NL2031044B1 - Regulatory gene for floral organ development of chinese cymbidium, and encoded protein and use thereof - Google Patents

Abstract

The present disclosure discloses a regulatory gene for floral organ development of Chinese Cymbidium and an encoded protein and use thereof. The regulatory gene for floral organ development of Chinese Cymbidium is isolated from a cDNA of a floral organ of Chinese orchid variety Cymbidium sinense "Dharma", with a nucleotide sequence as shown in SEQ ID NO: l; and a protein encoded by the regulatory gene for floral organ development of Chinese Cymbidium has an amino acid sequence as shown in SEQ ID NO: 2. In the present disclosure, it is found by a genetic engineering technology that the regulatory gene for floral organ development of Chinese Cymbidium is overeXpressed in Arabidopsis thaliana and Cymbidium, which can transform a plant flower type, resulting in petal curling, Cymbidium petals transforming into labella, and enhancing labellum traits.

Description

P1175/NLpd

REGULATORY GENE FOR FLORAL ORGAN DEVELOPMENT OF CHINESE CYMBIDIUM,

AND ENCODED PROTEIN AND USE THEREOF

TECHNICAL FIELD

The present disclosure relates to the technical field of plant genetic engineering, in particular to a regulatory gene for floral organ development of Chinese Cymbidium, and an encoded pro- tein and use thereof.

BACKGROUND ART

Cymbidium is a precious horticultural ornamental flower due to highly-specialized floral organs and rich biodiversity. More than 30,000 species in 801 genera of Cymbidium have been discov- ered all over the world. Most of terrestrial Cymbidium species are native to China, such that the Cymbidium is also called Chinese

Cymbidium. Cymbidium is listed as one of the top ten famous flow- ers in China, with an extremely high economic value. Plant molecu- lar biology studies have found that an expression region of a class B MADS-box gene expands a first round of floral organs. This is a molecular basis for the first and second rounds of floral or- gans of most monocotyledons to develop into a tepal-like struc- ture. However, unlike some other monocotyledons, the outermost three calyxes of Cymbidium can usually be divided into one dorsif- erous calyx and two lateral calyxes, which are similar in shape; while the inner three petals of the Cymbidium are divided into two lateral petals and another specialized curled labellum. The "Or- chid Code" proposed by Mondragon-Palomino et al. believes that AP3 genes in the class B MADS-box genes undergo two rounds of replica- tion in an early stage to form four subfamilies, which promote the separation of calyxes and petals, and the separation of lateral petals and labella, respectively, thereby specifically regulating the morphogenesis of different floral organs. However, some stud- ies have shown that class B MADS-box genes in different genera and species of the Cymbidium have different numbers of family members, and different subfamily members also have quite different expres-

sion patterns and functions.

The variation types of petals of the Chinese Cymbidium are far more abundant than those of the tropical epiphytic Cymbidium, and various variation types have been cultivated in the breeding, which significantly improves the ornamental and economic values.

However, the Chinese Cymbidium varieties mainly rely on the domes- tication and breeding of wild resources, and there are not many varieties suitable for large-scale industrialization; meanwhile, the Chinese Cymbidium has a long hybrid breeding cycle, high dif- ficulty in hybrid breeding, and unpredictable progeny traits, and modern molecular biotechnology-based breeding methods are serious- ly lagging behind in the Chinese Cymbidium. Therefore, it has be- come a scientific problem to be solved urgently at present to im- prove the ornamental traits, shorten the breeding cycle, and cul- tivate the novel varieties efficiently by means of molecular bio- technology.

SUMMARY

A purpose of the present disclosure is to overcome the long period and unpredictable traits in traditional Chinese Cymbidium cross-breeding, as well as to improve traits of plant petal types by using the latest molecular biology means to explore gene re- sources capable of being applied to improvement of the plant petal types.

