Application of phallus impudicus NCED3 protein in promoting plant flowering

Abstract

The invention belongs to the field of plant genetic engineering, and discloses application of phallus impudicus NCED3 protein in promoting plant flowering. The nucleotide sequence of the phallus impudicus NCED3 protein is as shown in SEQ ID NO.1, and the amino acid sequence of the phallus impudicus NCED3 protein is as shown in SEQ ID NO.2. After the gene is over-expressed in arabidopsis thaliana, the plant flowering phase of transgenic arabidopsis thaliana can be advanced, and the flowering phase of arabidopsis thaliana is influenced. Through systematic research, the biological function of the phallus impudicus NCED3 protein in promoting plant flowering is provided, an important theoretical basis can be provided for regulation and control research on the flowering phase of phallus impudicus, and the phallus impudicus NCED3 protein can be used as an excellent gene resource to be applied to flowering phase breeding of other flower plants.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and particularly relates to an application of Iris sanguinea NCED3 protein in promoting plant flowering. Background Art

[0002] In garden plants, flowering characteristics are one of the key factors that determine their ornamental value. Lush flowers, neat flowering periods, and bright flower colors directly affect the landscape effect and aesthetic value of plants. Traditionally, plant flowering regulation has relied heavily on cultivation and management measures (such as light and temperature control) or exogenous hormone treatments (such as gibberellins and growth regulators). With the development of molecular biology techniques, genetic engineering has provided a new technical path for flowering regulation, which can break through environmental factors and seasonal restrictions and achieve precise control of flowering time, flowering duration, and flower organ development at the molecular level. Therefore, using genetic engineering technology to improve plant flowering traits has become an important research direction for enhancing the landscape value of ornamental plants.

[0003] Iris sanguinea (Iris sanguinea Donn ex Hornem.), a perennial herbaceous plant in the Iridaceae family, holds a prominent position among ornamental plants for its unique floral structure and rich color variation. This species possesses not only exceptional cold tolerance but also excellent tolerance to a wide range of soil conditions, making it a valuable material for landscaping in northern China. It is widely used in wetland landscapes, flower borders, and cut flower production. In recent years, research on Iris sanguinea has focused on the following areas: germplasm collection and genetic evaluation, physiological mechanisms of stress resistance, establishment of hybrid breeding systems, optimization of in vitro rapid propagation techniques, and improvement of ornamental traits. Notably, regulating flowering time and improving flowering quality have become important breeding goals in both landscape applications and cut flower production. To achieve this goal, functional research on flowering-related genes and molecular breeding are crucial. Identifying key flowering-regulating genes and elucidating their mechanisms of action will provide theoretical and technical support for improving the flowering time of Iris sanguinea, ultimately leading to the development of new varieties with greater ornamental value.

[0004] In recent years, plant physiology research has revealed the core role of the abscisic acid (ABA) signaling pathway in the regulation of growth and development. Among them, NCED3, as the key rate-limiting enzyme in the ABA biosynthesis pathway, affects the endogenous ABA level by precisely regulating the carotenoid cleavage reaction, and plays an important role in regulating the flowering time of plants. Studies have shown that the ABA signaling network mediated by NCED3 can integrate environmental factors and endogenous signals, dynamically regulate the expression of flowering-related genes, and thus optimize the flowering pattern of plants. Based on this mechanism, the present invention provides a NCED3 protein from Cnidium spp., and uses the Arabidopsis model system to confirm that it has a significant flowering-promoting function. This research result not only provides a new perspective for in-depth analysis of the molecular mechanism of Cnidium spp. flowering regulation, but more importantly, it provides valuable genetic resources and theoretical basis for the targeted improvement of the flowering period of ornamental plants, which is of great significance to promoting the development of molecular breeding technology for garden plants. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of the NCED3 protein of Ciliata strychnifolia in promoting plant flowering. The present invention provides a new approach for regulating the flowering period of garden plants, is of great significance for exploring the function of the NCED3 protein of Ciliata strychnifolia and revealing the mechanism of plant flowering promotion, and provides an important reference for the molecular breeding of Arabidopsis thaliana plants.

