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CN114317474B - Glutathione S-transferase mutant with improved enzyme activity and application thereof - Google Patents

Glutathione S-transferase mutant with improved enzyme activity and application thereof Download PDF

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Publication number
CN114317474B
CN114317474B CN202111341341.1A CN202111341341A CN114317474B CN 114317474 B CN114317474 B CN 114317474B CN 202111341341 A CN202111341341 A CN 202111341341A CN 114317474 B CN114317474 B CN 114317474B
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slgst
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glutathione
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leu
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CN114317474A (en
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肖璐
段学武
江薰垣
万凯
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Institute Of Agricultural Quality Standards And Monitoring Technology Guangdong Academy Of Agricultural Sciences
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Institute Of Agricultural Quality Standards And Monitoring Technology Guangdong Academy Of Agricultural Sciences
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Abstract

The invention discloses a glutathione S-transferase mutant with improved enzyme activity and application thereof. Mutant SlGST T1 of glutathione-S-transferase K228A,R230A The amino acid sequence is shown in SEQ ID NO. 3. The invention obtains a mutant SlGST T1 of glutathione S-transferase by mutating lysine (Lys) at the 228 th site and arginine (Arg) at the 230 th site into alanine (Ala) through site-specific mutagenesis K228A,R230A Glutathione S-transferase and mutant SlGST T1 thereof are obtained by prokaryotic expression and protein purification K228A,R230A Protein, and the mutant SlGST T1 is detected and found by enzyme activity K228A,R230A The enzyme activity is obviously improved. The invention can provide technical reference for further research aiming at glutathione S-transferase in the aspects of biochemistry and molecular biology.

Description

Glutathione S-transferase mutant with improved enzyme activity and application thereof
The technical field is as follows:
the invention belongs to the technical field of biology, and particularly relates to a glutathione S-transferase mutant SlGST T1 with improved enzyme activity K228A,R230A And applications thereof.
The background art comprises the following steps:
glutathione-S-transferase (GST) is a large multifunctional gene family, widely distributed in eukaryotes and prokaryotes, is a key enzyme participating in glutathione catalytic reaction, and plays a biological function in multiple aspects of plant growth and development, response to biotic and abiotic stress, detoxification, biosynthesis of hormones, redox, metabolism and the like, and the improvement of the level of GSTs is helpful for maintaining the steady state of cellular redox and protecting organisms from being damaged by pressure and endotoxin.
The invention content is as follows:
the first purpose of the invention is to provide a mutant SlGST T1 of glutathione-S-transferase K228A,R230A The amino acid sequence is shown in SEQ ID NO. 3.
The second purpose of the invention is to provide a mutant SlGST T1 coding for the mutant K228A,R230A The gene of (1).
The third purpose of the invention is to provide a mutant containing coding SlGST T1 K228A,R230A A recombinant expression plasmid of the gene of (1).
Preferably, the expression plasmid is the expression vector pET28a (+).
The fourth purpose of the invention is to provide a host expression cell containing the recombinant expression plasmid.
Preferably, the host expression cell is E.coli.
A fifth object of the invention is a mutant SlGST T1 K228A,R230A Application in catalyzing glutathione combination reaction.
The sixth purpose of the invention is to provide a method for obtaining mutant SlGST T1 K228A,R230A The method of encoding a gene of (1), comprising the steps of:
(1) Carrying out PCR by taking an SlGST T1 gene as a template and taking sequences shown in SEQ ID NO.4 and SEQ ID NO.5 as primers to obtain a first PCR product, wherein the nucleotide sequence of the SlGST T1 gene is shown in SEQ ID NO. 1;
(2) Carrying out PCR by taking the first PCR product as a template and taking the sequences shown in SEQ ID NO.4 and SEQ ID NO.6 as primers to obtain a mutant SlGST T1 containing an enzyme cutting site K228A,R230A The coding gene of (4).