To achieve the above purpose, the present disclosure provides a regulatory gene for floral organ development of Chinese Cymbidi- um, where the gene is isolated from a cDNA of a floral organ of a

Chinese orchid variety Cymbidium sinense "Dharma", with a nucleo- tide sequence including 669 bases, as shown in SEQ ID NO: 1.

A second object of the present disclosure is to provide a protein encoded by the regulatory gene for floral organ develop- ment of Chinese Cymbidium, including 222 amino acid residues, as shown in SEQ ID NO: 2.

The present disclosure further provides an expression vector, a transgenic cell line, host bacteria and a transgenic material including the regulatory gene for floral organ development of Chi- nese Cymbidium.

In the present disclosure, it is found by a genetic engineer- ing technology that the regulatory gene for floral organ develop- ment of Chinese Cymbidium is overexpressed in Arabidopsis thaliana and Cymbidium, which can transform a plant flower type, resulting in petal curling, Cymbidium petals transforming into labella, and enhancing labellum traits. Therefore, the gene and the encoded protein thereof can be used in studies of regulatory pathways for the development of the floral organ of the Cymbidium, and improve- ment of a petal type for plant flowering-related traits.

The present disclosure has the following beneficial effects over the prior art:

In the present disclosure, the regulatory gene of floral or- gan development is isolated from the cDNA of the floral organ of the Chinese orchid variety Cymbidium sinense "Dharma"; by increas- ing expression of this gene, four-round floral organs of Arabidop- sis thaliana can be transformed from radial symmetry to bilateral symmetry, and petals curl upwards like a labellum structure; after the high expression of the regulatory gene for floral organ devel- opment of Chinese Cymbidium, the petals are transformed to the la- bella, and the number of labella is increased. In the present dis- closure, the provided regulatory gene of floral organ development of the Cymbidium and an encoded regulatory protein thereof can be used for the study of a molecular mechanism of floral organ devel- opment of the Cymbidium, and improvement of a petal type for plant flowering-related traits.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a phylogenetic tree analysis and multiple- sequence comparison of CsAP3 and AP3 derived from other species in

Example 1 of the present disclosure;

FIG. 2 shows a CsAP3 expression analysis of transgenic Ara- bidopsis thaliana plants in Example 2 of the present disclosure, where OE1l to OE6 represent transgenic Arabidopsis thaliana plants, respectively;

FIG. 3 shows a phenotype analysis of transgenic Arabidopsis thaliana in Example 2 of the present disclosure: petals are curl- ing, where WT represents wild type, CsAP3-0E1, CsAP3-0E2 and

CsAP3-0E3 represent transgenic plants; and

FIG. 4 shows transient high expression and a phenotype analy- sis of a CsAP3 gene in Chinese Cymbidium in Example 3 of the pre- sent disclosure, where CK represents an Agrobacterium solution- injected plant containing an empty vector, and CsAP3-0E represents an Agrobacterium solution-injected plant containing a plasmid

PCAM1301-CsAP3.

DETAILED DESCRIPTION OF THE EMBODIMENTS

To make the objectives, technical solutions and beneficial technical effects of the present disclosure clearer, the present disclosure is described in more detail below with reference to the accompanying drawings and specific examples. It should be under- stood that the specific examples described herein are merely to explain the present disclosure, rather than to limit the present disclosure. The parameters, ratios of the examples can be selected according to local conditions and have no substantial impact on the results.

Example 1 Cloning and sequence analysis of CsAP3 gene 1. RNA extraction 2 g of a floral organ tissue of a cultivar Cymbidium sinense "Dharma" was taken, a total RNA was extracted with a plant Trizol reagent (Invitrogen), followed by reverse transcription into a cDNA (Thermo Scientific Revert Aid First Strand cDNA Synthesis

Kit). 2. Acquisition of a target gene CsAP3

PCR was conducted using CsAP3-Fl: ATGGGGAGAGGGAAGATAGAGAT (SEQ ID NO: 3) and CsAP3-R1l: TCAAGCGAGACGCAGATCAT (SEQ ID NO: 4) as primers and the cDNA obtained in step 1 as a template by an Ex-

Tag enzyme (TaKaRa Biotechnology Co.) according to the following conditions: at 94°C for 4 min, followed by 34cycles of (94°C for 40 sec, 59.5°C for 40 sec, 72°C for 1.5 min, and 72°C for 10 min).

