A fusion protein based on optogenetics to regulate endogenous ABA signaling and its applications

Abstract

This invention belongs to the field of plant regulation and protein engineering technology, specifically relating to a fusion protein that regulates endogenous ABA signaling based on optogenetics and its applications. Specifically, this invention, based on optogenetics, fuses the protein kinase OST1 from the ABA signal transduction component with the optogenetic element cpLOV2, which can be activated / inactivated through dark / light treatment. This achieves the goal of using light to transmit and block ABA signals within the cell to regulate stomatal movement, thereby increasing plant biomass and achieving high and sustainable crop yields, thus possessing significant practical application value.

Description

Technical Field

[0001] This invention belongs to the field of plant regulation and protein engineering technology, specifically relating to a fusion protein based on optogenetics to regulate endogenous ABA signaling and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] With the continued growth of the global population and the increasingly severe climate change, the contradiction between water scarcity and food demand is becoming increasingly prominent, posing a huge challenge to agricultural production. In agricultural production, increasing crop biomass while reducing water loss, and thus improving water use efficiency (WUE), has become a key focus of current agricultural technological innovation. Plant carbon assimilation faces two major barriers: gas exchange through stomata and the binding of ribulose-1,2-bisphosphate carboxylase (RUBISCO) to CO2. As a key channel regulating CO2 exchange and water transpiration, stomata directly affect plant yield and WUE. However, this process involves a certain physiological contradiction: increasing stomatal conductance can promote carbon assimilation, but it also exacerbates water loss; and vice versa. To overcome this physiological contradiction, developing "intelligent stomata" has become an important breakthrough direction in current agricultural biotechnology. By precisely controlling stomatal movement and optimizing its response to environmental factors such as light and temperature, high crop yields and sustainability can be achieved.

[0004] Abscisic acid (ABA), a plant hormone, plays a crucial role in plant growth and development. When ABA synthesis or ABA signaling components are deficient, plants exhibit severe drought sensitivity. In recent years, significant progress has been made in understanding ABA signaling through genetic, physiological, biochemical, and chemical biological methods. ABA signaling has also been shown to play a vital role in abiotic stresses, particularly in regulating stomatal movement.

[0005] The core ABA signal transduction pathway consists of three parts: the ABA receptor, PP2C phosphatase, SNRK2 protein kinase, and downstream transcription factors and ion channels. In the absence of ABA, PP2C phosphatase interacts with SNRK2 and inhibits the activity of SNRK2S protein kinase, blocking ABA signal transmission. However, in the presence of ABA, ABA binds to the ABA receptor PYR / PYL / RCAR to form a binary complex, which then forms a ternary complex with PP2C phosphatase. This releases the inhibitory effect of PP2C on SNRK2S, and the activated SNRK2S phosphorylates downstream ion channels and transcription factors to transmit ABA signals. This is the core ABA signal transduction pathway and a target for drought resistance research in genetics and agrochemicals.

[0006] ABA-induced stomatal closure is very rapid, so chemical and genetic regulation is often used to artificially intervene in ABA signaling to achieve drought resistance. This includes designing ABA receptor agonists and antagonists, and regulating ABA signal transduction components at the gene and protein levels. However, current methods have limited spatiotemporal precision and specificity in terms of the selectivity and permeability of applied chemicals, the pleiotropic nature of gene modifications, and the time lag between gene manipulation and analysis. These limitations lead to significant side effects. Summary of the Invention

[0007] To address the shortcomings of the existing technologies, the inventors, through long-term technical and practical exploration, have provided a fusion protein based on optogenetics to regulate endogenous ABA signaling and its applications. This invention regulates the endogenous ABA signaling pathway in plants using optogenetics, thereby ultimately increasing plant biomass. Based on the above research findings, this invention has been completed.

[0008] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a fusion protein comprising at least a protein kinase OST1 in an ABA signal transduction component, and an optogenetic element cpLOV2 connected to the protein kinase OST1; wherein the protein kinase OST1 is located at the N-terminus of the fusion protein, and the cpLOV2 is located at the C-terminus of the fusion protein.

[0010] In a second aspect, the present invention provides a nucleic acid molecule capable of encoding the aforementioned fusion protein.

[0011] A third aspect of the present invention provides a recombinant expression vector containing the above-mentioned nucleic acid molecules.

[0012] In a fourth aspect, the present invention provides a host cell containing the above-described nucleic acid molecules, a recombinant expression vector containing the above-described nucleic acid molecules, or a fusion protein capable of expressing the above-described fusion protein.

[0013] A fifth aspect of the present invention provides the use of the above-described fusion protein, nucleic acid molecule, recombinant expression vector and / or host cell in any one or more of the following:

[0014] (1) Regulate the plant’s endogenous ABA signaling pathway;

[0015] (2) Regulating plant stomatal movement;

[0016] (3) Increase plant biomass.

[0017] A sixth aspect of the present invention provides a method for increasing plant biomass, the method comprising: transferring the nucleic acid molecule into the plant for overexpression.

