CN109321536A - The extracting method of oxidizing ferment enzyme activity in a kind of raising leaf tobacco extract - Google Patents

The extracting method of oxidizing ferment enzyme activity in a kind of raising leaf tobacco extract Download PDF

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CN109321536A
CN109321536A CN201811346921.8A CN201811346921A CN109321536A CN 109321536 A CN109321536 A CN 109321536A CN 201811346921 A CN201811346921 A CN 201811346921A CN 109321536 A CN109321536 A CN 109321536A
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enzyme activity
tobacco
pbs
oxidizing ferment
extracting solution
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徐国云
翟妞
周会娜
张慧
陈千思
刘萍萍
郑庆霞
许亚龙
王晨
李晶
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Zhengzhou Tobacco Research Institute of CNTC
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
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    • C12Y109/03—Oxidoreductases acting on a heme group of donors (1.9) with oxygen as acceptor (1.9.3)
    • C12Y109/03001—Cytochrome-c oxidase (1.9.3.1)
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    • C12Y—ENZYMES
    • C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
    • C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
    • C12Y115/01001—Superoxide dismutase (1.15.1.1)

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Abstract

The application belongs to tobacco metabolin technical field, and in particular to a kind of extracting method patent application matters for improving oxidizing ferment enzyme activity in leaf tobacco extract.This method is specifically included for the extraction of oxidizing ferment in fresh tobacco leaves: preparing extracting solution (using lysate RIPA, buffer NEB or PBS), tobacco leaf pretreatment, solwution method are extracted.Preliminary Determination the result shows that, SOD enzyme activity has obtained preferable raising in extracting solution, and up to 3.7U/ μ g holoprotein, POD enzyme activity is up to 6.3 × 10‑3U/ μ g holoprotein, and COX enzyme activity is up to 5.1 × 10‑3U/ μ g holoprotein has preferably achieved the purpose that reduce impurity protein interference, has promoted oxidizing ferment content.Simultaneously because associated extraction method is simple and quick, time-consuming shorter (only 1.5 h or so), so that the application has preferable scientific research value, technical foundation can be established for related enzyme activity analysis and the analysis of other GAP-associated protein GAPs and research.

