Biochemical activity and application of soybean gibberellin 2-site oxidase GmGA2ox17

Abstract

The invention discloses biochemical activity of soybean gibberellin 2-site oxidase GmGA2ox17 and application of the soybean gibberellin 2-site oxidase GmGA2ox17 and belongs to the technical field of biology. The technical problem to be solved by the invention is how to prepare GA34. The protein disclosed by the invention is named as GmGA2ox17, and is A1), A2) or A3) as follows: A1) a protein with an amino acid sequence of SEQ ID No.1; a2) a protein which is obtained by substitution and / or deletion and / or addition of amino acid residues on the amino acid sequence as shown in SEQ ID No.1 in the sequence table, has more than 98% of identity with A1) and has the same function as A1); and A3) a fusion protein obtained by connecting a label to the N end or / and C end of A1) or A2). Experiments prove that the GmGA2ox17 can catalyze GA4 to generate GA34, has the effect of gibberellin 2-position oxidase, and can be used for soybean molecule assisted breeding.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the biochemical activity and application of soybean gibberellin 2-oxidase GmGA2ox17. Background Art

[0002] Gibberellin (GA) is an important class of hormones in plants, which controls various aspects of plant growth and development, such as promoting seed germination, leaf growth, stem elongation, inducing flowering and fruit growth, etc. More than 130 types of gibberellins have been discovered and named so far, and the biologically active gibberellins reported are mainly GA1, GA3, GA4, and GA7. In agricultural production, the application of gibberellin is very extensive. For example, in vegetable cultivation, gibberellin can promote the growth of plants and increase yields. In fruit cultivation, gibberellin can promote the growth and ripening of fruits. GA 34 is an intermediate in the gibberellin metabolic pathway. Studying its synthesis and decomposition helps to understand the plant hormone regulation network. Plants lacking gibberellin usually show phenotypes such as short plants, late flowering, dark green and tufted leaves. It is precisely this dwarf phenotype that has important applications in crop breeding. The "Green Revolution" of rice and wheat characterized by semi-dwarfism that emerged in the 1960s utilized mutations in GA synthesis and signal transduction genes. Soybean (Glycine max (L.) Merr.) is an important food and cash crop originated from China, providing the main vegetable oil and protein resources for humans. However, in the past 60 years, the average yield per unit of soybeans has not had a significant breakthrough compared with other major food crops, and there is an urgent need for a "Green Revolution" in soybean production. However, the gibberellin synthesis and metabolism pathway in soybeans and its regulation are still poorly understood, which greatly limits the application of gibberellin to improve soybean yield and plant type.

[0003] The biosynthesis of gibberellin in plants is a complex metabolic process involving the regulation of multiple enzymes. These include geranylgeranyl pyrophosphate synthase (GGPPS), copalyl pyrophosphate synthase (CPS), ent-kaurene synthase (KS), gibberellin oxidase, etc. Gibberellin oxidase is a key enzyme in the processes of gibberellin biosynthesis and catabolism. According to the different action sites, gibberellin oxidase can be divided into gibberellin 20-oxidase (GA20ox), gibberellin 3-oxidase (GA3ox), gibberellin 13-oxidase (GA13ox), and gibberellin 2-oxidase (GA2ox). Based on the differences in substrate specificity, GA2ox enzymes are further divided into two major groups: C20-GA2ox and C19-GA2ox. C20-GA2oxs mainly catalyze the intermediates in the gibberellin biosynthesis pathway, such as GA 12 、GA 53The oxidation reaction, while C19-GA2ox is involved in the catabolism of active gibberellins and their direct precursors, including GA1, GA 49 and other key molecules. C19-GA2ox affects plant growth and development by regulating the gibberellin levels in plants, such as plant height, seed germination, flowering, and fruiting. However, relatively few studies have been conducted on the activity of C19-GA2ox enzymes in soybeans, which limits the comprehensive understanding of the soybean growth and development regulation network. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to prepare GA 34 .

