Application of a xizangia OST48 protein in plant tiller promotion

Abstract

The application belongs to the field of plant genetic engineering, and discloses application of a Juncus setchuenensis OST48 protein in plant tillering promotion. The nucleotide sequence of the Juncus setchuenensis OST48 protein is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2. After overexpression of the gene in Arabidopsis thaliana, the tillering of the transgenic Arabidopsis thaliana is increased, and the plant type of the transgenic Arabidopsis thaliana is affected. Through systematic research, the application proposes the biological function of the Juncus setchuenensis OST48 protein in plant tillering promotion. The application not only provides an important theoretical basis for Juncus setchuenensis tillering regulation research, but also can be applied to plant type breeding of other flower plants as a kind of excellent gene resource.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to a *Fragaria sibirica* OST48 protein and its application in promoting plant tillering. Background Technology

[0002] Tillering, as an important morphological characteristic of garden plants, directly affects their ornamental value. Studies have shown that graceful and uniform tillering morphology is a key factor in enhancing the aesthetic value of ornamental plants. Traditional tillering regulation methods mainly rely on conventional breeding techniques and the application of exogenous nutrients. While these methods have achieved some success, they suffer from limitations such as long cycles and low efficiency. With the rapid development of molecular biology techniques, genetic engineering technology has provided a new avenue for the precise regulation of plant tillering. By regulating the expression of tillering-related genes, targeted improvement of plant tillering traits can be achieved at the molecular level. This not only breaks through the limitations of traditional breeding techniques but also provides important technical support for improving the ornamental value of ornamental plants. Currently, tillering regulation based on genetic engineering has become one of the important research directions for improving the traits of ornamental plants.

[0003] *Iris sanguinea* Donn ex Hornem., a perennial herbaceous plant belonging to the genus *Iris* in the family Iridaceae, is highly valued in landscaping due to its unique flower structure, rich flower color phenotypes, strong environmental adaptability, and wide habitat tolerance. This species is suitable not only for wetland ecosystem restoration, such as creating aquatic landscapes along lakes and rivers, but also as an excellent flowering plant for flower beds and borders, and has potential for cut flower development. Particularly in cold-climate landscaping, *Iris sanguinea* is highly favored for its outstanding cold resistance. In recent years, research on *Iris sanguinea* has focused on several key areas, including germplasm resource conservation and innovative utilization, physiological response mechanisms to stress, construction of hybridization breeding systems, optimization of in vitro rapid propagation technology, and improvement of ornamental quality. Notably, in the fields of ornamental and cut flower applications, improving tillering ability has become a significant market demand. To meet the specific needs of landscaping applications for the tillering trait of *Iris sanguinea*, conducting research on tillering trait improvement based on genetic engineering has important practical significance. Among them, the discovery and functional analysis of tillering-related regulatory genes are an important theoretical basis for constructing an efficient genetic engineering breeding system, and also one of the key research directions at present.

[0004] This invention provides a *Fragaria spp.* OST48 protein and verifies its tillering regulatory function in the model plant *Arabidopsis thaliana*, aiming to provide theoretical reference and important genetic resources for *Fragaria spp.* tillering breeding. Summary of the Invention

[0005] The purpose of this invention is to provide an application of the OST48 protein of *Arabidopsis thaliana* in promoting plant tillering. This invention provides a new approach to promoting tillering in garden plants, and is of great significance for exploring the function of the OST48 protein of *Arabidopsis thaliana*, revealing the mechanism of plant tillering, and providing an important reference for molecular breeding of *Arabidopsis thaliana* plants.

[0006] One of the objectives of this invention is to provide a *Cymbidium goeringii* OST48 protein.

[0007] The second objective of this invention is to provide a gene encoding the OST48 protein of *Cymbidium goeringii*.

[0008] A third objective of this invention is to provide biomaterials related to the OST48 protein of *Dictyophora indica* as described above.

[0009] The fourth objective of this invention is to obtain Arabidopsis thaliana transformants containing the OST48 gene of *Cymbidium goeringii*.

[0010] The fifth objective of this invention is to provide an application of the OST48 protein from *Dictyophora indica* in promoting tillering in plants.

[0011] The objective of this invention will be achieved through the following technical solutions:

[0012] A *Cymbidium goeringii* OST48 protein is provided, characterized by having the nucleotide sequence shown in SEQ ID NO.1 of the sequence listing.

