Application of transcription factor CGA1 in improving photosynthetic rate and biomass of plants

Abstract

The invention belongs to the technical field of agricultural biology, and relates to application of a transcription factor CGA1 with an amino acid sequence of SEQ ID NO: 1 in improving the photosynthetic rate and biomass of plants. Expression of the transcription factor CGA1 in vascular bundle sheath cells of plant leaves can improve the photosynthetic rate and biomass of plants, and is beneficial to improvement of plant traits and cultivation of new varieties of plants.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, and more specifically, relates to the use of a transcription factor CGA1 in improving plant photosynthetic rate and biomass. Background Technology

[0002] CGA1 is the transcription factor with the strongest response to low CO2, and it has been verified in both rice and Arabidopsis thaliana as a key transcription factor regulating chlorophyll synthesis and chloroplast development (Cackett L, Luginbuehl LH, Schreier TB, et al. Chloroplast development in green plant tissues: The interplay between light, hormone, and transcriptional regulation[J]. New Phytologist, 2022, 233(5): 2000-2016.; Lee DY, Hua L, Khoshravesh R, et al. Engineering chloroplast development in rice through cell-specific control of endogenous genetic circuits[J]. Plant Biotechnology Journal, 2021, 19(11): 2291-2303.). This suggests that CGA1 may play an important role in the evolutionary history of C4 plants.

[0003] Overexpression of CGA1 using the BSC (bundle sheath cell) specific promoter GLDPA resulted in an increase in the area of ​​Arabidopsis BSCs, an increase in the number and area of ​​chloroplasts in BSCs and leaf veins, and an increase in starch accumulation. Figure 1 This aligns with the characteristics of Proto-Kranz during evolution and is consistent with previous reports (Chiang YH, Zubo YO, Tapken W, et al. Functional characterization of the gata transcription factors gnc and cga1 reveals their key role in chloroplast development, growth, and division in arabidopsis[J]. Plant Physiol, 2012, 160(1):332-348.). The increase in the number of mitochondria and peroxisomes ( Figure 3Enhanced development of A, C) and plasmodesmata ( Figure 3 The E and F values ​​are likely indirect results of chloroplast development activation. Proto-Kranz specifically refers to: increased BSC size, increased number of chloroplasts and mitochondria in BSC, and increased enzymes required for photosynthesis and photorespiration metabolism in BSC (Sage RF, Khoshravesh R, Sage TL. From proto-kranz to c4kranz: Building the bridge to c4 photosynthesis[J]. J Exp Bot, 2014, 65(13):3341-3356.).

[0004] Previous studies have shown that there may be a certain coordinated regulatory mechanism between chloroplasts and mitochondria. For example, by systematically analyzing the repressor of the mod1 mutant, key genes regulating chloroplast-mitochondrial communication were identified, revealing the important role of the malate cycle pathway in chloroplast energy metabolism and mitochondrial reactive oxygen species production (Zhao Y, Yu H, Zhou JM, et al. Malate circulation: Linking chloroplast metabolism tomitochondrial ros[J]. Trends Plant Sci, 2020, 25(5): 446-454.), ensuring the normal functioning of cells. Furthermore, the direct connection between chloroplasts, mitochondria, and plasmodesmata has been confirmed. The loss of RNA helicase function located in mitochondria and chloroplasts increases the number of plasmodesmata (Burch-Smith TM, Zambryski PC. Loss of increased size exclusion limit (ise)1 or ise2 increases the formation of secondary plasmodesmata[J]. Curr Biol, 2010, 20(11): 989-993.; Kobayashi K, Sasaki D, Noguchi K, et al. Photosynthesis of root chloroplasts developed in arabidopsislines overexpressing golden2-like transcription factors[J]. Plant and Cell Physiology, 2013, 54(8): 1365-1377.). These observations suggest that there is a regulatory mechanism within the cell that coordinates the development of chloroplasts, mitochondria, peroxisomes, and plasmodesmata. This also suggests that during C4 engineering, it may be possible to promote the development of other organelles and plasmodesmata, and thus promote the formation of wreath structures, simply by activating the development of chloroplasts in the BSC.

[0005] The CO2 compensation point is an important indicator for measuring photorespiration, representing the magnitude of respiration and photorespiration. Plants in the Proto-Kranz stage of C4 evolution have a lower CO2 compensation point and reduced photorespiration. This is of great biological significance in providing carbon sources for plants under harsh conditions such as high temperatures and low CO2, thereby improving plant adaptability and competitiveness. Summary of the Invention

[0006] In our research on the mechanism of photosynthesis in rice, our research group experimentally verified that the transcription factor CGA1 is specifically expressed in the bundle sheath cells of leaves, while it is not expressed or expressed at very low levels in other cell types. This promotes chloroplast development, increases chlorophyll content, promotes mitochondrial and peroxisome development, and lowers the CO2 compensation point, thereby enhancing the photosynthetic rate and biomass of rice. Based on this research, the present invention includes the following technical solution.

[0007] The first aspect of the present invention provides the use of transcription factor CGA1 (NCBI number Q9SZI6.1) with an amino acid sequence as shown in SEQ ID NO:1 or a conserved variant polypeptide thereof in improving photosynthetic rate and biomass in plants, wherein the conserved variant polypeptide is a polypeptide having more than 95% homology with CGA1, preferably more than 96% homology, preferably more than 97% homology, preferably more than 98% homology, more preferably more than 99% homology, and having the function of CGA1.

[0008] MGSNFHYTIDLNEDQNHQPFFASLGSSLHHHLQQQQQQQHFHHQASSNPSSLMSPSLSYFPFLINSRQDQVYVGYNNNTFHDVLDTHISQPLETKNFVSDGGSSSSDQMVPKKETRLKLTIKKKDNHQDQTDLPQSPIKDMTGTNSLKWISSKVRLMKKKKAIITTSDSSKQHTNND QSSNLSNSERQNGYNNDCVIRICSDCNTTKTPLWRSGPRGPKSLCNACGIRQRKARRAAMATATATAVSGVSPPVMKKKMQNKNKISNGVYKILSPLPLKVNTCKRMITLEETALAEDLETQSNSTMLSSSDNIYFDDLALLLSKSSAYQQVFPQDEKEAAILLMALSHGMVHG(SEQ ID NO:1).

[0009] The complete genome sequence of the transcription factor CGA1, including the 5'UTR region, 3'UTR region, introns, and exons, is SEQ ID NO:2; the nucleotide sequence of the exons, i.e. the coding region CDS, is SEQ ID NO:3.

[0010] The aforementioned plants can be crops, preferably grasses. These crops include, but are not limited to, rice, wheat, corn, soybeans, barley, oats, rye, sorghum, cotton, vegetables, and cruciferous plants such as Arabidopsis thaliana.

[0011] In one embodiment, the above-mentioned use is achieved by making the transcription factor CGA1 specifically expressed in bundle sheath cells, while not expressed or expressing it at very low levels in other cell types.

[0012] Preferably, the bundle sheath cells are bundle sheath cells in the leaf.

[0013] For example, the GLDPA promoter (nucleotide sequence SEQ ID NO:4) derived from yellow daisy can drive the specific expression of the transcription factor CGA1 in bundle sheath cells.

[0014] In one specific embodiment, the specific expression of the transcription factor CGA1 in bundle sheath cells can be achieved by assembling the GLDPA promoter derived from yellow chrysanthemum with the CGA1 gene (nucleotide sequence, for example, SEQ ID NO:2 or SEQ ID NO:3) into an expression vector to form a recombinant expression vector. The recombinant expression vector is then introduced into a plant, such as rice, using transgenic technology, such as Agrobacterium-mediated transformation, to obtain a transgenic plant that overexpresses the transcription factor CGA1 in bundle sheath cells.

