Use of a protein encoded by gibberellin synthesis pathway gene ga7ox in regulating plant development

Abstract

The present application belongs to the technical field of plant molecular biology, and particularly relates to application of a protein encoded by gibberellin synthesis pathway gene GA7ox in regulating plant development. The present application aims to determine the role of GA7ox in potato development, and provide a new option for breeding high-quality potato varieties. The technical scheme of the present application is that the protein encoded by gibberellin synthesis pathway gene GA7ox is applied in regulating plant development; the amino acid sequence of the protein encoded by gene GA7ox is shown in SEQ ID No. 9. The present application provides a new option for creating potato varieties with short or tall height, more branches, early tuber formation and / or high yield.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology technology, specifically relating to the application of the protein encoded by the gibberellin synthesis pathway gene GA7ox in regulating plant development. Background Technology

[0002] Potato (Solanum tuberosum L.) is a plant belonging to the genus Solanum in the family Solanaceae, and is an important food resource. As a tuberous crop, the significant economic value of potatoes is mainly reflected in their tubers. Tubers are both the vegetative and reproductive organs of potatoes, serving as the primary edible part and an important industrial raw material. The formation of potato tubers involves the induction and elongation of stolons, the cessation of stolon growth, and the swelling and tuber formation in the apical region. Various molecular signals regulate the formation of potato tubers; revealing the mechanisms of tuber formation and development, and understanding the mechanisms of action of these molecular signals, provides valuable reference for potato breeding methods and improvement efforts.

[0003] The biosynthetic pathway of gibberellins has been extensively studied in plants. The biochemical pathway of GA synthesis begins with geranyl diphosphate (GGPP) and proceeds via isopentenyl diphosphate (IPP), the 5-carbon building block of all terpenoid / isoprene compounds. In the green tissues of most plants, the basic isopentenyl unit IPP is generated via two pathways: the mevalonate (MVA) pathway in the cytoplasm and the methylerythritol phosphate (MEP) pathway in the plastid. The entire pathway can be divided into three stages based on its subcellular compartmentalization and the enzymes involved. The first stage is catalyzed by soluble enzymes located in the protoplast, leading to the production of endoroot-kaurene. In the second stage, endoroot-kaurene is oxidized to GA12-aldehyde, a common GA precursor, and further catalyzed by cytochrome P-450 monooxygenase in the endoplasmic reticulum. The third stage is catalyzed by 2-ketoglutarate-dependent dioxygenases in the cytoplasm.

[0004] GA7ox (Gibberellin 7-oxidase), also known as gibberellin 7-oxidase, is an enzyme involved in the biosynthesis of gibberellins (GAs) in plants. They are particularly present in developing seeds, but are also expressed in vegetative tissues, including roots. The species distribution of GA7ox appears to be limited; besides the Cucurbitaceae family, two other reports indicate its presence in potatoes. Studies in cucumbers suggest that the expansion of gene families such as GA2ox, GA3ox, and GA7ox may be related to improved plant adaptability to environmental conditions. GA7ox enzymes have been reported in species such as pumpkins and cucumbers to participate in the conversion of GA12-aldehyde to GA12, exhibiting monooxygenase 7-oxidase activity. In potatoes, research on GA7ox is relatively limited. Summary of the Invention

[0005] The technical problem to be solved by this invention is to clarify the role of GA7ox in potato development and provide a new option for breeding high-quality potato varieties.

[0006] The technical solution of the present invention is the application of the protein encoded by the gibberellin synthesis pathway gene GA7ox in regulating plant development; the amino acid sequence of the protein encoded by the gene GA7ox is shown in SEQ ID No. 9.

[0007] Specifically, the plant in question is a tuberous plant.

[0008] Furthermore, the tuber plant is potato.

[0009] The regulation of plant development includes regulating plant height, regulating the number of plant branches, and / or regulating tuber development.

[0010] Furthermore, the regulation of tuber development aims to promote earlier tuber formation and / or increase tuber yield.

