Soybean gibberellin 3-site oxidase GmGA3ox5 and application thereof

Abstract

The invention discloses soybean gibberellin 3-site oxidase GmGA3ox5 and application thereof, and belongs to the technical field of biology. The technical problem to be solved by the invention is how to prepare GA4. The soybean gibberellin 3-site oxidase GmGA3ox5 disclosed by the invention is A1), A2) or A3) as follows: A1) a protein of which the amino acid sequence is 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 GmGA3ox5 can catalyze GA9 to generate GA4, has the effect of gibberellin 3-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 soybean gibberellin 3-oxidase GmGA3ox5 and its application. Background Art

[0002] Gibberellins (GAs), as a class of important plant hormones, can regulate various stages of plant growth and development, including seed dormancy and germination, fruit ripening, induction of flowering, and the growth of stems and even whole plants. More than 130 types of gibberellins have been discovered and named so far, and the biologically active gibberellins reported mainly include GA1, GA3, GA4, and GA7. In agricultural production, gibberellins are widely used. For example, in vegetable cultivation, gibberellins can promote plant growth and increase yields. In fruit cultivation, gibberellins can promote fruit growth and ripening. GA4 is one of the main active gibberellins in plants, especially playing a leading role in Arabidopsis thaliana and cucurbitaceae plants (such as cucumbers and pumpkins). As an efficient plant growth regulator, GA4 has wide applications in agriculture, horticulture, and forestry, such as inducing fruit setting in cucumbers, pears, and apples, and promoting fruit enlargement. The biosynthesis of gibberellins is a complex metabolic pathway involving multiple enzymatic reactions, and gibberellin 3-oxidase (GA3ox) is one of the key enzymes in this pathway, responsible for catalyzing the 3β-hydroxylation reaction on the gibberellin precursor molecule, thus directly affecting the biosynthesis rate and efficiency of gibberellins.

[0003] In soybean (Glycine max (L.) Merr.), gibberellin 3-oxidase is a key enzyme in the gibberellin biosynthesis pathway. Its activity determines the synthesis rate of gibberellins, which in turn affects the growth and development of soybeans. At present, the specific biochemical characteristics of soybean gibberellin 3-oxidase have not been fully analyzed. This is of great significance for understanding the mechanism of action of gibberellin 3-oxidase in soybean gibberellin biosynthesis, optimizing its activity, and its application in agricultural biotechnology. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to prepare GA4.

[0005] To solve the above technical problem, the present invention first provides a protein, which is derived from soybean (Glycine max (L.) Merr.), named GmGA3ox5, and GmGA3ox5 is as follows A1), A2), or A3):

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

[0007] A2) A protein having 98% or more identity and the same function as that of A1), 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;

[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 A2) above is a protein having 98% or more identity and the same function as the amino acid sequence of the protein shown in SEQ ID No.1. Identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web page on 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, 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 A2) above can be artificially synthesized, or its coding gene can be synthesized first and then expressed biologically.

[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 connecting the coding sequence of a tag to its 5′-end and / or 3′-end. Among them, the DNA molecule shown in SEQ ID No.2 encodes the GmGA3ox5 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 using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag can be an 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 GmGA3ox5.

[0014] In this article, the substance that regulates the content or activity of GmGA3ox5 can be a substance that regulates the expression of the gene encoding GmGA3ox5.

[0015] In this article, the substance that regulates the expression of the gene encoding GmGA3ox5 can be a substance that performs at least one of the following six regulations: 1) regulation at the transcriptional level of the gene; 2) regulation after gene transcription (i.e., regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of the RNA transport of the gene (i.e., 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 mRNA degradation of the gene; 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

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

[0017] B1) A nucleic acid molecule encoding GmGA3ox5;

[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] Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the protein GmGA3ox5 of the present invention by using known methods, such as directed evolution or point mutation methods. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of the protein GmGA3ox5 isolated from the present invention, as long as they encode the protein GmGA3ox5 and have the function of the protein GmGA3ox5, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0026] The above-mentioned 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 by 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, 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 the amino acid sequence, and then the identity value (%) can be obtained.

[0028] The above-mentioned more than 75% identity can be 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% identity.

[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 GmGA3ox5 protein (GmGA3ox5 gene expression cassette) refers to DNA that can express the GmGA3ox5 protein in a host cell. This DNA may not only include a promoter that initiates the transcription of the GmGA3ox5 gene, but also include a terminator that terminates the transcription of the GmGA3ox5 gene. Further, the expression cassette may also include an enhancer sequence.