[0018] Furthermore, the nucleic acid molecules are expressed in plant guard cells.

[0019] The increase in plant biomass is specifically manifested in at least the following ways: increasing the leaf area of ​​the plant, and increasing the fresh weight and dry weight of the plant line.

[0020] Compared with existing technical solutions, one or more of the above technical solutions have the following beneficial effects:

[0021] The above technical solution is based on optogenetics, which fuses the protein kinase OST1 in the ABA signal transduction component with the optogenetic element cpLOV2. Dark / light treatment can activate / inactivate OST1. This allows the use of light to transmit and block intracellular ABA signals to regulate stomatal movement, thereby increasing plant biomass and achieving high and sustainable crop yields. Therefore, it has significant practical application value. Attached Figure Description

[0022] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0023] Figure 1 This explains the working principle of LCOST1.5 in this invention.

[0024] Figure 2 To identify truncated OST1 cells with kinase activity but not inhibited by ABI1. A represents the approach to constructing light-controlled OST1; B shows schematic diagrams of the structural domains of different truncated OST1 cells; C shows the activity screening of various truncated OST1 cells in the Xenopus oocyte system; D shows the screening of truncated OST1 cells not inhibited by ABI1 in the Xenopus oocyte system.

[0025] Figure 3 The evaluation results of the three designed high-throughput screening systems are presented.

[0026] Figure 4 The schematic diagram (A) and identification results (B) of the "yeast one-hybrid" screening system are shown.

[0027] Figure 5 Design scheme (A) with optogenetic elements located at the N-terminus of the fusion protein and T1 located at the C-terminus, and screening results (B).

[0028] Figure 6 The design scheme (A) and screening results (B) for modifying the Jα length of LJT1 are presented.

[0029] Figure 7 The design scheme (A) and the selection results (B) for placing AsLOV2 within the T1 structural domain are shown.

[0030] Figure 8 Design scheme (A) with T1 placed at the N-terminus and optogenetic element placed at the C-terminus, and screening results (B).

[0031] Figure 9 The flowchart shows the construction and screening process of the random mutant library LCOST1.

[0032] Figure 10 Structure and light-controlling properties of LCOST1 activated in darkness for the purpose of blue light suppression.

[0033] Figure 11 For the detection of the light control properties of LCOST1.5.

[0034] Figure 12 In vitro phosphorylation validation of LCOST1.5 protein under blue light and darkness (A) and under different blue light treatments (B).

[0035] Figure 13 The expression specificity (A), expression level (B), leaf area (C and D), fresh weight (E), and dry weight (F) of the KST1:LCOST1.5 transgenic Arabidopsis thaliana were compared with the control. In B, the endogenous OST1 expression level of the KST1:GFP transgenic strain was 1.

[0036] Figure 14 The image shows the stomatal movement phenotype of the KST1:LCOST1.5 transgenic Arabidopsis thaliana. A represents stomatal aperture during day and night; B represents continuous stomatal conductance during the dark-light-dark transition; C represents continuous stomatal conductance during the light-dark transition; and KST1:GFP represents the control.

[0037] Figure 15 The expression level of the RD29A gene in Arabidopsis thaliana guard cells of KST1:LCOST1.5 under dark and light conditions.

[0038] Figure 16 The expression specificity (A), expression level (B), leaf area (C and D), fresh weight (E), and dry weight (F) of KST1:LCOST1.5 transgenic tobacco were compared with the control. Among them, B is the result of RT-PCR electrophoresis. Detailed Implementation

[0039] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0040] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0041] In this invention, "fusion protein" refers to oligopeptides, peptides, protein sequences or fragments thereof, and specifically to molecules that are actually formed, recombinant, synthetic, or semi-synthetic. It should be noted that the term "fusion protein" and similar terms do not imply that the amino acid sequence is limited to a full-length molecule containing the complete natural amino acid sequence. It should be understood that the various references to "fusion protein" and similar terms herein will include full-length sequences as well as any fragments, derivatives, or variants thereof. More specifically, the fusion protein described in this invention is a protein obtained by fusing protein kinase OST1 with the optogenetic element cpLOV2.

[0042] In this invention, for sequence comparison, a sequence is typically used as a reference sequence and compared with the detection sequence. When using a sequence comparison algorithm, the detection and reference sequences are input into a computer, and the coordinates of the subsequences are specified if necessary, along with the parameters of the sequence algorithm program. Then, based on the selected program parameters, the sequence comparison algorithm calculates the percentage sequence identity (consistency) of the detection sequence relative to the reference sequence.

