KR100595060B1 - GENE FOR FLORAL INDUTION IN Pharbitis PROTEIN ENCODING BY THE SAME EXPRESSION VECTOR CONTAINING THE SAME - Google Patents

Abstract

The present invention relates to a cotyledon specific flowering induction gene, a protein encoded by the morning glory having a nucleotide sequence of SEQ ID NO: 1, the expression vector comprising the gene, the gene of the present invention can be effectively used in connection with induction of morning glory have.

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Description

Cotyledon-specific flowering induction gene of morning glory, protein encoded by it, expression vector comprising same {GENE FOR FLORAL INDUTION IN Pharbitis, PROTEIN ENCODING BY THE SAME, EXPRESSION VECTOR CONTAINING THE SAME}

The present invention relates to a cotyledon-specific flowering induction gene of morning glory, a protein encoded thereby, and an expression vector comprising the same.

Induction of flowering at the stage of development of higher plants is one of the most important and complex stages. Blooming in plants depends on several environmental factors, including photoperiod. In many plants, the change of photoperiod begins flowering, the stage of sexual reproduction, which is called Gwangju. Photogenic species of plants are regulated by the duration of light and dark over a period of approximately 24 hours (Garner and Allard, 1920; Vince-Prue, 1975). The photoperiod response occurs in at least two separate events: the detection of induced photoperiods in leaves and cotyledons, and the shift from the trophic to the reproductive stages in apical meristem (Bernier, 1988). . This includes signal transduction, flowering stimulation, or flowering-inducing hormones from apical leaves and cotyledon. Despite advances in biochemical research, the molecular components of the signal stimulus and the subjects of flowering stimuli that lead to apical tip movements remain enigmatic. Physical evidence and activity for the induction of flowering stimulation and inhibition have been studied by many researchers. The photogenic step of leaves and cotyledons is induced at the apex of the flower with the aim of including changes in gene expression. Some reports provide indirect supply for some changes and provide evidence (Bernier, 1988). Molecular genetics have approached the genetic regulation of flowering time for 10 years, the search for genes that regulate the onset of flowering has increased, and these genes have been thought of as potential transcriptional regulators, mainly with Arabidopsis ( CONSTANS , LUMINIDE PENDENDENS) (Lee et al ., 1994; Putterill et al ., 1995), (RNA linking proteins) (FCA) (Macknight et al ., 1997). Recently, genes ( FT and TFL1 ) homologous to acting down stream of CONSTANS involved in flowering in Arabidopsis have been identified (Kardailsky et al ., 1999; Kobayashi et al ., 1999). Modulations of processes associated with photogenic induction in morning glory or in other plant systems will probably occur numerically, including the amount of gene expression. Changes in several proteins and mRNAs in cotyledon can be expected during memorization induction in flowering response (Bassett et al ., 1991; Gressel et al ., 1978; Lay-Yee et al ., 1987; Li and Tan, 1991; Maeng, 1982; Ono et al ., 1991, 1993, 1996; Stiles and Davies, 1976). Several studies at the molecular level have attempted to identify several genes associated with flowering (Bassett et al ., 2000; Lay-Yee et al ., 1987; O'Neill et al ., 1994; Ono et al , 1993; Sage-On et al ., 1998; Zheng et al ., 1993, 1998). Recently, the differential display of mRNA has been used to explore the PnCO gene, which has homology with the CONSTANS gene involved in photoperiod during the mesmerizing stage. However, there is no known evidence for the operation of this gene in the beginning of flowering.

The present invention focuses on the isolation of genes related to flowering induction during photoperiod reactions. In the morning glory, induction of flowering occurs when the child is young by at least 14 hours of memorization after the cotyledons have grown (Takimoto, 1969). Physical evidence suggests flowering stimulation during memorization produced in cotyledons, and below it also in apical meristem (Zeevaart, 1962). This physical feature has made morning glory a popular plant during the photogenic induction of flowering from single (Vince-Prue and Gressel, 1985).

In the present invention, we isolated and identified genes in the morning glory that express specific expression during a single induced memorization. And this may be related to the induction of flowering

An object of the present invention is to provide a cotyledon-specific flowering induction gene of morning glory.

Another object of the present invention is to provide a cotyledon-specific flowering induction protein encoded by the gene.

Another object of the present invention to provide an expression vector containing the gene.                         

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In accordance with the above object, the present invention provides a cotyledon specific flowering induction gene PnFL 2 encoding the amino acid sequence of SEQ ID NO: 2. In the present invention, the gene may be characterized by having the nucleotide sequence of SEQ ID NO: 1.

According to the above another object, the present invention provides a cotyledon-specific flowering induction protein having the amino acid sequence of SEQ ID NO: 2.

According to another object, the present invention provides an expression vector containing the gene.

