CN105039500A - Method for determining enzyme activity of plant ribulose-1,2-bisphosphate carboxylase/oxygenase - Google Patents

Abstract

The invention discloses a method for measuring the enzyme activity of plant ribulose-1,5-bisphosphate carboxylase/oxygenase. The method for measuring plant ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme activity provided by the present invention comprises: using ribulose-1,5-bisphosphate carboxylase/oxygenase to catalyze RuBP React with _CO2 to obtain glyceric acid-3-phosphate; use wheat phosphoglycerate kinase 1 to catalyze the reaction of glyceric acid-3-phosphate with ATP to obtain 1,3-bisphosphoglycerate; use GAPDH to catalyze 1, 3-bisphosphoglycerate reacts with NADH, and calculates ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme activity according to the rate of change of NADH; wheat phosphoglycerate kinase 1 is sequence 2 in the sequence listing Proteins indicated at positions 97-504.

Description

Method for Determination of Plant Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Enzyme Activity

technical field

The invention relates to a method for measuring plant ribulose-1,5-bisphosphate carboxylase/oxygenase activity in the field of biotechnology.

Background technique

As a key enzyme in the glycolysis process, phosphoglycerate kinase (PGK) has been studied in depth in animals. It is related to many human diseases such as cancer, anemia, and nerve damage. Compared with animal research results, PGK has few research results in the field of plants. It is a key enzyme in the Calvin cycle and catalyzes ribulose-1,5-bisphosphate carboxylase/oxygenase (Ribulosebisphosphatecarboxylaseoxygenase, Rubisco) The carbon fixation reaction product 3-phosphoglycerate is converted into 1,3 diphosphoglycerate. In recent years, with the great development of the new generation of green revolution, in order to meet the needs of the international people's livelihood, scientists and breeders have been paying attention to the improvement of plant carbon sequestration efficiency. In plants, the enzymatic activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) directly affects the carbon sequestration efficiency of plants.

TaPGK1 in wheat is localized in the chloroplast and encoded by the nuclear genome. Therefore, the protein is expressed in the cytoplasm, translated into a precursor protein, and transported to the chloroplast through the transmembrane. During the translocation process, the TaPGK1 precursor protein will be cut off the signal peptide to form the mature protein.

Contents of the invention

The technical problem to be solved by the present invention is how to measure the enzyme activity of plant ribulose-1,5-bisphosphate carboxylase/oxygenase (Ribulosebisphosphatecarboxylaseoxygenase, Rubisco), and evaluate the carbon fixation efficiency of plant Rubisco and plants.

In order to solve the above-mentioned technical problems, the present invention firstly provides the application of wheat phosphoglycerate kinase 1 in the determination of Rubisco enzyme activity.

In the above application, the wheat phosphoglycerate kinase 1 can be the protein shown in the 97th-504th position of the sequence 2 in the sequence listing.

In the above application, the determination of the Rubisco enzyme activity can specifically be the determination of the Rubisco enzyme activity of plants or microorganisms.

The invention also provides a method for measuring Rubisco enzyme activity.

The method for measuring Rubisco enzyme activity provided by the present invention comprises following M1), M2) and M3):

M1) using Rubisco to catalyze the reaction of ribulose-1,5-bisphosphate (RuBP) with CO ₂ to obtain glycerate-3-phosphate;

M2) using the wheat phosphoglycerate kinase 1 to catalyze the reaction of the glyceric acid-3-phosphate with ATP to obtain 1,3-bisphosphoglycerate (glycerate-1,3-bisphosphate);

M3) GAPDH is used to catalyze the reaction of the 1,3-diphosphoglycerate and NADH, and the Rubisco enzyme activity is calculated according to the rate of change of the NADH content in the reaction system;

The name of the wheat phosphoglycerate kinase 1 is TaPGK1, which is the protein shown in the 97th-504th position of the sequence 2 in the sequence listing.

Among them, sequence 2 consists of 504 amino acids. Rubisco enzyme activity is the number of molecules of substrate (ribulose-1,5-bisphosphate, ribulose 1,5-bisphosphate) that each Rubisco molecule can catalyze in the carbon fixation reaction per minute.

In the above method, the Rubisco enzyme activity may be plant Rubisco enzyme activity; the method may also include extracting the plant protein to obtain the Rubisco or an extract containing the Rubisco. The determination of Rubisco enzyme activity can directly measure the Rubisco enzyme activity to obtain the Rubisco enzyme activity, and can also measure the Rubisco enzyme activity in the Rubisco-containing extract to obtain the Rubisco enzyme activity. The extraction of the vegetable protein can be carried out using an extraction buffer; the extraction buffer is composed of water and a solute, and the solute and its concentration are respectively 50mM Tris-HCl (pH7.6), 20mMMgCl ₂ , 20mMNaHCO ₃ and 0.2mM EDTA .

In the above method, the plant may be a monocot or a dicot. The monocot can be wheat, such as Konon 199.