A PCR product was recovered from agarose gel, and ligated to a vector pMD19-T (TaKaRa Biotechnology Co.), followed by sequencing in BGI. An analysis of sequencing results shows that an amplified fragment includes a complete CDs sequence of the target gene

CsAP3, which includes 669 bases and has a nucleotide sequence as shown in SEQ ID NO: 1, named as a Cymbidium sinense floral organ development regulatory gene (CsAP3 gene); an encoded protein by the gene has an amino acid sequence including 222 amino acid resi- dues, as shown in SEQ ID NO: 2, named as a Cymbidium sinense flo- 5 ral organ development regulatory protein (CsAP3 protein). The ob- tained FE. coli containing CsAP3-pMD19-T was currently stored in the Environmental horticulture Research Institute of Guangdong

Academy of Agricultural Sciences. 3. Sequence analysis of the CsAP3 gene

AP3-like genes were selected in Orchidaceae plants Oncidium,

Phalaenopsis equestris (Schauer) Rchb. f., Dendrobium crumenatum and other species for homology comparison with AP3 obtained from the Cymbidium sinense; GenBank accession numbers corresponding to amino acid sequences of each protein are as follows:

OMADS5(HM140840), OMADS3(AY196350), OMADS9(HM140841), PeM-

ADS2 (AY378149), PeMADS3(AY378150), PeMADS4(AY378147), PeM-

ADS5{AY378151), DcOAP3A{DQ119838) and DcOAP3B (DQ119839); the ho- mology comparison results show that the homology between the CsAP3 and OMADS3, OMADS5, OMADS9, PeMADS2, PeMADS3, PeMADS4, PeMADSS,

DcOAP3A and DcOAP3B were 51%, 64%, 95%, 62%, 93%, 77%, 56 %, 62% and 92%, respectively; it is mainly conserved at a 5'-end, while the conservation at a 3'-end is relatively low, and all genes have a conserved MADS domain and a conserved K domain of the MADS-box gene, as well as a typical Paleo-AP3 motif of the AP3 genes.

MEGA software was used to align the amino acids encoded by the CsAP3 gene of Cymbidium sinense with the class B MADS-box genes in Arabidopsis thaliana, rice, maize, Oncidium, Phalaenop- sis, Phalaenopsis equestris (Schauer) Rchb. f. and Dendrobium crumenatum to construct a phylogenetic tree (FIG. 1). The results show that monocotyledons and dicotyledons are clustered into sub- clades, respectively; amino acid sequences encoded by AP3 genes of different species are relatively different, and are scattered in 4 small subclades, cladel/2/3/4; CsAP3 belongs to the clade3 sub- clade.

Example 2 Functional analysis of the CsAP3 gene in Arabidop- sis thaliana 1. Construction of high expression vector for transformation of Arabidopsis thaliana

A CDs sequence of the Cymbidium sinenseCsAP3 gene (namely the

CsAP3 gene, with a nucleotide sequence as shown in SEQ ID NO: 1) was amplified using primers CsAP3-Fl: ATGGGGAGAGGGAAGATAGAGAT and