[0043] As mentioned earlier, developing "intelligent stomata" has become a crucial breakthrough direction in current agricultural biotechnology. This involves precisely regulating stomatal movement and optimizing its response to environmental factors such as light and temperature to achieve high crop yields and sustainability. ABA signaling has been proven to play a vital role in abiotic stress, particularly in regulating stomatal movement. However, ABA-induced stomatal closure is very rapid, so chemical and genetic regulation is often used to artificially intervene in ABA signaling to achieve drought resistance. However, current methods have limited spatiotemporal precision and specificity in terms of the selectivity of applied chemicals, permeability, pleiotropic effects of gene modification, and the time lag between gene manipulation and analysis, all of which lead to significant side effects.

[0044] Optogenetics is a novel biological technology that combines genetics and optics, utilizing light-responsive proteins to achieve precise regulation of physiological functions in cells, tissues, organs, and animals. Currently, optogenetics is widely used in research on various fundamental questions, including immunology, cell biology, developmental biology, and neuroscience. It involves expressing naturally occurring light-sensitive proteins or fusing proteins of interest into light-sensitive proteins through genetic engineering within specific living cells, followed by precise regulation of intracellular physiological activities using light of specific wavelengths. Compared to other traditional biophysical or chemical methods for manipulating cellular functions and processes, optogenetics offers advantages such as being non-invasive, reversible, low-toxicity, and possessing high spatiotemporal precision. Therefore, using optogenetics to regulate the endogenous ABA signaling pathway in plants is a feasible approach.

[0045] In view of this, in a typical embodiment of the present invention, a fusion protein is provided, the fusion protein including at least a protein kinase OST1 in the ABA signal transduction component, and an optogenetic element cpLOV2 connected to the protein kinase OST1; wherein the protein kinase OST1 is located at the N-terminus of the fusion protein, and the cpLOV2 is located at the C-terminus of the fusion protein.

[0046] Furthermore, the fusion protein is any one of the following (a1)-(a3):

[0047] (a1) The amino acid sequence shown in any one of SEQ ID NO. 1-5;

[0048] (a2) A protein derived from (a1) by substitution and / or deletion and / or addition of one or more amino acid residues and having the same function;

[0049] (a3) Other genes encode proteins that have at least 80% (including but not limited to 80%, 85%, 90%, 95%, 99%) the amino acid sequence composition shown in (a1) and have the same or similar activity as the fusion protein shown in (a1).

[0050] The proteins in (a1)-(a3) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically. The fusion proteins composed of the amino acid sequences described in SEQ ID NO. 1-5 are named LCOST1.1, LCOST1.2, LCOST1.3, LCOST1.4, and LCOST1.5, respectively, in this invention. It can be seen that LCOST1.5 is actually obtained by fusing a GFP to the N-terminus of the fusion protein LCOST1.4. Therefore, the fusion protein can be obtained by adding a reporter protein to any of the amino acid sequences shown in LCOST1.1-LCOST1.4. The reporter protein can be a fluorescent protein, and the fluorescent protein is located at the N-terminus or C-terminus of LCOST1.1-LCOST1.4, preferably at the N-terminus.

[0051] In another specific embodiment of the present invention, a nucleic acid molecule is provided, which is capable of encoding the above-mentioned fusion protein.

[0052] The nucleic acid molecule has a nucleotide sequence shown in any one of (b1)-(b4):

[0053] (b1) Nucleotide sequences as shown in SEQ ID NO. 6-10;

[0054] (b2) A sequence formed by replacing, deleting or inserting one or more nucleotides as shown in (b1);

[0055] (b3) has at least 80% (including but not limited to 80%, 85%, 90%, 95%, 99%) identity with the nucleotide sequence defined in (b1) or (b2) and encodes the same or similar functional protein.

[0056] (b4) A nucleotide sequence complementary to any of the nucleotide sequences described in (b1)-(b3).

[0057] In another specific embodiment of the present invention, a recombinant expression vector is provided, wherein the recombinant expression vector contains the above-mentioned nucleic acid molecules.

[0058] The recombinant expression vector is obtained by effectively linking the aforementioned nucleic acid molecules to an expression vector. The expression vector is any one or more of a viral vector, plasmid, bacteriophage, granule, or artificial chromosome. The viral vector may include adenovirus vector, retrovirus vector, or adeno-associated virus vector. The artificial chromosome includes bacterial artificial chromosome, bacteriophage P1-derived vector, yeast artificial chromosome, or mammalian artificial chromosome. More preferably, it is a plasmid. Furthermore, the plasmid may be a plant plasmid expression vector (such as the pCAMBIA series plasmid expression vector).

[0059] In another specific embodiment of the present invention, a host cell is provided, wherein the host cell contains the above-mentioned nucleic acid molecules, a recombinant expression vector containing the above-mentioned nucleic acid molecules, or is capable of expressing the above-mentioned fusion protein.

[0060] The host cells include bacterial cells, fungal cells, or plant cells;

[0061] The bacteria can be any one or more of Escherichia coli, Agrobacterium, Bacillus, Streptomyces, Pseudomonas, or Staphylococcus.