The PCR program of steps (1) and (2) is: pre-denaturation at 98 ℃ for 3min; denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 20s, extension at 72 ℃ for 60s,35 cycles; extension at 72 ℃ for 5min. The PCR system of the present invention is not particularly limited, and a conventional PCR system in the art may be used.
At present, no relevant report is available for improving the enzyme catalytic activity of glutathione S-transferase through site-directed mutagenesis, and the mutant SlGST T1 of the glutathione S-transferase is obtained by mutating lysine (Lys) at the 228 th site and arginine (Arg) at the 230 th site into alanine (Ala) through the site-directed mutagenesis K228A,R230A The glutathione S-transferase and the mutant SlGST T1 thereof are obtained by prokaryotic expression and protein purification K228A,R230A Protein, and the mutant SlGST T1 is detected and found by enzyme activity K228A ,R230A The enzyme activity is obviously improved. The invention can provide technical reference for further research aiming at glutathione S-transferase in the aspects of biochemistry and molecular biology.
Description of the drawings:
FIG. 1 shows SlGST T1-His and its mutant SlGST T1 in the examples of the present invention K228A,R230A His purified protein SDS-PAGE;
FIG. 2 is a comparison graph of enzyme activity measured by spectrophotometry in the examples of the present invention (a and b show that there is significant difference between the two groups of enzyme activity measured values, p is less than 0.05).
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Mutant SlGST T1 of glutathione S-transferase of the invention K228A,R230A The amino acid sequence is shown in SEQ ID NO. 3. Mutant SlGST T1 K228A,R230A The amino acid sequence of the SlGST T1 is mutated into Ala from Lys at the 228 th site and Arg at the 230 th site; the amino acid sequence of the SlGST T1 is shown in SEQ ID NO. 2. The method of the mutation is not particularly limited in the present invention, and a point mutation is preferable. The SlGST T1 is preferably derived from tomatoes, the gene sequence is shown as SEQ ID No.1, 711 base sequences are totally obtained, the protein encoded by the gene contains 236 amino acids, and the corresponding gene number in the tomato genome is Solyc12g056250.2.
The construction of a mutant of glutathione S-transferase, slGST T1, of the present invention is described in detail below K228A,R230A And a method ofAnd (4) testing the activity.
Example 1
Mutant SlGST T1 of glutathione S-transferase K228A,R230A The gene is obtained by a PCR mode, and the primer for PCR comprises a primer pair containing a restriction enzyme cutting site and a downstream primer containing a mutation site; the sequences of the primer pairs containing the enzyme cutting sites are shown as SEQ ID NO.4 and 5 (the amino acids of the enzyme cutting sites are thickened and the lower horizontal lines are marked); the sequences of the primer pairs containing the mutation sites are shown as SEQ ID NO.4 and 6 (amino acids at the mutation sites are bold).
1. Sample preparation: the tomato variety selected is AC (Solanum lycopersicum. Mill. Cv. Ailsa-Craig).
2. Cloning of the SlGST T1 Gene
Extracting total RNA in tomato fruits by a conventional hot boric acid method, carrying out reverse transcription on the extracted total RNA into cDNA by a method provided by a kit PrimeScriptTM RT Master Mix (Perfect Real Time, RR036A, taKara), and reacting: mixing X μ L RNA (< 500 ng), 2 μ L5 Xmix, and Y μ L RNase Free water to make total volume 10 μ L, placing in 37 deg.C constant temperature water bath for 15min in water bath, heating in 85 deg.C metal bath for 5s, and cooling at 4 deg.C.
Designing a primer containing a nde I enzyme cutting site and a primer containing a mutation site according to the sequence of an SlGST T1 gene in a tomato genome (https:// solgenomics. Net/tools/blast), wherein the sequences of the primers are shown in a table 1;
TABLE 1 cloning of SlGST T1 K228A,R230A Primers required for genes
Figure BDA0003352178100000041
Using tomato cDNA as a template, firstly respectively carrying out PCR by using an upstream primer (SEQ ID NO. 4) containing an enzyme cutting site and a downstream primer (SEQ ID NO. 5) to obtain a product 1, then using the product 1 as the template, carrying out PCR by using the upstream primer (SEQ ID NO. 4) containing an enzyme cutting site gene and the downstream primer (SEQ ID NO. 6) containing a mutation site to obtain a mutant SlGST T1 containing the enzyme cutting site K228A,R230A And (4) PCR products.
