Application of taurine and hypotaurine in inhibiting synthesis of plant cysteine

Abstract

The invention provides application of taurine and hypotaurine in inhibiting synthesis of plant cysteine. According to the invention, taurine and hypotaurine are used for directly inhibiting the catalytic function and activity of two cysteine synthesis key enzymes, namely SAT and OAS, in plants, so that synthesis of plant cysteine is hindered. The technology can be used for preparing a plant cysteine inhibitor, so that the amino acid metabolism process of plants is seriously disturbed, the growth of the plants is hindered, and then the technology has the potential of developing novel safe and non-toxic herbicides, can enrich the biological application in the field of agricultural weeding, and further lays a foundation for the application of exogenous compounds to plant amino acid synthesis inhibition. Meanwhile, the invention also provides a method for rapidly synthesizing the taurine and the hypotaurine in the nicotiana benthamiana leaves by utilizing an instantaneous conversion method, so that the synthesis cost of the taurine and the hypotaurine is reduced, and a basis is provided for later wide application.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to application of taurine and hypotaurine in inhibiting plant cysteine ​​synthesis. Background Art

[0002] Amino acids are essential substances in plants that can only be synthesized by themselves. Once the synthesis of amino acids is blocked, plants will have difficulty surviving. Therefore, among the currently commercialized herbicides, those that work by inhibiting the activity of key enzymes in plant amino acid biosynthesis account for a large proportion.

[0003] At present, the technology of using exogenous compounds to inhibit plant amino acid synthesis has been applied in many ways, and the most famous example is glyphosate (EPSPS inhibitor) herbicide. It has stable physical and chemical properties, low residue, does not damage the soil environment, is lethal to most plants, and has advantages that many herbicides on the market do not have. Among them, glyphosate plays a key role in blocking amino acid synthesis. Glyphosate can combine with EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) to form a stable complex, occupying the site of the enzyme's second substrate PEP (phosphoenolpyruvate), thereby effectively inhibiting the activity of the EPSP enzyme, making it impossible for other substrates to participate in the synthesis of aromatic amino acids and some secondary metabolic products, thereby achieving the purpose of hindering plant growth.

[0004] Cysteine, an aliphatic amino acid in plants, also plays an indispensable role in plant metabolism. Some studies have shown that the plant cysteine ​​metabolic pathway has a great influence on the synthesis and accumulation of some major metabolites in plants (such as amino acids, proteins and polyphenols, etc.), and is also closely related to the plant's response to adversity. It is an important sulfur donor molecule for many biosyntheses and redox. In addition, the synthesis pathway of plant cysteine ​​is also relatively specific. The two key enzymes in its synthesis, serine acetyltransferase (SAT) and O-acetylserine thiol lyase (OAS), are encoded by a multi-gene family and can reversibly bind to the two-enzyme complex for effective regulation. O-acetylserine thiol lyase is contained in the mitochondria, chloroplasts and cytoplasm of plant cells, thus forming a series of complex cysteine ​​pools. Therefore, inhibiting the synthesis of cysteine ​​will have a huge negative impact on plant metabolism and even hinder its growth. However, inhibitors for these two key enzymes in plant cysteine ​​synthesis have never been reported. Therefore, finding inhibitors that can inhibit these two enzymes will bring great prospects in the development and application of herbicides. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide a technical solution for the application of taurine and hypotaurine in inhibiting plant cysteine ​​synthesis.

[0006] The present invention is specifically implemented by the following technical solutions:

[0007] A first aspect of the present invention provides a use of taurine and hypotaurine in inhibiting plant cysteine ​​synthesis.

[0008] Furthermore, the taurine and hypotaurine can inhibit the biosynthesis of cysteine ​​in plants.

[0009] Furthermore, the taurine and hypotaurine can inhibit the activity of key enzymes in cysteine ​​synthesis in plants.

[0010] Furthermore, the key enzymes for cysteine ​​synthesis in the plant are serine acetyltransferase and O-acetylserine thiol lyase.

