Method for improving self-luminous intensity of plant

Abstract

The invention discloses a method for improving the self-luminous intensity of a plant, which is characterized in that an AroGL175Q gene or an RgTAL gene and a PtC3H gene or the AroGL175Q gene, the RgTAL gene and the PtC3H gene are expressed in a plant containing a fungus bioluminescence system FBP, so that a transgenic plant of which the caffeic acid content, the milk tree alkali content and the luminous intensity are obviously increased is obtained. According to the invention, through metabolic network optimization and transformation, phenylpropane metabolites required by middle and upper streams of a fungus bioluminescence pathway are increased, and synthesis of caffeic acid and milk tree alkali in plants is increased, so that bioluminescence enhancement is realized. The precursor supply bottleneck of a fungus bioluminescence system is broken through, and an innovative solution is provided for creating efficient self-luminous plants.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and specifically, discloses a method for increasing the autoluminescence intensity of plants. Background Technology

[0002] The phenylpropanoid pathway is a crucial metabolic pathway in plants. Its core product, L-phenylalanine (Phe), is not only an essential amino acid for protein synthesis but also a precursor to many secondary metabolites. These secondary metabolites play key roles in plant growth, development, and defense mechanisms. For example, lignin provides mechanical support for plant cell walls, flavonoids and isoflavones participate in antioxidant and antipathogenic defenses, while volatile phenylpropanoids help attract pollinators and transmit signals. Furthermore, many phenylpropanoid derivatives have important medicinal and industrial value, such as natural products like salicylic acid.

[0003] Bioluminescence is a light phenomenon produced by living organisms through natural chemical reactions. It is widely distributed in marine and terrestrial ecosystems and is hailed as one of the most amazing phenomena in nature. As a highly efficient biological signaling system, bioluminescence has become a research hotspot in many disciplines, including basic biology, applied biology, chemistry, and medicine (Tsarkova et al., 2016). The fungal bioluminescence pathway (FBP) is a bioluminescent pathway that uses caffeic acid as a substrate and ultimately produces green fluorescence at a wavelength of approximately 520 nm. This pathway involves a caffeic acid metabolic cycle composed of four enzymes: caffeic acid is converted to milkweed alkaloid by hispidin synthase (HispS), which is then catalyzed by hispidin-3-hydroxylase (H3H) to produce fungal luciferin. Luciferin is oxidized to caffeoylpyruvate by fungal luciferase (Luz), producing photons. Finally, caffeoylpyruvate hydrolase (CPH) converts caffeoylpyruvate back to caffeic acid, thus generating continuous, visible light and achieving the recycling of caffeic acid. It is noteworthy that in fungal bioluminescence systems, the phenylpropane pathway, as a key upstream metabolic pathway, has a metabolic flux that becomes a limiting factor for the entire luminescence system. With the acceleration of modern urbanization, the application prospects of luminescent plants with ornamental value in urban landscape construction and home gardening are becoming increasingly prominent. Plant systems, due to their unique advantages, have become ideal research platforms for bioluminescence: on the one hand, they possess complete energy production and storage mechanisms (Giraldo et al., 2014); on the other hand, they have strong self-repair and regeneration capabilities. These characteristics make the research and modification of plant bioluminescent systems not only of significant scientific importance but also hold immense application value. The brightness of plant bioluminescent systems is mainly limited by the supply efficiency of the luminescent substrate. Optimizing key metabolic pathways such as the phenylpropanoid pathway may break through the efficiency bottleneck of existing bioluminescent systems, laying the foundation for the development of novel sustainable luminescent plants. Summary of the Invention

