# Patent

## Title
Gene encoding protein involved in cytokinin synthesis

## Abstract

A gene is provided that encodes an amino acid sequence indicated in SEQ ID NO. 2, 4, 6, 8, 10, 12 or 14 originating in, for example, Arabidopsis thaliana.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional application of U.S. application Ser. No. 10/471,040, filed Sep. 8, 2003, abandoned, which is a National Phase Application of International Appl. No. PCT/JP02/02315, with an International Filing Date of Mar. 12, 2002 and designating the United States of America, which claims the benefit of Japanese Appl. No. 2001 69489, filed Mar. 12, 2001. Japanese Appl. No. 2001 69489, filed Mar. 12, 2001 is incorporated herein by reference in its entirety for all purposes.


REFERENCE TO A SEQUENCE LISTING
Incorporated herein by reference in its entirety is a Sequence Listing containing SEQ ID NOs: 1-35.
FIELD OF THE INVENTION
The present invention relates to a gene encoding a protein involved in cytokinin synthesis, a method of utilizing that gene, and a method for acquiring that gene.
BACKGROUND ART
Cytokinins are an important type of plant hormone. They have various effects including induction of cell division, formation of new buds, overcoming dormancy of axillary buds, prevention of aging and promotion of enlargement of fruit. Cytokinins have a structure in which a dimethylallyl group (isopentenyl group) bonds to a nitrogen atom at position 6 of adenine or adenosine, or has a structure in which the isopentenyl group is hydroxylated as their basic skeleton. Some bacteria that are pathogenic to plants are known to have cytokinin synthesis enzymes and among these, the cytokinin synthases of IPT and TZS of Agrobacterium are known to have activity that transfers the dimethylallyl group of dimethylallyl pyrophosphoric acid (DMAPP) to the nitrogen atom at position 6 of adenosine monophosphate (AMP). This reaction is considered to be the most important step in cytokinin synthesis. However, cytokinin synthesis enzymes possessed by plants and the proteins that encode them have yet to be identified.
DISCLOSURE OF THE INVENTION
Therefore, an object of the present invention is to provide a gene that encodes an enzyme that catalyzes cytokinin synthesis, a protein encoded thereby, and its application. In addition, an object of the present invention is to provide a method for identifying that gene.
The inventors of the present invention found a method for obtaining a novel gene that encodes an enzyme that catalyzes cytokinin synthesis from Arabidopsis thaliana, and obtained a novel gene that encodes an enzyme that catalyzes cytokinin synthesis.
Thus, the present invention provides a gene that encodes a protein involved in cytokinin synthesis. More specifically, the protein is a previously unreported enzyme in plants that catalyzes the reaction in which a side chain is introduced at position N6 of the adenine skeleton of cytokinins.
More specifically, the present invention provides a gene that encodes a protein involved in cytokinin synthesis having the amino acid sequence described in SEQ ID NO. 2, 4, 6, 8, 10, 12 or 14. In addition, the present invention provides a gene that encodes a protein involved in cytokinin synthesis having a modified amino acid sequence resulting from the addition and/or deletion of one or a plurality of amino acids and/or substitution by other amino acids in SEQ ID NO. 2, 4, 6, 8, 10, 12 or 14. Moreover, the present invention provides a gene encoding a protein involved in cytokinin synthesis that hybridizes with a nucleic acid described in SEQ ID NO. 1, 3, 5, 7, 9, 11 or 13, and particularly DNA or a portion thereof, under stringent conditions.
The present invention also provides a vector that contains that gene.
Moreover, the present invention provides a host that has been transformed by that vector. This host may be a plant cell or a plant body.
The present invention is also able to provide a production method of a protein involved in cytokinin synthesis by culturing and cultivating the aforementioned host.
In addition, the present invention is able to provide a method for regulating the growth of a plant or plant cells by introducing the aforementioned gene into a plant or plant cells and expressing said gene. Namely, various physiological actions in which cytokinins are involved, such as promotion of the formation of adventitious buds, overcoming the dormancy of lateral buds, prevention of the aging of flowers and leaves and the ripening of fruit, improving the longevity of flowers, maintaining photosynthesis function, promoting the enlargement of fruit, prevention of dropping and control of flowering, can be regulated by expressing this gene.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a drawing showing the structure of plasmid pTK015.
FIG. 2 is a photograph of a plant body regenerated from Arabidopsis thaliana transformed by pHM4-AtIPT5.