[0015] In another embodiment, the specific expression of the transcription factor CGA1 in bundle sheath cells can be achieved by providing a transcription factor CGA1 expression cassette / expression box (hereinafter referred to as GLDPA+CGA1 expression cassette) containing a GLDPA promoter derived from yellow chrysanthemum and a CGA1 gene (nucleotide sequence such as SEQ ID NO:2 or SEQ ID NO:3) located downstream therefrom. The GLDPA+CGA1 expression cassette is cloned into the plant chromosome genome using gene editing technology to obtain a transgenic plant that overexpresses the transcription factor CGA1 in bundle sheath cells.

[0016] The gene editing technologies mentioned above can be selected from the following group: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, and MuGENT.

[0017] A second aspect of the present invention provides a DNA molecule as a transcription factor CGA1 gene expression cassette, abbreviated as GLDPA+CGA1 expression cassette, the DNA molecule comprising a GLDPA promoter derived from yellow daisy and a CGA1 gene located downstream therefrom.

[0018] A third aspect of the present invention provides a recombinant plasmid containing the aforementioned DNA molecule, namely the transcription factor CGA1 gene expression cassette / expression box, and suitable for expression in Agrobacterium. The recombinant plasmid is an overexpression vector formed by cloning the aforementioned GLDPA+CGA1 gene expression cassette onto a plasmid vector suitable for expression in Agrobacterium, wherein the plasmid vector is, for example, a plant transgenic vector or a modified vector such as pHB-YFP, pHB-FLAG, pBin19, fluorescent reporter vector pGreenII0800-LUC, pCAMBIA3300, pCAMBIA1301, pCAMBIA2301, pBI121, or pTF102.

[0019] A fourth aspect of the present invention provides a recombinant Agrobacterium engineered strain, which is an Agrobacterium transformant containing the recombinant plasmid described above, used to mediate the transfer of the recombinant plasmid described above into plants such as rice and other crops. Preferably, the Agrobacterium is Agrobacterium tumefaciens, Agrobacterium EHA105, or Agrobacterium GV3101. For example, the above-mentioned recombinant plasmid is transferred into an Agrobacterium host strain using a heat shock method or a freeze-thaw method to form a recombinant Agrobacterium engineered strain.

[0020] The fifth aspect of the invention provides the use of the DNA molecules described above, the recombinant plasmids described above, and the recombinant Agrobacterium engineered bacteria described above in improving plant traits, and in cultivating new plant varieties with increased photosynthetic rate and / or biomass.

[0021] This invention discovers that when the CGA1 gene is specifically expressed in the bundle sheath cells of plants such as Arabidopsis thaliana leaves, while it is not expressed or is expressed at very low levels in other cell types, it achieves an increase in biomass and a higher photosynthetic rate, promotes chloroplast development, and lowers the CO2 compensation point, among other important phenotypes of improved photosynthetic efficiency. The technical solution of this invention can be used to improve crops and cultivate new plant varieties with high photosynthetic efficiency. Attached Figure Description

[0022] Figure 1 This shows the localization verification of the GLDPA promoter in Arabidopsis thaliana. A: Pro GLDPA :GUS and Pro GLDPA - Schematic diagram of the vector structure of Venus-N7; B and C: showing the localization of GUS signal in the BSC (Bundle Sheath Cells, Fig. B) and veins (Fig. C) of Arabidopsis thaliana leaves; D and E: Venus signal localization in the BSC (Bundle Sheath Cells, Fig. D) and veins (Fig. E) observed by fluorescence microscopy.

[0023] Figure 2The diagram shows that CGA1 expression in BSCs increases chlorophyll content. A: Schematic diagram of the expression vector; B: CGA1 gene expression in wild-type (WT) and three transgenic lines (Pro). GLDPA The expression levels of RNA in CGA1 (L3, L4, and L9) (n=3) were detected using specific primers CGA1-F and CGA1-R in leaves; C: chlorophyll content (n=5), error bars represent standard deviation (SD), t-test showed significant differences, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n represents the number of independent biological samples; D: Arabidopsis leaf phenotypic photographs, scale bar, 0.5 cm.

[0024] Figure 3 The study showed that specific expression of CGA1 increased the number and size of chloroplasts in the leaf sclera (BSC) and veins. Figures A to F show wild-type WT lines (Figures A, C, and E) and transgenic Arabidopsis plants (Pro). GLDPA : Ultrathin structural diagram of CGA1 leaf, transmission electron micrographs of mesophyll cells (MC), bundle sheath cells (BSC), and veins, scale bars 20 μm (Fig. A, B) and 10 μm (Fig. CF); GN: Violin diagram showing the area of ​​BSC cells and the changes in chloroplasts in BSC and veins; G: Average area per BSC (n≥39); H: Average amount of starch per BSC (n≥39); I: Average number of chloroplasts per BSC (n≥39); J: Area of ​​chloroplasts in BSC. (n≥39); K: Total chloroplast area in each BSC (n≥39); L: Ratio of chloroplast area to total BSC area in each BSC (n≥39), (n≥39 cells, at least 3 biological replicates per line); M: Number of chloroplasts per vein (n≥7); N: Average number of starch granules per vein (n≥7), at least three biological replicates per line. An asterisk indicates a significant difference compared to the control group, **P<0.01, ***P<0.001, ****P<0.0001.

[0025] Figure 4 The results show that CGA1 expression in BSCs increases the number of mitochondria and peroxisomes. Where: A: average number of mitochondria per BC cell (n≥39); B: average area per mitochondria (n≥39); C: average number of peroxisomes per BSC (n≥39); D: average area per peroxisome (n≥39); E and F: WT (Figure E) and Pro GLDAPImmunogold assay results of GDCP protein in mitochondria of CGA1 strain (Figure F). This gene is located in mitochondria; Figures E and F are schematic diagrams of mitochondria. Scale bar: 250 nm. Asterisks indicate significant differences compared to the control group using t-tests: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0026] Figure 5 The results showed that CGA1 expression in BSCs increased the photosynthetic rate of plants. A: Net photosynthetic light response curve (AQ curve), measured using a LI-6800 (Li-Cor, Inc., USA) at 22℃ and a CO2 concentration of 400 ppm. Data are presented as mean ± standard deviation (n = 6). B: Net photosynthetic CO2 response curve (A-Ci curve), measured using a LI-6800 (Li-Cor, Inc., USA) at a light intensity of 600 μmol / m². -2 s -1 The temperature was 22℃, and the data are expressed as mean ± standard deviation (n=4); C: Light response curve of stomatal conductance, measured at 22℃ and CO2 concentration of 400ppm, and the data are expressed as mean ± variance (n=6). For Figures A, B, and C, asterisks indicate that transgenic L3, L4, and L9 showed significant differences compared to WT using a t-test, *P<0.5; D: Maximum carboxylation rate (V cmax E: Maximum electron transport rate (J) max Figures D and E were calculated by fitting the A-Ci curves using the FvCB model, with data expressed as mean ± variance (n = 4); F: Leaf protein content, data expressed as mean ± standard deviation (n = 4), asterisks indicate significant differences compared to WT, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (G) SDS-PAGE analysis of RuBisCO protein in leaves, samples were taken from the same leaf area.

