Application of Escherichia coli EcPAPR gene in improvement of arsenic tolerance of organisms

Abstract

The invention discloses application of an Escherichia coli EcPAPR gene to improvement of arsenic tolerance of organisms, and belongs to the field of genetic engineering. Research finds that EcPAPR can improve the content of glutathione in corn seedlings; ecPAPR is overexpressed in corn leaves, and the tolerance of seedlings to heavy metal arsenic can be improved. Overexpression of the gene in Escherichia coli and Arabidopsis thaliana can also improve the tolerance of Escherichia coli and Arabidopsis thaliana to heavy metal arsenic, which shows that the gene has the effect of improving the tolerance of organisms to arsenic.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to the application of Escherichia coli EcPAPR gene in improving the tolerance of organisms to arsenic. Background Art

[0002] Sulfur is a key nutrient for plant growth and development and is essential for the growth and development of all plants. It plays a central role in plant defense responses to biotic as well as abiotic stresses and is a versatile platform for plants to initiate defense actions. It is a component of the amino acids cysteine ​​and methionine, a cofactor, metal cluster, and various primary and secondary metabolites such as glutathione, phytochelatins, and glucosinolates that protect plants from oxidative and environmental stresses. Sulfur can reduce arsenic accumulation by regulating amino acid and thiol metabolism in leaves, thereby alleviating arsenic poisoning reactions.

[0003] After being absorbed, sulfur is reduced to cysteine, methionine and cysteine, which are mainly divided into SO4 2- Restore to S 2- , S 2- Together with OAS, cysteine ​​is synthesized in two steps. First, SO4 2- After entering the plant body, it is activated by ATP sulfurylase (ATPS) to convert ATP into 5'-phosphoadenylic acid sulfate (APS), and under the catalysis of 5'-phosphoadenylic acid sulfate reductase (APR), APS is reduced to sulfite (SO3 2- ). APS can also be phosphorylated by APS kinase (APK) to 3' phospho-adenosine 5' phosphorylated sulfate (PAPS), which provides active sulfate for many sulfidation reactions in secondary metabolism and is used to synthesize glucosinolates, participating in plant stress resistance. It can be seen that PAPR and PAPS play a key role in plant resistance to abiotic stress.

[0004] Overexpression of the Escherichia coli PAPR gene (EcPAPR) in maize increased the level of maize sulfur reduction, thereby increasing the synthesis of cysteine ​​in leaves, thereby increasing the downstream content of methionine and glutathione. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for improving the tolerance of organisms to arsenic.

[0006] The technical scheme of the present invention is: application of Escherichia coli EcPAPR gene in improving biological tolerance to arsenic, wherein the nucleotide sequence of the Escherichia coli EcPAPR gene is as shown in SEQ ID No.1 or a degenerate sequence encoding the same protein as the nucleotide sequence shown in SEQ ID No.1.

[0007] Furthermore, the application is to overexpress the Escherichia coli EcPAPR gene in an organism, thereby improving the organism's tolerance to arsenic.

[0008] Furthermore, the organism is corn, Arabidopsis thaliana or Escherichia coli.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] The present invention found that EcPAPR can increase the glutathione content in corn seedlings; overexpression of EcPAPR in corn leaves can improve the tolerance of seedlings to heavy metal arsenic. Overexpression of the gene in Escherichia coli and Arabidopsis can also improve the tolerance of Escherichia coli and Arabidopsis to heavy metal arsenic, indicating that the gene has a significant effect on improving the tolerance of organisms to arsenic. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Analysis of EcPAPR enzyme activity and protein enrichment in transgenic lines, a is EcPAPR enzyme activity analysis, b is EcPAPR immunoblot analysis.

[0012] Figure 2 Glutathione content of transgenic lines.

[0013] Figure 3 Seedling phenotypes after sodium arsenite (AsIII) treatment, WT is the wild type; 39 is the EcPAPR overexpression line.

