TW201305339A - Gene, protein and method for controlling gynostemium perianth development - Google Patents

Abstract

The invention relates to a nucleic acid molecule for controlling gynostemium development, and a vector and cell comprising the nucleic acid molecule. A protein and method for controlling gynostemium development and a method for producing transgenic plants are also provided.

Description

Genes, proteins and methods for controlling the development of the core

The present invention relates to a technique for a plant gene. In particular, the present invention relates to genes, proteins and methods for controlling the development of a plexus column.

For plants, transcriptional regulators containing MADS-box have become the focus of flower-specific, developmental and evolutionary studies (M Nster et al, 1997, Proc. Natl. Acad. Sci. USA 94: 2415-2420; Purugganan et al, 1995, Genetics 140: 345-356; Weigel and Meyerowitz, 1994, Cell 78: 203-209). According to the "ABCDE model", the specificity of each inflorescence is determined by the unique combination activity of the organ characteristic genes A, B, C, D and E (Weigel and Meyerowitz, 1994; Theissen and Saedler, 2001; Zahn et al. 2005), in which the sepals are determined by the activity of A and E; the petals are determined by the activities of A, B and E; the stamens are determined by the activities of B, C and E; the carpels are determined by the activity of C and E; and the ovules are composed of C, The activity of D and E is determined. On the other hand, studies of protein-protein interactions have also demonstrated that MADS-box transcription factors act in combination at the molecular level (Egea-Cortines et al, 1999, EMBO J. 18: 5370-5379; Honma and Goto , 2001, Nature 409: 525-529; Ferrario et al, 2003, Plant Cell 15: 914-925). The diversity of the MADS-box gene during evolution is considered to be the main driver of floral diversity in terrestrial plants (Theissen et al, 2000, Plant Mol. Biol. 42: 115-49; Zahn et al, 2005, J Hered. 96: 225-240).

Orchidaceae belongs to the Lipopsida Asparagales and is currently known to contain more than 20,000 species and is one of the largest families in the plant kingdom. Known for its rich variety, the family seems to have a never-ending singular variation that represents the advanced and final line of flower evolution in monocots, and is presumably at a particularly high rate of species formation (Gill, 1989 "Fruiting failure," Pollinator inefficiency, and speciation in orchids, "Sinauer Associates, Sunderland, MA", shows that orchids are still actively evolving and are associated with flower diversity.

Although the flower-shaped staggered changes of orchids have long attracted the attention of evolutionary biologists, orchids have different flower shapes from other monocots, but have several common-source traits, including the combination of carpels (or styles) with at least some of the stamens. Gynostemium or column, lip or lip and highly evolved petals serve as landing platforms for pollinators (Rudall and Bateman, 2002, Biol. Rev. Camb. Philos. Soc. 77: 403- 41). The two pillars and the lips are the two most obvious flower organs of the orchid, which make the flower shape of the orchid symmetrical, and its fine and subtle development makes the orchid flower specialized into a precision instrument for animal pollination. At the same time, the arrangement of the two flower organs makes the cross-pollination within the species extremely effective, thereby promoting the formation and diversification of orchid species.

Despite its unique developmental reproductive biology and special pollination and ecological strategies, orchids are still at the molecular level compared to other species (Peakall, 2007, Mol. Ecol. 16: 2834-2837). Recently, the selection, performance and functional analysis of B-group genes have been carried out on several orchid species (Tsai et al, 2004, Plant Cell Physiol. 45: 831-844; Tsai et al, 2005, Plant Cell Physiol. 46: 1125 -1139; Tsai et al, 2008, Plant Cell Physiol. 49: 814-824; Tsai and Chen, 2006, The Scientific World Journal 6: 1933-1944; Kim et al, 2007, Plant Sci. 172: 319-326). The B group gene of orchid has a differential expression pattern in the flower part, and has duplicated four-clades. The divergent proteins encoded by these group B genes are also shown in orchids, group B genes. The evolution is related to the development of unique floral organs.

According to the ABCDE model, the C gene not only determines the development of stamens and carpels, but also determines the development of meristems. Because the C group mutants cannot develop the fourth inflorescence normally, and replace the carpels to form the C group mutations. In monocots, group C genes are repetitive and functionally diverse and appear partially redundant (Mena et al, 1996, Science 274: 1537-1540; Yamaguchi et al, 2006, Plant Cell 18: 15-28). In maize, the function of group C ZMM2 is mainly to determine the development of the organs . The function of ZAG1 is mainly to determine the development of flower meristems (Mena et al, 1996, Science 274: 1537-1540); in rice, OsMADS3 is in the inhibition 2 The inflorescence has a strong role in the development of the pulp and the stamen characteristics, while OsMADS58 determines the development of the meristem and regulates carpel development (Yamaguchi et al, 2006, Plant Cell 18: 15-28; Kater et al, 2006) , J. Exp. Bot. 57: 3433-3444); also in Antirrhinum (Davies et al, 1999, EMBO J. 18: 4023-4034) and Morning Glory ( Ipomoea nil ) (Nitasaka, 2003, The phenotype of C-functional mutants can be observed in Plant J. 36: 522-531); the phenotype of silencing C-function genes in petunia and gerbera is similar to that of C-function mutants (Kapoor et al, 2002, Plant J. 32: 115-127; Yu et al, 1999), Plant J. 17: 51-62). Nowadays grouped Phalaenopsis (Phalaenopsis), and Dendrobium (of Dendrobium) orchids identified gene cluster C (Skipper et al, 2006, Gene 366: 266-274; Song et al, 2006, Dev Genes Evol 216: 301-.. 313; Xu et al, 2006, Plant J. 46: 54-68), however, no functional analysis was performed on the identified Group C genes, nor was the relevant mutant strain obtained, and the actual flowering device was actually exemplified. The role of development, so the function of orchid C gene in controlling flower development is still unknown.

Therefore, in order to increase the quality of orchids, it is necessary for the industry to control the development of floral organs at the molecular level to carry out flower shape improvement.

Summary of invention

In the present invention, two C-group genes, namely CeMADS1 and CeMADS2 , are isolated from Cymbidium ensifolium to control the development of the plexus column.

The present invention provides an isolated nucleic acid molecule comprising a population consisting of the following polynucleotides:

(a) the nucleotide sequence of SEQ ID NO: 1 or 3;

(b) a polynucleotide encoding a polypeptide represented by the amino acid sequence of SEQ ID NO: 2 or 4;

(c) a polynucleotide complementary to (a) or (b);

(d) Polynucleotides heterozygous with (a), (b), (c), (d) or (e) under highly stringent conditions.

The invention further provides a vector comprising a nucleic acid molecule according to the foregoing.

The invention further provides a cell comprising the aforementioned vector.

The invention also provides a protocorn-like body comprising the nucleic acid molecule described above.

