geodetic inhibitory gene from the adiabatic chrysanthemum, the geodetic chrysanthemum 95B1-49 was obtained by crossing phoenix and Inga, and was cultivated at 25 ° C for 12 hours in a glass greenhouse at Chungnam National University.

Chrysanthemum seedlings were used to extract total RNA and genomic DNA, and apical part, root, stem and leaf were extracted for RNA extraction. Treatment of 1000ppm of etepon at the apical end of apodic chrysanthemum (95B1-49) before and after treatment for 1, 2, 4, 8, 16, 24 hours and every 1, 3, 6, 9, 12, 15 days The portions were extracted over time and stored at -70 ° C.

To clone the cDNA of the geodetic inhibitor gene in chrysanthemum, the cDNA library first extracted total RNA using Trizol (Invitrogen, USA). RT-PCR was performed with RevertAid ™ H Minus First Strand cDNA Synthesis Kit (MBI Fermentas, USA) using 5 μg of RNA. The first-strand cDNA synthesis reaction product was amplified by PCR using DgLsL specific primers (forward: 5'-ACCGCTAATCAAGCGATT-3 '; reverse: 5'-AGCTTCCAGTGAATCAAA-3') prepared using the tomato Ls gene.

The cDNA library was screened using the product 524bp of the PCR as a probe. cDNA libraries were prepared at 8.1 × 10 ⁷ pfu / μl using the ZAP-cDNA Synthesis Kit (Stratazine, USA). Among them, 4.5 × 10 ⁶ plaques were screened using 524 bp as a probe. The resulting phage was infected with XL1-Blue MRF (stratazine, USA) and changed to bacterial state to obtain plasmid DNA. The plasmid DNA was then analyzed using the T7 and T3 primers, and then analyzed for sequencing.

As a result of cDNA library screening, only a portion of the gene was obtained, so 5 'rapid amplification of cDNA ends (5' RACE) was performed using the 5 '/ 3'-RACE kit (Roche Diagrotics, USA). ) Method was performed. Nine specific primers of chrysanthemum were used to perform 5 'RACE, and PCR products were cloned using pGEM-T easy vector (Promega, USA) and sequenced.

As a result, the DgLsL cDNA encoded an 1686 bp open reading frame, and the predicted protein consisted of 561 amino acids, 103 bp 5'-untranslated region, 57 bp 3'-non-coding region and poly A tail ( Not shown).

The homology assay revealed that the DgLsL gene belongs to the plant-specific GRAS family protein. The GRAS group includes common VHIID motifs, leucine heptad repeats, and LXXLL motifs, and GAI , RGA , Rht -B1 / Rht - D1 , d8 , PAT1 , NLS , which are negative regulators of gibberellin signal transduction. , SCR , SHR and Ls genes belong.

The C-terminal portion of DgLsL contains leucine heptad repeat and LXXLL motifs, both of which mediate the reaction between protein-proteins. The LXXLL motif has been found to mediate the binding of receptors in the nucleus to transcriptional interactivators, and the DgLsL protein is likely to interact with factors involved in transcriptional regulation by these protein domains that regulate the level of transcriptional activation.

In addition, as shown in Figure 1, the gene of DgLsL shows 40% homology with Ls of the carrot, 38% homology with the known carrot. Due to the varying lengths of the N-terminal regions, the Ls of carrots, rice, tomatoes, and Arabidopsis, which have been previously identified, have low homology with each other by 30-50%, suggesting a large difference in plant species of the Ls gene.

The expected amino acid sequences were subjected to phylogenetic analysis using Clsustal W. Then we ran the TreeView software program. The genes used for this analysis were LAS (AY196482; A. thaliana ), Ls (AF098674; L. esculentum ), MOC1 (AY242058; O. sativa ), DcLSLP (AB189844; D. carota ), GAI (Y15193; A. thaliana ), LeGAI (AY269087; L. esculentum ), OsGAI (AB030956; O. sativa ), rht - D1a (CAB51555; Triticum aestivum ), GAIP (AAQ96164; Cucurbita maxima ), GAI1 (AF378125; Vitis vinifera ), d8 (AJ242530; Zea mays ), RGA1 (Y11336; A. thaliana ), RGA2 (Y11337; A. thaliana ), SHR (AF233752; A. thaliana ), PAT1 (NM_203® A. thaliana ), SCR (AF263457; Z. mays ), OsSCR (AB180961; O. sativa ), PsSCR (AB048713; Pisum sativum ), SCARECROW1 (U62798; A. thaliana ), SLN1 (AF460219; Hordeum vulgare ), LOB (NM_125703; A. thaliana), rs2 (AF143447; Z . mays ), LOB (AY542798; Z. mays ), STM (U32344 A. thaliana ) and PhSTM (AY112704; Petunia hybrida ).