[0011] Furthermore, the taurine and hypotaurine as cysteine ​​synthetase inhibitors can disrupt the process of plant amino acid metabolism, thereby affecting plant growth.

[0012] The second aspect of the present invention provides a method for rapidly synthesizing taurine and hypotaurine of animal origin in plants.

[0013] Furthermore, the method for rapidly synthesizing taurine and hypotaurine of animal origin in plants is to rapidly synthesize taurine and hypotaurine in Nicotiana benthamiana leaves by using a transient conversion method.

[0014] Furthermore, the rapid synthesis of taurine and hypotaurine in Nicotiana benthamiana leaves using the transient transformation method requires the simultaneous overexpression of animal cysteine ​​dioxygenase and cysteine ​​sulfenic acid decarboxylase in the plant.

[0015] Furthermore, the transient transformation method is used to rapidly synthesize taurine and hypotaurine in Nicotiana benthamiana leaves. After transient expression, the Nicotiana benthamiana leaves are collected for extraction to obtain high-content taurine and hypotaurine.

[0016] The present invention utilizes taurine and hypotaurine to directly inhibit the catalytic function and activity of two key enzymes for cysteine ​​synthesis in plants, SAT and OAS, thereby hindering the synthesis of plant cysteine. The technology can be used to prepare plant cysteine ​​inhibitors, thereby seriously disrupting the amino acid metabolism process of plants and hindering their growth. It has the potential to develop new safe and non-toxic herbicides, which can enrich the application of biology in the field of agricultural weed control and further lay a solid foundation for the application of exogenous compounds to inhibit plant amino acid synthesis. At the same time, the present invention also provides a method for rapidly synthesizing taurine and hypotaurine in Nicotiana benthamiana leaves using an instantaneous conversion method, which reduces the synthesis cost of taurine and hypotaurine and provides a basis for future widespread application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 2 shows the synthesis of taurine and hypotaurine in Nicotiana benthamiana leaves based on two modules; (a) is an integrated diagram of the two modules for the synthesis of taurine and hypotaurine in Nicotiana benthamiana leaves; the cysteine ​​synthesis module involves the enzymes SAT and OAS, which convert serine, which is present in high concentrations in the plant, into cysteine; the taurine synthesis module contains the enzymes CDO and CSAD, which convert cysteine ​​into hypotaurine, which is then formed into taurine through spontaneous oxidation; (b) is an LC / MS chromatogram and quantitative analysis of cysteine ​​in Nicotiana benthamiana leaves, showing that cysteine ​​increased significantly after the cysteine ​​synthesis module was expressed alone, and increased expression of the cysteine ​​synthesis module increased cysteine ​​expression. There was no significant change after the expression of the taurine synthesis module; (c) and (d) LC / MS chromatograms and quantitative data demonstrated that hypotaurine and taurine were successfully detected in Nicotiana benthamiana leaves after the expression of the taurine synthesis module, and the levels of these two compounds did not show additional increases after the simultaneous expression of the cysteine ​​synthesis module; the data provided are the mean ± SEM of three biological replicates, and each infected Nicotiana benthamiana plant was regarded as a separate biological replicate; the unpaired t-test was used to determine the statistical significance between different groups, and the asterisk indicates the significance level (**p<0.01, ***p<0.001);

[0018] Figure 2 Taurine and hypotaurine inhibit plant cysteine ​​biosynthesis by inhibiting the activities of SAT and OAS enzymes; wherein, (a) is a schematic diagram of taurine and hypotaurine inhibiting the key enzymes SAT and OAS in cysteine ​​biosynthesis in plants; (b) shows that the application of 100 μM hypotaurine or 50 μM taurine can significantly reduce the level of plant cysteine ​​synthesis compared with leaves expressing only the cysteine ​​synthesis module; (c) shows that in a one-pot enzymatic assay of the enzymes SlSAT and SlOAS, the synthesis of cysteine ​​is significantly reduced after the addition of taurine or hypotaurine; (d) shows that the activity of SlSAT is reduced after the addition of taurine or hypotaurine because the synthesis of its product coenzyme A (CoA) is reduced; (e) shows that the activity of SlOAS is reduced in the presence of taurine or hypotaurine, and the yield of cysteine ​​synthesized by it is reduced; data are expressed as the mean ± SEM of three biological replicates. Asterisks indicate significant differences between treatment groups as determined by unpaired t-test (*p<0.05, **p<0.01, ***p<0.001);

