CN115927375A - Chinese orchid flowering regulatory gene AP1 coding sequence and application thereof - Google Patents

Abstract

The invention discloses a Chinese orchid flowering regulation gene AP1 coding sequence and application thereof. The method separates the orchid floral organ development regulating gene from the floral organ cDNA of the cymbidium of China orchid, improves the expression quantity of the floral organ development regulating gene, and can cause the top dominance loss of arabidopsis thaliana, the lateral branch inflorescence is increased, the arrangement sequence of small flowers on the inflorescence axis is irregular, the petals in the flowers are not normally developed, and little petals or the petals are converted into sepals; the gene expression level is found to be obviously and positively correlated with the sepal increase in the cymbidium, and the expression level is obviously increased in the crotch-type variant, which shows that the gene has the promotion effect on the development of the cymbidium sepals. The cymbidium flowering regulation gene can be used for researching the molecular mechanism of the development of the orchid floral organ and improving the flowering character of plants.

Description

Chinese orchid flowering regulatory gene AP1 coding sequence and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a Chinese orchid flowering control gene AP1, and a coding protein and application thereof.

Background

The orchids have highly specialized flower organs and rich biodiversity and are precious gardening ornamental flowers. The flower organ of the orchid is obviously different from the traditional perianth model of the angiosperm, 6 perianth pieces are arranged into 2 rounds, petals are positioned on an inner wheel, sepals are an outer wheel, 1 petal in the center is characterized as a lipped, and stamens and pistils are fused into 1 stamen column, so that the postovary development mode which can be started after pollination is realized, a peak of the phylogeny of the seed plant in the monocotyledon is represented, and the method is an ideal material for researching the floral development and the phylogeny of the monocotyledon. At present, a batch of MADS related genes are annotated through whole genome sequencing, and a molecular hypothesis of orchid floral formation based on a tetrad model is provided through researches of phalaenopsis, oncidium and the like. However, the petal variation types of the national orchid are far more abundant than tropical aerial orchids such as butterfly orchid, oncidium orchid and the like, in the process of artificial domestication and breeding, a plurality of sepals and petal form variations such as plum petals, lotus petals, narcissus petals, butterfly petals and the like, and a plurality of variation types such as little petals or no petals, multi-petals and tree-shaped flowers and the like are bred, so that the ornamental value and the economic value of the national orchid are obviously improved, but the national orchid varieties are mainly domesticated and bred by virtue of wild resources at present, the national orchid is suitable for large-scale and industrialized varieties, the cross breeding period is long, the difficulty is high, the offspring characters are difficult to expect, and the modern molecular biotechnology means is seriously lagged. However, how to improve ornamental properties and efficiently cultivate new species by modern molecular biotechnology means is a scientific problem to be urgently solved at present.

Disclosure of Invention

The invention aims to: the invention solves the problems of long period, difficult character expectation and the like of the traditional crossbreeding, and adopts the latest molecular biology means to dig the gene resource which can be applied to the flower type improvement of plants, thereby realizing the character improvement of the Chinese orchid flowering.

In order to realize the aim, the invention provides a Cymbidium flowering regulating gene which is separated from a floral organ cDNA of a Cymbidium margaritae variety 'white ink' (Cymbidium sinense) of Cymbidium sinense, and a nucleotide sequence of the gene consists of 744 bases and is shown as SEQ ID NO. 1.

The second purpose of the invention is to provide a protein coded by the cymbidium flowering regulatory gene, which consists of 247 amino acid residues and is shown as SEQ ID NO. 2.

The third purpose of the invention is to provide an expression vector, a transgenic cell line, a host bacterium or transgenic material containing the above cymbidium flowering control gene.

The fourth purpose of the invention is to provide the application of the Chinese orchid flowering regulation gene in plant flowering trait improvement. Preferably, the improvement of the flowering character of the plant is an improvement of increasing the inflorescence of lateral branches, and/or the occurrence of petals in a small number of petals, and/or promoting transformation of the petals to sepals.