[0004] The purpose of this invention is to overcome the aforementioned defects and deficiencies in the prior art and provide a method for increasing the content of caffeic acid and milkweed alkaloids in plants and improving the autoluminescence intensity of plants. This is achieved through metabolic pathway redesign, introducing... AroG L175Q , RgTAL , PtC3HMultiple heterologous genes increase the production of phenylpropane metabolites required in the upstream of the fungal bioluminescence pathway, and increase the synthesis of caffeic acid and milk alkaloids in plants, thereby enhancing bioluminescence.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution: This invention optimizes and modifies the endogenous metabolic network of plants through a systematic metabolic engineering strategy. Specifically, it introduces [a specific method / mechanism] to regulate the upstream metabolic network of phenylpropanoid metabolites required in the fungal bioluminescence pathway. AroG L175Q It is a feedback-insensitive 3-deoxy-D-arabinohepantrolactone-7-phosphate synthase (DAHPS) from *E. coli*, capable of bypassing the feedback inhibition of phenylalanine precursor branched acid, which is normally encountered by DAHPS in plants, allowing a continuous flow of carbon to the phenylpropanoid metabolic pathway. Through the analysis of… AroG L175Q Plant codons were optimized, and the optimized ones were... AroG L175Q The CDS sequence of the gene is shown in SEQ ID No. 1. Furthermore, to further optimize the gene circuit design, tyrosine was introduced into the caffeic acid synthesis pathway. RgTAL Genes (derived from) Rhodotorula glutinis (as shown in SEQ ID No. 2). Also found in plants originating from poplar. PtC3H The gene C3H is a key enzyme in the phenylpropane metabolic pathway, responsible for hydroxylating p-coumaric acid to produce caffeic acid. Its CDS sequence is shown in SEQ ID No. 3. This invention utilizes... AroG L175Q 、RgTAL, PtC3H Combined with fungal bioluminescent systems FBP Co-expression was performed in co-infected plants, and the results showed that when... AroG L175Q and FBP When co-expressed with the system, the luminescence intensity increased significantly, approximately doubling. Metabolic data showed that... AroG L175Q and FBP After co-expression in the system, the contents of caffeic acid and milk alkaloids also increased significantly by about 100%. Furthermore, when... RgTAL , PtC3H common and FBP When expressed in the system, luminescence was also significantly enhanced, based on metabolic data. RgTAL , PtC3H The addition of [a specific ingredient] resulted in an approximately 1-fold increase in the luminescent substrate caffeic acid and an approximately 1.5-fold increase in milk alkaloids, indicating that the two synergistically optimized the precursor supply. Meanwhile... FBP and AroG L175Q , RgTAL ,PtC3H When co-expressed, the luminescence intensity is approximately the same as when expressed alone. FBP The results also showed that when all three genes worked together, the contents of caffeic acid and milk alkaloids both increased by about two times; this indicates that the combined action of the three genes maximizes precursor accumulation, providing more abundant precursor substances for the fungal bioluminescence pathway, thereby significantly improving bioluminescence efficiency. AroG L175Q Gene or RgTAL Genes and PtC3H Gene co-expression or AroG L175Q Gene, RgTAL Genes and PtC3H Gene co-expression can increase the content of caffeic acid and milk alkaloids in plants and enhance the intensity of plant bioluminescence.

[0006] Therefore, this invention first protects AroG L175Q Gene or RgTAL Genes and PtC3H Gene co-expression or AroG L175Q Gene, RgTAL Genes and PtC3H The application of gene co-expression in increasing the content of caffeic acid and milk alkaloids in plants and enhancing the intensity of plant bioluminescence. AroG L175Q The coding sequence of the gene is shown in SEQ ID No. 1; RgTAL The coding sequence of the gene is shown in SEQ ID No. 2; PtC3H The gene coding sequence is shown in SEQ ID No. 3; the plant is a self-luminescent plant containing a fungal bioluminescent system (FBP), wherein the FBP contains... HispS Gene, H3H Gene, Luz Genes and CPH Gene.

[0007] Furthermore, the fungal bioluminescent system FBP also includes NPGA Genes. NPGA can perform post-translational modifications on HispS, which enhance HispS activity and thus increase the strength of FBP.

[0008] Preferably, the NPGA The gene comes from Aspergillus nidus ( Aspergillus nidulans ),Right now AnNPG A.

[0009] This invention also provides a method for increasing the content of caffeic acid and milkweed alkaloids in plants and increasing the bioluminescence intensity of plants, for the purpose of... AroG L175QGenes and fungal bioluminescent systems (FBP) co-infect plants for co-expression or RgTAL Gene, PtC3H Genes and fungal bioluminescent systems (FBP) co-infect plants for co-expression or AroG L175Q Gene, RgTAL Gene, PtC3H The gene was co-expressed in plants by co-infecting them with the fungal bioluminescence system FBP, resulting in transgenic plants with significantly increased caffeic acid and milk alkaloid content and luminescence intensity; AroG L175Q The coding sequence of the gene is shown in SEQ ID No. 1; RgTAL The coding sequence of the gene is shown in SEQ ID No. 2; PtC3H The coding sequence of the gene is shown in SEQ ID No. 3; the fungal bioluminescent system FBP contains HispS Gene, H3H Gene, Luz Genes and CPH Gene.