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

[0033] B3) Specifically, the recombinant vector can be pMAL-c2x-MBP-GmGA3ox5. The pMAL-c2x-MBP-GmGA3ox5 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 GmGA3ox5 gene shown in SEQ ID No.2 in the sequence listing.

[0034] Among the above substances, the microorganism can be yeast, bacteria, algae or fungi. Among them, the bacteria can 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 GmGA3ox5 as a gibberellin 3β-hydroxylase also belongs to the protection scope of the present invention.

[0037] The application of GmGA3ox5, or a substance that regulates the content or activity of GmGA3ox5, in the preparation of gibberellin 3β-hydroxylase also belongs to the protection scope of the present invention.

[0038] The application of GmGA3ox5 or the substance that regulates the content or activity of GmGA3ox5 in the production of GA4, or in the preparation of a product for the production of GA4, 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 producing GA4, the method comprising: using GA9 as a substrate and performing a catalytic reaction with GmGA3ox5 to obtain GA4.

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

[0041] M1) 25-60°C;

[0042] M2) 25-50°C;

[0043] M3) 30-50°C;

[0044] M4) 30 - 42 °C;

[0045] M5) 30 - 37 °C.

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

[0047] N1) 7.5 - 8.5;

[0048] N2) 7.5 - 8.

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

[0050] In the above method, the reaction system may 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, 3.2 μM GA9, and 28.5 μg GmGA3ox5, made up to 100 μL with water.

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

[0053] Experimental results show that GmGA3ox5 of the present invention can catalyze the formation of GA4 from GA9, and it has the function of gibberellin 3 - hydroxylase. GmGA3ox5 of the present invention and substances that regulate the content or activity of GmGA3ox5 can be used for soybean molecular - assisted breeding.

[0054] The present invention will be further described in detail below in conjunction with specific embodiments. The embodiments given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following embodiments provided 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 1Expression of soybean gene GmGA3ox5 in different tissues. Among them, Cotyledon-1 to Cotyledon-2 are cotyledon tissues at the germination stage and seedling stage of soybean, Stem-1 to Stem-2 are stem tissues at the germination stage and seedling stage of soybean, Leaf Bud-1 to Leaf Bud-3 are leaf bud tissues at the germination stage, seedling stage and flower bud differentiation stage of soybean, Leaf-1 to Leaf-2 are mature leaf tissues at the seedling stage and flower bud differentiation stage of soybean, Flower-1 to Flower-3 are flower bud, flower bud and open flower tissues, Seed-bearing Pod is the seed-bearing pod tissue 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 the seed tissue at six weeks of pod development, Shoot Apical Meristem is the shoot apical meristem at the flower bud differentiation stage, and Root is the root tissue at the seedling stage.

[0056] Figure 2 Purification SDS-PAGE analysis of MBP-GmGA3ox5.

[0057] Figure 3 Results of the enzymatic reaction of GmGA3ox5. LC-MS was used to analyze the production of the end product GA4 in the enzymatic reaction (using GA9 as the substrate). High-resolution mass spectrometry was used to extract ions. The mass-to-charge ratio m / z 315.1602 corresponds to GA9, and the mass-to-charge ratio m / z 331.1551 corresponds to GA4.

[0058] Figure 4 In vitro biochemical parameters of GmGA3ox5 protein.

[0059] Figure 5 Effect of temperature on the enzyme activity of soybean GmGA3ox5. The enzyme activity at 37°C was defined as 100%.

[0060] Figure 6 Effect of pH value on the enzyme activity of soybean GmGA3ox5. The enzyme activity at pH 7.5 was defined as 100%. Specific Embodiments

[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 sources 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 GmGA3ox5 Protein and Its Encoding Gene

[0063] Using the cDNA of the stem tissue of soybean cultivar Williams 82 as a template, PCR amplification was carried out with primers GmGA3ox5 F (5′-ATGCCTTCACTCTCAGAAGCCT-3′) and GmGA3ox5 R (5′-CTAGCTAGGTGCACAAAGCCGA-3′) to obtain a PCR product of approximately 1 Kb (i.e., the coding region of the GmGA3ox5 gene). After sequencing, this PCR product was 1059 bp, and the coding sequence of the GmGA3ox5 gene is shown in SEQ ID No.2, encoding the GmGA3ox5 protein shown in SEQ ID No.1.