The fifth purpose of the invention is to provide the application of the Chinese orchid flowering regulation protein in the improvement of plant flowering traits. Preferably, the improvement of the flowering character of the plant is an improvement of increasing the inflorescence of lateral branches, and/or the occurrence of petals in a small number of petals, and/or promoting transformation of the petals to sepals.

The sixth object of the present invention is to provide a method for increasing the inflorescence of lateral branches of plants, and/or for reducing the number of petals, and/or for promoting the transformation of petals to sepals, which comprises the following steps: constructing a recombinant expression vector for over-expressing the cymbidium flowering control gene, and transforming the recombinant expression vector into plants.

Preferably, said transforming the recombinant expression vector into a plant is transforming agrobacterium with the recombinant expression vector, and infecting the plant with the obtained recombinant agrobacterium.

According to the invention, through the discovery of a gene engineering technology, the cymbidium organ development regulation gene is overexpressed in arabidopsis thaliana, the flowering character of a plant can be changed, apical dominance loss, lateral branches are increased, petals are abnormally developed, and the petals are transformed into sepals, so that the gene and the protein coded by the gene can be used for research on flowering character regulation ways such as the development of floral organs, the number of lateral branches and the like, and the flowering character of the plant is improved.

The beneficial effects of the invention are as follows:

the invention separates the orchid floral organ development regulating gene from the floral organ cDNA of the Cymbidium goeringii (Cymbidium sinense) of Cymbidium goeringii, which can cause the top end advantage loss of arabidopsis, the increase of lateral branch inflorescence, the irregular arrangement sequence of small flowers on the inflorescence shaft, the abnormal development of petals in the flowers, the appearance of few petals or the transformation of the petals to sepals after the expression quantity is improved; the gene expression level is found to be obviously and positively correlated with the sepal increase in the cymbidium, and the expression level is obviously increased in the crotch-type variant, which shows that the gene has the promotion effect on the development of the cymbidium sepals. The cymbidium flowering regulation gene can be used for researching the molecular mechanism of orchid floral organ development and improving the flowering character of plants.

Drawings

FIG. 1 is a graph of the phylogenetic tree analysis of CsAP1 and other species in example 1.

FIG. 2 is the expression pattern analysis of CsAP1 in different tissues of Mecca in example 2 of the present invention.

FIG. 3 is the analysis of the tissue expression pattern of CsAP1 in different developmental stages of cymbidium in example 2 of the present invention.

FIG. 4 is a phenotypic analysis of transgenic Arabidopsis thaliana in example 3 of the present invention.

FIG. 5 is the expression profile of AP1 gene in different rosette variants of the national orchid in example 4 of the present invention. WT _1 to WT _4 represent general patterns; laPV1_ 1-LaPV 1_4 represent the variety of the petal flower, namely 'Huaxi Hepiai'; laPV2_ 1-LaPV 2_4 represent the petal flower variety of 'Samsung butterfly'; MPV _ 1-MPV _4 represent petaloid flower variety 'Jade lion'; NLV _1 to NLV _4 represent the six-petal flower variety 'fragrant'.

FIG. 6 shows the verification of AP1 gene expression in different petal variants in Orlandia in example 4 of the present invention. WT means: common species 'silver needle'; var1 represents: the tree Y flower mutation material 'Cuiyu peony'.

Detailed Description

In order to make the objects, technical solutions and advantageous technical effects of the present invention clearer, the present invention is further described in detail with reference to the following embodiments. It should be understood that the examples described in this specification are for the purpose of illustration only and are not intended to limit the invention, and that the parameters, proportions, etc. of the examples may be selected appropriately without materially affecting the results.

Example 1 cloning and sequence analysis of CsAP1 Gene

1. Extraction of RNA

2g of floral organ tissue of the M.meyeriana cultivar ` Baimo `, was extracted with plant Trizol (Invitrogen) reagent for total RNA and reverse transcribed into cDNA (Thermo Scientific RevertAID First Strand cDNAsSynthesis Kit).