[0005] To solve the above technical problem, the present invention first provides a protein, which is derived from Glycine max (L.) Merr. and is named GmGA2ox17. GmGA2ox17 is one of the following A1), A2), or A3):

[0006] A1) A protein with an amino acid sequence of SEQ ID No.1;

[0007] A2) A protein that has undergone substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in SEQ ID No.1 in the sequence listing, has more than 98% identity with A1), and has the same function;

[0008] A3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0009] The protein in the above A2) is a protein that has 98% or more identity with the amino acid sequence of the protein shown in SEQ ID No.1 and has the same function. Identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively and performing a search to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained. The 98% or more identity means 98% or 99% identity.

[0010] The protein in the above A2) can be artificially synthesized or can be obtained by first synthesizing its coding gene and then performing biological expression.

[0011] The coding gene of the protein in A2) above can be obtained by deleting the codons of one or several amino acid residues in the DNA sequence shown in SEQ ID No. 2, and / or performing missense mutations of one or several base pairs, and / or ligating the coding sequences of tags at its 5′ end and / or 3′ end. Among them, the DNA molecule shown in SEQ ID No. 2 encodes the GmGA2ox17 protein shown in SEQ ID No. 1.

[0012] The tag in A3) can be a polypeptide or protein that is fused and expressed with the target protein by using in vitro DNA recombination technology, so as to facilitate the expression, detection, tracing and / or purification of the target protein. The tag can be MBP tag, Poly-Arg, Poly-His, FLAG, Strep-tag II, c-myc, HA tag, GST tag and / or SUMO tag, etc.

[0013] The present invention also provides a substance for regulating the content or activity of GmGA2ox17.

[0014] In this article, the substance for regulating the content or activity of GmGA2ox17 can be a substance for regulating the expression of the GmGA2ox17 coding gene.

[0015] In this article, the substance for regulating the expression of the GmGA2ox17 coding gene can be a substance that performs at least one of the following six regulations: 1) regulation at the gene transcription level; 2) regulation after gene transcription (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of the RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) regulation after translation of the gene (that is, regulation of the activity of the protein translated by the gene).

[0016] The above substance can be any one of the following B1) to B7):

[0017] B1) A nucleic acid molecule encoding GmGA2ox17;

[0018] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0019] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0020] B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0021] B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2);

[0022] B6) A transgenic plant tissue containing the nucleic acid molecule described in B1), or a transgenic plant tissue containing the expression cassette described in B2);

[0023] B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2).

[0024] Among the above substances, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0025] A person of ordinary skill in the art can easily mutate the nucleotide sequence encoding the protein GmGA2ox17 of the present invention by using known methods, such as directed evolution or site-directed mutagenesis. Those nucleotides that have been artificially modified and have 75% or more identity with the nucleotide sequence of the protein GmGA2ox17 isolated from the present invention, as long as they encode the protein GmGA2ox17 and have the function of the protein GmGA2ox17, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0026] The above 75% or more identity can be 80%, 85%, 90% or more than 95% identity.

[0027] Identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI homepage website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and performing a search to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0028] The identity of more than 75% may be an identity of at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0029] B1) The nucleic acid molecule may be a DNA molecule whose coding sequence is SEQ ID No.2 in the sequence listing.

[0030] Specifically, the nucleic acid molecule in B1) may be the DNA molecule shown in SEQ ID No.2.

[0031] B2) The expression cassette containing the nucleic acid molecule encoding the GmGA2ox17 protein (GmGA2ox17 gene expression cassette) refers to DNA that can express the GmGA2ox17 protein in a host cell. This DNA may not only include a promoter that initiates the transcription of the GmGA2ox17 gene, but also include a terminator that terminates the transcription of the GmGA2ox17 gene. Further, the expression cassette may also include an enhancer sequence.

[0032] Among the above substances, the vector may be a plasmid, cosmid, phage or viral vector. Specifically, the plasmid may be the pMAL-c2x vector.

[0033] B3) The recombinant vector may specifically be pMAL-c2x-MBP-GmGA2ox17. The pMAL-c2x-MBP-GmGA2ox17 is a recombinant vector obtained by replacing the DNA fragment between the BamH I and HindⅢ recognition sequences of the pMAL-c2x vector with the GmGA2ox17 gene shown in SEQ ID No.2 in the sequence listing.