CsAP3-R1: TCAAGCGAGACGCAGATCAT, cloned into a pMDl19-Tvector (Takara), followed by sequencing in to BGI. After sequencing cor- rectly, double enzyme digestion was conducted using EcoRI and SacI {Thermo Fisher Scientific), and a target fragment was recovered and purified; the target fragment was ligated into a pBIl21 vector that was digested with the EcoRI and the SacI and purified, and named as pBI-AP3. 2. Transformation of the Arabidopsis thaliana 2.1. Transformation of Agrobacterium GV3101 1) Competent cells stored at -80°C were completely thawed; about 1 ug of a plasmid (pBI-AP3) was added to mix gently, fol- lowed by standing on ice for 30 min. 2) Quick-freezing was conducted in liquid nitrogen for 1 min, followed by standing at 37°C for 1 min, the above step was repeat- ed once, 1 mL of an LB was added, followed by shaking incubation at a constant temperature of 28°C and 200 rpm for 4-6 h. 3) A bacterial solution was centrifuged at 4,000 rpm for 5 min at room temperature; a supernatant was discarded, followed by resuspending pellets with 100 uL of an LB medium. 4) All the mixtures were spread onto a LB plate containing 50 pg/mL kanamycin and 25 pg/mL gentamicin, followed by incubation in a constant-temperature incubator at 28°C for 48-72 h; after colo- nies were grown, bacteria were selected and re-streaked to ensure that it was a single clone, thereby obtaining Agrobacterium trans- formed with pBI-CsAP3. 2.2. Transformation of Arabidopsis thaliana by inflorescence infection

Arabidopsis thaliana inflorescence was soaked in an Agrobac- terium solution transformed with pBI-CsAP3 at OD=0.3-0.5 for 2-3 sec, taken out, sealed in the dark for 16 h, followed by continu- ing culture. After about 3-4 weeks, seeds were collected and stored at 4°C in the dark. 2.3. Screening of Arabidopsis thaliana transformants

1) A 0.1% mercuric chloride solution was prepared. 2) The collected seeds were put into a 1.5 mL Eppendorf tube, 1 mL of 75% alcohol was added to shake well, followed by rotating on a Blood Tube Rotator for 5 min and sterilizing with 0.1% mercu- ric chloride for 5 min. 3) Centrifugation was conducted at 6,000 rpm for 2 min, a su- pernatant was discarded, 1 mL of sterilized water was added to mix well, followed by rotating on the Blood Tube Rotator for 2 min; the above process were repeated 3 times. 4) A supernatant was removed, the seeds were suspended with 1 mL of the sterile water, spread on a surface of a 1/2 MS plate me- dium containing 50 ug/ml kanamycin, followed by sealing the plate; vernalization was conducted in the dark at 4°C for 3 d, and the seeds were transferred to a tissue culture room, followed by seed- ling raising under the conditions of illumination of 16 h light/8 h dark at 23°C. 5) After the seedlings grew green cotyledons, the number of seedlings with green cotyledons were observed and counted, fol- lowed by cultivation in a substrate. 6) After a life cycle of about 60 d, first-generation seeds

Tl of transgenic Arabidopsis thaliana were obtained, and second- generation homozygous seeds T2 (transgenic plants) were harvested for subsequent experiments. 3. Analysis of CsAP3 expression level in transgenic Arabidop- sis thaliana

To determine the biological function of CsAP3 gene in Ara- bidopsis thaliana, cDNAs of a wild-type Arabidopsis thaliana and a transformant (T2) obtained in step 2 were used as templates, and primers CsAP3QRT-F: AAGGCAAGCGAGCTGACTGT and CsAP3QRT-R:

CCATCCTCTGCCTGATCTCC were used to detect the expression level of

CsAP3 gene in transgenic Arabidopsis thaliana, where PCR was con- ducted according to the following procedure: pre-denaturation at 95°C for 30 sec, followed by 40 cycles (95°C for 10 sec, 59.5°C for 10 sec, 72°C for 30 sec), and extension at 72°C for 10 min.

The amplification was conducted using an iCycler IQ Real-time PCR

Detection System (Bio-Rad, USA) according to instructions of a

Hiscript EORT SuperMix for gPCR (+gDNA wiper) kit (Vazyme Biotech

Co., Ltd). The results show that the expression level of CsAP3 is increased in obtained transgenic plants, and three lines of the plants were selected for subsequent analysis (FIG. 3). 4. Phenotypic analysis of transgenic Arabidopsis thaliana

Harvested T3-generation homozygous transgenic plant seeds were surface-sterilized and plated on an MS medium containing 50 ng/ml kanamycin, cultured at 4°C in the dark for 2 d, followed by culture with illumination of 16 h light/8 h dark at 23°C. During the flowering period of Arabidopsis thaliana, it is found that the floral organs of the transgenic plants have curled petals, and a stigma of the wild type grows straight upward; however, the inter- nal stigma of the transgenic plants grows curvedly, and it is found that the development of the two ventricles of the fruit pods is inconsistent (FIG. 3).