DESCRIPTION OF THE PREFERRED EMBODIMENTS
The inventors of the present invention surmised that the reaction that catalyzes the introduction of an isopentenyl (dimethylallyl) side chain at the N6 position of the adenine skeleton is the rate-limiting step of cytokinin synthesis. Examples of genes that are known to encode isopentenyl group transferases involved in cytokinin synthesis include the ipt (gene4) gene encoded by T-DNA and the tzs gene present in the vir region of the Ti-plasmid of Agrobacterium tumefaciens, the ptz gene present in several species of Pseudomonas, the ipt gene of Rhodococcus faciens, and the etz gene of Erwinia herbicola. Among these, the gene products of the tzs and ipt genes of Agrobacterium, the etz gene of Pseudomonas and the ipt gene of Rhodococcus have been demonstrated to have DMAPP:AMP dimethylallyl transferase activity in vitro. In addition to these, many living organisms have enzymes that transfer an isopentenyl group from DMAPP to rRNA. If plant cytokinins are assumed to be synthesized by isopentenylation of the adenine skeleton, then there is the possibility that enzymes that catalyze this reaction may have amino acids in common with the isopentenyl group transferases mentioned above. Therefore, the amino acid sequences of these gene products were first compared to find the amino acid residues preserved therein. The resulting sequence was determined to be GxTxxGK[ST]xxxxx[VLI]xxxxxxx[VLI][VLI]xxDxxQx[57,60][VLI][VLI]xGG[ST] (SEQ ID NO: 35). Here, x indicates an arbitrary amino acid, amino acid residues enclosed in brackets [ ] indicate which one of the amino acid residues contained therein, and [a,b] indicates the number of arbitrary types of amino acid residues greater than or equal to a but less than or equal to b.
The genome sequence of Arabidopsis thaliana was searched using a TAIR Pattern Matching program to find the possible genes or estimated gene regions based on this amino acid sequence pattern. The resulting eight genes consisted of AT4g24650 (number of the estimated gene region as determined by the Genome Project), T20010—210 (number of the estimated gene region as determined by the Genome Project), 29375-30301 bp of T16G12 (accession number: AC068809) genome clone, MDB19.12 (number of the estimated gene region as determined by the Genome Project), MVI11.6 (number of the estimated gene region as determined by the Genome Project), T26J14.3 (number of the estimated gene region as determined by the Genome Project), F2J7.12 (number of the estimated gene region as determined by the Genome Project) and AF109376.
Among these eight genes, an estimated gene, AF109376, has been cloned as the cDNA and annotated as being tRNA isopentenyl transferase mRNA. Among the seven remaining genes, T20010—210, MDB19.12, AT4g24650, MVI11.6, T26J14.3 and F2J7.12 have not being isolated as full length cDNA but estimated genes, and annotated to be likely tRNA isopentenyl transferases. 29375-30301 bp of T16G12 (accession number: AC0699089) genome clone is not even annotated.
The genes or estimated genes of AT4g24650, T20010—210, cDNA corresponding to 29375-30301 bp of T16G12 (accession number: AC068809) genome clone, MDB19.12, MVI11.6, T26J14.3 and F2J7.12 are designated AtIPT4, AtIPT3, AtIPT5, AtIPT7, AtIPT8 and AtIPT6. In addition, each of their nucleotide sequences are shown with SEQ ID NOs. 1, 3, 5, 7, 9, 11 and 13, and their corresponding amino acid sequences are indicated with SEQ ID NOs. 2, 4, 6, 8, 10, 12 and 14.
Although the calli of Arabidopsis thaliana normally form leaves and buds (to be referred to as shoots) when cytokinins are present in the medium, if cytokinins are not present, it does not form any shoots or even if they are formed, the frequency of formation is extremely low. Therefore, if callus efficiently forms shoots even in the absence of cytokinins when a gene has been introduced and expressed in the callus, the introduced gene can be considered to encode a cytokinin synthase or protein involved in cytokinin reactions.
Alternatively, by expressing the resulting gene using a gene expression system such as E. coli or yeast and then measuring the enzyme activity, the resulting gene can be confirmed to encode a cytokinin synthase or protein involved in cytokinin reactions.
On the basis of the above, in the present invention, a gene that encodes a cytokinin synthase or protein involved in cytokinin synthesis of plant origin was isolated and identified for the first time.
In addition, in the present invention, plant cytokinin synthase was found to transfer the DMA group of DMAPP to ATP and ADP.
Examples of a gene of the present invention include that coding for the amino acid sequence described in SEQ ID NO. 2, 4, 6, 8, 10, 12 or 14. However, proteins having an amino acid sequence that has been modified by addition or deletion or a plurality of amino acids, and/or substitution by other amino acids are known to maintain activity similar to the original protein. Thus, a modified gene that encodes a cytokinin synthase or protein involved in cytokinin synthesis activity and has an amino acid sequence that has been modified with respect to an amino acid sequence described in SEQ ID NO. 2, 4, 6, 8, 10, 12 or 14 by addition or deletion of one or a plurality of amino acids and/or substitution by other amino acids, is also included in the present invention.
Here, the degree of this modification is the degree which is possible by means that were commonly known technologies prior to filing of the present patent application, examples of which include site-specific mutagenesis, PCR method and so on. The number of amino acids subject to modification while maintaining the activity of the cytokinin synthase or that relating to cytokinin synthesis is, for example 100 or less, for example 50 or less, preferably 25 or less, and for example 10 or less.