Description

The extracting method of oxidizing ferment enzyme activity in a kind of raising leaf tobacco extract
Technical field
The application belongs to tobacco metabolin technical field, and in particular to a kind of to improve oxidizing ferment enzyme activity in leaf tobacco extract Extracting method patent application matters.
Background technique
For tobacco as a kind of more special industrial crops, yield and quality is directly related to the strong of Chinese national economy Kang Fazhan.In order to study tobacco gene function, new breeding material is formulated, tobacco metabolin is furtherd investigate and is analyzed is aobvious So have a very important significance.
SOD is the enzyme for being widely present in the intracellular family's confactor containing metal of aerobe, is catalyzed O2 -Disproportionation it is anti- It answers, generates H2O and O2, and H2O2It is converted under catalase (CAT) and glutathione peroxidase (GSH-Px) catalysis It is removed for water.Therefore, SOD is the key that organism Oxidative Stress enzyme, and activity determines O2 -And H2O2It is dense Degree.The environment-stress such as arid, low temperature are frequently encountered in tobacco leaf growth and development process, under these stress conditions, in tobacco seedlings The content of endogenous antioxidant can be remarkably decreased, while cell leakage increases, O2 -It is a large amount of to generate, accumulate film rouge peroxide Change product, and increases with the extension of environment-stress time.With the aggravation of peroxidation of membrane lipids, active oxygen is generated and is removed Mechanism it is unbalance, the growth and development of tobacco seedlings and quality of tobacco can generate irreversible adverse effect.
And peroxidase (peroxidase, POD) and cytochrome oxidase are other two kinds and oxygen in organism Change highly relevant biological enzyme, wherein peroxidase (peroxidase, POD) can be catalyzed fatty acid, aromatic amine and phenolic material The oxidation of matter is one of key enzyme of lignin synthesis, also the elimination reaction of the biosynthesis of participation ethylene and oxygen radical. Meanwhile the disease resistance of POD and plant also has close relationship.Cytochrome oxidase is the end in a kind of higher organism respiratory chain Oxidizing ferment is held, that is responsible for catalysis passes to O from cromoci (cytc) for electronics2Reaction be main in energetic supersession Can not inverse step and mitochondrial respiratory control in main portions.
Since oxidases albumen is in the importance of plant growth and development process, for oxidizing ferment in growth course The accurate analysis of class enzyme activity screens plants physical growth research and new variety of plant, new material and identification all has weight Want meaning.
In the prior art, there are mainly two types of the extracting methods of vegetable protein (including biological enzyme): solwution method and the precipitation method.Its The middle precipitation method will lead to part protease structure and change and lose activity, therefore is heavy because using denaturant in its extraction process Shallow lake method is not appropriate for the extraction of tobacco leaf holoprotein.And when solution application method extracts, due to different albumen (biology Enzyme) characteristic is different, therefore specifically extracts selected extracting solution and extraction process also tends to lack referentiability, often it is both needed to weight New design.And in the prior art there is not yet preferably extracting oxidases extracting method report.
Summary of the invention
The application is designed to provide oxidizing ferment enzyme activity enzyme activity extracting method in a kind of raising leaf tobacco extract, to be cigarette Certain methodology basis is established in careless growth metabolism research.
Details are as follows for the technical solution that the application is taken.
The extracting method of oxidizing ferment enzyme activity in a kind of raising leaf tobacco extract, this method is for oxidizing ferment in fresh tobacco leaves It extracts, the oxidizing ferment refers mainly to: superoxide dismutase (SOD), peroxidase (peroxidase, POD) and cell color Plain C oxidizing ferment (COX)；Specifically comprise the following steps:
(1) extracting solution is prepared, lysate RIPA, NEB or PBS specifically can be used；Formula composition are as follows:
Lysate RIPA: Tris-Cl(pH 8.0), 50 mM, EDTA, 1mM, SDS, 0.1%, NaCl, 150 mM, NP-40,1%, PMSF,1 mM；
Buffer NEB:Hepes(pH 7.5), 20mM, KCl, 40 mM, EDTA, 1 mM, PMSF, 1 mM；
The PBS:NaCl of pH 8.0,137 mM, KCl, 2.7 mM, Na2HPO49.5 mM, NaH2PO4, 0.5 mM, PMSF, 1 mM；
The PBS:NaCl of pH7.2,137 mM, KCl, 2.7 mM, Na2HPO47.2 mM, NaH2PO4, 2.8 mM, PMSF, 1 mM；
The PBS:NaCl of pH 6.0,137 mM, KCl, 2.7 mM, Na2HPO41.2 mM, NaH2PO4, 8.8 mM, PMSF, 1 mM；
(2) tobacco leaf pre-processes
Tobacco K326(or NC82 will be cultivated) fresh tobacco leaves of 8-12 leaf phase, after liquid nitrogen flash freezer, pulverize.
(3) solwution method is extracted
It pulverizes the extracting solution (lysate) for being added in tobacco sample and being prepared in step (1) in step (2), after concussion mixes, It stands on ice and is no less than 1h；
In terms of specific dosage, pulverize tobacco sample 100mg, and extracting solution (lysate) dosage is 500 μ l-1mL；
After the completion of standing, 5000 ~ 15000rpm centrifugation be no less than 20min(centrifugal speed specifically be, for example, 5000 rpm, 9000rpm or 13000rpm etc.), retain supernatant, as containing higher oxygen enzyme (superoxide dismutase (SOD), and/or Peroxidase (peroxidase, POD) and/or cytochrome C oxidase (COX)) enzyme activity extracting solution；