In the above method, the reaction of M1), the reaction of M2) and the reaction of M3) can be carried out in the same reaction system. The change rate of NADH can be obtained by measuring the change amount of NADH absorbance per minute at 340 nm in the reaction system. The reaction system consists of the ribulose 1,5-diphosphate, the CO ₂ , the Rubisco or the extract containing the Rubisco, the wheat phosphoglycerate kinase 1, the ATP, the Said GAPDH, said NADH and M1) said reaction, M2) said reaction and M3) said reaction required other reagent composition. The CO ₂ can be obtained from the reaction of substances that can generate CO ₂ , for example, CO ₂ can be obtained from the reaction of NaHCO ₃ and H ⁺ . The other reagents may include all or part of the seven of Mops-KOH, KCl, MgCl ₂ , NaHCO ₃ , DTT, phosphocreatine and creatine kinase, and the other reagents may only be composed of the Mops-KOH, The seven components of the KCl, the MgCl ₂ , the NaHCO ₃ , the DTT, the creatine phosphate, and the creatine kinase.

In the reaction system, the concentration of the RuBP can be 2.5mM, the concentration of the wheat phosphoglycerate kinase 1 can be 10U/ml, the concentration of the ATP can be 2mM, and the concentration of the GAPDH can be 20U/ml. ml, the concentration of NADH may be 1 mM. In the reaction system, the concentration of the Mops-KOH can be 20mM, the concentration of the _KCl can be 10mM, the concentration of the MgCl can be 10mM, the concentration _of the NaHCO can be 30mM, and the concentration of the DTT can be 10mM. The concentration may be 0.5mM, the creatine phosphate concentration may be 10mM, and the creatine kinase concentration may be 10U/ml.

The RuBP can be a Sigma product with a product number of R0878. The ATP can be a product of Sigma Company, the article number is A3377. The GAPDH can be a Sigma product with a product number of G2267. The NADH can be a product of Sigma Company, the article number is N6785. The creatine phosphate can be a Sigma product, the product number is P7936. The creatine kinase can be a product of Sigma, the product number is C3755.

When utilizing described reaction system to measure Rubisco enzyme activity, the calculating formula of Rubisco enzyme activity can be:

Among them, ΔΔ340/min is the change of NADH absorbance per minute at 340nm; 6.22 is the absorption constant of NADH at 340nm; x is the concentration of Rubisco in the reaction system (μM).

In the above method, the method may also include the preparation method of TaPGK1;

The preparation method of the wheat phosphoglycerate kinase 1 may include the following 1) and 2):

1) introducing the coding gene of TaPGK1 into microorganisms to obtain recombinant microorganisms expressing TaPGK1;

2) culturing the recombinant microorganism to obtain TaPGK1.

In the above method, introducing the gene encoding TaPGK1 into the microorganism is introducing an expression vector containing the gene encoding TaPGK1 into the microorganism.

In the above method, the gene encoding TaPGK1 can be the gene shown in a1) or a2) or a3) as follows:

a1) The nucleotide sequence is a cDNA molecule or a DNA molecule of sequence 1 in the sequence listing;

a2) It has 75% or more identity with the nucleotide sequence defined in a1) and encodes a cDNA molecule or genomic DNA molecule of TaPGK1;

a3) A cDNA molecule or a genomic DNA molecule that hybridizes to the nucleotide sequence defined in a1) under stringent conditions and encodes TaPGK1.

Among them, sequence 1 consists of 1227 nucleotides, and encodes the protein shown in the 97th-504th position of sequence 2.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes nucleotide sequences that are 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher identical to the nucleotide sequence of sequence 1 of the present invention . Identity can be assessed visually or with computer software. Using computer software, identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

In the above method, the stringent condition is to hybridize and wash the membrane twice at 68°C in a solution of 2×SSC and 0.1% SDS, and to wash the membrane twice for 5 minutes each time, and then in a solution of 0.5×SSC and 0.1% SDS to Hybridize and wash the membrane twice at 68°C, 15 min each time; or, hybridize and wash the membrane at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

The identity of 75% or more may be 80%, 85%, 90% or more.

In one embodiment of the present invention, the gene encoding TaPGK1 (ie, the DNA molecule shown in Sequence 1) is introduced into Escherichia coli BL21 (DE3) through a recombinant vector containing the expression cassette of the gene encoding TaPGK1. The recombinant vector is the recombinant vector pHUE-TaPGK1 obtained by replacing the DNA fragment between the SacII and SacI recognition sequences of the carrier pHUE with the DNA molecule shown in sequence 1, and expresses the fusion protein shown in sequence 2 of pHUE-TaPGK1.

Among them, the 5th-10th position of sequence 2 is the amino acid sequence of the His tag, which is encoded by the coding sequence of the His tag in the carrier pHUE; the 21st-96th position of the sequence 2 is the amino acid sequence of Ub, which is encoded by the coding sequence of Ub in the carrier pHUE Encoding; the 97th-504th position of sequence 2 is the amino acid sequence of TaPGK1, which is encoded by sequence 1.

In the above method, said 2) may include the following 21) and 22):

21) cultivating the recombinant microorganism to obtain a recombinant microorganism culture;

22) Purifying the recombinant microbial culture to obtain TaPGK1.