[0019] Figure 3The expression of the taurine synthesis module severely disrupted the amino acid profile of Nicotiana benthamiana leaves; (a) Nicotiana benthamiana leaves expressing the taurine synthesis module showed a significant increase in the levels of 16 free amino acids, showing more significant changes compared with the leaves expressing the cysteine ​​synthesis module; (b) The changes in the amino acid profile produced by the co-expression of the cysteine ​​synthesis module and the taurine synthesis module were very similar to the amino acid profile observed when the taurine synthesis module was expressed alone, and most of them had no significant differences; the data shown in the figure are expressed as the mean ± SEM of three biological replicates, and each infected Nicotiana benthamiana plant was regarded as a single biological replicate; asterisks indicate significant differences between the comparison groups determined by unpaired t-test (*p<0.05, **p<0.01, ***p<0.001). DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with specific embodiments.

[0021] 1. Plant Materials and Growth Conditions

[0022] This experiment uses Nicotiana benthanmiana leaves as biological materials to evaluate the efficacy of synthetic modules expressed in plants. The seedlings of Nicotiana benthanmiana were cultured in a plant growth chamber set at a temperature of 21-25°C, a light intensity of 6000Lux, and a photoperiod scheme of 16 hours of light alternating with 8 hours of darkness. After the cotyledons appear about 7-10 days after sowing, the seedlings are carefully transferred to separate pots and watered regularly to maintain adequate nutrient supply. The morphological criteria for plant health are the basis for the selection of experimental materials, such as vibrant green and robust leaves at the 5-6 week development stage.

[0023] 2. Construction of plant transient transformation vector and protein expression vector

[0024] The non-plant-derived genes used in this experiment were chemically synthesized, including cysteine ​​dioxygenase (CcCDO:BAC73112) and cysteine ​​sulfinic acid decarboxylase (CcCSAD:BAC73113) from carp (Cyprinuscarpio). At the same time, the serine acetyltransferase (SlSAT:Solyc02g082850) and O-acetylserine thiol lyase (SlOAS:Solyc09g082060) from tomato (Solanum lycopersicum) were amplified by PCR using the primers described in detail in Table S1. pEAQ and pET28b vectors were linearized using PCR with their respective primers, and the primers were also listed in Table S1. According to the manufacturer's homologous recombination instructions, a one-step fusion cloning hybrid kit (TOROIVD, FCM-050) was used to obtain a vector fused to the gene fragment and the vector backbone.

[0025] 3. Transient Expression in Nicotiana benthamiana

[0026] The effect of synthetic module expression in plants was evaluated using the Nicotiana benthamiana transient expression system. The pEAQ vector carrying the correct gene fragment was introduced into the Agrobacterium tumefaciens GV3101 strain, and then the positive strains were screened on LB plate medium containing 50 mg / L kanamycin and 25 mg / mL rifampicin, and the positive colonies were confirmed by PCR using the primers listed in Table S1. The positive Agrobacterium strains were then cultured at 28°C in LB liquid medium containing the same antibiotics until the OD600 value reached 1.0 to 1.2. The strains of each gene that needed to be expressed together were mixed in a centrifuge tube, and the volume of each strain was calculated based on the actual OD600 value of each strain to ensure that the final OD600 of each strain was adjusted to 0.5. The combined bacterial solution was centrifuged at 5000g for 10 minutes at room temperature. The precipitate was washed with washing buffer (10mM MES, pH 5.6; 10mM MgCl2) and reprecipitated by centrifugation. The resulting precipitate was resuspended in infection buffer (10 mM MES, pH 5.6, 10 mM MgCl2 and 200 mM acetosyringone) to prepare the infection solution. After incubation at room temperature for 2 hours, the infection solution was loaded into a syringe and gently infiltrated into the back of the Nicotiana benthamiana leaves. 5-6 week-old Nicotiana benthamiana was selected for transformation, focusing on infecting 3 to 5 leaves below the top. After infection, the leaves showed a short wet appearance. Infected leaves were harvested 4-5 days after infection, immediately frozen in liquid nitrogen, and stored at -80 ° C for subsequent analysis.