Further, protein concentration in supernatant can be detected using BCA method, using ELISA method to oxygen in supernatant The activity for changing enzyme is detected.
In the prior art, for superoxide dismutase (SOD), peroxidase (POD), cell in extracted holoprotein When cytochrome C oxidase (COX) enzyme activity carries out analysis measurement, since extracted protein impurities are more, thus accurate enzyme activity is surveyed Surely larger impact is caused.In the application, part has been done for holoprotein extracting mode and has been optimized, to improve corresponding enzyme activity effect It is living.
Preliminary Determination the result shows that, after associated extraction process parameter optimizing, superoxide dismutase in extracting solution (SOD) enzyme activity has obtained preferable raising, and up to 3.7U/ μ g holoprotein, peroxidase (POD) enzyme activity is up to 6.3 × 10-3U/μg Holoprotein, and cytochrome C oxidase (COX) enzyme activity is up to 5.1 × 10-3U/ μ g holoprotein, has preferably reached reduction impurity egg The purpose of oxidizing ferment content is disturbed, promoted to white spirit.Simultaneously because associated extraction method is simple and quick, time-consuming shorter (only 1.5 h are left It is right) so that the application has preferable scientific research value, it can be SOD, POD, COX activity analysis and other GAP-associated protein GAPs point Technical foundation is established in analysis and research.
Detailed description of the invention
The SOD activity of Fig. 1 difference extracting solution difference centrifugation rate；
Fig. 2 difference extracts the SOD activity of liquid proportional；
The SOD activity that Fig. 3 different cultivars obtains；
The POD activity that Fig. 4 difference extracting solution difference centrifugation rate obtains；
Fig. 5 difference extracts the POD activity that liquid proportional obtains；
The POD activity that Fig. 6 different cultivars obtains；
The COX activity that Fig. 7 difference extracting solution obtains；
The COX activity that the PBS of Fig. 8 difference pH is obtained；
Fig. 9 difference extracts the COX activity of liquid proportional；
The COX activity that PBS is obtained in Figure 10 difference tobacco bred；
In each figure: K326, NEB, RIPA respectively indicate corresponding lysate, and P6, P7, P8 respectively indicate pH=6.0,7.2,8.0 PBS, it is 5000,13000 that number 5,13, which respectively indicates centrifugal speed, and 1:5,1:10 respectively indicate extraction liquid proportional.
Specific embodiment
Explanation is further explained to the application below with reference to embodiment, before introducing specific embodiment, with regard to following realities It applies the backgrounds such as part Experiment material, experimental method involved in example and briefly introduces and be described as follows.
Experimental material:
Tobacco K326 and NC82 are cultivated, laboratory culture takes fresh tobacco leaves to the 8-12 leaf phase.
Experimental method:
1, when BCA method detection protein concentration, with specific reference to following operating procedure:
(1) supernatant is taken, dilutes 10 times；According to sample size, add 1 volume BCA reagent B(50:1 by 50 volume BCA reagent As) match BCA working solution processed, mixes well；
(2) blank well, standard sample wells and sample well, each sample is arranged to repeat three times, 20 μ l protein extracts of every empty addition, 200 μ L BCA working solutions；
(3) ELISA Plate is put and vibrates 30sec on the oscillator, 37 DEG C are placed 30 minutes, and then microplate reader detects at 562nm, With protein content (μ g) for abscissa, light absorption value is ordinate, draws standard curve；
According to the light absorption value of institute's sample, establishing criteria curve obtains corresponding protein content (μ g), total divided by sample diluting liquid Volume (20 μ L) is sample actual concentrations (unit: μ g/ μ L) multiplied by sample extension rate；
2, when ELISA method detection enzymatic activity, with reference to following steps:
(1) equilibrium at room temperature 20min Enzyme assay reagent；
(2) standard sample wells is set and sample aperture, standard sample wells respectively add the 50 μ L of standard items of various concentration；It is added in sample aperture to be measured 50 μ L of sample, blank well are not added；
In addition to blank well, the detection antibody 100 of horseradish peroxidase (HRP) label is added in every hole in standard sample wells and sample aperture μ L seals reacting hole with sealing plate film, and 37 DEG C of water-baths or insulating box incubate 60min；
(3) liquid is discarded, is patted dry on blotting paper, every hole is filled it up with cleaning solution (350 μ L), is stood 1min, is got rid of cleaning solution, blotting paper On pat dry, so repeat board-washing 5 times (can also use board-washing machine-wash plate)；
Substrate A in kit, each 50 μ L of B is added in every hole, and 37 DEG C are protected from light incubation 15min；
Every hole is added in terminate liquid 50 μ L, 15min, and the OD value in each hole is measured at 450nm wavelength；
Using the OD value of surveyed standard items as abscissa, the concentration value of standard items is ordinate, draws standard curve, and obtain straight line The OD value of sample is substituted into equation, calculates the concentration of sample by regression equation.
Embodiment 1
The present embodiment is improved for superoxide dismutase (SOD) enzyme activity in leaf tobacco extract is improved, and specific steps are brief It is described below.
(1) extracting solution is prepared, lysate RIPA, NEB or PBS specifically can be used；Formula composition are as follows:
Lysate RIPA: Tris-Cl(pH 8.0), 50 mM, EDTA, 1mM, SDS, 0.1%, NaCl, 150 mM, NP-40,1%, PMSF,1 mM；