[0011] Specifically, the above-mentioned regulation of tuber development is achieved by reducing the expression of the GA7ox gene in plants.

[0012] Specifically, the method of reducing the expression of the GA7ox gene in plants is to knock out the GA7ox gene or interfere with the expression of the GA7ox gene.

[0013] Furthermore, the expression of the interfering gene GA7ox is silenced using the VIGS method.

[0014] Furthermore, the regulation of plant height refers to reducing plant height.

[0015] Specifically, the above-mentioned method of reducing plant height is to reduce the expression of the GA7ox gene in the plant.

[0016] Specifically, the method of reducing the expression of the GA7ox gene in plants is to knock out the GA7ox gene or interfere with the expression of the GA7ox gene.

[0017] Furthermore, the expression of the interfering gene GA7ox is silenced using the VIGS method.

[0018] Furthermore, the regulation of plant branch number is to increase the number of plant branches.

[0019] Specifically, the above-mentioned method of increasing the number of plant branches is to reduce the expression of the GA7ox gene in the plant.

[0020] Specifically, the method of reducing the expression of the GA7ox gene in plants is to knock out the GA7ox gene or interfere with the expression of the GA7ox gene.

[0021] Furthermore, the expression of the interfering gene GA7ox is silenced using the VIGS method.

[0022] The present invention also provides a method for creating high-yielding and / or early-tuberizing plants by reducing the expression of the gene GA7ox in the plant; the amino acid sequence of the protein encoded by the gene GA7ox is shown in SEQ ID No. 9.

[0023] Specifically, the plant in question is a tuberous plant.

[0024] Furthermore, the tuber plant is potato.

[0025] Specifically, the method of reducing the expression of the GA7ox gene in plants is to knock out the GA7ox gene or interfere with the expression of the GA7ox gene.

[0026] Furthermore, the expression of the interfering gene GA7ox is silenced using the VIGS method.

[0027] Specifically, the operation of using the VIGS silencing method is as follows: construct a VIGS silencing expression vector for the GA7ox gene, and transform the vector into a plant.

[0028] The method of conversion into the plant is injection.

[0029] The present invention also provides a method for creating dwarf plants by reducing the expression of the gene GA7ox in the plant; the amino acid sequence of the protein encoded by the gene GA7ox is shown in SEQ ID No. 9.

[0030] Specifically, the plant in question is a tuberous plant.

[0031] Furthermore, the tuber plant is potato.

[0032] Specifically, the method of reducing the expression of the GA7ox gene in plants is to knock out the GA7ox gene or interfere with the expression of the GA7ox gene.

[0033] Furthermore, the expression of the interfering gene GA7ox is silenced using the VIGS method.

[0034] Specifically, the operation of using the VIGS silencing method is as follows: construct a VIGS silencing expression vector for the GA7ox gene, and transform the vector into a plant.

[0035] The method of conversion into the plant is injection.

[0036] The present invention also provides a method for creating multi-branched plants by reducing the expression of the gene GA7ox in the plant; the amino acid sequence of the protein encoded by the gene GA7ox is shown in SEQ ID No. 9.

[0037] Specifically, the plant in question is a tuberous plant.

[0038] Furthermore, the tuber plant is potato.

[0039] Specifically, the method of reducing the expression of the GA7ox gene in plants is to knock out the GA7ox gene or interfere with the expression of the GA7ox gene.

[0040] Furthermore, the expression of the interfering gene GA7ox is silenced using the VIGS method.

[0041] Specifically, the operation of using the VIGS silencing method is as follows: construct a VIGS silencing expression vector for the GA7ox gene, and transform the vector into a plant.

[0042] The method of conversion into the plant is injection.

