Corn gene ZmFDH1 as well as encoding protein and application thereof

Abstract

The invention relates to a corn gene ZmFDH1 as well as an encoded protein and application thereof, and relates to the technical field of plant genetic engineering, the nucleotide sequence of the corn gene ZmFDH1 is shown as SEQ ID NO.1, and the amino acid sequence of the ZmFDH1 protein encoded by the corn gene ZmFDH1 is shown as SEQ ID NO.7. The corn gene ZmFDH1 has a nucleotide sequence shown as SEQ ID NO.3. The corn gene ZmFDH1 has a nucleotide sequence shown as SEQ ID NO.4. It is found for the first time that ZmFDH1 protein coded by the ZmFDH1 gene in corn is unique high-expression formate dehydrogenase which efficiently and specifically catalyzes formic acid degradation and is wide in optimal action temperature and pH range, and the formate dehydrogenase is crucial for subsequent expansion of the ZmFDH1 gene and the ZmFDH1 protein coded by the ZmFDH1 gene in enzyme engineering research and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and particularly relates to a maize gene ZmFDH1, its encoded protein, and applications thereof. Background Art

[0002] Enzymes capable of oxidizing formate in organisms are divided into two types in microorganisms and higher plants. The first type is formate dehydrogenase in anaerobic microorganisms and archaea. These enzymes are characterized by high molecular mass, complex quaternary structure, the presence of various prosthetic groups, and instability to oxygen. The second type is NAD+-dependent formate dehydrogenase, which consists of two identical subunits, both having two active sites, and neither containing metal ions nor prosthetic groups in the protein globule. This group of formate dehydrogenases belongs to the 2-oxoacid d-specific dehydrogenase superfamily. Formate dehydrogenase catalyzes the reversible reaction of oxidizing formate to carbon dioxide while reducing NAD+ to NADH. This reaction has no proton release or extraction step, requires cleavage of a carbon-hydrogen bond in the substrate, and forms a new carbon-hydrogen bond in the product.

[0003] Previous reports have shown that formate dehydrogenase (FDH) is a mitochondrial enzyme present in the mitochondrial matrix and plays a crucial role in the metabolism of higher plants under various environmental stress conditions. In a study on the role of VuFDH in Vigna umbellata in aluminum (Al) tolerance and low pH (H + ) tolerance, the expression of VuFDH in the root tip was induced by Al and H + stress. Overexpression of VuFDH in Nicotiana tabacum led to a decrease in formate production under Al and H + stress, thereby reducing the sensitivity to Al and H + stress. The FDH gene (GmFDH) encoded by Danba black bean was induced by Al stress, and overexpression of GmFDH could alleviate the growth of tobacco roots under Al stress and reduce the accumulation of Al and ROS in the roots.

[0004] Barley FDH is induced under iron deficiency and hypoxia conditions in roots. In potato tubers grown in the dark, the concentration of FDH increases sharply. In the model plant Arabidopsis thaliana, AtFDH is localized in mitochondria and chloroplasts. It was observed that the expression level of AtFDH was lower in formate-treated samples, while it was higher in formaldehyde- and methanol-treated samples. Therefore, it is reasonable to conclude that to a greater extent, the synthesis of FDH is not caused by the formate substrate, but by its reduced form (formaldehyde). The researchers also found that the overexpression of AtFDH regulated by the Rubisco small subunit promoter in tobacco chloroplasts increased the absorption and metabolism of formaldehyde in tobacco leaves. The FDH activity and the accumulation of FDH transcripts in the roots of rice plants fed with formate were greatly enhanced, and the length and fresh weight of the aerial parts of the seedlings also increased.

[0005] In maize, formate dehydrogenase participates in the last step of oxalic acid metabolism and, together with formyl-CoA synthetase, oxalyl-CoA and formyl-CoA hydrolase, completes the degradation of oxalic acid. However, there is currently little research and analysis on the gene encoding formate dehydrogenase FDH in maize. Summary of the Invention

[0006] The object of the present invention is to provide a maize gene ZmFDH1, its encoded protein and applications in order to solve the above problems.

[0007] The present invention achieves the above object through the following technical solutions:

[0008] The present invention provides a maize gene ZmFDH1, and the nucleotide sequence of the maize gene ZmFDH1 is shown as SEQ ID NO.1.

[0009] As a further optimized scheme of the present invention, the maize variety is the B73 maize inbred line.

[0010] The present invention also provides a ZmFDH1 protein encoded by the above maize gene ZmFDH1, and the amino acid sequence of the ZmFDH1 protein is shown as SEQ ID NO.7.

[0011] The present invention also provides an application of the above maize gene ZmFDH1 or the ZmFDH1 protein as described above in the degradation of formate in maize.