Buffer NEB:Hepes(pH 7.5), 20mM, KCl, 40 mM, EDTA, 1 mM, PMSF, 1 mM；
The PBS:NaCl of pH 8.0,137 mM, KCl, 2.7 mM, Na2HPO49.5 mM, NaH2PO4, 0.5 mM, PMSF, 1 mM；
The PBS:NaCl of pH7.2,137 mM, KCl, 2.7 mM, Na2HPO47.2 mM, NaH2PO4, 2.8 mM, PMSF, 1 mM；
The PBS:NaCl of pH 6.0,137 mM, KCl, 2.7 mM, Na2HPO41.2 mM, NaH2PO4, 8.8 mM, PMSF, 1 mM；
(2) tobacco leaf pre-processes
Tobacco K326 and NC82 are cultivated, laboratory culture to 8-12 leaf phase acquires fresh tobacco leaves.After liquid nitrogen flash freezer, it is ground into Powder.
(3) solwution method is extracted
It pulverizes the extracting solution (lysate) for being added in tobacco sample and being prepared in step (1) in step (2), after concussion mixes, It stands on ice and is no less than 1h；
In terms of specific dosage, pulverize tobacco sample 100mg, and extracting solution (lysate) dosage is that 500 μ l-1mL(are specially 500 μ l or 1mL)；
After the completion of standing, 5000 ~ 15000rpm centrifugation be no less than 20min(centrifugal speed specifically be, for example, 5000 rpm, 9000rpm or 13000rpm etc.), retain supernatant, as contains the extracting solution of higher superoxide dismutase (SOD) enzyme activity；
Further, protein concentration in supernatant is detected using BCA method, the work using ELISA method to SOD in supernatant Property is detected.
To in supernatant after the PBS of different extracting solutions, difference pH, different centrifugal speeds, different extraction liquid proportional processing SOD activity is detected, as a result as shown in Figure 1, Figure 2, Figure 3 shows respectively.Analysis it can be seen that using PBS(pH 8.0) processing when, Being obtained in the case of enzyme activity effect is obviously higher namely high speed centrifugation under high speed centrifugation parameter (13000 rpm) preferably to remove Impurity protein influences enzyme activity determination；In the PBS processing of different pH, SOD activity is higher when the PBS of pH 8.0 is extracted；? When the processing of different extracting solution usage ratios, when 1:10 ratio, SOD activity is higher.And for different cultivar NC82, equally The activity for the SOD that processing method obtains is also higher.
In general, with regard to SOD enzyme activity determination or for extracting, using PBS (pH 8.0), liquid proportional 1:10, centrifugation are extracted Rate 13000rpm, processing result is optimal, main reason is that, after cracking processing, can preferably it be removed in the case of high speed centrifugation Impurity protein influences enzyme activity determination, and suitable ph also makes the activity of SOD more preferable.
Embodiment 2
The present embodiment is specifically improved for improving peroxidase in leaf tobacco extract (POD) enzyme activity, detailed process with Embodiment 1 is similar, has only done appropriate adjustment with regard to part operation parameter.
To in supernatant after the PBS of different extracting solutions, difference pH, different centrifugal speeds, different extraction liquid proportional processing POD activity is detected, as a result respectively as shown in Fig. 4, Fig. 5, Fig. 6.Analysis it can be seen that using PBS(pH 8.0) processing when, Being obtained in the case of enzyme activity effect is obviously higher namely high speed centrifugation under high speed centrifugation parameter (13000 rpm) preferably to remove Impurity protein influences enzyme activity determination；In the PBS processing of different pH, POD activity is higher when the PBS of pH 8.0 is extracted；? When difference extracts liquid proportionals processing, when 1:10 ratio, POD activity is higher.And for different cultivar NC82, it is same to handle The activity for the POD that method obtains is also higher.
In general, with regard to POD enzyme activity determination or for extracting, using PBS (pH 8.0), liquid proportional 1:10, centrifugation are extracted Rate 13000rpm, processing result is optimal, main reason is that, after cracking processing, can preferably it be removed in the case of high speed centrifugation Impurity protein influences enzyme activity determination, and suitable ph also makes the activity of POD more preferable.
Embodiment 3
The present embodiment is specifically improved, specifically for improving cytochrome C oxidase in leaf tobacco extract (COX) enzyme activity Process is similar to Example 1, has only done appropriate adjustment with regard to part operation parameter.
To in supernatant after the PBS of different extracting solutions, difference pH, different centrifugal speeds, different extraction liquid proportional processing COX activity is detected, as a result respectively as shown in Fig. 7, Fig. 8, Fig. 9, Figure 10.Analysis is it can be seen that using PBS(pH 7.2) When RIPA and NEB is handled, low-speed centrifugal result is relatively preferable, according to the analysis, this is mainly the interaction between impurity protein There is certain counteracting, reduces the influence for COX enzyme activity determination, and the extraction effect of PBS is more preferable；At the PBS of different pH When reason, COX activity is higher when the PBS of pH 8.0 is extracted；In the processing of different extraction liquid proportionals, when 1:10 ratio, COX activity is more It is high.And it is also higher for the activity of the obtained COX of different cultivar NC82, same processing method.
In general, with regard to COX enzyme activity determination or for extracting, using PBS (pH 8.0), liquid proportional 1:10, centrifugation are extracted Rate 5000rpm, processing result is optimal, main reason is that, after cracking processing, the interaction between impurity protein has centainly It offsets, reduces for COX enzyme activity determination, and suitable ph also makes the activity of COX more preferable.