In addition, the present invention also provides a gene composed of DNA that encodes a cytokinin synthase or protein having activity involved in cytokinin synthesis, and which is capable of hybridizing with a nucleic acid having a nucleotide sequence described in SEQ ID NO. 1, 3, 5, 7, 9, 11 or 13, or portion thereof, under stringent conditions. Here, stringent conditions refer to hybridization conditions consisting of 5×SSC and 50° C. Furthermore, as the suitable hybridization temperature varies according to the particular nucleotide sequence and the length of that nucleotide sequence, hybridization can be carried out by suitable selecting the hybridization temperature.
A cDNA library, genomic DNA library and so forth prepared from a plant or microorganism, etc. having cytokinin synthase activity or activity involved in cytokinin synthesis can be used for the source of the gene subjected to the aforementioned hybridization, examples of which include plants such as Arabidopsis thaliana, corn, poplar, petunia, tobacco, rice, tomato and eucalyptus plants.
The nucleotide sequence of a gene encoding a cytokinin synthase or protein involved in cytokinin synthesis obtained in this manner has homology of 50% or more, 60% or more, preferably 70% or more or 80% or more, and for example 90% or more, with respect to a nucleotide sequence indicated in SEQ ID NO. 1, 3, 5, 7, 9, 11 or 13.
As will be concretely indicated in the examples, the subject gene encoding a protein having an amino acid sequence indicated in SEQ ID NO. 2, 4, 6, 8, 10, 12 or 14 can be obtained from Arabidopsis thaliana in the form of cDNA or genomic DNA.
In addition, DNA encoding a protein having a modified amino acid sequence can be synthesized using commonly used site-specific mutagenesis or PCR method by using DNA having the inherent nucleotide sequence as a base. For example, a DNA fragment containing a desired modification can be obtained by obtaining a DNA fragment in which a modification is desired to be introduced by restriction enzyme treatment of the inherent cDNA or genomic DNA, and then performing site-specific mutagenesis or PCR method using this DNA as a template and a primer containing the desired mutation. Subsequently, this DNA fragment into which the mutation has been introduced should then be coupled with a DNA fragment that encodes another portion of the target protein.
Alternatively, in order to obtain DNA that encodes protein composed of a shortened amino acid sequence, DNA that encodes an amino acid sequence longer than the target amino acid sequence, such as the entire amino acid sequence, should be digested by a desired restriction enzyme and, if the resulting DNA fragment does not encode the entire target amino acid sequence, a DNA fragment composed of the portion of the sequence that is lacking should be synthesized and then coupled to that fragment.
Alternatively, cytokinin synthase or a protein having activity involved in cytokinin synthesis can also be obtained by using antibody to a protein having an amino acid sequence described in SEQ ID NO. 2, 4, 6, 8, 10, 12 or 14, and cytokinin synthase or protein having activity involved in cytokinin synthesis of other organisms can be cloned using antibody.
Thus, the present invention also relates to a recombinant vector, and particularly an expression vector, that contains the aforementioned gene, and to a host transformed by said vector. Procaryotic organisms or eucaryotic organisms can be used as hosts. Examples of prokaryotic organisms include bacteria such as Escherichia coli and other Escherichia species, Bacillus subtilis and other Bacillus species as well as other commonly used host microorganisms.
Examples of eucaryotic organisms include lower eucaryotic organisms such as eucaryotic microorganisms in the form of yeasts and molds. Examples of yeasts include Saccharomyces cerevisiae and other Saccharomyces species, while examples of molds include Aspergillus oryzae, Aspergillus niger and other Aspergillus species as well as Penicillium species. Moreover, plant cells and animal cells may also be used as hosts, examples of which include cells systems of animals cells such as mouse, hamster, monkey or human cells, and more specifically, COS cells, Vero cells, CHO cells, L cells, C127 cells, BALB/c 3T3 cells and Sp-2/0 cells. Examples of plant cells include tobacco and Arabidopsis cultured cells as well as cultured cells of poplar, eucalyptus and acacia species.
Moreover, insect cells such as silkworm (Bombyx mori) cells or adult silkworms themselves can be used as hosts. In addition, yoga (Spodoptera frugiprd) or cabbage looper (Trichoplusiani) cells can also be used.
Plasmids, phages, phagemids and viruses (such as Baculovirus (expression in insects) or Vaccinia virus (expression in animal cells)) can be used as expression vectors.
Expression vectors of the present invention contain expression control regions such as promoters, terminators, replication origins and so forth depending on the type of host into which they are to be introduced. Examples of promoters of bacterial expression vectors include lac promoter, examples of yeast promoters include glyceraldehyde 3-phosphate dehydrogenase promoter, PHO5 promoter, adhI promoter and pqk promoter, and examples of mold promoters include amylase promoter and trpC promoter.
In addition, examples of insect promoters include Baculovirus polyhedron promoter, and examples of animal cell promoters include Simian Virus 40 early and late promoters, CMV promoter, HSV-TK promoter or SRα promoter. Examples of plant promoters include the 35S promoter of cauliflower mosaic virus and nopaline synthase promoter, while examples of inductive promoters include glutathione-S-transferase II gene promoter, hsp80 promoter and ribulose 2-phosphate carboxylase small subunit gene promoter.
In addition, preferable modes of the expression vector include, in addition to those described above, those containing enhancers, splicing signals, poly A addition signals or selection markers (for example, dihydrofolic acid reductase genes (methotrexate resistant) and neo genes (G418 resistant)). Furthermore, in the case of using an enhancer, SV40 enhancer, for example, is inserted upstream or downstream from the gene.