And carrying out PCR amplification by using tomato cDNA as a template and using an upstream primer (SEQ ID NO. 4) and a downstream primer (SEQ ID NO. 5) containing enzyme cutting sites to obtain an amplification product of the SlGST T1 gene.
The PCR conditions were: pre-denaturation at 98 ℃ for 3min, followed by 35 cycles (denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 20s, and extension at 72 ℃ for 60 s), and extension at 72 ℃ for 5min. After the PCR reaction was completed, the detection was performed by 1% agarose gel electrophoresis.
3. Construction of SlGST T1 expression Strain
Respectively amplifying product of SlGST T1 gene and SlGST T1 K228A,R230A The PCR product is recycled and is connected with a pET28a (+) vector to construct a recombinant expression plasmid, and the reaction system selected by the connection is an In-fusion reaction system (TaKara) optimization: purification RCR fragment,100ng; linear vector:200ng;5 XIn-Fusion HD Enzyme Premix, 2. Mu.L, according to the above volume plus Deionized water to 10. Mu.L. Corresponding negative controls: linear vector:1 mu L of the solution; 5 XIn-Fusion HD Enzyme Premix, 2. Mu.l; deinized water,7 μ L. pUC19 positive control: purification RCR fragment, 2. Mu.L of 2kb control insert; linear vector:1 μ L of pUC19control vector;5 × InFussion HD Enzyme Premix,2 μ L; deinized water:5 μ L. The connection program of the present invention: and (3) slightly and uniformly mixing the reaction system, placing the mixture at 50 ℃ for incubation for 15min, and then placing the mixture on ice to obtain a ligation product, wherein the ligation product can be subjected to next conversion or stored at-20 ℃ for later use. Transforming the ligation product into escherichia coli DH5 alpha competent cells, culturing for 1h (37 ℃,200 rpm) in 700 mu LLB culture medium, taking 400 mu L of bacterial liquid, coating the bacterial liquid on a solid LB plate containing 0.05mg/mL kanamycin, culturing overnight at 37 ℃, selecting positive colonies, carrying out PCR identification, sending the positive bacterial liquid to Guangzhou Ongke sequencing company for sequencing, transforming the plasmid with correct sequencing into escherichia coli BL21 (DE 3) competent cells, adding 50% sterile glycerol according to the proportion of 1 to PCR identification of the positive bacterial liquid, and preserving at low temperature (-80 ℃), so as to respectively obtain SlGST T1 and SlGST T1 K228A,R230A And (3) expressing the strain.
4. SlGST T1-His and SlGST T1 K228A,R230A Prokaryotic expression and purification of His
(1) Prokaryotic expression
Respectively will be respectively provided withSlGST T1 and SlGST T1 K228A,R230A The expression strain is subjected to amplification culture (37 ℃,200 rpm), when the concentration of the bacterial liquid reaches OD600 of 0.4-0.6, the bacterial liquid is cooled to 15 ℃, then protein inducer IPTG with the final concentration of 1mM is added for low-temperature induction (15 ℃,100 rpm), and the bacterial body is collected after culture for 18-20 h.