Claims (5)

1. a kind of extracting method for improving oxidizing ferment enzyme activity in leaf tobacco extract, which is characterized in that this method is directed to fresh tobacco leaves The extraction of middle oxidizing ferment, specifically comprises the following steps:
(1) extracting solution is prepared, lysate RIPA, buffer NEB or PBS are specifically used；Formula composition are as follows:
Tris-Cl, 50 mM of lysate RIPA: pH 8.0, EDTA, 1mM, SDS, 0.1%, NaCl, 150 mM, NP-40,1%, PMSF,1 mM；
Hepes, 20mM of buffer NEB:pH 7.5, KCl, 40 mM, EDTA, 1 mM, PMSF, 1 mM；
(2) tobacco leaf pre-processes
The fresh tobacco leaves for cultivating the tobacco 8-12 leaf phase are ground；
(3) solwution method is extracted
It is stood on ice after concussion mixes in step (2) extracting solution for being added in tobacco sample and being prepared in step (1) of pulverizing No less than 1h；In terms of specific dosage, pulverize tobacco sample 100mg, and extracting solution dosage is 500 μ l ~ 1mL；
After the completion of standing, 5000 ~ 15000rpm centrifugation is no less than 20min, retains supernatant, the as extracting solution of oxidizing ferment.
2. improving the extracting method of oxidizing ferment enzyme activity in leaf tobacco extract as described in claim 1, which is characterized in that the oxidation Enzyme refers to: superoxide dismutase, peroxidase and cytochrome C oxidase.
3. improving the extracting method of oxidizing ferment enzyme activity in leaf tobacco extract as claimed in claim 2, which is characterized in that step (2) In, the cultivation tobacco is K326 NC82 kind.
4. improving the extracting method of oxidizing ferment enzyme activity in leaf tobacco extract as claimed in claim 3, which is characterized in that step (1) PH=8.0 of middle PBS, 7.2 or 6.0, concrete composition are as follows:
The PBS:NaCl of pH=8.0,137 mM, KCl, 2.7 mM, Na2HPO49.5 mM, NaH2PO4, 0.5 mM, PMSF, 1 mM；
The PBS:NaCl of pH=7.2,137 mM, KCl, 2.7 mM, Na2HPO47.2 mM, NaH2PO4, 2.8 mM, PMSF, 1 mM；
The PBS:NaCl of pH=6.0,137 mM, KCl, 2.7 mM, Na2HPO41.2 mM, NaH2PO4, 8.8 mM, PMSF, 1 mM.
5. improving the extracting method of oxidizing ferment enzyme activity in leaf tobacco extract as claimed in claim 4, which is characterized in that
When being extracted for superoxide dismutase and/or peroxidase, in step (1), using the PBS of ph=8.0, step (3) In, extracting solution dosage is 1mL, and 13000 rpm are centrifuged 20min；
When being extracted for cytochrome C oxidase, in step (1), using the PBS of ph=8.0；In step (3), extracting solution dosage 20min is centrifuged for 1mL, 5000 rpm.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115354013A (en) * 2022-08-17 2022-11-18 中国烟草总公司郑州烟草研究院 Preparation method of tobacco protoplast