In the above method, the protein expressed by the expression vector can be a fusion protein obtained by linking the amino-terminus or carboxy-terminus of the protein shown in the 97-504th position of sequence 2 in order to facilitate the purification of TaPGK1 as shown in the following table. Purification The recombinant microbial culture can be purified by conventional protein purification methods using the tags shown in the table below.

list, sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

The purification process may also include removing tags and/or other sequences in the fusion protein to obtain TaPGK1 shown in positions 97-504 of Sequence 2.

In one embodiment of the present invention, TaPGK1 is obtained as follows: Purify the fusion protein by using the His tag in the fusion protein shown in Sequence 2, and then cut off the His tag and Ub to obtain TaPGK1.

The above method can be carried out at low temperature (0-10°C), such as 4°C.

In order to solve the above technical problems, the present invention also provides the application of biological materials related to TaPGK1 in the determination of Rubisco enzyme activity;

The biological material is any one of the following A1) to A20):

A1) a nucleic acid molecule encoding TaPGK1;

A2) an expression cassette containing the nucleic acid molecule of A1);

A3) a recombinant vector containing the nucleic acid molecule of A1);

A4) a recombinant vector containing the expression cassette described in A2);

A5) a recombinant microorganism containing the nucleic acid molecule of A1);

A6) a recombinant microorganism containing the expression cassette described in A2);

A7) A recombinant microorganism containing the recombinant vector described in A3);

A8) a recombinant microorganism containing the recombinant vector described in A4);

A9) a transgenic plant or animal cell line containing the nucleic acid molecule of A1);

A10) a transgenic plant or animal cell line containing the expression cassette described in A2);

A11) a transgenic plant or animal cell line containing the recombinant vector described in A3);

A12) a transgenic plant or animal cell line containing the recombinant vector described in A4);

A13) Transgenic plant or animal tissue containing the nucleic acid molecule of A1);

A14) transgenic plant or animal tissue containing the expression cassette described in A2);

A15) Transgenic plant or animal tissue containing the recombinant vector described in A3);

A16) Transgenic plant or animal tissue containing the recombinant vector described in A4);

A17) A transgenic plant or animal organ containing the nucleic acid molecule of A1);

A18) a transgenic plant or animal organ containing the expression cassette described in A2);

A19) A transgenic plant or animal organ containing the recombinant vector described in A3);

A20) A transgenic plant or animal organ containing the recombinant vector described in A4).

In the above application, the nucleic acid molecule in A1) may be the gene shown in a1), a2) or a3).

Wherein, 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 mRNA or hnRNA.

Those skilled in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence encoding TaPGK1 of the present invention. Those nucleotides that have been artificially modified and have 75% or higher identity with the nucleotide sequence of TaPGK1 isolated in the present invention, as long as they encode TaPGK1 and have the function of TaPGK1, are all derived from the nucleotide sequence of the present invention And is equivalent to the sequence of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or more, or 85% or more, or 90% or more of the nucleotide sequence of the protein composed of the amino acid sequence shown in the 97-504 position of the coding sequence 2 of the present invention Nucleotide sequences with high, or 95% or greater identity. Identity can be assessed visually or with computer software. Using computer software, identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

In the above-mentioned application, the stringent conditions are in a solution of 2×SSC and 0.1% SDS, hybridize at 68° C. and wash the membrane twice, each time for 5 minutes, and then in a solution of 0.5×SSC and 0.1% SDS, in Hybridize and wash the membrane twice at 68°C, 15 min each time; or, hybridize and wash the membrane at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

The identity of 75% or more may be 80%, 85%, 90% or more.

In the above-mentioned application, the expression cassette (TaPGK1 gene expression cassette) described in B2) that contains the nucleic acid molecule encoding TaPGK1 refers to the DNA that can express TaPGK1 in the host cell, and the DNA can not only include the promoter that starts TaPGK1 gene transcription, A terminator that terminates transcription of the TaPGK1 gene may also be included. Further, the expression cassette may also include an enhancer sequence.

The existing expression vector can be used to construct the recombinant vector containing the TaPGK1 gene expression cassette.

In the above application, the vector can be a plasmid, cosmid, phage or viral vector.

In the above applications, the microorganisms can be yeast, bacteria, algae or fungi, such as Escherichia coli.

In the above applications, the transgenic plant or animal cell lines, transgenic plant or animal tissues and transgenic plant or animal organs do not include propagation materials.

In one embodiment of the present invention, the gene encoding TaPGK1 (ie, the DNA molecule shown in Sequence 1) is introduced into Escherichia coli BL21 (DE3) through a recombinant vector containing the expression cassette of the gene encoding TaPGK1. The recombinant vector is the recombinant vector pHUE-TaPGK1 obtained by replacing the DNA fragment between the SacII and SacI recognition sequences of the carrier pHUE with the DNA molecule shown in sequence 1, and expresses the fusion protein shown in sequence 2 of pHUE-TaPGK1. The difference between the pHUE-TaPGK1 and the pHUE is only that the pHUE-TaPGK1 is a DNA molecule shown in Sequence 1 in which the DNA between the SacII and SacI recognition sequences of the pHUE is replaced.

