Application of protein coded by gibberellin synthetic pathway gene GA7ox in regulation and control of plant development

Abstract

The invention belongs to the technical field of plant molecular biology, and particularly relates to application of a protein coded by a gibberellin synthetic pathway gene GA7ox in regulation and control of plant development. The technical problem to be solved by the invention is to clarify the effect of GA7ox in potato development and provide a new choice for cultivating high-quality potato varieties. The technical scheme of the invention is the application of the protein coded by the gibberellin synthetic pathway gene GA7ox in regulation and control of plant development. The amino acid sequence of the protein coded by the gene GA7ox is as shown in SEQ ID No. 9. The invention provides a new choice for creating potato varieties with high and short plants, more branches, early tuberization and / or high yield.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant molecular biology, and particularly relates to the application of a protein encoded by a gibberellin synthesis pathway gene GA7ox in regulating plant development. Background Art

[0002] Potato (Solanum tuberosum L.), a plant of the Solanaceae family, is an important food resource. As a tuber crop, potato's important economic value is primarily reflected in its tubers. The tuber is both the potato's nutritional and reproductive organ, its primary edible part and an important industrial raw material. The formation of potato tubers progresses through the induction and elongation of runners, the cessation of runner growth, and the swelling and tuber formation of the top region. The formation of potato tubers is regulated by various molecular signals. Uncovering the mechanisms of potato tuber development and understanding the mechanisms of action of these various molecular signals is valuable for potato cultivation and improvement.

[0003] The gibberellin biosynthesis pathway has been extensively studied in plants. The biochemical pathway for GA synthesis begins with geranyl geranyl diphosphate (GGPP) and proceeds through isopentenyl diphosphate (IPP), the five-carbon building block of all terpenoid / isoprene compounds. In the green tissues of most plants, the fundamental isoprene unit, IPP, is generated via two pathways: the mevalonate (MVA) pathway in the cytoplasm and the methylerythritol phosphate (MEP) pathway in the plastids. 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 protoplasts, leading to the production of ent-kaurene. In the second stage, ent-kaurene is oxidized to GA12-aldehyde, a general GA precursor, which is further catalyzed by cytochrome P-450 monooxygenases in the endoplasmic reticulum. The third stage is catalyzed by 2-oxoglutarate-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. GA7ox appears to be species-restricted; in addition to the Cucurbitaceae, there are two reports of its presence in potato. Studies in cucumber suggest that the expansion of gene families such as GA2ox, GA3ox, and GA7ox may be associated with improved environmental adaptability. In species such as pumpkin and cucumber, GA7ox enzymes have been reported to be involved in the conversion of GA12-aldehyde to GA12, exhibiting monooxygenase 7-oxidase activity. In potato, GA7ox has been less well-studied. Summary of the Invention

[0005] The technical problem to be solved by the present 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] Particularly, the plant is a tuberous plant.

[0008] Furthermore, the tuberous plant is potato.

[0009] Wherein, the regulating plant development is regulating plant height, regulating the number of plant branches and / or regulating tuber development.

[0010] Furthermore, the regulation of tuber development is 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 gene GA7ox in the plant.

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

[0013] Furthermore, the expression of the interference gene GA7ox is silenced by VIGS.

[0014] Furthermore, the regulating plant height is to reduce the plant height.

[0015] Specifically, the above-mentioned reduction in plant height is achieved by reducing the expression of the gene GA7ox in the plant.

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

[0017] Furthermore, the expression of the interference gene GA7ox is silenced by VIGS.

[0018] Furthermore, the regulating the number of plant branches 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 gene GA7ox in the plant.

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

[0021] Furthermore, the expression of the interference gene GA7ox is silenced by VIGS.

[0022] The present invention also provides a method for creating high-yield and / or early-tube-bearing 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] Particularly, the plant is a tuberous plant.

[0024] Furthermore, the tuberous plant is potato.

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

[0026] Furthermore, the expression of the interference gene GA7ox is silenced by VIGS.

