A PhERF110 gene from petunia and its application in regulating plant architecture

Abstract

This invention discloses a petunia PhERF110 gene and its application in regulating plant architecture, belonging to the field of genetic engineering technology. This invention provides a petunia PhERF110 gene, whose nucleotide sequence is shown in SEQ ID NO.1, with a full length of 1386 bp. This invention constructed overexpression vectors and RNAi vectors for the petunia PhERF110 gene, and conducted plant transformation experiments. The experimental results showed that overexpression of the PhERF110 gene significantly increased the number of branches in transgenic plants, while PhERF110 RNAi significantly reduced the number of branches. That is, the PhERF110 gene provided by this invention positively regulates plant branching development. This invention provides a new gene resource for cultivating transgenic plants with increased branching numbers and is of great significance for regulating plant architecture.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a petunia PhERF110 gene and its application in regulating plant architecture. Background Technology

[0002] Petunia is a herbaceous plant belonging to the genus Petunia in the family Solanaceae. It is mostly cultivated as an annual or biennial flowering plant. Due to its high ornamental value and strong adaptability, it is widely used in landscaping. There are numerous horticultural varieties, which can be classified according to plant characteristics as: tall varieties, dwarf varieties, clump-forming varieties, creeping varieties, and upright varieties. Furthermore, because petunias have a clear genetic background and their phenotype is easy to observe, they are a model plant for plant development research.

[0003] Ethylene is one of the earliest plant regulatory substances established as a plant hormone. It promotes seed germination, leaf expansion, root hair elongation, lateral root growth, induces flower bud differentiation and breaks dormancy, and participates in processes such as fruit ripening and leaf abscission. Ethylene-responsive transcription factors (ERFs) belong to the AP2 / ERF subfamily and play an important role in the ethylene signaling pathway, participating in various plant life activities and responses to various abiotic stresses.

[0004] Currently, there is limited research on the role of the ERF gene in petunia in regulating plant development, especially in the development of plant branches. Summary of the Invention

[0005] The purpose of this invention is to provide a petunia PhERF110 gene and its application in regulating plant architecture, so as to solve the problems existing in the prior art. The petunia PhERF110 gene provided by this invention can efficiently positively regulate the branching development of plants.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a petunia PhERF110 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The present invention provides a recombinant vector containing the aforementioned petunia PhERF110 gene.

[0009] The present invention also provides an engineered bacterium comprising the recombinant vector described above.

[0010] The present invention also provides the application of the aforementioned petunia PhERF110 gene, the aforementioned recombinant vector, or the aforementioned engineered bacteria in regulating plant architecture.

[0011] Preferably, the regulation of plant architecture includes regulating the branching development of the plant.

[0012] Preferably, the expression level of the petunia PhERF110 gene is upregulated in the plant to promote branching development and increase the number of branches.

[0013] Preferably, the plant is Arabidopsis thaliana or petunia.

[0014] The present invention also provides a method for promoting branching development in plants, comprising the step of upregulating the expression level of the PhERF110 gene of petunia in plants to increase the number of branches in the plants.

[0015] The present invention also provides a method for inhibiting branching development in plants, comprising the step of downregulating the expression level of the PhERF110 gene of petunia in the plant to reduce the number of branches of the plant.

[0016] Preferably, the plant is Arabidopsis thaliana or petunia.

[0017] The present invention discloses the following technical effects:

[0018] This invention provides a petunia PhERF110 gene, the nucleotide sequence of which is shown in SEQ ID NO.1, with a full length of 1386 bp. Overexpression vectors and RNAi vectors of the petunia PhERF110 gene were constructed, and plant transformation experiments were conducted. The results showed that overexpression of the PhERF110 gene significantly increased the number of branches in transgenic plants, while PhERF110 RNAi significantly reduced the number of branches. This indicates that the PhERF110 gene provided by this invention positively regulates branching development in plants. This invention provides a new gene resource for cultivating transgenic plants with increased branching numbers and is of great significance for plant architecture regulation. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1PhERF110 overexpression transgenic plants in Arabidopsis thaliana are shown in the following diagrams: A compares the phenotypes of WT and PhERF110 overexpression transgenic plants; B shows the expression level of PhERF110, with OE 1, OE 2, and OE 3 representing different transgenic lines; C shows the branch number statistics of WT and transgenic Arabidopsis plants (n=12); and D shows the plant height statistics of WT and transgenic Arabidopsis plants.

