Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8237—Externally regulated expression systems
  • C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline

Definitions

• a new flavor of a genetically engineered tomato that suppresses the expression of the gene for polygalacturonic acid degrading enzyme in tomato to suppress the ripening of tomato is well known. Attempts have been made to suppress the expression of the enzyme gene, suppress the generation of ethylene, and suppress the ripening of tomatoes.
• ethylene response control involves recombination of a target gene of a transcription amplification factor gene, ethylene, which stimulates a promoter of an ethylene-responsive gene.
• This group of ethylene-responsive transcription factors (ERF s) is known to be a factor that positively regulates the expression of a group of ethylene-responsive plant genes (Ohme-Takagi, M. and Shinshi, H., ( 1995) Ethylene-inducible DNA binding proteins that interact with an ethylene-; responsive element.Plant Cell 7: 173-182 .; Suzuki, K., Suzuki, N., Ohme-Takagi, M. and Shinshi, H., ( 1998) Immediate early induction of mRNAs for ethylene-responsive transcription factors in tobacco leaf strips after cutting. Plant J. 15: 657-665.
• JP-A-2000-50877 discloses a method for imparting resistance to environmental stress to plants such as tobacco by introducing a transcription factor that controls an ethylene-inducible gene.
• U.S. Pat. No. 5,824,868 discloses a method for reducing the ethylene responsiveness of a plant transformed with a modified ethylene responsive DNA and controlling the expression of such a transformed nucleic acid. It has been disclosed. Problems to be solved by the invention
• An object of the present invention is to specify a transcriptional coactivator (transcriptional cofactor, MBF) of an ethylene-responsive transcription factor group (ERF s), and to specifically control the expression of an ethylene-responsive plant gene group.
• MBF transcriptional cofactor
• EEF s ethylene-responsive transcription factor group
• the gene used to control the ethylene response according to the present invention encodes multiprotein bridging 'factor-1 (MB F 1), which is one of the transcriptional regulators required for the ethylene response.
• MBF1 multiprotein bridging 'factor-1
• Transcriptional regulators have only a small number of molecules in cells, but they are important factors that control the intracellular information network that they constitute. It can affect biological response. Therefore, the expression level of the MBF1 gene originally in the plant body is changed by using a gene that is subsequently introduced into the plant, It provides a means to alter the ethylene responsiveness of plants and control the freshness of crops.
• Enhancing the ethylene response of a plant may be confirmed to directly enhance the ethylene response of the plant, or it may be confirmed that it enhances the expression of an ethylene-responsive transcription factor (ERF s;), for example, ERF2 described below. You may.
• ERF s ethylene-responsive transcription factor
• the gene which is considered to have the same function as the amino acid sequence of rice MBF1 cloned by the inventors is also found in other plants.
• Arabidopsis AtMBFla 81.69%, AtMBFlb; 79.58%, AtMBFlc; 47.97%, Satsuma Remono StMBFl; 78.17%, Castor RcMBFl; 82.39% ⁇ Tomato LeMBFl; 44.90.
• the least homologous tomato, LeMBFl is induced by ethylene, suggesting that it is also a gene involved in the ethylene response.
• the present invention is a gene comprising the DNA of the following (a) or (b).
• the present invention is a polynucleotide comprising a part of this gene.
• promoter used here examples include a cauliflower mosaic virus 35S open motor, a heat shock promoter, a chemically-conductive promoter, and the like.
• the present invention is a plasmid containing the above polynucleotide.
• the plasmid used here include binary vectors such as a Ti plasmid and a pBI-121 plasmid.
• the present invention is a plant transformed with the polynucleotide.
• Plants to which the present invention can be applied include monocots such as rice, maize and wheat, and dicots such as tomato.
• FIG. 1 shows the nucleotide sequence of the DNA probe used in Reference Example 1.
• the figure shows the wild-type ERE (top) and the mutant ERE (mERE, bottom).