2. Acquisition of target Gene CsAP1

With CsAP1-F1:5 '(GTCAATAGAGAAAGTACAATG) -3' (SEQ ID NO: 3) and CsAP1-R1:5 'TTATCCATTCATTCATTGATGGAGTGAGC-3' (SEQ ID NO: 4) as a primer and cDNA obtained in the above step 1 as a template, PCR was performed using Ex-Taq enzyme (TaKaRa Biotechnology Co.) under the following conditions: 94 ℃ for 4min, then 34cycles (94 ℃ 40s, 59.5 ℃ 40s, 72 ℃ 1.5min, 72 ℃ 10 min). The PCR product was recovered from the agarose gel, and then ligated to pMD19-T vector (TaKaRa Biotechnology Co.) and sent to the Huada institute for sequencing. The analysis of the sequencing result shows that the amplified fragment contains the complete CDs sequence of the target gene CsAP1, the complete CDs sequence consists of 744 bases, the nucleotide sequence is shown as SEQ ID NO:1 and is named as a cymbidium organ development regulatory gene (CsAP 1 gene), the amino acid sequence of the encoded protein consists of 247 amino acid residues, and the amino acid sequence is shown as SEQ ID NO:2 and is named as a cymbidium organ development regulatory protein (CsAP 1 protein). The obtained CsAP1-pMD 19-T-containing Escherichia coli is currently stored in environmental gardening institute of Guangdong province academy of agricultural sciences.

The CsAP1 gene has a nucleotide sequence shown in SEQ ID NO.1, and the specific sequence is shown as follows: <xnotran> ATGGGAAGAGGGAGGGTTCAGTTGAAGCGAATTGAGAATAAAATTAACCGGCAGGTGACTTTCTCTAAGCGAAGATCGGGGTTGCTTAAGAAGGCTCACGAGATATCGGTGCTCTGCGACGCTGAGGTCGCTCTAATCGTGTTTTCTAACAAGGGAAAACTCTATGAGTATTCCACCGAAGCCAGTATGGAGAAGATTCTTGAACGGTATGAGCGCCATTCATACGCTGAAAGAGCATTATTTTCCAATGAGGCTAACTCACAGGCTGATTGGCGCCTTGAATATAATAAATTGAAGGCAAGGGTCGAAAGCTTACAGAAGAGCAAACGGCACCTTATGGGGGAGCAACTTGATTCCTTGAGCACTAAAGAACTTCAACATCTAGAGCAACAGCTTGAAAGTTCCTTGAAACATATACGATCTAGAAAGACCCAGCTCATGCTCGATTCAATTTCCGAGCTACAAAAAAAGGAAAAATTATTGCTGGATCAAAACAAGACCTTAGAGAAAGAGATTATGGCTAAAGAGAAGGCCAAAGCTTTGGTGCAGAATGCACCTTGGGAAAAGCAAAACCAATGCCAGTATAGCTCTGCTCCATCTCATGCCGAAATTTCGAATTTTGGTTCAACTCCTGCCAGCAGAACTCTTCGAGCAAGAGCCAGTGAAGAAGAATCACCCCAGCCACAGTTAAGATTAGGCAACACTTTACTGCCGCCATGGATGCTCACTCATATGAATGGATAA. </xnotran>

The coding protein of the CsAP1 gene has an amino acid sequence shown in SEQ ID NO.2, and the specific sequence is shown as follows: <xnotran> MGRGRVQLKRIENKINRQVTFSKRRSGLLKKAHEISVLCDAEVALIVFSNKGKLYEYSTEASMEKILERYERHSYAERALFSNEANSQADWRLEYNKLKARVESLQKSKRHLMGEQLDSLSTKELQHLEQQLESSLKHIRSRKTQLMLDSISELQKKEKLLLDQNKTLEKEIMAKEKAKALVQNAPWEKQNQCQYSSAPSHAEISNFGSTPASRTLRARASEEESPQPQLRLGNTLLPPWMLTHMNG. </xnotran>