[0027] Figure 6 This study demonstrates that CGA1 expression in leaf stalks (BSCs) increases biomass. A and D: Arabidopsis phenotypes under normal light (Fig. A) and strong light (Fig. D); B, C, E, F: biomass (Fig. B, E) and leaf number (Fig. C, F) under normal light (Fig. B and C) and strong light (Fig. E and F); G: schematic diagram. CGA1 expression in BSCs and leaf veins activates organelles in BSCs, increases photosynthetic rate, and reduces photorespiration, thereby improving the overall photosynthetic capacity of the plant and increasing biomass. Data are presented as mean ± standard deviation (n = 4). An asterisk indicates a significant difference compared to WT. A t-test was performed: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns: indicates no significant difference. Detailed Implementation

[0028] Our research confirms that the specific expression of the transcription factor CGA1 in bundle sheath cells can increase the photosynthetic rate and biomass of rice because CGA1 promotes chloroplast development, increases chlorophyll content, promotes mitochondrial and peroxisome development, lowers the CO2 compensation point, and increases leaf photosynthetic rate. By improving light energy utilization efficiency (the ratio of leaf photosynthetic rate to incident light quantum flux density), it increases the biomass of plants such as rice (the dry weight of all aboveground stems, leaves, and panicles).

[0029] The specific expression mentioned in this article refers to the overexpression of the CGA1 gene only, or mainly, in the bundle sheath cells of leaves, while it is not expressed or is expressed at very low levels in other cell types. This specific expression is a necessary condition for the CGA1 gene to achieve the aforementioned functions.

[0030] One specific method to achieve this specific expression is to introduce the GLDPA promoter (represented as the FtGLDPA promoter) from the yellow daisy (Flaveria trinervia) species into plants such as rice to regulate the expression of the transcription factor CGA1. The transcription factor CGA1 can be derived from rice (Oryza sativa L.), represented as OsCGA1, or from Arabidopsis thaliana, represented as AtCGA1. For example, the FtGLDPA promoter is first assembled with the OsCGA1 gene or AtCGA1 to form an expression cassette. This cassette is then cloned into an expression vector, and this vector is introduced into rice using transgenic technology to obtain transgenic rice. The CGA1 gene is driven by the GLDPA promoter, achieving specific expression in the bundle sheath cells of the transgenic plant.

[0031] As used in this article, the term "wild type" refers to native plants such as rice or Arabidopsis thaliana that have not undergone genetic modification or mutagenesis.

[0032] Correspondingly, the terms “(plant) mutant,” “transgenic plant,” and “genetically engineered plant” in this article have the same meaning, referring to plants that have been genetically modified from wild-type plants, especially those with increased light energy utilization and / or biomass.

[0033] In the description of the technical solutions of this invention, the term "and / or" used in terms such as "A and / or B" or "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0034] In this document, for the sake of simplicity, a protein such as CGA1 may sometimes be used interchangeably with the name of its encoding gene (DNA), CGA1. Those skilled in the art should understand that these terms represent different types of substances in different descriptive contexts. Their meanings are readily understood by those skilled in the art based on the context. For example, when describing the function or category of a transcription factor, CGA1 refers to a protein; when used as a gene description, it refers to the gene encoding that protein.

[0035] Those skilled in the art will expect that conserved variants of the transcription factor CGA1, which share high homology (identity) with the amino acid sequence SEQ ID NO:1 (e.g., more than 95%), may have the same function.

[0036] As used herein, the term "conserved variant polypeptide" refers to a polypeptide that substantially retains the same biological function or activity as the polypeptide. The variant specifically refers to minor amino acid mutations, such as (1) a polypeptide formed by substitution, deletion, or addition of one or more (e.g., 1-20, preferably 1-10; more preferably 1-5; more preferably 1-3) amino acid residues of the amino acid sequence represented by transcription factor CGA1, and having the function of wild-type transcription factor CGA1; (2) a polypeptide having more than 95%, more preferably more than 98%, more preferably more than 99% identity with the sequence represented by transcription factor CGA1, and having the function of wild-type transcription factor CGA1; or (3) a polypeptide formed by adding a tag sequence to the N or C terminus of the sequence represented by transcription factor CGA1, or by adding a signal peptide sequence to its N terminus. Such fragments, derivatives, and analogs are well known to those skilled in the art in accordance with the teachings herein. The term "mutation" includes, but is not limited to, the substitution, deletion, insertion, or chemical modification of amino acid residues, preferably positive mutations, i.e., mutations that enhance function. The substitution can be a non-conservative substitution, a conservative substitution, or a combination of both. A "conservative" amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and therefore generally includes the substitution of amino acids in polypeptides with amino acids from the same or similar amino acid definition class. However, as used herein, a conserved mutation does not include hydrophilic-hydrophilic, hydrophobic-hydrophobic, hydroxyl-containing-hydroxyl-containing, or small residue-small residue substitutions if a conserved mutation can be alternatively expressed as aliphatic-to-aliphatic, nonpolar-to-nonpolar, polar-to-polar, acidic-to-acidic, basic-to-basic, aromatic-to-aromatic, or restriction residue-to-restriction residue substitutions. As is known in this technical field, common examples of conservative substitutions include: substitutions between aromatic amino acids F, W, and Y; substitutions between hydrophobic amino acids L, I, and V; substitutions between polar amino acids Q and N; substitutions between basic amino acids K, R, and H; substitutions between acidic amino acids D and E; and substitutions between hydroxyl amino acids S and T. Furthermore, A, V, L, or I can be conservatively mutated to another aliphatic residue or another nonpolar residue. Exemplary conservative substitutions can be performed according to the table below.

[0037]

[0038]

[0039] "Non-conservative substitution" refers to the substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitution can be performed between, rather than within, the amino acids defined above. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the substituted region (e.g., proline replacing glycine), (b) charge or hydrophobicity, or (c) side chain volume.

[0040] "Deletion" refers to a modification of a peptide by removing one or more amino acids from a reference peptide. Deletions may include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids constituting the reference peptide sequence, while preserving transcription factor CGA1 activity and / or the modified properties of engineered transcription factor CGA1. Deletions may target the interior and / or ends of the peptide. In various embodiments, deletions may comprise continuous segments or may be discontinuous.

[0041] "Insertion" refers to a modification of a polypeptide by adding one or more amino acids to a reference polypeptide. In some embodiments, the modified engineered transcription factor CGA1 includes inserting one or more amino acids into the naturally occurring transcription factor CGA1 and into other modified transcription factor CGA1 polypeptides. The insertion can be internal to the polypeptide, or at the carboxyl terminus or amino terminus. Insertions as used herein include fusion proteins as known in the art. The insertion can be a continuous amino acid segment or separated by one or more amino acids in the naturally occurring polypeptide.

[0042] This invention also includes analogs of the claimed protein CGA1. These analogs may differ from the natural SEQ ID NO:1 in amino acid sequence, in form of modification that does not affect the sequence, or both. These protein analogs include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis through radiation or exposure to a mutagen, site-directed mutagenesis, or other known biochemical techniques. Analogs also include those having residues different from the natural L-amino acid (e.g., D-amino acids), and those having non-naturally occurring or synthetic amino acids (e.g., β, γ-amino acids). It should be understood that the proteins of this invention are not limited to the representative proteins exemplified above.

[0043] The construction of the aforementioned transgenic plants can be achieved using traditional Agrobacterium-mediated transformation with recombinant plasmids or gene editing technology.

[0044] In a specific implementation, a GLDPA+CGA1 expression cassette and nucleic acid construct, or expression construct, are constructed based on the gene CGA1 with nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3 and the GLDPA promoter with nucleotide sequence SEQ ID NO:4. Then, a GLDPA+CGA1 expression plasmid is constructed, and finally, the GLDPA promoter and CGA1 as exogenous genes are expressed in wild plants.