[0034] Among the above substances, the microorganism may be yeast, bacteria, algae or fungi. Among them, the bacteria may be from the genus Escherichia (such as Escherichia coli BL21(DE3)), Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus, etc.

[0035] Among the above substances, the transgenic plant cell line, transgenic plant tissue and transgenic plant organ do not include propagation materials.

[0036] The application of GmGA2ox17 as a gibberellin 2-oxidase also belongs to the protection scope of the present invention.

[0037] The application of GmGA2ox17, or a substance that regulates the content or activity of GmGA2ox17, in the preparation of gibberellin 2-oxidase also belongs to the protection scope of the present invention.

[0038] The application of GmGA2ox17 or the substance that regulates the content or activity of GmGA2ox17 in the production of GA 34 or in the preparation of a product for the production of GA 34 or in the breeding of plants (such as soybeans) also belongs to the protection scope of the present invention.

[0039] The present invention also provides a method for the production of GA 34 The method includes: using GA4 as a substrate and conducting a catalytic reaction with GmGA2ox17 to obtain GA 34 .

[0040] In the above method, the reaction can be carried out under any one of the conditions of M1)-M5):

[0041] M1) 20 - 60 °C;

[0042] M2) 20 - 50 °C;

[0043] M3) 20 - 42 °C;

[0044] M4) 20 - 37 °C;

[0045] M5) 25 - 30 °C.

[0046] In the above method, the reaction can be carried out in a system with the following pH of N1) or N2):

[0047] N1) 7.5 - 8.5;

[0048] N2) 7.5 - 8.

[0049] In the above method, the reaction can be carried out in a Tris buffer solution (such as 100 mM Tris buffer solution).

[0050] In the above method, the reaction system can also contain FeSO4, α-ketoglutaric acid, ascorbic acid, and / or DTT.

[0051] In an embodiment of the present invention, the reaction system is: 100 mM Tris-HCl (pH 7.5), 1 mM FeSO4, 10 mM α-ketoglutaric acid, 10 mM ascorbic acid, 5 mM DTT, 3 μM GA4, and 13.6 μg GmGA2ox17, and water is added to make up to 100 μL.

[0052] In an embodiment of the present invention, the reaction time is 2 hours.

[0053] Experimental results have shown that GmGA2ox17 of the present invention can catalyze the formation of GA from GA4 34 , which has the function of gibberellin 2-oxidase. Therefore, GA can be produced in vitro using GmGA2ox17 34 , laying a foundation for the in vitro synthesis of its downstream compounds and the like. On the other hand, GmGA2ox17 of the present invention and substances that regulate the content or activity of GmGA2ox17 can be used for soybean molecular-assisted breeding to prepare lines with altered endogenous gibberellin levels. The obtained lines can be used as breeding resources to accelerate the cultivation process of related plant varieties. The obtained lines can also be used to study which other genes are involved in the synthesis and metabolism of gibberellin after the change in the expression level of the GmGA2ox17 gene. The present invention provides new germplasm resources for genetic breeding work, provides new materials for soybean variety breeding, and has a positive effect on accelerating the improvement of soybean varieties.

[0054] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not limit the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 . Expression of soybean gene GmGA2ox17 in different tissues. Among them, cotyledon-1 to cotyledon-2 are cotyledon tissues of soybean during germination and seedling stages, stem-1 to stem-2 are stem tissues of soybean during germination and seedling stages, leaf bud-1 to leaf bud-3 are leaf bud tissues of soybean during germination, seedling stages, and flower bud differentiation stage, leaf-1 to leaf-2 are mature leaf tissues of soybean during seedling stage and flower bud differentiation stage, flower-1 to flower-3 are flower bud, flower bud, and open flower tissues, pod with seeds is pod tissue with seeds at four weeks of pod development, pod-1 to pod-2 are pod tissues at three weeks and four weeks of pod development, seed is seed tissue at six weeks of pod development, apical meristem is apical meristem during flower bud differentiation, and root is root tissue during seedling stage.

[0056] Figure 2 For SDS-PAGE analysis of MBP-GmGA2ox17 purification.

