Sugarcane gibberellin oxidase gene GA2ox1 and application thereof in regulation and control of plant height

Abstract

The invention discloses a sugarcane gibberellin oxidase gene GA2ox1 and application thereof in regulation and control of plant height, and relates to the technical field of plant genetic engineering. The nucleotide sequence of the sugarcane gibberellin oxidase gene GA2ox1 is shown as SEQ ID NO: 1, and the encoded protein of the sugarcane gibberellin oxidase gene GA2ox1 can catalyze the oxidation reaction of active gibberellin and reduce the content of endogenous gibberellin in plants. By constructing an overexpression vector of the gene and transforming sugarcane plants, extension of sugarcane stalks can be remarkably inhibited, so that dwarf varieties are cultivated to improve lodging resistance. The gene and the method provided by the invention provide a new technical means for improving the plant type of the sugarcane.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, in particular to a sugarcane gibberellin oxidase gene GA2ox1 and an application thereof in regulating plant strain growth. Background Art

[0002] Gibberellic acid oxidase (GA2ox) is a key enzyme in gibberellin metabolism. Studies have shown that gibberellin oxidase is involved in many processes of plant growth and development, such as internode elongation, root growth, fruit development, and abiotic stress responses. Gibberellin oxidase inactivates active gibberellins (such as GA1 and GA4) by oxidizing them, thereby regulating plant height. Currently, research on the GA2ox1 gene has focused on crops such as rice, corn, and carrots. For example, patent CN 116083452 B, "A carrot gibberellin oxidase gene and its expression and application," discloses an isolated carrot gibberellin oxidase gene, DcGA20ox2, which can increase the biomass of carrot plants aboveground, change the structure of carrot fleshy roots, and regulate the degree of lignification of carrot fleshy roots. Patent CN 111778265 B, "A corn gibberellin oxidase mutant gene, mutant, expression vector, and application," discloses a corn gibberellin oxidase that can improve drought tolerance in Arabidopsis. However, there are currently no reports on the isolation and application of gibberellin oxidase-related functional genes in sugarcane. Existing sugarcane varieties generally have the problem of lodging due to their stems being too high, which restricts the increase in yield. Summary of the Invention

[0003] To address these shortcomings, the present invention provides a sugarcane gibberellin oxidase gene, GA2ox1, and its application in regulating sugarcane growth. The GA2ox1 gene was cloned from sugarcane for the first time and verified to significantly inhibit sugarcane stem elongation and improve lodging resistance. The specific technical solution is as follows:

[0004] A sugarcane gibberellin oxidase gene GA2ox1, the nucleotide sequence of the sugarcane gibberellin oxidase gene GA2ox1 is shown in SEQ ID NO: 1:

[0005]

[0006] The present invention also provides a sugarcane gibberellin oxidase, which is encoded by the sugarcane gibberellin oxidase gene GA2ox1, and has an amino acid sequence as shown in SEQ ID NO: 2:

[0007] MVAITAPSSIEQIPLVQCPRANVGQQAGAAAIPCVDLSAPGAAAAVADACRSVGFFRATNHGVPARVAEALEARAMAFFALPAQEKLDMSGAARPMGYGSKSIGSNGDVGWLEYLLLSVSANTVKVSSLPPSLRAALEEYTAAVREVCGRVLELIAEGLGVDRSLLRAMVVGREGSDELVRVNHY PPCPLLPPVDCGVTGFGEHTDPQIISVLRSNRTAGLQIKLRDGRWVPVPPAPDSFFVNVGDALQVLTNGRFKSVKHRVVAPEGAQSRLSVIYFGGPAPSQRIAPLPQVMRDGEQSLYREFTWAEYKTAMYKTRLADHRLGPFELRATAATNTNGCMPPPPSADPHCNGSGTCLPPPQQQQVAEVH.

[0008] The encoded protein contains 314 amino acids and has a conserved 2-oxoglutarate-dependent oxidase domain.

[0009] The present invention also provides a recombinant expression vector containing the sugarcane gibberellin oxidase gene GA2ox1.

[0010] Furthermore, the recombinant expression vector comprises a constitutive promoter or a tissue-specific promoter.

