Method for producing GA15 and used protein

Abstract

The invention discloses a method for producing GA15 and used protein, and belongs to the technical field of biology. The technical problem to be solved by the invention is how to prepare GA15. The method for producing the GA15 disclosed by the invention comprises the following steps: by taking GA12 as a substrate, carrying out catalytic reaction by utilizing GmGA20ox7 to obtain the GA15, the GmGA20ox7 is the following A1), A2) or A3): A1) a protein with an amino acid sequence represented by 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 represented by SEQ ID No.1 in a sequence table, has more than 98% of identity with A1) and has the same function as A1), and A3) a fusion protein which is obtained by connecting a label to an N terminal or / and a C terminal of A1) or A2). The GmGA20ox7 can catalyze GA12 to generate GA15.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for producing GA 15 and the protein used. Background Art

[0002] Gibberellins (GAs), as an important class of 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. Currently, more than 130 types of gibberellins have been discovered and named, and the mainly reported bioactive gibberellins are GA1, GA3, GA4, and GA7. In agricultural production, gibberellins are widely used. For example, in vegetable cultivation, gibberellins can promote plant growth and increase yield. In fruit cultivation, gibberellins can promote fruit growth and ripening. GA 15 is an intermediate in the gibberellin biosynthesis pathway and is usually converted from precursor substances (such as GA 12 ) through a series of enzymatic reactions. GA 15 can be further metabolized into the bioactive gibberellin GA4. The biosynthesis of gibberellins is a complex metabolic pathway involving multiple enzymatic reactions, and gibberellin 20 oxidase (GA20ox) is at a key node position. It catalyzes the key steps of gibberellin biosynthesis, that is, converting precursor substances such as GA 12 and GA 53 into GA9 and GA 20 etc., and then generating the active gibberellins GA4 and GA1 through the action of gibberellin 3 oxidase, thus playing a decisive role in the active level and balance of gibberellins in plants. Summary of the Invention

[0003] The technical problem to be solved by the present invention is how to prepare GA 15 .

[0004] To solve the above technical problem, the present invention first provides a method for producing GA 15 , the method comprising: using GA 12 as a substrate and performing a catalytic reaction with a protein to obtain GA 15 ; the protein is derived from Glycine max ( Glycine max (L.) Merr.), and its name is GmGA20ox7, and GmGA20ox7 is one of the following A1), A2), or A3): A1) a protein with an amino acid sequence of SEQ ID No. 1; A2) A protein with the amino acid sequence shown as SEQ ID No.1 in the sequence listing, which has undergone substitution and / or deletion and / or addition of amino acid residues and has more than 98% identity with A1) and has the same function; A3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0005] In the above method, the reaction can be carried out at 25 - 42 °C.

[0006] Furthermore, the reaction can be carried out at 30 °C.

[0007] In the above method, the reaction can be carried out in a system with the following pH of N1) or N2): N1) 7.5 - 8.5; N2) 8.

[0008] In the above method, the reaction can be carried out in Tris buffer (such as 100 mM Tris buffer).

[0009] In the above method, the reaction system can also contain FeSO4, α-ketoglutaric acid, ascorbic acid and / or DTT.

[0010] In one embodiment of the present invention, the reaction system can be: 100 mM Tris-HCl (pH 8.0), 1 mM FeSO4, 10 mM α-ketoglutaric acid, 10 mM ascorbic acid, 5 mM DTT, 3 μM GA 12 and 14.4 μg GmGA20ox7, and make up to 100 μL with water.

[0011] In one embodiment of the present invention, the reaction time is 6 hours.

[0012] The protein in A2) above is a protein having 98% or more identity with the amino acid sequence of the protein shown in SEQ ID No.1 and having the same function. 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 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, 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 "having 98% or more identity" means having 98% or 99% identity.

[0013] The protein in A2) above can be artificially synthesized, or its coding gene can be synthesized first and then obtained by biological expression.

[0014] 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 sequences of tags at its 5′ end and / or 3′ end. Among them, the DNA molecule shown in SEQ ID No.2 encodes the GmGA20ox7 protein shown in SEQ ID No.1.

[0015] The tag in A3) can be a polypeptide or protein that is fused and expressed with the target protein by 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.

[0016] The above-mentioned GmGA20ox7 also belongs to the protection scope of the present invention.

[0017] The present invention also provides substances for regulating the content or activity of GmGA20ox7.

[0018] In this article, the substances for regulating the content or activity of GmGA20ox7 can be substances for regulating the expression of the GmGA20ox7 coding gene.