[0014] Figure 4 Seedling phenotypes after sodium arsenate (AsV) treatment, WT is the wild type; 39 is the EcPAPR overexpression line.

[0015] Figure 5 RNAseq analysis of EcPAPR overexpression maize, a is the PCA principal component analysis; b is the volcano plot of up- and down-regulated expression of differentially expressed genes; c is the KEGG analysis of the main metabolic pathways; d is the GO term analysis related to the sulfur reduction pathway.

[0016] Figure 6Metabolic pathway diagram corresponding to the upregulated genes in RNAseq analysis of EcPAPR-overexpressed corn. a is the pathway diagram of the sulfur reduction pathway. Light blue represents genes that are significantly upregulated in EcPAPR-overexpressed corn leaves (P value<0.05); b is the metabolic pathway and log2fold change value corresponding to the upregulated genes marked in a. Aprl1: 5'-phosphoadenylate sulfate reductase 1, Aprl2: 5'-phosphoadenylate sulfate reductase 2, Sultr1;2: sulfate transporter 1.2, Sir1: sulfite reductase 1, Sat2: acetylserine transferase 2, Sat4: acetylserine transferase 4, Trxf1: thioredoxin 1, Sam: S-adenosyl-L-methionine-dependent methyltransferase superfamily protein, Oms: O-methyltransferase, Gst2, Gst4, Gst7, Gst14, Gst21, Gst37, Gst38: glutathione transferase 2, 4, 7, 14, 21, 37, 38, Gln2: glutamine synthetase 2, Gln3: glutamine synthetase Hsp3, Sod4:, superoxide dismutase 4, Sod9: superoxide dismutase 9, Grx1: glutaredoxin 1, Grx-C13: glutaredoxin C13, Grx: glutaredoxin, Hipp45: heavy metal-related prenylated plant protein 45, Nakr1: protein sodium-potassium root defective 1, Vc2: vacuolar cation / proton exchanger 2, tip2a: vacuolar membrane protein 2, Hsp4, Hsp6, Hsp7, Hsp8, Hsp9, Hsp12, Hsp22, Hsp101, Hsp18f, Hsp18c: heat shock protein 4, 6, 7, 8, 9, 12, 22, 101, 18f, 18c, Hsftf8: heat shock protein transcription factor 8.

[0017] Figure 7 Analysis of arsenic tolerance in EcPAPR-overexpressing Arabidopsis, a is the construction of the EcPAPR gene overexpression vector; b is the immunoblot analysis of EcPAPR protein accumulation in single-copy homozygous transgenic lines 36, 23; c is the in vitro determination of EcPAPR enzyme activity in transgenic lines 36, 23; d, e, f, g are the As(III) and As(V) tolerance analysis of transgenic lines 36, 23.

[0018] Figure 8 Overexpression of EcPAPR in Escherichia coli can improve the strain's tolerance to arsenic. DETAILED DESCRIPTION

[0019] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from commercial channels unless otherwise specified.

[0020] The culture medium formula is as follows:

[0021] Liquid

[0022] N6 Salts and vitamins, 1.5 mg L -1 2,4-Dichlorophenoxyacetic acid (2,4-D), 0.7 g L -1 L-Proline, 68.4 g L -1 Sucrose, 36 g L -1 Glucose (pH 5.2), acetosyringone final concentration was 100uM.

[0023] 100mM acetosyringone: Dissolve AS in DMSO to make 200mM and then dilute with water 1:1 to 100mM.

[0024] Co-culture medium

[0025] N6 Salts and vitamins, 1.5 mg L -1 2,4-D, 0.7 g L -1 L-Proline, 30g L -1 Sucrose, 3 g L -1 Gellan gum (pH 5.8). Silver nitrate 0.85 mg L -1 , acetosyringone (100uM), L-cysteine ​​(400mg L -1 ).