[0021] Figure 2 PhERF110 overexpressing transgenic plants are shown in the following diagrams: A shows a comparison of the phenotypes of control plants and PhERF110 overexpressing transgenic plants, with OE 2, OE 4, and OE 15 representing different transgenic lines; B shows the detection of PhERF110 expression levels; C shows the statistics of the number of branches in control and transgenic plants (n=24); and D shows the statistics of the plant height in control and transgenic plants.

[0022] Figure 3 The diagram shows the phenotypic analysis of PhERF110 RNAi transgenic plants. In the diagram, A is a comparison of the phenotypic characteristics of the control and PhERF110 RNAi transgenic plants, and Line 1, Line 2, and Line 3 represent different transgenic lines. B shows the expression level of PhERF110 RNAi. C shows the number of branches in the control and transgenic plants (n=24). D shows the plant height in the control and transgenic plants. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] The plant materials used in this invention, petunia (Petunia × hybrida cv. Mitchell Diploid) and Arabidopsis thaliana, were both cultivated in the plant growth and culture room of the College of Horticulture, Anhui Agricultural University. The photoperiod for plant cultivation was 16h / 8h (light / dark), the temperature was 25℃±2℃, and the light intensity was 3000Lx.

[0029] Example 1

[0030] This invention analyzed the transcriptome data of petunia 1 hour after decapitation and found that members of the ERF family were upregulated by a large fold. One member was selected for full-length cloning, resulting in a single gene. A phylogenetic tree was constructed using this gene and members of the Arabidopsis ERF family, showing that it clustered with Arabidopsis AtERF110. Therefore, this gene was named PhERF110.

[0031] The full-length PhERF110 of petunia is 1386 bp, and its sequence is shown in SEQ ID NO.1, encoding a protein containing 461 amino acids.

[0032] SEQ ID NO.1:

[0033]

[0034] Example 2: PhERF110 Phenotypic Regulatory Function Analysis

[0035] 1. Experimental Methods

[0036] The experimental methods used in this embodiment are as follows.

[0037] 1.1 Carrier Construction and Transformation Methods

[0038] 1.1.135S::PhERF110 vector construction and transformation

[0039] (1) The target gene was amplified using primers PhERF110-1300-F / PhERF110-1300-R (primer sequences are shown in SEQ ID NO.2-3 in Table 1), and then recovered by gel extraction.

[0040] (2) pSuper1300-GFP was double-digested with HindIII and SalI. The system was as follows: 3 μL plasmid DNA, 1 μL each of the restriction enzymes, 2 μL 10×Qcut Buffer, and 13 μL ddH2O. The incubation period was 37℃ for 40 min, followed by 50℃ for 5 min.

[0041] (3) The target gene fragment and the vector double-digested fragment are then processed according to... The recombination reaction is performed according to the instructions of the Onestep PCRCloning Kit.

[0042] (4) Add 5 μL of recombinant product to 50 μL of LDH5α competent cells, mix well, and place on ice for 30 min; incubate in a 42℃ water bath for 30 s - 1 min, and immediately place on ice for 2 - 3 min; add 500 μL of LB solution, and place in a shaker at 37℃ for 1 h until the bacterial culture becomes turbid; centrifuge at 6000 rpm for 3 min, leave 250 μL of supernatant, mix well again, spread evenly on an LB plate, seal tightly with a sealing strip, and incubate upside down at 37℃ overnight.

[0043] (5) Select positive plaques for culture and sequencing. Add the correctly sequenced recombinant plasmid to 100 μL of GV3101 Agrobacterium competent cells and incubate on ice for 5 min; incubate in liquid nitrogen for 5 min, incubate in water at 28℃ for 5 min, and incubate on ice for 5 min; add 500 μL of LB liquid medium and incubate at 28℃ with shaking for 2-3 h; centrifuge at 6000 rpm for 1 min to collect the bacterial cells, spread them evenly on YEB solid medium containing rifampicin or kanamycin resistance, seal tightly with a sealing strip and incubate at 28℃ for 2 days to identify positive clones for later use.