[0057] Figure 3 For the results of the enzymatic reaction of GmGA2ox17. LC-MS / QTOF analyzes the formation of the final product GA (using GA4 as the substrate) in the enzymatic reaction. High-resolution mass spectrometry extracts ions, with a mass-to-charge ratio of m / z 331.1551 corresponding to GA4 and a mass-to-charge ratio of m / z 347.15 corresponding to GA 34 ; 34 .

[0058] Figure 4 They are the in vitro biochemical parameters of the GmGA2ox17 protein.

[0059] Figure 5 They are the effects of temperature on the enzyme activity of soybean GmGA2ox17. The enzyme activity at 25 °C was defined as 100%.

[0060] Figure 6 They are the effects of pH on the enzyme activity of soybean GmGA2ox17. The enzyme activity at pH 7.5 was defined as 100%. Detailed implementation manners

[0061] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples can be obtained from commercial channels unless otherwise specified. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5'-terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3'-terminal nucleotide of the corresponding DNA / RNA.

[0062] Example 1. Obtaining of the GmGA2ox17 protein and its encoding gene

[0063] Using the cDNA of the stem tissue of soybean variety Williams 82 as a template, PCR amplification was carried out with primers GmGA2ox17 F (5'-ATGGTGTTGTTGTCCAAAGCAAC-3') and GmGA2ox17 R (5'-TTACGAAGCTGCAATTCTTTCAAAATGT-3') to obtain a PCR product of about 1 Kb (i.e., the coding region of the GmGA2ox17 gene). After sequencing, this PCR product was 984 bp. The coding sequence of the GmGA2ox17 gene is shown in SEQ ID No. 2 and encodes the GmGA2ox17 protein shown in SEQ ID No. 1.

[0064] The expression of the GmGA2ox17 gene in different development stages and different tissue organs of soybean was detected, and the results are shown in Figure 1 , and the GmGA2ox17 gene was highly expressed in the pod tissue, shoot apical meristem and root at three weeks of pod development.

[0065] Example 2. GmGA2ox17 has gibberellin 2-oxidase enzyme activity

[0066] 1. Obtaining of the recombinant plasmid pMAL-c2x-MBP-GmGA2ox17

[0067] 1) Total RNA was extracted from the stem tissues of the soybean cultivar Williams 82, reverse transcribed into cDNA, and using this cDNA as a template, PCR amplification was performed with the primer pair consisting of EGmGA2ox17 F and EGmGA2ox17 R to obtain an amplification product. The primers used are as follows:

[0068] EGmGA2ox17 F: 5′-AAGGATTTCAGAATTC GGATCC ATGGTGTTGTTGTCCAAAGCAAC-3′ (the BamH I restriction enzyme recognition sequence is underlined);

[0069] EGmGA2ox17 R: 5′-AACGACGGCCAGTGCC AAGCTT TTACGAAGCTGCAATTCTTTCAAAATGT-3′ (the HindⅢ restriction enzyme recognition sequence is underlined).

[0070] 2) The amplification product from step 1) was ligated to the vector backbone obtained by double digestion of the pMAL-c2x vector (NEB, catalog number #E8000S) with BamH I and HindⅢ to obtain the recombinant plasmid pMAL-c2x-MBP-GmGA2ox17. According to the sequencing results, the correctly sequenced recombinant plasmid was structurally described as follows: pMAL-c2x-MBP-GmGA2ox17 is a recombinant vector obtained by replacing the DNA fragment between the BamH I and HindⅢ recognition sequences of the pMAL-c2x vector (as indicated by the position in the vector instruction manual, the insertion section is between positions 2706 and 2727) with the GmGA2ox17 gene shown in SEQ ID No.2 in the sequence listing. pMAL-c2x-MBP-GmGA2ox17 can express the MBP-GmGA2ox17 fusion protein with an MBP tag (expected molecular weight is approximately 80.4 kDa).