In order to solve the above technical problems, the present invention also provides a Rubisco enzyme activity assay system.

The Rubisco enzyme activity assay system provided by the present invention may include R1, which is a reagent and/or instrument required for measuring Rubisco enzyme activity. The required reagent consists of the ribulose 1,5-bisphosphate, CO ₂ or substances that can generate CO ₂ , the wheat phosphoglycerate kinase 1 or the biological material, the ATP, the GAPDH, the NADH and other reagents required for the determination of Rubisco enzyme activity. The substances capable of generating CO ₂ may be NaHCO ₃ and H ⁺ . The other reagents may include all or part of the seven of the Mops-KOH, the KCl, the MgCl ₂ , the NaHCO ₃ , the DTT, the creatine phosphate and the creatine kinase, The other reagents may also be only all or all of the seven of the Mops-KOH, the KCl, the MgCl ₂ , the NaHCO ₃ , the DTT, the creatine phosphate and the creatine kinase part. The instrument required for measuring Rubisco enzyme activity can be a spectrophotometer. The spectrophotometer is a product of Beckman Coulter, Inc., model DU730.

In the above system, the Rubisco enzyme activity can be plant Rubisco enzyme activity; the system can also include R2 and/or R3; the R2 is the reagent needed to extract Rubisco from the plant; the R3 is the Reagents required for wheat phosphoglycerate kinase 1 described above.

The system can be composed of only the R1, or only the R1 and the R2, or only the R1 and the R3, or only the R1, the R2 and the R2 Said R3 composition.

The reagent required for extracting Rubisco from the plant can be the extraction buffer.

The reagents required for the preparation of the wheat phosphoglycerate kinase 1 can be the reagents required for the preparation of the wheat phosphoglycerate kinase 1 by using the preparation method of TaPGK1. Reagents required can be vectors required for protein expression, microorganisms and other reagents required for protein expression, and various reagents required for protein purification.

The carrier may be the carrier pHUE. The microorganism may be Escherichia coli BL21(DE3). The reagents required for protein purification may include dialysis buffer, Ni-NTA purification medium, protease and imidazole solution composition. The reagents required for the protein purification may also only consist of all or part of the four of the dialysis buffer, the Ni-NTA purification medium, the protease and the imidazole solution composition.

The dialysisbuffer is composed of water and solute, and the solute and its concentration in the dialysisbuffer are 20mM Tris-HCl (pH8.0), 30mMNaCl and 0.5mMKCl respectively. The Ni-NTA purification medium can be a product of Merck KGaA with a product number of 70666.

The protease may be Usp2-cc protease. The Usp2-cc protease can specifically be a product of Beijing Yishihe Biotechnology Co., Ltd., with the article number G11502.

The imidazole solution composition is composed of imidazole solutions with different concentrations. The imidazole solutions of different concentrations are all composed of water and solute, and the solutes are respectively NaH ₂ PO ₄ , NaCl and imidazole, and the concentrations of NaH ₂ PO ₄ in the imidazole solutions of different concentrations are all 50mM, and the The concentration of NaCl in the imidazole solutions with different concentrations is 300mM, and the imidazole concentrations in the imidazole solutions with different concentrations are 10mM, 25mM or 250mM respectively.

In the above system, the plant can be a monocot or a dicot. The monocot can be wheat, such as Konon 199.

The above system can also be a kit including the reagents required for the determination of Rubisco enzyme activity, the reagents required for extracting Rubisco from the plant and/or the reagents required for the preparation of the wheat phosphoglycerate kinase 1.

In order to solve the above technical problems, the present invention also provides any application in the following X1-X3:

X1, the application of the method for measuring Rubisco enzyme activity in crop breeding;

X2. Application of the wheat phosphoglycerate kinase 1 in crop breeding;

X3. The application of the biological material in crop breeding;

The plant breeding includes measuring Rubisco enzyme activity.

In the above applications, the crops may be monocotyledonous plants or dicotyledonous plants. The monocot can be wheat, such as Konon 199.

In order to solve the above technical problems, the present invention also provides any application in the following Y1-Y3:

Y1, the application of the method for measuring Rubisco enzyme activity in the selection and breeding of high enzyme activity crop varieties;

Y2. Application of the wheat phosphoglycerate kinase 1 in breeding crop varieties with high enzyme activity;

Y3. The application of the biological material in breeding high enzyme activity crop varieties.

In the above application, the crops with high enzyme activity are crops with improved enzyme activity compared with conventional crops.

In the above applications, the crops may be monocotyledonous plants or dicotyledonous plants. The monocot can be wheat, such as Konon 199.