[0012] The present invention also provides an application of the above maize gene ZmFDH1 or the ZmFDH1 protein as described above in enhancing the formate dehydrogenase activity in the catalytic formate dehydrogenation reaction.

[0013] As a further optimization solution of the present invention, the reaction system for catalyzing the formic acid dehydrogenation reaction comprises the ZmFDH1 protein, 50 mM substrate and 100 mM sodium phosphate buffer solution, and the substrate is formic acid or formate.

[0014] As a further optimization solution of the present invention, the reaction conditions for catalyzing the formic acid dehydrogenation reaction are that pH = 5 - 11 and the temperature is 4 - 80 °C.

[0015] As a further optimization solution of the present invention, the reaction conditions for catalyzing the formic acid dehydrogenation reaction are that pH = 7 - 9 and the temperature is 50 - 60 °C.

[0016] The beneficial effects of the present invention are as follows:

[0017] The present invention discovers for the first time that the ZmFDH1 protein encoded by the ZmFDH1 gene in maize is the only formic acid dehydrogenase with high expression, efficient and specific catalysis of formic acid degradation, and a relatively wide range of optimal action temperature and pH, which is crucial for subsequent expansion of the ZmFDH1 gene and its encoded ZmFDH1 protein in enzyme engineering research and applications. Description of the Drawings

[0018] Figure 1 It is a diagram of the amino acid sequence alignment results of the ZmFDHs proteins (s = 1 - 6) encoded by the screened ZmFDHs genes (s = 1 - 6);

[0019] Figure 2 It is a phylogenetic tree of the ZmFDHs genes;

[0020] Figure 3 It is a diagram of the gene structure analysis results of the ZmFDHs genes (3A) and a diagram of the protein motif analysis results of the ZmFDHs proteins (3B);

[0021] Figure 4 It is a diagram of the transcriptome analysis results of the ZmFDHs genes (4A) and a diagram of the qRT-PCR analysis results (4B);

[0022] Figure 5 It is a subcellular localization map of the ZmFDHs proteins;

[0023] Figure 6 It is a diagram of the electrophoresis analysis results of the ZmFDHs proteins (6A) and a diagram of the catalytic activity experimental results of the ZmFDHs proteins (6B - E). Detailed Embodiments

[0024] The present application will be further described in detail below with reference to the accompanying drawings. It is necessary to point out here that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0025] I. Materials

[0026] Unless otherwise specified, the methods used in this embodiment are all conventional methods known to those skilled in the art. The reagents and other materials used are all commercially available products unless otherwise specified.

[0027] II. Methods

[0028] 2.1 Screening of candidate genes

[0029] 2.1.1 Identification of ZmFDH gene family members in the maize genome

[0030] Based on the maize B73 genome version 4, screening revealed that there are 6 genes in maize potentially encoding FDH proteins, named ZmFDH1-ZmFDH6 genes respectively. According to the cloning results of these 6 ZmFDHs (s = 1-6) genes, it was found that ZmFDH2 matched the annotation of the maize B73 genome version 3, which is different from version 4. The nucleotide sequences of the cloned ZmFDH1-ZmFDH6 genes are shown in SEQ ID NO.1-6 in sequence, and the amino acid sequences of the corresponding encoded ZmFDHs (s = 1-6) proteins are shown in SEQ ID NO.7-12 in sequence. The amino acid sequence alignment results of ZmFDH1-ZmFDH6 proteins are as Figure 1 shown.

[0031] 2.1.2 Phylogenetic relationship and gene structure analysis of ZmFDH family members

[0032] Phylogenetic analysis of FDH proteins was performed on four gramineous plants (maize, wheat, rice, and sorghum) and the model plant Arabidopsis thaliana. There is only one FDH (AtFDH) protein in the Arabidopsis thaliana genome, while all four gramineous plants contain two or more FDH proteins, including six in maize (ZmFDH1-6), seven in wheat (TaFDH2-6B, TaFDH2-6D, TaFDH6A, TaFDH7B, TaFDH7A, TaFDH7D, TaFDH4D), two in rice (OsFDH1 and OsFDH2), and two in sorghum (SbFDH1 and SbFDH2). The FDH proteins can be divided into three groups (I-III). Zm FDH1, 3, 4, 5, and 6 proteins are classified into group I, and ZmFDH2, together with AtFDH, SbFDH2, TaFDH6A, TaFDH2-6B, and TaFDH2-6D, is classified into group II. As Figure 2 shown, the results indicate that there is a conserved evolutionary relationship between ZmFDH1 and SbFDH1, between ZmFDH2 and SbFDH2, and between ZmFDH3-6.