[0027] Specifically, the operation of adopting the VIGS silencing method is: constructing a VIGS silencing expression vector of the gene GA7ox, and transforming the vector into plants.

[0028] Wherein, the method of transformation into plants is injection.

[0029] The present invention also provides a method for creating a dwarf plant, which is achieved 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] Particularly, the plant is a tuberous plant.

[0031] Furthermore, the tuberous plant is potato.

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

[0033] Furthermore, the expression of the interference gene GA7ox is silenced by VIGS.

[0034] Specifically, the operation of adopting the VIGS silencing method is: constructing a VIGS silencing expression vector of the gene GA7ox, and transforming the vector into plants.

[0035] Wherein, the method of transformation into plants is injection.

[0036] The present invention also provides a method for creating multi-branched plants, which is achieved 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] Particularly, the plant is a tuberous plant.

[0038] Furthermore, the tuberous plant is potato.

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

[0040] Furthermore, the expression of the interference gene GA7ox is silenced by VIGS.

[0041] Specifically, the operation of adopting the VIGS silencing method is: constructing a VIGS silencing expression vector of the gene GA7ox, and transforming the vector into plants.

[0042] Wherein, the method of transformation into plants is injection.

[0043] Beneficial effects of the present invention: The present invention provides the use of a protein encoded by the gibberellin synthesis pathway gene GA7ox in regulating plant development, specifically for regulating plant height, the number of plant branches and / or tuber development. By reducing the expression of the gene GA7ox in tuber plants, plants with short plant height, many branches, early tuber formation and / or high yield can be obtained. The present invention provides a new option for creating potato varieties with short plant height (short plants are beneficial for lodging resistance while improving nutrient utilization efficiency, especially nitrogen utilization rate), many branches (many branches can increase potential tuber formation sites, improve ground cover, and increase leaf photosynthetic capacity, and the base of each branch has the potential to produce runners and tuber; a large number of branches usually means an increase in the potential tuber formation sites and the number of runners of a single plant), early tuber formation and / or high yield. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0046] Figure 3 These are the potato phenotypes 20 days after injection with pTRV2-StGA7ox; a: WT; b: pTRV2; c: StGA7ox.

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

[0048] Figure 5 The average number of tubers per plant for pTRV2-StGA7ox injection 90d.

[0049] Figure 6 The structure diagram of pTRV2-StGA7ox recombinant plasmid. DETAILED DESCRIPTION

[0050] Gibberellins are closely related to the growth and development of plants, and plants mainly regulate the level of gibberellins in vivo by controlling the expression of genes related to the gibberellin synthesis pathway. GA7ox is an important gibberellin synthase, which is proved to be involved in catalyzing multi-step oxidation reactions and finally synthesizing gibberellins in other plants, but there are few reports on its role in the process of potato tuber formation. The present application uses the method of virus induced gene silencing (VIGS) to reveal the regulation mode of gibberellin synthesis gene GA7ox in the process of potato tuber formation, which can provide certain reference for potato breeding work, and has important significance for improving potato yield and quality.

[0051] The kits and materials used in the following examples are as follows: 1M MES: take 10.66g MES, and make up to 50mL with ddH2O; 1M MgCl2: take 4.76g MgCl2, and make up to 50mL with ddH2O; 200 mM As: take As 0.65g, and make up to 10mL with DMSO; The above mother liquor is filtered with a 0.22μm PES filter to remove bacteria.

[0052] Infection solution: take 2mL of MES mother liquor, 2mL of MgCl2 mother liquor, and 200μL of As in the super-clean bench, and make up to 200mL with sterilized ddH2O. The remaining part of the required instruments and reagents for the experiment are shown in Table 1 and Table 2.

[0053] Table 1 Required reagents for the experiment .

[0054] Table 2 Required instruments for the experiment .

[0055] In order to further illustrate the technical means and effects adopted by the present application to achieve the predetermined object of the application, the specific embodiments, structures, features and effects according to the present application will be described in detail below in combination with the drawings and preferred embodiments.