[0006] One of the objectives of the present invention is to provide a NCED3 protein from Cnidium monnieri.

[0007] A second object of the present invention is to provide a gene encoding the NCED3 protein of Iris fasciata.

[0008] The third object of the present invention is to provide biological materials related to the NCED3 protein of Cnidium monnieri.

[0009] The fourth object of the present invention is to obtain Arabidopsis transformed plants containing the NCED3 gene of Iris serrata.

[0010] A fifth object of the present invention is to provide a use of the NCED3 protein from Iris sanguinea in promoting plant flowering.

[0011] The purpose of the present invention is achieved through the following technical solutions:

[0012] Provided is a Ciliata strychnifolia NCED3 protein, characterized in that it has a nucleotide sequence shown as SEQ ID NO.1 in the sequence table.

[0013] Provided is a gene encoding the NCED3 protein of Cnidium monnieri, characterized in that it has the amino acid sequence shown in SEQ ID NO.2 in the sequence table.

[0014] Provided is a biological material related to the NCED3 protein of Cnidium monnieri, characterized by comprising any one of the following (A1) to (A5):

[0015] (A1) A recombinant cloning vector containing the gene encoding the NCED3 protein of Cnidium sibiricum;

[0016] (A2) a recombinant plant overexpression vector containing a gene encoding the NCED3 protein of Cnidium monnieri;

[0017] (A3) a recombinant plant overexpression vector obtained by connecting a tag to the N-terminus and / or C-terminus of the gene described in (A2);

[0018] (A4) a bioengineered bacterium containing the recombinant plant overexpression vector described in (A2) or (A3);

[0019] (A5) A transgenic plant containing the recombinant plant overexpression vector described in (A2) or (A3).

[0020] The plant overexpression vector containing the gene encoding the above-mentioned NCED3 protein of Cnidium monnieri described in (A2) and / or (A3) refers to a DNA capable of overexpressing the above-mentioned NCED3 protein of Cnidium monnieri in host cells. The DNA may not only contain a promoter for initiating transcription of the NCED3 gene, but also a terminator for terminating transcription of the NCED3 gene.

[0021] The plant expression vector is pCAMBIA1300-GFP.

[0022] The bioengineering bacteria is Agrobacterium EHA105 (Weidi, China).

[0023] The use of the NCED3 protein from Iris strychnifolia in promoting plant flowering;

[0024] A method for regulating the flowering period of a plant comprises the following steps:

[0025] (B1) introducing a recombinant plant overexpression vector containing the NCED3 gene of C. serrata into a recipient plant;

[0026] The method for introducing a plant expression vector into a recipient plant is characterized by:

[0027] The plant expression vector containing the NCED3 gene of Iris sanguinea is transformed into plant tissues through the inflorescence infection method, and the transformed plant tissues are cultivated into plants.

[0028] According to the technical solution of the invention, the recipient plant is a monocotyledonous plant or a dicotyledonous plant, preferably Iris sanguinea or Arabidopsis thaliana.

[0029] The present invention has the following beneficial effects:

[0030] (1) The present invention cloned the NCED3 gene from the flower buds of Ilex serrata, constructed the GV1300-NCED3-GFP plant expression vector, transformed Agrobacterium EHA105 (Weidi, China) by the freeze-thaw method, and transformed the GV1300-NCED3-GFP plant expression vector into Arabidopsis thaliana by the inflorescence infection method. The results showed that overexpression of the NCED3 gene in Arabidopsis thaliana advanced the flowering period of Arabidopsis thaliana plants.

[0031] (2) The flowering period of transgenic Arabidopsis plants was advanced, indicating that the NCED3 gene of Cnidium sylvatica has the function of promoting plant flowering. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 Shown is the electrophoresis diagram of PCR identification of Arabidopsis thaliana by the NCED3 gene of the present invention, wherein M is DL2000Maker, 1-2 are wild-type Arabidopsis thaliana, 3 is the GV1300-NCED3-GFP recombinant plasmid, and 4-10 are transgenic Arabidopsis lines.