The invention further provides a method for producing a transgenic plant, the steps comprising:

(a) directing the aforementioned nucleic acid molecule to a plant cell to obtain a plant transgenic cell;

(b) regenerating the plant transgenic cells to produce the transgenic plant.

The invention further provides a protein encoded by the nucleic acid molecule described above.

The present invention further provides a method of controlling the development of a spheroid in a orchid plant comprising altering the amount of expression of the aforementioned protein in the plant. The present invention also provides a method for controlling flower shape development in orchid plants, which comprises the aforementioned method for controlling the development of a spheroid.

Detailed description of the invention

The invention aims to study the key genes controlling flower shape development in plants, and to control flower shape development by molecular breeding methods.

The present invention provides an isolated nucleic acid molecule comprising a population consisting of the following polynucleotides:

(a) the nucleotide sequence of SEQ ID NO: 1 or 3;

(b) a polynucleotide encoding a polypeptide represented by the amino acid sequence of SEQ ID NO: 2 or 4;

(c) a polynucleotide complementary to (a) or (b);

(d) Polynucleotides heterozygous with (a), (b), (c), (d) or (e) under highly stringent conditions.

The invention utilizes wild type four season orchids and clumps of four-season mutant plants as materials to explore the role of group C genes in orchid flower shape development. According to the present invention, a full-length cDNA sequence of two C-group genes of Four Seasons is provided and designated as CeMADS1 (SEQ ID NO. 1) which can translate 233 amino acids (SEQ ID NO. 2) and CeMADS2 (SEQ ID NO) 3) It can translate 234 amino acids (SEQ ID NO. 4). The nucleotide sequence identity of CeMADS1 and CeMADS2 was 83%; the consistency and similarity of CeMADS1 protein and CeMADS2 protein were 85% and 96%, respectively. Multiple sequence alignments with other Group C proteins of plants showed that both proteins have a typical MIKC type domain structure, and CeMADS1 and CeMADS2 contain AG motifs I and II at their C-terminus. On the other hand, both CeMADS1 and CeMADS2 are presented as single-replica genes in the genus of the genus Cymbidium.

The results of spatial performance pattern analysis showed that CeMADS1 was only expressed in the column and not in other flower organs and vegetative tissues. CeMADS2 was mainly expressed in the column and also in other flower organs, but not Will be expressed in nutrition organizations. During the development of flower buds, CeMADS1 showed a greater amount of performance in the first stage, while CeMADS2 showed more obvious performance in the second stage and later stages. Comparing the results of CeMADS1 and CeMADS2 in the wild-type and plexus-type mutants, the CeMADS1 was expressed in the wild-type four-season orchid, but not in the plexus-type mutant, while the CeMADS2 was consistently expressed in the wild-type and The plexus-type mutant strain is calyx. Although not wishing to be bound by theory, Xianxin CeMADS1 can initiate the development of the core column, and then CeMADS2 plays a maintenance role to complete the morphogenesis of the core column; in addition, the performance of the CeMADS2 RNA transcript in the peduncle is much higher than that of CeMADS1 , indicating CeMADS2 can also participate in pedicel/ovule development.

In terms of protein-protein interaction, the interaction strength of CeMADS1 homodimer is similar to that of CeMADS1-CeMADS2 heterodimer, but stronger than CeMADS2 homodimer; CeMADS1 interacts with E group gene (eg PeMADS8) The interaction strength of the -PeMADS8 heterodimer is also stronger than that of the CeMADS2-PeMADS8 heterodimer. These results indicate that the CeMADS1 homodimer, CeMADS1-CeMADS2 heterodimer and/or C-E complex play a key role in the development of the four-phase blue column.

In addition, the large expression of CeMADS1 or CeMADS2 in Arabidopsis by Agrobacterium tumefaciation results in the growth of primary inflorescences, and the primary inflorescences are abnormally terminated by a cluster of flower buds. However, in the CeMADS1 and CeMADS2 transgenic Arabidopsis, the branches of the primary inflorescence did not end with the top flower. These results suggest that the function of CeMADS1 or CeMADS2 may contribute to the meristematic characteristics of the flower in the primary flowering inflorescence.

As used herein, "isolated nucleic acid molecule" refers to a nucleic acid molecule (1) that is not associated with all or part of another nucleic acid molecule (covalent or non-covalent), but the nucleic acid molecule is naturally and otherwise The nucleic acid molecule is related; (2) is not associated with a molecule that is not related to the molecule in nature; or (3) is not associated with other nucleic acid molecules in nature. The nucleic acid molecule can be a genomic DNA, cDNA, mRNA, or other artificial RNA, or a combination thereof. An isolated nucleic acid molecule according to the invention may contain a leader sequence, a coding region or a non-exon and an intron, and it may comprise additional nucleotides that do not affect the function of the nucleic acid molecule. For example, its 5' and 3' untranslated regions can contain different numbers of nucleotides.

As used herein, "polynucleotide" refers to a single or double stranded nucleic acid polymer of at least 10 bases in length. In some aspects, the polynucleotide can be in the form of ribonucleic acid or deoxyribonucleic acid or a modified form thereof. Such modifications include: bromouridine and inosine derivatives, ribose modifications such as 2', 3'-dideoxyribose, such as phosphorus An internucleotide linkage modification of phosphorodiselenoate, phosphoroanilothioate, phosphoranladate or phosphoroamidate, and analogs thereof.

Specific examples of the nucleic acid molecule of the present invention include a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or 3, but the present invention is not limited to a polynucleotide having such a specific nucleotide sequence; the nucleic acid molecule of the present invention may Having a nucleotide sequence selected from any combination of crypto residues relative to individual amino acid residues, the selection of the cipher is accomplished using conventional methods, for example, selecting a password can take into account the frequency with which the host uses the cryptographic code [Nucleic Acids Res., 9, 43 (1981)].

According to the sequence information in the specific examples of the genes disclosed herein, the nucleic acid molecules of the present invention can be easily prepared or obtained by conventional genetic engineering methods [Reference Molecular Selection Second Edition, Cold Spring Bay Laboratory, 1989; Zoku Seikagaku Jikken Koza "Idenshi Kenkyu-ho I, II, III", published by Nippon Seikagakkai (1986)].

The nucleic acid molecules of the present invention are also included under highly stringent conditions (e.g., 0.2 x SSC containing 0.1% SDS at 60 ° C, or 0.1 x SSC containing 0.1% SDS at 60 ° C), and can be SEQ ID NO: 1 or 3 or any polynucleotide which encodes a polynucleotide of the polypeptide represented by the amino acid sequence of SEQ ID NO: 2 or 4, or a polynucleotide which is heterozygous for the polynucleic acid complementary to the polynucleotide.

