Highlight-induced photosynthetic electron transfer loop based on recombinase and application

Abstract

The invention discloses a recombinase-based highlight induced photosynthetic electron transfer loop and application thereof, the loop uses a highlight induced ELIP1 promoter to drive FLP recombinase expression, and regulates and controls the expression of a photosynthetic electron transfer key component gene PETE or PETC through an FLP / FRT site specific recombination system. The problem of light inhibition caused by overload of a plant photosynthetic electron transfer chain in a highlight environment is solved, light intensity dependent gene expression regulation and control are realized, the photosynthetic electron transfer characteristic under highlight stress is effectively improved, and the photoadaptability and photosynthetic efficiency of plants are enhanced. The system provides a new tool for crop stress resistance molecular breeding and synthetic biology research.

Description

Technical Field

[0001] This invention belongs to the field of plant synthetic biology and genetic engineering. By combining the engineering design concept of synthetic biology with the technical means of plant genetic engineering, a gene regulatory circuit with environmental response characteristics is constructed to achieve precise regulation of the photosynthetic electron transfer process in plants. Specifically, it involves constructing a high-light-inducible promoter. ELIP1 FLP recombinase-driven gene expression circuit regulating photosynthetic electron transport and its application in plants. Background Art

[0002] The light reactions of photosynthesis rely on photosystem II (PSII), photosystem I (PSI), and a series of electron transport carriers to drive electron transport, producing ATP and NADPH for photosynthetic carbon fixation. Under high-light conditions, plant photosynthetic machinery is susceptible to photoinhibition due to absorption of light energy exceeding its utilization capacity. This leads to overload of the photosynthetic electron transport chain, accumulation of reactive oxygen species, and decreased photosynthetic efficiency, which in turn affects crop yield. The use of constitutive promoters to express exogenous genes in specific species has been widely used in plant genetic improvement. However, constitutive promoters only enable continuous expression of the target gene and are unable to respond to specific environmental conditions. Furthermore, overexpression of the target gene can lead to energy depletion, cytotoxicity, or unintended phenotypes. Therefore, the development of inducible or conditional genetic circuits is crucial for improving the safety and precision of gene expression.

[0003] The FLP / FRT system is a site-specific recombination system derived from yeast. The FLP recombinase can recognize and cut FRT sites, achieving deletion, inversion or integration of DNA fragments. It is a commonly used tool for constructing gene expression circuit logic gates in synthetic biology. ELIP1 The Early Light-Induced Protein 1 (PETE) gene promoter is a high light-inducible promoter. Its expression activity is very low under weak light or dark conditions, but it is rapidly activated under strong light conditions and drives gene expression. b 6 f complex subunits) are key components of photosynthetic electron transport, but there is currently no ELIP1 The promoter is combined with the FLP / FRT recombinase system and used to optimize plant photosynthetic electron transport or enhance stress resistance. Summary of the Invention

[0004] The purpose of the present invention is to provide a ELIP1The gene promoter drives the expression of FLP recombinase, ultimately outputting a high-light-inducible gene expression circuit of PETE or PETC, a key component of photosynthetic electron transport, which is used to achieve light-intensity-dependent regulation of gene expression of photosynthetic elements in plants, improve photosynthetic electron transport characteristics under high light stress, and thus enhance plant photosynthesis and light adaptability.

[0005] To achieve the above object, the present invention adopts the following technical solutions: A highly light-inducible photosynthetic electron transport circuit based on a recombinase, the highly light-inducible photosynthetic electron transport circuit comprising: (1) High light-inducible promoter; (2) FLP recombinase, driven by the high light-inducible promoter; (3) a gene expression cassette comprising an FRT site, wherein the expression cassette comprises a gene for a key component of photosynthetic electron transport; The high-light-inducible promoter activates the expression of FLP recombinase under strong light conditions and regulates the expression of photosynthetic electron transfer key component genes through FRT site recombination. The nucleotide sequence of the FLP recombinase is shown in SEQ ID NO.1.