Experiments have shown that when utilizing the method for measuring Rubisco enzyme activity of the present invention and the wheat phosphoglycerate kinase 1 (TaPGK1) used in the method to measure the enzyme activity of Rubisco, each Rubisco can catalyze 315 RuBP molecules per minute; When yeast-derived PGK is commercially available, each Rubisco can catalyze 83 RuBP molecules per minute. When detecting Rubisco enzyme activity, it is more sensitive to use TaPGK1 to detect wheat Rubisco enzyme activity than yeast-derived PGK. Therefore, in the experiment of detecting the Rubisco enzyme activity of large crops (such as wheat), the TaPGK1 of the present invention can be used to measure the Rubisco enzyme activity. Since in plants, the enzyme activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) directly affects the carbon fixation efficiency of plants, TaPGK1 of the present invention can also be used to breed high carbon fixation efficiency of crop varieties.

Description of drawings

Figure 1 is the SDS-PAGE detection result of the first round of protein purification. Among them, WC represents whole cell lysate, S represents supernatant, FT represents flow through night, W1 represents washing solution 1, W2 represents washing solution 2, and E represents eluent.

Figure 2 is the absorbance value of the reaction system at 340nm during the determination of TaPGK1 enzyme activity. Here, TaPGK means TaPGK1, and PGKCK means yeast-derived PGK.

Figure 3 is the absorbance value of the reaction system at 340nm during the determination of Rubisco enzyme activity. Here, TaPGK represents TaPGK1, and CK represents yeast-derived PGK.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The carrier pHUE in the following examples is described in the literature (ProteinScience magazine, 2004, volume 13, pages 1331 to 1339), the public can obtain the biological material from the applicant, and the biological material is only for repeating the present invention. It is used for relevant experiments and cannot be used for other purposes.

Escherichia coli BL21 (DE3) in the following examples is a product of Tiangen Biochemical Technology (Beijing) Co., Ltd., and the product catalog number is CB105-02.

The Ni-NTAlysis buffer (imidazole solution 1) in the following examples is composed of water and solute. The solute and its concentration in the Ni-NTAlysis buffer are 50mM NaH ₂ PO ₄ , 300mM NaCl and 10mM imidazole, respectively, and the pH is 8.0.

The 10mM imidazole washing solution (imidazole solution 2) in the following examples is composed of water and solute, the solute and its concentration in the 10mM imidazole eluent are 50mM NaH ₂ PO ₄ , 300mM NaCl and 10mM imidazole, respectively, and the pH is 8.0.

The 25mM imidazole washing solution (imidazole solution 3) in the following examples is composed of water and solute, the solute and its concentration in the 25mM imidazole eluent are 50mM NaH ₂ PO ₄ , 300mM NaCl and 25mM imidazole, respectively, and the pH is 8.0.

The 250mM imidazole eluent (imidazole solution 4) in the following examples is composed of water and solute. The solute and its concentration in the 250mM imidazole eluate are 50mM NaH ₂ PO ₄ , 300mM NaCl and 250mM imidazole, respectively, and the pH is 8.0.

The dialysisbuffer in the following examples is composed of water and solute, and the solute and its concentration in the dialysisbuffer are respectively 20mM Tris-HCl (pH8.0), 30mMNaCl and 0.5mMKCl.

The Usp2-cc protease in the following examples is a product of Beijing Yishihe Biotechnology Co., Ltd., and the article number is G11502. Usp2-cc protease is an enzyme that efficiently cuts ubiquitin-like proteins, which can cut Ub of the fusion protein expressed by the pHUE expression vector.

The spectrophotometer in the following examples is a product of Beckman Coulter, Inc., model DU730.

Embodiment 1, the preparation of TaPGK1 and its enzymatic activity assay

The TaPGK1 gene (GenebankAccessionNO.X15233) in this example is derived from wheat, its nucleotide sequence is sequence 1, and it encodes the protein represented by amino acids 97-504 of sequence 2.

1. Construction of recombinant vectors and recombinant bacteria

The DNA fragment between the SacII and SacI recognition sequences of the vector pHUE was replaced with the DNA molecule shown in Sequence 1, and other sequences were kept unchanged to obtain a recombinant vector, which was named pHUE-TaPGK1. The pHUE-TaPGK1 was introduced into Escherichia coli BL21(DE3) to obtain a recombinant bacterium, which was named BL21/pHUE-TaPGK1. BL21/pHUE-TaPGK1 expresses the fusion protein shown in Sequence 2.

Among them, the 5th-10th position of sequence 2 is the amino acid sequence of the His tag, which is encoded by the coding sequence of the His tag in the carrier pHUE; the 21st-96th position of the sequence 2 is the amino acid sequence of Ub, which is encoded by the coding sequence of Ub in the carrier pHUE Encoding; the 97th-504th position of sequence 2 is the amino acid sequence of TaPGK1, which is encoded by the DNA molecule shown in sequence 1.

2. Preparation of TaPGK1

Pick the single clone of BL21/pHUE-TaPGK1 in step 1 and insert it into 50ml of LB-resistant medium (LB-resistant medium is a medium in which the concentration of ampicillin obtained by adding ampicillin to LB liquid medium is 100 μg/ml) , shake culture overnight at 37°C and 220 rpm to obtain BL21/pHUE-TaPGK1 culture solution 1. Transfer BL21/pHUE-TaPGK1 culture solution 1 to 1 liter of ampicillin-resistant LB medium at a volume ratio of 1:40, and culture it with shaking at 37 degrees and 220 rpm to OD=0.8-1.0 to obtain BL21 /pHUE-TaPGK1 culture solution 2, add IPTG to BL21/pHUE-TaPGK1 culture solution 2 until the concentration of IPTG is 0.5mM to obtain a pre-protein induction solution, and shake the pre-protein induction solution at 16°C and 220 rpm After 24 hours, the protein induction solution was obtained, and the protein induction solution was centrifuged at 4000 rpm to collect BL21/pHUE-TaPGK1 cells.