(2) Protein purification
Adding sterile water to suspend the thalli, centrifuging for 10min at 4 ℃ at 6 000g to remove a culture medium, adding a proper volume of protein extracting solution (20 mM Tris-HCl,500mM NaCl, pH 7.5) to suspend the thalli, and mixing the components according to the volume of bacterial liquid in a ratio of 1:1000 Triton-100 was added to improve the efficiency of protein extraction. After the thalli is fully suspended, the thalli is crushed for 1h (30 s of ultrasonic treatment and 30s of stopping treatment) by using a low-temperature ultrahigh-pressure continuous flow cell crusher, and a container containing bacterial liquid needs to be kept in a full ice bath during the ultrasonic treatment so as to prevent the activity of protein from being reduced due to heating of a probe in the ultrasonic treatment process. PMSF with the final concentration of 1mM is added after the ultrasonication is finished to inhibit the degradation of protein. 9 000g at 4 ℃ for 30min, the supernatant was passed through a membrane (0.45 μm), then Ni NTASuperflow Cartridges (QIAGEN) equilibrated with the protein extract were added to the protein solution, finally imidazole was added to a final concentration of 10mM, and the protein was allowed to bind to the filler by spinning at low temperature 4 ℃ for more than 2 h.
The supernatant of the protein after standing is firstly passed through a nickel column, finally, the filler is also transferred into the nickel column, after the supernatant is completely passed through the nickel column, 40mM imidazole (containing 20mM Tris-HCl,500mM NaCl and pH 7.5) is added to wash the impurity protein until the solution under flowing is detected to be protein-free by Coomassie brilliant blue G250 (Biyun day). The target protein was then eluted with 250mM imidazole (containing 20mM Tris-HCl,500mM NaCl, pH 7.5). Since imidazole has an effect on protein activity, the collected target protein solution was immediately centrifuged (4 ℃ C., 4 000g) with 50mM potassium phosphate buffer solution at pH7 through Sephadex G-25 desalting column (GE)) to replace imidazole in the protein. Finally storing the purified target protein in an ultra-low temperature refrigerator at-80 ℃, reserving part of purified protein and unpurified protein for running gel detection, and carrying out SlGST T1-His and mutant SlGST T1 thereof K228A,R230A the-His SDS-PAGE is shown in FIG. 1.
5. Activation of protein by spectrophotometryAnd (5) detecting sex. The kit of Suzhou Keming biotechnology limited company is selected to determine SlGST T1-His and mutant SlGST T1 thereof K228A,R230A The specific procedures for the activity of His are described in the kit (GST-2-W) of glutathione S-transferase (GST).
Definition of activity units: 1nmol/L CDNB is catalyzed to bind to GSH at 25 ℃ or 37 ℃ per minute per mg protein to 1 unit of enzyme activity. The calculation formula is as follows:
GST (nmol/min/mg prot) = (A2-A1) ÷ ε ÷ d × 109 × V anti-total ÷ (Cpr × V-like) ÷ T =230 × (A2-A1) ÷ Cpr
The results are shown in FIG. 2, and it can be seen from FIG. 2 that the mutant SlGST T1 K228A,R230A The activity of the protein is obviously higher than that of SlGST T1, and the mutation improves the enzyme activity of glutathione S-transferase by 14.35%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Sequence listing
<110> institute of agricultural quality standards and monitoring technology of Guangdong province academy of agricultural sciences
<120> glutathione S-transferase mutant with improved enzyme activity and application thereof
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 711
<212> DNA
<213> tomato (Solanum lycopersicum)
<400> 1