[0043] The beneficial effects of this invention are as follows: This invention provides the application of the protein encoded by the gibberellin synthesis pathway gene GA7ox in regulating plant development, specifically regulating plant height, number of branches, and / or tuber development. By reducing the expression of the GA7ox gene in tuber plants, plants with short stature, many branches, early tuber formation, and / or high yield can be obtained. This invention provides a new option for creating potato varieties with short stature (short stature is beneficial for lodging resistance and improves nutrient utilization efficiency, especially nitrogen utilization), many branches (many branches increase potential tuber formation points, improve ground cover, increase leaf photosynthetic capacity, and each branch base has the potential to produce stolons and tubers; a large number of branches usually means an increase in the potential tuber formation sites and the number of stolons per plant), early tuber formation, and / or high yield. Attached Figure Description

[0044] Figure 1 PCR verification image of pTRV2-StGA7ox transformed DH5α colonies; M: 2000 marker, 1-5: PCR bands of pTRV2-StGA7ox recombinant plasmid.

[0045] Figure 2 The graph shows the relative expression level of the StGA7ox gene; *: P<0.05; **: P<0.01.

[0046] Figure 3 Phenotypic images of potatoes 20 days after injection of pTRV2-StGA7ox; a: WT; b: pTRV2; c: StGA7ox.

[0047] Figure 4 Statistical chart of potato plant height 20 days after injection of pTRV2-StGA7ox; **: P<0.01.

[0048] Figure 5 A statistical chart showing the average number of tubers per plant 90 days after injection of pTRV2-StGA7ox.

[0049] Figure 6 This is a structural diagram of the pTRV2-StGA7ox recombinant plasmid. Detailed Implementation

[0050] Gibberellins are closely related to plant growth and development. Plants mainly regulate gibberellin levels by controlling the expression of genes related to the gibberellin synthesis pathway. GA7ox is an important gibberellin synthase, and has been shown in other plants to participate in catalyzing multi-step oxidation reactions and ultimately synthesizing gibberellins. However, its role in potato tuber formation has been less reported. This invention utilizes virus-induced gene silencing (VIGS) to reveal the regulatory pattern of the gibberellin synthesis gene GA7ox during potato tuber formation. This can provide a certain reference for potato breeding and is of great significance for improving potato yield and quality.

[0051] The reagent kits and materials used in the following examples:

[0052] 1M MES: Take 10.66g of MES and dilute to 50mL with ddH2O;

[0053] 1M MgCl2: Take 4.76g MgCl2 and dilute to 50mL with ddH2O;

[0054] 200 mM As: Take 0.65 g of As and dilute to 10 mL with DMSO;

[0055] All the above mother liquors were sterilized by filtration using a 0.22μm PES filter.

[0056] Infection solution: In a clean bench, take 2 mL of MES stock solution, 2 mL of MgCl2 stock solution, and 200 μL of As, and bring the volume to 200 mL with sterile ddH2O. The instruments and reagents required for the remaining experiments are shown in Tables 1 and 2.

[0057] Table 1 Reagents required for the experiment

[0058] .

[0059] Table 2. Instruments required for the experiment

[0060] .

[0061] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0062] Example 1: Construction of gene silencing vector

[0063] Total RNA was extracted from potatoes according to the instructions of the MolPure® Plant Plus RNA Kit: 0.1 g of young potato leaves were placed in a 1.5 mL RNase-free centrifuge tube, and liquid nitrogen was added. The leaves were then thoroughly ground with a sterile, enzyme-free glass pestle. 1 mL of lysis buffer (LB) was added to the centrifuge tube, and the mixture was manually shaken for 15 seconds, followed by vortexing for 30 seconds to ensure thorough mixing. The mixture was centrifuged at 12000 rpm for 10 min, and 480 μL of the supernatant was transferred to a new 1.5 mL RNase-free centrifuge tube. 240 μL of anhydrous ethanol was added, and the mixture was mixed by pipetting. The mixture was then transferred to an RNA adsorption column collection tube, centrifuged at 12000 rpm for 1 min, and the waste liquid was discarded. 350 μL of protein removal buffer (PL) was added, and the mixture was incubated at room temperature for 1 min, then centrifuged at 12000 rpm for 30 seconds, and the waste liquid was discarded. 50 μL of DNase I working solution was added to the center of the adsorption column membrane, and the mixture was incubated at room temperature for 15 min. Add 350 μL of protein removal buffer (PL), centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid. Add 500 μL of wash buffer, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid; repeat once. Centrifuge the empty column at 12000 rpm for 2 minutes to prevent residual ethanol from affecting the elution effect. Transfer the adsorption column to a new 1.5 mL RNase-free centrifuge tube, add 30 μL of RNase-free H2O to the center of the adsorption column, incubate at room temperature for 2 minutes, and centrifuge at 12000 rpm for 1 minute. Add the liquid back to the adsorption column, incubate at room temperature for 2 minutes, and centrifuge at 12000 rpm for 1 minute. Store the RNA solution at -80°C.