[0019] In this article, the substance that regulates the expression of the GmGA20ox7-encoding gene 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 transcription of the gene (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) regulation after translation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0020] The above-mentioned substance can be any one of the following B1) to B7): B1) A nucleic acid molecule encoding GmGA20ox7; 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).

[0021] Among the above-mentioned 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.

[0022] Those skilled in the art can easily mutate the nucleotide sequence encoding the protein GmGA20ox7 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 GmGA20ox7 isolated from the present invention, as long as they encode the protein GmGA20ox7 and have the function of the protein GmGA20ox7, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0023] The above-mentioned identity of 75% or more may be an identity of 80%, 85%, 90% or 95% or more.

[0024] Identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site 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, the identity value (%) of the amino acid sequence can be calculated.

[0025] The above-mentioned 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%.

[0026] B1) The nucleic acid molecule may be a DNA molecule whose coding sequence is SEQ ID No. 2 in the sequence listing.

[0027] Specifically, the nucleic acid molecule in B1) may be the DNA molecule shown in SEQ ID No. 2.

[0028] B2) The expression cassette containing the nucleic acid molecule encoding the GmGA20ox7 protein (GmGA20ox7 gene expression cassette) refers to DNA that can express the GmGA20ox7 protein in a host cell. This DNA may not only include a promoter that initiates the transcription of the GmGA20ox7 gene, but also include a terminator that terminates the transcription of the GmGA20ox7 gene. Further, the expression cassette may also include an enhancer sequence.

[0029] Among the above-mentioned substances, the vector may be a plasmid, cosmid, phage or viral vector. Specifically, the plasmid may be the pMAL-c2x vector.

[0030] B3) The recombinant vector may specifically be pMAL-c2x-MBP-GmGA20ox7. The pMAL-c2x-MBP-GmGA20ox7 is a recombinant vector obtained by replacing the DNA fragment between the BamH I and Hind III recognition sequences of the pMAL-c2x vector with the GmGA20ox7 gene shown in SEQ ID No.2 in the sequence listing.

[0031] Among the above substances, the microorganism may be yeast, bacterium, alga or fungus. Among them, the bacterium may be from the genus Escherichia ( Escherichia ), such as Escherichia coli BL21 (DE3), Erwinia ( Erwinia ), Agrobacterium tumefaciens ( Agrobacterium) ), Flavobacterium ( Flavobacterium) ), Alcaligenes ( Alcaligenes ), Pseudomonas ( Pseudomonas ), Bacillus ( Bacillus ), etc.

[0032] Among the above substances, the transgenic plant cell line, transgenic plant tissue and transgenic plant organ do not include propagation materials.

[0033] The application of GmGA20ox7 as a gibberellin 20-oxidase also belongs to the protection scope of the present invention.

[0034] The application of GmGA20ox7, or the substance that regulates the content or activity of GmGA20ox7 in the preparation of gibberellin 20-oxidase also belongs to the protection scope of the present invention.

[0035] The application of GmGA20ox7, or the substance that regulates the content or activity of GmGA20ox7 in the production of GA 15 , or in the preparation of products for the production of GA 15 , or in the breeding of plants (such as soybean) also belongs to the protection scope of the present invention.

[0036] Experimental results show that the GmGA20ox7 of the present invention can catalyze GA 12 to generate GA 15 . Therefore, GmGA20ox7 can be used for the in vitro production of GA 15 , laying a foundation for the in vitro synthesis of its downstream GA4, etc. On the other hand, the GmGA20ox7 of the present invention and the substance that regulates the content or activity of GmGA20ox7 can be used for soybean molecular assisted breeding.

[0037] 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 constitute any limitation to the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 . Soybean genes GmGA20ox7 Expression in different tissues. Among them, cotyledons are cotyledon tissues during soybean germination, stems - 1 to stems - 2 are stem tissues during soybean germination and seedling stages, leaf buds - 1 to leaf buds - 3 are leaf bud tissues during soybean germination, seedling stage and flower bud differentiation stage, leaves - 1 to leaves - 2 are mature leaf tissues during soybean seedling stage and flower bud differentiation stage, flowers - 1 to flowers - 3 are flower bud, flower bud and open flower tissues, pods are pod tissues at three weeks of pod development, seeds are seed tissues at six weeks of pod development, apical meristems are apical meristems during flower bud differentiation period, and roots are root tissues during seedling stage.

[0039] Figure 2 For the purified SDS - PAGE analysis of MBP - GmGA20ox7.