[0006] In one embodiment, the high light inducible promoter is ELIP1 Gene promoter, the ELIP1 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0007] The gene expression cassette containing FRT sites includes two co-directional FRT sites, a transcription terminator located between the FRT sites, and an expression unit of a photosynthetic electron transport key component gene PETE or PETC.

[0008] In specific applications, the Arabidopsis genome data published on the TAIR official website (http: / / www.arabidopsis.org / ) was obtained. ELIP1 The gene promoter sequence (gene number: AT3G22840) has unique light-responsive properties and can specifically initiate the expression of downstream genes under light conditions; the protein coding sequence of the PETE gene (gene number: AT1G20340) is obtained. This gene encodes the plastocyanin electron transporter, which is responsible for transferring electrons from the cytochrome of photosystem II (PSII) to the cytochrome of photosystem II (PSII). b 6 f The complex is delivered to photosystem I (PSI); the protein coding sequence of the PETC gene (gene number: AT4G03280) is obtained, which encodes the Rieske Fe / S protein, which is a cytochrome b 6 fComponents of a complex.

[0009] Using molecular biology and genetic engineering techniques, a gene expression circuit vector regulated by light-inducible recombinase was constructed. ELIP1 The gene promoter sequence is connected to the FLP recombinase coding sequence to construct a light-inducible recombinase expression vector, which can specifically drive the expression of FLP recombinase under the induction of light; an expression cassette containing an FRT site is designed, and a terminator is placed between two synorientated FRT sites. Its upstream sequence is the strong promoter 35S, and the downstream sequence is the PETE or PETC gene.

[0010] More specifically, the method for constructing an expression vector for a recombinase-based highly light-inducible photosynthetic electron transport circuit comprises the following steps: (1) Obtaining gene coding sequences: ELIP1 Gene promoter sequence, FLP recombinase coding sequence, and coding sequences of photosynthetic electron transport key component genes PETE and PETC; (2) Construction of recombinase expression module: ELIP1 After PCR amplification and restriction enzyme digestion of the gene promoter, the FLP recombinase coding sequence and terminator were connected to construct a high-light-inducible recombinase expression cassette. ELIP1::NLS-FLP::NOS ; (3) Design of target gene expression module: construct an expression cassette containing two synorientated FRT sites, insert a transcription terminator between the FRT sites, with a strong 35S promoter upstream and the PETE or PETC gene coding sequence downstream; before recombination occurs, the PETE or PETC gene transcription is silent due to terminator blocking; (4) Vector splicing: Through vector construction technology, the recombinase expression module and the target expression module containing FRT regulation are cloned into the plant expression vector. The recombinant vector is amplified in Escherichia coli, and positive clones are screened. After enzyme digestion identification, sequencing is sent to confirm that there are no mutations in each module, indicating that the vector construction is successful.

[0011] In order to verify the effectiveness of this gene expression circuit in enhancing photosynthetic electron transfer efficiency and plant adaptability to high light stress, the inventors further transformed tobacco with the constructed gene expression circuit vector regulated by the light-inducible recombinase through Agrobacterium-mediated transformation to obtain a stably transformed tobacco strain. When the plant is under low light, the PETE or PETC gene cannot be expressed due to the presence of the terminator; when the plant is exposed to high light, ELIP1The gene promoter is activated, driving the expression of FLP recombinase. FLP recombinase recognizes and binds to the FRT recombination site, deleting the terminator sequence through FLP-mediated recombination, thereby achieving expression of the target gene, PETE or PETC. This highly photocontrolled gene expression circuit exhibits high spatiotemporal specificity, enabling precise expression of specific genes at the desired time and in the desired tissue.

[0012] Therefore, the present invention also protects the use of the above-mentioned recombinase-based high-light-inducible photosynthetic electron transport circuit or its expression vector in improving the high-light adaptability of plants.

[0013] Based on the above application, the present invention also protects a method for improving the high light adaptability of plants, which is to introduce the recombinase-based high light-inducible photosynthetic electron transport circuit into plants, so that the plants express high light-inducible photosynthetic electron transport component genes, improve photosynthetic electron transport under high light stress, and thus improve the high light adaptability of plants.