Host transformation by an expression vector can be carried out in accordance with ordinary methods well known among persons with ordinary skill in the art, and these methods are described in, for example, Current Protocols in Molecular Biology, John Wiley & Sons Publishing, 1995. Culturing of the transformant can also be carried out in accordance with ordinary methods. Purification of protein involved in cytokinin synthesis from the culture can be carried out in accordance with ordinary methods for isolating and purifying proteins, examples of which include ultrafiltration and various types of column chromatography such as chromatography using Sepharose.
On the basis of the current level of technology, adventitious bud formation can be promoted even in plants such as roses, for which individual regeneration is difficult even by artificial regulation using plant hormones externally added to the medium, by inserting and expressing this gene in a plant by coupling this cDNA or gene under the control of a composite or inductive promoter with a system that uses Agrobacterium or a system that uses a particle gun or electroporation and so forth. Moreover, the various physiological actions exhibited by cytokinins in plants, such as lateral bud elongation, prevention of aging, flowering time, promotion of fruit enlargement and prevention of fruit dropping, can be regulated by controlling the expression of a gene that encodes a protein involved in cytokinin synthesis.
The following provides a detailed description of the present invention according to examples. Unless stated otherwise, molecular biological techniques are in accordance with Molecular Cloning (Sambrook, et al., 1989).
Example 1
Search for and Isolation of Genes Encoding Proteins Involved in Cytokinin Synthesis
As a result of analyzing the amino acid sequences of the gene products of the tzs gene of Agrobacterium tumefaciens (Accession No. X03933-1, Protein ID No. CAA27572.1), the ipt gene of Agrobacterium tumefaciens (Accession No. AB025109-1, Protein ID No. BAA76344.1), the ptz gene of Pseudomonas syringae (Accession No. X03679-1, Protein ID No. CAA27315.1), the ipt gene of Rhodococcus faciens (Accession No. Z29635-4, Protein ID No. CAA82744.1), the dimethylallyl transferase gene of Erwinia herbicola (Accession No. Z46375-2, Protein ID No. CAA86510.1) and the delta-2-isopentenyl phosphate (IPP) transferase gene of Escherichia coli tRNA (Accession No. U14003-83, Protein ID No. AAA97067.1) using the amino acid sequence comparison software, Clustal V of Macvector 6.5.3, the preserved sequence of GxTxxGK[ST]xxxxx[VLI]xxxxxxx[VLI][VLI]xxDxxQx [57,60][VLI][VLI]xGG[ST] (SEQ ID NO: 35) was found. Here, x indicates an arbitrary amino acid, amino acid residues enclosed in brackets [ ] indicate which one of the amino acid residues contained therein, and [a,b] indicates the number of arbitrary types of amino acid residues greater than or equal to a but less than or equal to b.
Next, in order to isolate those genes or estimated gene regions considered to have this preserved amino acid sequence pattern, a search was made of the genomic database of Arabidopsis thaliana using the TAIR Pattern Matching Program. As a result, eight genes consisting of the estimated gene region numbers as designated by the National Genome Project of AT4g24650, T20010—210, MDB19.12, MVI11.6, T26J14.3, F2J7.12, AF109376 and the 29375-30301 bp region of the T16G12 genome clone (Accession No. AC068809) were determined to have the aforementioned preserved amino acid sequence pattern.
Among these eight genes, cDNA has previously been cloned for AF109376 only, and is annotated as tRNA isopentenyl transferase mRNA. However, since the total length cDNA of AT4g24650, T20010—210, MDB19.12, MVI11.6, T26J14.3 and F2J7.12 have not been isolated, they are annotated as probably being tRNA isopentenyl transferases. With respect to the 29375-30301 bp region of the genome clone of T16G12 (Accession No. AC068809), there are even no annotations regarding its estimated function. Namely, the functions of these eight genes are only estimated on the basis of computer analyses, while there have been no experimental analyses whatsoever, and their functions were not known. In addition, their enzyme activity has also not been measured, and their substrates have not been identified.
The nucleotide sequences of AT4g24650 (AtIPT4), T2000—210 (AtIPT3), the cDNA corresponding to the 29375-30301 bp region of the genome clone of T16G12 (Accession No. AC068809) (AtIPT5), MDB19.12 (AtIPT7), MVI11.6 (AtIPT8), T26J14.3 (AtIPT1) and F2J7.12 (AtIPT6) are shown in SEQ. ID NOs. 1, 3, 5, 7, 9, 11 and 13.
In addition, a molecular phylogenetic tree was produced with the Clustal W Program (retrieved from the internet:<URL: http://www.ddbj.nig.ac.jp/E-mail/clustalw-e.htm>, Thompson, et al., 1994, Nucl. Acids Res., 22, 4673-4680), including each of the amino acids encoded by them (SEQ ID NOs. 2, 4, 6, 8, 10, 12 and 14) and homologous amino acid sequences obtained as a result of searching DNA databases (such as the DNA Databank of Japan (DDBJ) (retrieved from the internet:<http://www.ddbj.nig.ac.j>). As a result, the sequence indicated with SEQ ID NOs. 2, 4, 6, 8, 10, 12 and 14 were demonstrated to form a family with eucaryotic or bacterial DMAPP:tRNA isopentenyl transferase or isopentenyl transferase involved in cytokinin synthesis of plant pathogens such as Agrobacterium that form gall. Moreover, the amino acid sequences indicated in SEQ ID NOs. 2, 10, 12 and 14 formed a single subgroup. Those genes that were related to this subgroup consisted of AAL83819 (DDBJ accession no.) of petunia origin and BAB86364 (DDBJ accession no.) of rice origin. In addition, the amino acid sequences indicated in SEQ ID NOs. 4, 6 and 8 also formed a single subgroup, and AW720363 (DDBJ accession no.) of bird's-foot trefoil Lotus japonicus origin was positioned in this subgroup.