• Each DNA probe contains two copies of the GC box (bold) or the mutant GC box.
• FIG. 2 shows a gel electrophoresis showing specific binding of ERF 2 to ERE in gel shift assay.
• F indicates free DNA probe with no binding
• C indicates DNA-ERF2 complex.
• FIG. 3 shows the structure of plasmid DNA type II (pERE) used for in vitro transcription.
• FIG. 5 shows the electrophoresis of transcripts from plasmid DNA type II (pERE) in the presence of the purified recombinant protein indicated at the top of each lane.
• FIG. 6 corresponds to the nucleotide sequence of cDNA of rice MBF1 (oMBF1) (SEQ ID NO: 2) and the predicted amino acid sequence (SEQ ID NO: 1, 85-510 of SEQ ID NO: 2). ).
• the underlined section indicates the poly-A addition signal.
• FIG. 7 shows the effect on transcription of reporter plasmid DNA (pERE-GUS) in transient 'Atsusei' using tobacco leaves.
• the vertical axis indicates an increase in GUS activity depending on the amount of reporter 'plasmid DNA (pERE-GUS) introduced into the tobacco leaves. Values are the average of the results of two independent experiments.
• FIG. 8 shows the effects of the addition of a transcription factor (ERF2) and a transcriptional co-activator (oMBFl) on transient 'Atsusei' using tobacco leaves.
• ERF2 transcription factor
• oMBFl transcriptional co-activator
• FIG. 9 shows oMBF 1 -dependent amplification of the transcriptional activation ability of ERF2 in transient atssie using tobacco leaves.
• the effect of oMBF1 addition on the transcription of reporter plasmid DNA in an ERF2-dependent manner is shown.
• each reaction contained 2 g pERE-GUS, 1 g p35S-LUS, 0.2 ig p35S-ERF2, and various amounts of p 35S—MBF 1 was added.
• the figure shows the average of the results of two independent experiments.
• FIG. 10 shows the oMBF1-dependent amplification of the transcriptional activation ability of ERF2 in the transient 'Atsusei' using tobacco leaves.
• ERF2 shows transcription amplification depending on the nucleotide sequence of ERE in the presence.
• the reporter and effector 'plasmid DNAs shown below each bar graph were mixed and then introduced into tobacco leaves. The figure shows the average of the results of three independent experiments.
• FIG. 12 shows the oMBF1-dependent amplification of the transcription activation ability of ERF4 in transient 'Atsusei using tobacco leaves.
• reporter 1 and effector 'plasmid DNA were mixed and then introduced into tobacco leaves.
• the figure shows the average of the results of three independent experiments. The invention's effect
• the transcriptional coactivator gene of the present invention can be used.
• the target gene that responds to ethylene is changed, or the gene that synthesizes ethylene is changed.
• the gene that encodes a transcriptional cofactor as an information molecule is changed as in the system of the present invention.
• the expression of the target gene group can be controlled collectively, so that a greater effect can be exerted than with the conventional method.
• each recombinant E. coli-derived E. coli was overexpressed in E. coli and purified.
• the protein coding region of each of the four tobacco ERFs was inserted into the expression plasmid pET15b (Novagen, Madison, WI), and the recombinant ERF protein was transformed into E. coli (BL21 / DE3 / pLysS). It was expressed in large quantities.
• the four types of recombinant proteins were purified by using a carrier (His-bind resin, Novagen) in which Ni was individually incorporated.
• Recombinant TBP derived from octopus (t TBP) was purified by the method already reported. (Biosci. Biotech.
• the DNA fragment containing 53 bp of length wild-type ethylene-responsive element (ERE) ( ⁇ "32 P ) A ⁇ ⁇ and ⁇ 4 -polynucleotide kinase (TA ARA shuzo No. Ltd., Kyoto, JAPAN) using the The DNA probe was radiolabeled, and the multiple copies of ERE (SEQ ID NOS: 3 and 4) or mERE (SEQ ID NOs: 5 and 6) fragments (Fig. 1) were ligated to wild-type or mutant, respectively.