[0045] The coding sequence of the polypeptide CGA1 or fragments thereof of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, conventional techniques can be used to amplify the CGA1 gene from genomic DNA, and primers can be designed based on the nucleotide sequence disclosed in the present invention, especially the open reading frame sequence.

[0046] As used herein, the terms "expression cassette," "expression box," "gene expression cassette," or "nucleic acid construct" refer to a gene expression system containing all the necessary elements required for expressing the target polypeptide CGA1. Typically, it includes the following elements: a GLDPA promoter, a gene sequence encoding the polypeptide, and a terminator; optionally, it may also include a signal peptide coding sequence, etc.; these elements are operatively linked. In this invention, the preferred GLDPA promoter for regulating CGA1 gene expression is polynucleotide SEQ ID NO:4.

[0047] As used herein, the term "expression construct" or "expression building block" refers to a recombinant DNA molecule that may contain one or more gene expression cassettes. These "constructs" are typically contained within an expression vector (plasmid vector).

[0048] As used herein, “operationally linked” or “operationally connected” refers to a functional spatial arrangement of two or more nucleic acid regions or sequences. For example, the GLDPA promoter region is positioned at a specific location relative to the target gene CGA1 nucleic acid sequence SEQ ID NO:2 or SEQ ID NO:3, such that transcription of the nucleic acid sequence is guided by the promoter region, thereby the promoter region is “operationally linked” to the nucleic acid sequence.

[0049] The nucleic acid constructs described in this invention can be manipulated in various ways to ensure the expression of the polypeptide or transcription factor CGA1. The nucleic acid constructs can be manipulated according to the different expression vectors or requirements before insertion into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0050] In some embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or a gene knock-in vector. The nucleic acid sequences SEQ ID NO:2-4 of the present invention can be cloned into many types of vectors, such as plasmids, phage particles, phage derivatives, animal viruses, and granules. Cloning vectors can be used to provide the coding sequence of the protein or polypeptide of the present invention. Expression vectors can be provided to cells in the form of bacterial or viral vectors. Specific expression of the CGA1 gene of the present invention is typically achieved by operably linking the nucleic acid sequences SEQ ID NO:2 or 3 of the present invention to the GLDPA promoter and incorporating the construct into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. Typical expression vectors contain expression control sequences that can be used to regulate the expression of the desired nucleic acid sequence.

[0051] Gene knock-in vectors can be used to integrate the GLDPA+CGA1 described herein into a region of interest in the host genome. Typically, gene knock-in vectors contain the polynucleotide sequence described herein, as well as 5' and 3' homologous arms required for genomic homologous recombination. In some embodiments, the nucleic acid constructs described herein contain 5' homologous arms, the polynucleotide sequence described herein, and 3' homologous arms. When using gene knock-in vectors, CRISPR / Cas9 technology can be used simultaneously to homologously recombine the polynucleotide sequence into the site of interest. CRISPR / Cas9 technology guides the Cas9 nuclease to modify the genome at the insertion site by designing guide RNAs targeting the target gene, resulting in increased homologous recombination efficiency in the modified region, thus homologously recombinating the target fragment GLDPA+CGA1 contained in the gene knock-in vector into the target site. The steps of CRISPR / Cas9 technology and the reagents used, such as the Cas9 nuclease, are well known in the art.

[0052] Methods well known to those skilled in the art can be used to construct nucleic acid constructs. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Furthermore, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline, ampicillin resistance, or chloramphenicol for *E. coli*, *Agrobacterium*, etc.

[0053] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0054] Vectors containing appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

[0055] When constructing transgenic plants using the traditional Agrobacterium-mediated transformation method, the methods for constructing transgenic plants include:

[0056] 1) Provide Agrobacterium carrying an expression vector, wherein the expression vector contains the coding sequence of the GLDPA promoter and the polypeptide CGA1;

[0057] 2) Contact plant cells, tissues, or organs with Agrobacterium in step 1) to transfer the coding sequence into the plant cells and integrate it into the chromosomes of the plant cells;

[0058] 3) Select plant cells or tissues into which the coding sequence has been introduced; and

[0059] 4) Regenerate plants from the plant cells or tissues in step 3).

[0060] The method described herein can be used to construct transgenic plants with different uses.

[0061] We identified transcription factors that play a key role in chloroplast development regulation through transcriptome sequencing and gene regulatory network construction, and verified their functions through transgenic experiments. Among these transcription factors, we found that specific expression of the CGA1 transcription factor using the GLDPA promoter of *Chrysanthemum indicum* promotes chloroplast development, increases chlorophyll content, promotes mitochondrial and peroxisomal development, lowers the CO2 compensation point, increases leaf photosynthetic rate, improves light energy utilization (the ratio of leaf photosynthetic rate to incident light quantum flux density), and increases biomass (the dry weight of all aboveground stems, leaves, and spikelets).

[0062] The methods used in this study for transgenic vector design, data analysis, and experimental verification are reasonable and can serve as a reference for research on other biological processes.

[0063] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0064] Example

[0065] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0066] In the embodiments described herein, unless otherwise specified, the temperature generally refers to room temperature (15-30°C).

[0067] The molecular biology experiments in this embodiment, including plasmid construction, enzyme digestion, competent cell preparation, and transformation, were mainly conducted in accordance with *Molecular Cloning: A Laboratory Manual* (3rd Edition), edited by J. Sambrook and DW. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. For example, the methods for competent cell transformation and competent cell preparation were both performed according to Chapter 1, page 96 of *Molecular Cloning: A Laboratory Manual* (3rd Edition). Specific experimental conditions could be determined through simple experiments if necessary.

[0068] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0069] The primer synthesis and gene sequencing in this embodiment were outsourced to Sangon Biotech (Shanghai) Co., Ltd.

[0070] The PCR primers used in the examples are listed in Table 1 below.

[0071] Table 1. Primer sequences used for gene identification and quantitative PCR primer sequences used for gene expression determination.

[0072]

[0073] Note: In primer names, "-F" indicates forward and "-R" indicates reverse.

[0074] Example 1: Construction of transgenic Arabidopsis thaliana

[0075] Previous literature (Engelmann S, Wiludda C, Burscheidt J, et al. The gene for the p-subunit of glycine decarboxylase from the c4 species flaveria trinervia: Analysis of transcriptional control in transgenic flaveria bidentis (c4) and arabidopsis (c3) [J]. Plant Physiol, 2008, 146(4): 1773-1785.) reported that the GLDPA promoter derived from Flavelia trinervia can be specifically expressed in Flavelia trinervia BSC cells. In order to investigate the expression sites of exogenous genes driven by this GLDPA promoter, such as the commonly used reporter genes GUS (β-glucuronidase gene), Venus (fluorescent protein), and N7 (nuclear localization signal peptide) in Arabidopsis, we constructed Pro GLDPA -GUS and Pro GLDPA -Venus-N7 vector and perform transgenic verification.

[0076] With Pro GLDPA Taking GUS transgenic Arabidopsis thaliana as an example, the construction method is as follows.

[0077] 1. Plasmid Pro GLDPA -GUS build

[0078] 1.1 Gene Cloning

[0079] The genome of yellow chrysanthemum (Flaveria trinervia) was extracted, and the promoter GLDPA sequence was cloned.

[0080] Leaves of *Chrysanthemum indicum* were picked, ground into powder with liquid nitrogen, and the genome of *Arabidopsis thaliana* was extracted using a plant genomic DNA extraction kit (DP321) (Tiangen Biotech (Beijing) Co., Ltd.).