As shown in FIG. 2, phylogenetic analysis shows that DgLsL is closely related to the latent suppressor of transcriptional regulator proteins belonging to the GRAS group known as transcription factors.

To elucidate the genomic composition of the DgLsL gene, a genome clone was isolated by PCR by constructing a forward primer (ch5u) in the 5'-UTR portion and a reverse primer (ch3u) in the 3'-UTR portion of the DgLsL gene.

Specifically, the genomic DNA of chrysanthemum was extracted from the leaves of chrysanthemum using the DNeasy Plant Mini kit (Qiagen, USA). Using the extracted genomic DNA, the genome of the geodetic inhibitory gene (DgLsL) of the chrysanthemum was PCR, isolated from 1% agarose gel and cloned into pGEM-T easy vector (Promega, USA), and then analyzed for sequencing.

Sequencing revealed no sequence deviation between the cDNA and its genomic counterpart. This proves that the DgLsL gene does not contain introns.

Southern blot was performed to measure the copy number of the DgLsL gene in chrysanthemum. For this purpose, the genomic DNA of chrysanthemum was extracted from the leaves of chrysanthemum using DNeasy Plant Mini kit (Qiagen, USA). 40 μg genomic DNA restriction enzyme Eco R V, Bam H I, Sac I was cut using I, separated on a 0.8% agarose gel and transferred to a nylon membrane (Hybond N +, Amersham, UK) to genomic DNA and fixed with UV. The blotted membrane was pre-hybridized at 65 ° C. using Church buffer method 7% SDS, 0.5M EDTA, 0.5M sodium phosphate and 1% bovine serum albumin. After prehybridization at 65 ° C. for about 4 hours, an open reading frame (0.6 kb) of the geodetic inhibitory gene (DgLsL) labeled with P ³² was hybridized at 65 ° C. for overnight with a probe. The filter was then washed twice at 65 ° C. for 20 minutes using 2 × SSC (30mM sodium citrate, 0.3M NaCl, pH7.0) and 0.1% SDS solution, then at 1 × at 65 ° C. for about 10 minutes. Final washes were performed using SSC (30 mM sodium citrate, 0.3M NaCl, pH7.0) and 0.1% SDS. The membrane filter was developed for about 1 day using the PERSONAL MOLECULAR IMAGER FX system (Biorad, USA).

As shown in FIG. 3, the DgLsL gene had one copy in the genome of chrysanthemum (see FIG. 3).

Experimental Example  One: A geodetic  Chrysanthemum Ethephon  Phenotype analysis after processing

Phenotypes were analyzed after etepon treatment to investigate the effects of etepon on aphrodisiac in atrial chrysanthemum.

To this end, by treating 1000ppm of etepon at the apical end of the geodetic chrysanthemum (95B1-49), the apical growth point was extracted every 1, 3, 6, 9, 12 and 15 days before and after treatment. Then precipitated in fixed solution (FAA: 10% (v / v) formaldehyde, 5% (v / v) glacial acetic acid, 50% (v / v) ethanol, 35% (v / v) dH ₂ O) Fixed. Then infiltration with paraffin. It was then cut into 10 μm of lamella cutting machine (microtome) for speculum, and the specimen was placed on a slide glass coated with silicon (Makenami glass, Japan) and observed with a scanning electron microscope (SEM, Hitachi, Japan).

As shown in FIG. 4, it was confirmed that a solution was observed 12 days after the etepon treatment.

Experimental Example  2: Chrysanthemum side ground suppressor  Gene expression pattern

The existing Ls mRNA was found to be very low in expression level, and thus, Northern pattern analysis did not confirm the expression pattern and confirmed by RT-PCR. Chrysanthemum DgLsL was also performed in Northern, but the expression level was so small that the expression pattern was confirmed by RT-PCR. Since the apocalyptic chrysanthemum generates a solution after the etepon treatment, RT-PCR was performed to determine whether the DgLsL gene is also expressed after the etepon treatment.