[0062] In one or more specific embodiments of the present invention, the bacteria include, but are not limited to, Escherichia coli (such as Escherichia coli DH5α), Agrobacterium tumefaciens (such as GV3101), Agrobacterium rhizogenes, Lactococcus lactis, Bacillus subtilis, Bacillus cereus, or Pseudomonas fluorescens.

[0063] The fungal cells include yeast;

[0064] The plant cells can be monocotyledonous or dicotyledonous plant cells. Furthermore, the plant can be a crop, including food crops and cash crops, without specific limitations.

[0065] In another specific embodiment of the present invention, the use of the above-mentioned fusion protein, nucleic acid molecule, recombinant expression vector and / or host cell in any one or more of the following:

[0066] (1) Regulate the plant’s endogenous ABA signaling pathway;

[0067] (2) Regulating plant stomatal movement;

[0068] (3) Increase plant biomass.

[0069] The plant can be a monocotyledonous plant or a dicotyledonous plant. Furthermore, the plant can be a crop, including food crops and cash crops, without specific limitations.

[0070] In another specific embodiment of the present invention, a method for increasing plant biomass is provided, the method comprising: transferring the nucleic acid molecule into the plant for expression.

[0071] Furthermore, the nucleic acid molecules are expressed in plant guard cells.

[0072] The increase in plant biomass is specifically manifested in at least the following ways: increasing the leaf area of ​​the plant, and increasing the fresh weight and dry weight of the plant line.

[0073] The present invention will be further illustrated below with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified, all materials and reagents used in the following embodiments are commercially available. Unless otherwise specified, all experimental methods used are conventional methods.

[0074] In this invention, different photoreceptor proteins were linked to variants of OST1 using different linkers to obtain a large number of fusion proteins. Through screening, a photocontrolled OST1 variant that is activated in the dark but inactivated under blue light was obtained and named LCOST1.5. Its structure and working principle are as follows: Figure 1 As shown.

[0075] Example 1: Construction of the light control element LCOST1

[0076] 1) Filtering of OST1 that is not controlled by ABA

[0077] Previous studies have shown that activation and inactivation of OST1 are key steps in ABA signaling. In the absence of ABA, PP2C interacts with OST1 and inhibits its activity. However, in the presence of ABA, PP2C interacts with the ABA-ABA receptor complex to form a ternary complex, thereby relieving the inhibitory effect of PP2C on OST1 and enabling ABA signaling. When OST1 lacks Domain II, its interaction with ABI1 is affected, and it loses its ABA response. To construct a photoregulated OST1 (LCOST1), we designed a process, as follows... Figure 2 As shown in Figure A, by truncating Domain II, a minimal functional domain with a simpler structure and more flexible regulation is obtained while maintaining protein kinase activity. Using this strategy, we aim to obtain a minimal OST1 structural unit that is not inhibited by ABI1 and fuse it with an optogenetic element. Based on the structure and function of OST1, this invention truncated OST1 to four different lengths, with the results shown below. Figure 2As shown in Figure B, the amino acids are 1-318aa, 12-318aa, 21-318aa, and 1-227aa, named T1, T2, T3, and T4, respectively. Simultaneously, this invention also reconstructed the core ABA signaling pathway (ABA receptor PYL1, PP2C phosphatase ABI1, OST1 protein kinase, and the downstream ion channel SLAC1) in Xenopus oocytes. Since the ion channel SLAC1 requires kinase activation to have channel activity in Xenopus oocytes, this invention co-injected different truncated OST1 molecules with SLAC1 into Xenopus oocytes and detected the kinase activity of each OST1 molecule by recording the SLAC1 current. The results are as follows... Figure 2 As shown in C, T1, T2, and T3 can all activate SLAC1, indicating that these three truncated OST1s all have kinase activity.

[0078] Since ABA activates OST1 by breaking the interaction and inhibition of PP2C on OST1, to screen for OST1 with kinase activity but not controlled by ABA, we used ABI1 as a representative of PP2C and co-injected ABI1 with truncated kinase-active OST1 and SLAC1 into Xenopus laevis. The results are as follows: Figure 2 As shown in D, only T1 is not inhibited by ABI1. In summary, T1 is an OST1 with kinase activity that is not inhibited by ABI1.

[0079] 2) Establish a high-throughput screening system for LCOST1

[0080] To obtain photoregulated OST1, this invention plans to fuse T1 with an optogenetic element. Since there are many ways for two proteins to fuse, a low-cost, high-throughput screening system is needed. However, the Xenopus oocyte system is not suitable for high-throughput screening. Therefore, we designed the following four screening systems, and the screening principles of each system are as follows:

[0081] a. Tobacco screening system: Since the expression of RD29A is significantly upregulated by ABA, RD29A is often used as a marker gene for ABA signal response. The promoter of RD29A drives the expression of firefly luciferase LUC, and RD29A:LUC is used as a reporter gene to determine whether the fused OST1 has kinase activity.

