Application of phallus impudicus OST48 protein in plant tillering promotion

Abstract

The invention belongs to the field of plant genetic engineering, and discloses application of phallus impudicus OST48 protein in plant tillering promotion. The nucleotide sequence of the phallus impudicus OST48 protein is as shown in SEQ ID NO.1, and the amino acid sequence of the phallus impudicus OST48 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 OST48 protein in plant tillering promotion is provided, an important theoretical basis can be provided for regulation and control research on phallus impudicus tillering, and the phallus impudicus OST48 protein can serve 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 OST48 protein of Iris sanguinea and application thereof in promoting plant tillering. Background Art

[0002] Tillering is one of the important morphological characteristics of garden plants, and its growth state directly affects the ornamental effect of plants. Studies have shown that beautiful and neat tillering morphology is a key factor in improving the aesthetic value of ornamental plants. Traditional tillering regulation methods mainly rely on conventional breeding techniques and the application of exogenous nutrients. Although these methods have achieved certain results, they have limitations such as long cycles and low efficiency. With the rapid development of molecular biology technology, genetic engineering technology has provided a new way to accurately regulate plant tillering. By regulating the expression of tillering-related genes, the directional improvement of plant tillering traits can be achieved at the molecular level, which not only breaks through the limitations of traditional breeding technology, but also provides important technical support for improving the ornamental value of ornamental plants. At present, tillering regulation based on genetic engineering has become one of the important research directions for the improvement of ornamental plant traits.

[0003] Iris sanguinea Donn ex Hornem. is a perennial herbaceous plant of the genus Iris in the family Iridaceae. It has important application value in landscaping due to its unique flower structure, rich flower color phenotype, strong environmental adaptability and wide habitat tolerance. This species is not only suitable for wetland ecosystem restoration, such as lakeside, riverbank and other water landscape construction, but also can be used as an excellent ornamental plant in flower beds and flower borders, and has the potential for cut flower development. Especially in cold-region garden construction, Iris sanguinea Donn ex Hornem. is highly favored for its outstanding cold-resistant characteristics. In recent years, research on Iris sanguinea has formed several key directions, mainly including germplasm resource conservation and innovative utilization, physiological response mechanism of adversity, construction of hybrid breeding system, optimization of in vitro rapid propagation technology and improvement of ornamental quality. It is worth noting that in the field of ornamental and cut flower applications, improving tillering ability has become an important market demand orientation. In order to meet the specific needs of tillering traits of Iris sanguinea in landscape applications, it is of great practical significance to carry out research on tillering traits improvement based on genetic engineering. Among them, the discovery of tillering-related regulatory genes and their functional analysis are important theoretical foundations for building an efficient genetic engineering breeding system, and are also one of the key research directions at present.

[0004] The present invention provides a OST48 protein of Iris sanguinea and verifies its tillering regulation function in a model plant Arabidopsis thaliana, aiming to provide theoretical reference and important gene resources for the tillering breeding of Iris sanguinea. Summary of the invention

[0005] The purpose of the present invention is to provide an application of the OST48 protein of the spatholobus in promoting plant tillering. The present invention provides a new way for tillering of garden plants, is of great significance for exploring the function of the OST48 protein of the spatholobus and revealing the tillering formation mechanism of plants, and provides an important reference for the molecular breeding of Arabidopsis plants.

[0006] One of the purposes of the present invention is to provide a OST48 protein from Trichosanthes kirilowii.

[0007] The second object of the present invention is to provide a gene encoding the OST48 protein of Iris sanguisorba officinalis.

[0008] The third object of the present invention is to provide biological materials related to the OST48 protein of Iris sanguisorba officinalis.

[0009] The fourth object of the present invention is to obtain Arabidopsis transformed plants containing the OST48 gene of Ilex strychnifolia.

[0010] The fifth object of the present invention is to provide an application of Iris sanguinea OST48 protein in promoting plant tillering.

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

[0012] Provided is a smilax china OST48 protein, characterized in that it has a nucleotide sequence shown in SEQ ID NO.1 in the sequence table.

[0013] Provided is a gene encoding the OST48 protein of Iris sanguinea, characterized in that it has an amino acid sequence shown in SEQ ID NO.2 in the sequence table.

[0014] Provided is a biological material related to the OST48 protein of Iris strychnifolia, characterized in that it comprises any one of the following (A1) to (A5):

[0015] (A1) a recombinant cloning vector containing a gene encoding the OST48 protein of Cnidium monnieri;

[0016] (A2) a recombinant plant overexpression vector containing a gene encoding the OST48 protein of Ilex strychnifolia;

[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 OST48 protein of the radix strychnifolia described in (A2) and / or (A3) refers to a DNA capable of overexpressing the above-mentioned OST48 protein of the radix strychnifolia in host cells, and the DNA may contain not only a promoter for initiating transcription of the OST48 gene, but also a terminator for terminating transcription of the OST48 gene.