Example 2
Excessive Expression in Plants of Genes Encoding Proteins Involved in Cytokinin Synthesis
i) Production of Gene Insertion Vector for Plants
pBI35T (WO 01/16332) was treated with EcoRI and HindIII to obtain a DNA fragment containing a promoter of cauliflower mosaic virus 35S RNA gene, a multi-cloning site and the terminator of 35S RNA gene. This was then treated with HindIII and EcoRI of pGPTV-KAN (Becker, R., et al., Plant Molecular Biology, 20, 1195-1197, 1992), and among the two fragments formed, the longer fragment was ligated to obtain pTK015 (FIG. 1). Similarly, this DNA fragment containing a promoter of cauliflower mosaic virus 35S RNA gene, a multi-cloning site and the terminator of 35S RNA gene was then treated with HindIII and EcoRI of pGPTV-Bar (Becker, R., et al., Plant Molecular Biology, 20, 1195-1197, 1992), and among the two fragments formed, the longer fragment was ligated to obtain pTK016.
For the predicted open reading frame of AF109376, DNA was amplified by incubating at 94° C. for 2 minutes followed by 40 cycles consisting of 15 seconds at 94° C., 30 seconds at 53° C. and 2 minutes and 40 seconds at 68° C. using the cDNA library of Arabidopsis thaliana for the template DNA of the PCR reaction, primer 398 (5′-TCCCCCGGGCGATGATGATGTTAAACCCTAGC-3′) (SEQ ID NO. 15) and primer 399 (5′-TCCCCCGGGTC AATTTACTTCTGCTTCTTGAACTTC) (SEQ ID NO. 16) as primers and pfx DNA polymerase (Gibco BRL), and purifying the amplified DNA followed by treatment with SmaI and repeated purification of the DNA. This was then cloned to the SmaI site of pTK015, after which the cloned product in the sense direction downstream from the 35S RNA gene promoter (35S promoter) of cauliflower mosaic virus was selected and designated as pTK015-AF109376.
AtIPT4 was amplified by incubating at 94° C. for 2 minutes followed by 42 cycles consisting of 15 seconds at 94° C., 20 seconds at 53° C. and 1 minute at 68° C. using the genomic DNA of Arabidopsis thaliana for the template of the PCR reaction, primer 421 (AAAATGAAGTGTAATGACAAAATGGTTGTG-3′) (SEQ ID NO. 17) and primer 407 (5′-GTCCAAACTAGTTAAGACTTAAAAATC-3′) (SEQ ID NO. 18) as primers and pfx DNA polymerase (Gibco BRL), followed by purification and cloning to the SmaI site of pTK015. The cloned product in the sense direction downstream from the 35S promoter was designated as pTK015-AtIPT4.
For AtIPT3, DNA was amplified using genomic DNA of Arabidopsis thaliana for the template, primer 703 (5′-CACCAGCAAGTTTATATTGCAAAGCGT-3′) (SEQ ID NO. 19) and primer 705 (5′-GTTGTAACCACGTAAAAGATAAGGGTG-3′) (SEQ ID NO. 20) as primers and Herculase (trade name, Stratagene) as heat-resistant DNA synthase. The PCR reaction was carried out for 1 minute at 92° C. followed by 35 cycles consisting of 30 seconds at 92° C., 30 seconds at 55° C. and 2 minutes and 30 seconds at 70° C. Following purification of this product, it was cloned at the blunt terminal to the SmaI site of pTK016, and the cloned product of AtIPT3 in the sense direction downstream from the 35S promoter was selected and designated as pTK016-AtIPT3.
For pTK015, after digesting with SmaI and KpnI, the DNA was purified using the QUIAquick PCR Purification Kit (Qiagen). Here, the sequence of the multi-cloning site was altered by cloning primer 852(5′-CTCGAGTTGGCGCGCCACCCGGGATTAATTAAGAC TAGTGGGGTAC-3′) (SEQ ID NO. 27) and primer 853 (5′-CCCACTAGTCTTAATTAA TCCCGGGTGGCGCGCCAACTCGAG-3′) (SEQ ID NO. 28). Here, since primer 852 and primer 853 are synthetic DNA having mutually complementary sequences, this procedure was carried out by incubating the three elements consisting of a fragment obtained by digesting pTK015 with SmaI and KpnI, primer 852 and primer 853 in the presence of ligase under ordinary conditions. The plasmid produced in this manner was designated as pHM4. The only difference between pHM4 and pTK015 is the sequence of the multi-cloning site. Those unique sites present in the multi-cloning site of pTK015 consist of XbaI, XhoI, SmaI, PacI, SpeI, KpnI and SalI.
4 μg of pHM4 were digested with 20 units of BamHI. The terminals were blunted by incubating half the amount for 30 minutes at 70° C. in the presence of 200 μM deoxyATP, deoxyTTP, deoxyCTP, deoxyGTP and 1 unit of pfu DNA polymerase (Stratagene). After treating this for 1 hour at 37° C. with 20 units of calf intestine alkaline phosphatase (Takara), the DNA was purified using the QUIAquick PCR Purification Kit (Qiagen). Here, the cloned DNA fragment that was amplified (for 35 cycles consisting of 20 seconds at 94° C., 30 seconds at 55° C. and 1 minute at 72° C.) from genomic DNA of Arabidopsis thaliana using Herculase heat-resistant DNA polymerase (Stratagene), primer 918 (5′-ATG ACA GAA CTC AAC TTC CAC CT-3′) (SEQ ID NO. 29) and primer 879 (5′-CAAAAAAAAGATCTAATTTTGCACCAAATGCCGCTT-3′) (SEQ ID NO. 30) was cloned and designated as pHM4-AtIPT1.
A cloned DNA fragment amplified from the genomic DNA of Arabidopsis thaliana using primer 533 (5′-ATTATGCAAAATCTTACG TCCACATTCGTC-3′) (SEQ ID NO. 31) and primer 881 (5′-ACAGGATCCTCACACTTTGTCTTTCACCAAG-3′) (SEQ ID NO. 32) was cloned in the same manner as the production of pHM4-AtIPT1 and designated as pHM4-AtIPT8.
A sequence containing the entire code region of SEQ ID NO. 6 starting 66 bps upstream from the translation starting point ATG described in SEQ ID NO. 5 was amplified by PCR using genomic DNA extracted from a Columbia wild strain (Takara Shuzo) as a template and using primer 856 (5′-CCGCTCGAGA TGAAGCCATGCATGACGGCTC-3′) (SEQ ID NO. 33) and primer 857 (5′-GGACTAGTCACCGGGAAATCGCCGCCA-3′) (SEQ ID NO. 34). These primers contain restriction enzyme sites and were treated with XhoI and SpeI following PCR. This DNA fragment was cloned to pHM4, a vector excessive expression in plants, and designated as pHM4-AtIPT5.
ii) Gene Insertion into Plants