The invention further provides a vector comprising the aforementioned nucleic acid molecule. The vector can be used to store, produce, or otherwise introduce the nucleic acid molecule into a prokaryotic, eukaryotic, plant or plant cell; preferably, the vector is a shuttle vector. As used herein, the term "shuttle vector" refers to a vector that can be manipulated and selected in both plants and in cells that are susceptible to replication, typically prokaryotes. Typically, the shuttle vector comprises a selectable marker, such as an anti-kanamycin marker that can be selected from plant cells or an actinomycin marker that can be selected from prokaryotes. In addition, the vector also contains an origin that can be replicated in prokaryotes and a restriction enzyme position that can be used for genetic manipulation. Preferably, the nucleic acid molecule according to the present invention is under the control of a promoter, and more preferably, the promoter can drive the expression of a gene in a reproductive tissue or a part of a plant, such as a cauliflower mosaic virus 35S protein promoter (cauliflower). Mosaic virus 35S protein promoter), α-1 and β-1 tubulin promoter and histone promoter. In some embodiments of the invention, the promoter is an inducible promoter comprising, but not limited to, a heat shock protein promoter, a light-driven promoter comprising chlorophyll a/b light. Those of ordinary skill in the art to which the present invention pertains can accomplish the construction of such carriers in accordance with the teachings of the present invention.

In another aspect, the present invention provides a cell comprising the aforementioned vector, which is obtained by introducing the vector into a cell by transformation. As used herein, the term "transfer" refers to the alteration of genetic material in a cell by introduction into a cell by a nucleic acid molecule. Those skilled in the art can accomplish this transfection through the disclosure of the present invention and the general knowledge of molecular biology. For example, when introducing a vector into a bacterium, a heat shock method can be used, and when the vector is introduced into a plant cell, the carrier can be used. Gene gun or vacuum infiltration. If the plant is regenerated from a transgenic cell, all cells of the plant have the same genetic material, which is usually referred to as a transgenic plant; if only one part of the plant (such as only germ cells or tissues) is transferred In colonization, only the transformed cell exhibits a different phenotype than its parent, which is commonly referred to as a mosaic plant, which can be used to remove the cells to be transformed from the parent for transcription. The invention also provides a protocorn-like body comprising the nucleic acid molecule described above.

The invention further provides a method for producing a transgenic plant, the steps comprising:

(a) directing a nucleic acid molecule having the foregoing to a plant cell to obtain the plant transgenic cell;

(b) regenerating the plant transgenic cells to produce the transgenic plant.

Transgenic plants are usually obtained through pseudo-spheres, which are tissues with strong differentiation ability, proliferation ability and rapid proliferation rate. They are separated from the pseudo-spheres during the induction or aseptic sowing of plant vegetative bodies. After individual cells, they can regenerate into a new pseudo-sphere and develop into a plant (Arditti, J., 1992, Fundamentals of Orchid Biology, John Wiley and Sons, New York). The term "regeneration" as used herein refers to a method of growing from a plant cell, a group of plant cells, or a part of a plant to a plant, the method of regeneration being well known to those of ordinary skill in the art to which the invention pertains. .

In a preferred aspect of the method for producing a transgenic plant of the present invention, the step (a) introduces the nucleic acid molecule into the cell by gene gun or vacuum intrusion, and the certain cells in the pseudo-sphere are transformed into a transgenic cell. The selection marker on the vector screens and distinguishes the transfected and non-transfected cells, and then proceeds to step (b) to regenerate the transfected cells into the transgenic plants.

The invention further provides a protein encoded by the aforementioned nucleic acid molecule. According to a preferred aspect of the invention, the protein is a protein having the sequence set forth in the amino acid of SEQ ID NO: 2 or 4.

The protein of the present invention can be obtained by a conventional recombinant technique, and preferably the nucleotide sequence of SEQ ID NO: 1 or 3 is used as a template, and if necessary, the amino acid residue relative to the protein can be appropriately selected and modified. The code is used to design the nucleotide sequence.

The protein of the present invention can also be produced according to the amino acid sequence of SEQ ID NO: 2 or 4 by a general chemical synthesis method, and examples of the synthesis method include a general liquid phase synthesis method or a solid phase synthesis method.

Specific examples of the peptide synthesis method include a stepwise lengthening method of sequentially bonding an amino acid to another amino acid residue according to amino acid sequence information, thereby lengthening the amino acid chain; A condensation method in which a fragment is composed of a plurality of previously synthesized amino acids, and a fragment is bonded to another fragment via a coupling reaction. The protein of the present invention can be synthesized by any method.

In such peptide synthesis, condensation can be carried out by a conventional method. Examples of the condensation method include an azide compound method, a mixed acid anhydride method, a DCC method, an activated ester method, an oxidation-reduction method, a DPPA (diphenylphosphoryl azide) method, a DCC+ additive (1-hydroxybenzotriazole, N-hydroxy succinylamine, N-hydroxy-5-nor -2,3-Dicarboxyliminium) method and Woodward method.

The solvent used in this method may be selected from solvents widely used in peptide condensation reactions, and examples of the solvent include dimethylformamide (DMF), dimethylhydrazine (DMSO), decylamine hexaphosphate, and Alkane, tetrahydrofuran (THF), ethyl acetate, and solvent mixtures thereof.

In the foregoing peptide synthesis, esterification may be used to esterify a carboxyl group on a non-reactive amino acid or peptide, and to protect it, for example, a lower alkyl ester; for example, a methyl ester, an ethyl ester, Tri-butyl ester, and aryl alkyl ester; for example, benzyl ester, p-methoxybenzyl ester, and p-nitrobenzyl ester.

An amino acid having a side chain functional group (e.g., a hydroxyl group of tyrosine) can be protected by a group such as an ethenyl group, a benzyl group, a benzyloxycarbonyl group, or a third-butyl group. However, protection is carried out as appropriate. In addition to this, for example, a mercapto group on arginine may be a suitable protecting group (such as a nitro group, a toluenesulfonyl group, a p-methoxyphenylsulfonyl group, a methyl-2-sulfonyl group). Benzyloxycarbonyl, different Protection by oxycarbonyl or adamantyloxycarbonyl).

The protecting group included on the aforementioned amino acid, peptide and protein of the present invention can be deprotected by a routine method; for example, catalytic reduction or utilization of liquid ammonia/sodium, hydrogen fluoride, hydrogen bromide, hydrogen chloride, trifluoroacetic acid, Reagents such as acetic acid, formic acid or methanesulfonic acid.

If necessary, depending on the physical and chemical properties of the protein, various purified separation and purification techniques can be used to obtain purified proteins [Ref. "Biochemistry Data Book II", pp. 1175-1259, first edition, first print, Tokyo Kagaku Dojin, June 23, 1980; Biochemistry, 25, (25), 8274 (1986); Eur. J. Biochem., 163, 313 (1987)].