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

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

[0023] The use of the Iris sanguinea OST48 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 OST48 gene of Ilex strychnifolia 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 OST48 gene of Ilex sanguinea was transformed into plant tissues through the inflorescence infection method, and the transformed plant tissues were 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 OST48 gene from the flower buds of Ilex strychnifolia, constructed the GV1300-OST48-GFP plant expression vector, transformed Agrobacterium EHA105 (Weidi, China) by the freeze-thaw method, and transformed the GV1300-OST48-GFP plant expression vector into Arabidopsis thaliana by the inflorescence infection method. The results showed that overexpression of the OST48 gene in Arabidopsis thaliana increased the tillering of Arabidopsis thaliana plants.

[0031] (2) The transgenic Arabidopsis plants tillered, indicating that the OST48 gene of the cultivar of Trichosanthes 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 drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 It shows the electrophoresis diagram of PCR identification of Arabidopsis thaliana with the OST48 gene of the present invention, wherein M is DL2000Maker, 1 is wild-type Arabidopsis thaliana, 2 is the GV1300-OST48-GFP recombinant plasmid, and 3-10 are transgenic Arabidopsis thaliana strains.

[0034] Figure 2 The figure shows the comparison of tillering phenotypes of IsOST48 transgenic Arabidopsis, where WT is wild-type Arabidopsis, EV is empty-transfected Arabidopsis, and OE is transgenic Arabidopsis. DETAILED DESCRIPTION

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

[0036] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of OST48 protein of Iris sanguinea in promoting plant tillering.

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

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

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

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

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

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

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

[0044] Example 1 Cloning of the OST48 gene of Iris strychnifolia

[0045] 1. Plant Total RNA Extraction

[0046] The total RNA of plants was extracted using the OminiPlant RNAKit (DNase I) (Kangwei Century, China) kit, and the operation steps were carried out according to the 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 instructions. The cDNA was diluted 10 times and used as a template for gene cloning.

[0049] 3. Gene cloning

[0050] Primer 5 was used to design specific primers for cloning the OST48 gene of C. scinii. The primer sequences are as follows:

[0051] OST48-F1:5'-GCTGTCATCTCGTGCTCTAAAG-3'

[0052] OST48-R1: 5'-CTACTTCCTGACTCTCTTCG-3'

[0053] Prepare 50 μL of PCR reaction system, including 2 μL template cDNA, 25 μL 2×PCR buffer for KODFX, 10 μL 2mM dNTPs, 1 μL each of upstream and downstream primers, 1 μL KOD FX 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: 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] The amplified product was obtained by PCR amplification; the amplification result was detected by 1% agarose gel electrophoresis, and the target band was recovered from the gel; the recovered product was then connected to the cloning vector pEASY-Blunt Zero Cloning Vector (Quanshijin, China), transformed into Escherichia coli TOP10 (TIANGEN, China) and coated on LB solid containing 100 mg / L Amp resistance, and inverted in a 37°C incubator for 10-12 hours, and the single clone strain was picked for bacterial liquid PCR identification, and the positive clone was expanded and cultured in LB liquid culture medium containing 100 mg / L Amp resistance for 10 mL, and then sent to a biological company for sequencing.

[0056] The nucleotide sequence of the OST48 gene of C. strychnifolia was obtained as follows:

[0057]

[0058] The ORF region of the OST48 gene of C. strychnifolia contains 1326 bases, encoding 441 amino acids. The amino acid sequence is as follows:

[0059] MASNHPLLQTLAVTLTLISLLPSLSSFSAETPTDRRILVLLDDLAVRSSHSSFFSSLSSRGFDLDFRLADDPKLSLQRYGQHLYDALVLFCPSTTRFGGLLDHQAILDF VDSGHDLILAADSSASDLIKGIATECGVDFDEDSEAVVIDHNGYAVLDTEGDHTLIAADDFIRSDVILGEKPIEAPVLFRGIGGHSLNTANSLILKVLSASPSSYSANPTTK LSSPPSLTGSAISLVSVVQARNNARILISGSLDLFSNRFFKSAVRKVGTSTEHQKTGNEQFVTEISKWVFHERGHLRAVNVSHHKAGESSEPAMYRINDDLEYSVEIYEW SGKTWKPYVADDVQVQFYMMSPYVLKTMSNDQKGLYSTSFKVPDVYGVFQFKVEYRRLGYTSLSLSKQIPVRPFRHNEYERFITAAYPYYGASFSTMAAFFIFSIVYLYHK

[0060] Example 2 Construction of Plant Overexpression Vector of Iris sanguinea OST48 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 OST48 gene cloning vector 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. The sequences of the vector homology arm primers (the underlined parts are the Sal I and BamH I restriction sites) are as follows:

[0062] OST48-F2:5'- TTGATACATATGCCCGTCGAC ATGGCGAGCAACCAC-3'

[0063] OST48-R2:5'- CCCTTGCTCACCATGGATCC CTTGTGATAAAGATAGAC-3'

[0064] (2) The expression vector GV1300-GFP plasmid was digested with Sal I and BamH I restriction endonucleases, the vector fragment was recovered, the linearized vector was connected to the OST48 gene fragment with the vector homology arm added, the competent E. coli DH5α was transformed and coated on LB solid containing 100 mg / L Amp resistance, and inverted in a 37°C incubator for 10-12 hours, and the single clone strain was picked for bacterial liquid PCR identification, and the positive clone was expanded and cultured in LB liquid medium containing 100 mg / L Amp resistance for 10 mL, and then sent to a biological company for sequencing. The vector construction used the ClonExpress II One Step Cloning Kit (Novozyme, China) homologous recombination kit according to the instructions.