[0033] Through gene structure analysis of the ZmFDHs genes (s = 1-6), it was found that both ZmFDH1 and ZmFDH2 consist of 6 exons, ZmFDH3, ZmFDH4, and ZmFDH6 all have 4 exons, while ZmFDH5 consists of only 2 exons (as Figure 3 shown in A); through amino acid sequence analysis of the ZmFDHs proteins (s = 1-6), it was found that they have shared conserved domains. Subsequently, protein motif analysis was also performed on the ZmFDHs members, and a total of 9 conserved motifs were found (as Figure 3 shown in B); both ZmFDH1 and ZmFDH2 contain all 9 motifs, ZmFDH3 contains 7 motifs, ZmFDH4 and ZmFDH6 both contain 8 motifs, and ZmFDH5 contains only 6 motifs.

[0034] 2.1.3 Transcriptome analysis of candidate genes

[0035] By analyzing the transcript data obtained from public RNA-seq data, the specific expression patterns of the ZmFDH1-ZmFDH6 genes in different tissues and different developmental stages (S: seeds; En: endosperm; Em: embryo) were analyzed (as Figure 4 shown in A); among them, the ZmFDH1 gene is highly expressed in both embryos and endosperm after pollination, with the highest expression in the endosperm 10 days after pollination, and the highest expression in all tissues such as roots. Compared with the ZmFDH1 gene, the other 5 ZmFDH genes are expressed at lower levels or not expressed at all in each tissue.

[0036] 2.1.4, qRT-PCR Analysis of Candidate Genes

[0037] The expression profiles of ZmFDH1-ZmFDH6 genes in multiple plant organs (including roots, shoots, leaves, silks, husks, ears, kernels, embryos and endosperms after pollination) were verified by qRT-PCR respectively, and it was found that some genes were consistent with the RNA-seq data (as shown in Figure 4 Figure B); The ZmFDH1 gene was highly expressed in all tissues after pollination, as well as in embryos and endosperms. Compared with the ZmFDH1 gene, the expression frequencies of ZmFDH2 and ZmFDH3 genes were lower in each tissue, while the expressions of ZmFDH4, ZmFDH5 and ZmFDH6 were not detected.

[0038] 2.2 Subcellular Localization Analysis

[0039] To determine the subcellular localization, nucleotide fragments of ZmFDHs genes and ZmO7 gene were amplified from the endosperm cDNA of maize B73 (the nucleotide sequence of ZmO7 gene is shown in SEQ ID NO.13), p35S-ZmFDHs-GFP and p35S-ZmO7-RFP were constructed, the vectors were transformed into Agrobacterium tumefaciens strain GV3101, and co-injected into the leaves of Nicotiana benthamiana. After 48 hours, observation was carried out with a Zeiss LSM880 confocal microscope, and the results were as shown in Figure 5 Figure, and the results showed that the ZmFDHs protein was localized in the cytoplasm.

[0040] 2.3 Expression and Purification of Encoded Proteins of Candidate Genes

[0041] The coding sequences of ZmFDHs genes were amplified and inserted into the expression vector PcoldTF vector to construct the ZmFDHs-PcoldTF recombinant plasmid. The recombinant plasmid was transformed into Escherichia coli DH5α cells for DNA sequencing. After sequencing verification, the recombinant plasmid was transformed into the expression strain Escherichia coli BL21 cells (Rosetta strain), induced expression with IPTG and purified. The purified proteins were analyzed by SDS-PAGE electrophoresis, as shown in Figure 6 Figure A, and the results showed that both Pcold-TF protein and ZmFDHs-PcoldTF protein met the target band size. Using BSA as the standard, the purity of the protein was quantified by SDS-PAGE and A280 absorbance. The analysis results showed that the protein purity was high and the next experiment could be carried out.

[0042] 2.4 Catalytic Activity Experiment of Encoded Proteins of Candidate Genes

[0043] 2.4.1, Protein Catalytic Activity Test with Sodium Formate as Substrate

[0044] The purified ZmFDHs protein and the control Pcold-TF protein were respectively reacted with the sodium formate substrate for testing. The reaction system: The ZmFDHs protein and the control Pcold-TF protein (1000 ng each) were incubated in a dark room-temperature solution for 30 min: 100 mM sodium phosphate buffer, pH = 7.0, 50 mM substrate, 0.8 mM NAD+, 0.03 mg / mL phenazine methosulfate, 0.4 mg / mL NBT. (It was according to the method of Uotila and Koivusalo) (Uotila L., Koivusal oM. Purification of formaldehyde and formate dehydrogenases from pea seeds byaffinit y chromatography and S-formylglutathione as the intermediate offormaldehyde metabolis m[J]. Arch Biochem Biophys, 1979, 196(1): 33-45.), and the test results were as Figure 6 shown in B. Only the ZmFDH1-PcoldTF protein showed high catalytic activity towards the substrate (sodium formate / formic acid) (the higher the catalytic activity, the darker the blue color), while the other ZmFDH2 / 3 / 4 / 5 / 6-PcoldTF proteins showed almost no catalytic activity when reacting with sodium formate. Therefore, the ZmFDH1 protein encoded by the ZmFDH1 gene has high formate dehydrogenase activity.