[0011] The present invention also provides a transgenic plant comprising the gene GA2ox1 or the vector.

[0012] Furthermore, the transgenic plant is sugarcane or Arabidopsis thaliana.

[0013] The present invention also provides a method for inhibiting plant height, comprising introducing the gene GA2ox1 or the vector into a plant, and screening to obtain plants expressing sugarcane gibberellin oxidase.

[0014] Furthermore, the gene GA2ox1 or the vector is introduced into the plant through Agrobacterium-mediated method.

[0015] The present invention also provides an application of the gene in inhibiting plant growth.

[0016] Furthermore, the plant is sugarcane or Arabidopsis thaliana.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This study cloned the GA2ox1 gene from sugarcane for the first time. The protein encoded by this gene catalyzes the oxidation of active gibberellins, reducing the plant's endogenous gibberellin content. By constructing an overexpression vector for this gene and transforming sugarcane plants, the transgenic plants showed a 35%-50% decrease in endogenous GA1 content, a 25%-30% decrease in plant height, a 15% increase in stem diameter, and significantly improved lodging resistance. This suggests that this gene can significantly inhibit sugarcane stem elongation, thereby enabling the development of dwarf varieties with improved lodging resistance. The gene and method of this invention provide a new technical approach for improving sugarcane plant type and have broad application prospects.

[0019] 2. Compared with chemical dwarfing agents, the present invention achieves plant type improvement through gene editing, with stable effects and no residual risk. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0021] Figure 1 This is the electrophoresis diagram of the PCR amplification of the GA2ox1 gene in Example 1;

[0022] Figure 2 This is the electrophoresis diagram of 16 colony PCR tests in Example 1;

[0023] Figure 3 This is the bacterial cloning and detection diagram of the GA2ox1-F1R1 product in Example 2;

[0024] Figure 4 This is a gel image of the colony PCR test in Example 3, where M is Marker DL2000, 1-4 are GA2ox1 Agrocybe single colonies, CK is a blank control (water), and P is a positive control (plasmid);

[0025] Figure 5 This is a comparison of the plant heights of transgenic sugarcane plants and non-transgenic sugarcane plants screened in Example 4. CK represents non-transgenic sugarcane plants, and the treatment group represents transgenic sugarcane plants.

[0026] Figure 6 This is a comparison of the plant heights of the transgenic Arabidopsis plants and non-transgenic Arabidopsis plants screened in Example 5. The left side shows the non-transgenic Arabidopsis plants, and the right side shows the transgenic Arabidopsis plants. DETAILED DESCRIPTION

[0027] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0028] Example 1 Cloning of the coding region sequence of the sugarcane gibberellin oxidase gene GA2ox1

[0029] (1) Total RNA extraction from sugarcane leaves

[0030] Sugarcane seedling leaves were cut and ground into powder under liquid nitrogen. Total RNA was extracted according to the instructions of the TaKaRa MiniBEST Universal RNA Extraction Kit column-based RNA extraction kit (Bao Ri Yi Biotechnology (Beijing) Co., Ltd.).

[0031] (2) cDNA synthesis

[0032] Reference cDNA synthesis reagent PrimeScript TM IV 1st strand cDNA Synthesis Mix: Prepare the reverse transcription mixture according to the following reverse transcription system and place it in a 200 μL RNase-free PCR tube. Mix thoroughly, incubate at 30°C for 10 minutes, then incubate at 42°C for 20 minutes. Heat at 95°C for 5 minutes to inactivate the enzyme and cool on ice. The resulting cDNA should be stored at -20°C. All reagents used in the reaction were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd. The reverse transcription system is shown in Table 1:

[0033] Table 1

[0034]

[0035] (3) Amplification of target gene

[0036] Design the following primers for amplification:

[0037] F:5'-GGGATCCTCTAGAGATTGAGTAGAGTCCATACGCTCGCTC-3' (as shown in SEQ ID NO: 3 in the sequence listing)

[0038] R:5'-CCTGCAGGTCGACGATTTCTCCAAGCTAGGCAGTAGGCAC-3' (as shown in SEQ ID NO: 4 in the sequence listing)

[0039] Using sugarcane cDNA sequence as template, the cDNA sequence was amplified according to the following PCR reaction system. The electrophoresis diagram of GA2ox1 gene amplification is shown in the figure below. Figure 1 shown.