[0082] b. Functional complementation in potassium-deficient yeast: This experiment is based on the phenomenon of OST1 inhibiting KAT1. Theoretically, the potassium ion channel KAT1 can complement the growth of potassium-deficient yeast CY162. After co-transforming different fused OST1s with KAT1, the growth status of the yeast is used to determine whether the fused OST1 has kinase activity.

[0083] c. Yeast Three-Hybrid System: The principle of this experiment is based on OST1 promoting the interaction between AKS1 and 14-3-3 protein. OST1, AKS1, and 14-3-3 are co-transformed into yeast AH109. When the fused OST1 has kinase activity, AKS1 can interact with the 14-3-3 protein, allowing the yeast to grow normally. The growth status of the yeast is used to determine whether the fused OST1 has kinase activity.

[0084] d. Yeast one-hybrid system: This experimental principle is based on the phenomenon of OST1 phosphorylating transcription factor ABI5. Transcription factor ABI5 has no self-activating activity; its transcriptional activity depends on phosphorylation. OST1 and ABI5 are co-transformed into yeast AH109, ​​and the growth of the yeast is used to determine whether the fused OST1 has kinase activity.

[0085] Next, we will verify these four systems one by one:

[0086] In the tobacco screening system, the results are entered Figure 3 As shown in Figure A, both OST1 and T1 can induce the expression of RD29A:LUC under light, indicating that the ABA signal is activated at this time. However, their activity is relatively weak in the dark, which may be related to the regulation of RD29A expression by the biological clock. Therefore, this system is not suitable for subsequent screening of LCOST1 activity.

[0087] In the "potassium absorption-deficient yeast functional complementation" system, empty EV represented the negative control, and KAT1 represented the positive control. The results are as follows: Figure 3 As shown in B, KAT1 can complement the growth of CY162, but neither OST1 nor T1 inhibits yeast growth, so this system is not suitable for screening.

[0088] In the yeast three-hybrid system, T1 and OST1 were both positive controls, and T4 was a negative control. Results are as follows: Figure 3 As shown in C, the positive controls all promoted the interaction between AKS1 and 14-3-3 protein, while the negative controls did not. Therefore, this system is suitable for screening, but it has the disadvantage of poor reproducibility.

[0089] In the yeast one-hybrid system (the principle is as follows) Figure 4 A), the result is as follows Figure 4 As shown in B, transcription factor ABI5 is linked to BD. When inactive T4 and empty EV are co-transformed with it, yeast growth in the defective medium (SD / -LWAH) is inhibited because BD-ABI5 cannot be activated to express the reporter gene. However, when active OST1 and T1 are used, yeast growth is promoted. The reproducibility is good and it is not affected by light. Therefore, this system will be used for screening LCOST1.

[0090] 3) Design of LCOST1

[0091] Among numerous optogenetic elements, those with a light-oxygen-electric domain (LOV domain) stand out due to their simple composition, small molecular weight, convenience, and good reversibility. Therefore, this invention primarily selects light-responsive proteins with the LOV2 domain as optogenetic elements.

[0092] Because protein-protein fusion occurs in various ways, including the positional relationship between the two proteins, the type of linker, and the length of the linker, this invention mainly focuses on the following two strategies:

[0093] 1. To design the positional relationship between T1 and LOV2, we primarily used AsLOV2 and cpLOV2 (cpLOV2 is derived from AsLOV2 through a cyclic substitution method, by exchanging the N-terminus and C-terminus of AsLOV2 to obtain a new optogenetic element), and secondarily included LOV2 domains from Arabidopsis thaliana AtPhot1 and AtPhot2. The optogenetic elements were then fused into the N-terminus, C-terminus, and middle of T1, respectively.

[0094] 2. Design of linkers between proteins. The primary linker used is a flexible linker composed of glycine and serine (Gly-Gly-Gly-Gly-Ser), and fusion is achieved by changing the length of the linker between two proteins. In addition, different linkers were used, including the linker used in the LOV2 domain of AtPhot1 and AtPhot2.

[0095] Based on these two strategies, we designed a total of 30 fused OST1 variants across four main categories and screened them using a high-throughput screening system. Based on the stomatal movement characteristic of opening during the day and closing at night, our screening target was LCOST1 variants that are activated in darkness but inactivated under blue light.

[0096] The specific design is as follows:

[0097] 1. The optogenetic element is located at the N-terminus of the fusion protein, and T1 is located at the C-terminus. This class utilizes the LOV2 domain from oat-derived AsLOV2-Jα, AtPhot1, and AtPhot2, and uses different linkers between LOV2 and T1. The specific fusion method is as follows: Figure 5 As shown in Figure A. The filtering results are as follows. Figure 5As shown in Figure B, when the optogenetic element is placed at the N-terminus, the fused OST1 remains active under both darkness and blue light. A notable exception is LJT1 (directly linked to T1 by AsLOV2-Jα without an intermediate linker), which exhibits different growth patterns under darkness and blue light, indicating that AsLOV2 indeed regulates T1 activity. Subsequently, we attempted to modify the length of Jα to achieve further improvement, with the following results... Figure 6 As shown, regardless of the truncation method, its activity under blue light and darkness is not significantly different.