Specific examples of protein separation techniques include general restitution treatment, protein precipitation treatment (salting out), centrifugation, penetrating piezoelectric impact method, ultrasonic, ultrafiltration, molecular sieve chromatography (film filtration), adsorption chromatography , ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis, and combinations thereof. More preferably, affinity chromatography containing a column capable of specifically binding an antibody to the protein of the present invention can be employed. Alternatively, the protein of the invention may be purified using, for example, ion exchange resins, partition chromatography, film chromatography, and countercurrent distribution.

The present invention further provides a method of controlling the development of a spheroid in a orchid plant comprising altering the amount of expression of the aforementioned protein in the plant.

The present invention is applicable to plants and cells thereof, preferably monocotyledons or Arabidopsis and its cells; more preferably orchids and cells thereof; more preferably, Silybum and its cells. According to the invention, the plant comprises a plant of wild species and a mutant produced by an artificial method comprising genetic material or X-ray induced random mutagenesis or genetic modification of the plant using recombinant techniques.

Preferably, the method according to the invention comprises varying the number of sets of nucleic acid molecules encoding the protein in at least one cell of the plant; more preferably, the cell line is derived from a pseudo-sphere.

The present invention also provides a method for controlling flower shape development in orchid plants, which comprises the aforementioned method for controlling the development of a spheroid.

Preferably, the method according to the invention further controls the development of the florets, inflorescences, meristems, pedicels or ovules.

The invention is illustrated by the following examples, which are not intended to limit the invention to the invention.

Instance Materials and methods Plant material

Wild type four season orchids and plexus mutants were supplied by Chun-Rong Hu's orchid nursery (Guanling Vil., Baihe Township, Tainan County, Taiwan) and grown in natural light and a greenhouse controlled temperature of 23 ° C to 27 ° C.

scanning electron microscope

The sample was fixed in FAA (18:1:1 ethanol [50%], glacial acetic acid, formaldehyde). After dehydration in a series of ethanol-acetone, the sample was subjected to critical point drying, and sputter-coated with platinum, and observed under a scanning electron microscope (Hitachi S-4200, Tokyo) at an accelerating voltage of 15 kV. Photographs were taken using a Verichrome panchromatic film (Kodak, Rochester, NY, U.S.A.).

RNA preparation

For RNA extraction, collect the various flower buds of the wild type Four Seasons orchids (including sepals, petals, lips and pillars) and the complete buds of the two plexus mutants and soak them in liquid nitrogen and store at -80 °C. Until the RNA is extracted. Total RNA was extracted according to the method described by Tsai et al. (Tsai et al, 2004, Plant Cell Physiol. 45: 831-844).

CeMADS  3' and 5' rapid amplification of cDNA ends (RACE)

The full-length cDNA was obtained by extending the 3' and 5' ends of the cDNA using a SMART RACE cDNA amplification kit (Clontech, Palo Alto, CA). According to the manufacturer's instructions, 300 ng of total RNA of the flower bud was used as the first cDNA for the synthesis of 3'- and 5'-RACE. The CeMADS- pure line containing the 3'-end cDNA was amplified by PCR and carried out using the 3' and 5'-specific universal primers (Clontech) and the 5' and 3' gene-specific primers of the different CeMADS genes. The gene-specific primers CeMADS1 and CeMADS2 for CeMADS-N are 5'-TGCCATCAACAAGCACTGAGTATGA-3' (SEQ ID NO. 5), 5'-CTATTACACAACTGTATCACCAACTCTCGAGC-3' (SEQ ID NO. 6) and 5'-CTCTACAGATAAAACTAACTTGGCAAGACAGAG-, respectively. 3' (SEQ ID NO. 7). The thermal cycle was initially denatured at 94 °C for 5 minutes, followed by 25 cycles at 94 °C for 30 seconds, at 60 °C for 30 seconds, and at 72 °C for 2 minutes, and finally at 72 °C for a final extension of 5 minutes. The RACE product was re-amplified using the CeMADS gene-specific nested primer and the nested universal primer provided in the RACE kit.

The gene-specific nested primer CeMADS-N2 is 5'-CAAGAAGCCWCAAAACTGCGTCA-3' (SEQ ID NO. 8), and CeMADS1 and CeMADS2 are 5'-TCATACTCAGTGCTTGTTGATGGCA-3' (SEQ ID NO. 9). The PCR was initially denatured at 94 °C for 5 minutes, followed by 30 cycles of 30 seconds at 94 °C, 30 seconds at 60 °C and 2 minutes at 72 °C, and a final extension of 5 minutes at 72 °C. The PCR product was cloned into the pGEM-T Easy vector (Promega, Madison, WI) and two strands from 10 positive lines were sequenced.

Sequence data analysis

The original DNA sequence data was edited using Sequencher V. 4.1 (GeneCode, Ann Arbor, MI) to remove vector and poly A sequences as well as poor quality data. Manually inspect the computer-processed sequence and further edit as necessary to improve the quality and reliability of the data compared to the electropherogram. Each edited sequence translates six reading frames and uses the BLASTX program to compare with the National Center for Biotechnology Information (NCBI) non-redundant (nr) database (Altschul et al, 1997). , Nucleic Acids Res. 25: 3389-3402), and uses preset BLAST parameter values.

Systematic analysis

The 41 gene lines used in the phylogenetic analysis were downloaded from GenBank and sequence aligned with Clustal W (Thompson et al, 1994, Nucleic Acids Res. 22: 4673-4680). Non-conservative I and C regions were excluded from this alignment. The phylogenetic tree was constructed by the neighbor-joining method and evaluated by the bootstrap value estimated by 1,000 parallel operations.

Isolation of genomic DNA and Southern blot analysis

The genomic DNA was isolated from the leaves according to the method described by Tsai et al. (Tsai et al, 2004, Plant Cell Physiol. 45: 831-844). A sample of the genomic DNA was digested with restriction enzymes Eco R I and Dra I, resolved in a 0.8% agarose gel, and transferred to a Hybond-N ⁺ nylon membrane using a vacuum transfer system (Amersham Pharmacia Biotech) (Amersham Pharmacia) Biotech, Piscataway, NJ). A partial C-terminal region and a 3'-UTR sequence were selected as probes. Generation of CeMADS1- specific probe (258 bp) by PCR using the CeMADS1- specific internal primer pair 5'-CTATTACACAACTGTATCACCAACTCTCGAGC-3' (SEQ ID NO. 10) and 5'-TGCCATCAACAAGCACTGAGTATGA-3' (SEQ ID NO. 11) . Use CeMADS2 specific primer internal to the 5'-CTCTACAGATAAAACTAACTTGGCAAGACAGAG-3 '(SEQ ID NO. 12) and 5'-TGCCATCAACAAGCACTGAGTATGA-3' (SEQ ID NO. 13) amplified CeMADS2 specific probe (292 bp). The Southern blot is hybridized to the probe labeled α- ^32P . Pre-hybridization and hybridization were performed according to standard procedures (Sambrook et al, 2001, Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