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Hereinafter, the present invention will be described in detail.

The gene of the present invention is a specific flowering induction gene ( PnFL 2) represented by the nucleotide sequence of SEQ ID NO: 1, and various modifications may be made to the coding region without changing the amino acid sequence of the protein expressed from the coding region. In addition, various modifications or modifications may be made within a range that does not affect the expression of genes in parts other than coding regions, and such modified genes are also included in the scope of the present invention. Accordingly, the present invention also encompasses polynucleotides having fragment sequences that are substantially identical to those of the genes. Substantially identical polynucleotides refer to those having sequence homology of at least 80%, preferably at least 90% and most preferably at least 95%.

The protein expressed from the gene of the present invention consists of 206 amino acid residues, has an amino acid sequence of SEQ ID NO: 2, and has a size of 22.8 Kd. However, even in the amino acid sequence of the protein, substitution, addition or deletion of amino acids may be made within a range that does not affect the function of the protein, and only a part of the protein may be used depending on the purpose. Such modified amino acid sequences are also within the scope of the present invention. Thus, the present invention also encompasses polypeptides and fragments thereof having substantially the same amino acid sequence as said protein, wherein substantially identical polypeptides have at least 80%, preferably at least 90%, most preferably at least 95% sequence homology. It means things that have.

The genes and proteins of the present invention may be isolated from morning glory or synthesized according to known DNA or peptide synthesis methods. For example, the gene of the present invention can be obtained by screening and cloning according to a conventional method based on the nucleotide sequence information of SEQ ID NO: 1. The genes thus produced can be used to replicate DNA in large quantities or to produce proteins in bulk.

The blooming of morning glory is controlled by photoperiod. This single plant is induced by flowering at least 14 hours after cotyledon growth (Imamura, 1967). Physical evidence suggests that during stimulation, flowering stimuli are produced in the cotyledons and underlying them in apical meristem (Zeevaart, 1962).

In order to extract the gene of the present invention, a differential display method by PCR was used. We obtained a cDNA clone designed with PnFL-2 and found that it was a single copy gene in the morning glory genome. In the database search, the nucleotide of the PnFL-2 gene was a novel gene with little homology with previous genes. The gene of the present invention has a 50% homology with the polypeptide of the Arabidopsis unknown protein (GenBank accession no. NP_173352) or other protein (GenBank accession no. NP_177633). It has a 53% homology with the unknown protein of rice (GenBank accession no. AC091734). The amino acid sequence of PnFL-2 contains a mitochondrial energy transfer motif. However, the direct evidence that this motif is related to flowering induction could not be detected.

The time -dependent change in the amount of PnFL-2 mRNA in cotyledon during photoperiod treatment may explain the close association of this gene with flowering. The amount of PnFL-2 mRNA slowly increases, starting at 10 hours and reaching peak at 16 hours of memorization. In the light cycle, the amount of PnFL-2 mRNA is markedly reduced. The induction of morning glory blooms is well analyzed as a developmental response that is facilitated by a single treatment (Imamura, 1967). A six-day-old morning glory can induce flowering by a single 14-hour memorization. Omitted experiments show that the cotyledons are fully induced by memorizing or specifying the cotyledons for 4 hours and at that time the amount of flowering stimulus is saturated (Zeevaart, 1962). Therefore, if the expression of one or more genes changes with photoperiod induction in morning glory cotyledons, it will be detected within 14 hours of memorization. The correlation between the night length of flowering induction and the expression pattern of PnFL-2 gene in morning glory is notable. The expression of PnFL-2 transcripts decreased with 15 minutes of light in the middle of the cancer cycle (Fig. 5B), indicating that flowering induction was inhibited by night break treatment. Other identified genes of morning glory ( PnGLP , PnC401, PnTCTP, PnZIP, PnCO ) increase or decrease expression during the night break reaction and show good circadian expression (Bassett et al ., 2000; Liu et al ., 2001; Ono et al ., 1996; Sage-Ono et al ., 1998; Zheng et al , 1998). However, it is not clear whether increased expression during memorization and low expression during night break reactions are involved in flowering induction. Because this expression pattern is accompanied by other biochemical and developmental processes, the genes involved in it (Millar and Kay, 1991; O'neil et al ., 1994; Pepper and Chory, 1997; Sage-Ono, 1998). ). Therefore, our results indicate that the presence of the time mechanism in the morning glory is possible by observation of the regulation of the genes of night length and photoperiodic control. Organ- specific accumulation of PnFL-2 mRNA reflects the involvement of this gene in flowering, and is known to synthesize organ induction and flowering-inducing substances in the detection of photoperiodic signals in cotyledons and leaves.