[0034] Figure 2 The figure shows a comparison of flowering phenotypes of IsNCED3 transgenic Arabidopsis, where WT is wild-type Arabidopsis, EV is empty-transfected Arabidopsis, and OE is transgenic Arabidopsis. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below with reference to the examples and accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. The experimental methods in the following embodiments are all conventional methods, and the materials and reagents used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0036] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of the NCED3 protein from Cnidium monnieri in promoting plant flowering.

[0037] The relevant culture medium preparation method in the embodiment is as follows:

[0038] LB liquid medium: 5 g / L yeast extract + 10 g / L tryptone + 10 g / L sodium chloride;

[0039] LB solid medium: 5 g / L yeast extract + 10 g / L tryptone + 10 g / L sodium chloride + 15 g / L agar;

[0040] YEP liquid medium: 10 g / L yeast extract + 10 g / L tryptone + 5 g / L sodium chloride;

[0041] YEP solid medium: 10 g / L yeast extract + 10 g / L tryptone + 5 g / L sodium chloride + 15 g / L agar;

[0042] 1 / 2MS solid medium: 2.47 g / L 1 / 2MS + 20 g / L sucrose + 7.8 g / L agar.

[0043] The specific test plan of this embodiment is as follows:

[0044] Example 1 Cloning of the NCED3 gene from Cnidium monnieri

[0045] 1. Plant Total RNA Extraction

[0046] Plant total RNA was extracted using the OminiPlant RNAKit (DNase I) (Kangwei Century, China) kit, and the operation steps were carried out according to the manufacturer's instructions.

[0047] 2. cDNA Synthesis

[0048] cDNA synthesis using PrimeScript TM RT reagent Kit with gDNA Eraser (Perfect RealTime) (Takara, Japan) was used according to the manufacturer's instructions. cDNA was diluted 10-fold and used as a template for gene cloning.

[0049] 3. Gene Cloning

[0050] Primer 5 was used to design specific primers for cloning the NCED3 gene of C. scindapsus. The primer sequences are as follows:

[0051] NCED3-F1: 5'-CTCCTCATCCTTTCTCGCATTCCTT-3'

[0052] NCED3-R1: 5'-CGGGGACCTCAGCAGCATATC-3'

[0053] Prepare a 50 μL PCR reaction system, including 2 μL template cDNA, 25 μL 2× PCR buffer for KODFX, 10 μL 2 mM dNTPs, 1 μL each of upstream and downstream primers, 1 μL KODFX and 10 μL ddH2O. Prepare the above reaction solution on ice, mix the reaction solution evenly and then centrifuge at low speed (2500 rpm).

[0054] The PCR reaction program was as follows: 95°C for 5 min; 95°C for 30 s, 59°C for 45 s, 72°C for 90 s, 35 cycles; 72°C for 10 min.

[0055] Amplified products were obtained by PCR amplification; the amplification results were detected by 1% agarose gel electrophoresis, and the target bands were recovered from the gel. The recovered products were then ligated into the cloning vector pEASY-Blunt Zero Cloning Vector (Quanshijin, China), transformed into Escherichia coli TOP10 (TIANGEN, China), and plated on LB solid medium containing 100 mg / L Amp resistance. The cells were incubated upside down in a 37°C incubator for 10-12 hours. Monoclonal strains were selected for bacterial liquid PCR identification, and positive clones were expanded to 10 mL in LB liquid medium containing 100 mg / L Amp resistance before being sent to a biological company for sequencing.