[0010] Furthermore, the method specifically includes the following steps: S1. Construct respectively containing AroG L175Q Gene, RgTAL Gene or PtC3H Plant binary expression vectors for genes; S2. Simultaneous transient conversion of fungal bioluminescent systems FBP and... AroG L175Q Plant binary expression vectors or simultaneous transient transformation of fungal bioluminescent systems FBP and RgTAL Gene, PtC3H Gene-plant binary expression vectors or simultaneous transient transformation of fungal bioluminescent systems FBP and AroG L175Q , RgTAL Gene, PtC3H Gene-plant binary expression vector; S3. Cultivation resulted in transgenic plants with significantly increased caffeic acid and milk alkaloid content, as well as increased luminescence intensity.

[0011] Furthermore, the fungal bioluminescent system FBP also includes NPGA Gene.

[0012] Furthermore, the aforementioned AroG L175Q Gene, RgTAL Gene or PtC3H In plant binary expression vectors for genes, the upstream of the target gene contains a 35S promoter sequence.

[0013] Furthermore, the plant binary expression vector is pCAMBIA1300.

[0014] Furthermore, the instantaneous conversion is performed using the Agrobacterium-mediated method.

[0015] Furthermore, the plant in question is a higher plant.

[0016] Preferably, the higher plant is chrysanthemum, petunia, rapeseed, orchid, or tobacco.

[0017] Preferably, the HispS Gene, H3H Gene, Luz Genes and CPH The gene comes from the fungus Ganoderma lucidum ( Neonothopanus nambi ),Right now NnHispS , NnH3H , NnLuz, NnCPH .

[0018] Furthermore, the fungal bioluminescent system FBP uses Cre / loxP Marker-mediated self-deletion expression vectors can be used to obtain transgenic plants without selection markers by employing a marker self-deletion system.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention redesigns metabolic pathways to... AroG L175Q Gene or RgTAL Genes and PtC3H Gene or AroG L175Q Gene, RgTAL Genes and PtC3H Gene expression in plants containing the fungal bioluminescence system (FBP) resulted in transgenic plants with significantly increased caffeic acid and galactoside content, as well as enhanced bioluminescence intensity. This invention enhances bioluminescence by optimizing the metabolic network, increasing the production of phenylpropanoid metabolites required upstream in the fungal bioluminescence pathway, and thus increasing the synthesis of caffeic acid and galactoside in the plant. This invention overcomes the precursor supply bottleneck of the fungal bioluminescence system, providing an innovative solution for creating highly efficient self-luminescent plants. Attached Figure Description

[0020] Figure 1 Based on FBP Carrier schematic diagram, including Cre / loxP Mediated marker-assisted self-deletion expression vectors and their co-assembly NnHispS , NnH3H , NnLuz, NnCPH and AnNPG Expression cassette of gene A.

[0021] Figure 2 for FBP System addition AroG L175Q Post-genetic optimization of performance. Among them, Figure 2 A in the middle is FBP and AroG L175Q Bioluminescence performance and intensity analysis of *Nicotiana benthamiana* leaves were performed after 72 hours of co-expression. Scale bar: 1 cm. Error represents the standard deviation of nine replicates. Statistical significance was determined by two-tailed sampling. t Inspection and evaluation (**) p ≤0.01). B and C represent the contents of caffeic acid (B) and milkweed alkaloid (C) in the leaves of the above samples analyzed by LC-MS / MS. Statistical significance was determined by two-tailed... t Inspection and evaluation (***) p ≤ 0.001).

[0022] Figure 3 for FBP System addition RgTAL and PtC3H Post-genetic optimization of performance. Among them, Figure 3 A in the middle is FBP and RgTAL （T） , PtC3H (C) Bioluminescence performance and intensity analysis of *Nicotiana benthamiana* leaves were performed after 72 hours of co-expression. Scale bar: 1 cm. Error represents the standard deviation of twelve replicates. Statistical significance was determined by two-tailed sampling. t Inspection and evaluation (**) p ≤0.01). B and C represent the contents of caffeic acid (B) and milkweed alkaloid (C) in the leaves of the above samples analyzed by LC-MS / MS. Statistical significance was determined by two-tailed... t Inspection and evaluation (**) p ≤ 0.01, *** p ≤ 0.001).

