Application of phallus impudicus AP2 protein in plant tillering promotion

Abstract

The invention belongs to the field of plant genetic engineering, and discloses application of phallus impudicus AP2 protein in plant tillering promotion. The nucleotide sequence of the phallus impudicus AP2 protein is as shown in SEQ ID NO.1, and the amino acid sequence of the phallus impudicus AP2 protein is as shown in SEQ ID NO.2. After the gene is over-expressed in arabidopsis thaliana, the plant tillering of the transgenic arabidopsis thaliana can be increased, and the plant type of the arabidopsis thaliana is influenced. Through systematic research, the biological function of the phallus impudicus AP2 protein in plant tillering promotion is provided, an important theoretical basis can be provided for phallus impudicus tillering regulation and control research, and the phallus impudicus AP2 protein can be used as an excellent gene resource to be applied to plant type breeding of other flower plants.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and particularly relates to an AP2 protein of Sanguisorba officinalis and an application thereof in promoting plant tillering. Background Art

[0002] In landscape design, tillering characteristics directly influence the overall aesthetic appeal of a plant. A beautiful, symmetrical, and well-balanced tillering morphology is crucial for enhancing the aesthetic value of ornamental plants. Traditionally, the regulation of plant tillering traits has relied primarily on conventional breeding methods or the exogenous application of nutrients and plant growth regulators. With the rapid development of modern biotechnology, genetic engineering has provided a new path for improving plant tillering, potentially overcoming the technical bottlenecks of traditional breeding and enabling precise control of plant tillering characteristics at the molecular level. Therefore, the use of genetic engineering to improve plant tillering traits has become an important research direction and technical approach for enhancing the aesthetic value of ornamental plants.

[0003] Iris sanguinea Donn ex Hornem. is a perennial herbaceous plant of the genus Iris in the family Iridaceae. It boasts unique flower shapes, rich colors, strong cold tolerance, and wide adaptability. It is suitable for landscapes in wetlands and shallow waters such as lakesides, riverbanks, and ponds, as well as in flower beds, borders, and as a cut flower. Its outstanding ornamental value makes it an excellent ornamental plant for cold-region landscaping in my country, with broad application prospects. In recent years, research on Iris sanguinea has focused on germplasm conservation and innovation, stress tolerance, hybrid breeding, tissue culture and rapid propagation, and quality improvement. As an important ornamental and cut flower material, there is significant market demand for improved tillering ability in Iris sanguinea. To meet the demand for optimized tillering traits in Iris sanguinea for landscape applications, genetic engineering techniques are crucial for improving tillering traits. The identification and functional verification of key genes regulating tillering will provide important theoretical foundations and technical support for genetic engineering breeding of Iris sanguinea.

[0004] As an important member of the AP2 / ERF transcription factor family, the AP2 gene affects the activity of the apical meristem by regulating the WUS-CLV3 pathway, thereby promoting the tillering development of the plant. Studies have shown that in rice, SNB / OsIDS1 (AP2 subfamily) can maintain meristem activity to increase the number of tillers, confirming that the AP2 gene has a tillering-promoting function in monocotyledons. Based on this, the present invention verifies the tillering regulatory function of the AP2 protein of the scutellaria baicalensis in the model plant Arabidopsis thaliana, which not only provides a theoretical basis for analyzing the molecular mechanism of scutellaria baicalensis tillering, but also provides important genetic resources for the molecular breeding of scutellaria baicalensis tillering traits. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of the AP2 protein of Iris sanguinea in promoting plant tillering. The present invention provides a new approach for tillering of garden plants, is of great significance for exploring the function of the AP2 protein of Iris sanguinea and revealing the mechanism of tillering formation of plants, and provides an important reference for molecular breeding of Arabidopsis plants.

[0006] One of the objectives of the present invention is to provide an AP2 protein from Sanguisorba officinalis.

[0007] A second object of the present invention is to provide a gene encoding the AP2 protein of Sanguisorba officinalis.

[0008] The third object of the present invention is to provide biological materials related to the AP2 protein of Sanguisorba officinalis.

[0009] The fourth object of the present invention is to obtain Arabidopsis transformed plants containing the AP2 gene of Ichthyophthirius schoenostoma.

[0010] A fifth object of the present invention is to provide an application of the AP2 protein of Iris sanguinea in promoting tillering of plants.

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

[0012] Provided is a Ciliata sanguinea AP2 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 AP2 protein of Sanguisorba officinalis, 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 AP2 protein of Sanguisorba officinalis, characterized by comprising any one of the following (A1) to (A5):

[0015] (A1) a recombinant cloning vector containing a gene encoding AP2 protein from Sanguisorba officinalis;

[0016] (A2) a recombinant plant overexpression vector containing a gene encoding the AP2 protein of Sanguisorba officinalis;

[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 sanguinea AP2 protein described in (A2) and / or (A3) refers to a DNA capable of overexpressing the above-mentioned sanguinea AP2 protein in a host cell, and the DNA may contain not only a promoter for initiating transcription of the AP2 gene, but also a terminator for terminating transcription of the AP2 gene.

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

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

[0023] The application of the Iris sanguinea AP2 protein in promoting plant tillering;

[0024] A method for regulating plant tillering comprises the following steps:

[0025] (B1) introducing a recombinant plant overexpression vector containing the AP2 gene of Iris sanguinea 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 AP2 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 AP2 gene from the flower buds of Ilex serrata, constructed the GV1300-AP2-GFP plant expression vector, transformed Agrobacterium EHA105 (Weidi, China) by the freeze-thaw method, and transformed the GV1300-AP2-GFP plant expression vector into Arabidopsis thaliana by the inflorescence infection method. The results showed that overexpression of the AP2 gene in Arabidopsis thaliana increased the tillering of Arabidopsis thaliana plants.