[0014] The present invention also includes recombinant vectors, transgenic plant cells, tissues, plants, and their progeny containing the aforementioned elements. These transgenic plants are capable of expressing specific genes under high-light conditions, thereby exhibiting improved traits, such as enhanced light adaptability. Furthermore, products or food additives derived from these transgenic plants are also within the scope of protection of the present invention. These products have higher quality and safety, providing new options for agricultural breeding and production.

[0015] The "plant" referred to in the present invention includes the entire tobacco plant, its parent and offspring plants, and different parts of the plant, including seeds, fruits, buds, stems, leaves and other tissues and organs. The gene expression circuits constructed by the present invention can play a role in these different parts.

[0016] Advantages of the present invention: The present invention utilizes ELIP1 The high-light-inducible promoter achieves the spatiotemporal specific expression of the FLP recombinase, thereby initiating the expression of the target gene, and realizing a logic gate circuit with high light as input and photosynthetic electron transfer protein as output. The tobacco leaves were transformed using the Agrobacterium-mediated method and a stable transformed tobacco strain was obtained. By simulating a high-light stress experiment in an incubator, it was revealed that the gene expression circuit can optimize the expression of the photosynthetic electron transport chain elements PETE or PETC, improve the photosynthetic electron transfer characteristics under high light stress, enhance the adaptability of plants under high light, and provide new tools and resources for crop molecular breeding and synthetic biology research. The present invention functionally integrates a high-light-responsive promoter, a site-specific recombination system and a photosynthetic key gene to achieve a precise response to a high-light environment, which has very important application value for improving the high-light adaptability of plants and optimizing photosynthetic efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the construction of the high-light-inducible gene expression circuit vector of the present invention.

[0018] Figure 2 This is a gene expression test and electron transfer characteristic test of tobacco leaves in a high-light-inducible photosynthetic electron transport circuit (PETE as the output element). In the figure, "GL-PETE" represents plants with PETE as the output element circuit transferred under normal growth light, "HL-PETE" represents plants with PETE as the output element circuit transferred after high-light treatment, and "HL-WT" represents wild-type control plants after high-light treatment.

[0019] Figure 3 This is a gene expression test and electron transfer characteristic test of tobacco leaves in a high-light-inducible photosynthetic electron transport circuit (PETC as the output element). In the figure, "GL-PETC" represents plants with PETC as the output element circuit transferred under normal growth light, "HL-PETC" represents plants with PETC as the output element circuit transferred after high-light treatment, and "HL-WT" represents wild-type control plants after high-light treatment.

[0020] Figure 4 Schematic diagram of the operation of the logic gate formed by the high-light-induced gene expression circuit of the present invention. DETAILED DESCRIPTION

[0021] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. However, the specific experimental methods involved in the following examples, unless otherwise specified, are all conventional methods or are performed under the conditions recommended by the manufacturer's instructions.

[0022] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The experimental methods in the following examples are all conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be purchased from the market.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0024] As used herein, the terms "nucleic acid," "nucleic acid sequence," "nucleotide," "nucleic acid molecule," or "polynucleotide" are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-stranded or double-stranded structures. These nucleic acids or polynucleotides include, but are not limited to, gene coding sequences, antisense sequences, and regulatory sequences in non-coding regions. These terms include a gene. "Gene" or "gene sequence" is widely used to refer to a functional DNA nucleic acid sequence. Thus, a gene may include introns and exons in a genomic sequence, and / or include coding sequences in a cDNA, and / or include cDNA and its regulatory sequences. In specific embodiments, such as with respect to isolated nucleic acid sequences, it is preferably assumed to be cDNA.

[0025] In addition, in order to have a more intuitive understanding of the technical solution of the present invention, some professional terms involved in the present invention are explained as follows: "Gene expression circuit" is a gene regulation system designed in synthetic biology that can produce predetermined gene expression patterns based on specific signals, thereby achieving precise control of cell physiological behavior.