[0064] The expression of the GmGA3ox5 gene in different developmental stages and different tissues and organs of soybean was detected, and the results are shown in Figure 1 , and the GmGA3ox5 gene was highly expressed in the cotyledon tissue and roots during the germination period of soybean.

[0065] Example 2, GmGA3ox5 has gibberellin 3β-hydroxylase activity

[0066] 1. Obtaining of the recombinant plasmid pMAL-c2x-MBP-GmGA3ox5

[0067] 1) Total RNA was extracted from the stem meristem of soybean cultivar Williams 82 and reverse-transcribed into cDNA. Using this cDNA as a template, PCR amplification was carried out with a primer pair composed of EGmGA3ox5 F and EGmGA3ox5 R to obtain an amplification product. The primers used are as follows:

[0068] EGmGA3ox5 F: 5′-AAGGATTTCAGAATTC GGATCC ATGCCTTCACTCTCAGAAGCCT-3′ (the BamH I restriction enzyme recognition sequence is underlined);

[0069] EGmGA3ox5 R: 5′-AACGACGGCCAGTGCC AAGCTT CTAGCTAGGTGCACAAAGCCGA-3′ (the HindⅢ restriction enzyme recognition sequence is underlined).

[0070] 2) The amplification product obtained in step 1) was ligated to the vector backbone obtained by double digestion of pMAL-c2x vector (NEB, catalog number #E8000S) with BamH I and HindⅢ to obtain the recombinant plasmid pMAL-c2x-MBP-GmGA3ox5. According to the sequencing results, the correctly sequenced recombinant plasmid was structurally described as follows: pMAL-c2x-MBP-GmGA3ox5 is a recombinant vector obtained by replacing the DNA fragment between the BamH I and HindⅢ recognition sequences of the pMAL-c2x vector (the insertion site is between positions 2706 and 2727 according to the position indicated in the vector instruction manual) with the DNA molecule shown by nucleotides 1 to 1059 from the 5'-end of SEQ ID No.2 in the sequence listing. pMAL-c2x-MBP-GmGA3ox5 can express the MBP-GmGA3ox5 fusion protein with an MBP tag (expected molecular weight is about 82.3 kDa).

[0071] 2. Obtaining of recombinant strain BL21-pMAL-c2x-MBP-GmGA3ox5

[0072] The recombinant plasmid pMAL-c2x-MBP-GmGA3ox5 obtained in step 1 above was introduced into Escherichia coli BL21(DE3) to obtain the recombinant strain BL21-pMAL-c2x-MBP-GmGA3ox5. The pMAL-c2x vector was introduced into Escherichia coli BL21(DE3) to obtain the control strain BL21-pMAL-c2x.

[0073] 3. Preparation of MBP-GmGA3ox5 fusion protein

[0074] 1) The recombinant strain BL21-pMAL-c2x-MBP-GmGA3ox5 obtained in step 2 was inoculated into LB liquid medium (containing 100 μg / ml ampicillin) and cultured with shaking at 37°C (200 rpm) until OD 600 = 0.6, then IPTG was added (to make its final concentration in the culture system 0.2 g / l) and the culture conditions were changed to 16°C, and cultured with shaking at 200 rpm for 16 hours.

[0075] 2) The culture system completed in step 1) was collected, and the cells were collected by centrifugation.

[0076] 3) The cells obtained in step 2) above were sonicated, and the protein was purified by affinity chromatography using Amylose resin to obtain the MBP-GmGA3ox5 protein solution containing the purified MBP-GmGA3ox5 fusion protein. The SDS-PAGE electrophoresis results of the MBP-GmGA3ox5 protein solution are shown in Figure 2A band of approximately 82 kDa was obtained between protein markers 70 and 100, which was consistent with the expected molecular weight of the MBP-GmGA3ox5 fusion protein (82.3 kDa).

[0077] 4. Analysis of MBP-GmGA3ox5 fusion protease activity

[0078] MBP-GmGA3ox5 fusion protein enzyme catalyzed reaction system: The total reaction volume is 100 μL, including: 100 mmol / L Tris-HCl (pH 7.5), 1 mmol / L FeSO4, 10 mmol / L α-ketoglutarate, 10 mmol / L ascorbic acid, 5 mmol / L DTT, 3.2 μM GA9 and 28.5 μg purified MBP-GmGA3ox5 fusion protein, and water is added to make up to 100 μL.

[0079] The resulting reaction system was subjected to enzymatic reaction at 37°C for 2 hours, and then 100 μl of methanol was added to terminate the reaction to obtain the MBP-GmGA3ox5 product.