Northern ink dot hybridization

For northern blot analysis, total RNA from different flower buds or different tissues (10 μg per lane) was isolated on a 1% agarose gel and transferred to Hybond-N ⁺ nylon membrane (Amersham Pharmacia, Biotech) on. For pre-hybridization, the membrane was placed in a glass test tube and hybridization buffer (5 M NaCl, 50 mM Tris (pH 7.5), 10% SDS, 1% Na ₂ P ₂ O ₄ , 10 mM EDTA) was added at 42 °C. 100×Denhard's solution (0.1 mg/ml), 50% dextran sulfate and 50% formamide. 0.1 mg/ml salmon sperm DNA was added to the prehybridization solution, denatured at 95 ° C for 5 minutes, kept on ice for 5 minutes and at 42 ° C for 2 hours. The RNA dots were hybridized to the same probe as described above. Pre-hybridization and hybridization were performed according to standard protocols (Sambrook et al, 2001, Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The filter was washed twice with 2 X SSC, 0.1% SDS for 15 minutes at room temperature, then finally washed with 0.1 X SSC, 0.1% SDS for 30 minutes at 42 ° C, followed by autoradiography at -80 ° C and Stay for 3 days. Signals were detected using a FLA-7000 fluorescence image analyzer (Fujifilm, Stamford, CT).

Real-time quantitative RT-PCR

RNA samples were treated with RQ1 DNase (Promega) to remove residual DNA, and the first strand of cDNA was synthesized using the Superscript II kit (Invitrogen). Primers for real-time RT-PCR were designed using Primer Express (Applied Biosystems, Foster City, CA). The gene-specific primers used for the CeMADS1 5'-GGTAAGGGAAGATTGTTGCAGTGA-3 '(SEQ ID NO. 14) and 5'-TTTCCATCCATACTTTTCTTACAAAACATA-3' (SEQ ID NO. 15). The gene-specific primers for CeMADS2 as 5'-AAATCTGGAGTGAATTGGAATGGT-3 '(SEQ ID NO. 16) and 5'-AAGTAGAGTCTCTACAGATAAAACTAACTTG-3' (SEQ ID NO. 17). The ubiqutin gene was used as an internal quantitative control group. Ubiqutin-specific primers for the gene of 5'-CCGGATCAGCAAAGGTTGA-3 '(SEQ ID NO. 18) and 5'-AAGATTTGCATCCCTCCCC-3' (SEQ ID NO. 19). Each RT-PCR reaction contained 5 ng cDNA, 20 μM primer and 12.5 μl SYBR GREEN PCR mix (Applied Biosystems) and water was added to 25 μl. Real-time PCR was performed using an ABI 7500 real-time PCR instrument (Applied Biosystems). PCR was initially denatured at 94 ° C for 10 minutes, followed by 40 cycles at 94 ° C for 15 seconds, at 60 ° C for 1 minute, and at 95 ° C for 15 seconds, at 60 ° C for 1 minute, at 95 ° C for 15 seconds. The dissociation phase consists of. For each of the real-time RT-PCR reactions, each sample was subjected to three replicate analyses. The data was analyzed using the sequencing detection software version 1.2.2 (Applied Biosystems).

In situ hybridization

The four season orchids were fixed in 4% (v/v) trioxane and 0.5% (v/v) glutaraldehyde at 4 ° C for 24 hours. It was dehydrated via a series of ethanol, embedded in Histoplast and 8 μm sections were obtained using a Shandon Finesse 325 inflorescence microtome (Thermo, Waltham, MA). Synthetic and anti-strand RNA probes containing Dichoxigenin-labeled partial C-terminal regions and 3'-UTR as described above were synthesized and used according to the manufacturer's instructions (Roche Applied Science, Penzberg, Germany). Alkaline phosphatase for signal hybridization and immunological detection (Boehringer Mannheim, Mannheim, Germany).

Yeast two-hybrid analysis

Yeast two-hybrid (Y2H) analysis was performed using the MATCHMAKER system (Clontech). The Eco RI fragment containing the full-length coding region of the two C-group genes CeMADS1 and CeMADS2 , and the E-group gene PeMADS8 of Hydrangea , was amplified by PCR and cloned into the binding domain of the vector pGBKT7 and the vector pGADT7. Activation domain. Primer for cloning full-length coding region of the CeMADS1 5'-GAATTCATGGAGCCCAAGGAGAAGATGG-3 '(SEQ ID NO. 20) and 5'-GAATTCTTACCCAAGCTGCAGAGCAGTCTG-3' (SEQ ID NO. 21). Primer for cloning full-length coding region of the CeMADS2 5'-GAATTCATGGGAAGGGGAAAGATAGAGATCAAGAG-3 '(SEQ ID NO. 22) and 5'-GAATTCCTAACCTAATTGGAGGGCAGTCTGCTG-3' (SEQ ID NO. 23). Primer for cloning full-length coding region of the PeMADS8 5'-GAATTCATGGGAAGAGGGAGAGTGGAGC-3 '(SEQ ID NO. 24) and 5'-GAATTCGGGGGAAAGCCAGAAATTTGTA-3' (SEQ ID NO. 25). Whether the construct is properly fused is confirmed by sequence analysis. Transformation using yeast strain AH109, lithium acetate method and community lift filtration test (Gietz et al, 1992, Nucleic Acids Res. 20: 1425). The transformants containing the binding domain and the activating domain plastid were screened on selection medium lacking histidine, leucine and tryptophan (-3) according to the manufacturer's instructions (Clontech).

Quantification of the yeast two-hybrid assay was performed using yeast strain Y187 (Clontech). The positive transformant grown on the selection medium was further grown and resuspended in O-nitrophenol-bD-galactopyranoside (4 mM in Z buffer) as the substrate Z buffer (100 mM). NaPO ₄ , 10 mM KCl, 1 mM MgSO ₄ , 50 mM β-mercaptoethanol (pH 7.0)). For intensity quantification of protein-protein interactions, β-galactosidase activity was measured at OD ₄₂₀ using a DU 640B spectrophotometer (Beckman Coulter Inc., Fullerton, CA) and according to the method described by Miller (Miller, 1992, A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

Construction of transformational fusion

The Sma I fragment containing the full-length cDNA of CeMADS1 and CeMADS2 was separately selected into the shuttle vector pBI121 (Clontech) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The positive strand construct was tested using PCR and used for plant transformation. The primer pair for the full-length CeMADS1 cDNA was 5'-CCCGGGGAGTGAAATAGATCGAGAAAGCTA-3' (SEQ ID NO. 26), 5'-CCCGGGGGAAGATTAAAGTTCCATGAAATA-3' (SEQ ID NO. 27). The primer pair for the full length CeMADS2 cDNA was 5'-CCCGGGGAAAACTGGCTTTCTTTCAGGCAG-3' (SEQ ID NO. 28) and 5'-CCCGGGCTTGTATTTTATTTCTTCTACAAAATATAGAGT-3' (SEQ ID NO. 29).