Example 3 Functional analysis of CsAP3 gene in Cymbidium 1. Construction of high expression vector

A CDs sequence of the Cymbidium sinenseCsAP3 gene was ampli- fied using primers CsAP3-Fl: ATGGGGAGAGGGAAGATAGAGAT and CsAP3-R1:

TCAAGCGAGACGCAGATCAT, cloned into a pMD19-Tvector (Takara), fol- lowed sequencing correctly. Double enzyme digestion was conducted using EcoRI and SacI and a target fragment was recovered and puri- fied; the target fragment was ligated into a pCAM1301 vector that was digested with the EcoRI and the SacI and purified, and ob- tained pCAM1301-CsAP3. 2. Transformation of Cymbidium leaves 2.1. Transformation of Agrobacterium GV3101 1) Competent cells stored at -80°C were completely thawed on ice, about 1 pg of a plasmid (pCAM1301-CsAP3) added to mix gently, followed by standing on ice for 30 min. 2) Quick-freezing was conducted in liquid nitrogen for 1 min, followed by standing at 37°C for 5 min, the above step was repeat- ed once, 1 mL of an LB was added, followed by shaking incubation at a constant temperature of 28°C and 200 rpm for 4-6 h. 3) A bacterial solution was centrifuged at 4,000 rpm for 5 min at room temperature; a supernatant was discarded, followed by resuspending Agrobacterium pellets with 100 pL of an LB medium. 4) All the mixtures were spread onto a LB plate containing 50 pg/mL kanamycin and 32 pg/mL gentamicin, followed by incubation in a constant-temperature incubator at 28°C for 48-72 h; after colo- nies were grown, bacteria were selected and re-streaked to ensure that it was a single clone, thereby obtaining Agrobacterium with a plasmid pCAM1301-CsAP3. 2.2. Chinese Cymbidium buds transformed by direct injection of Agrobacterium

The young leaf buds and flower buds of Chinese Cymbidium va- riety "Yin Zhen" were directly injected with an Agrobacterium so- lution containing the plasmid pCAM1301-CsAP3 at OD=0.3-0.5, and an empty vector pCAM1301 was used as a control. The injection was conducted once a week for three consecutive weeks, followed by continuing growing in a culture room. After about 8 to 12 weeks, the inoculated floral organs were collected for gene expression detection. 3. Analysis of CsAP3 gene expression level in Cymbidium after inoculation with Agrobacterium

To determine the biological function of CsAP3 gene in Chinese

Cymbidium, cDNAs of an uninoculated wild type of the Chinese Cym- bidium variety "Yin Zhen", the empty vector and the inoculated leaves were used as templates, and primers CsAP3QRT-F: AAGGCAA-

GCGAGCTG ACTGT and CsAP3QRT-R: CCATCCTCTGCCTGATCTCC were used to detect the expression level of CsAP3 gene in Cymbidium after in- jection of Agrobacterium, where PCR was conducted according to the following procedure: pre-denaturation at 95°C for 30 sec, followed by 40 cycles (95°C for 10 sec, 57°C for 10 sec, 72°C for 26 sec).