pTK015, pTK015-AF109376, pTK015-AtIPT4 and pTK016-AtIPT3 were inserted into the callus of Arabidopsis thaliana using Agrobacterium. The method for inserting genes using Agrobacterium was in accordance with the method of Akama, et al. (Akama, K. et al., 1992 Plant Cell Rep., 12, 7-11). The calli containing the inserted genes were cultured in two types of media consisting of cytokinin-free medium [GM medium (Akama, K. et al., 1992 Plant Cell Rep., 12, 7-11) containing 50 μg/ml of kanamycin sulfate, 100 μg/ml of cefotaxime, 100 μg/ml of vancomycin and 0.3 μg/ml of indole acetate] and cytokinin-containing medium (cytokinin-free medium containing 0.5 μg/ml of trans-zeatin). When observed two weeks later, neither the calli transformed with pTK015 or calli transformed with pTK015-AF109376 formed shoots in the cytokinin-free medium, and only formed shoots in the cytokinin-containing medium. In contrast, the calli transformed with pTK015-AtIPT4 formed shoots in both the cytokinin-free and cytokinin-containing medium. In addition, calli transformed with pTK016-AtIPT3 similarly formed shoots in both the cytokinin-free and cytokinin-containing media.
In addition, calli of Arabidopsis thaliana respectively inserted with pHM4, pHM4-AtIPT1, pHM4-AtIPT8 and pHMR-AtIPT5 were cultured in cytokinin-free medium containing 0.2 μg/ml of indole acetate, 50 μg/ml of kanamycin and 100 μg/ml of claforan. The procedure was the same as the example in which AtIPT4 was inserted into calli. Although the calli containing pHM4 did not form shoots, the calli containing pHM4-AtIPT1, pHM4-AtIPT8 and pHM4-AtIPT5 formed shoot tissue.
On the basis of these findings, AtIPT4 and AtIPT3 were suggested to have the ability to induce shoots and the ability to synthesis cytokinins.
In addition, it was also indicated that excessive expression of AtIPT1, AtIPT5 and AtIPT3 is capable of causing a cytokinin response.
iii) Excessive Expression of pHM4-AtIPT5 in Plants
pHM4-AtIPT5 was transformed in Arabidopsis thaliana using the vacuum infiltration Agrobacterium infection method (O. Araki, Shujunsha Publishing, Cell Engineering Supplement, Plant Cell Engineering Series 4, Experimental Protocols in Model Plants, p. 109-113). The resulting seeds were cultivated in MS agar medium containing 50 μg/ml of kanamycin followed by selection of transformants. When the transformants were cultivated in vermiculite containing one-half the concentration of MS medium, an extremely large number of lateral buds were formed in several of the plants (causing the plants to appear bushy) (FIG. 2). This phenotype was not observed in pHM4 transformants cultivated as a control. As a result, it was determined that when AtIPT5 is expressed in excess, terminal bud dominance diminishes and lateral bud formation is promoted.
Example 3
Measurement of Enzyme Activity of Proteins Involved in Cytokinin Synthesis
i) Plasmid Production for Measuring Enzyme Activity
The code region was amplified by using the pTK015-AtIPT4 produced in Example 2 as a template, using primer 480 (5′-GGAATTCCATATGAAGTGTAATGACAAAATGGTTGA^3′) (SEQ ID NO. 21) and primer 481 (5′-GAAGATCTGTCCAAACTAGTTAAGACTTAAAAA TC-3′) (SEQ ID NO. 22) as primers, and using LA taq (Takara Shuzo). After purifying the amplified region, it was treated with NdeI and BglII followed by again purifying the DNA. This DNA fragment was cloned between the NdeI and BamHI sites of pET16b (Novagen) to produce pET16b-AtIPT4.
In addition, the coding region was amplified using pTK015-AF109376 as the template for the PCR reaction, using primer 550 (5′-GATCCCCGGCATATGATGATGTTAAACCCTAGC-3′) (SEQ ID NO. 23) and primer 551 (5′-ACGGTACCCATA TGTCAATTTACTTCTGCTTCTTGAAC-3′) (SEQ ID NO. 24) as primers, and using Herculase (Stratagene) as heat-resistant DNA polymerase. This was then treated with NdeI and cloned to the NdeI site of pET16b to produce pET16b-AF109376.
Moreover, the coding region was amplified using genomic DNA of Arabidopsis thaliana as the template for the PCR reaction, using primer 741 (5′-TTATACATATGAAGCCATGCATGACGGCTCTAAG-3′) (SEQ ID NO. 25) and primer 742 (5′-CGGGATCCTCACCGGG AAATCGCCGCCA-3′) (SEQ ID NO. 26) as primers, and using LA taq (Takara Shuzo) as heat-resistant enzyme. Following purification, the DNA was treated with NdeI and BamHI and cloned between the NdeI and BamHI sites of pET15b (Novagen) to produce pET15b-AtIPT5.
ii) Measurement of Enzyme Activity of E. Coli Extract
As was previously described, AtIPT1, AtIPT4, AtIPT8 and AtIPT6 form a single subgroup, while AtIPT3, AtIPT5 and AtIPT7 form a different subgroup. Enzyme activity in E. coli was measured from each for a single gene.
After culturing E. coli strain AD494(DE3)pLysS containing pET16b-AtIPT4, pET16b-AF109376 or pET15b-AtIPT5 for 12 hours at 20° C. in the presence of 1 mM IPTG, the microorganisms were collected by centrifugation, and after adding Buffer A (25 mM Tris-HCl, 50 mM KCl, 5 mM β-mercaptoethanol, 1 mM PMSF and 20 μg/ml of leupeptin) to an OD600 of 100, the E. coli were disrupted by freezing and thawing. The disrupted E. coli were then centrifuged for 10 minutes at 300000 g followed by recovery of the supernatants. 10 μl of these supernatants were mixed with Buffer A containing 60 μM DMAPP, 5 μM [3H]AMP (722 GBq/mmol) and 10 mM MgCl2 followed by incubation for 30 minutes at 25° C. Subsequently, 50 mM of Tris-HCl (pH 9) was added to this reaction liquid followed by the addition of calf intestine alkaline phosphatase to a concentration of 2 units/30 μl and incubating for 30 minutes at 37° C. to carry out a dephosphatization reaction. As a result of developing the reaction liquid by C18 reversed-phase thin layer chromatography (mobile phase: 50% methanol) and detecting the reaction products by autoradiography, formation of isopentenyl adenosine was confirmed in the reaction liquids containing extracts of E. coli having pET16b-AtIPT4 and pET15b-AtIPT5. However, formation activity of isopentenyl adenosine was not observed in the extract of E. coli containing pET16b-AF109376.
iii) Measurement of Enzyme Activity of Purified Proteins