atgtctctca aagtttacgt cgatcgtctt tctcaacctt ctcgtgctat tctcatcttt 60
tgcaaattaa atggaattga atttgaagag gttaatattg atcttgctaa aggtcaacat 120
cgcactcctg aatatcaaga agttaatatc atgaagcaag taccagccat agttcatgat 180
acttttaagc ttttcgaaag tcatgcgatt cttagatatc tagcttctgc atttccggaa 240
actgctgatc attggtatcc aaaagatctg cagaaaagag caaatgtaga atgtgtcttg 300
gattggcacc atgctaactt acgtcgtggt tcagcgggat atgtctttaa cactatactg 360
gcacctgcat ttggattgcc tttgaatcca caagctgctg cagaaggtaa aaatctcctt 420
tctgcatctc ttgcaacgat tgacacctat tggctgcaga aagacggaag ctttttgctt 480
ggaaattccc aaccttccct tgcagatctt agcttagttt gtgagattat gcaactccag 540
tttcttgacg agaaggatcg tgagggtcta ttaagcccgc acaagaacgt gttgaagtgg 600
attgacgatg tgaagagtgc cacagcacct tatttcgatg aaatacatgc aactctcttc 660
aaagttagcg agatttttca gaagcagcga gctggtggag caagcagcta a 711
<210> 2
<211> 236
<212> PRT
<213> tomato (Solanum lycopersicum)
<400> 2
Met Ser Leu Lys Val Tyr Val Asp Arg Leu Ser Gln Pro Ser Arg Ala
1 5 10 15
Ile Leu Ile Phe Cys Lys Leu Asn Gly Ile Glu Phe Glu Glu Val Asn
20 25 30
Ile Asp Leu Ala Lys Gly Gln His Arg Thr Pro Glu Tyr Gln Glu Val
35 40 45
Asn Ile Met Lys Gln Val Pro Ala Ile Val His Asp Thr Phe Lys Leu
50 55 60
Phe Glu Ser His Ala Ile Leu Arg Tyr Leu Ala Ser Ala Phe Pro Glu
65 70 75 80
Thr Ala Asp His Trp Tyr Pro Lys Asp Leu Gln Lys Arg Ala Asn Val
85 90 95
Glu Cys Val Leu Asp Trp His His Ala Asn Leu Arg Arg Gly Ser Ala
100 105 110
Gly Tyr Val Phe Asn Thr Ile Leu Ala Pro Ala Phe Gly Leu Pro Leu
115 120 125
Asn Pro Gln Ala Ala Ala Glu Gly Lys Asn Leu Leu Ser Ala Ser Leu
130 135 140
Ala Thr Ile Asp Thr Tyr Trp Leu Gln Lys Asp Gly Ser Phe Leu Leu
145 150 155 160
Gly Asn Ser Gln Pro Ser Leu Ala Asp Leu Ser Leu Val Cys Glu Ile
165 170 175
Met Gln Leu Gln Phe Leu Asp Glu Lys Asp Arg Glu Gly Leu Leu Ser
180 185 190
Pro His Lys Asn Val Leu Lys Trp Ile Asp Asp Val Lys Ser Ala Thr
195 200 205
Ala Pro Tyr Phe Asp Glu Ile His Ala Thr Leu Phe Lys Val Ser Glu
210 215 220
Ile Phe Gln Lys Gln Arg Ala Gly Gly Ala Ser Ser
225 230 235
<210> 3
<211> 236
<212> PRT
<213> tomato (Solanum lycopersicum)
<400> 3
Met Ser Leu Lys Val Tyr Val Asp Arg Leu Ser Gln Pro Ser Arg Ala
1 5 10 15
Ile Leu Ile Phe Cys Lys Leu Asn Gly Ile Glu Phe Glu Glu Val Asn
20 25 30
Ile Asp Leu Ala Lys Gly Gln His Arg Thr Pro Glu Tyr Gln Glu Val
35 40 45
Asn Ile Met Lys Gln Val Pro Ala Ile Val His Asp Thr Phe Lys Leu
50 55 60
Phe Glu Ser His Ala Ile Leu Arg Tyr Leu Ala Ser Ala Phe Pro Glu
65 70 75 80
Thr Ala Asp His Trp Tyr Pro Lys Asp Leu Gln Lys Arg Ala Asn Val
85 90 95
Glu Cys Val Leu Asp Trp His His Ala Asn Leu Arg Arg Gly Ser Ala
100 105 110
Gly Tyr Val Phe Asn Thr Ile Leu Ala Pro Ala Phe Gly Leu Pro Leu
115 120 125
Asn Pro Gln Ala Ala Ala Glu Gly Lys Asn Leu Leu Ser Ala Ser Leu
130 135 140
Ala Thr Ile Asp Thr Tyr Trp Leu Gln Lys Asp Gly Ser Phe Leu Leu
145 150 155 160
Gly Asn Ser Gln Pro Ser Leu Ala Asp Leu Ser Leu Val Cys Glu Ile
165 170 175
Met Gln Leu Gln Phe Leu Asp Glu Lys Asp Arg Glu Gly Leu Leu Ser
180 185 190
Pro His Lys Asn Val Leu Lys Trp Ile Asp Asp Val Lys Ser Ala Thr
195 200 205
Ala Pro Tyr Phe Asp Glu Ile His Ala Thr Leu Phe Lys Val Ser Glu
210 215 220
Ile Phe Gln Ala Gln Ala Ala Gly Gly Ala Ser Ser
225 230 235
<210> 4
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ccgcgcggca gccatatgat gtctctcaaa gtttacgtcg atcgtctttc tc 52
<210> 5
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
agtcatgcta gccatatgtt agctgcttgc tccaccagct c 41
<210> 6
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
agtcatgcta gccatatgtt agctgcttgc tccaccagct gcctgcgcct gaaa 54

Claims (9)

1. Mutant SlGST T1 of glutathione-S-transferase K228A,R230A The polypeptide is characterized in that the amino acid sequence is shown as SEQ ID NO. 3.