Resuspend the above-mentioned BL21/pHUE-TaPGK1 cells with Ni-NTAlysis buffer to obtain a cell suspension; place the cell suspension on ice for ultrasonication (ultrasound conditions: power 55W, ultrasonic for 5 seconds, stop for 30 seconds, 50 cycles in total) to obtain the whole cell lysate, centrifuge the whole cell lysate at 4°C, 20000rpm for 1 hour, collect the supernatant; filter the supernatant through a 0.22 μm filter membrane to obtain a protein filtrate.

The protein filtrate was purified by the Ni-NTA column for the first round of protein purification. The Ni-NTA purification medium used in the protein purification process was the product of Merck KGaA with a product number of 70666. The protein was purified according to the conventional Ni-NTA The steps of column purification of protein are carried out. After the protein filtrate is passed through the Ni-NTA column, the 10mM imidazole washing solution, 25mM imidazole washing solution and 250mM imidazole eluting solution are respectively passed through the column, and the effluent is collected to obtain the flow-through solution, washing solution 1 and washing solution respectively. 2 and the eluate, SDS-PAGE was performed on the flow-through (F), wash 1 (W1), wash 2 (W2) and eluate (E), respectively, and the results are shown in Figure 1. The results showed that the eluate (E) contained a higher quality target protein.

Add a sufficient amount of Usp2-cc protease to the eluate and incubate at 4°C for 24 hours to obtain reaction solution 1. Dialyze reaction solution 1 in dialysis buffer overnight (12 hours) to obtain a dialyzed protein solution; dialyze The final protein solution was filtered with a 0.22 μm filter membrane to obtain a dialyzed protein filtrate. The dialyzed protein filtrate was purified by Ni-NTA column for the second round of protein purification (the Ni-NTA purification medium used in the protein purification process is the product of Merck KGaA with product number 70666): the dialyzed protein filtrate was passed through Ni-NTA -NTA column, collecting the effluent to obtain a TaPGK1 solution. The TaPGK1 solution was detected by SDS-PAGE, and the results showed that the TaPGK1 solution contained relatively high-purity TaPGK1 (ie, the protein shown in positions 97-504 of Sequence 2). The protein concentration in the TaPGK1 solution was detected, and the concentration of TaPGK1 in the TaPGK1 solution was 1 mg/mL.

3. Enzyme activity determination of TaPGK1

The enzymatic activity of TaPGK1 in the TaPGK1 solution of step 2 is determined by using the following enzyme activity reaction principle:

NADH has a stable absorption peak at 340nm in the spectrophotometer, so the enzymatic activity of TaPGK1 can be calculated according to the change of the 340nm spectrophotometric count value in the reaction system. A standard enzymatic activity unit (1U) of TaPGK1 is defined as: under the following measurement conditions, The amount of enzyme required to consume 1 μmol glycerate-3-phosphate (glyceric acid-3-phosphate) within 1 minute reaction time. The enzyme activity calculation formula is as follows:

Among them, ΔΔ340/min is the variation of NADH absorbance per minute at 340nm; 0.1 is the volume (ml) of the reaction system; df. is the dilution of the measured enzyme TaPGK1; 6.22 is the absorption constant of NADH at 340nm; Volume (ml) of TaPGK1 used.

The experiment was repeated three times, and the specific steps of each repeated experiment were as follows:

The reaction system for measuring TaPGK1 enzyme activity contained 100mM Triethanolamine-HCl (pH7.2), 0.35mM NADH, 10mMATP (product of Sigma Company, product number A3377), 13mMglycerate-3-phosphate (Santasc-214793), 10mM MgCl ₂ , 3U/mlGAPDH (Sigma product, product number is G2267) and 5 μl of the TaPGK1 solution in step 2, make up 100 μl with deionized water, and the concentration of TaPGK1 in the reaction system is 0.05 mg/ml. The reaction system was reacted at 25°C, and the absorbance value of the reaction system at 340 nm was measured every 10 seconds for a total of 10 minutes. The results are shown in Table 1 and FIG. 2 . The absorbance value of the reaction system in the first 4 minutes of reaction was selected to calculate the TaPGK enzyme activity, and the TaPGK1 enzyme activity in the TaPGK1 solution was obtained as 694±1.3U/mL.

According to the above method, replace the TaPGK1 solution in step 2 with PGK solution (PGK solution is the solution obtained by adding PGK (Sigma product, product number P7634) derived from PGK yeast to deionized water), and the other steps remain unchanged. PGK The enzyme activity of the solution was 638±1.3U/mL, indicating that the enzyme activities of TaPGK1 derived from wheat and PGK derived from yeast were basically the same.