[0081] PCR primers (5'-3'):

[0082] Forward 1381-ProGLDPA-F:

[0083] GTCGACCTGCAGCCAAGCTTAAGCTTTACTCCTCTCAACT;

[0084] Reverse 1381-ProGLDPA-R:

[0085] GTGGACTCCTCTTAAAGCTTAGTGTAAGATGGGGTCTAAT.

[0086] High-fidelity enzyme KOD-FX-Neo (toyobo, product number: KFX-201)

[0087] PCR conditions: 94℃ for 2 min; 98℃ for 10 s, 58℃ for 30 s, 68℃ for 3 min, 35 cycles; 68℃ for 10 min.

[0088] Sequencing of the gene fragment amplified by RT-PCR confirmed that the GLDPA sequence is SEQ ID NO:4, which is correct.

[0089] 1.2 Construction of plant carriers

[0090] 1.2.1 The 2000bp fragment of the promoter GLDPA sequence (SEQ ID NO:4) was amplified by PCR using the fidelity enzyme kod-FX-Neo, and the sequence was confirmed to be correct.

[0091] PCR primers (5'-3'):

[0092] Forward 1381-ProGLDPA-F:

[0093] GTCGACCTGCAGCCAAGCTTAAGCTTTACTCCTCTCAACT;

[0094] Reverse 1381-ProGLDPA-R:

[0095] GTGGACTCCTCTTAAAGCTTAGTGTAAGATGGGGTCTAAT.

[0096] High-fidelity enzyme KOD-FX-Neo (toyobo, product number: KFX-201)

[0097] PCR conditions: 94℃ for 2 min; 98℃ for 10 s, 58℃ for 30 s, 68℃ for 3 min, 35 cycles; 68℃ for 10 min.

[0098] 1.2.2 The plant expression vector pCAMBIA1381 was ligated into the HindIII restriction site to form a vector.

[0099] proGLDPApCAMBIA1381 build

[0100] The restriction enzyme HindIII-HF (NEB, catalog number: R3104) was used.

[0101] Enzyme digestion conditions: 37℃ for 1.5h, 80℃ for 25min.

[0102] Using the ClonExpress II One Step Cloning Kit (Novizan, item number: C112-01)

[0103] Recombination reaction conditions: 37℃ for 30 min, and immediately placed on ice to cool after the reaction is completed.

[0104] Take 1 μg of the correctly sequenced plasmid proGLDPApCAMBIA1381 and TOP10 heat-shock competent cells (Shanghai Weidi Biotechnology Co., Ltd., catalog number: DL1010), incubate on ice for 30 minutes, heat shock at 42°C for 5 minutes, place on ice for 2 minutes, add 200 μL of LB medium, and incubate at 37°C on a shaker for 1 hour. Spread all bacterial cells onto kanamycin-resistant LB agar plates and incubate upside down at 37°C for 1 day.

[0105] Colonies were selected for PCR identification. After the PCR reaction, the PCR products were identified using agarose gel electrophoresis. If a band of the correct size was found, the corresponding numbered bacteria were considered positive clones and sent to the company for sequencing verification.

[0106] After the above steps, plasmid Pro was constructed. GLDPA -GUS.

[0107] 2. Plasmid transformation into Agrobacterium and plant transformation

[0108] 2.1 Take 1 mg of the correctly sequenced plasmid proGLDPApCAMBIA1381 and Agrobacterium GV3101 competent cells (Shanghai Weidi Biotechnology Co., Ltd., catalog number: AC1001) constructed above, incubate on ice for 30 min, flash freeze in liquid nitrogen for 5 min, heat shock at 37℃ for 5 min, place on ice for 5 min, add 1 mL of LB medium, and incubate at 28℃ on a shaker for 4 hours. Spread all bacterial cells on kanamycin-resistant and rifamycin-resistant LB agar plates, and incubate at 28℃ for 2-4 days to obtain the engineered Agrobacterium.

[0109] 2.2 Arabidopsis transformation was performed using Agrobacterium-mediated inflorescence infection. Removing the main stem after Arabidopsis enters the flowering stage promotes the production of more lateral stems. Before transformation, the plant was thoroughly watered, and fully opened flowers and siliques were removed, leaving only tender or newly emerging flower buds. Transformation solution: MS 0.4g, sugar 10g, MES 0.1g, pH 5.7, Silwet 90μL (added before transformation), with the remainder made up to 200mL with water.

[0110] Monoclonal Agrobacterium bacteria preserved in GV3101 were cultured overnight at 28°C and 200 rpm in 3 mL of antibiotic-resistant LB broth until OD600 > 1.5. The bacterial culture was then transferred to approximately 200 mL of LB broth containing the appropriate antibiotic at a 1:100 volume ratio and cultured overnight at 28°C and 200 rpm until OD600 > 0.8. After collecting the bacterial culture, the liquid was resuspended in an equal volume of transformation medium, and Arabidopsis inflorescences were immersed in the transformation medium for 50 seconds. The inflorescences were covered with a black plastic bag, kept moist overnight, and protected from light for approximately 20 hours. Seeds were harvested, dried, and positive seedlings were selected on the appropriate medium. Transgenic plant identification: After obtaining seeds, selection was performed on 1 / 2 MS solid medium containing the appropriate antibiotic. After approximately 10 days of growth, positive plants were selected and transplanted into cultivation soil. After a period of growth, DNA was extracted from rosette leaves, amplified by PCR using vector-specific primers, and further identified at the transcriptional and protein levels.

[0111] After the above steps, Pro is constructed. GLDPA -GUS transgenic Arabidopsis thaliana plants.

[0112] Using the same method, we can build Pro. GLDPA -Venus-N7 transgenic Arabidopsis thaliana plants.

[0113] Example 2: Verification of the localization of the promoter GLDPA in Arabidopsis thaliana

[0114] Using the Pro constructed in Example 1 GLDPA -GUS transgenic Arabidopsis plants and Pro GLDPA -Venus-N7 transgenic Arabidopsis plants were used to investigate the localization of the promoter GLDPA in Arabidopsis.

[0115] ProGLDPA-GUS leaves were placed in β-glucuronidase (GUS) staining solution (0.1M Na2HPO4, 0.1M NaH2PO4, 5mM K3[Fe(CN)6], 5mM K4[Fe(CN)6], 1% Triton X-100, 20% methanol, 0.6 mg / ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid, cyclohexylammonium salt (X-glucose)). After incubation overnight in the dark at 37°C, the leaves were washed several times with 70% (v / v) ethanol for destaining. Unsectioned leaves were imaged under a Leica MZ12.5 stereomicroscope.

[0116] The results are shown in Figure 1 GUS (GUS) was observed using fluorescence microscopy. Figure 1 (B, C) and Venus signal ( Figure 1 The D and E components were mainly distributed in the veins and BSCs of Arabidopsis leaves, confirming the conservation of the GLDPA promoter function among different species.

[0117] Example 3: Construction of CGA1-expressing transgenic Arabidopsis

[0118] The following steps were taken to construct transgenic Arabidopsis thaliana overexpressing CGA1.

[0119] 1. Plasmid Pro GLDPA -CGA1 build

[0120] Following the method described in Example 1, plasmid Pro was constructed. GLDPA -CGA1 includes the following steps.

[0121] 1.1 Gene Cloning

[0122] 1.1.1 Extract the genome of Flavoreria trinervia and clone the promoter GLDPA sequence.

[0123] Leaves of *Chrysanthemum indicum* were picked, ground into powder with liquid nitrogen, and the genome of *Arabidopsis thaliana* was extracted using a plant genomic DNA extraction kit (DP321) (Tiangen Biotech (Beijing) Co., Ltd.).