[0071] 2. Obtaining the recombinant bacterium BL21-pMAL-c2x-MBP-GmGA2ox17

[0072] The recombinant plasmid pMAL-c2x-MBP-GmGA2ox17 obtained in the above step 1 was introduced into Escherichia coli BL21(DE3) to obtain the recombinant bacterium BL21-pMAL-c2x-MBP-GmGA2ox17. The pMAL-c2x vector was introduced into Escherichia coli BL21(DE3) to obtain the control bacterium BL21-pMAL-c2x.

[0073] 3. Preparation of the MBP-GmGA2ox17 fusion protein

[0074] 1) Inoculate the recombinant bacterium BL21-pMAL-c2x-MBP-GmGA2ox17 obtained in step 2 into an LB liquid medium (containing 100 μg / mL ampicillin), and culture it with shaking at 37 °C (200 rpm) until OD 600 = 0.6, then add IPTG (to make its final concentration in the culture system 0.2 g / L), and at the same time change the culture conditions to 16 °C and culture with shaking at 200 rpm for 16 hours.

[0075] 2) Collect the culture system completed in step 1), and collect the bacterial cells by centrifugation.

[0076] 3) Ultrasonically disrupt the bacterial cells obtained in step 2) above, and perform affinity chromatography purification of the protein using Amylose resin to obtain an MBP-GmGA2ox17 protein solution containing the purified MBP-GmGA2ox17 fusion protein. The SDS-PAGE electrophoresis result of the MBP-GmGA2ox17 protein solution is shown in Figure 2 , and a band with a size of approximately 80 kDa is obtained between the protein marker 70 and 100, which is consistent with the expected molecular weight of the MBP-GmGA2ox17 fusion protein (80.4 kDa).

[0077] According to the methods of steps 1)-3) above, replace "recombinant bacterium BL21-pMAL-c2x-MBP-GmGA2ox17" with "control bacterium BL21-pMAL-c2x" to obtain a control protein.

[0078] 4. Activity analysis of MBP-GmGA2ox17 fusion protein

[0079] The enzymatic reaction system of MBP-GmGA2ox17 fusion protein: The total reaction volume is 100 μL, including: 100 mM Tris-HCl (pH 7.5), 1 mM FeSO4, 10 mM α-ketoglutaric acid, 10 mM ascorbic acid, 5 mM DTT, 3 μM GA4, and 13.6 μg of the purified MBP-GmGA2ox17 fusion protein, and add water to make up to 100 μL.

[0080] Incubate the obtained reaction system at 25 °C for an enzymatic reaction for 2 hours, then add 100 μL of methanol to terminate the reaction to obtain an MBP-GmGA2ox17 product.

[0081] According to the above method, replace "purified MBP-GmGA2ox17 fusion protein" with "control protein" to obtain a control product.

[0082] Detect each product using LC-qTOF-MS / MS, using GA4 (Sigma-Aldrich, catalog number G7276) and GA 34(Shanghai Zhenzhun Biotechnology Co., Ltd., product number ZTR-G377370)) was used as the standard, and the LC-qTOF-MS / MS detection conditions were as follows:

[0083] The liquid chromatography model was Agilent 1290, and the mass spectrometry model was Agilent 6550 QTOF;

[0084] The chromatographic column was a BEH C18 column (pore size 1.7 microns, length 2.1 × 100 mm);

[0085] Mobile phase: Phase A was water (containing 0.1% (volume percentage) formic acid), and Phase B was acetonitrile (containing 0.1% (volume percentage) formic acid);

[0086] The elution program was as follows:

[0087] 0 - 0.1 minute: 99% (volume percentage) of Phase B and 1% (volume percentage) of Phase A;

[0088] 0.1 - 15.5 minutes: Uniformly changed from 99% (volume percentage) of Phase B and 1% (volume percentage) of Phase A to 99.5% (volume percentage) of Phase B and 0.5% (volume percentage) of Phase A;

[0089] 15.5 - 17 minutes: 99.5% (volume percentage) of Phase B and 0.5% (volume percentage) of Phase A;

[0090] Flow rate 0.3 mL / min; column temperature 35 °C. Capillary voltage 4000 V; carrier gas temperature, 225 °C; drying gas flow rate, 13 L / min; sheath gas temperature 350 °C; sheath gas flow rate 12 L / min. Injection volume was 1 μL, and data acquisition was in the negative ion mode (m / z 50–1700).