3. CsAP1 Gene sequence analysis

And (3) carrying out homologous sequence search on the CsAP1 gene of the cymbidium in NCBI, carrying out homologous sequence comparison by using software MEGA to obtain coded amino acid, and constructing an evolutionary tree. The result shows that CsAP1 has high conservation in different species, and has MADS structural domain and K structural domain which are conserved in MADS-box gene. Wherein the homology with cymbidium MADS1 (KC 148540) and cymbidium MADS-box (MF 474256) is 99 percent, and the homology with the KX347442 gene of cymbidium/FUL is 98.9 percent. The sequence identity of the protein has 82.7-88.0% with DoMADS2 (AF 198175), butterfly orchid (DQ 104327) CAL (XM _ 020737650) and helmet-shaped lip orchid MADS genes (KF 914211) in dendrobium, and has 80% with CAL genes (XM _024167505, XM_024167506) in Moraceae Chuansang (FIG. 1).

Example 2 expression Pattern of CsAP1 in orchid

1. Extraction of RNA

2g of plant tissues of four-wheel flower organs including sepals, petals, lipflaps and synanthus pillars, inflorescence axes, small flower stalks, outer buds, inner buds and the like of the cymbidium cultivar 'white ink' in the flowering period are taken, total RNA of the plant tissues is extracted by using a plant Trizol (Invitrogen) reagent, and 2 mu L of the total RNA is taken and is subjected to reverse transcription into cDNA by using a reverse transcription Kit Thermo Scientific revertAid First Strand cDNAsynthesis Kit.

2. Quantitative PCR

The primer CsAP1QRT-F is utilized: 5 'GCTGAGGTCGCTCTAATCGT-3' (SEQ ID NO: 5) and CsAP1QRT-R:5 'AGGAATCAAGTTGCTCTCCCCC-3' (SEQ ID NO: 6), and the real-time quantitative PCR detection is carried out on the expression level of the CsAP1 gene in different tissues of orchid. With an Actin QRT-F:5 'ATGTCGCCATCCAAGCTGTT-3' (SEQ ID NO: 7) and Actin QRT-R:5 'CACGTCCCAGCAAGGTCAAGA-3' (SEQ ID NO: 8) as a primer and Actin as an internal reference. The following procedure was followed: pre-denaturation at 95 ℃ for 30s, followed by 40 cycles (95 ℃ 10s, 59.5 ℃ 10s, 72 ℃ 30 s) of extension at 72 ℃ for 10min. Amplification was performed using the iCycler IQreal-time PCR Detection System (Bio-Rad, USA) according to the instructions of the Hiscript II QRT SuperMix for qPCR (+ gDNA wiper) (Vazyme Biotech Co., ltd.).

3. Expression analysis

PCR results were analyzed using an ICYCLER REALALDIME DETECTION SYSTEM software (version 7.0). It was found that the expression level of CsAP1 gene was lower in four floral organs of cymbidium, petal, labial valve and synanthotamus in flowering stage, while the expression level in rachis, petiolus floridum, outer bract and inner bract was significantly increased (FIG. 2). Further, expression quantity detection is carried out on the above tissues in different stages of the development process of the cymbidium sinense from a 9-month flower bud semi-dormant stage, a 12-month flower stalk rapid elongation stage and a 1-month flower blooming stage, and from 9-month 8 days to the next 1-month 14 days, material drawing is carried out for 9 times to detect the change condition of the expression quantity of the CsAP1, and the expression quantity of the CsAP1 is gradually reduced along with the development of flower buds and the development of flowers (figure 3).

Example 3 analysis of function of CsAP1 Gene in Arabidopsis thaliana

1. Construction of high expression vector for transforming Arabidopsis thaliana

With primers CsAP1-F1:5 'GTCAATAGAGAAAGTACAATG-3' (SEQ ID NO: 3) and CsAP1-R1:5 'TTATCCATTCATTCATATGAGTGAGC-3' (SEQ ID NO: 4) the CDs sequence of the cymbidium CsAP1 gene (i.e., csAP1 gene, nucleotide sequence shown in SEQ ID NO: 1) was amplified and cloned into pMD19-Tvector (Takara) for sequencing of the SmaDagene. After sequencing, the purified target fragment was recovered by double digestion with EcoRI and SacI (Thermo Fisher Scientific) and ligated to the pBI121 vector purified by the same two enzymes, designated pBI-AP1.

