Petunia hybrida PhERF110 gene and application thereof in regulation and control of plant type

Abstract

The invention discloses a petunia hybrida PhERF110 gene and application of the petunia hybrida PhERF110 gene in regulation and control of plant types, and belongs to the technical field of gene engineering. The invention provides a petunia PhERF110 gene, the nucleotide sequence of the petunia PhERF110 gene is shown as SEQ ID NO.1, and the full length of the petunia PhERF110 gene is 1386bp. An overexpression vector and an RNAi vector of the Petunia hybrida PhERF110 gene are respectively constructed, a plant transformation experiment is carried out, and experiment results show that after the PhERF110 gene is overexpressed, the number of branches of a transgenic plant is remarkably increased, but PhERF110 RNAi causes the number of branches of the transgenic plant to be remarkably reduced, that is, the PhERF110 gene provided by the invention is capable of positively regulating and controlling the branch development of the plant. The invention provides a new gene resource for cultivating transgenic plants with increased branches, and has great significance in plant type regulation and control.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a Petunia hybrida PhERF110 gene and its application in regulating plant architecture. Background Art

[0002] Petunia hybrida is a herbaceous plant of the genus Petunia in the Solanaceae family. It is mostly cultivated as an annual or biennial ornamental flower. Due to its high ornamental value and strong adaptability, it is widely used in landscape gardening, and there are extremely many horticultural varieties. According to plant traits, it can be divided into: tall varieties, dwarf varieties, tufted varieties, creeping varieties, and upright varieties. At the same time, because Petunia hybrida has a clear genetic background and its phenotypes are easy to observe, it is a model plant for plant development research.

[0003] Ethylene is one of the earliest established plant regulatory substances as a plant hormone. It can promote seed germination, leaf expansion, root hair elongation, lateral root growth, induce flower bud differentiation and break dormancy, and participate 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 signal transduction pathway, participating in various plant life activities and various stress responses.

[0004] Currently, there are few studies on the ERF genes in Petunia hybrida during the regulation of plant development, especially there is no report on the research in plant branching development. Summary of the Invention

[0005] The object of the present invention is to provide a Petunia hybrida PhERF110 gene and its application in regulating plant architecture to solve the problems existing in the above-mentioned prior art. The Petunia hybrida PhERF110 gene provided by the present invention can efficiently and positively regulate the branching development of plants.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a Petunia hybrida PhERF110 gene, and the nucleotide sequence of the Petunia hybrida PhERF110 gene is as shown in SEQ ID NO.1.

[0008] The present invention provides a recombinant vector containing the Petunia hybrida PhERF110 gene described above.

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

[0010] The present invention also provides an application of the Petunia hybrida PhERF110 gene, the recombinant vector or the engineered bacterium described above in regulating plant architecture.

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

[0012] Preferably, increasing the expression level of the petunia PhERF110 gene in the plant promotes the branching development of the plant and increases the number of branches of the plant.

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

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

[0015] The present invention also provides a method for inhibiting the branching development of a plant, which includes the step of decreasing the expression level of the petunia PhERF110 gene 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] The present invention provides a petunia PhERF110 gene, whose nucleotide sequence is shown in SEQ ID NO.1 and is 1386bp in full length. The present invention respectively constructs an overexpression vector and an RNAi vector of the petunia PhERF110 gene and conducts plant transformation experiments. The experimental results show that after overexpressing the PhERF110 gene, the number of branches of the transgenic plant increases significantly, while PhERF110 RNAi results in a significant decrease in the number of branches of the transgenic plant, that is, the PhERF110 gene provided by the present invention positively regulates the branching development of the plant. The present invention provides a new gene resource for cultivating transgenic plants with an increased number of branches and is of great significance for the regulation of plant architecture. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0020] Figure 1Phenotypic analysis diagram of transgenic plants overexpressing PhERF110 in Arabidopsis thaliana; among them, A shows the comparison of phenotypes between WT and transgenic plants overexpressing PhERF110; B shows the detection of the expression level of PhERF110, and OE 1, OE 2, and OE 3 represent different transgenic lines; C shows the statistics of the number of branches of Arabidopsis thaliana WT and transgenic plants (n = 12); D shows the statistics of the plant height of Arabidopsis thaliana WT and transgenic plants.