[0026] Recovery medium

[0027] N6 Salts and vitamins, 1.5 mg L -1 2,4-D, 0.7 g L -1 L-Proline, 30g L -1 Sucrose, 0.5 g 2-(4-morpholinyl)-ethanesulfonic acid (MES), 3 g L -1 Gellan gum, (pH 5.8), silver nitrate 0.85 mg L -1 , thiophanate-methyl (250 mg L -1 ).

[0028] First screening medium

[0029] N6 Salts and vitamins, 1.5 mg L -1 2,4-D, 0.7 g L -1 L-Proline, 30g L -1 sucrose, 0.5 g MES, 3 g L -1 Gellan gum, (pH 5.8), silver nitrate 0.85 mg L -1 , thiophanate-methyl (250 mg L -1 ), 1.5 mg L -1Glyphosate.

[0030] Second screening medium

[0031] N6 salts and vitamins, 1.5 mg L -1 2,4-D, 0.7 g L -1 L-Proline, 30g L -1 sucrose, 0.5 g MES, 3 g L -1 Gellan gum (pH 5.8), silver nitrate 0.85 mg L -1 , thiophanate-methyl (250 mg L -1 ), 3.0 mg L -1 Glyphosate.

[0032] First differentiation medium

[0033] MS Salt and MS Vitamins, 60g L -1 Sucrose, 100 mg L -1 Inositol, 3 g L -1 Gellan gum (pH 5.8). Thioproterenol (250 mg L -1 ), glyphosate (3 mg L -1 ) Add after sterilization.

[0034] Second differentiation medium

[0035] MS salt and MS vitamins, 100 mg L -1 Inositol, 30 g L -1 Sucrose, 3 g L -1 Gellan gum, (p H 5.8).

[0036] Example 1 Overexpression of EcPAPR in Maize

[0037] 1) Target gene cloning and vector construction

[0038] The promoter used is Bundle sheath-specific Rubisco small subunit 1 (rbcS1 gene), referred to as rbcS1 (GenBank: AH005359.3). This promoter is from corn and is 1030 bp long. There is a 20 bp signal peptide behind the promoter, which targets the expressed protein to the vascular bundle sheath cells, achieving tissue cell-specific expression and avoiding the toxicity of exogenous proteins to plants due to accumulation in non-functional tissues. The sequence can be found at http: / / www.ncbi.nlm.nih.gov / nuccore / 339635306.

[0039] The Escherichia coli 3'-phospho-5'-phosphoadenylate sulfate reductase EcPAPR gene (SEQ ID No. 1) was cloned, and the promoter and EcPAPR gene were connected to the corresponding positions of the plant binary expression vector pTF102, and the vector was named pTF102 / rbcS1P:EcPAPR.

[0040] Table 1 All primers used in the experiment

[0041]

[0042] 2) Obtaining transgenic lines

[0043] Introduce the expression vector pTF102 / rbcS1P:EcPAPR into the Agrobacterium strain EHA101; take the immature embryos (1.5-2.0mm) of maize HiII hybrids that have been pollinated for about 13 days, and infect the immature embryos with pre-activated Agrobacterium. Aspirate the bacterial solution and transfer the immature embryos to the co-cultivation medium. After three days of co-cultivation at 20℃, transfer to the recovery medium. After 7 days of dark cultivation at 28℃, transfer the immature embryos with growth ability to the first screening medium and culture them in the dark at 28℃ for 2 weeks. Transfer to the second screening medium and culture them in the dark for 2 weeks. Transfer the well-growing callus to the first differentiation medium for differentiation, and differentiate them in the dark at 28℃ for 3 weeks. Transfer to the second differentiation medium and differentiate them into plants under light at 24℃.

[0044] 38 transgenic T0 generation strains were obtained. After sowing the T1 generation seeds of the strains with high enzyme activity, they were test-crossed with B73 as the male parent. After six generations of backcrossing and self-pollination, BC6F1 was obtained. From them, two strains with high EcPAPR expression and homozygous were selected for subsequent experiments.