2. Transformation of Arabidopsis plants

2.1 transformation of Agrobacterium GV3101

1) Thawing competent cells stored at-80 deg.C on ice, adding about 1 μ g plasmid (pBI-AP 1) after the cells are completely dissolved, gently mixing, and standing on ice for 30min.

2) Quickly freezing with liquid nitrogen for 1min, thawing at 37 deg.C for 5min, and repeating once. Adding 1mL of LB at 28 ℃ for constant temperature shaking culture for 4-6 h,200rpm.

3) After shaking and concentrating the bacterial liquid, centrifuging at 4000rpm for 5min at room temperature. The supernatant was discarded and the Agrobacterium pellet was resuspended in 100. Mu.L of LB medium.

4) All the mixtures were plated on LB plates containing 50. Mu.g/mL kanamycin and 25. Mu.g/mL gentamicin, incubated at 28 ℃ for 48 to 72 hours in an incubator, and after colonies were grown out, the colonies were picked and restreaked to ensure monoclonality, thereby obtaining Agrobacterium transformed with pBI-AP1.

2.2 transformation of Arabidopsis thaliana by inflorescence infection.

Soaking an arabidopsis inflorescence for 2-3 s by using agrobacterium liquid transformed with pBI-AP1 and having OD = 0.3-0.5, taking out, sealing for 16 hours in a dark place, and then moving to a growth chamber for continuous growth. After about 3-4 weeks, the seeds were collected and stored at 4 ℃ in the dark.

2.3 Arabidopsis transformants selection

1) Preparing a mercuric chloride solution with the mass fraction of 0.1%.

2) The collected seeds were placed in a 1.5mL Eppendorf Tube, shaken well with 1mL of 75% by volume alcohol, and rotated on a Blood Tube Rotator for 5min. And (3) sterilizing the mixture for 5 minutes by using 0.1 mass percent of mercuric chloride.

3) Centrifuging at 6000rpm,2min. The supernatant was discarded, 1mL of sterilized water was added, mixed well, and spun on a Blood Tube Rotator for 2min. Repeat 3 times.

4) The supernatant was removed and the seeds were suspended in 1mL of sterile water and plated on the surface of 1/2MS medium containing 50. Mu.g/mL kanamycin. And (3) sealing plates, performing vernalization in the dark at 4 ℃ for 3 days, transferring the plates into a tissue culture chamber (16 h L/8h D) for illumination, and performing the condition at 23 ℃.

5) And observing and counting the number of green cotyledon seedlings after the seedlings grow out of the green cotyledon. Removing the culture medium adhered to the root, and transferring to the matrix for culture.

6) Obtaining the first generation seeds of the transgenic arabidopsis after a life cycle of about 60 days. T-generation homozygous seeds (transgenic plants) were harvested for subsequent testing.

3. Analysis of expression level of CsAP1 in transgenic Arabidopsis

In order to determine the biological function of the CsAP1 gene in Arabidopsis, taking Arabidopsis wild type and cDNA of a transformant (a transgenic plant after the growth of a second generation homozygous seed) obtained in the step 2 as templates, and adopting a primer CsAP1QRT-F:5 'GCTGAGGTCGCTCTAATCGT-3' (SEQ ID NO: 5) and CsAP1QRT-R:5 'AGGAATCAAGTTGCTCTCCCCC-3' (SEQ ID NO: 6) the expression level of CsAP1 gene in transgenic Arabidopsis thaliana was determined. The following procedure was followed: pre-denaturation at 95 ℃ for 30s, followed by 40 cycles (95 ℃ 10s, 59.5 ℃ 10s, 72 ℃ 30 s) of extension at 72 ℃ for 10min. Amplification was performed using the iCycler IQreal-time PCR Detection System (Bio-Rad, USA) according to the instructions of the Hiscript II QRT SuperMix for qPCR (+ gDNA wiper) (Vazyme Biotech Co., ltd.). The results show that the expression quantity of CsAP1 in the obtained transgenic plants is improved, and three lines are selected for subsequent phenotype analysis.