[0056] The nucleotide sequence of the NCED3 gene of C. serrata was obtained as follows:

[0057]

[0058] The ORF region of the NCED3 gene of C. serrata contains 1680 bases and encodes 559 amino acids. The amino acid sequence is shown below:

[0059] MIRSSYSSHIPSSTTTTGVNRQIQEKHQQKKGMTSPILKESKSKPQWNIFQQAAAAALDKVEEAFIT NVLENKHPLPKTADPAVQIAGNFAPVGEQAPHSNLPVSGRIPPFINGVYVRNGANPHFAPVAGHHLFDGDGMVHAVHLRNGQASYACRYTETERLRQERAIGRPMFPKAIGELHGHSGIARLM LFCARGLVGLIDPSHGTGVANAGLIYFHDRLLAMSEDDLPYHVRISPSGDLETVGRYDFGGQLDSAMIAHPKLDPASNELFALSYDVVKKPYLKYFRFSPSGDKSPDVEIPLDQPTMMHDFAIT ENLVVVPDQQVVFKLQEMIRGGSPVVFDKEKTSRFGILPKYATDASEMQWVDVPECFCFHLWNAWEDNESGEVVVIGSCMTPADSIFNESDESLKSVLSEIRLDPRTGKSTRRPILSTADHFN LEAGMVNRNRLGRKTRFAYLAIAEPWPKVSGFAKVDLSTGGVEKFIYGENKYGGEPYFVPREGSTREDDGYVLAFVHNEETSESELQIVNGTDMRLEATVKLPSRVPYGFHGTFIGSKDLETQA

[0060] Example 2 Construction of a Plant Overexpression Vector for the NCED3 Gene of Iris serrata

[0061] (1) Using the homologous recombination method, vector homology arm primers with Sal I and BamH I restriction sites were designed, and PCR amplification was performed using the plasmid connected to the NCED3 gene cloning vector of Xisun as a template. The PCR reaction system and reaction procedure were the same as those for gene cloning. After the PCR was completed, the target fragment was recovered by gel gel. The sequences of the vector homology arm primers (the underlined parts are the Sal I and BamH I restriction sites) are as follows:

[0062] NCED3-F2:5'- TTGATACATATGCCCGTCGAC ATGATTCGCTCTTCTTATTCCTCT-3'

[0063] NCED3-R2:5'- CCCTTGCTCACCATGGATCC TGCCTGCGTCTCCAAATC-3'

[0064] (2) The expression vector GV1300-GFP plasmid was digested with Sal I and BamH I restriction endonucleases, and the vector fragment was recovered. The linearized vector was ligated with the NCED3 gene fragment with the vector homology arms added, and transformed into Escherichia coli competent DH5α and spread on LB solid containing 100 mg / L Amp resistance. The culture was inverted and cultured in a 37°C incubator for 10-12 hours. Single clones were selected for bacterial liquid PCR identification. Positive clones were expanded to 10 mL in LB liquid medium containing 100 mg / L Amp resistance and then sent to a biological company for sequencing. Vector construction was performed using the ClonExpress II One Step Cloning Kit (Novozymes, China) homologous recombination kit according to the manufacturer's instructions.

[0065] Example 3: Application of the NCED3 gene from Ilex serrata in regulating flowering time

[0066] 1. Cultivation of Arabidopsis thaliana

[0067] In a clean bench, appropriate amounts of vector-transfected Arabidopsis thaliana and wild-type Arabidopsis thaliana seeds (Colombia type Col-0) were placed in 1.5 mL centrifuge tubes, disinfected with 75% alcohol for 1 minute, rinsed three times with sterile water, then sterilized with 0.8% sodium hypochlorite solution (Xilong) for 10 minutes, and rinsed five times with sterile water. The disinfected wild-type Arabidopsis seeds were inoculated onto 1 / 2 MS solid culture medium and vernalized in a 4°C refrigerator in the dark for 2-3 days before being cultured in a plant incubator. After 7-9 days, the germinated Arabidopsis seedlings were transplanted into pots filled with a culture medium (a 5:3:2 mixture of peat soil, vermiculite, and perlite, sterilized by high temperature and high pressure). Culture was continued in the plant incubator for another 3 weeks. Transformation was performed when the Arabidopsis thaliana flower stalks formed and a large number of buds were produced. The plant incubator maintained environmental conditions: 16 h light / 8 h dark, 20-22°C.