[0040] The PCR reaction system is shown in Table 2:

[0041] Table 2

[0042] Reagents Dosage Sugarcane cDNA 1ul 2×Gflex PCR Buffer 25ul Tks Gflex DNA Polymerase 1ul F(10uM) 1ul R(10uM) 1ul <![CDATA[dH2O]]> Make up 50uL

[0043] The corresponding PCR reaction procedures are as follows:

[0044] Pre-denaturation: 94°C for 1 min;

[0045] Denaturation: 98°C for 10 s, annealing: 55°C for 15 s, extension: 68°C for 60 s) × 35 cycles;

[0046] Extension: 72°C for 5 min; keep warm at 16°C.

[0047] The amplified gene sequence is shown in SEQ ID NO: 1.

[0048] (4) Connect the product to the pMD19-T vector for sequencing verification

[0049] ① Infusion cloning: (using the In-Fusion HD Cloning Kit)

[0050] Connection system Dosage pMD19-T 25ng snippet 25ng 5×In-Fusion HD Enzyme Premix 2ul <![CDATA[H2O]]> 10ul

[0051] Reaction conditions: 50°C, 15 min.

[0052] Take an appropriate amount of the ligation product and heat-transform it into E. coli Competent Cell DH5a (Cat. No. 9057), spread it on Amp-resistant plates, and culture it at 37°C overnight.

[0053] ② Bacteria PCR: Pick 16 colonies on each plate and perform PCR test. The electrophoresis results of PCR products are as follows: Figure 2 shown.

[0054] ③Product sequencing: Select an appropriate amount of the above PCR products for sequencing:

[0055] ④ Plasmid extraction: After analyzing and comparing the product sequencing results, errors and redundant sequences are eliminated, and the extracted plasmids are selected for sequencing after identification.

[0056] Example 2: Construction of recombinant expression vector

[0057] (1) Linearization of vector p5941-GFP-flag: Double-digest the plasmid p5941-GFP-flag with XhoI and XbaI. After digestion, perform agarose gel electrophoresis to detect the results. Cut the gel corresponding to the large vector fragment and use the Biotek gel recovery kit to recover the digestion product. The double-digestion reaction system is as follows:

[0058]

[0059] Reaction conditions: 37℃: enzyme digestion for 40 min; 65℃: inactivation for 20 min.

[0060] (2) Ligation of the target fragment and the vector: Add reaction buffer to the PCR tube, add the target DNA fragment (GA2ox1) and the above-mentioned linearized vector at a certain molar ratio, and finally add homologous recombinase. Mix well and keep warm at 37°C for 30 minutes.

[0061] (3) Transformation: Thaw a 50 μL tube of competent E. coli cells (competent cell efficiency > 5 × 106 cfu / μg) on ​​ice, gently tap the tube wall to resuspend the cells, add 10 μL of the previous step reaction solution to the competent cells, flick a few times, and incubate on ice for 30 min; heat shock in a 42°C water bath for 50 s and quickly place on ice for 2 min; add 700 μL LB liquid culture medium and incubate on a shaker at 37°C for 60 min; take 400 μL of bacterial solution in a clean bench and evenly spread it on a plate (kana resistance, final concentration is 50 mg / L). After the bacterial solution is absorbed by the solid culture medium, invert and incubate at 37°C overnight.

[0062] (4) Identification of positive clones: Colony PCR was used to identify positive clones. The detection primers are as follows:

[0063] Primer name Sequence (5'-3') Sequence Listing PF AGAAGACGTTCCAACCACG SEQ ID NO:5 PR CGGTAAGGATCTGAGCTACAC SEQ ID NO:6

[0064] GA2ox1-F1R1 product clone test bacteria such as Figure 3 As shown, the recombinant expression vector was preserved after successful identification.