[0023] Figure 4 for FBP System addition AroG L175Q , RgTAL and PtC3H The luminescence of plants was significantly enhanced after gene administration. Among them, Figure 4 A in the middle is FBP and AroG L175Q （A） , RgTAL (T) , PtC3H (C) Bioluminescence performance and intensity analysis of *Nicotiana benthamiana* leaves were performed after 72 hours of co-expression. Scale bar: 1 cm. Error represents the standard deviation of twenty-four replicates. Statistical significance was determined by two-tailed sampling. tInspection and evaluation (****) p ≤0.0001). B and C represent the contents of caffeic acid (B) and milkweed alkaloid (C) in the leaves of the above samples analyzed by LC-MS / MS. Statistical significance was determined by two-tailed... t Inspection and evaluation (***) p ≤ 0.001, **** p ≤ 0.0001).

[0024] Figure 5 for FBP System addition AroG L175Q , RgTAL and PtC3H A schematic diagram of metabolic pathway optimization that significantly enhances luminescence in plants after gene therapy. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0027] The plant expression vector was pCAMBIA1300-35S-Flag-NOS, preserved in the laboratory: the backbone vector pCAMBIA1300, the MCS region containing a 35S overexpression promoter, a 3×Flag tag and a NOS terminator, Kan resistance, and hygromycin plant selection markers.

[0028] Example 1: Design and Construction of Related Carriers Based on the sequence information of the vector pCAMBIA1300-35S-Flag-NOS, the plant codons were optimized and modified. AroG L175Q Gene (CDS sequence as shown in SEQ ID No. 1). RgTAL Genes (derived from) Rhodotorula glutinis, (CDS sequence shown in SEQ ID No. 2) PtC3H Gene (CDS sequence as shown in SEQ ID No. 3). Designed according to the instructions of the one-step rapid cloning kit. AroG L175Q , RgTAL and PtC3H The relevant primers were used to amplify and remove the stop codon using KOD One high-fidelity DNA polymerase.AroG L175Q , RgTAL and PtC3H The gene coding region sequence was ligated into the pCAMBIA1300-35S-Flag-NOS vector, which already contained a 3×Flag tag, using homologous recombination to construct the pCAMBIA1300-AroG gene. L175Q pCAMBIA1300-RgTAL and pCAMBIA1300-PtC3H vectors.

[0029] Example 2: Verification of the transient expression effect of related vectors in tobacco 1. Method The above pCAMBIA1300-AroG L175Q After transformation of EHA105 Agrobacterium with pCAMBIA1300-RgTAL and pCAMBIA1300-PtC3H vectors, and verification of correct expression, transient expression was achieved in tobacco. Simultaneous transient transformation of the FBP system in tobacco was then used to further demonstrate the results. FBP Carrier schematic diagram as follows Figure 1 (as shown) and the aforementioned carrier. The specific method is as follows: 1) Activate in LB medium containing Kana+Rif and incubate for 1 day at 200 rpm in a shaker at 28°C; 2) Transfer 1 mL of bacterial culture to 20 mL of LB medium containing Kana + Rif + 15 μM acetylsyringone for expansion culture. Incubate at 28℃ and 200 rpm until OD reaches 100%. 600 =0.8-1.0; 3) Centrifuge at 5000 rpm for 10 min to collect bacteria. Wash Agrobacterium with infiltration buffer (10 mM MES, 10 mM MgCl2, 150 μM AS) and resuspend at OD. 600 =0.8-1.0, let stand at room temperature for 2-3 hours; 4) Use a 1 mL syringe with the needle removed to draw up the bacterial solution. Gently poke a small hole (without piercing the leaf) on the lower epidermis of a tender ZY100 tobacco leaf that is about 3 weeks old. Press the back with your thumb and slowly inject Agrobacterium into the tobacco leaf with a syringe. Mark the water-soaked area of ​​the tobacco leaf. 5) The plants were cultured at 25℃ under normal growth conditions for approximately 72 hours. Subsequently, images of the plant bioluminescence signals and quantitative analysis of photon flux were performed using the NIGHTSHADE LB985 system manufactured by Berthold GmbH, Germany. Bioluminescence images were captured with a 60-second exposure, and then the region used for photon calculation was selected. Finally, the data was exported for analysis.

[0030] 6) After taking the photos, cut off the marked leaf area, put it into liquid nitrogen for quick freezing, and store it in a -80℃ freezer.

[0031] 7) Perform LC-MS / MS (liquid chromatography-mass spectrometry) analysis on the samples.

[0032] 2. Results when AroG L175Q and FBP When the system is injected alone, the luminescence intensity increases significantly, approximately doubling. Figure 2 A), Metabolic data show that, AroG L175Q and FBP After co-expression in the system, the contents of caffeic acid and milk alkaloids also increased significantly by about 100%. Figure 2 (B and 2C).