[0098] 2. AsLOV2-Jα was placed between the T1 domains. Based on the analysis of the OST1 crystal structure, the position of the αC domain was found to be crucial to the kinase activity; therefore, AsLOV2-Jα was inserted into the loop region preceding the αC domain. Figure 7 One design places the OST1 fusion before amino acid 52, and the other before amino acid 56. However, the fusion OST1 obtained by this design completely loses its activity, which may be because AsLOV2-Jα disrupts the structure of the OST1 protein kinase.

[0099] 3. T1 was placed at the N-terminus of the fusion protein, and cpLOV2 at the C-terminus. Since AsLOV2-Jα tends to regulate downstream linkers of the C-terminal Jα helix, limiting its application, its cyclic replacement, cpLOV2, was introduced here, swapping the N-terminus and C-terminus of AsLOV2. T1 and cpLOV2 were then fused using flexible linkers of different lengths, as shown in the results. Figure 8 As shown, when there is no linker between T1 and cpLOV2 (n=0, T1-cpLOV2), there is no significant difference in its activity under darkness and blue light. However, when there is a flexible linker between T1 and cpLOV2 (n=1), its activity is completely lost. When there are two linkers connected (n=2), its activity is restored, indicating that the regulation of T1 by cpLOV2 is effective.

[0100] In summary, although we did not find LCOST1 with dark activation and blue light suppression through various fusion methods, LJT1 and T1-cpLOV2 are special cases. Therefore, we will further optimize based on LJT1 and T1-cpLOV2.

[0101] 4) Optimization of LCOST1

[0102] In protein modification, common methods include constructing random mutant libraries, semi-rational design, and computer-aided protein design. We adopted the traditional method of constructing random mutant libraries, using LJT1 and T1-cpLOV2 as templates and randomly mutagenizing them using Error-Prone PCR. By adjusting the PCR reaction system, we constructed two libraries with a capacity of 10... 4 A high-quality random mutant library containing CFU and mutation rates of 1–4 amino acids was constructed. The random mutant library was screened using a high-throughput screening system. The construction and screening process of the mutant library is as follows: Figure 9 As shown, by screening 20,000 clones, we identified three LCOST1 variants that are activated in the dark and inactivated by blue light in a T1-cpLOV2-based mutant library, named LCOST1.1, LCOST1.2, and LCOST1.3, respectively. DNA was extracted from yeast and sequenced, and the results are shown below. Figure 10 As shown in Figure A, LCOST1.1 is formed by the mutation of cysteine ​​at position 405 to serine (C405S), LCOST1.2 is formed by the mutation of proline at position 282 to serine and methionine at position 332 to valine (P282S, M332V), and LCOST1.3 is formed by the mutation of glutamic acid at position 114 to valine and lysine at position 335 to methionine (E114V, K335M). Among these, LCOST1.1 exhibits the lowest activity under blue light, but growth curve measurements after 102 hours of blue light irradiation revealed that it still retained weak activity under blue light. Figure 10 B), therefore, LCOST1.1 needs further optimization.

[0103] Subsequently, we analyzed the mutation sites of LCOST1.2 and LCOST1.3 separately, and the results are as follows: Figure 10 As shown in Figure C, LCOST1.2-1 loses its photocontrollability when only the M332V site is mutated, while LCOST1.2-2 retains its photocontrollability when only the P282S site is mutated, indicating that the P282S mutation is crucial for photocontrollability. Therefore, based on mutation site analysis, we performed a site-directed P282S mutation on LCOST1.1, obtaining a new LCOST1 that is inactivated by blue light and activated by darkness, named LCOST1.4. Figure 10 C). To facilitate subsequent observation of protein expression intensity and specificity, we fused a GFP to the N-terminus of the LCOST1.4 protein, naming the fused protein LCOST1.5, and tested its light-controlled properties. The results are as follows. Figure 11As shown in Figure A, using red light as a control, LCOST1.5 was found to be inactive under blue light but active in the dark, indicating that the fusion of GFP with LCOST1.4 does not affect its photosensitive properties. To further determine its activity under blue light, we irradiated it with blue light for 102 hours and measured its growth curve, as shown in Figure A. Figure 11 As shown in B, LCOST1.5 is essentially inactive under blue light.

[0104] In summary, this invention, by fusing optogenetic elements with T1, obtained three LCOST1 molecules that are inactivated by blue light and activated in the dark through rational design and random mutation, named LCOST1.1, LCOST1.2, and LCOST1.3, respectively. LCOST1.1 has the best light controllability, but it still has weak activity under prolonged blue light. Therefore, based on LCOST1.1, a site-directed mutation of P282S was performed to obtain LCOST1.5, which is strictly controlled by blue light and activated in the dark.