Plant transformation

Invasion by vacuum was carried out by adding a transformation medium containing 0.02% (v/v) Silwet L-77 (Lehle Seeds, Round Rock, Tx) (Bechtold et al, 1993, CR Acad. Sci. Ser. III Sci. Vie. 316: 1194-1199) Transforming Arabis mustard. Arabidopsis transformation was selected, supplemented with 50 μg ml ^-1 screening seeds (T ₀₎ on Connor neomycin (Sigma-Aldrich) of media. After 2 weeks of selection, the kanamycin-resistant seedlings (T ₁ ) were transferred to soil and grown under the above conditions. The connamycin separation in the T ₁ generation was analyzed by using a Chi-square test. Connor neomycin resistance using homozygous T ₂ of each construct was confirmed by substituting the integration fragment obtained from PCR. A transformation ratio of 3:1 was collected for further analysis.

result

Flower morphogenesis between wild type and plexus mutants was performed using wild type of Four Seasons Orchid (Fig. 1A) and two plexus mutants (Type A and Type B) (Figs. 1B and 1C) as plant material. The plant has an indeterminate inflorescence, wherein the wild-type flower has three sepals in the first inflorescence, two petals and one lip in the second inflorescence, and one in the center of the flower, which is a combination of male and female reproductive organs. Pillar. These flowers usually have a strong aroma. The shape and color of the sepals and petals are similar to each other except that the petals usually have a strong central band and reddish-brown spots toward the base. The lip (the highly evolved petal) is light yellow or green with red edges. The shape of the lip is oval to triangular and has a kinked to wavy edge. The column is light yellow and has a short red line at the bottom. The column or column has four pollen blocks on a semi-circular viscidium. Inflorescence meristems produce floral meristems in spiral leaf order, while flower meristems produce flower primordia in inflorescence leaf order (Fig. 1D and 1E). Type A (Fig. 1B) and Type B (Fig. 1C) plexus mutants have indefinite flowers with repeating plate patterns from the outermost inflorescence to the center. There is no colloidal column in the type A and type B mutants (Figs. 1B, 1C and 1F).

Identification and sequence analysis of the C-group MADS-box gene from Four Seasons

In order to select the C gene, the full-length cDNA was obtained by rapid amplification (RACE) of the cDNA end, and the two full-length cDNAs of the C gene in the four seasons were selected and named as CeMADS1 and CeMADS2 . The nucleotide sequence identity of CeMADS1 and CeMADS2 was 83%. ORF translation resulted in 233 aa protein (CeMADS1) and 234 aa protein (CeMADS2) with consistency and similarity of 85% and 96%, respectively. Multiple sequence alignments with other Group C proteins of plants showed that both proteins have a typical MIKC type domain structure (Figure 2). In addition, CeMADS1 and CeMADS2 contain AG motifs I and II at their C-terminus (Fig. 2). These motifs are conserved in many AG homologs in angiosperms (Kramer et al, 2004, Genetics 166: 1011-1023).

Phylogenetic relationship between CeMADS paralogs and other AGAMOUS genes

To determine the phylogenetic relationship between CeMADS1 , CeMADS2, and other AGAMOUS genes, the amino acid sequence of each gene was used as input to reconstruct the phylogeny of the known AGAMOUS subfamily of the MADS-box gene. Phylogenetic analysis of these genes revealed that the Orchidaceae C group is unimodal and has two subfamilies of the C gene in orchids. This indicates that the orchid C gene line caused by gene duplication occurs before the genus of the subfamily species (Fig. 3).

Genomic distribution of CeMADS gene

In order to understand the distribution of the genomic distribution of CeMADS1 and CeMADS2 in the genome of the four seasons, Southern blot analysis was carried out. A probe containing 20 μg of the genomic DNA of the Four Seasons genomic DNA digested with Eco R I (lanes 1 and 3) and Dra I (lanes 2 and 4) under stringent conditions and probes derived from the 3' specific region of the CeMADS gene Hybridization (Figure 4). The results showed that both CeMADS1 and CeMADS2 were expressed as single-replicon genes in the Cymbidium genome. In addition, the probe used in the Southern dot method is a gene specific for the difference between CeMADS1 and CeMADS2 .

Performance profile of two CeMADS genes

Since these two C group paralogs exist in the genus, the roles of the two genes are further explored. First, the spatial expression patterns of CeMADS1 and CeMADS2 genes between wild-type and plexus mutant flower buds were compared by northern blot analysis (Fig. 5). The 3'-end gene-specific region of the CeMADS gene was used as the above probe.

The performance of CeMADS1 was only detected in the column (Fig. 5, column 1, lanes 1-4). However, in all flower organs, mainly the column, the performance of CeMADS2 was detected (Fig. 5, column 2, lanes 1-4). These results indicate that these two CeMADS genes play an important role in the development of the core column. In addition, the CeMADS2 gene product can have multiple functions in different aspects of orchid flower development.

The differential function of these two CeMADS genes in flower morphogenesis in the plexus mutant was further analyzed (Fig. 5). A strong signal of CeMADS1 expression was detected in wild-type flower buds. In contrast, no CeMADS1 transcript was detected in RNA samples from A-type or B-type bud flower buds (Fig. 5, column 1, lanes 5-7). However, no significant difference in the detection signal of CeMADS2 was observed between wild-type and type A or B-type plexus mutants (Fig. 5, column 2, lanes 5-7). These results indicate that in the flower morphogenesis of orchids, the performance of CeMADS1 is significantly related to the development of normal spheroids .

In addition, in the various developmental stages of flower buds (1st stage: 0.5-1 cm, stage 2: 1-2 cm, stage 3: 2-3 cm) and determination of CeMADS1 and CeMADS2 in different vegetative tissues of Four Seasons Time performance (Figures 6 and 7). The results showed that CeMADS1 and CeMADS2 were detected in all flower bud development stages, and they still had very special performance patterns. CeMADS1 performed more strongly in the first stage; compared to CeMADS1 , CeMADS2 performed more significantly in the subsequent stages (Fig. 6). These results indicate that CeMADS1 can initiate the development of the core column, and then CeMADS2 plays a maintenance role to complete the morphogenesis of the core column. However, CeMADS1 and CeMADS2 do not appear in vegetative tissues such as roots, leaves and shoots (Fig. 7). Interestingly, the CeMADS2 RNA transcript performed much better in peduncle than CeMADS1 (Figure 7), suggesting that CeMADS2 can also be involved in pedicel/ovule development.