[0045] 3) Analysis of EcPAPR enzyme activity and protein enrichment in EcPAPR-transgenic corn

[0046] (1) EcPAPR enzyme activity assay

[0047] The principle of enzyme activity assay is as follows:

[0048] Adenosine 3′-phosphate 5′-phosphosulfate lithium salt hydrate (PAPS) generates APS under the action of PAPR enzyme, and APS then undergoes the following reaction:

[0049] 5'-adenylylsulfate(APS)+DTT red→SO3 2- (sulfite)+DTT ox+AMP

[0050] Reagents used in this experiment

[0051] Adenosine 3′-phosphate 5′-phosphosulfate lithium salt hydrate(PAPS)SIGMA A1651-1MG,

[0052] Tri-n-octylamine (Sigma T-8631), Nuclease P1 (Sigma N-8630), [35S]PAPS (New England Nuclear#NEG-010), Adenosine 5'-phosphosulfate (APS) (SigmaA-5508)

[0053] The radioactivity of [35S]PAPS usually purchased is about 50-70GBq per mmol, 30MBq / ml, 0.45mM PAPS.

[0054] The EcPAPR enzyme activity assay system is 50mM Tris pH 8.0, 1mM EDTA (pH 8.0), 1mM EDTA (pH8.0), 25μM [35S]PAPS, 10mM DTT, 1mM Thioredoxin; the total reaction volume is 100μl. The reaction starts after adding DTT or enzyme solution and is incubated at 30℃ for 15 to 30 minutes.

[0055] After the enzyme reaction is completed, open the centrifuge tube cap and place it in a 6 ml scintillation vial containing 1 ml trioctylamine (Sigma # T-8631). Add 5 μl 0.84M Na2SO3 (final concentration 40 mM) as a carrier, and then add 45 μl concentrated sulfuric acid (final concentration 3.0 M). Quickly cover the scintillation vial cap and place it in a fume hood overnight.

[0056] After acidification, SO3 2- It is converted into gaseous sulfur dioxide, which is collected in trioctylamine.

[0057] After leaving overnight, carefully remove the enzyme reaction tube from the scintillation vial, taking care not to spill the reaction solution into the scintillation vial. Add 3 ml of Ready-Safe (Beckman) scintillation fluid into the scintillation vial, cover tightly, and mix thoroughly with a vortexer. Use a scintillation counter to read the 35S radioactivity value (cpm) and calculate the enzyme activity. The calculation method is as follows:

[0058] 1. Subtract the blank value (the CPM value obtained from the reaction without adding protein solution);

[0059] 2. The CPM value / nmol APS obtained for a known concentration of APS is obtained by converting the CPM value / nmol APS to the CPM / nmol APS value;

[0060] 3. Divide the CPM value of the sample by CPM / nmol APS to get the number of APS nmols in the reaction. Divide the number of APS nmols by the reaction time to get nmol APS / min;

[0061] 4. Divide the value obtained in 3 by the number of mg of protein added to obtain the enzyme activity in nmol / min / mg protein. The endogenous APR enzyme activity of Arabidopsis thaliana is generally 1-5 nmol / min / mg.

[0062] The enzyme activities of the 10 transgenic lines were Figure 1 As shown in a, compared with the wild type, the enzyme activity of EcPAPR in different strains varies greatly, ranging from 0.01-0.6nmol SO3 2- / min / mg varies.

[0063] (2) EcPAPR immunoblot analysis

[0064] Immunoblotting analysis with EcPAPR antibody revealed that ( Figure 1 In b), protein accumulation and enzyme activity are positively correlated.