[0033] Furthermore, when RgTAL , PtC3H common and FBP When expressed in the system, luminescence is also significantly enhanced. Figure 3 A) From the perspective of metabolic data... RgTAL , PtC3H The addition of this compound increased the luminescent substrate caffeic acid by approximately 1 time and milk alkaloids by approximately 1.5 times. Figure 3 (B and 3C) indicates that the two synergistically optimize precursor supply.

[0034] and FBP and AroG L175Q , RgTAL , PtC3H When co-expressed, the luminescence intensity is approximately the same as when expressed alone. FBP 3 times ( Figure 4 A), metabolic results also showed that when the three genes worked together, the content of caffeic acid and milk alkaloids increased by about 2 times. Figure 4 B and 4C); its metabolic pathway optimization diagram is shown below. Figure 5 As shown. Therefore, when the three work together, precursor accumulation can be maximized, providing more sufficient precursor substances for the fungal bioluminescence pathway, thereby significantly improving bioluminescence efficiency.

Claims

1. AroG L175Q Gene or RgTAL Genes and PtC3H Gene co-expression or AroG L175Q Gene, RgTAL Genes and PtC3H The application of gene co-expression in increasing the content of caffeic acid and milkweed alkaloids in plants and enhancing the intensity of plant bioluminescence is characterized by, The AroG L175Q The coding sequence of the gene is shown in SEQ ID No. 1; RgTAL The coding sequence of the gene is shown in SEQ ID No. 2; PtC3H The gene coding sequence is shown in SEQ ID No. 3; the plant is a self-luminescent plant containing a fungal bioluminescent system (FBP), wherein the FBP contains... HispS Gene, H3H Gene, Luz Genes and CPH Gene.

2. The application according to claim 1, characterized in that, The fungal bioluminescent system FBP also includes NPGA Gene.

3. A method for increasing the content of caffeic acid and milkweed alkaloids in plants and increasing the intensity of plant bioluminescence, characterized in that, Will AroG L175Q Genes and fungal bioluminescent systems (FBP) are used to transiently transform plants for co-expression or RgTAL Gene, PtC3H Genes and fungal bioluminescent systems (FBP) are used to transiently transform plants for co-expression or AroG L175Q Gene, RgTAL Gene, PtC3H Genes and the fungal bioluminescence system FBP were transiently transformed into plants for co-expression, resulting in transgenic plants with significantly increased caffeic acid and milk alkaloid content and luminescence intensity; AroG L175Q The coding sequence of the gene is shown in SEQ ID No. 1; RgTAL The coding sequence of the gene is shown in SEQ ID No. 2; PtC3H The coding sequence of the gene is shown in SEQ ID No. 3; the fungal bioluminescent system FBP contains HispS Gene, H3H Gene, Luz Genes and CPH Gene.

4. The method according to claim 3, characterized in that, The method specifically includes the following steps: S1. Construct respectively containing AroG L175Q Gene, RgTAL Gene or PtC3H Plant binary expression vectors for genes; S2. Simultaneous transient conversion of fungal bioluminescent systems FBP and... AroG L175Q Plant binary expression vectors or simultaneous transient transformation of fungal bioluminescent systems FBP and RgTAL Gene, PtC3H Gene-plant binary expression vectors or simultaneous transient transformation of fungal bioluminescent systems FBP and AroG L175Q , RgTAL Gene, PtC3H Gene-plant binary expression vector; S3. Cultivation resulted in transgenic plants with significantly increased caffeic acid and milk alkaloid content, as well as increased luminescence intensity.

5. The method according to claim 3 or 4, characterized in that, The fungal bioluminescent system FBP also includes NPGA Gene.

6. The method according to claim 4, characterized in that, The AroG L175Q Gene, RgTAL Gene or PtC3H In plant binary expression vectors for genes, the upstream of the target gene contains a 35S promoter sequence.

7. The method according to claim 4, characterized in that, The plant binary expression vector is pCAMBIA1300.

8. The method according to claim 3 or 4, characterized in that, The instantaneous conversion was achieved using the Agrobacterium-mediated method.

9. The method according to any one of claims 3 to 8, characterized in that, The plant in question is a higher plant.

10. The method according to claim 9, characterized in that, The higher plants mentioned are chrysanthemum, petunia, rapeseed, orchid, or tobacco.

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