[0068] 2. Preparation of infection solution

[0069] Transform competent Agrobacterium EHA105 (Weidi, China) according to the instructions. Then, spread the culture onto YEP solid medium containing 50 mg / L Kana and 25 mg / L Rif and incubate at 28°C for 36 h. Pick a single clone for bacterial liquid PCR verification. Expand the positive colony containing the target gene in 10 mL of YEP (or LB) liquid medium containing Kana at 28°C and 180 rpm for 12-16 h. Pipette 1 ml of the cultured bacterial liquid and add it to 50 mL of fresh YEP (or LB) liquid medium containing the corresponding antibiotics. Continue to shake and culture until the OD reaches 0. 600 The cultured bacterial solution was centrifuged at 5000 rpm for 5 min, the supernatant was discarded in a clean bench to collect the bacteria, and the bacteria were resuspended in an equal volume (50 mL) of infection solution (pH 5.8) containing 5% sucrose and 3% silwet-77, and then infect Arabidopsis thaliana.

[0070] 3. Infection of Arabidopsis thaliana

[0071] Take wild-type Arabidopsis plants to be transformed, remove opened flowers, and use the appearance of white flower buds as the standard. Soak the stalks in the infection solution for 1 minute, then cover with an opaque black plastic bag for 24 hours before uncovering and cultivating in a plant culture room. Also, keep wild-type Arabidopsis plants that have not been infected with Agrobacterium as a negative control. DNA from transgenic Arabidopsis plants is extracted and identified by PCR using primers targeting the homology arms of the NCED3 gene to obtain transgenic-positive Arabidopsis plants.

[0072] 4. Identification of Transgenic-Positive Arabidopsis Plants

[0073] DNA was extracted from the seven transgenic Arabidopsis plants obtained and PCR identification was performed using homology arm primers (NCED3-F2 and NCED3-R2). Wild-type Arabidopsis served as a negative control and the GV1300-NCED3-GFP recombinant plasmid served as a positive control. The results showed that seven transgenic Arabidopsis lines contained the target band, indicating that the NCED3 gene had been successfully inserted into the seven Arabidopsis genomes containing the target fragment ( Figure 1 ).

[0074] 5. Observation of Flowering Phenotype of Transgenic Arabidopsis Plants

[0075] By comparing the flowering phenotypes of empty vector Arabidopsis, wild-type Arabidopsis and transgenic Arabidopsis overexpressing IsNCED3 gene ( Figure 2) found that neither the wild-type nor the empty vector control group displayed a flowering phenotype, while three independent IsNCED3 transgenic lines flowered significantly earlier than the control group by 5, 7, and 6 days, respectively. This result clearly demonstrates that heterologous expression of the IsNCED3 gene in this model plant promotes flowering, effectively shortening the vegetative growth period and accelerating the transition to reproductive growth, providing direct experimental evidence for the use of the NCED3 gene to regulate flowering time.

Claims

1. A NCED3 protein from Trichosanthes kirilowii, the gene sequence of which is shown in SEQ ID NO.

1.

2. The gene encoding the NCED3 protein of claim 1, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

3. The biological material related to the NCED3 protein of Cnidium monnieri according to claim 1, characterized in that: Including any one of the following (A1) to (A2): (A1) A recombinant plant overexpression vector containing a gene encoding the NCED3 protein of Cnidium monnieri; (A2) A bioengineered bacterium containing the recombinant plant overexpression vector described in (A1).

4. The biomaterial related to the NCED3 protein of Cnidium monnieri according to claim 3, characterized in that: The plant expression vector is pCAMBIA1300-GFP.

5. The biomaterial related to the NCED3 protein of Cnidium monnieri according to claim 3, characterized in that: The bioengineering bacteria is Agrobacterium EHA105.

6. Use of the biomaterial related to the NCED3 protein of Cnidium monnieri according to any one of claims 3 to 5 in promoting plant flowering, characterized in that: A recombinant plant overexpression vector containing a gene encoding the NCED3 protein of Cnidium monnieri was introduced into Arabidopsis thaliana.

7. The use according to claim 6, characterized in that Plant tissues are transformed by using Agrobacterium-mediated transformation and then grown into plants.

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