[0026] "Recombinant vector" refers to a vector that adds expression elements (such as promoters, protein coding sequences, terminators, etc.) to the basic skeleton of a cloning vector to achieve the expression of the target gene.

[0027] 1. Construction of a High-Light-Inducible Gene Expression Circuit Vector like Figure 1 As shown, the construction of the high-light-inducible gene expression circuit vector is as follows: 1.1 Acquisition and design of promoter and gene sequences First, we retrieved and obtained the Arabidopsis thaliana database TAIR (The Arabidopsis Information Resource, http: / / www.arabidopsis.org / ). ELIP1 (Early Light-Induced Protein 1) promoter sequence (Gene ID: AT3G22840). ELIP1The promoter exhibits remarkable high-light induction properties, rapidly activated under intense light stress to drive expression of downstream genes, making it an ideal regulatory element for implementing high-light-responsive gene circuits. Subsequently, the coding sequences for two key photosynthetic electron transport proteins were obtained: PETE (plastocyanin, gene ID AT1G20340) and PETC (Rieske iron-sulfur protein, gene ID AT4G03280). These proteins participate in electron transfer between photosystems I and II, and their expression levels significantly influence photosynthetic efficiency.

[0028] In order to achieve precise recombinant regulation of target genes in plant cells, the present invention introduces a Saccharomyces cerevisiae ) FLP site-specific recombinase system. The recombinase coding sequence was codon-optimized for plant cells to improve its expression efficiency in plant cells. To further ensure the correct localization and function of the recombinase protein, a nuclear localization signal (NLS) was added to the N-terminus of the coding sequence to ensure that the FLP protein can efficiently enter the cell nucleus and recognize and act on the FRT site.

[0029] 1.2 Construction of recombinase expression module The core of the recombinase expression module is the high light-inducible promoter driving the expression of FLP recombinase. First, high-fidelity PCR was used to amplify the recombinase from Arabidopsis genomic DNA. ELIP1 Promoter fragment. To improve cloning efficiency and subsequent splicing accuracy, specific restriction endonuclease sites (such as XbaI and BamHI) and homologous recombination linker sequences (20 bp of complementary sequence between the ends of the DNA fragment and the target vector) are introduced at the 5' end of the primer. PCR products are separated and purified by agarose gel electrophoresis and then double-digested with the corresponding restriction endonucleases. The digested products are then purified using a gel recovery kit.

[0030] The purified ELIP1 The promoter fragment was ligated to the plant expression vector backbone, which had been digested with the same enzymes. The ligation system used T4 DNA ligase and the ligation was carried out overnight at 16°C. The ligation product was transformed into competent Escherichia coli DH5α, plated on LB plates containing the appropriate antibiotic, and screened for positive clones. A single colony was selected for expansion and plasmid extraction. The correctness of the inserted sequence was verified by restriction enzyme digestion and sequencing.

[0031] On this basis, the FLP recombinase coding sequence (shown in SEQ ID NO.1) that has been plant codon optimized (using conventional codon optimization online tools) and added with NLS is synthesized and cloned into the intermediate vector. Homologous recombination arms that match the vector are also introduced upstream and downstream of this sequence. Using a homologous recombination enzyme kit (such as ClonExpress® MultiS OneStep Cloning Kit), the FLP sequence is cloned with ELIP1 The promoter and Nos terminator were spliced ​​to construct a complete recombinase expression cassette ( ELIP1::NLS-FLP::NOS The function of this expression cassette is to express FLP recombinase under strong light induction, thereby achieving light-dependent gene recombination events.

[0032] 1.3 Target gene expression module design The design of the target gene expression module is crucial for achieving recombination-dependent gene expression. This module consists of the following elements: a strong 35S promoter upstream to provide basal transcriptional drive; two FRT recombination recognition sites arranged in the same direction; a transcription terminator sequence (such as the Nos terminator from Agrobacterium tumefaciens) inserted between the two FRT sites to block transcriptional elongation; and the coding sequence of the target gene (PETE or PETC) ligated downstream of the second FRT site.