2. A mutant SlGST T1 encoding the mutant of claim 1 K228A,R230A The gene of (1).
3. A recombinant plasmid containing the coding mutant SlGST T1 of claim 2 K228A,R230A The recombinant expression plasmid of (1).
4. The recombinant expression plasmid of claim 3, wherein the expression plasmid is the expression vector pET28a (+).
5. A host expression cell comprising the recombinant expression plasmid of claim 3 or 4.
6. The host expression cell of claim 5, wherein the host expression cell is E.coli.
7. Mutant SlGST T1 as claimed in claim 1 K228A,R230A Application in catalyzing glutathione combination reaction.
8. Mutant SlGST T1 is obtained K228A,R230A The method of encoding a gene of (1), comprising the steps of:
(1) Carrying out PCR by taking an SlGST T1 gene as a template and taking sequences shown in SEQ ID NO.4 and SEQ ID NO.5 as primers to obtain a first PCR product, wherein the nucleotide sequence of the SlGST T1 gene is shown in SEQ ID NO. 1;
(2) Carrying out PCR by taking the first PCR product as a template and taking the sequences shown in SEQ ID NO.4 and SEQ ID NO.6 as primers to obtain a mutant SlGST T1 containing an enzyme cutting site K228A,R230A The coding gene of (1).
9. The method of claim 8, wherein the PCR of steps (1) and (2) is programmed by: pre-denaturation at 98 ℃ for 3min; denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 20s, extension at 72 ℃ for 60s,35 cycles; extension at 72 ℃ for 5min.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6136605A (en) * 1994-08-26 2000-10-24 Wisconsin Alumni Research Foundation Glutathione S-transferase isoforms
CN104328092A (en) * 2014-09-28 2015-02-04 邦泰生物工程(深圳)有限公司 Glutathione synthetase mutant, encoding gene and application
CN104630172A (en) * 2014-11-06 2015-05-20 南京大学 Mutant of schistosoma japonicum glutathione-S-transferase and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6136605A (en) * 1994-08-26 2000-10-24 Wisconsin Alumni Research Foundation Glutathione S-transferase isoforms
CN104328092A (en) * 2014-09-28 2015-02-04 邦泰生物工程(深圳)有限公司 Glutathione synthetase mutant, encoding gene and application
CN104630172A (en) * 2014-11-06 2015-05-20 南京大学 Mutant of schistosoma japonicum glutathione-S-transferase and application thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
A0A3Q7JBS2;UniProt;《UniProt》;20190410;序列部分 *
NCBI:XP_004252589.1;NCBI;《GenBank》;20180808;序列部分 *
Proteome-wide identification of non-histone lysine methylation in tomato during fruit ripening;Lu Xiao等;《Journal of Advanced Research》;20220224;表1，图5 *
家蚕谷胱甘肽-S-转移酶E4基因的组织表达和序列特征及重组蛋白的酶活性分析;谭祥等;《蚕业科学》;20120815(第04期);摘要 *

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