[0091] The results were as Figure 3 shown, and the MBP-GmGA2ox17 fusion protein could catalyze the formation of GA from GA4 34 .

[0092] In the LC-MS analysis of the enzymatic reaction, using GA4 as the substrate, the formation of the final product GA 34 was as follows: For high-resolution mass spectrometry extracted ions, the mass-to-charge ratio m / z 331.1551 corresponded to GA4, and the mass-to-charge ratio m / z 347.15 corresponded to GA 34 ( Figure 3 ).

[0093] According to the above steps, the substrate GA4 concentration in the reaction system was changed to 0.30, 0.60, 1.50, 2.40, 3.00, 6.02, 12.03 μM respectively to determine the in vitro biochemical parameters of the enzyme, and the determination results are shown in Figure 4, indicating that GmGA2ox17 has a strong affinity for the substrate GA4.

[0094] Example 3. Effects of Temperature and pH on the Enzyme Activity of Soybean GmGA2ox17

[0095] According to the steps of "Activity Analysis of MBP-GmGA2ox17 Fusion Protein" in Example 2, when changing the reaction temperature (25 °C) to 20, 25, 30, 37, 42, 50, and 60 °C respectively, the activity of the MBP-GmGA2ox17 fusion protein was detected, and other steps remained unchanged.

[0096] The results are as Figure 5 shown, and the activity of soybean GmGA2ox17 is the highest at 25 °C.

[0097] According to the steps of "Activity Analysis of MBP-GmGA2ox17 Fusion Protein" in Example 2, when changing the pH value of Tris-HCl (pH 7.5) in the enzyme activity analysis system to 6.5, 7.0, 7.5, 8.0, and 8.5 respectively, the activity of the MBP-GmGA2ox17 fusion protein was detected, and other steps remained unchanged.

[0098] The results are as Figure 6 shown, and the activity of soybean GmGA2ox17 is the highest at pH 7.5.

[0099] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific examples of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including those that deviate from the scope disclosed in this application but are made using conventional techniques known in the art. Some basic features can be applied according to the scope of the appended claims below.

Claims

1. A protein, which is any one of A1), A2) or A3) as follows: A1) A protein having an amino acid sequence of SEQ ID No.1; A2) A protein which is obtained by substitution and / or deletion and / or addition of amino acid residues to the amino acid sequence shown in SEQ ID No.1 in the sequence listing, has an identity of more than 98% with A1) and has the same function; A3) A fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of A1) or A2).

2. A substance for regulating the content or activity of the protein according to claim 1.

3. The substance according to claim 2, wherein: The substance is any one of the following B1) to B7): B1) A nucleic acid molecule encoding the protein according to claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) A transgenic plant tissue containing the nucleic acid molecule described in B1), or a transgenic plant tissue containing the expression cassette described in B2); B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2).

4. The substance according to claim 2, wherein: The nucleic acid molecule described in B1) is a DNA molecule having a coding sequence of SEQ ID No.2 in the sequence listing.

5. Use of the protein according to claim 1 as a gibberellin 2-oxidase; Or, use of the protein according to claim 1, or any substance described in claims 2-4 in the preparation of a gibberellin 2-oxidase.

6. Use of the protein according to claim 1 or the substance according to any one of claims 2-4 in the production of GA 34 or in the preparation of a product for the production of GA 34 or in plant breeding.

7. Method for producing GA 34 comprising: Using GA4 as a substrate, a catalytic reaction is carried out with the protein described in claim 1 to obtain GA 34 .

8. The method according to claim 7, characterized in that: The reaction is carried out under any one of the conditions of M1)-M5): M1)20～60℃； M2)20～50℃； M3)20～42℃； M4)20～37℃； M5)25～30℃； And / or, the reaction is carried out in a system with a pH of the following N1) or N2): N1)7.5～8.5； N2)7.5～8。 9. The method according to claim 7 or 8, characterized in that: The reaction is carried out in Tris buffer.

10. The method according to any one of claims 7-9, characterized in that: The reaction system further contains FeSO4, α-ketoglutaric acid, ascorbic acid and / or DTT.

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