[0056] Example 1 Construction of gene silencing vector Total potato RNA was extracted according to the instructions for 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 ground thoroughly with liquid nitrogen using a sterile, enzyme-free glass pestle. 1 mL of Lysis Buffer (LB) was added to the tube and mixed manually for 15 seconds, followed by vortexing for 30 seconds. The tube was centrifuged at 12,000 rpm for 10 minutes. 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 mixed thoroughly by pipetting. The mixture was added to an RNA adsorption column / collection tube and centrifuged at 12,000 rpm for 1 minute. The waste solution was discarded. 350 μL of Deproteinization Buffer (PL) was added, the mixture was incubated at room temperature for 1 minute, and then centrifuged at 12,000 rpm for 30 seconds. 50 μL of DNase I working solution was added to the center of the adsorption column membrane and incubated at room temperature for 15 minutes. Add 350 μL of deproteinizing solution PL and centrifuge at 12,000 rpm for 30 seconds, discarding the waste solution. Add 500 μL of rinse solution and centrifuge at 12,000 rpm for 30 seconds, discarding the waste solution; repeat once. Centrifuge the empty column at 12,000 rpm for 2 minutes to prevent residual ethanol from affecting the elution effect. Place the adsorption column in 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 12,000 rpm for 1 minute. Add the liquid back to the adsorption column, incubate at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute. Store the RNA solution at -80°C.

[0057] Reverse transcribe potato total RNA into cDNA: Prepare the reverse transcription reaction mixture according to Table 3 (prepare the reaction mixture on ice). Reverse transcription reaction conditions: 37°C for 15 minutes (reverse transcription reaction), 85°C for 5 seconds (reverse transcriptase inactivation reaction), 4°C. Detect the reverse transcription product by agarose gel electrophoresis and store at -20°C.

[0058] Table 3 Reverse transcription reaction system .

[0059] Potato cDNA was used as a template, StGA7ox-F was used as a forward primer (primer sequences are shown in Table 4 ), and StGA7ox-R was used as a reverse primer to amplify a 342 bp StGA7ox gene fragment. StGA7ox The gene sequence is available in the Potato Genome Database, http: / / spuddb.uga.edu / , with the sequence accession number Soltu.DM.03G030400.2; the amplified 342 bp sequence is the aforementioned StGA7oxThe 7323-7577 of the gene sequence. The PCR product was recovered and purified according to the instruction of the universal DNA purification recovery kit (Table 1).

[0060] SEQ ID No. 9 Amino acid sequence of the protein encoded by StGA7ox gene:

[0061] Table 4 Primer sequences used in virus-induced gene silencing experiment .

[0062] The StGA7ox 342bp fragment was ligated to the intermediate vector pMD19-T using T4 ligase to obtain the StGA7ox-T recombinant plasmid. The StGA7ox-T recombinant plasmid was transformed into E. coli DH5a competent cells, and single colony PCR was performed using gene primers. The single colony with positive detection result was picked and inoculated in LB liquid medium containing 50 ng / mL Amp at 37°C for 15 h. Five mL of bacterial solution was taken to extract the StGA7ox-T recombinant plasmid, which was sent to Shanghai SunGene Bioengineering Co., Ltd. for sequencing, and the result was as expected.

[0063] The StGA7ox 342bp fragment with homologous arms was amplified from the StGA7ox-T recombinant plasmid using primers vStGA7ox-F and vStGA7ox-R. The PCR product was purified using a universal DNA purification and recovery kit. The pTRV2 vector (provided by the laboratory of Mr. Ha Da of Inner Mongolia University) 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 is shown in Table 5. The system was gently mixed and reacted at 50°C for 15 min. After the reaction, the centrifuge tube was cooled on ice for a few seconds. The recombination product was stored at -20°C.

[0064] Table 5 Seamless cloning system .