[0045] 2.4.2. Protein catalytic activity test with different carboxylate sodium salts as substrates

[0046] The ZmFDH1-PcoldTF protein (1000 ng) was respectively reacted with a variety of different carboxylate sodium salts and the control sodium chloride substrate for testing. As Figure 6 shown in C, the results showed that the ZmFDH1-PcoldTF protein only exhibited high catalytic activity when the substrate was sodium formate / formic acid, while for the remaining other carboxylate sodium salt substrates (such as oxalate, malate, acetate, maleate, citrate, sodium chloride), the ZmFDH1-PcoldTF protein showed almost no catalytic activity when reacting with them. Therefore, it was verified that the ZmFDH1 protein encoded by the ZmFDH1 gene has specific and exclusive activity towards formic acid, and the above subcellular localization verified that the ZmFDH1 protein is localized in the cytoplasm, indicating that the ZmFDH1 protein can more widely participate in intracellular formate metabolism.

[0047] 2.4.3. Optimal reaction temperature test of the protein

[0048] Under the reaction conditions with a pH of 7, the ZmFDH1-PcoldTF protein was used to catalyze the formic acid dehydrogenation reaction under different temperature conditions, and the formic acid dehydrogenase activities corresponding to the reactions at each temperature were measured. The results are as Figure 6 shown in D. The optimal reaction temperature of the ZmFDH1 protein is 60 °C. When the reaction temperature is between 50 °C and 60 °C, the relative enzyme activity can reach more than 90%, indicating that the temperature range of action of this enzyme is relatively wide, and the relative enzyme activity is relatively weak under the reaction conditions of 4 °C. When the temperature reaches 70 °C, the relative enzyme activity decreases rapidly, and when the reaction temperature is 80 °C, it drops below 20%. At this time, the protein structure of formic acid dehydrogenase may be severely damaged. This shows that the catalytic enzyme activity of the ZmFDH1 protein is the highest at 50 °C - 60 °C, but it is relatively sensitive to temperatures above 70 °C.

[0049] 2.4.4. Testing the Optimal Reaction pH Value of the Protein

[0050] Under the condition of a temperature of 60 °C, the enzyme activities under the reaction conditions with different pH values were measured. The results are as Figure 6 shown in E. The formic acid dehydrogenase activity of the ZmFDH1 protein is the highest between pH 7 and 8. When the pH is between 8 and 11, the relative activity of the formic acid dehydrogenase of the ZmFDH1 protein gradually decreases, but there is still more than 60% relative activity of the formic acid dehydrogenase when the pH is 11. When the pH is 4, the relative activity of the formic acid dehydrogenase of the ZmFDH1 protein is 0%, but when the pH is 5, the relative activity of the formic acid dehydrogenase of the ZmFDH1 protein reaches more than 60%. This shows that the pH range of action of the ZmFDH1 protein is very wide, and the relative enzyme activity can reach more than 60% when the pH is between 5 and 11.

[0051] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A corn gene ZmFDH1, characterized in that: The nucleotide sequence of the corn gene ZmFDH1 is shown in SEQ ID NO.

1.

2. A ZmFDH1 protein encoded by the maize gene ZmFDH1 according to claim 1, characterized in that: The amino acid sequence of the ZmFDH1 protein is shown in SEQ ID NO.

7.

3. Use of the maize gene ZmFDH1 as claimed in claim 1 or the ZmFDH1 protein as claimed in claim 2 in the degradation of maize formate.

4. Use of the maize gene ZmFDH1 according to claim 1 or the ZmFDH1 protein according to claim 2 to improve the activity of formate dehydrogenase in catalyzing formate dehydrogenation reaction.

5. The use according to claim 4, characterized in that: The reaction system for catalyzing formic acid dehydrogenation reaction comprises the ZmFDH1 protein, 50 mM substrate and 100 mM sodium phosphate buffer, wherein the substrate is formic acid or formate.

6. The use according to claim 4, characterized in that: The reaction conditions of the catalytic formic acid dehydrogenation reaction are pH=5-11 and temperature 4-80°C.

7. The use according to claim 6, characterized in that: The reaction conditions of the catalytic formic acid dehydrogenation reaction are pH=7-9 and temperature 50-60°C.

Images