[0124] PCR primers (5'-3'):

[0125] Forward 1300-ProGLDPA-F:

[0126] TATGACATGATTACGAATTCAAGCTTTACTCCTCTCAACTTT

[0127] Reverse 1300-ProGLDPA-R:

[0128] CGGGTACCGAGCTCGAATTCAGTGTAAGATGGGGTCTAATGG

[0129] High-fidelity enzyme KOD-FX-Neo (toyobo, product number: KFX-201)

[0130] PCR conditions: 94℃ for 2 min; 98℃ for 10 s, 58℃ for 30 s, 68℃ for 3 min, 35 cycles; 68℃ for 10 min.

[0131] Sequencing of the gene fragment amplified by RT-PCR confirmed that the GLDPA sequence is SEQ ID NO:4, which is correct.

[0132] 1.1.2 RNA was extracted from Arabidopsis thaliana, and the CGA1 transcription factor sequence was cloned by RT-PCR.

[0133] Wild-type Arabidopsis thaliana leaves (Co1-0) were collected, ground into powder using liquid nitrogen, and total RNA was extracted using the GeneJET Plant RNA Purification Kit (Thermo Scientific, catalog number K0802). The extracted RNA was then directly reverse transcribed to produce cDNA.

[0134] Using a reverse transcription kit ( One-Step gDNA Removal and cDNA Synthesis SuperMix (Catalog No.: AT311) for reverse transcription to produce cDNA.

[0135] PCR primers (5'-3'):

[0136] Forward 1300-CGA1-F:

[0137] ATCTTACACTGAATTCGAGCTCATGGGTTCCAATTTTCATTACA;

[0138] Reverse 1300-CGA1-R: AAGTTCTTCTCCTTTACTCATCCCGTGAACCATTCCGTG.

[0139] High-fidelity enzyme KOD-FX-Neo (toyobo, product number: KFX-201)

[0140] PCR conditions: 94℃ for 2 min; 98℃ for 10 s, 58℃ for 30 s, 68℃ for 90 s, 35 cycles; 68℃ for 10 min.

[0141] Sequencing of the gene fragment amplified by RT-PCR confirmed that the CGA1 sequence is SEQ ID NO:3, confirming its correctness.

[0142] 1.2 Construction of plant carriers

[0143] 1.2.1 The 2000bp fragment of the promoter GLDPA sequence (SEQ ID NO:4) was amplified by PCR using the fidelity enzyme kod-FX-Neo, and the sequence was confirmed to be correct.

[0144] PCR primers (5'-3'):

[0145] Forward 1300-ProGLDPA-F:

[0146] TATGACATGATTACGAATTCAAGCTTTACTCCTCTCAACTTT

[0147] Reverse 1300-ProGLDPA-R:

[0148] CGGGTACCGAGCTCGAATTCAGTGTAAGATGGGGTCTAATGG

[0149] High-fidelity enzyme KOD-FX-Neo (toyobo, product number: KFX-201)

[0150] PCR conditions: 94℃ for 2 min, 98℃ for 10 s, 58℃ for 30 s, 68℃ for 3 min, 35 cycles, 68℃ for 10 min.

[0151] Sequencing of the gene fragment amplified by RT-PCR confirmed that the GLDPA sequence is SEQ ID NO:4, which is correct.

[0152] 1.2.2 The GLDPA promoter sequence was ligated into the plant expression vector pCAMBIA1300 using the EcoRI restriction site to form the vector proGLDPApCAMBIA1300.

[0153] Use the restriction enzyme EcoRI-HF restriction enzyme (NEB, catalog number: R3101).

[0154] Enzyme digestion conditions: 37℃ for 1.5h, 80℃ for 25min.

[0155] Using the ClonExpress II One Step Cloning Kit (Novizan, item number: C112-01)

[0156] Recombination reaction conditions: 37℃ for 30 min, and immediately placed on ice to cool after the reaction is completed.

[0157] 1.2.3 The CGA1 transcription factor sequence was ligated into the plant expression vector proGLDPApCAMBIA1300 using the SacI restriction site, forming the vector proGLDPA-CGA1-pCAMBIA1300.

[0158] Use the restriction enzyme SacI-HF (NEB, catalog number: R3156).

[0159] Enzyme digestion conditions: 37℃ for 1.5h, 80℃ for 25min.

[0160] Using the ClonExpress II One Step Cloning Kit (Novizan, item number: C112-01)

[0161] Recombination reaction conditions: 37℃ for 30 min, and immediately placed on ice to cool after the reaction is completed.

[0162] Take 1 μg of the correctly sequenced plasmid proGLDPA-CGA1-pCAMBIA1300 and TOP10 heat-shock competent cells (Shanghai Weidi Biotechnology Co., Ltd., catalog number: DL1010), incubate on ice for 30 minutes, heat shock at 42°C for 5 minutes, place on ice for 2 minutes, add 200 μL of LB medium, and incubate at 37°C on a shaker for 1 hour. Spread all bacterial cells onto kanamycin-resistant LB agar plates and incubate upside down at 37°C for 1 day.

[0163] Colonies were selected for PCR identification. After the PCR reaction, the PCR products were identified using agarose gel electrophoresis. If a band of the correct size was found, the corresponding numbered bacteria were considered positive clones and sent to the company for sequencing verification.

[0164] After the above steps, the construction plasmid Pro is constructed. GLDPA -CGA1.

[0165] 2.Pro GLDPA Construction of CGA1 transgenic Arabidopsis

[0166] 2.1 Plasmid transformation into Agrobacterium and plant transformation

[0167] 2.1.1 Take 1 mg of the correctly sequenced plasmid proGLDPA-CGA1-pCAMBIA1300 and Agrobacterium GV3101 competent cells (Shanghai Weidi Biotechnology Co., Ltd., catalog number: AC1001) and incubate on ice for 30 min, flash freeze in liquid nitrogen for 5 min, heat shock at 37℃ for 5 min, place on ice for 5 min, add 1 mL of LB medium, and incubate at 28℃ on a shaker for 4 hours. Spread all bacterial cells onto kanamycin-resistant and rifamycin-resistant LB agar plates and incubate at 28℃ for 2-4 days to obtain the engineered Agrobacterium.

[0168] 2.1.2 Arabidopsis transformation was performed by inoculating the inflorescences with Agrobacterium.

[0169] After Arabidopsis enters its flowering period, removing the main shoot can promote the production of more lateral shoots. Before transformation, water thoroughly and remove fully opened flowers and siliques, leaving only tender or newly emerging flower buds. Transformation solution: MS 0.4g, sugar 10g, MES 0.1g, pH 5.7, Silwet 90μL (added before transformation), and the remainder made up to 200mL with water.

[0170] Monoclonal Agrobacterium bacteria preserved in GV3101 were cultured overnight at 28°C and 200 rpm in 3 mL of antibiotic-resistant LB broth until OD600 > 1.5. The bacterial culture was then transferred to approximately 200 mL of LB broth containing the appropriate antibiotic at a 1:100 volume ratio and cultured overnight at 28°C and 200 rpm until OD600 > 0.8. After collecting the bacterial culture, the liquid was resuspended in an equal volume of transformation medium, and Arabidopsis inflorescences were immersed in the transformation medium for 50 seconds. The inflorescences were covered with a black plastic bag, kept moist overnight, and protected from light for approximately 20 hours. Seeds were harvested, dried, and positive seedlings were selected on the appropriate medium. Transgenic plant identification: After obtaining seeds, selection was performed on 1 / 2 MS solid medium containing the appropriate antibiotic. After approximately 10 days of growth, positive plants were selected and transplanted into cultivation soil. After a period of growth, DNA was extracted from rosette leaves, amplified by PCR using vector-specific primers, and further identified at the transcriptional and protein levels.