[0065] Example 3: Transformation of the recombinant expression vector into Agrobacterium EHA105

[0066] Take out 50uL of EHA105 competent cells (competent cell efficiency should be > 5x10 6 cfu / μg), thawed on ice;

[0067] Add 5uL of the recombinant expression vector of Example 2, mix gently, and place on ice for 5 minutes; freeze in liquid nitrogen for 5 minutes; place in a 37°C water bath for 5 minutes; place on ice for 5 minutes; add 700uL of resistance-free LB liquid medium, shake at 28°C for 2 hours; take 400uL

[0068] The bacterial solution was spread on the plate (kana + rif, final concentration of 50 mg / L), and cultured upside down at 28°C. After single colonies grew, 4 single colonies were selected for each gene for colony PCR verification.

[0069] Detection primers:

[0070] Primer name Sequence (5'-3') Sequence Listing PF AGAAGACGTTCCAACCACG SEQ ID NO:7 PR CGGTAAGGATCTGAGCTACAC SEQ ID NO:8

[0071] Detection glue picture Figure 4 shown.

[0072] Example 4: Sugarcane genetic transformation

[0073] Sugarcane embryogenic callus was infected with Agrobacterium EHA105 transformed as described in Example 3, and transgenic plants were obtained by hygromycin selection. The endogenous GA1 content, plant height, and stem diameter of the resulting transgenic sugarcane plants were measured. High-performance liquid chromatography (HPLC) coupled with mass spectrometry (e.g., LC-MS / MS) was used to determine endogenous GA1 content. Plant height is measured from the base of the sugarcane plant (at ground level) to the tip of the highest leaf or the top of the stem. Stem diameter is the cross-sectional diameter of the mid-section of the sugarcane stem.

[0074] The results showed that the endogenous GA1 content of the transgenic plants decreased by 35%-50%, the plant height decreased by 25%-30%, and the stem diameter increased by 15%, indicating that the lodging resistance of the transgenic sugarcane plants was significantly improved. Figure 5 As shown in the figure, the treated group (transgenic plants) are shorter than the CK (non-transgenic plants).

[0075] Example 5: Arabidopsis genetic transformation

[0076] The Arabidopsis embryogenic callus was infected with Agrobacterium EHA105 transformed in Example 3, and transgenic plants were obtained by hygromycin screening. The height comparison of the screened transgenic Arabidopsis plants and non-transgenic plants is shown in the figure below. Figure 6 As shown, transgenic Arabidopsis is shorter.

[0077] In summary, the protein encoded by the sugarcane gibberellin oxidase gene GA2ox1 can catalyze the oxidation of active gibberellins, reducing the plant's endogenous gibberellin content. By constructing an overexpression vector for this gene and transforming sugarcane plants, it is possible to significantly inhibit sugarcane culm elongation, thereby cultivating dwarf varieties with improved lodging resistance.

[0078] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A sugarcane gibberellin oxidase gene GA2ox1, characterized in that: The nucleotide sequence of the sugarcane gibberellin oxidase gene GA2ox1 is shown in SEQ ID NO:

1.

2. A sugarcane gibberellin oxidase, characterized in that The sugarcane gibberellin oxidase is encoded by the sugarcane gibberellin oxidase gene GA2ox1 according to claim 1, and its amino acid sequence is shown in SEQ ID NO:

2.

3. A recombinant expression vector, characterized in that: The recombinant expression vector contains the sugarcane gibberellin oxidase gene GA2ox1 according to claim 1.

4. A recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector comprises a constitutive promoter or a tissue-specific promoter.

5. A transgenic plant, characterized in that: The method comprises the gene GA2ox1 according to claim 1 or the vector according to claim 3.

6. A transgenic plant according to claim 5, characterized in that: The transgenic plant is sugarcane or Arabidopsis thaliana.

7. A method for suppressing plant height, characterized in that: The method comprises introducing the gene GA2ox1 according to claim 1 or the vector according to claim 3 into a plant body, and screening to obtain plants expressing sugarcane gibberellin oxidase.

8. The method for suppressing sugarcane plant height according to claim 6, characterized in that: The gene GA2ox1 or the vector is introduced into the plant through Agrobacterium-mediated method.

9. Use of the gene according to claim 1 in inhibiting plant growth.

10. The use according to claim 9, characterized in that The plant is sugarcane or Arabidopsis thaliana.

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