[0105] The amino acid and nucleotide sequences of LCOST1.1, LCOST1.2, LCOST1.3, LCOST1.4 and LCOST1.5 of the present invention are shown in SEQ ID NO.1-5 and SEQ ID NO.6-10, respectively;

[0106] The specific experimental steps are as follows:

[0107] 1. Experimental Materials

[0108] The KOD One PCR Master Mix kit was purchased from TOYOBO, the gel extraction kit from AXYGEN, the restriction endonuclease kit from NEB, and the ClonExpress II One Step Cloning Kit from Novizan.

[0109] 2. Experimental Procedure

[0110] 2.1 Target gene amplification (taking T1-cpLOV2 as an example)

[0111] 1) The CDS sequence of T1 was obtained by amplification of Arabidopsis cDNA, and its nucleotide and amino acid sequences are shown in SEQ ID NO. 11 and SEQ ID NO. 12, respectively. The sequence of the optogenetic element cpLOV2 was obtained by whole-genome synthesis by Shanghai Sangon Biotech, and its nucleotide and amino acid sequences are shown in SEQ ID NO. 13 and SEQ ID NO. 14, respectively. The two fragments were spliced ​​together by overlap extension PCR to obtain T1-cpLOV2. The amplification primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and purified by PAGE. The primer sequences are shown below, where the underlined parts are homologous arms.

[0112]

[0113] 2) Amplification primers for the T1-cpLOV2 sequence were designed and synthesized by Shanghai Sangon Biotech Co., Ltd. The purification method was PAGE. The primers were constructed on the pGADT7-1 vector (the vector obtained by removing the AD domain from pGADT7). The primer sequences are as follows, where the underlined parts are homologous arms.

[0114] The AD-T1-cpLOV2 used to construct the yeast screening system.

[0115]

[0116]

[0117] 3) Using the KOD One PCR Master Mix kit, add the following components to a 200 μL PCR tube to prepare a 40 μL reaction system.

[0118]

[0119] 4) Set up the PCR amplification reaction program according to the KOD One PCR Master Mix kit instructions.

[0120] 5) Perform agarose gel electrophoresis on the PCR amplification products and detect the target band using a UV gel imaging system. Cut the target band from the gel and recover the target fragment.

[0121] 2.2 Enzyme digestion and recovery of empty vector

[0122] 1) pGADT7-1 was double-digested with restriction endonucleases EcoRI and NdeI. The following components were added to 200 μL PCR tubes to prepare 50 μL reaction systems.

[0123]

[0124] Mix the above reaction system thoroughly, and then place the reaction system in a 37℃ incubator for 30 minutes to complete the linearization of the plasmid.

[0125] 2) The linearized plasmid was analyzed by agarose gel electrophoresis, and the target band was detected using a UV gel imaging system. The target band was then cut into gel fragments, and the target fragment was recovered.

[0126] 2.3 Ligation and transformation of vector and target gene

[0127] The recovered target gene and the linearized vector product were recombined using the ClonExpressII One Step Cloning Kit from Novizan. Specific procedures were followed according to the kit instructions. The ligation product was transformed into DH5α *E. coli*, cultured overnight, and single clones were picked and sequenced to obtain the vector pGADT7-1-T1-cpLOV2.

[0128] The construction methods for other vectors are the same as those described above.

[0129] Example 2: In vitro phosphorylation verification of the light controllability of LCOST1.5

[0130] After successfully constructing GST-LCOST1.5, it was expressed in prokaryotes. The obtained GST-LCOST1.5 protein was then subjected to in vitro phosphorylation experiments using blue light and darkness to verify the light-controlled properties of LCOST1.5.

[0131] First, GST-LCOST1.5 was inactivated by treating it under blue light for 60 min. Then, the protein was added to the following in vitro phosphorylation reaction mixture (10 μL):

[0132]

[0133] After mixing by blowing and suction, they were placed under blue light (488nm, 50μmol m) respectively. -2 s -1 The mixture was incubated in the dark at room temperature for 30 min, followed by the addition of 0.5 μL of PNBM (50 mM) and mixed by pipetting. The reaction was then continued at room temperature for another 2.5–3 h. After the reaction was complete, 10 μL of 2× loading buffer was added, and the mixture was heated at 99 °C for 5 min before Western blot analysis. The results are as follows: Figure 12 As shown in Figure A, the experimental results indicate that LCOST1.5 does not autophosphorylate under blue light, but can autophosphorylate and phosphorylate the substrate under darkness.

[0134] To further verify the photocontrollability of LCOST1.5, LCOST1.5 was inactivated by treating it under blue light for 30 min. The reaction solution was then mixed using the method described above, and then placed under blue light of different intensities (0, 1, 5, 10, 25, 50 and 85 μmol m). - 2 s -1 After treatment, add 0.5 μL of PNBM (50 mM) and mix by purge. Continue the reaction at room temperature for 2.5–3 h. After the reaction is complete, add 10 μL of 2× loading buffer, heat at 99 °C for 5 min, and then perform Western blot. The results are as follows: Figure 12 As shown in Figure B, the results indicate that the intensity of autophosphorylation and substrate phosphorylation of LCOST1.5 decreases with increasing light intensity, and the weak blue light (1 μmol m -2 s -1 This significantly reduces the autophosphorylation of LCOST1.5, indicating that the activity of LCOST1.5 protein is controlled by blue light, and it is activated in the dark and inactivated under blue light.