[0064] Total RNA from potatoes was reverse transcribed into cDNA: The reverse transcription reaction solution was prepared according to Table 3 (the reaction solution was prepared on ice). Reverse transcription reaction conditions: 37℃ for 15 min (reverse transcription reaction), 85℃ for 5 sec (reverse transcriptase inactivation reaction), 4℃. The reverse transcription products were detected by agarose gel electrophoresis and then stored at -20℃.

[0065] Table 3 Reverse transcription reaction system

[0066] .

[0067] Using potato cDNA as a template, StGA7ox-F as the forward primer (primer sequences are shown in Table 4), and StGA7ox-R as the reverse primer, a 342bp StGA7ox gene fragment was amplified. StGA7ox The gene sequence can be found in the Potato Genome Database, at http: / / spuddb.uga.edu / , accession number Soltu.DM.03G030400.2; the amplified 342bp sequence is as described above. StGA7ox The PCR products were recovered and purified according to the instructions of the universal DNA purification and recovery kit (Table 1). The positions 7323-7577 of the gene sequence were used.

[0068] The amino acid sequence of the protein encoded by the StGA7ox gene (SEQ ID No. 9):

[0069]

[0070] Table 4 Primer sequences used in virus-induced gene silencing experiments

[0071] .

[0072] The 342bp fragment of StGA7ox was ligated into the intermediate vector pMD19-T using T4 ligase, resulting in the StGA7ox-T recombinant plasmid. The StGA7ox-T recombinant plasmid was transformed into *E. coli* DH5α competent cells. Single-colony PCR was performed using gene primers. Positive colonies were picked and cultured in LB broth containing 50 ng / mL Amp at 37°C for 15 h. The StGA7ox-T recombinant plasmid was extracted from 5 mL of the bacterial culture and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The results were as expected.

[0073] Using the StGA7ox-T recombinant plasmid as a template, a 342bp fragment of StGA7ox with homologous arms was amplified using primers vStGA7ox-F and vStGA7ox-R. The PCR product was purified using a universal DNA purification and recovery kit. The pTRV2 vector (gifted from the laboratory of a professor at Harbin University of Inner Mongolia) was linearized by double digestion with QuickCut™ Bam HI and QuickCut™ Xho I. The StGA7ox 342bp fragment was seamlessly cloned into the linearized pTRV2 vector. The seamless cloning system components are shown in Table 5. The system was gently mixed and incubated at 50°C for 15 min. After the reaction, the centrifuge tubes were placed on ice for a few seconds to cool. The recombinant product was stored at -20°C.

[0074] Table 5 Seamless Cloning System

[0075] .

[0076] The ligation product pTRV2-StGA7ox was transformed into E. coli DH5α competent cells and cultured overnight at 37°C. Single colonies were picked for colony PCR (verification results are shown in [link to data]). Figure 1 Single colonies that successfully underwent PCR were picked and placed in LB liquid medium supplemented with a final concentration of 50 ng / μL Kana. After incubation at 37°C for 12 h, the pTRV2-StGA7ox recombinant plasmid was extracted. The recombinant plasmid was then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the results were as expected. The pTRV2-StGA7ox recombinant plasmid (vector structure shown in...) Figure 6 Transformed into Agrobacterium GV3101 competent cells.