Table 1. The absorbance value of the reaction system at 340nm during the determination of TaPGK1 enzyme activity

Response time (s) TaPGK1 PGK 30 1.37±0 1.41±0 60 1.26±0.001 1.30±0 90 1.15±0.002 1.18±0 120 1.04±0.004 1.089±0.001 150 0.94±0.006 1.00±0.001 180 0.849±0.007 0.909±0.003 210 0.75±0.009 0.82±0.003 240 0.66±0.009 0.74±0.004 270 0.57±0.009 0.65±0.004 300 0.48±0.009 0.57±0.004 330 0.40±0.01 0.50±0.005 360 0.32±0.01 0.42±0.005 390 0.25±0.009 0.35±0.005 420 0.18±0.009 0.29±0.005 450 0.15±0.004 0.23±0.004 480 0.13±0.001 0.18±0.003 510 0.12±0 0.15±0.002 540 0.16±0 0.13±0 570 0.12±0 0.13±0 600 0.11±0 0.12±0

Embodiment 2, the application of TaPGK1 in measuring wheat Rubisco enzyme activity

1. Determination of wheat Rubisco enzyme activity by TaPGK1

The chemical reaction for measuring ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubulosebisphosphatecarboxylaseoxygenase, Rubisco) enzyme activity by TaPGK1 is as follows:

NADH has a stable absorption peak at 340nm in the spectrophotometer, so the Rubisco enzyme activity can be calculated according to the change in the 340nm spectrophotometer value in the reaction system. Rubisco enzyme activity is the number of molecules of the substrate (ribulose-1,5-bisphosphate, 1,5-bisphosphate ribulose) catalyzed by each Rubisco molecule per minute in the carbon fixation reaction. The formula for calculating Rubisco enzyme activity is as follows:

Among them, ΔΔ340/min is the change of NADH absorbance per minute at 340nm; 6.22 is the absorption constant of NADH at 340nm; x is the concentration of Rubisco in the reaction system (μM).

1.1 Extraction of wheat leaf whole cell protein

Extraction of wheat leaf whole-cell protein: fully grind 500mg of Kenon 199 leaves in liquid nitrogen to obtain leaf powder, add 0.5ml extraction buffer to the leaf powder (extraction buffer is composed of water and solute, solute and its concentration are respectively 50mM Tris -HCl (pH7.6), 20mMMgCl2, 20mMNaHCO3 and 0.2mMEDTA), after fully mixing, centrifuge at 4 degrees at 16000g for 20 minutes, discard the precipitate, and obtain the supernatant, which is the extract of Kenon 199 containing Rubisco, The Rubisco in the extract was quantified by SDS PAGE and used for the detection of wheat Rubisco enzyme activity in the next step.

1.2 Determination of wheat Rubisco enzyme activity

100 μl TaPGK1 assay Rubisco enzyme activity reaction system contains: 20mMMops-KOH (pH7.5), 10mMKCl, 10mMMgCl ₂ , 30mMNaHCO ₃ (as the source of CO ₂ in this reaction), 0.5mMDTT, Rubisco-containing Conon 199 extraction in step 1.1 (the concentration of Rubisco in the reaction system is 0.5μM), 10mM phosphocreatine (Phosphocreatine) (Sigma product, product number is P7936), 10U/ml creatine kinase (Creatinekinase) (Sigma product, product number is C3755), 1mM NADH ( Sigam company product, article number N8129), 20U/mlGAPDH (Sigma company product, article number is G2267), the TaPGK1 solution of embodiment 1 step 2 (the concentration of TaPGK1 in this reaction system is 10U/ml), 2mMATP (Sigma company product, article number A3377 ) and 2.5mMRuBP (ribulose-1,5-bisphosphate) (Sigma product, catalog number R0878), make up 100 μl with deionized water. The reaction system was reacted at 25°C, and the absorbance value of the reaction system at 340 nm was measured every 10 seconds for a total of 10 minutes. The results are shown in Table 2 and Figure 3.

Measure the Rubisco enzyme activity reaction system according to the above-mentioned TaPGK1, replace the TaPGK1 solution in step 2 of Example 1 with the PGK solution of Example 1, and keep the others unchanged to obtain 100 μl PGK to measure the Rubisco enzyme activity reaction system (100 μl TaPGK1 measures the Rubisco enzyme activity reaction system) The TaPGK1 enzyme activity is the same as the TaPGK1 enzyme activity in the 100 μl PGK assay Rubisco enzyme activity reaction system). The reaction system was reacted at 25°C, and the absorbance value of the reaction system at 340 nm was measured every 10 seconds for a total of 10 minutes. The results are shown in Table 2 and Figure 3.