[0021] Figure 2 Phenotypic analysis diagram of transgenic plants overexpressing PhERF110; among them, A shows the comparison of phenotypes between control plants and transgenic plants overexpressing PhERF110, and OE 2, OE 4, and OE 15 represent different transgenic lines; B shows the detection of the expression level of PhERF110; C shows the statistics of the number of branches of control and transgenic plants (n = 24); D shows the statistics of the plant height of control and transgenic plants.

[0022] Figure 3 Phenotypic analysis diagram of PhERF110 RNAi transgenic plants; among them, A shows the comparison of phenotypes between control and PhERF110 RNAi transgenic plants, and Line 1, Line 2, and Line 3 represent different transgenic lines; B shows the detection of the expression level of PhERF110 RNAi; C shows the statistics of the number of branches of control and transgenic plants (n = 24); D shows the statistics of the plant height of control and transgenic plants. Detailed implementation mode

[0023] The various exemplary implementation modes 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, characteristics, and implementation schemes of the present invention.

[0024] It should be understood that the terms used in the present invention are only for describing specific implementation modes and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0025] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0026] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0027] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, that is, they are intended to include but not limited to.

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

[0029] Example 1

[0030] The present invention analyzed the transcriptome data of Petunia hybrida 1 h after decapitation and found that the up-regulation multiple of members of the ERF family was relatively large. One of the members was selected for full-length cloning, and finally a gene was obtained. An evolutionary tree was constructed with this gene and members of the Arabidopsis ERF family, showing that it clustered into one branch with Arabidopsis AtERF110. Therefore, this gene was named PhERF110.

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

[0032] SEQ ID NO.1:

[0033]

[0034] Example 2 Analysis of the Plant Phenotype Regulation Function of PhERF110

[0035] 1. Experimental Methods

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

[0037] 1.1 Vector Construction and Transformation Methods

[0038] 1.1.1 Construction and Transformation of the 35S::PhERF110 Vector

[0039] (1) The primer pairs PhERF110-1300-F / PhERF110-1300-R (the primer sequences are shown as SEQ ID NO.2 - 3 in Table 1) were used to amplify the target gene, and gel extraction was performed.

[0040] (2) pSuper1300-GFP was double digested with HindⅢ and Sal I, and the reaction system was as follows: 3 μL of plasmid DNA, 1 μL of each restriction enzyme, 2 μL of 10×Qcut Buffer, and 13 μL of ddH2O. Incubate at 37°C for 40 min, then at 50°C for 5 min.

[0041] (3) The target gene fragment and the vector double-digested fragment were subjected to a recombination reaction according to the instructions of the plus Onestep PCR Cloning Kit.

[0042] (4) 5 μL of the recombinant product was added to 50 μL of DH5α competent cells, pipetted and mixed well, and placed on ice for 30 min; incubated in a 42°C water bath for 30 s - 1 min, and immediately placed on ice for 2 - 3 min; 500 μL of LB solution was added, and the mixture was placed on a 37°C shaker for 1 h until the bacterial solution became turbid; centrifuged at 6000 rpm for 3 min, 250 μL of the supernatant was left and pipetted and mixed well, then evenly spread on the LB plate, sealed with a sealing strip, and incubated overnight at 37°C in an inverted position.

[0043] (5) Positive colonies were picked for culture and sequencing. The correctly sequenced recombinant plasmid was added to 100 μL of GV3101 Agrobacterium competent cells, and left on ice for 5 min; treated with liquid nitrogen for 5 min, incubated in a 28°C water bath for 5 min, and then in an ice bath for 5 min; 500 μL of LB liquid medium was added, and the mixture was shaken and cultured at 28°C for 2 - 3 h; centrifuged at 6000 rpm for 1 min to collect the bacteria, and evenly spread on the YEB solid medium containing rifampicin or kanamycin resistance, sealed with a sealing strip, and cultured at 28°C for 2 d to identify positive clones for standby.