4. Phenotypic analysis of transgenic Arabidopsis

The harvested T3 generation homozygous transgenic plant seeds are surface sterilized, spread on an MS culture medium containing 50 mug/mL kanamycin, cultured in the dark at 4 ℃ for 2 days, transferred to (16 h L/8h D) light and cultured at 23 ℃. In the flowering period of arabidopsis, the morphological structure of the flower organ of the transgenic plant is found to be abnormal, which shows that the petals are abnormally developed, the petals are transformed into sepals, and each flower has one petal curl. In addition, all transgenic lines showed apical dominance loss, increased lateral shoots, and developed more inflorescences (FIG. 4).

Example 4 CsAP1 Gene expression in cymbidium and Association analysis between different petal types

1. Different petal-shaped materials of Chinese orchid

The method is characterized in that the cymbidium in the national orchid is used as a material, common flower type scents and different petal type variant materials are selected, and the petal type scents comprise a petal flower variety 'Huaxi lotus' (labellum-like character variety 1, laPV1), 'Sanxing butterfly' (labellum-like character variety2, laPV 2), a petaloid flower variety 'Jade lion' (MPV) and a petaloid flower variety 'Wedelia' (null-lip variety, NLV) without a lip and a stamen combining column. 2g of four-wheel flower organs, namely sepals, petals, lipsticks and synanthus, of the flowers of each variety which are just bloomed are extracted by using plant Trizol (Invitrogen) reagent, and the purity, concentration and integrity of an RNA sample are detected. After the sample is detected to be qualified, library construction is carried out, and the main process is as follows:

(1) Enriching eukaryotic mRNA by magnetic beads with Oligo (dT);

(2) Adding Fragmentation Buffer to randomly break mRNA;

(3) Using mRNA as a template, synthesizing a first cDNA chain by using hexabasic random primers (random hexamers), then adding buffer solution, dNTPs, RNase H and DNA polymerase I to synthesize a second cDNA chain, and purifying cDNA by using AMPure XP beads;

(4) Carrying out end repair on the purified double-stranded cDNA, adding A tail and connecting a sequencing joint, and then carrying out fragment size selection by using AMPure XP beads;

(5) And finally, obtaining a cDNA library through PCR enrichment.

After the library is constructed, the concentration and the Insert Size (Insert Size) of the library are detected by using the Qubit2.0 and Agilent 2100 respectively, and the effective concentration of the library is accurately quantified by using a Q-PCR method so as to ensure the quality of the library.

2. Transcriptome sequencing

llumina Hiseq ^TM The resulting raw image data file was converted to the original sequencing sequence (Sequenced Reads) by CASAVA Base recognition (Base Calling) analysis. After analyzing the base composition and quality value of the data, filtering the data according to the analysis result of original data (Raw data), and removing a joint sequence and a pollution part; removing excess "N" base(s) (ii)>10%) and sequences containing too many low-quality bases (bases with a quality value below 20 account for more than 20% of the entire sequence).

3. Analysis of Gene expression

And (3) comparing the sequenced sequence after quality control with a reference genome by using HISAT2, grouping different sequenced samples according to RSeQC statistical comparison results, and comparing the gene expression levels of different groups according to expression quantity distribution maps and box type maps of all genes. For replicate samples under the same set, the final expression was the average of all replicates. And comparing the Reads obtained by sequencing with a Unigene library by adopting Bowtie, and carrying out expression level estimation by combining RSEM according to the comparison result. The expression abundance of the corresponding Unigene was expressed using FPKM values.

FPKM (Fragments Per kinase of transcript Per Million mapped Reads) is the number of Reads Per Million Reads from alignment to the length of each Kilobase of a gene, and is a common method of estimating gene expression levels in transcriptome sequencing data analysis. FPKM eliminates the effect of gene length and sequencing variation on the calculation of gene expression. The FPKM calculation is as follows:

in the formula, cDNA Fragments represent the number of Fragments aligned to a certain transcript, i.e., the number of Reads at both ends; mapped Fragments (Millons) represent the total number of Fragments aligned to the transcript, in units of 10^ 6; transcript Length (kb): transcript length, in units of 10^3 bases. The results show that CsAP1 is expressed in lower amount in each round of floral organs, and the expression amount in sepals is slightly higher. However, in hexapetalous flowers, which were sepalated, and lacked normal petals, labial petals and synaptera cordifolia, the expression was elevated, especially in the outermost round sepals, indicating that AP1 gene expression may be positively correlated with sepal development (fig. 5).