Table 2. The absorbance value of the reaction system at 340nm during the determination of Rubisco enzyme activity

Response time (s) TaPGK1 PGK 30 3.00±0 3.00±0 60 3.00±0 3.00±0 90 2.867±0.1 3.00±0.1 120 2.448±0.1 2.901±0.05 150 1.887±0.06 2.804±0.09 180 1.66±0.075 2.546±0.068 210 1.617±0.11 2.254±0.10 240 1.601±0.094 1.913±0.1 270 1.593±0.065 1.683±0.073 300 1.581±0.052 1.565±0.064 330 1.577±0.034 1.51±0.045 360 1.574±0.024 1.494±0.034 390 1.576±0.021 1.476±0.30

When using TaPGK1, each Rubisco can catalyze 315 RuBP molecules per minute; while using commercial yeast-derived PGK, each Rubisco can catalyze 83 RuBP molecules per minute. The absorbance of NADH at 340nm changed more rapidly than that of the control yeast-derived PGK. The reaction process can be completed within 2 minutes, and the reaction process using yeast-derived PGK can be completed within 3 minutes. Therefore, in the experiment of detecting the Rubisco enzyme activity of large crops (such as wheat), the TaPGK of the present invention can be used to measure the Rubisco enzyme activity.

Claims (10)

1. Application of wheat phosphoglycerate kinase 1 in the determination of ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme activity. 2. The application according to claim 1, characterized in that: the wheat phosphoglycerate kinase 1 is the protein shown in the 97th-504th position of the sequence 2 in the sequence listing. 3. The method for measuring ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme activity, including the following M1), M2) and M3): M1) using ribulose-1,5-bisphosphate carboxylase/oxygenase to catalyze the reaction of ribulose 1,5-bisphosphate with CO ₂ to obtain glyceric acid-3-phosphate; M2) using the wheat phosphoglycerate kinase 1 described in claim 1 or 2 to catalyze the reaction of the glyceric acid-3-phosphate with ATP to obtain 1,3-diphosphoglycerate; M3) GAPDH is used to catalyze the reaction between the 1,3-diphosphoglycerate and NADH, and the ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme activity is calculated according to the change rate of the NADH content in the reaction system. 4. the application of the biological material related to wheat phosphoglycerate kinase 1 described in claim 1 or 2 in the determination of ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme activity; The biological material is any one of the following A1) to A20): A1) the nucleic acid molecule encoding wheat phosphoglycerate kinase 1 described in claim 1 or 2; A2) an expression cassette containing the nucleic acid molecule of A1); A3) a recombinant vector containing the nucleic acid molecule of A1); A4) a recombinant vector containing the expression cassette described in A2); A5) a recombinant microorganism containing the nucleic acid molecule of A1); A6) a recombinant microorganism containing the expression cassette described in A2); A7) A recombinant microorganism containing the recombinant vector described in A3); A8) a recombinant microorganism containing the recombinant vector described in A4); A9) a transgenic plant or animal cell line containing the nucleic acid molecule of A1); A10) a transgenic plant or animal cell line containing the expression cassette described in A2); A11) a transgenic plant or animal cell line containing the recombinant vector described in A3); A12) a transgenic plant or animal cell line containing the recombinant vector described in A4); A13) Transgenic plant or animal tissue containing the nucleic acid molecule of A1); A14) transgenic plant or animal tissue containing the expression cassette described in A2); A15) Transgenic plant or animal tissue containing the recombinant vector described in A3); A16) Transgenic plant or animal tissue containing the recombinant vector described in A4); A17) A transgenic plant or animal organ containing the nucleic acid molecule of A1); A18) a transgenic plant or animal organ containing the expression cassette described in A2); A19) A transgenic plant or animal organ containing the recombinant vector described in A3); A20) A transgenic plant or animal organ containing the recombinant vector described in A4). 5. application according to claim 4, is characterized in that: A1) described nucleic acid molecule is the gene shown in following a1) or a2) or a3): a1) The nucleotide sequence is a cDNA molecule or a DNA molecule of sequence 1 in the sequence listing; a2) has 75% or more identity with the nucleotide sequence defined in a1), and encodes the cDNA molecule or genomic DNA molecule of wheat phosphoglycerate kinase 1 described in claim 1 or 2; a3) a cDNA molecule or a genomic DNA molecule that hybridizes to the nucleotide sequence defined in a1) under stringent conditions and encodes wheat phosphoglycerate kinase 1 according to claim 1 or 2. 6. Ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme activity assay system, comprising 1,5-bisphosphate ribulose, CO ₂ , wheat phosphoglycerate kinase described in claim 1 or 2 1. ATP, GAPDH and NADH. 7. Ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme activity assay system, comprising 1,5-bisphosphate ribulose, CO ₂ , the biological material described in claim 4 or 5, ATP, GAPDH and NADH. 8. Any one of the following X1-X3 applications: X1, the application of the method described in claim 3 in crop breeding; X2, the application of wheat phosphoglycerate kinase 1 described in claim 1 or 2 in crop breeding; X3, the application of the biological material described in claim 4 or 5 in crop breeding; The crop breeding includes measuring ribulose-1,5-bisphosphate carboxylase/oxygenase activity. 9. Application of the method according to claim 3 in breeding high enzyme activity crop varieties. 10. The application of the wheat phosphoglycerate kinase 1 according to claim 1 or 2 or the biological material according to claim 4 or 5 in breeding crop varieties with high enzyme activity.