The present inventors in order to accurately describe the function of PnFL-2 gene has PnFL-2 gene over-expressing Arabidopsis thaliana and subjected to the phenotype analysis. When the bolting time of overexpression baby pole and wild type was observed in long-term conditions, it was confirmed that there was a time difference of about 3-4 days, which confirmed that the PnFL-2 gene was indirectly related to induction of flowering. there was.

In the present invention, although there is no direct evidence of the induction of flowering of the PnFL-2 gene in the morning glory, as a result, this novel gene may be functionally related with the flowering induction process.

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

Example 1 Cloning of the PnFL-2 Gene

Total RNAs were extracted from 6-day cotyledons treated with 16 hours of continuous light and dark by the method of Chomczynski and Sacchi (1987). Poly (A ⁺ ) RNA was obtained using paramagnetic oligo (dT) beads (PolyAtract mRNA isolation II, Promega) according to the manufacturer's method. For reverse transcription, the genes they expressed differently using differential display (Liang and Pardee, 1992) were examined using the RNAimage kit (GenHunter). Differential display PCR was performed with 20 different combinations of primers and all four T12MN primers. In addition, one different cDNA band was obtained on 6% polyacrylamide gel. The cDNA fragments were then subcloned into the pBluescript SK + vector (Stratagen). The clone was divided from the same gene, which was later designed as the PnFL-1 gene, and used the cDNAs as a probe for cDNA library screening.

cDNA libraries were constructed using Poly (A ⁺ ) RNA from cotyledon tissue exposed to 16-hour memory. cDNAs were cloned using λZAPII phage vector (Stratagen) by the manufacturer's method. In the cDNA library, differential display PCR products were screened with probes (Sambrook et al . 1989) and subcloned into pBluescript SK + plasmids by in vivo excision pBluescript from the λZAPII phage vector depicted in the stratazine protocol and in full length PnFL-2 cDNA. Got.

Example 2: Sequencing

The sequencing of PnFL-2 cDNA was performed using BigDye terminator RR mix (PerkinElmer) and automated DNA sequencer (model 310 Genetic Analyzer, Perkin Elmer). Nucleotide and amino acid sequencing was performed using the DNASIS program (Hitachi Software Inc, Japan). Data was performed using the BLAST system (Altschu et al ., 1990) and the ExPASy system (Molecular Biology Center at Genova, University Hospital and University of Genova, Genova, Switzerland).

Example 3: Isolation of Genomic DNA and Southern Blot Analysis

Genomic DNA was isolated from morning glory cotyledons by the method of Rogers and Bendich (1988). And it cut out with Bam HI, Bg lII, Eco RI, and Hin dIII (DAKARA). The excised DNA was electrophoresed on 0.8% agarose gel and blotting on a nylon membrane filter (Schleicher & Schuell). The DNA was hybridized with ³² P-labeled full length PnFL-2 cDNA at 65 ° C. for 16 hours in a hybridization solution containing 6X SSPE, 5X Denhardt's solution, 0.1% SDS and 100 μg / ml salmon sperm DNA. And, washed twice with 2X SSPE and 0.1% SDS for 5 minutes at room temperature. And it carried out twice more for 30 minutes at 60 degreeC. After 3 days, the Kodak XAR-5 film was exposed at -80 ° C using an intensifying screen.

Example 4: Northern blot hybridization

RNA extraction and northern blot hybridization methods have already been described above. 30 μg of total RNAs were electrophoresed on 1% formaldehyde-agarose gel and transferred to nylon membrane filter (Schleicher & Schuell). RNAs were bound to the membrane by UV cross-linking. To electrophoretize the same amount of RNA, the gels were stained with ethidium bromide after electrophoresis. RNA on the filter was mixed with ³² P-labeled full-length PnFL-2 cDNA in a hybridization solution containing 50% formamide, 5X SSPE, 5X Denhardt's solution, 0.1% SDS and 100 μg / ml salmon sperm DNA for 16 hours at 42 ° C. Hybridized together. The filter was washed twice in 2X SSPE and 0.1% SDS for 5 minutes at room temperature and again for 30 minutes at 42 ° C. The filter was exposed to Kodak XAR-5 film at -80 ° C using an intensifying screen for 3 days.

Example 5: DNA construct preparation

A vector containing the CaMV 35S promoter was used to construct the PnFL2 gene, and the binary vector pCB302-1 vector (Xiang, 1999) containing the Bar gene and β-glucuronidase ( GUS ) gene. The Bar gene is resistant to Glufosinate ammonium (Basta) in plants, and the Bam HI- Pst I fragment (β-glucuronidase gene) in T-DNA containing Bar and GUS genes is expressed in pCB302-1 vector containing CaMV 35S promoter. Take it out. The coding sequence of the PnFL2 gene is cut into Bam HI and Pst I (Roche) and inserted into the pCB302-1 vector. The pCB302-1 vector containing the PnFL2 gene was transformed to Agrobacterium-tumefaciens strain GV3101 using the freeze and thaw method and grown in soil for 2 weeks before being selected as 0.15% Glufosinate ammonium (Basta).