[0171] Following the above steps, transgenic Arabidopsis plants overexpressing CGA1 were constructed. After resistance screening and PCR verification, three homozygous T3 lines were obtained, labeled L3, L4, and L9, respectively. These are three distinct lines corresponding to different insertion sites. Because vector insertion is random, having the same phenotype at three different insertion sites ensures the stability and consistency of gene expression.

[0172] Example 4: Investigation of CGA1 gene expression level, protein content, and plant biomass in transgenic Arabidopsis plants

[0173] 1. Assessment of CGA1 expression levels in transgenic materials

[0174] RNA was extracted from Arabidopsis thaliana, and the expression level of CGA1 was identified by qPCR. The steps are as follows:

[0175] Leaves from wild-type Arabidopsis thaliana Co1-0 and transgenic Arabidopsis thaliana plants were collected, ground into powder using liquid nitrogen, and total RNA was extracted from the Arabidopsis thaliana using the GeneJET Plant RNA Purification Kit (Thermo Scientific, catalog number K0802). The extracted RNA was then directly reverse transcribed to produce cDNA.

[0176] Using a reverse transcription kit ( One-Step gDNA Removal and cDNA Synthesis SuperMix (Catalog No.: AT311) for reverse transcription to produce cDNA.

[0177] Real-time qPCR was performed using the CFX 96 system (Bio-Rad) and SYBR Green Real-time PCR Master Mix (Yisheng Biotechnology).

[0178] qPCR program: 95℃ for 30s; 95℃ for 5s, 60℃ for 30s, 35 cycles.

[0179] See Figure 2 Compared to the wild type (WT), the transgenic material showed a significant increase in CGA1 expression (Figure B) and a marked increase in chlorophyll content (Figure C). The transgenic lines exhibited deeper and greener leaf veins, indicating that CGA1 expression in bundle sheath cells (BSCs) promoted chlorophyll synthesis in cells surrounding microtubules.

[0180] 2. Assessment of CGA1 protein content in transgenic materials

[0181] Slices of approximately 1 mm from fully expanded leaves of 28-day-old newborn plants were selected. The samples were then dehydrated and embedded according to a standard protocol (Wang H, Huang Y, Xiao Q, Huang X, Li C, Gao X, Wang Q, Xiang X, Zhu Y, Wang J, Wang W, Larkins BA, Wu Y (2020) Carotenoids modulate kernel texture in maize by influencing amyloplast envelope integrity. Nat Commun 11(1):5346. doi:10.1038 / s41467-020-19196-9.). The samples were cut into ultrathin sections using a diamond scalpel on a Leica EMUC6-FC6 ultramicroscope, and then imaged at 80 kV using a Hitachi H-7650 transmission electron microscope.

[0182] Transmission electron microscopy (TEM) images of wild-type and transgenic Arabidopsis thaliana leaves were obtained using ImageJ software. The number and size of planar cell area, chloroplasts, mitochondria, peroxisomes, and starch were quantitatively studied. (Schneider CA, Rasband WS, Eliiceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7):671-675. doi:10.1038 / nmeth.2089.)

[0183] See Figure 3 Further observation of the transverse anatomical structure of leaves through ultrathin section analysis revealed that the transgenic line's BSCs and veins exhibited significantly higher numbers and sizes of chloroplasts than the WT line (CF in the figure). The number of chloroplasts in the veins and BSCs increased at least twofold compared to WT (M in the figure), and the chloroplast area increased by approximately 1.4 times (J in the figure). Although the area of ​​the transgenic line's BSCs also increased significantly (G in the figure), the proportion of the total chloroplast area in the BSCs to the total BSC area increased significantly (L in the figure). Moreover, the number of starch granules in the chloroplasts of the transgenic line's BSCs and veins increased by 3.5-5.5 times compared to WT (H and M in the figure). This indicates that the specific expression of the CGA1 gene in BSCs and veins effectively promotes the differentiation and development of chloroplasts within BSC cells, and the photosynthetic function of BSCs has been activated.

[0184] 3. Biomass assessment of transgenic plants

[0185] See Figure 4 In addition to the number and area of ​​chloroplasts, the transgenic BSC cell line also showed an increase in the number of mitochondria and peroxisomes. Compared with WT, the number of mitochondria increased by at least 1.6 times (Figure A), while the size of mitochondria varied inconsistently among different transgenic Arabidopsis lines. Specifically, the area of ​​individual mitochondria increased in the L3 line but decreased in the L9 line (Figure C). The number of peroxisomes in the transgenic BSC line increased by approximately 2 times compared with WT (Figure C), and their area also increased significantly (Figure D). These results indicate that the CGA1 gene promotes the development of other organelles while also promoting chloroplast development.

[0186] Example 5: Specific expression of CGA1 transcription factor reduces CO2 compensation point

[0187] The CO2 compensation point (Γ) shall be detected using the following method.

[0188] Measuring instrument: LI-6800 portable optical measurement instrument (Li-Cor, USA).

[0189] Seedling age: Materials were planted under short-day conditions (10h light / 14h darkness) and were 4 weeks old. When conducting measurements, a small leaf chamber (2cm) was used. 2 Measurements were taken alternately between transgenic plants and control groups, with 4-6 biological replicates per group.

[0190] Step 1: Set the light intensity in the leaf chamber to 250 μmol m⁻²s. -1 (Red to blue ratio 9:1), temperature 22℃, humidity 66%, ambient CO2 concentration 400ppm, flow rate 300μmol / s -1 .

[0191] Step 2: After a 12-minute acclimatization period, the CO2 concentration in the leaf chamber was gradually decreased in the order of 150, 100, 80, 50, and 30 ppm. The photosynthetic rate was then acclimatized for 2 minutes and an "Auto Match" operation was performed.

[0192] Step 3: End the measurement and calculate the CO2 compensation point.

[0193] The results are listed in Table 2.

[0194] Table 2. Expression of CGA1 in BSC can lower the CO2 compensation point in plants.

[0195]

[0196] Our analysis of gas exchange data showed that, compared to WT, the Γ values ​​of the three transgenic lines L3, L4, and L9 were significantly lower than WT (Table 2), decreasing by approximately 3 ppm. Since there were no significant differences in respiration among the transgenic lines (Table 2), this indicates that the decrease in the CO2 compensation point in the transgenic lines was caused by reduced photorespiration, meaning that specific expression of CGA1 in BSCs can reduce leaf photorespiration. Therefore, specific expression of the CGA1 gene not only activates BSCs, increases the content of organelles and related photosynthetic and photorespiration-related proteins, and promotes starch accumulation, but also significantly reduces plant photorespiration.

[0197] Example 6: Specific expression of CGA1 transcription factor enhances photosynthetic capacity and biomass in plants

[0198] The photosynthetic rate of the plant was detected using the following method, and light response curves and CO2 response curves were generated.

[0199] Measuring instrument: LI-6800 portable optical measurement instrument (Li-Cor, USA).

[0200] Seedling age: Materials were planted under short-day conditions (10h light / 14h darkness) and were 4 weeks old. When conducting measurements, a small leaf chamber (2cm) was used. 2 Measurements were taken alternately between transgenic plants and control groups, with 4-6 biological replicates per group.

[0201] AQ (Audiometric Response) Measurement:

[0202] Step 1: Set the light intensity inside the leaf chamber to 600 μmol m⁻²s. -1 (Red to blue ratio 9:1), temperature 22℃, humidity 60%, ambient CO2 concentration 400ppm, flow rate 300μmol / s -1 .