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001090318A2 (en) * 2000-05-26 2001-11-29 Bayer Aktiengesellschaft Regulation of human polyamine oxidase-like enzyme
JP2004033119A (en) * 2002-07-04 2004-02-05 Marine Biotechnol Inst Co Ltd Method for screening enzyme-inhibiting peptide
CN1884540A (en) * 2006-06-15 2006-12-27 武汉大学 Use of tobacco prosystemin gene in transgenic tobacco
CN101870998A (en) * 2010-07-13 2010-10-27 四川农业大学 Method for detecting activity of plant superoxide dismutase, catalase and peroxidase
CN103145817A (en) * 2013-02-28 2013-06-12 华南农业大学 Sickle-alfalfa chloroplast cold-response protein (MfcpCOR14) and coding gene and application thereof
CN104928317A (en) * 2015-06-09 2015-09-23 昆明理工大学 Plant expression vector with nicotiana tabacum gibberellins synthesis transcription regulation factor gene, and application of plant expression vector

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001090318A2 (en) * 2000-05-26 2001-11-29 Bayer Aktiengesellschaft Regulation of human polyamine oxidase-like enzyme
JP2004033119A (en) * 2002-07-04 2004-02-05 Marine Biotechnol Inst Co Ltd Method for screening enzyme-inhibiting peptide
CN1884540A (en) * 2006-06-15 2006-12-27 武汉大学 Use of tobacco prosystemin gene in transgenic tobacco
CN101870998A (en) * 2010-07-13 2010-10-27 四川农业大学 Method for detecting activity of plant superoxide dismutase, catalase and peroxidase
CN103145817A (en) * 2013-02-28 2013-06-12 华南农业大学 Sickle-alfalfa chloroplast cold-response protein (MfcpCOR14) and coding gene and application thereof
CN104928317A (en) * 2015-06-09 2015-09-23 昆明理工大学 Plant expression vector with nicotiana tabacum gibberellins synthesis transcription regulation factor gene, and application of plant expression vector

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CRISTINA PIGNOCCHI ET AL.: "The Function of Ascorbate Oxidase in Tobacco", 《PLANT PHYSIOLOGY》 *
王震等: "植物叶蛋白提取方法及研究进展", 《山西农业科学》 *
钟庆辉: "《烟草化学基本知识》", 31 December 1985 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115354013A (en) * 2022-08-17 2022-11-18 中国烟草总公司郑州烟草研究院 Preparation method of tobacco protoplast
CN115354013B (en) * 2022-08-17 2024-02-02 中国烟草总公司郑州烟草研究院 Tobacco protoplast preparation method

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Application publication date: 20190212