[0065] 4) Determination of glutathione content in leaves

[0066] In plants, glutathione is one of the main ways of sulfate reduction. TM The glutathione content in the leaves of corn overexpressing the EcPAPR gene was measured using a glutathione detection kit (Promega, catalog number V6911). Compared with non-transgenic wild-type corn, the glutathione content in the leaves of transgenic corn was significantly increased. Figure 2 As shown, T2 line 39 (588.7±59.9 nmol g -1 FW), line 40 (543.1±17.4nmol g-1 FW), and line 41 (443.1±17.4nmol g-1 FW), which were all higher than the glutathione levels in wild-type B73 plants (322.2±7.4nmol g -1 FW) high.

[0067] 5) Analysis of arsenic tolerance in corn seedlings

[0068] In the early stage of this study, wild-type three-leaf corn seedlings were treated with As(III) and As(V) at gradient concentrations of 0, 5mM, 10mM, 20mM, and 30mM. Finally, 10mM As(III) and As(V) were determined to be the arsenic poisoning reaction concentrations for wild-type corn.

[0069] The 10-day-old seedlings of wild-type and EcPAPR-overexpressing maize materials with the same growth were watered with the same amount of 10 mM sodium arsenite. The phenotypes were observed 3 days later and it was found that the transgenic line 39 showed arsenic tolerance ( Figure 3 ).

[0070] The 10-day-old seedlings of wild-type and EcPAPR-overexpressing maize materials with the same growth were watered with the same amount of 10 mM sodium arsenate. The phenotypes were observed 3 days later and it was found that the transgenic line 39 showed arsenic tolerance ( Figure 4 ).

[0071] 6) Transcriptome analysis of maize leaves overexpressing EcPAPR

[0072] In order to analyze the changes in leaf transcriptome in the EcPAPR overexpressing lines under normal growth conditions, total RNA was extracted from functional leaves before flowering, and RNAseq analysis was performed using the corresponding wild type as a control ( Figure 5 ),Depend on Figure 5 Cluster analysis in a showed good repetition among samples. 2959 down-regulated genes and 2597 up-regulated genes were obtained (P<0.05) ( Figure 5 (b) KEGG analysis found that there were differentially expressed genes in amino acid synthesis, cysteine, methionine metabolism and glutathione metabolism pathways ( Figure 5 c). The GO analysis results are similar to those of KEGG, with differentially expressed genes in the sulfur reduction pathway and related downstream metabolic pathways ( Figure 5 (d) This indicates that overexpression of EcPAPR in maize leaves alters the gene expression of the sulfur reduction pathway in leaves, thereby affecting the synthesis of downstream amino acids and the physiological and biochemical characteristics of the plant.

[0073] The specific major significantly up-regulated genes are annotated on the corresponding metabolic pathways ( Figure 6 a), the up-regulation factor is log2fold change ( Figure 6b). The results showed that the sulfur transporter (Sultr1:2) was up-regulated, indicating that EcPAPR overexpression promoted the transport of sulfur ions into the plant; 5'-phosphoadenylate sulfate reductase (Aprl1, April2), sulfite reductase 1 (SiR1), acetylserine transferase (Sat2, Sat4) and PAPR substrate thioredoxin (Trxf1) were all up-regulated. Arsenic stimulates the production of free radicals and reactive oxygen species (ROS) in plants, which can cause oxidative damage to important macromolecular lipid membranes, proteins and DNA. Overexpression of the EcPAPR gene increased the superoxide dismutase (Sod4, Sod9) of the plant, which helps to clear harmful substances. Under heat shock at a temperature 5°C higher than the normal growth temperature, the synthesis of most normal proteins and the transcription of mRNA in the organism are inhibited, and some new proteins are rapidly synthesized, which are called heat shock proteins (Hsps). This reaction is called heat shock reaction. In addition to temperature, many other factors such as heavy metal ions and arsenite can induce heat shock reactions, and the synthesized Hsp participates in many metabolic processes of organisms. Heat shock proteins have the function of molecular chaperones, protecting cells from damage and repairing damaged proteins. In the leaves of corn overexpressing the EcPAPR gene, heat shock protein family genes (Hsp4, Hsp6, Hsp7, Hsp8, Hsp9, Hsp12, Hsp22, Hsp101, Hsp18f, Hsp18c) are also upregulated accordingly. Glutathione S-transferases are enzymes that catalyze the synthesis of glutathione complexes from GSH. Its expression is induced by biotic stress (fungal inducers and pathogen attacks) and abiotic stress (cold, drought, hydrogen peroxide and heavy metals, etc.). Many studies have shown that the Gsts gene family plays an important role in the stress response and detoxification of heavy metal arsenic. Glutathione transferases (Gst2, Gst4, Gst7, Gst14, Gst21, Gst37, Gst38) were upregulated in corn overexpressing the EcPAPR gene, showing enhanced active detoxification ability to arsenic in the absence of arsenic stress.