[0065] The ligation product pTRV2-StGA7ox was transformed into E. coli DH5a competent cells, which were incubated overnight at 37°C. Single colonies were picked and subjected to colony PCR (the verification result is shown in Figure 1 ). The single colonies with successful colony PCR were inoculated in LB liquid medium containing 50 ng / μL Kana at a final concentration, and incubated at 37°C for 12 h. Then, the pTRV2-StGA7ox recombinant plasmid was extracted. The recombinant plasmid was sent to Shanghai SunGene Bioengineering Co., Ltd. for sequencing, and the result was as expected. The pTRV2-StGA7ox recombinant plasmid (the vector structure is shown in Figure 6 ) was transformed into Agrobacterium GV3101 competent cells.

[0066] Example 2 Transformation of gene silencing vector into potato The agrobacterium infection liquid containing pTRV2-StGA7ox was mixed with the agrobacterium infection liquid containing pTRV1, and was injected into potato leaves. The agrobacterium infection liquid containing pTRV2 empty vector was mixed with the pTRV1 agrobacterium infection liquid, and was injected into potato leaves as a control group. After 20 days, the gene silencing effect was detected by qRT-PCR, and the gene silencing plant was evaluated by combining phenotype analysis.

[0067] After 20 days of TRV injection, the leaf tissue near the agrobacterium injection point was taken, the total RNA of potato was extracted according to the instructions of MolPure® Plant Plus RNA Kit polysaccharide polyphenol plant RNA extraction kit, and then the fluorescence quantitative PCR of each RNA sample was carried out by using Hifair® Advanced One Step RT-qPCR SYBR Green Kit reagent kit, and the primers were StGA7ox-F and StGA7ox-R. The qRT-PCR reaction system is shown in Table 6. The qRT-PCR reaction program is shown in Table 7.

[0068] Table 6 qRT-PCR reaction system .

[0069] Table 7 qRT-PCR reaction program .

[0070] The qRT-PCR results are shown in Table 6, and the expression amount of the target silencing gene of StGA7ox transgenic strain is significantly lower than that of wild type and empty vector control group, which proves that the StGA7ox silencing strain is successfully constructed. After 20 days of TRV injection, the plant height and branching of StGA7ox VIGS silencing strain are observed as shown in Table 7; the plant height data analysis is shown in Table 8. Figure 2 Figure 3 Figure 4 The results show that compared with wild type plants and TRV empty vector infected strains, StGA7ox gene silencing strains show significant decrease in plant height and increase in branching.

[0071] After 90 days of TRV injection, the average number of tubers per plant of StGA7ox VIGS silencing strain of each different treatment group is counted as shown in Table 9. Compared with wild type plants, StGA7ox gene silencing strains form tubers earlier and more. Figure 5

[0072] ​​​The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Application of a protein encoded by the gibberellin biosynthesis pathway gene GA7ox in regulating plant development, characterized in that: The amino acid sequence of the protein encoded by the gene GA7ox is shown in SEQ ID No. 9; the regulating plant development is regulating plant height, regulating the number of plant branches and / or regulating tuber development; and the plant is a tuberous plant.

2. The application according to claim 1, characterized in that: The tuberous plant is potato.

3. The application according to claim 1, characterized in that: One of the following: a) regulating plant height is reducing plant height; b) regulating tuber development to promote earlier tuber formation and / or increase tuber yield; c) the regulating the number of plant branches is to increase the number of plant branches.

4. The application according to claim 1, characterized in that: The control of plant height, the control of plant branch number and / or the control of tuber development is achieved by reducing the expression of the gene GA7ox in the tuber plant.

5. A method for producing high-yield and / or early-bearing tuber 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.

6. A method for producing dwarf 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.

7. A method for producing a multi-branched tuberous plant, 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.

8. The method according to any one of claims 5 to 7, characterized in that: The method of reducing the expression of the gene GA7ox in the tuber plant is to knock out the gene GA7ox or interfere with the expression of the gene GA7ox.

9. The method according to claim 8, characterized in that: The expression of the interference gene GA7ox was silenced by VIGS.

10. The method according to claim 9, characterized in that: The operation of adopting the VIGS silencing method is as follows: constructing a VIGS silencing expression vector of the gene GA7ox, and transforming the vector into tuber plants; the method of transforming into the tuber plants is injection.

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