In situ localization of CeMADS transcripts

The spatial and temporal expression patterns of the CeMADS1 and CeMADS2 genes were studied using in situ hybridization of anti-strand RNA probes. The results showed that CeMADS1 and CeMADS2 transcripts have highly similar expression patterns (Fig. 8). CeMADS1 and CeMADS2 were detected at very early stages of the flower primordium (Figs. 8A, 8E). At a later stage, it is more focused on the paraxial plane of the developing column (Figs. 8B, 8C, 8F). These results also prove that CeMADS1 and CeMADS2 may be related to the development of the core column. A negative control was performed using a positive strand RNA probe (Fig. 8D, 8G).

Interaction behavior of two CeMADS proteins

To study protein-protein interactions between Group C and Group E genes in orchids, yeast two-hybrid assays including colony-lift filter and liquid culture assays were performed. CeMADS1 , CeMADS2 and the SEPALLATA (E group) gene PeMADS8 (Hymenoptera MADS8 , Fig. 11) were separately selected into the binding domain vector pGBKT7 and the activation domain vector pGADT7. The results showed that both CeMADS1 and CeMADS2 have the ability to form homodimers and heterodimers (Fig. 9). Alternatively, it can interact with the E group PeMADS8 independently (Fig. 9). The interaction strength of the CeMADS1 homodimer was similar to that of the CeMADS1-CeMADS2 heterodimer, but it was stronger than the CeMADS2 homodimer (Fig. 9). In addition, the interaction strength of the CeMADS1-PeMADS8 heterodimer was also stronger than that of the CeMADS2-PeMADS8 heterodimer (Fig. 9). These results indicate that the CeMADS1 homodimer, CeMADS1-CeMADS2 heterodimer and/or CE complex play a key role in the morphogenesis of the four-phase blue column.

Functional analysis of two CeMADS genes by ectopic expression in Arabidopsis

To study the function of the C-group CeMADS gene, the Agrobacterium- mediated transformation constructs showed the Arabidopsis thaliana of CeMADS1 and CeMADS2 under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Of the 11 independent CeMADS1 transgenic strains obtained, 5 showed finite growth of primary inflorescences (Fig. 10). A similar phenotype was also observed in 6 of the 19 independent transgenic Arabidopsis thaliana expressing CeMADS2 . As shown in Figures 10A to 10C, the primary inflorescence tip abnormally terminates with a cluster of flower buds. However, in the CeMADS1 and CeMADS2 transgenic Arabidopsis, the branches of the primary inflorescence did not end with the top flower. These results suggest that the function of CeMADS1 or CeMADS2 may contribute to the meristematic characteristics of the flower in the primary flowering inflorescence.

In addition to its role in determining the characteristics of floral meristems, the function of AG is also to form stamen and carpel characteristics. The ectopic expression of AG in Arabidopsis reached the homeomorphic transformation of carpelloid and staminoid parts in the first inflorescence and the second inflorescence, respectively (Mizukami and Ma, 1992, Cell 71: 119 -131). However, the Arabidopsis thaliana, which overexpresses the transgenic gene of CeMADS1 or CeMADS2 , did not alter flower morphology (Fig. 10).

The above-described embodiments are merely illustrative of the principles and effects of the invention, and are not intended to limit the invention. Modifications and variations of the embodiments described above will be apparent to those skilled in the art without departing from the spirit of the invention. The scope of the invention should be as set forth in the appended claims.

<110> National Cheng Kung University

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Figure 1 shows wild-type flowers and plexus mutant flowers of the four seasons. (A) Wild-type flowers of the Four Seasons Orchid. The flower has three sepals and three petals. A petal is structurally distinct and is referred to as a lip or lip. Male and female reproductive parts are born in the structure of the flower center, that is, the core column. The pollen grains stick together to form a pollen block, which is located at the top of the pegs and under the pollen cover. (B) and (c) Four-season orchid plexus mutant type A and type B flowers. The plexus mutant flower represents the spheroidal column, and the male and female genitals are replaced by new germinated flowers, and the ectopic flower continues to produce sepals and petals. (D, E) Scanning electron micrograph (SEM) of the initial flower development stage of wild type Four Seasons. Scale bar = 200 μm. (F) SEM of the initial flower development stage of the Four Seasons orchid plexus mutant. Scale bar = 500 μm. Inflorescence meristem; Fm, floral meristem; Se, bract primordium; Pe, petal primordium; Li, lip primordium; Co, primordium. Te, was filmed.

Figure 2 shows an alignment of the deduced amino acid sequence of the CeMADS gene and other class C genes. Multiple comparisons are generated by the computer program PILEUP and presented by the PRETTYBOX. Common consistency is represented by a black box. The common similarity is indicated by gray, the difference is indicated by white, the comparison gap is indicated by a dot, and the position not occupied by the amino acid is indicated by "~". The MADS, I, K, and C fields are indicated at the top of each column. The AG primitive is indicated by a black line. AGAMOUS (CAA16753) is from Arabidopsis thaliana ; CsAG1 (AAS67610) is from Crocus sativus ; DcOAG1 (AAZ95250) is from pigeon sarcophagus; FARINELLI (CAB42988) is from Antirrhinum majus ; and OSMADS3 (Q40704) From rice ( Oryza sativa ).