[0040] Figure 3 For the results of the enzymatic reaction of GmGA20ox7. LC - MS was used to analyze the production of the final product GA 12 (as the substrate) in the enzymatic reaction. 15 High - resolution mass spectrometry was used to extract ions. The mass - to - charge ratio m / z 331.1915 corresponds to GA 12 and the mass - to - charge ratio m / z 329.1758 corresponds to GA 15 . DETAILED DESCRIPTION OF THE EMBODIMENTS

[0041] In the following embodiments, the experimental methods 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 embodiments can be obtained from commercial sources unless otherwise specified.

[0042] Example 1. GmGA20ox7 is a protein with gibberellin 20 - oxidase enzyme activity The present invention discovers that the GmGA20ox7 protein derived from soybean Williams 82 has gibberellin 20 - oxidase enzyme activity. In soybean Williams 82, its amino acid sequence is as shown in SEQ ID No.1, and its CDS sequence is as shown in SEQ ID No.2. GmGA20ox7 The expression of the gene in different development stages and different tissues and organs is shown in Figure 1 ,GmGA2ox17 The gene is highly expressed in the mature leaf tissues of soybean seedlings and during flower bud differentiation.

[0043] 1. Obtaining the recombinant plasmid pMAL-c2x-MBP-GmGA20ox7 (1) Total RNA was extracted from the stem tissues of the soybean cultivar Williams 82 and reverse-transcribed to obtain cDNA. Using this cDNA as a template, PCR amplification was performed with the primer pair consisting of EGmGA20ox7 F and EGmGA20ox7 R to obtain an amplification product.

[0044] EGmGA20ox7 F: 5′-AAGGATTTCAGAATTC GGATCC ATGGCCATAGAGTGCATAACAAATATAC-3′ (the BamH I restriction enzyme recognition sequence is underlined); EGmGA20ox7 R: 5′-AACGACGGCCAGTGCC AAGCTT TCAGCTCATTTTCCGTTGAAGAAGC-3′ (the Hind Ⅲ restriction enzyme recognition sequence is underlined).

[0045] (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 Hind Ⅲ to obtain the recombinant plasmid pMAL-c2x-MBP-GmGA20ox7. According to the sequencing results, the structure of the correctly sequenced recombinant plasmid is described as follows: pMAL-c2x-MBP-GmGA20ox7 is a recombinant vector obtained by replacing the DNA fragment between the BamHI and Hind Ⅲ recognition sequences of the pMAL-c2x vector (the insertion segment position is between positions 2706 and 2727 according to the position indicated in the vector instruction manual) with the DNA molecule shown in SEQ ID No.2 in the sequence listing. pMAL-c2x-MBP-GmGA20ox7 can express the MBP-GmGA20ox7 fusion protein with an MBP tag (the expected molecular weight is approximately 86.5 kDa).

[0046] 2. Obtaining the recombinant strain BL21-pMAL-c2x-MBP-GmGA20ox7 The recombinant plasmid pMAL-c2x-MBP-GmGA20ox7 obtained in the above step 1 was introduced into Escherichia coli BL21(DE3) to obtain the recombinant strain BL21-pMAL-c2x-MBP-GmGA20ox7. The pMAL-c2x vector was introduced into Escherichia coli BL21(DE3) to obtain the control strain BL21-pMAL-c2x.

[0047] The construction steps are as follows: First, extract the recombinant plasmid obtained in Step 1. Pipette 100 ng of the recombinant plasmid into 100 μL of BL21(DE3) competent cells, gently mix, and place on ice for 30 minutes. Then, perform heat shock in a 42°C water bath for 90 seconds, quickly ice-bath for 5 minutes. Add 1 mL of LB medium in a laminar flow hood and incubate in a 37°C shaker for 1 hour. Then, centrifuge the bacterial solution at 6000 g rpm, discard the supernatant, re-add 100 μL of LB medium to resuspend the bacterial cells, evenly spread the resuspended bacterial cells on an LB plate with ampicillin resistance, and incubate at 37°C for 12 hours. Pick the grown colonies for PCR identification. If there are bands with appropriate sizes in the electrophoresis result, it proves that the recombinant strain construction is successful.

[0048] 3. Preparation of the fusion protein of soybean gibberellin 20-oxidase GmGA20ox7 1) Inoculate the recombinant bacteria obtained in Step 2 into LB liquid medium (containing 100 μg / mL ampicillin), and incubate 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 incubate with shaking at 200 rpm for 16 hours.

[0049] 2) Collect the culture system that has completed Step 1), and centrifuge to collect the bacterial cells.