[0074] Heavy metal-related transporters heavy metal-related prenylated plant protein 45 (Hipp45), protein sodium-potassium root defective 1 (Nakr1), vacuolar cation / proton exchanger 2 (Vc2), and tonoplast protein 2 (tip2a) were all generally upregulated.

[0075] Another class of upregulated genes is glutaredoxins (Grxs), which are ubiquitous small heat-stable disulfide oxidoreductases and members of the thioredoxin (Trx) fold protein family, whose currently known function is to protect cells from protein oxidative damage.

[0076] The above results indicate that overexpression of EcPAPR confers the plant with the ability to actively resist stress.

[0077] Example 2 Overexpression of EcPAPR in Arabidopsis can improve the tolerance of plants to arsenic

[0078] Construction of Arabidopsis thaliana lines overexpressing the EcPAPR gene ( Figure 7 a), single-copy homozygotes were screened, and four transgenic lines with different exogenous protein accumulation were selected based on the results of immunoblotting ( Figure 7 b), the EcPAPR enzyme activity in the transgenic strains is as follows Figure 7 As shown in c.

[0079] Seedlings grown on 1 / 2MS medium for one week were selected and transplanted to 1 / 2MS medium supplemented with As(III) or As(V) (pH 5.8) at different concentrations (0, 100 μM 250 μM, 500 μM, 1000 μM, 2000 μM) under the following conditions: light intensity of 300 μmol m -2 s -1 , relative humidity 80%, temperature 24℃ under light, temperature 20℃ under dark, cycle 16h light, 8h dark. One week later, the phenotype was observed. When As(III) or As(V) reached 1000μM, the wild-type Arabidopsis phenotype was severe arsenic poisoning, and growth was basically stagnant, while the EcPAPR overexpressing material showed obvious arsenic tolerance (such as Figure 7 d, e, f, g), but the growth was also significantly slower than when no arsenic was added. This indicates that overexpression of the EcPAPR gene in Arabidopsis can improve its tolerance to arsenic.

[0080] Example 3 Overexpression of EcPAPR in Escherichia coli can improve its tolerance to arsenic

[0081] The EcPAPR was constructed into the E. coli prokaryotic expression vector pET30b and transferred into wild-type E. coli and EcPAPR mutant strain cysH, with the empty vector as the control. The same amount of bacterial liquid was inoculated into 200 ml LB liquid medium, and sodium arsenate with a final concentration of 75 μmol was added. The OD was measured at 0 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours and 72 hours. 600 The results showed that the mutant strain grew slowly, while the mutant strain grew faster after the EcPAPR gene was transferred into it. The wild type strain grew fastest after overexpressing EcPAPR ( Figure 8 ).

Claims

1. Application of the Escherichia coli EcPAPR gene in improving the tolerance of organisms to arsenic, wherein the nucleotide sequence of the Escherichia coli EcPAPR gene is shown in SEQ ID No. 1 or a degenerate sequence encoding the same protein as the nucleotide sequence shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that: The application is to overexpress the Escherichia coli EcPAPR gene in an organism, thereby improving the organism's tolerance to arsenic.

3. The use according to claim 1, characterized in that: The organism is corn, Arabidopsis thaliana or Escherichia coli.

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