[0044] Table 1 Primer Information

[0045]

[0046] 1.1.2 Construction and transformation of PhERF110 RNAi vector

[0047] Based on the PhERF110 gene sequence, primers with a target fragment of approximately 150 bp were designed, and Xho I and Kpn I restriction sites were added. The primer sequences are shown in Table 1, SEQ ID NO. 4-5. Amplification was performed using cDNA as a template, and the amplification products were recovered from the gel.

[0048] PhERF110 gel-recovered products and the pJL10 vector plasmid were double-digested with Xho I and Kpn I restriction endonucleases, followed by ligation with T4 ligase. Positive clones were identified and sequenced to obtain recombinant plasmids containing the positive and negative insert fragments. The recombinant plasmids were then double-digested with Xba I and Cla I, and the same method was used to finally obtain RNAi recombinant plasmids containing both positive and negative insert fragments.

[0049] 1.2 Arabidopsis inflorescence staining and identification of positive plants

[0050] 1.2.1 Arabidopsis inflorescence infection

[0051] Weigh 0.948 g of 4.74 g / L MS solution, add 10 g of sucrose and 200 mL of sterile water, and sterilize in a 500 mL glass bottle. Transfer 30 mL of the prepared Agrobacterium to a 50 mL centrifuge tube and centrifuge at 5000 rpm for 5 min. Add 30 mL of sterile water and centrifuge for 5 min. Add 15 mL of the invasion solution to the 50 mL centrifuge tube and gently mix with a pipette tip. Adjust the OD value using a spectrophotometer. 600 =0.8; Immerse the newly emerging Arabidopsis inflorescences in the infection solution for 2 minutes; Place them flat in a culture dish and cover them to keep them moist, and culture them in the dark for 24 hours, then place them under normal conditions; One week later, use the same infection solution to re-infect the Arabidopsis inflorescences, wait for the seeds to mature, collect the T0 generation seeds, dry them, and store them for later use.

[0052] 1.2.2 Identification of transgenic Arabidopsis thaliana

[0053] T0 Arabidopsis seeds obtained by the Arabidopsis inflorescence inoculation method were placed in sterilized 5 mL test tubes and soaked in 75% ethanol for 1.5 min, then rinsed three times with sterile water. 2% NaClO was added for sterilization with shaking for 5 min, followed by rinsing three times with sterile water. The seeds were then resuspended in anhydrous ethanol and pipette-dried onto filter paper. Subsequently, they were evenly sown in MS medium (containing 30 mg / L hygromycin) and treated at 4°C for 2 days before being cultured in a culture room.

[0054] When the Arabidopsis thaliana plants reached four leaves, they were transplanted to a growth chamber. When the plants were 30 days old, leaves were harvested to extract RNA, which was then reverse-engineered into cDNA and detected by PCR to determine successful transfection. Positive transgenic plants were selected for T3 for subsequent phenotypic analysis.

[0055] 1.3 Genetic transformation of petunias

[0056] 1.3.1 Cultivation of aseptic petunia seedlings

[0057] Soak the seeds in 75% alcohol for 1.5 minutes, then rinse three times with sterile water. Add 2% NaClO, shake thoroughly for about 10 minutes, rinse three times with sterile water, and then use 95% alcohol to extract the seeds. Drain the moisture from the seed surface and inoculate them onto 1 / 2 MS solid medium. Incubate at 25°C in the dark. Once the seeds show signs of sprouting, move them to an environment with a photoperiod of 16 hours / day and a light intensity of 2000-3000 Lx for further cultivation.

[0058] 1.3.2 Activation and culture of Agrobacterium

[0059] Activation of PhERF110 RNAi Agrobacterium tumefaciens culture, OD of the culture solution 600 At 0.3, the bacterial suspension was dispensed into 50 mL centrifuge tubes, centrifuged at 5000 rpm and 4 °C for 10 min, the supernatant was discarded, and an appropriate amount of resuspension was taken with a pipette to resuspend the bacterial cells.