[0013] A gene encoding the OST48 protein of *Cymbidium goeringii* is provided, characterized in that it has the amino acid sequence shown in SEQ ID NO.2 of the sequence listing.

[0014] Provide biomaterials related to the OST48 protein of *Dictyophora indica*, characterized in that they comprise any one of the following (A1) to (A5):

[0015] (A1) A recombinant cloning vector containing the gene encoding the OST48 protein of *Cymbidium goeringii*;

[0016] (A2) A recombinant plant overexpression vector containing a gene encoding the OST48 protein of *Dictyophora indica*;

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

[0018] (A4) Bioengineered bacteria 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 *Fragaria spp.* OST48 protein as described in (A2) and / or (A3) refers to DNA capable of overexpressing the above-mentioned *Fragaria spp.* OST48 protein in host cells, which may contain not only a promoter to initiate transcription of the OST48 gene, but also a terminator to terminate transcription of the OST48 gene.

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

[0022] The bioengineered bacteria is Agrobacterium EHA105 (Video, China).

[0023] The application of the aforementioned *Dictyophora indica* OST48 protein in promoting plant tillering;

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

[0025] (B1) The recombinant plant overexpression vector containing the OST48 gene of *Dictyophora indica* was introduced into the recipient plant;

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

[0027] Plant expression vectors containing the OST48 gene of *Dictyophora indica* were transformed into plant tissues via inflorescence infection, and the transformed plant tissues were then cultured into plants.

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

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

[0030] (1) The present invention cloned the OST48 gene from the flower bud of *Fructus scutellariae* and constructed the GV1300-OST48-GFP plant expression vector. The vector was transformed into *Agrobacterium tumefaciens* EHA105 (Weidi, China) by freeze-thaw method. The GV1300-OST48-GFP plant expression vector was transformed into *Arabidopsis thaliana* by inflorescence infection method. The results showed that overexpression of the OST48 gene in *Arabidopsis thaliana* increased tillering of *Arabidopsis thaliana* plants.

[0031] (2) The tillering of transgenic Arabidopsis plants indicates that the OST48 gene of *Arabidopsis thaliana* has the function of promoting plant tillering. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 The image shown is an electrophoresis diagram of PCR identification of Arabidopsis thaliana with the transgenic OST48 gene in this invention. M is DL2000Maker, 1 is wild-type Arabidopsis thaliana, 2 is GV1300-OST48-GFP recombinant plasmid, and 3-10 are transgenic Arabidopsis thaliana lines.

[0034] Figure 2 The image shows a comparison of tillering phenotypes of IsOST48 transgenic Arabidopsis thaliana, where WT represents wild-type Arabidopsis thaliana, EV represents transgenic Arabidopsis thaliana, and OE represents transgenic Arabidopsis thaliana. Detailed Implementation

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

[0036] The purpose of this invention is to overcome the shortcomings of the prior art and provide an application of the OST48 protein of *Fragaria sibirica* in promoting tillering in plants.

[0037] The preparation methods for the relevant culture media in the examples are 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 plan for this embodiment is as follows:

[0044] Example 1: Cloning of the OST48 gene from *Dictyophora indica*

[0045] I. Extraction of total RNA from plants

[0046] Total RNA was extracted from plants using the OminiPlant RNAKit (Dnase I) (Kangwei Century, China), and the procedure was performed according to the instructions.

[0047] II. cDNA Synthesis

[0048] cDNA synthesis was performed using PrimeScript. TM The RT reagent kit with gDNA Eraser (Perfect RealTime) (Takara, Japan) was used. The operation steps were performed according to the instructions. The cDNA was diluted 10 times and used as a template for gene cloning.

[0049] III. Gene Cloning

[0050] Cloning-specific primers for the *Cymbidium goeringii* OST48 gene were designed using Primer 5. The primer sequences are as follows:

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

[0052] OST48-R1: 5'-CTACTTCCTGACTCTCTTCG-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 forward and reverse primers, 1 μL KODFX and 10 μL ddH2O. Prepare the reaction solution on ice, mix the reaction solution thoroughly and centrifuge at low speed (2500 rpm).