[0065] Example 3 Application of OST48 gene from Trichosanthes strychnifolia in regulating tillering

[0066] 1. Cultivation of Arabidopsis thaliana

[0067] In the clean bench, place an appropriate amount of Arabidopsis thaliana with empty vectors and wild-type Arabidopsis thaliana seeds (Colombia type Col-0) in a 1.5mL centrifuge tube, disinfect with 75% alcohol for 1min, rinse with sterile water 3 times, add 0.8% sodium hypochlorite solution (Xilong) for 10min, and rinse with sterile water 5 times. The disinfected wild-type Arabidopsis thaliana seeds were inoculated on 1 / 2MS solid culture medium, placed in a 4℃ refrigerator in the dark for 2-3d of vernalization, and then placed in a plant culture room for culture. After 7-9d, the germinated Arabidopsis seedlings were transplanted into pots filled with culture medium (peat soil: vermiculite: perlite mixed at 5:3:2, sterilized by high temperature and high pressure), placed in the plant culture room for further culture for 3 weeks, and when Arabidopsis thaliana has flower scapes and produces more flower buds, genetic transformation is ready. The environmental conditions in the plant culture room are: 16h light / 8h dark, 20-22℃.

[0068] 2. Preparation of infection solution

[0069] Transform Agrobacterium competent EHA105 (Weidi, China), refer to the instructions for transformation steps, then spread on YEP solid medium containing 50 mg / L Kana and 25 mg / L Rif, and invert and culture at 28°C for 36 hours; pick a single clone for bacterial liquid PCR verification, expand the positive colony containing the target gene in YEP (or LB) liquid medium containing Kana to 10 mL, the culture conditions are 28°C, 180 rpm shaking culture for 12-16 hours; take 1 ml of the cultured bacterial liquid, add it to 50 mL of fresh YEP (or LB) liquid medium containing the corresponding antibiotics, and continue shaking culture until OD 600About 0.6-0.8; centrifuge the cultured bacterial solution at 5000rpm for 5min, discard the supernatant in an ultra-clean workbench to collect the bacteria, and resuspend the bacteria in an equal volume (50mL) of infection solution (pH 5.8) containing 5% sucrose and 3% silwet-77, and wait for infection of Arabidopsis thaliana.

[0070] 3. Infection of Arabidopsis thaliana

[0071] Take the wild-type Arabidopsis plants to be transformed, and remove the opened flowers with the flower buds turning white as the standard. Soak the Arabidopsis stalks to be transformed in the infection solution for 1 minute, then cover them with an opaque black plastic bag for 24 hours, then uncover them and culture them in a plant culture room. At the same time, set up wild-type Arabidopsis that has not been infected with Agrobacterium as a negative control. Extract the DNA of the transgenic Arabidopsis plants, and use the OST48 gene homology arm primers for PCR identification to obtain transgenic positive Arabidopsis plants.

[0072] 4. Identification of Transgenic Arabidopsis Plants

[0073] DNA was extracted from the eight transgenic Arabidopsis plants and PCR was performed using homology primers (OST48-F2 and OST48-R2). The wild-type Arabidopsis was used as a negative control and the GV1300-OST48-GFP recombinant plasmid was used as a positive control. The results showed that eight transgenic Arabidopsis lines contained the target bands, indicating that the OST48 gene had been successfully inserted into the eight Arabidopsis genomes containing the target fragments ( Figure 1 ).

[0074] 5. Observation of 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 Arabidopsis and empty-transgenic Arabidopsis did not show tillering phenotype, while Arabidopsis plants overexpressing the IsOST48 gene significantly induced tillering. The number of tillers in the three independent transgenic lines was 2, 3, and 3, respectively, which were significantly increased compared with the control group. This result shows that overexpression of the IsOST48 gene in Arabidopsis can effectively promote the formation and growth of tillers.

Claims

1. A OST48 protein from Ilex strychnifolia, the gene sequence of which is shown in SEQ ID NO.

1.

2. The gene encoding the OST48 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 OST48 protein of 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 OST48 protein of Ilex strychnifolia; (A2) A bioengineered bacterium containing the recombinant plant overexpression vector described in (A1).

4. The biomaterial related to the OST48 protein of Cnidium monnieri according to claim 3, characterized in that: The plant expression vector is pCAMBIA1300-GFP.

5. The biomaterial related to the OST48 protein of Cnidium monnieri according to claim 3, characterized in that: The bioengineering bacteria is Agrobacterium EHA105.

6. The use of the biomaterial related to the OST48 protein of Iris sanguinea in promoting plant tillering according to any one of claims 3 to 5, characterized in that: The recombinant plant overexpression vector containing the gene encoding the OST48 protein of Cistanche deserticola 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 cultivated into plants.

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