[0027] 4. Extraction and detection of amino acids and their derivatives

[0028] About 100 mg of ground Nicotiana benthamiana sample was extracted using 400 μL of isotope-labeled amino acid extraction buffer (detailed components are shown in Table S2). After the sample was vortexed for 10 seconds to mix, it was extracted at 90°C for 10 minutes. The extraction solution was then cooled on ice for 5 minutes and centrifuged at 13,000 g for 10 minutes at 4°C. The supernatant was transferred to a new 1.5 mL centrifuge tube and filtered using a 0.45 μm hydrophilic polytetrafluoroethylene (PTFE) centrifugal filter to prepare samples for liquid chromatography and temporarily stored at 4°C if necessary.

[0029] The amino acids and their derivatives were separated and quantified using an Agilent 6495Triple Quadrupole LC / MS system equipped with a Waters ACQUITY UPLC BEH amide column (1.7 μm particle size, 2.1×100 mm). The chromatographic separation was started with a mobile phase consisting of 10% solvent A (10 mM ammonium acetate and 0.2% formic acid) and 90% solvent B (acetonitrile). The column temperature was maintained at 40° C. and the flow rate was set at 0.4 mL / min. The elution gradient was as follows: first, 1 minute at 90% solvent B, then 1 to 4 minutes to reduce solvent B to 50% concentration, then maintained at 50% solvent B for 5 minutes, at 5.1 minutes, the solvent B concentration was increased again to 90%, and maintained at 90% solvent B for 7 minutes.

[0030] The amino acids and their derivatives in Nicotiana benthamiana leaf samples were quantitatively analyzed by isotope dilution in multiple reaction monitoring (MRM) mode. Isotope-labeled standards in the extraction buffer facilitated the quantification of peaks by MRM, and the dwell time, collision voltage, fragmentation voltage, precursor ion, and product ion were optimized and listed in Table S3. The system calculated the concentrations of 20 standard amino acids and taurine and hypotaurine based on the peak area and internal standards. Each tobacco plant was considered as a biological replicate, and each treatment had three replicates.

[0031] 5. Protein expression and purification using E. coli system

[0032] In order to study the effects of taurine and hypotaurine on the activity of plant serine acetyltransferase (SAT) and O-acetylserine thiol lyase (OAS), the present invention carried out protein expression and in vitro enzyme activity determination. The tomato SlSAT and SlOAS genes were cloned into the pET28b expression vector and transformed into Escherichia coli Rosetta (DE3) cells. The positive strains were screened on LB plate medium containing 50mg / L kanamycin and 34mg / L chloramphenicol using the primers detailed in Table S1. The positive Escherichia coli strains were cultured at 37°C in liquid LB medium containing the above-mentioned antibiotics until OD600 reached 0.5-0.6. After adding 0.05mM isopropyl β-D-1-thiogalactopyranoside (IPTG), the culture was shaken and cultured at 16°C for about 14 hours at 120rpm to induce protein expression. The Escherichia coli cells were harvested by centrifugation at 4°C for 15 minutes at 5000g. The precipitate was resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10% glycerol, pH 8.0), the resuspended cells were lysed by ultrasonic treatment, and then the E. coli cell debris was removed by centrifugation at 20000 g for 30 minutes at 4 ° C. The supernatant was combined with Ni-NTA resin pre-equilibrated with lysis buffer at 4 ° C for 2 hours. The resin was then collected by gentle centrifugation at 1000 g for 5 minutes at 4 ° C, transferred to a gravity column, washed with lysis buffer to remove unbound proteins, and eluted with imidazole buffer containing increasing concentrations. Protein purity was assessed using SDS-PAGE and Western blot analysis. For short-term storage, the purified protein was mixed with glycerol to a final concentration of 40% and stored at -20 ° C for subsequent use.