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

[0055] The amplification product was obtained by PCR amplification; the amplification results were detected by 1% agarose gel electrophoresis, and the target band was recovered from the gel; then the recovered product was ligated into the cloning vector pEASY-Blunt Zero Cloning Vector (TransGen, China), transformed into Escherichia coli TOP10 (TIANGEN, China) and plated on LB solid containing 100 mg / L Amp resistance. It was incubated upside down in a 37°C incubator for 10-12 h, and single clones were picked for bacterial PCR identification. Positive clones were expanded in 10 mL of LB liquid medium containing 100 mg / L Amp resistance and then sent to a biotechnology company for sequencing.

[0056] The nucleotide sequence of the *Cirsium japonicum* OST48 gene is shown below:

[0057]

[0058] The ORF region of the *Cymbidium goeringii* OST48 gene contains 1326 bases and encodes 441 amino acids. The amino acid sequence is shown below:

[0059] MASNHPLLQTLAVTLTLISLLPSLSSFSAETPTDRRILVLLDDLAVRSSHSSFFSSLSSRGFDLDFRLADDPKLSLQRYGQHLYDALVLFCPSTTRFGGLLDHQAILDF VDSGHDLILAADSSASDLIKGIATECGVDFDEDSEAVVIDHNGYAVLDTEGDHTLIAADDFIRSDVILGEKPIEAPVLFRGIGGHSLNTANSLILKVLSASPSSYSANPTTK LSSPPSLTGSAISLVSVVQARNNARILISGSLDLFSNRFFKSAVRKVGTSTEHQKTGNEQFVTEISKWVFHERGHLRAVNVSHHKAGESSEPAMYRINDDLEYSVEIYEW SGKTWKPYVADDVQVQFYMMSPYVLKTMSNDQKGLYSTSFKVPDVYGVFQFKVEYRRLGYTSLSLSKQIPVRPFRHNEYERFITAAYPYYGASFSTMAAFFIFSIVYLYHK

[0060] Example 2: Construction of plant overexpression vector for the OST48 gene of *Dictyophora indica*

[0061] (1) Using homologous recombination, vector homologous arm primers with Sal I and BamHI restriction sites were designed. PCR amplification was performed using the plasmid ligated with the *Cymbidium oxysporum* OST48 gene as a template. The PCR reaction system and procedure were the same as those for gene cloning. After PCR, the target fragment was recovered by gel extraction. The sequences of the vector homologous arm primers (underlined parts are Sal I and BamHI 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 BamHI restriction endonucleases, the vector fragment was recovered, and the linearized vector was ligated with the OST48 gene fragment with added homologous arms. This was transformed into competent E. coli DH5α cells and plated on LB agar containing 100 mg / L Amp resistance medium. The cells were incubated upside down at 37°C for 10–12 h. Single clones were picked for colony PCR identification. Positive clones were expanded in 10 mL of LB liquid medium containing 100 mg / L Amp resistance medium and then sent to a biotechnology company for sequencing. Vector construction was performed using the ClonExpress II One Step Cloning Kit (Novizan, China) according to the manufacturer's instructions.

[0065] Example 3: Application of the *Cymbidium goeringii* OST48 gene in tillering regulation

[0066] I. Cultivation of Arabidopsis thaliana

[0067] In a clean bench, appropriate amounts of empty Arabidopsis thaliana vector and wild-type Arabidopsis thaliana seeds (Columbia type Col-0) were placed in 1.5 mL centrifuge tubes. The tubes were sterilized with 75% alcohol for 1 min, rinsed three times with sterile water, and then sterilized with 0.8% sodium hypochlorite solution (Xilong) for 10 min, followed by rinsing five times with sterile water. The sterilized wild-type Arabidopsis thaliana seeds were inoculated onto 1 / 2 MS solid medium and vernalized in the dark at 4°C for 2-3 days, followed by cultivation in a plant culture chamber. After 7-9 days, the germinated Arabidopsis thaliana seedlings were transplanted into pots containing a cultivation substrate (a mixture of peat moss, vermiculite, and perlite in a 5:3:2 ratio, sterilized by high temperature and autoclave) and placed in the plant culture chamber for further cultivation for 3 weeks. Genetic transformation was then prepared when the Arabidopsis thaliana produced flower stalks and numerous flower buds. The environmental conditions in the plant culture chamber were: 16 h light / 8 h dark, 20-22°C.