Similar to Example 3, Part (i), AtIPT4 was cloned in pET32b (Novagen) and an extract was prepared from E. coli in the same manner as Example 3, Part (ii). This was designated as Sample A. 400 μl of Ni-NTA agarose suspension (containing 110 μl of Ni-NTA agarose as precipitate, 30 mM NaH2PO4 (pH 8), 15 mM indazole, 0.9 M NaCl, 7.5 mM β-mercaptoethanol, 0.5 mM PMSF and 30 μg/ml of leupeptin) were added to 800 μl of Sample A. This suspension was designated as Sample B. Sample B was then centrifuged to separate into supernatant (Sample C) and precipitate. Washing liquid (consisting of 20 mM NaH2PO4 (pH 8), 10 mM indazole, 0.3 M NaCl, 5 mM β-mercaptoethanol, 0.5 mM PMSF and 10 μg/ml of leupeptin) was added to the precipitate, and the Ni-NTA agarose was washed four times using a procedure in which the precipitate was recovered by centrifugation. This Ni-NTA agarose was then suspended in 500 μl of washing liquid and designated as Sample D. 50 μl of Sample D were then mixed with 50 μl of 2× reaction liquid (25 mM Tris-HCl (pH 7.5), 75 mM KCl, 10 mM MgCl2, 10 μg/ml of leupeptin, 1 mM PMSF and 66 μM DMAPP) containing one of the nucleotides of ATP, ADP or AMP or adenosine or adenine at 0.25 μM, which were labeled with 3H, and allowed to react for 30 minutes at 23° C. After adding 700 μl of ethyl acetate to this reaction liquid followed by stirring and centrifuging, 550 μl of the ethyl acetate layer were recovered followed by the addition of 500 μl of distilled water. After stirring and centrifugal separation, 350 μl of the ethyl acetate layer were recovered followed again by the addition of 500 μl of distilled water. After again stirring and separating by centrifugation, 0.5 ml of ACSII (Pharmacia) were added to 50 μl of the ethyl acetate layer followed by measurement of radioactivity with a liquid scintillation counter. As a result, the dimethylallyl group of DMAPP was found to have efficiently transferred to ATP and ADP. Thus, it was shown that the AtIPT4 product has activity that transfers a dimethylallyl group to ATP and ADP.
When the Km for ATP was measured in the presence of 0.4 mM DMAPP using purified recombinant AtIPT4 protein (2 ng/ml), the resulting value was comparable to the Km of 11.1 μM (Morris, et al., Aust. J. Plant Physiol., 20, 621-637, 1993) of tzs for AMP. In addition, when the Km for DMAPP was measured in the presence of 200 μM ATP, the value was 6.5 μM.
Similarly, AtIPT1 also encoded protein having activity that transfers a dimethylallyl group to ATP and ADP.
iv) Identification of Reaction Products
The aforementioned Sample D was mixed with an equal volume of 2× reaction liquid (containing 1 mM ATP and 1 mM DMAPP) and allowed to react for 1 hour at 25° C. After centrifuging, the supernatant was divided into two equal portions, and one of the portions was treated with calf intestine alkaline phosphatase in the same manner as previously described. After diluting each portion with 3 volumes of acetone and holding for 30 minutes at −80° C., they were centrifuged for 30 minutes at 17,000×g to remove the protein. After drying the supernatant to a solid under reduced pressure, it was dissolved in methanol. A portion of the dried supernatant was fractionated with the Chemocobond ODS-W column (Chemco). Elution was carried out using a linear concentration gradient by first eluting for 15 minutes with 20 mM KH2PO4 followed by 30 minutes with an 80% aqueous acetonitrile solution containing K2HPO4 ranging from 20 mM to 4 mM. The sample not treated with calf intestine alkaline phosphatase exhibited two main peaks in Chemocobond ODS-W column chromatography. The retention time of the peak that eluted first coincided with the retention time of ATP. The retention time of the peak (Peak A) that eluted later did not coincide with any of the retention times of ATP, adenosine or isopentenyl adenosine. The sample treated with calf intestine alkaline phosphatase also exhibited two main peaks in Chemocobond ODS-W column chromatography. The retention time of the peak that eluted first coincided with the retention time of adenosine, while the retention time of the peak that eluted later (Peak B) coincided with that of isopentenyl adenosine.
After drying the fractions of Peaks A and B, they were dissolved in ethanol and analyzed by fast atom bombardment mass spectrometry (JMS-SX102 or JEOL Mstation, JOEL Datum Ltd.). As a result, a signal originating in the compound of Peak A was unable to be obtained, because of inhibition of ionization by the triphosphate group. Signals originating in the compound of Peak B were observed at m/z values of 336 and 204, with the former corresponding to isopentenyl adenosine, and the latter corresponding to a decomposition product of isopentenyl adenosine. On the basis of the above, Peak A was thought to be isopentenyl ATP (also referred to as iPTP), which is a compound resulting from the phosphatization of isopentenyl adenosine.
When the genes indicated with SEQ ID NOs. 2 and 6 were expressed in E. coli, both exhibited cytokinin synthesis activity. In addition, a cytokinin response was evoked in the case of excessive expression in plants of enzymes of the genes indicated in SEQ ID NOs. 2, 4, 6, 10 and 12. In addition, SEQ ID NOs. 2, 10, 12 and 14 or SEQ ID NOs. 4, 6 and 8 were clearly shown to be extremely closely related in terms of their respective molecular systems. Thus, each of these are considered to be cytokinin synthesis enzymes. Thus, it was possible to control cell division, differentiation, axillary bud length, regulation of nutrient distribution, inhibition of aging, reproductive growth and seed growth by controlling the expression of the genes of the present invention and their analogous genes. In addition, since they are plant genes, the appearance of toxicity and so forth of proteins expressed in plants into which these genes have been inserted is unlikely.
Since ATP can be efficiently used as a substrate of cytokinin synthesis, these genes are expected to function more effectively in plants than cytokinin synthesis genes originating in bacteria using AMP as substrate.
EXPLANATION OF SYMBOLS