[0060] 1.3.3 Screening and Rooting of Petunia Resistant Buds

[0061] Using a sterilized scalpel, cut the leaves of sterile petunia seedlings into 0.5cm x 0.5cm cubes and place them on a symbiotic solid culture medium for pre-culture. After two days of dark incubation, place the leaf discs in the prepared infection solution and gently stir with tweezers to ensure full contact between the leaves and the bacterial solution. After 8 minutes, remove the petunia leaves from the bacterial solution and blot dry with sterilized absorbent paper. Place the cleaned leaves, leaf-side down, on the co-culture medium, arranging them closely on the symbiotic solid culture medium for further culture. Leave a portion of uninfected leaves as a control, loosely arranged with leaf-side up on the callus induction medium, maintaining the same culture conditions. After 2 days of dark culture, rinse the petunia leaves 5-6 times with sterile water. After 2-3 days, transfer the petunia leaves that have undergone dark culture to the selection medium (Kan 50mg / L, Tim 200mg / L), seal and label them, and place them on a light-controlled tissue culture rack at 25℃. Change the medium every 12 days. When the resistant buds grow to about 1.5cm, cut them off and place them in the rooting medium (Kan 50mg / L, Tim 200mg / L). After the petunia tissue culture seedlings have grown stably and large, harden them off and transplant them.

[0062] 2. Experimental Results

[0063] 2.1 PhERF110 overexpression leads to an increase in the number of branches in Arabidopsis thaliana.

[0064] To investigate the function of PhERF110, a 35S::PhERF110 overexpression vector was constructed and transformed into wild-type Arabidopsis thaliana. Transgenic plants were screened, yielding 21 independent transgenic lines. After screening to the T3 generation, three lines were selected for phenotypic analysis. Results are as follows: Figure 1 As shown, compared with the wild type, the number of branches in PhERF110-overexpressing transgenic plants increased from 7.9 to 31, 23.6, and 21, respectively. However, plant height decreased from 27.72 cm to 25.3, 22.3, and 20 cm, respectively. These results indicate that PhERF110 overexpression promotes increased branching in Arabidopsis thaliana and reduces plant height.

[0065] 2.2 Phenotypic analysis of PhERF110 overexpression transgenic plants

[0066] To investigate the function of PhERF110, a 35S::PhERF110 overexpression vector was constructed and transformed into petunias, resulting in 30 lines. Three lines were selected and screened to the T3 generation for phenotypic analysis.

[0067] The results are as follows Figure 2 As shown, the number of basal branches in the transgenic lines increased from 1.08 to 7.30, 5.60 and 5.80, respectively, while the plant height decreased from 37.5 cm to 22.8, 22.7 and 22.9 cm, respectively, indicating that overexpression of PhERF110 caused changes in the number of branches and a decrease in plant height.

[0068] 2.3 PhERF110 RNAi plant phenotypic analysis

[0069] To further investigate the function of PhERF110, the pJL10-PhERF110 vector was constructed and transformed into petunias. Transgenic plants were screened, and a total of 18 independent transgenic lines were obtained. After screening up to the T3 generation, three lines were selected for phenotypic analysis.

[0070] The results are as follows Figure 3 As shown, qRT-PCR analysis revealed that RNAi led to a decrease in PhERF110 expression levels to 0.21, 0.19, and 0.31 times that of the control, respectively. Compared to the control plants, the number of basal branches in the three PhERF110 RNAi transgenic lines decreased from 1.96 to 1.03, 1.02, and 1.05, respectively, while plant height remained largely unchanged. These results indicate that the decrease in PhERF110 expression levels leads to a reduction in the number of branches in petunias, further validating that PhERF110 positively regulates branching development in plants.

[0071] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A PhERF110 gene for petunias, characterized in that, The nucleotide sequence of the petunia PhERF110 gene is shown in SEQ ID NO.

1.

2. A recombinant vector comprising the petunia PhERF110 gene as described in claim 1.

3. An engineered bacterium comprising the recombinant vector of claim 2.

4. The application of the petunia PhERF110 gene as described in claim 1, the recombinant vector as described in claim 2, or the engineered bacteria as described in claim 3 in regulating plant architecture, characterized in that, The regulation of plant architecture refers to the regulation of branching development in the plant. Upregulating the expression level of the PhERF110 gene in the petunia in the plant promotes branching development and increases the number of branches. The plant in question is either Arabidopsis thaliana or Petunia spp.

5. A method for promoting branching development in plants, characterized in that, The steps include upregulating the expression level of the PhERF110 gene of petunia as described in claim 1 in plants to increase the number of branches in the plants; The plant in question is either Arabidopsis thaliana or Petunia spp.

6. A method for inhibiting branching development in plants, characterized in that, The steps include downregulating the expression level of the PhERF110 gene of petunia as described in claim 1 in plants to reduce the number of branches in the plants; The plant in question is a petunia.

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