• MERE mutant type
• MERE mutant type
• ERE wild type
• ERF2 amplifies ERE-dependent transcription in HeLa nuclear extract (HNE).
• HNE HeLa nuclear extract
• the plasmid DNA for in vitro transcription used here was constructed as follows. In order to construct the plasmid DNA type II (pERE) in FIG. 3, the previously reported plasmid pU35 (Pro. Natl. Acad. Sci. USA 87: 7035-7039 (1990)) ⁇ DB g1 II site Then, two copies of the ERE DNA fragment (Fig. 1) were inserted. For pmERE plasmid DNA ⁇ M, two copies of mERE were inserted instead of ERE in the same manner as above. PHSE 200 TA and pH S E 200GA, which are control 'plasmid DNA alive', are as previously reported (Plant Mol. Biol. 34: 69-79 (1997)).
• the in vitro transcription reaction was examined according to the following procedure.
• the HeLa nuclear extract used for in vitro transcription was prepared according to a previous report (Meth. Enzymol-101: 582-598 (1983)).
• Table 2 shows the composition of the reaction solution for standard in vitro transcription.
• HNE human nuclear extract
• the 15 OmM sodium acetate transfer reaction was performed at 30 ° C for 60 minutes and stopped by adding a 75 t1 stop solution (Table 2).
• the reaction mixture was further added with 100 / X 1 PCIAA (50% phenol, 48% chloroform, 2% isoamyl alcohol) to recover the aqueous layer, and then added 1001 CIAA (96%) to the aqueous layer. % Chloroform and 4% isonoamyl alcohol), and the aqueous layer was collected.
• 1 ⁇ l of 3 M sodium acetate and 300 W of ethyl alcohol were added to this solution to recover nucleic acids.
• pHS E20 OTA is upstream of the gene encoding the Arabidopsis heat shock protein 2 It contains a 100 bp promoter sequence and a 200 bp transcription region that does not contain guanine residues in the sense strand.
• an effector 'plasmid DNA (p35S-MBF1) expressing MBF1 in tobacco cells was constructed in the following manner.
• p35S-ERF2 and p35S-MBF1 of the plasmid DNA the plasmid.
• the XbaI—SacI fragment of the vector pBI221 (CLONTECH Laboratories Inc., CA) was used.
• a part coding for a certain j3-gunorecuronidase was stripped, and there was a cDNA of ERF2 of tobacco ERF2 (accession No. ABO 16264) having both an upstream XbaI site and a downstream SacI site added by PCR. )
• a cDNA fragment of rice MBF1 SEQ ID NO: 2.
• Table 3 summarizes the plasmid DNA used in this example.
• Trigent 'Atsusy' using tobacco leaves was performed according to a previous report (Plant Mol, Biol. Reporter 18: 101-107 (2000)). 2 / zg of reporter-plasmid DNA (p ERE-GUS), 1 ⁇ g of control 'plasmid DNA (p 35 S-LUC) and various amounts of effector. g of gold microparticles (1.5 diameter, 3.0 / Xm, Aldrich Chem. l) and 30 ⁇ l of TE buffer. Next, 3 ⁇ l of sodium triacetate and 100 / X1 of ethyl alcohol were added thereto, and the DNA was adsorbed by centrifugation.
• reporter-plasmid DNA pERE-GUS
• p35S-LUC control 'plasmid DNA
• oMBF1 functions as a transcriptional coactivator for clones other than ERF2, for example, for ERF4, the same experiment as in Example 1 was performed using p35S-ERF2 to p35 S—Changed to ERF 4. The results are shown in FIGS. 11 and 12.

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• 2002
  • 2002-02-12 JP JP2002033512A patent/JP3848173B2/ja not_active Expired - Fee Related
• 2003
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