[0033] When no recombination occurs, RNA polymerase begins transcription under the drive of the 35S promoter due to the presence of a terminator between the FRT sites. However, transcription terminates prematurely upon encountering the terminator, preventing the expression of the downstream PETE or PETC genes. Only when the FLP recombinase is expressed and activated does it recognize the two FRT sites and perform specific recombination, excising the terminator fragment between them. This allows the 35S promoter to directly drive transcription of the target gene, thereby achieving gene activation.

[0034] To construct this module, a core backbone sequence containing two co-oriented FRT sites was first synthesized, and a terminator fragment was cloned between the FRTs. Subsequently, the coding sequences of PETE and PETC were amplified by PCR using Arabidopsis thaliana cDNA as a template. Homologous arms matching the vector were incorporated into the primers, and the target genes were cloned downstream of the FRT using a homologous recombination enzyme kit. All constructs were verified by colony PCR, restriction enzyme analysis, and sequencing.

[0035] 1.4 Vector splicing In order to integrate the recombinase expression module and the target gene expression module into the same plant expression vector, the highly efficient homologous recombination cloning technology was used to complete the assembly of multiple fragments. ELIP1::NLS-FLP::NOSThe expression cassette and the target gene expression cassette containing the FRT are amplified separately by PCR. Primers are designed to include at least 20 bp of homology to the ends of the linearized vector. The selected plant expression vector (e.g., pCAMBIA1300) is linearized with an appropriate restriction endonuclease and dephosphorylated to minimize self-ligation. Subsequently, the linearized vector and the two expression module fragments are combined using a homologous recombination enzyme kit to achieve directional, seamless multi-fragment assembly.

[0036] The reaction products were directly transformed into competent Escherichia coli DH5α and plated on plates containing the appropriate antibiotic for screening. Positive clones were selected and expanded, and plasmids were extracted. The recombinant plasmid configuration was initially confirmed by restriction enzyme digestion, and the correct connection of each component was further verified by PCR across the splice junctions. Finally, the recombinant vector was fully sequenced to ensure that the sequences of each component were correct and that the homologous recombination process had not introduced unintended mutations.

[0037] 2. Agrobacterium-mediated genetic transformation of tobacco 2.1 Material Preparation Transformed varieties: Tobacco ( Nicotiana tabacum ), disease-free and healthy seedlings suitable for laboratory cultivation were selected.

[0038] Agrobacterium strain: Agrobacterium tumefaciens GV3101.

[0039] 2.2 Transformation vector into Agrobacterium The recombinant plasmid was introduced into Agrobacterium using the competence method, and positive strains containing resistance markers were selected.

[0040] 2.3 Tobacco leaf disc transformation process Collect 4-6 week old tobacco leaves and cut into pieces of about 1 cm 2 Leaf discs; soak the leaf discs in Agrobacterium suspension (OD600 approximately 0.6-1.0) for 10-15 minutes, remove excess liquid, place on co-culture medium, and incubate in the dark for 2-3 days; transfer to selective medium containing appropriate antibiotics (kanamycin) and fungicides (ampicillin) for tissue culture to promote differentiation and budding; regularly transfer the culture medium to promote bud growth and root induction; transplant the stably rooted seedlings into soil and grow them under greenhouse conditions; detect transgenic fragments in genomic DNA by PCR and screen positive plants.

[0041] 3. High light stress treatment and physiological index measurement 3.1 High light stress treatment The light intensity in the plant growth chamber was set to the growth light condition (about 60 µmol·m -2 ·s -1) was used as a normal growth control; the high light treatment was set to approximately 600 µmol·m -2 ·s -1 To simulate a natural high-light environment, transgenic positive plants and wild-type controls were placed under high-light conditions for 6 hours, after which growth light conditions were restored. This high-light treatment was continued for 3 consecutive days to fully activate the high-light-induced PETE or PETC expression circuit.