[0080] According to the above method, the "purified MBP-GmGA3ox5 fusion protein" was replaced with the "control protein" to obtain a control product.

[0081] Each product was detected by LC-qTOF-MS / MS, using GA9 (Shanghai Zhenzhun Biotechnology Co., Ltd., product number ZTR-G377525) and GA4 (Sigma-Aldrich, product number G7276) as standards. The LC-qTOF-MS / MS detection conditions were as follows:

[0082] The liquid chromatography model was Agilent 1290, and the mass spectrometry model was Agilent 6550QTOF;

[0083] The chromatographic column was a BEH C18 column (pore size 1.7 μm, length 2.1 × 100 mm);

[0084] Mobile phase: Phase A is water (containing 0.1% (volume percentage) formic acid), phase B is acetonitrile (containing 0.1% (volume percentage) formic acid);

[0085] The elution procedure is:

[0086] 0-0.1 minutes: 99% (volume percentage) phase B and 1% (volume percentage) phase A;

[0087] 0.1-15.5 minutes: 99% (volume percentage) of phase B and 1% (volume percentage) of phase A change at a uniform rate to 99.5% (volume percentage) of phase B and 0.5% (volume percentage) of phase A;

[0088] 15.5 - 17 minutes: 99.5% (volume percentage) of Phase B and 0.5% (volume percentage) of Phase A;

[0089] Flow rate: 0.3 mL / min; Column temperature: 35 °C. Capillary voltage: 4000 V; Carrier gas temperature: 225 °C; Dry gas flow rate: 13 L / min; Sheath gas temperature: 350 °C; Sheath gas flow rate: 12 L / min. Injection volume: 1 μL. Data acquisition was performed in negative ion mode (m / z 50–1700).

[0090] The results are as Figure 3 shown, and the MBP - GmGA3ox5 protein can catalyze the formation of GA4 from GA9.

[0091] In the LC - MS analysis of the enzymatic reaction, using GA9 as the substrate, the formation of the end - product GA4 is as follows: For high - resolution mass spectrometry extracted ions, the mass - to - charge ratio m / z 315.1602 corresponds to GA9, and the mass - to - charge ratio m / z 331.1551 corresponds to GA4 ( Figure 3 ).

[0092] Following the above steps, the substrate GA9 concentration in the reaction system was changed to 0.32, 0.63, 1.58, 2.53, 3.16, 6.32, 12.64 μM respectively to determine the in vitro biochemical parameters of the enzyme. The measurement results are shown in Figure 4 , indicating that GmGA3ox5 has a strong affinity for the substrate GA9.

[0093] Example 3. Effects of Temperature and pH on the Enzyme Activity of Soybean GmGA3ox5

[0094] Following the steps of "Activity Analysis of MBP - GmGA3ox5 Fusion Protein" in Example 2, the activity of the MBP - GmGA3ox5 fusion protein was detected when the reaction temperature (37 °C) was changed to 25, 30, 37, 42, 50, 60 °C respectively.

[0095] The results are as Figure 5 shown, and the activity of soybean GmGA3ox5 is the highest at 37 °C.

[0096] Following the steps of "Activity Analysis of MBP - GmGA3ox5 Fusion Protein" in Example 2, the activity of the MBP - GmGA3ox5 fusion protein was detected when the pH value of Tris - HCl in the enzyme activity analysis system (pH 7.5) was changed to 6.5, 7.0, 7.5, 8.0, 8.5 respectively, and other steps remained unchanged.

[0097] The results are as Figure 6 shown, and the activity of soybean GmGA3ox5 is the highest at pH 7.5.

[0098] The above has described the present invention in detail. For those skilled in the art, without departing from the gist and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments 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 cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some basic features can be applied within the scope of the following appended claims.

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 substituting and / or deleting and / or adding 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, characterized in that: 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 3-hydroxylase; or, use of the protein according to claim 1, or any of the substances according to claims 2-4 in the preparation of a gibberellin 3-hydroxylase.

6. Use of the protein according to claim 1 or any of the substances according to claims 2-4 in the production of GA4, or in the preparation of a product for producing GA4, or in plant breeding.

7. A method for producing GA4, comprising: Using the protein according to claim 1 to carry out a catalytic reaction with GA9 as a substrate to obtain GA4.

8. The method according to claim 7, wherein: The reaction is carried out under any one of the conditions of M1)-M5): M1)25～60℃； M2)25～50℃； M3)30～50℃； M4)30～42℃； M5)30～37℃； 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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