[0031] (2) The transgenic Arabidopsis plants tillered, indicating that the AP2 gene of Ilex serrata has the function of promoting plant tillering. 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 PCR identification electrophoresis diagram of Arabidopsis thaliana transfected with the AP2 gene of Ciliata strychnifolia according to the present invention, wherein M is DL2000Maker, 1-2 are wild-type Arabidopsis thaliana, 3 is the GV1300-AP2-GFP recombinant plasmid, and 4-10 are transgenic Arabidopsis thaliana lines.

[0034] Figure 2 The figure shows the comparison of tillering phenotypes of IsAP2 transgenic Arabidopsis thaliana, 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 AP2 protein of Sanguisorba officinalis in promoting plant tillering.

[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 experimental scheme of this embodiment is as follows:

[0044] Example 1 Cloning of the AP2 gene from Sanguisorba officinalis

[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 AP2 gene of C. serrata. The primer sequences are as follows:

[0051] AP2-F1: 5'-ATGTTGGATCTCAACGACTCTCCTGAG-3'

[0052] AP2-R1: 5'-TTCTTCTACAGCCAATTCGGTGCTGC-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 AP2 gene of Iris serrata was obtained as follows:

[0057]

[0058] The ORF region of the AP2 gene of C. serrata contains 1311 bases and encodes 436 amino acids. The amino acid sequence is shown below:

[0059] MLDLNDSPEGEETNNKASSSSSPSDIDDSTANILVTRQFFPVVDGTVDEAPSTGNLPLPIAHWMGVQFRQPEKPVVETSQPTKKSRRGPRSRSSQYRGVTFYRRTGRWE SHIWDCGKQVYLGGFDTAHAAARAYDRAAIKFRGVEADINFTLEDYDDDLKQIGNLTKEEFIHVLRRQSTGFPKGSSKYRGVTLHKCGRWEARMGQFLGKKYVYLGLFD TEVEAARAYDKAVIKCNGKDAVTNFDPSIYADELNFTPAHIDNQSLDLSLGSSGSKSRSFERMAEESPVGMDQRVPMVFGQDWRSDSRNTKAKFDDKLKEQGSILNHPF AQSQMPTNELHKYPLHLRIAEVPPMFQITQHHQFNPYGYHQSPSSNDGAKAGGGLSLAMGGERQQRQRQTEWGGGVAIGPATSQLFATATAASSGFQPQLPAKAAPNWL

[0060] Example 2 Construction of Plant Overexpression Vector of Iris sanguinea AP2 Gene

[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 AP2 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] AP2-F2:5'- TTGATACATATGCCCGTCGAC ATGTTGGATCTCAACGACTCTCCT-3'

[0063] AP2-R2:5'- CCCTTGCTCACCATGGATCC CAGCCAATTCGGTGCTGCT-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 AP2 gene fragment with the vector homology arms added, and the vector was transformed into Escherichia coli competent DH5α and spread on LB solid containing 100 mg / L Amp resistance. The cells were incubated upside down 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 AP2 gene from Sanguisorba officinalis in regulating tillering

[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. 600The 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, using 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 incubating 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 AP2 gene to obtain transgenic-positive Arabidopsis plants.

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

[0073] DNA was extracted from the 7 transgenic Arabidopsis plants obtained and PCR identification was performed using homology arm primers (AP2-F2 and AP2-R2). The wild-type Arabidopsis was used as a negative control and the GV1300-AP2-GFP recombinant plasmid was used as a positive control. The results showed that 7 transgenic Arabidopsis lines contained the target band, indicating that the AP2 gene had been successfully inserted into the 7 Arabidopsis genomes containing the target fragment ( Figure 1 ).

[0074] 5. Observation of the Tillering Phenotype of Transgenic Arabidopsis Plants

[0075] By comparing the tillering of empty vector Arabidopsis, wild type Arabidopsis and transgenic Arabidopsis plants, we found that ( Figure 2 ). Wild-type and untransfected Arabidopsis plants showed no tillering phenotype, while Arabidopsis plants overexpressing the IsAP2 gene significantly induced tillering. The number of tillers in three independent transgenic lines was 2, 3, and 2, respectively, all showing a significant increase compared to the control group. This result demonstrates that overexpression of the IsAP2 gene in Arabidopsis can effectively promote tiller formation and growth.

Claims

1. An AP2 protein from Sanguisorba officinalis, the gene sequence of which is shown in SEQ ID NO.

1.

2. The gene encoding the AP2 protein of claim 1, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

3. The biomaterial related to the AP2 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 AP2 protein from Sanguisorba officinalis; (A2) A bioengineered bacterium containing the recombinant plant overexpression vector described in (A1).

4. The biomaterial related to the AP2 protein of Iris sanguinea according to claim 3, characterized in that: The plant expression vector is pCAMBIA1300-GFP.

5. The biomaterial related to the AP2 protein of Iris sanguinea according to claim 3, characterized in that: The bioengineering bacteria is Agrobacterium EHA105.

6. Use of the biomaterial related to the AP2 protein of Iris sanguinea according to any one of claims 3 to 5 in promoting plant tillering, characterized in that: A recombinant plant overexpression vector containing a gene encoding AP2 protein from Sanguisorba officinalis 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.

Images