[0077] Example 2: Transformation of potatoes with gene silencing vector

[0078] Agrobacterium infection solutions containing pTRV2-StGA7ox and pTRV1 were mixed and injected into potato leaves. An additional control group was created by mixing Agrobacterium infection solutions containing the empty pTRV2 vector and pTRV1. Gene silencing effects were assessed using qRT-PCR after 20 days, and the gene-silencing lines were evaluated based on phenotypic analysis.

[0079] Twenty days after TRV injection, leaf tissue near the Agrobacterium injection site was collected, and total RNA was extracted from potatoes according to the instructions of the MolPure® Plant Plus RNA Kit. Then, quantitative real-time PCR was performed on each RNA sample using the Hifair® Advanced One Step RT-qPCR SYBR Green Kit, with primers StGA7ox-F and StGA7ox-R. The qRT-PCR reaction system is shown in Table 6. The qRT-PCR reaction procedure is shown in Table 7.

[0080] Table 6 qRT-PCR reaction system

[0081] .

[0082] Table 7 qRT-PCR reaction procedure

[0083] .

[0084] qRT-PCR results are as follows Figure 2 As shown, the expression level of the target silent gene in the StGA7ox transgenic line was significantly lower than that in the wild-type and empty vector control groups, proving the successful construction of the StGA7ox silent line. The plant height and branching of the StGA7ox VIGS silent line were observed 20 days after TRV injection. Figure 3 Plant height data analysis, such as Figure 4 The results showed that, compared with wild-type plants and TRV empty vector-infected plants, the StGA7ox gene-silenced plants exhibited significantly reduced plant height and increased branching.

[0085] Ninety days after TRV injection, the average number of tubers per plant in different treatment groups of the StGA7ox VIGS silent strain was statistically analyzed. Figure 5 Compared to wild-type plants, the StGA7ox gene-silenced plants form tubers earlier and in greater quantities.

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The application of the protein encoded by the gibberellin synthesis pathway gene GA7ox in regulating tuber plant development, characterized by: The amino acid sequence of the protein encoded by the gene GA7ox is shown in SEQ ID No. 9; the regulation of tuber plant development is to regulate plant height, regulate the number of plant branches, and / or regulate tuber development, which is achieved by reducing the expression of the gene GA7ox in tuber plants; the regulation of plant height is to reduce plant height; the regulation of tuber development is to promote earlier tuber formation and / or increase tuber yield; the regulation of the number of plant branches is to increase the number of plant branches; the tuber plant is potato.

2. A method for creating high-yielding and / or early-forming tuber plants, characterized by: This is achieved by reducing the expression of the gene GA7ox in tuber plants; the amino acid sequence of the protein encoded by the gene GA7ox is shown in SEQ ID No. 9; the tuber plant is potato.

3. A method for creating dwarf tuberous plants, characterized by: This is achieved by reducing the expression of the gene GA7ox in tuber plants; the amino acid sequence of the protein encoded by the gene GA7ox is shown in SEQ ID No. 9; the tuber plant is potato.

4. A method for creating multi-branched tuberous plants, characterized in that: This is achieved by reducing the expression of the gene GA7ox in tuber plants; the amino acid sequence of the protein encoded by the gene GA7ox is shown in SEQ ID No. 9; the tuber plant is potato.

5. The method according to any one of claims 2 to 4, characterized in that: The method for reducing the expression of the GA7ox gene in tuber plants is to knock out the GA7ox gene or interfere with the expression of the GA7ox gene.

6. The method according to claim 5, characterized in that: The expression of the interfering gene GA7ox was silenced using the VIGS method.

7. The method according to claim 6, characterized in that: The operation using the VIGS silencing method is as follows: construct a VIGS silencing expression vector for the GA7ox gene, and transform the vector into tuber plants; the transformation into tuber plants is performed by injection.

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