[0203] Step 2: After 12 minutes of acclimatization, the light intensity in the leaf chamber is adjusted according to the following values: 1200, 800, 600, 400, 300, 200, 150, 100, 50, and 0 μmol m⁻²s. -1 The order gradually decreases. Allow 2 minutes for acclimatization and perform an "Auto Match" operation before recording the photosynthetic rate.

[0204] Step 3: Restore default settings and end the measurement.

[0205] Photoresponse porosity: The photoresponse porosity curve is obtained based on the photoresponse curve.

[0206] Constructing the CO2 response curve (ACi):

[0207] Step 1: The light intensity in the leaf chamber was set to 600 μmol m⁻² s⁻¹ (red to blue ratio 9:1), the temperature to be 22℃, the humidity to be 66%, the ambient CO₂ concentration to be 400 ppm, and the flow rate to be 300 μmol s⁻¹.

[0208] Step 2: After 12 minutes of acclimatization, the CO2 concentration in the leaf chamber was gradually decreased in the order of 400, 250, 150, 100, 80, 50, and 30 ppm. The plant was allowed to acclimatize for 2 minutes before recording the photosynthetic rate and then the "Auto Match" function was performed.

[0209] Step 3: Set the ambient CO2 concentration to 400 ppm and allow it to acclimatize for another 12 minutes.

[0210] Step 4: The CO2 concentration in the leaf chamber was gradually increased in the order of 400, 600, 900, 1200, 1400, and 1600 ppm. Allow the chamber to acclimate for 2 minutes and perform an "Auto Match" operation before recording the photosynthetic rate.

[0211] Step 5: Set the ambient CO2 concentration to 400 ppm and end the measurement.

[0212] Vcmax and Jmax: Using the R package "plantecophys", the maximum rate of RuBP carboxylation (Vcmax), maximum photosynthetic electron transport rate (Jmax), and respiration value were calculated by fitting the Farquhar-von Caemmerer-Berry (FvCB) model.

[0213] See Figure 5 We further measured the light response curves and CO2 response curves of the photosynthetic rates of transgenic and wild-type plants. The results showed that the net photosynthetic rate of transgenic Arabidopsis was significantly higher than that of the wild type, especially under high light and high CO2 conditions (Figures A and B). The stomatal conductance of the transgenic lines was also significantly increased (Figure C). After fitting the A-Ci curve using the Farquhar model, the maximum carboxylation rate (Vcmax) and maximum electron transport rate (Jmax) of RuBisCO in transgenic Arabidopsis were significantly higher in all three transgenic lines compared to wild-type plants (Figures D and E). Furthermore, the protein content in the whole leaves of transgenic Arabidopsis was significantly higher than that of wild-type plants (Figure F), and SDS-PAGE results showed a significant increase in the RuBisCO content of the whole leaves (Figure G). This may be due to the increased number of chloroplasts and the higher RuBisCO content in the BSCs of transgenic Arabidopsis. These results collectively indicate that promoting chloroplast development in Arabidopsis BSCs through specific expression of CGA1 can improve leaf photosynthetic capacity.

[0214] See Figure 6We observed the phenotypes of Arabidopsis thaliana under normal and high light conditions and statistically analyzed their biomass. Normal light treatment involved growing the plants for 30 days at a light intensity of 150 μmol m⁻² s⁻¹, and then measuring their biomass. High light treatment involved partially transferring Arabidopsis plants grown to 21 days under normal light to treatments of 200 μmol m⁻² s⁻¹ for 2 days, 300 μmol m⁻² s⁻¹ for 3 days, and 450 μmol m⁻² s⁻¹ for 4-5 days, and then measuring their biomass. The experimental results are shown below. Figure 6 (Figures A and D) Compared to WT, transgenic lines showed a greater number of leaves under both normal and strong light conditions. Under normal light, the biomass of transgenic plants, except for the L3 line, was higher in both L4 and L9 than in WT (Figures B and C). Under strong light, the increase in biomass of transgenic lines was more significant, increasing by 35%-50% compared to WT; the increase in the number of leaves was also greater than under normal light, increasing by approximately 50% compared to WT (Figures E and F). Furthermore, the biomass and number of leaves under strong light were significantly higher than under normal light (Figures BC and EF), which is consistent with the finding that transgenic Arabidopsis leaves have a higher photosynthetic rate under strong light (Figure A). In conclusion, specific expression of CGA1 in BSC enhances the activation of photosynthetic mechanisms in BSC, significantly increasing the photosynthetic rate of leaves and promoting the accumulation of Arabidopsis biomass.

[0215] Although the above embodiments only use Arabidopsis thaliana as an example to illustrate the technical solution of the present invention, the technical solution of the present invention is also applicable to other plant species such as rice and corn, based on the disclosure of the present invention. Therefore, without departing from the spirit of the present invention, those skilled in the art can make various modifications or alterations to the present invention, and the equivalent forms of various variations or modifications should also fall within the scope of the present invention.

[0216] It should be noted that the listing and discussion of previously disclosed documents in this specification should not be construed as an admission that such documents are prior art or common general knowledge.

Claims

1. The use of transcription factor CGA1 (NCBI number Q9SZI6.1) with the amino acid sequence shown in SEQ ID NO:1 or its conserved variant polypeptide in improving plant photosynthetic rate and biomass, wherein the conserved variant polypeptide is a polypeptide with more than 95% homology to CGA1 and having the function of CGA1.

2. The use as described in claim 1, characterized in that, The plants are selected from the following group of crops: rice, wheat, corn, soybean, barley, oats, rye, sorghum, cotton, vegetables, and cruciferous plants.

3. The use as described in claim 1, characterized in that, This induces the expression of transcription factor CGA1 in bundle sheath cells.

4. The use as described in claim 3, characterized in that, The GLDPA promoter (nucleotide sequence SEQ ID NO:4) derived from yellow daisy was used to drive the expression of transcription factor CGA1 in bundle sheath cells.

5. The use as described in claim 4, characterized in that, The expression of the transcription factor CGA1 in bundle sheath cells is achieved by: inserting the GLDPA promoter from yellow chrysanthemum and the CGA1 genome into an expression vector to form a recombinant expression vector; and using transgenic technology to introduce the recombinant expression vector into plants such as rice to obtain transgenic plants that overexpress the transcription factor CGA1 in bundle sheath cells.

6. The use as described in claim 4, characterized in that, The expression of the transcription factor CGA1 in bundle sheath cells is achieved by providing a transcription factor CGA1 expression cassette containing a GLDPA promoter derived from yellow chrysanthemum and a CGA1 gene located downstream therefrom, and cloning the expression cassette into the plant chromosome genome using gene editing technology to obtain a transgenic plant that overexpresses the transcription factor CGA1 in bundle sheath cells.

7. A DNA molecule, a transcription factor CGA1 gene expression cassette, characterized in that, It contains the GLDPA promoter derived from yellow daisy and the CGA1 gene located downstream of it.

8. A recombinant plasmid, characterized in that, It is a plasmid containing the aforementioned DNA molecule, namely the transcription factor CGA1 gene expression cassette, and is suitable for expression in Agrobacterium.

9. A recombinant Agrobacterium engineered strain, characterized in that, It is an Agrobacterium transformant containing the recombinant plasmid as described in claim 8.

10. The application of the DNA molecule as described in claim 7, the recombinant plasmid as described in claim 8, and the recombinant Agrobacterium engineered bacteria as described in claim 9 in improving plant traits and in cultivating new plant varieties with increased photosynthetic rate and / or biomass.

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