[0042] 3.2 Target gene expression detection Total RNA was extracted from the treated leaves and cDNA was synthesized. The expression of PETE or PETC was analyzed by qRT-PCR. The internal reference was a commonly used stable expression gene of tobacco (such as ACTIN). The results showed that ( Figure 2 A and Figure 3 (A) High light as input effectively outputs PETE or PETC expression. All measurements were repeated at least three times, and statistical analysis (t-test) was performed to determine if differences were significant. "***" indicates p < 0.0005.

[0043] 3.3 Determination of photosynthetic electron transport performance The electron transport rates (ETRs) of photosystem I and photosystem II were measured using a pulse amplitude modulation fluorimeter (Dual-PAM); The differences in electron transport indicators between the transgenic lines and the wild type under high light conditions were compared to evaluate the regulatory effect of the gene circuit on high light stress. Figure 2 B, C and Figure 3 (B and C) Under high light stress, the electron transport rates of both photosystem I and photosystem II in the transgenic lines were significantly increased compared to those in the wild-type plants. All measurements were repeated at least three times, and statistical analysis (t-test) was performed to determine if the differences were significant. * and ** indicate p < 0.05 and p < 0.005, respectively.

[0044] Through the above steps, the ELIP1 The promoter drives the high-light-inducible gene expression circuit of FLP recombinase. When the plant is under low light, the PETE or PETC gene cannot be expressed due to the presence of the terminator; when the plant is exposed to high light, ELIP1The gene promoter is activated, driving the expression of FLP recombinase. FLP recombinase can recognize and bind to the FRT recombination site, and delete the terminator sequence through FLP-mediated recombination, thereby achieving conditional expression of key photosynthetic electron transport components of PETE or PETC under high light conditions. This circuit removes the terminator sequence mediated by FLP / FRT, relieves the downstream gene expression blockade, and precisely controls the time and space of gene expression. This high-light-controlled gene expression circuit has a high degree of spatiotemporal specificity and can accurately achieve specific gene expression at the required time and tissue ( Figure 4 ), stably transformed tobacco plants showed induced expression of target genes and improved photosynthetic performance after high light induction, proving that this high-light-inducible gene circuit effectively enhanced the efficiency of photosynthetic electron transfer and the adaptability of plants to high light stress.

[0045] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, it is of course possible to easily make other implementation methods by replacing or changing the technical content disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A highly light-inducible photosynthetic electron transport circuit based on a recombinase, characterized in that: The high light-inducible photosynthetic electron transport circuit comprises: (1) High light-inducible promoter; (2) FLP recombinase, driven by the high light-inducible promoter; (3) a gene expression cassette comprising an FRT site, wherein the expression cassette comprises a gene for a key component of photosynthetic electron transport; The high-light-inducible promoter activates the expression of FLP recombinase under strong light conditions and regulates the expression of photosynthetic electron transfer key component genes through FRT site recombination. The nucleotide sequence of the FLP recombinase is shown in SEQ ID NO.

1.

2. The highly light-inducible photosynthetic electron transport circuit based on a recombinase according to claim 1, characterized in that The high light inducible promoter is ELIP1 Gene promoter, the ELIP1 The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. The highly light-inducible photosynthetic electron transport circuit based on a recombinase according to claim 1, characterized in that The gene expression cassette containing the FRT site includes a strong promoter 35S, two co-directional FRT sites, a transcription terminator located between the FRT sites, and an expression unit of a photosynthetic electron transport key component gene PETE or PETC.

4. An expression vector comprising the recombinase-based highly light-inducible photosynthetic electron transport circuit according to claim 1.

5. Use of the recombinase-based high-light-inducible photosynthetic electron transport circuit according to any one of claims 1 to 3 or the expression vector according to claim 4 in improving the high-light adaptability of plants.

6. A method for improving plant adaptability to high light, characterized in that: The method is to introduce the recombinase-based high-light-inducible photosynthetic electron transport circuit described in claim 1 into plants, so that the plants express high-light-inducible photosynthetic electron transport component genes and improve photosynthetic electron transport under high light stress.

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