[0033] 6. In vitro enzyme activity assay of SlSAT and SlOAS

[0034] In vitro enzyme activity assays of SlSAT and SlOAS were performed to elucidate the function of these enzymes in cysteine ​​biosynthesis. For the SlSAT assay, a reaction mixture containing 50 mM Tris-HCl (pH 7.6), 1 mM EDTA, 20 mM L-serine, and 0.1 mM acetyl-CoA was prepared. Catalysis was initiated by the addition of purified recombinant SlSAT protein, and the reaction was catalyzed for 25 min at 20 °C. The content of product coenzyme A (CoA) was measured by adding 50 μl of 1 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and the reaction was continued for an additional 5 min, monitoring the formation of yellow 5-thio-2-nitrobenzoic acid spectrophotometrically at 412 nm. A standard curve was established using known concentrations of CoA (0, 12.5, 25, 50, 100, and 200 μM) in the reaction mixture. To evaluate the effects of hypotaurine and taurine, 100 μM hypotaurine or 50 μM taurine were added to the reactions and analyzed along with control experiments.

[0035] The SlOAS enzyme activity reaction mixture consisted of 50 mM Tris-HCl (pH 7.5), 5 mM DTT, 5 μM PLP, 5 mM O-acetylserine, and 10 mM Na2S. The reaction was started after the addition of purified SlOAS protein, maintained at 25°C for 10 min, and then 100 μl of acetic anhydride was added to terminate the reaction. The mixture was then treated with 200 μl of ninhydrin reagent (25 mg / ml in acetate:HCl, 60:40, v / v) and heated at 95°C for 10 min, followed by rapid cooling, and finally 200 μl of anhydrous ethanol was added to stop the reaction. The generation of cysteine ​​was quantified by measuring the absorbance at 560 nm. To explore the effects of hypotaurine and taurine on SlOAS enzyme activity, 100 μM hypotaurine or 50 μM taurine was added to the reaction. Each treatment was repeated 3 times, and each experiment was repeated 3 times independently.

[0036] 7. Experimental Results

[0037] Taurine and hypotaurine produced by the taurine module inhibit the action of cysteine ​​biosynthetic enzymes in Nicotiana benthamiana

[0038] Utilizing an existing dual-module synthesis method for increasing carnosine production, the present application turns its attention to synthesizing taurine, an aminosulfonic acid that is not normally present in plant metabolism. Using Nicotiana benthamiana as a host, the aim is to direct the naturally high levels of serine into the production of cysteine, the direct precursor of taurine. The present application introduces a cysteine ​​synthesis module, including tomato serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS6), to facilitate this conversion. Subsequently, the animal-specific enzymes contained in the taurine module - cysteine ​​dioxygenase (CDO) and cysteine ​​sulfenic acid decarboxylase (CSAD) from carp - are used to convert cysteine ​​into hypotaurine, and ultimately into taurine ( Figure 1 a). First, expression of a single cysteine ​​biosynthesis module in Nicotiana benthamiana leaves increased cysteine ​​levels 11-fold, from 1.23 μg / g to 14.40 μg / g ( Figure 1 b); After expressing the taurine synthesis module, the content of hypotaurine was significantly increased, and an appropriate amount of taurine was produced ( Figure 1 c, 1d). However, when both modules were expressed simultaneously, the levels of hypotaurine and taurine were slightly lower than those obtained by expressing the taurine synthesis module alone, while the change in cysteine ​​level was not significant ( Figure 1 b).

[0039] Secondly, the application of 100 μM hypotaurine or 50 μM taurine can effectively inhibit the function of the cysteine ​​synthesis module in Nicotiana benthamiana leaves. Compared with the expression of the cysteine ​​module alone, when hypotaurine was injected at the same time, the cysteine ​​content was significantly reduced from 14.40 μg / g to 5.57 μg / g ( Figure 2 b); When taurine was injected simultaneously, the cysteine ​​content decreased to 2.30 μg / g, close to the level of the pEAQ control group ( Figure 2 b).