The same cDNAs were used as templates, primers Ubiquitin QRT-F:

CAAAGAAGGCATTCCAC CAGAT and Ubiquitin QRT-R: CCGAGTCCCCAACTTT-

GTAGAA were used for amplifying Ubiquitin as an internal refer- ence, where PCR was conducted according to the same procedure as

Part 3 in Example 2. The results show that the expression level of

CsAP3 gene is increased in plants injected with the Agrobacterium solution containing the plasmid pCAM1301-CsAP3. 4. Phenotypic analysis of Cymbidium with high expression lev- el of CsAP3

The flower buds of the plants inoculated with the Agrobacte- rium solution containing the plasmid pCAM1301-CsAP3 grew for 3 weeks, and the flowers bloomed; compared with the uninoculated wild-type or empty vector, the plants injected with the Agrobacte- rium solution containing the plasmid pCAM1301-CsAP3 transforms the petals to labella, and have enhanced labellum traits to produce more labellum structures (FIG. 4).

The above described are merely preferred implementations of the present disclosure. It should be pointed out that the pre- ferred implementations should not be construed as a limitation to the present disclosure, and the protection scope of the present disclosure should be subject to the claims of the present disclo- sure. Those of ordinary skill in the art may make several improve- ments and modifications without departing from the spirit and scope of the present disclosure, but the improvements and modifi- cations should fall within the protection scope of the present disclosure,

SEQUENCE LISTING

<110> Environmental horticulture Research Institute of Guangdong

Academy of Agricultural Sciences <120> REGULATORY GENE FOR FLORAL ORGAN DEVELOPMENT OF CHINESE

CYMBIDIUM, AND ENCODED PROTEIN AND USE THEREOF <130> HKJP202111905 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 669 <212> DNA <213> Cymbidium sinense Dharma <400> 1 atggggagag ggaagataga gataaagaag atagagaacc ctactaacag gcaagtcact 60 tactctaaga ggagagctgg gatcatgaag aaggcaagcg agctgactgt tctctgcgac 120 gctcagctct ctcttgtaac gttctcgagc actggcaagt tctctgagta ttgtagtcca 180 accactgaca ccaagagcat atatgatcgt taccagcagg tgtccggcat aaatctatgg 240 agctcgcagt acgagaagat gcagaatacg ttgaatcatt taaaggagat aaaccaaacc 300 ctgagaaggg agatcaggca gaggatgggg gaggaccttg atgggctgga aatcaaggaa 360 ctgcgtggtc ttgagcaaaa tatggatgag tccctgaagc ttgtaaggaa tcggaagtat 420 catgtcatca gtacccagac agatacctac aaaaagaagc tgaagaactc tcaagaaacc 480 cacaggaact tactgcggga gctggaagcg gagcacgcag tctattatgt ggatgatgat 540 ccgaacagct atgatggtgc acttgcacta ggaaatgggc cttcctacct gtactcattc 600 cgtagccaac caagccagcc aaaccttcaa ggaatgggat atggccctca tgatctgcgt 660 ctcgcttga 669 <210> 2 <211> 222 <212> PRT <213> Cymbidium sinense Dharma <400> 2