4. Gene expression verification

In order to further determine the expression mode of the AP1 gene in the national orchid, the general variety 'silver needle' and the tree Y flower mutation material 'emerald peony' in the national orchid are used, the whole flower cDNA is used as a template, and a primer CsAP1QRT-F is adopted: 5 'GCTGAGGTCGCTCTAATCGT-3' (SEQ ID NO: 5) and CsAP1QRT-R:5 'AGGAATCAAGTTGCTCTCCCCC-3' (SEQ ID NO: 6) and the expression level of the AP1 gene in orchid was determined. The following procedure was followed: pre-denaturation at 95 ℃ for 30s, followed by 40 cycles (95 ℃ 10s, 57 ℃ 10s, 72 ℃ 26 s). The same cDNA was also used as template, using Actin QRT-F:5 'ATGTCGCCATCCAAGCTGTT-3' (SEQ ID NO: 7) and Actin QRT-R:5 'CACGTCCAAGGTCAAGA-3' (SEQ ID NO: 8) amplified Actin as an internal reference. The following procedure was followed: pre-denaturation at 95 ℃ for 30s, followed by 40 cycles (95 ℃ 10s, 59.5 ℃ 10s, 72 ℃ 30 s) of extension at 72 ℃ for 10min. Amplification was performed using the iCycler IQreal-time PCR Detection System (Bio-Rad, USA) according to the instructions of the Hiscript II QRT SuperMix for qPCR (+ gDNA wiper) (Vazyme Biotech Co., ltd.). The results show that the expression level of AP1 is remarkably increased in an acra type only developing sepal quilt without petals and lipsticks and synanthus columns (figure 6). The method shows that the cymbidium AP1 can control the growth of sepals and has important regulation and control effect on the structure of flowering branches.

Appropriate variations and modifications of the embodiments described above will occur to those skilled in the art, in light of the above disclosure and teachings. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present invention should fall within the scope of the claims of the present invention. Furthermore, although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims (10)

1. A cymbidium flowering regulation gene is characterized in that the nucleotide sequence of the cymbidium flowering regulation gene is shown as SEQ ID NO. 1.

2. A cymbidium flowering regulatory protein is characterized in that the amino acid sequence of the cymbidium flowering regulatory protein is shown as SEQ ID NO. 2.

3. The cymbidium flowering regulatory protein according to claim 2, wherein the cymbidium flowering regulatory protein is encoded by the cymbidium flowering regulatory gene according to claim 1.

4. An expression vector, transgenic cell line, host bacterium or transgenic material comprising the cymbidium flowering regulatory gene of claim 1.

5. The use of the cymbidium flowering gene of claim 1 for improving flowering characteristics of plants.

6. The use as claimed in claim 5, wherein the improvement in flowering characteristics of the plant is an improvement in flowering of the side branches, and/or in the appearance of fewer petals, and/or in the promotion of transformation of petals into sepals.

7. Use of the cymbidium flowering regulating protein according to claim 2 or 3 for improving flowering characteristics of plants.

8. The use as claimed in claim 7, wherein the improvement in flowering is an improvement in flowering of the plant by increasing the inflorescence of the lateral branches and/or by reducing the number of petals and/or by promoting transformation of the petals into sepals.

9. A method for increasing the inflorescence of lateral branches of plants, and/or the petals of the plants have fewer petals, and/or the petals of the plants are promoted to be transformed into sepals, which is characterized by comprising the following steps: constructing a recombinant expression vector for over-expressing the Chinese orchid flowering control gene of claim 1, and transforming the recombinant expression vector into plants.

10. The method according to claim 9, wherein the recombinant expression vector is transformed into Agrobacterium and the plant is infected with the obtained recombinant Agrobacterium.

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