[0044] Table 1 Primer Information Table

[0045]

[0046] 1.1.2 Construction and Transformation of PhERF110 RNAi Vector

[0047] According to the PhERF110 gene sequence, primers with a target fragment of about 150 bp were designed, and Xho I and Kpn I restriction sites were added. The primer sequences are shown as SEQ ID NO.4 - 5 in Table 1. Using cDNA as a template for amplification, and the amplified product was recovered by gel extraction.

[0048] The PhERF110 gel-extracted product and the pJL10 vector plasmid were double-digested with Xho I and Kpn I restriction endonucleases, then ligated with T4 ligase. Positive clones were identified and sequenced to obtain a recombinant plasmid containing the sense insert fragment. The recombinant plasmid was further double-digested with Xba I and Cla I, and finally an RNAi recombinant plasmid containing forward and reverse insert fragments was obtained using the same method as above.

[0049] 1.2 Arabidopsis Inflorescence Infiltration and Identification of Positive Plants

[0050] 1.2.1 Arabidopsis Inflorescence Infiltration

[0051] Weigh 0.948 g of 4.74 g / L MS, add 10 g of sucrose and 200 mL of sterile water, put it into a 500 mL glass bottle for sterilization and reserve; pour 30 mL of well-shaken Agrobacterium into a 50 mL centrifuge tube, centrifuge at 5000 rpm for 5 min with a high-speed centrifuge. Add 30 mL of sterilized sterile water and centrifuge for 5 min. Add 15 mL of infiltration solution into a 50 mL centrifuge tube, and gently pipette and mix with a pipette tip. Use a spectrophotometer to adjust OD 600 = 0.8; Immerse the just-emerged white inflorescence of Arabidopsis into the infiltration solution for 2 min; lay it flat in a culture dish and cover it with moisture, and perform dark culture for 24 h, then place it under normal conditions for cultivation; after one week, infiltrate the inflorescence of Arabidopsis with the same infiltration solution again. Wait for the seeds to mature, harvest the T0 generation seeds, and perform drying treatment for storage and reserve.

[0052] 1.2.2 Identification of Transgenic Arabidopsis

[0053] The T0 Arabidopsis seeds obtained by the Arabidopsis inflorescence infiltration method were placed in a sterilized 5 mL test tube, soaked in 75% alcohol for 1.5 min, and rinsed 3 times with sterile water; add 2% NaClO and shake for sterilization for 5 min, rinse 3 times with sterile water, then suspend with absolute ethanol, and then pipette and blow dry on filter paper. Then sow them evenly on MS medium (containing 30 mg / L hygromycin), place them in a 4°C refrigerator for low-temperature treatment for 2 d, and then place them in a culture room for cultivation.

[0054] When Arabidopsis thaliana grows to 4 leaves, transplant it to the growth chamber. When Arabidopsis thaliana grows to 30 days old, cut its leaves to extract RNA, reverse transcribe it into cDNA, and detect it by PCR to determine whether it has been successfully introduced into Arabidopsis thaliana. Screen the positive transgenic plants to T3 for subsequent phenotypic analysis.

[0055] 1.3 Petunia hybrida genetic transformation

[0056] 1.3.1 Culture of Petunia hybrida sterile seedlings

[0057] Soak with 75% alcohol for 1.5 min, then rinse 3 times with sterile water, pour in 2% NaClO, shake well for about 10 min, rinse 3 times with sterile water, then suck out the seeds with 95% alcohol, dry the water on the surface of the seeds, and then inoculate them on 1 / 2 MS solid medium and culture in the dark at 25℃. After the seeds show white, transfer them to an environment with a light intensity of 2000 - 3000 Lx and a light time of 16 h / d for culture.

[0058] 1.3.2 Activation and culture of Agrobacterium

[0059] Activate the PhERF110 RNAi Agrobacterium liquid. When the OD of the bacterial liquid 600 reaches 0.3, aliquot the bacterial liquid into 50 mL centrifuge tubes, centrifuge at 5000 rpm and 4℃ for 10 min, pour out the supernatant, and use a pipette to aspirate an appropriate amount of resuspension to resuspend the bacterial cells.