Met Gly Arg Gly Lys Ile Glu Ile Lys Lys Ile Glu Asn Pro Thr Asn 1 5 10 15

Arg Gln Val Thr Tyr Ser Lys Arg Arg Ala Gly Ile Met Lys Lys Ala

Ser Glu Leu Thr Val Leu Cys Asp Ala Gln Leu Ser Leu Val Thr Phe

Ser Ser Thr Gly Lys Phe Ser Glu Tyr Cys Ser Pro Thr Thr Asp Thr 60

Lys Ser Ile Tyr Asp Arg Tyr Gln Gln Val Ser Gly Ile Asn Leu Trp 65 70 75 80

Ser Ser Gln Tyr Glu Lys Met Gln Asn Thr Leu Asn His Leu Lys Glu 85 90 95

Ile Asn Gln Thr Leu Arg Arg Glu Ile Arg Gln Arg Met Gly Glu Asp 100 105 110

Leu Asp Gly Leu Glu Ile Lys Glu Leu Arg Gly Leu Glu Gln Asn Met 115 120 125

Asp Glu Ser Leu Lys Leu Val Arg Asn Arg Lys Tyr His Val Ile Ser 130 135 140

Thr Gln Thr Asp Thr Tyr Lys Lys Lys Leu Lys Asn Ser Gln Glu Thr 145 150 155 160

His Arg Asn Leu Leu Arg Glu Leu Glu Ala Glu His Ala Val Tyr Tyr 165 170 175

Val Asp Asp Asp Pro Asn Ser Tyr Asp Gly Ala Leu Ala Leu Gly Asn 180 185 190

Gly Pro Ser Tyr Leu Tyr Ser Phe Arg Ser Gln Pro Ser Gln Pro Asn 195 200 205

Leu Gln Gly Met Gly Tyr Gly Pro His Asp Leu Arg Leu Ala 210 215 220

<2105 3

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence of forward primer CsAP3-F1

<400> 3 atggggagag ggaagataga gat 23 <2105 4

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence of reverse primer CsAP3-R1

<400> 4 tcaagcgaga cgcagatcat 20 <216> 5

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence of forward primer CsAP3QRT-F

<400> 5 aaggcaagcg agctgactgt 20 <210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence of reverse primer CsAP3QRT-R

<400> 6 ccatcctctg cctgatctcc 20 <210> 7

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence of forward primer UbiquitinActin QRT-F

<400> 7 caaagaaggc attccaccag at 22 <2105 8

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence of reverse primer UbiquitinActin QRT-R

<400> 8 ccgagtcccc aactttgtag aa 22

Claims (7)

CONCLUSIESCONCLUSIONS 1. Expressievector, omvattende een regulerend gen voor de ontwik- keling van bloemorganen van Chinese Cymbidium, met een nucleotide- sequentie zoals getoond in SEQ ID NO: 1; waarbij een eiwit dat wordt gecodeerd door het regulerende gen voor de ontwikkeling van bloemorganen van Chinese Cymbidium een aminozuur- sequentie heeft zoals getoond in SEQ ID NO: 2.An expression vector comprising a regulatory gene for the development of floral organs of Chinese Cymbidium, having a nucleotide sequence as shown in SEQ ID NO: 1; wherein a protein encoded by the floral organ development regulatory gene of Chinese Cymbidium has an amino acid sequence as shown in SEQ ID NO: 2. 2. Transgene cellijn, omvattende het regulerende gen voor de ont- wikkeling van bloemorganen van Chinese Cymbidium volgens conclusie2. Transgenic cell line comprising the regulatory gene for the development of floral organs of Chinese Cymbidium according to claim 1.1. 3. Gastheerbacterie, omvattende het regulerende gen voor de ont- wikkeling van bloemorganen van Chinese Cymbidium volgens conclusie3. Host bacterium comprising the regulatory gene for the development of floral organs of Chinese Cymbidium according to claim 1.1. 4. Transgeen materiaal, omvattende het regulerende gen voor de ontwikkeling van bloemorganen van Chinese Cymbidium volgens con- clusie 1.4. Transgenic material comprising the regulatory gene for the development of floral organs of Chinese Cymbidium according to claim 1. 5. Gebruik van het regulerende gen voor de ontwikkeling van bloem- organen van Chinese Cymbidium volgens conclusie 1 bij de verbete- ring van een bloembladtype voor plantbloei gerelateerde eigen- schappen.Use of the flower organ development regulatory gene of Chinese Cymbidium according to claim 1 in the improvement of a petal type for plant flowering related properties. 6. Gebruik volgens conclusie 5, waarbij de verbetering van een bloembladtype het transformeren van een bloemblad in een labellum is om het aantal labellum te vergroten.Use according to claim 5, wherein the improvement of a petal type is to transform a petal into a labellum to increase the number of labellum. 7. Gebruik van het eiwit dat wordt gecodeerd door het regulerende gen voor de ontwikkeling van bloemorganen van Chinese Cymbidium volgens conclusie 1 bij de verbetering van een bloembladtype voor plantbloei gerelateerde eigenschappen.Use of the protein encoded by the floral organ development regulatory gene of Chinese Cymbidium according to claim 1 in the improvement of a petal type for plant flowering related properties.