    PAg7:TERMINATOR OF T-DNA GENE 7
    NPTII:NEOMYCIN PHOSPHOTRANSFERASE II
    PNOS:NOPALINE SYNTHETASE PROMOTER
    P35S:PROMOTER OF CAULIFLOWER MOSAIC VIRUS 35S RNA GENE
    PA35S:TERMINATOR OF CAULIFLOWER MOSAIC VIRUS 35S RNA GENE
    LB:LEFT BORDER OF T-DNA
    RB:RIGHT BORDER OF T-DNA

## Claims

1. An isolated polynucleotide consisting of a nucleotide sequence encoding a protein having cytokinin synthesis activity and the amino acid sequence of SEQ ID NO: 6, or an isolated polynucleotide only encoding a protein having cytokinin synthesis activity and the amino acid sequence of SEQ ID NO: 6.

2. An isolated polynucleotide consisting of a nucleotide sequence encoding a protein having cytokinin synthesis activity wherein the protein has a modified amino acid sequence resulting from the addition, deletion, or substitution of one to 25 amino acids in SEQ ID NO:6 or an isolated nucleotide sequence only encoding a protein having cytokinin synthesis activity, wherein the protein has a modified amino acid sequence resulting from the addition, deletion, or substitution of one to 25 amino acids in SEQ ID NO:6.

3. A vector comprising the isolated polynucleotide of claim 1 or claim 2.

4. An isolated host cell transformed with the vector of claim 3.

5. The transformed host cell of claim 4, wherein the transformed host cell is a plant cell.

6. A plant cell into which the polynucleotide of claim 3 has been inserted.

7. A method for regulating the growth of a plant or plant cells by inserting the polynucleotide of claim 1 into a plant cell and expressing said polynucleotide to regulate the growth of the plant cells.

8. A method for inducing adventitious bud formation in a plant or plant cells by inserting the polynucleotide of claim 1 into a plant cell, and expressing the polynucleotide to induce adventitious bud formation in the plant cells.

9. A method for regulating the growth of a plant or plant cells by inserting the polynucleotide of claim 2 into a plant cell and expressing the polynucleotide to regulate the growth of the plant cells.

10. A method for inducing adventitious bud formation in a plant or plant cells by inserting the polynucleotide of claim 2 into a plant cell, and expressing the polynucleotide to induce adventitious bud formation in the plant cells.