[0060] 1.3.3 Screening and rooting of Petunia hybrida resistant buds

[0061] Use a sterilized blade to cut the leaves of Petunia hybrida sterile seedlings into small squares of 0.5 cm × 0.5 cm and place them on the symbiotic solid medium for pre - culture. After two days of dark culture, place the leaf discs in the prepared infection liquid, and gently stir with forceps to make the leaves fully contact with the bacterial liquid. After 8 min, clamp out the Petunia hybrida leaves from the bacterial liquid and dry the bacterial liquid on the leaves with sterilized absorbent paper. Place the dried leaves face - down on the co - culture medium, arrange them closely on the symbiotic solid medium for culture, leave a part of the non - infected leaves as a control, place them face - up and loosely on the callus induction medium, and keep the culture conditions unchanged. After two days of dark culture, rinse 5 - 6 times with sterile water. After 2 - 3 days, transfer the Petunia hybrida leaves after dark culture to the screening medium (kan 50 mg / L, Tim 200 mg / L), seal and label, and place them on the light tissue culture rack at 25℃ for culture; change the medium every 12 d; when the resistant buds grow to about 1.5 cm, cut them and put them into the rooting medium (kan 50 mg / L, Tim 200 mg / L) for culture. After the Petunia hybrida tissue - cultured seedlings grow stably and grow up, carry out acclimatization treatment and transplant.

[0062] 2. Experimental results

[0063] 2.1 Overexpression of PhERF110 leads to an increase in the number of branches in Arabidopsis

[0064] To study the function of PhERF110, a 35S::PhERF110 overexpression vector was constructed and transformed into wild-type Arabidopsis. Transgenic plants were screened, and a total of 21 independent transgenic lines were obtained. At the T3 generation, 3 lines were selected for phenotypic analysis. The results are as Figure 1 shown. Compared with the wild type, the number of branches in the PhERF110 overexpression transgenic plants increased from 7.9 to 31, 23.6, and 21, respectively. While the plant height decreased from 27.72 cm to 25.3, 22.3, and 20 cm, respectively. These results indicate that overexpression of PhERF110 can promote an increase in the number of branches in Arabidopsis and cause a decrease in plant height.

[0065] 2.2 Phenotypic analysis of PhERF110 overexpression transgenic plants

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

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

[0068] 2.3 Phenotypic analysis of PhERF110 RNAi plants

[0069] To further study the function of PhERF110, a pJL10-PhERF110 vector was constructed and transformed into petunias. Transgenic plants were screened, and a total of 18 independent transgenic lines were obtained. At the T3 generation, 3 lines were selected for phenotypic analysis.

[0070] The results are as Figure 3 shown: qRT-PCR detection showed that RNAi reduced the expression level of PhERF110 to 0.21, 0.19, and 0.31 times that of the control, respectively. Compared with 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 the plant height did not change significantly. These results indicate that a decrease in the expression level of PhERF110 causes a decrease in the number of branches in petunias, further verifying that PhERF110 positively regulates the branching development of plants.

[0071] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A petunia PhERF110 gene, 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 according to claim 1 .

3. An engineered bacterium comprising the recombinant vector according to claim 2.

4. Use of the petunia PhERF110 gene according to claim 1, the recombinant vector according to claim 2 or the engineered bacteria according to claim 3 in regulating plant type.

5. The use according to claim 4, characterized in that The regulating plant architecture includes regulating the branch development of the plant.

6. The use according to claim 5, characterized in that The expression level of the petunia PhERF110 gene is up-regulated in the plant, thereby promoting the branching development of the plant and increasing the number of branches of the plant.

7. The use according to any one of claims 4 to 6, characterized in that: The plant is Arabidopsis thaliana or petunia.

8. A method for promoting plant branching development, characterized in that: The method comprises the steps of up-regulating the expression level of the petunia PhERF110 gene according to claim 1 in a plant to increase the number of branches of the plant.

9. A method for inhibiting plant branching development, characterized in that: The method comprises the steps of down-regulating the expression level of the petunia PhERF110 gene according to claim 1 in a plant to reduce the number of branches of the plant.

10. The method according to claim 8 or 9, characterized in that The plant is Arabidopsis thaliana or petunia.

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