To this end, total RNA was extracted using TRIzol (Invitrogen, USA). For RT-PCR, cDNA was prepared using RevertAid ™ H Minus First Strand cDNA Synthesis Kit (MBI Fermentas, USA) using 5 μg of RNA. Chrysanthemum Geodetic Suppressor (DgLsL) gene was also identified with Etepon treatment to determine whether it was expressed when treated with etepon. PCR was confirmed using a primer (forward: 5'-CACCCCCTACAATAGTGACCGT-3 '; reverse: 5'-GCGTTCAAAAACTCGATGGT-3').

As shown in FIG. 5, the control group not treated with etepon was found to have a band after the etepon treatment but the band was not identified, and thus, DgLsL was expressed depending on the etepon treatment.

In addition, in order to investigate the organ-specific expression patterns of the chrysanthemum geodetic inhibitor (DgLsL) gene, PCR was performed by organs of flowers, leaves, stems, apical meristems, and roots with the cDNA. RT-PCR performed PCR 34 cycles for the former and 25 cycles for the latter. To confirm whether the same amount of cDNA was added to each PCR reaction, primers (forward: 5'-CTCATGGGATGTGGCTTCTT-3 '; reverse: 5'-GCGTTCAAAAACTCGATGGT-3') were used to amplify RNA genes of ribosomes as controls.

As shown in Figures 6A and B, transcripts were expressed at low levels in all tissues and slightly higher in stems.

Therefore, in order to quantify and quantify the difference in expression of Chrysanthemum Geodetic Suppressor (DgLsL) gene, real-time quantification using Rotor-Gene 2000 PCR machine (Korbet Research, Australia) and SYBR Premix Ex Taq mix (Takara Bio, Japan) Real-time PCR was performed. 40 cycle PCR amplification using Chrysanthemum Geodetic Suppressor (DgLsL) gene to see the expression based on the same cDNA used in RT-PCR and a ribosomal RNA which is a control gene used for gene expression analysis Was performed. Similar chrysanthemum geodetic inhibition (DgLsL) gene expression was 2 ^were digitized using a ^{△ Ct (Hung et al 2003.} ). This calculation is valid because the measured PCR efficiency is close to 100%.

As shown in FIG. 6C, it appeared in all organs but was highest in the stem. In chrysanthemums, germs appear irregularly between leaves and stems as well as at the top end. This phenomenon has been reported to be the result of some unknown effects in the vascular bundle of stems. Therefore, it may be because of the high expression in the stem of chrysanthemum. The origin of the blastocysts is not yet clear. But BRANCHED is involved in meristem SAM SILKLESS1 ( BD1 ) / FRIZZY PANICLE ( FZP ), CUPSHAPED COTYLEDON ( CUC ) / NO APICAL MERISTEM ( NAM ), LATERAL ORGAN BOUNDARIES ( LOB ), genes of STM have been identified.

In conclusion, the DgLsL gene from geodetic chrysanthemum 95B1-49 regulates the initiation of adipose meristem for geodetic formation.

In the future, the correlation between these genes involved in SAM and Ls should be investigated, and the functional analysis of the transformed chrysanthemum should be performed by deleting the gene of DgLsL.

As described through the above Examples and Experimental Examples, the present invention relates to a geodetic inhibitory gene derived from chrysanthemum, regulates the initiation of aquatic meristem for geodetic formation derived from a geodetic chrysanthemum, and exists as a copy in the genome In addition, there is an effect of providing a geodetic inhibitor gene without introns. In addition, the gene has an effect that is expressed when treated with etepon. Therefore, the present invention can be used to suppress the generation of geodes of chrysanthemum by using the gene, it is a very useful invention in the breed development industry because it has the effect of reducing the time and labor required to remove the geodes of chrysanthemum.

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Claims (2)

A cation inhibitory gene derived from a geodetic chrysanthemum 95B1-49 having the nucleotide sequence set forth in SEQ ID NO: 1. Squamous inhibitory protein derived from non-fatty chrysanthemum 95B1-49 having the amino acid sequence set forth in SEQ ID NO: 2.

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