[0050] 3) Ultrasonically disrupt the bacterial cells obtained in the above Step 2), and perform affinity chromatography purification of the protein using Amylose resin to obtain the purified MBP-GmGA20ox7 fusion protein solution. The SDS-PAGE electrophoresis result of the MBP-GmGA20ox7 protein solution is shown in Figure 2 , and a band with a size of approximately 86 kDa is obtained between the protein marker 70 and 100, which is consistent with the expected molecular weight of the MBP-GmGA20ox7 fusion protein (86.5 kDa).

[0051] 4. Activity determination of soybean gibberellin 20-oxidase GmGA20ox7 protein Enzymatic reaction system of soybean gibberellin 20-oxidase GmGA20ox7: The total reaction volume is 100 μL, including: 100 mmol / L Tris-HCl (pH 8.0), 1 mmol / L FeSO4, 10 mmol / L α-ketoglutaric acid, 10 mmol / L ascorbic acid, 5 mmol / L DTT, 3 μmol / L GA 12 and 14.4 μg of the purified MBP-GmGA20ox7 fusion protein, and make up to 100 μL with water.

[0052] The obtained reaction system was subjected to enzymatic reaction at 30 °C for 6 hours, and then 100 μL of methanol was added to terminate the reaction, yielding the MBP-GmGA20ox7 product.

[0053] According to the above method, "purified MBP-GmGA20ox7 fusion protein" was replaced with "control protein" to obtain the control product.

[0054] Each product was detected by LC-qTOF-MS / MS, using GA 12 (Sigma-Aldrich, 1164-45-0) and GA 15 (Shanghai Zhenzhun Biotechnology Co., Ltd., 13744-18-8) as standards. The LC-qTOF-MS / MS detection conditions were as follows: The liquid chromatography model was Agilent 1290, and the mass spectrometry model was Agilent 6550 QTOF; The chromatographic column was a BEH C18 column (pore size 1.7 μm, length 2.1 × 100 mm); 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); The elution program was as follows: 0 - 0.1 minute: 99% (volume percentage) of Phase B and 1% (volume percentage) of Phase A; 0.1 - 15.5 minutes: linearly changing 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; 15.5 - 17 minutes: 99.5% (volume percentage) of Phase B and 0.5% (volume percentage) of Phase A; Flow rate was 0.3 mL / min; column temperature was 35 °C. Capillary voltage was 4000 V; carrier gas temperature was 225 °C; drying gas flow rate was 13 L / min; sheath gas temperature was 350 °C; sheath gas flow rate was 12 L / min. The injection volume was 1 μL, and data acquisition was performed in the negative ion mode (m / z 50 - 1700).

[0055] The results were as Figure 3 shown. The MBP-GmGA20ox7 protein could catalyze GA 12 to generate GA 15 .

[0056] The above has described the present invention in detail. For those skilled in the art, without departing from the spirit 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 variations, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made by using conventional techniques known in the art. Some basic features can be applied within the scope of the following appended claims.

Claims

1. Method for producing GA 15 comprising: Using GA 12 as a substrate, a catalytic reaction is carried out using a protein to obtain GA 15 ; The protein is any of the following A1), A2), or A3): A1) A protein with an amino acid sequence of SEQ ID No. 1; A2) A protein that has more than 98% identity with A1) and has the same function after 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; A3) A fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of A1) or A2).

2. The method according to claim 1, wherein: The reaction is carried out at 25 - 42 °C; Furthermore, the reaction is carried out at 30 °C; And / or, the reaction is carried out in a system with a pH of N1) or N2) as follows: N1）7.5～8.5； N2）8。 3. The method according to claim 1 or 2, characterized in that: The reaction is carried out in Tris buffer.

4. The method according to any one of claims 1-3, characterized in that: The reaction system also contains FeSO4, α-ketoglutaric acid, ascorbic acid and / or DTT.

5. The protein described in claim 1.

6. A substance that regulates the content or activity of the protein described in claim 1.

7. The substance according to claim 6, characterized in that: The substance is any one of the following B1) to B7): B1) A nucleic acid molecule encoding the protein described in 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).

8. The substance according to claim 7, characterized in that: The nucleic acid molecule described in B1) is a DNA molecule with a coding sequence of SEQ ID No. 2 in the sequence listing.

9. The application of the protein described in claim 1 as a gibberellin 20-oxidase; Or, the application of the protein described in claim 1, or any of the substances described in claims 6 - 8 in the preparation of gibberellin 20-oxidase.

10. Use of the protein described in claim 1 or any of the substances described in claims 6 - 8 in the production of GA 15 or in the preparation of a product for the production of GA 15 or in plant breeding.

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