[0040] In order to clarify the inhibitory mechanism of hypotaurine and taurine on the cysteine ​​biosynthesis pathway, this application proposes the hypothesis that these metabolites can directly inhibit the enzyme activities of key enzymes SAT and OAS in the cysteine ​​module ( Figure 2 a). Introduction of 100 μM hypotaurine or 50 μM taurine into the one-pot reaction mixture of SlSAT and SlOAS resulted in a significant decrease in cysteine ​​production ( Figure 2 c). Single enzyme activity assays using SlSAT or SlOAS also showed that hypotaurine and taurine can indeed inhibit the catalytic activity of these key enzymes in plant cysteine ​​synthesis ( Figure 2 d, 2e).

[0041] Compared with leaves expressing only the cysteine ​​biosynthesis module, expression of the taurine biosynthesis module alone resulted in a significant upregulation of 15 amino acids ( Figure 3 a, Table S4). However, the amino acid profile changes produced by the co-expression of the cysteine ​​synthesis module and the taurine synthesis module were very similar to the amino acid profile observed when the taurine synthesis module was expressed alone ( Figure 3 b) These changes indicate that taurine and hypotaurine seriously disrupt the amino acid metabolism process of plants by acting as inhibitors of plant SAT and OAS enzyme activities, thereby affecting their normal growth.

[0042] Table S1: Primer sequences

[0043]

[0044]

[0045] Table S2: Composition of isotope-labeled amino acid extraction buffer

[0046] name English name Isotope labeling concentration Methanol Methanol - 5％ Formic acid Formic acid - 0.1％ Alanine L-Alanine 13C3,15N 8uM Arginine L-Arginine 13C6 8uM Aspartic acid L-Aspartic acid 13C4 8uM Glutamate L-Glutamic acid 13C5 8uM Glycine Glycine 13C2,15N 8uM Histidine L-Histidine 13C6 8uM Isoleucine L-Isoleucine 13C6,15N 8uM Leucine L-Leucine 13C6,15N 8uM Lysine L-Lysine 13C6 8uM Methionine L-Methionine 13C5,15N 8uM Phenylalanine L-Phenylalanine 13C6 8uM Proline L-Proline 13C5 8uM Serine L-Serine 13C3,15N 8uM Threonine L-Threonine 13C4 8uM Tyrosine L-Tyrosine 13C6 8uM Valine L-Valine 13C5 8uM Cystine L-Cystine 13C6,15N2 4uM

[0047] Table S3: MRM parameter settings: electrospray photoionization (positive ion mode)

[0048]

[0049] Table S4: Amino acid content in Nicotiana benthamiana leaves after expressing different modules μg / g (fresh weight)

[0050]

Claims

1. Application of taurine and hypotaurine in inhibiting plant cysteine ​​synthesis.

2. The use according to claim 1, characterized in that The taurine and hypotaurine can inhibit the biosynthesis of cysteine ​​in plants.

3. The use according to claim 1, characterized in that The taurine and hypotaurine can inhibit the activity of key enzymes in cysteine ​​synthesis in plants.

4. The use according to claim 3, characterized in that The key enzymes for cysteine ​​synthesis in the plant are serine acetyltransferase and O-acetylserine thiol lyase.

5. Use of taurine and hypotaurine as cysteine ​​synthetase inhibitors.

6. The use according to claim 1, characterized in that This application uses a transient conversion method to rapidly synthesize animal-derived taurine and hypotaurine in Nicotiana benthamiana leaves.

7. As claimed in claim 6, characterized in that The rapid synthesis of taurine and hypotaurine in Nicotiana benthamiana leaves using the transient transformation method requires the simultaneous overexpression of animal cysteine ​​dioxygenase and cysteine ​​sulfenic acid decarboxylase in the plant.

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