Example 6: Plant Transformation

Arabidopsis (Colombia spp.) Was subjected to vacuum infiltration transformation method (Bechtold et al ., 1994). Agrobacterium-tumefaciens containing pCB302-1 vector was 50 μg / ml kanamycin and 50 μg / ml rifampicin. Grow in liquid medium until OD600 reaches 2.0. At this point, pellets were obtained by centrifuging the cultured medium, followed by 5% sucrose, 44 nM benzylamino purine and 0.005% (V / V) Silwet L- in 1/2 Murashige and Skoog (MS) salts (Murashige and Skoog, 1962). Add 77 (Lehle Seeds) and immerse the Arabidopsis in medium adjusted to pH 5.7 to transform. The baby pole to be transformed is raised under normal light and cut off the first bolt. The Arabidopsis to be transformed is transformed into the Agrobacterium-tumefaciens mixture when the first flower blooms and the second bolt comes out. When transforming, Agrobacterium-tumefaciens solution is placed in a round container and vacuum is applied at 600 mmHg for 7 min. After transformation, the plants are placed in a cold room for two days and transferred back to the culture room until they receive the next generation of seeds. The transformed Arabidopsis was screened for one week in medium containing MS salts, 1.5% sucrose, 0.75% Phyto-agar (Duchefa) and 50 μg / ml kanamycin and 250 μg / ml carbenicillin. Other transformation lines are also screened in the same medium. Wild type controls use Colombian species.

1 is a Northern blot analysis of PnFL-2 mRNA and column 1 is amplified from total RNA extracted from cotyledons after single treatment for one cycle, row 3 for 3 cycles, column 4 for 5 cycles after continuous exposure to light. One PnFL-2 cDNA fragment. Each row is 30 μg of cotyledon total RNA, and the amount of rRNA is equivalent. This shows that expression increases after a single treatment.

Figure 2 is a diagram analyzing the nucleotide and amino acid sequence of PnFL-2 cDNA. Underscore is. Recognition sequence of restriction enzyme Bgl Ⅱ.

FIG. 3 is a diagram comparing the amino acid sequence of PnFL-2 with another homologous protein. (Genbank accession no.NP_173352), unknown protein 2 of Arabidopsis (Genbank accession no. NP_177633), and unknown protein of rice (Genbank accession no.AC091734) . In comparison to the figure, the gap is for optimal alignment. In the picture, you can see 50% homology with baby pole.

4 shows Southern blot analysis of PnFL-2 gene. Genomic DNA was extracted from cotyledons. Row 1 was Bam HI, row 2 Bgl II, row 3 Eco RI and row 4 Hin d, and DNA gel-blot. hybridization was carried out. Full-length PnFL-2 cDNA was labeled as ³² P-dCTP and used as a probe.

Fig. 5 is a Northern blot analysis of PnFL-2 transcripts. 5A is the total RNAs extracted from the cotyledons at a single interval every 2 to 4 hours, and 5B is the 15-minute night break treatment at 8 hours. .

FIG. 6 shows tissue specific expression of PnFL-2 gene. Total RNA was extracted from different tissues of morning glory young individuals, and RNA gel-blot hybridization was performed. Full-length PnFL-2 cDNA was labeled as ³² P-dCTP and used as a probe. Row L is the first leaf, row C is the cotyledons, row S is the stem, row R is the root. Each row is 30 μg total RNA.

Figure 7 is a phenotype analysis of overexpressing Arabidopsis and wild type, 7A is a wild-type Arabidopsis cultivated three weeks, 7B is a three-week PnFL-2 overexpressed Arabidopsis and 7C is a five-week wild-type and transformant RT It is a -PCR result and is shown using a primer having the following sequence. 7D is the northern blot of wild type and transformants 5 weeks old.

Forward 5`-cggcaagatatgggttcatcggaaa-3` (SEQ ID NO: 3)

Reverse 5`-cggcaaaccctataattcaggttca-3` (SEQ ID NO: 4)

The PnFL 2 gene of the present invention is specifically expressed in the cotyledon of morning glory and may be useful for plants through rapid flowering of plants as it is involved in induction of flowering.

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Claims (5)

Cotyledon specific flowering induction gene ( PnFL 2) encoding the amino acid sequence of SEQ ID NO: 2. The cotyledon specific flowering induction gene ( PnFL 2) according to claim 1, which has a nucleotide sequence of SEQ ID NO: 1. Cotyledon specific flowering induction protein having an amino acid sequence of SEQ ID NO: 2. An expression vector containing the gene of claim 1 or 2. delete