Figure 3 shows the phylogenetic analysis of the AG-like genes of C and D lines and gymnosperms. The published plant MADS-box protein sequence was retrieved from the GenBank database (PLENA{AAB25101}[金鱼草];FARINELLI{CAB42988}[金鱼草];FBP6{CAA48635}[ Petunia hybrida ];FBP7{CAA57311 }[小牛牛];FBP11{CAA57445}[Petunia];pMADS3{CAA51417}[Petunia];NAG1{AAA17033}[Tobacco ( Nicotiana tabacum )];TAG1{AAA34197}[ Solanum lycopersicon ]; TAG11{AAA34197}[category of tomato]; DcMADS4{CAC81071}[ Daucus carota ];GAGA1{CAA08800}[ Gerbera hybrida ];GAGA2{CAA08801}[gerbera];AGAMOUS{CAA37642}[ Arabidopsis thaliana]; SHP1{AAA32730}[Arabidopsis thaliana]; SHP2{AAA32735}[Arabidopsis thaliana]; STK{AAC49080}[Arabidopsis thaliana]; MdMADS10{CAA04324}[Apple (Musus domestica )];MdMADS14{CAC80857 }[Apple];GhMADS2{AAN15183}[ Gossypium hirsutum ];STAG1{AAD45814}[ Fragaria ananassa ];VvMADS1{AAK58564}[ Vitis vinifera ];VvMADS5{AAM21345}[Grape]; SLM1{CAA56655}[Siene latifolia ]; MpMADS2{BAB70737}[ Magnolia precossimina ]; McAG{AAO20104}[Morordica charantia ]; PeMADS1{AAL76415}[Hime phalaenopsis]; DcOAG1{AAZ95250}[ pigeon sarcophagus]; DcOAG2{AAZ95251}[ pigeon sarcophagus]; DthyrAG1{AAY86364}[ pigeon sarcophagus]; DthyrAG2 {AAY86365}[ pigeon sarcophagus]; Pha1AG1{BAE80120}[dwarf cultivar]; Pha1AG2{BAE80121}[dwarf cultivar];ApMADS2{BAC66963}[ Agapanthus praecox ];LLAG1{AAR98731}[musk lily ( Lilium longiflorum )]; LMADS2 {AAS01766} [musk lily]; HvAG1{AAL93196} [hordeum vulgare ]; HvAG2{AAL93197} [barley]; WAG{BAC22939} [wheat ( Triticum aestivum )]; OsMADS3{AAA99964 }[稻];OsMADS58{BAE54300}[Rice];OsMADS13{AAF13594}[Rice];ZAG1{AAA02933}[Malay ( Zea mays )];ZMM1{CAA57073}[Corn];DAL2{CAA55867}[European Spruce] ;CeMADS1{GU123626}[Four Seasons]; CeMADS2{GU123627}[Four Seasons]). The self-expanding values from 1000 parallel operations are indicated on most of the main nodes. CeMADS1 and CeMADS2 are highlighted by an empty frame. The single-source flower homeotype genome is marked by a black line at the right edge.

Figure 4 shows Southern blot analysis of the CeMADS1 and CeMADS2 genes in the Four Seasons blue genome. Using a probe derived from the 3'-specific region of the CeMADS1 and CeMADS2 genes, a DNA gel containing 30 μg of genomic DNA digested with Eco R I (lanes 1 and 3) and Dra I (lanes 2 and 4) was spotted. Hybridization under stringent conditions. The size of the DNA marker is shown at the left edge (kb).

Figure 5 shows the northern blot analysis of CeMADS1 and CeMADS2 in different flower organs and wild type and plexus mutant flower buds of Four Seasons. Each lane contained 10 μg of total RNA from the following: bracts (lane 1); petals (lane 2); lip (lane 3); column (lane 4); w bud: wild-type flower bud (lane 5); mA flower bud: plexus mutant type A flower bud (lane 6); mB flower bud: plexus mutant B type flower bud (lane 7). The 28S ribosomal RNA indicates that an equal amount of total RNA is loaded in each lane.

Figure 6 shows the relative performance of CeMADS1 and CeMADS2 at various developmental stages of flower buds. Stage 1 (0.5-1 cm), Stage 2 (1-2 cm), Stage 3 (2-3 cm). Three replicates of real-time PCR experiments were performed.

Figure 7 shows the relative performance of CeMADS1 and CeMADS2 in different vegetative tissues as determined by real-time RT-PCR experiments. Three replicates of real-time PCR experiments were performed.

Figure 8 shows the in situ localization of CeMADS1 and CeMADS2 in the longitudinal section of the developing flower bud. The sections were hybridized to a counter-strand 3' specific CeMADS1 (A, B, C) or CeMADS2 (E, F) RNA probe or a positive strand RNA probe (D, CeMADS1 ; G, CeMADS2 ). The CeMADS1 and CeMADS2 transcripts were detected in floral meristem (Fm), and the hybridization signal was then concentrated in the column (Co). Se, sepals; Pe, petals; Li, lip.

Figure 9 shows the protein-protein interaction behavior between CeMADS1, CeMADS2 and orchid SEPALLATA-like PeMADS8 proteins. The SEPALLATA (E group) gene PeMADS8 , such as CeMADS1 and CeMADS2 in Rhododendron and Hybrid Phalaenopsis, was cloned into the binding domain vector pGBKT7 and the activation domain vector pGADT7. Activation of HIS3 and lacZ is indicated by growth on selected medium lacking histidine, leucine and tryptophan (-3), respectively, and indicating blue. Yeast transformed with the vectors pGADT7+pGBKT7 and PeMADS4+PeMADS6 were used as negative and positive controls, respectively.

Figure 10 shows the phenotype of the transgenic gene Mustard genus showing CeMADS1 and CeMADS2 . (A) Wild type Brassica. (B) Transgenic gene mustard genus that expresses CeMADS1 . (C) A magnified white circle area in (B). (D) Transgenic gene mustard genus that expresses CeMADS2 . (E) The white circle area in the enlarged (D).

(no component symbol description)

Claims (18)

An isolated nucleic acid molecule comprising a population consisting of: (a) a nucleotide sequence of SEQ ID NO: 1 or 3; (b) an amine encoding SEQ ID NO: 2 or 4 a polynucleotide of a polypeptide represented by a base acid sequence; (c) a polynucleotide complementary to (a) or (b); and (d) under highly stringent conditions, with (a), (b), (c), (d) or (e) a heterozygous polynucleotide. A vector comprising the nucleic acid molecule according to claim 1. According to the carrier of claim 2, it is a shuttle vector which can be expressed in a plant body. According to the vector of claim 2, it comprises an inducible promoter. A cell comprising the vector of any one of claims 2 to 4. The cell according to claim 5, wherein the cell is a prokaryotic cell or a plant cell. The cell according to claim 6, wherein the cell is a orchid plant cell. A protocorn-like body comprising the nucleic acid molecule of claim 1. A method for producing a transgenic plant, the method comprising the steps of: (a) directing a nucleic acid molecule according to claim 1 to a plant cell to obtain a plant transgenic cell; and (b) regenerating the plant transgenic cell to produce the transfection plant. The method of claim 9, wherein the plant cell is a orchid plant cell. The method of claim 9, wherein the step (a) directs the nucleic acid molecule to the plant cell by a gene gun or vacuum infiltration. A protein encoded by the nucleic acid molecule according to claim 1. The protein according to claim 13 which has the amino acid sequence as shown in SEQ ID NO: 2. A method of controlling the development of a spheroid in a orchid plant comprising altering the amount of expression of the protein according to claim 13 or 14 in the plant. According to the method of claim 15, which comprises changing the number of sets of nucleic acid molecules encoding the protein in at least one cell of the plant. The method of claim 16, wherein the cell line is derived from a pseudo-sphere. A method of controlling flower shape development in a plant of the family Orchidaceae, comprising the method of controlling the development of a core column according to any one of claims 15 to 17. According to the method of claim 18, it controls the development of a petal flower, an inflorescence, a meristem, a peduncle or an ovule.