[0068] II. Preparation of Infected Bacterial Fluid

[0069] Transformation of Agrobacterium competent cells EHA105 (Weidi, China) was performed according to the manufacturer's instructions. The cells were then plated on YEP solid medium containing 50 mg / L Kana and 25 mg / L Rif and incubated upside down at 28°C for 36 h. Single colonies were picked for colony PCR verification. Positive colonies containing the target gene were expanded in 10 mL of YEP (or LB) liquid medium containing Kana and cultured at 28°C with shaking at 180 rpm for 12-16 h. One mL of the cultured culture was then added to 50 mL of fresh YEP (or LB) liquid medium containing the appropriate antibiotic and cultured with shaking until OD (Organic Dioxide) was reached. 600The pH is approximately 0.6-0.8. Centrifuge the cultured bacterial solution at 5000 rpm for 5 min, discard the supernatant in a clean bench and collect the bacterial cells. Resuspend the bacterial cells in an equal volume (50 mL) of infection solution (pH 5.8) containing 5% sucrose and 3% silwet-77, and wait for them to infect Arabidopsis thaliana.

[0070] III. Infection by Arabidopsis thaliana

[0071] Wild-type Arabidopsis thaliana plants to be transformed were selected, and the open flowers were removed as the standard for flower bud emergence. The flower stalks of the Arabidopsis thaliana plants to be transformed were immersed in the infection solution for 1 minute, then covered with an opaque black plastic bag for 24 hours before being uncovered and cultured in a plant culture room. Wild-type Arabidopsis thaliana plants uninfected with Agrobacterium tumefaciens were used as a negative control. DNA was extracted from the transgenic Arabidopsis thaliana plants, and PCR identification was performed using primers with the OST48 gene homologous arm, yielding transgenic positive Arabidopsis thaliana plants.

[0072] IV. Identification of transgenic positive Arabidopsis thaliana plants

[0073] DNA was extracted from eight transgenic Arabidopsis thaliana plants, and PCR identification was performed using homologous arm primers (OST48-F2 and OST48-R2). Wild-type Arabidopsis thaliana served as a negative control, and the GV1300-OST48-GFP recombinant plasmid served as a positive control. The results showed that eight transgenic Arabidopsis thaliana lines contained the target band, indicating that the OST48 gene had been successfully inserted into the genomes of eight Arabidopsis thaliana lines containing the target fragment. Figure 1 ).

[0074] V. Observation of tillering phenotype in transgenic positive Arabidopsis thaliana plants

[0075] A comparative observation of tillering in Arabidopsis thaliana plants with empty vector, wild-type Arabidopsis thaliana, and transgenic Arabidopsis thaliana revealed that ( Figure 2 In contrast, neither wild-type Arabidopsis nor transgenic Arabidopsis exhibited tillering phenotype, while Arabidopsis plants overexpressing the IsOST48 gene significantly induced tillering. The three independent transgenic lines produced 2, 3, and 3 tillers, respectively, all significantly more than the control group. This result indicates that overexpression of the IsOST48 gene in Arabidopsis can effectively promote tillering and growth.

Claims

1. A *Cymbidium goeringii* OST48 protein, the gene sequence of which is shown in SEQ ID NO.

1.

2. The gene encoding the *Dictyophora indica* OST48 protein as described in claim 1, characterized in that, The amino acid sequence is shown in SEQ ID NO.

2.

3. A biomaterial related to the *Dictyophora indica* OST48 protein as described in claim 1, characterized in that, Including any one of the following (A1) to (A2): (A1) A recombinant plant overexpression vector containing the gene encoding the OST48 protein of *Dictyophora indica*; (A2) Bioengineered bacteria containing the recombinant plant overexpression vector described in (A1).

4. The biomaterial related to the *Dictyophora indica* OST48 protein according to claim 3, characterized in that: The plant expression vector described is pCAMBIA1300-GFP.

5. The biomaterial related to the *Dictyophora indica* OST48 protein according to claim 3, characterized in that: The bioengineered bacteria is Agrobacterium EHA105.

6. The application of the biomaterials related to the OST48 protein of *Dictyophora indica* as described in any one of claims 3 to 5 in promoting tillering, characterized in that... A recombinant plant overexpression vector containing the gene encoding the OST48 protein of *Dictyophora indica* was introduced into *Arabidopsis thaliana*.

7. The application according to claim 6, characterized in that, Plant tissues were transformed using Agrobacterium-mediated transformation, and the transformed plant tissues were then cultured into plants.

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