[0135] Example 3: Genetic transformation of LCOST1.5 Arabidopsis thaliana

[0136] 1) Construction of plant overexpression vectors

[0137] Primers for amplifying the LCOST1.5 sequence were designed and synthesized by Shanghai Sangon Biotech Co., Ltd., and purified by PAGE. The primer sequences are shown below, with underlined homologous arms. pCAMBIA-KST1-LCOST1 and pCAMBIA-KST1-GFP were used to construct the plant guard cell expression vector, with pCAMBIA-KST1-GFP serving as a negative control. The KST1 sequence is shown in SEQ ID NO.15, and the vector construction method is the same as in "Example 1".

[0138]

[0139] 2) Arabidopsis genetic transformation

[0140] The successfully constructed vector was transformed into Agrobacterium GV3101, and transgenic Arabidopsis was constructed using the pollen infection method. The specific steps are as follows: a. Agrobacterium preparation: Positive Agrobacterium was streaked on solid LB medium (containing rifampin + kanamycin + gentamicin) and cultured at 28°C for 2-3 days. Clones from the plates were picked and inoculated into 2 mL of LB liquid medium (containing rifampin + kanamycin + gentamicin) and cultured overnight at 28°C. 1 mL of the overnight bacteria was transferred to 100 mL of LB liquid medium containing antibiotics for expansion culture. The culture was continued until the bacterial culture turned orange-yellow (OD). 600When approximately 2), centrifuge at 5000 rpm and discard the supernatant; b. Preparation of infection solution: 2.15 g MS salt, 50 g sucrose, add 1 L ddH2O, dissolve, then add 400 μL silwet-77, shake well, and adjust pH to 5.7-5.8. c. Infection of fluff: Resuspend the Agrobacterium precipitate using the above infection solution, and adjust OD... 600 The concentration was around 0.8. Select Arabidopsis thaliana plants in good flowering condition, cut off the existing pods, immerse the flower buds in the dye solution for 1 min 30 s, and then culture them in the dark for 24 hours before normal culture.

[0141] 3) Identification of transgenic Arabidopsis thaliana

[0142] a. The collected T0 generation seeds were placed in 1 / 2 MS medium containing hygromycin for screening positive seedlings. Positive seedlings with developed roots were then transferred to soil for culture. After 2-3 weeks, laser confocal microscopy was used to observe whether LCOST1.5 was specifically expressed in guard cells. The results were as follows: Figure 13 As shown in Figure A, LCOST1.5 is indeed specifically expressed in guard cells.

[0143] b. To determine the expression level, qRT-PCR was used to analyze the expression level of the LCOST1.5 gene in positive seedlings. The qRT-PCR was performed using the Vazyme Taq Pro Universal SYBR qPCR Master Mix, with the Arabidopsis housekeeping gene ACTIN2 / 8 as an internal control. Primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and purified by PAGE. The primer sequences are as follows:

[0144]

[0145] The results are as follows Figure 13 As shown in B, with the expression level of endogenous OST1 as 1, the expression levels of the KST1:LCOST1.5 lines were higher than those of the control, with expression levels exceeding 5.6-fold and 10.5-fold, indicating that these two lines are independent LCOST1.5 overexpression transgenic lines.

[0146] Example 4: LCOST1.5 increases Arabidopsis biomass.

[0147] 1) Determination of leaf area of ​​KST1:LCOST1.5 transgenic Arabidopsis thaliana

[0148] Leaf area was measured in Arabidopsis thaliana plants at 4 weeks of age. The method involved selecting two independent lines, photographing the plant with a ruler placed alongside the Arabidopsis thaliana, and then measuring the leaf area using ImageJ software. The results are shown below. Figure 13 As shown in C and 13D. The data indicate that the leaf area of ​​both transgenic lines was significantly higher than that of the control.

[0149] 2) Determination of fresh and dry weight of KST1:LCOST1.5 transgenic Arabidopsis thaliana

[0150] Fresh weight of Arabidopsis thaliana plants at 4 weeks of age was measured. The method involved selecting two independent lines, cutting the above-ground parts of the Arabidopsis thaliana with scissors, weighing them using an electronic balance, and recording the weight as the fresh weight. Fresh leaves were then dried in an 80℃ oven for 2-3 days, and weighed using an electronic balance; this was the dry weight. Results are as follows: Figure 13 As shown in E and 13F, the fresh weight and dry weight of the two transgenic lines were significantly higher than those of the control.

[0151] Example 5: LCOST1.5 Regulation of Stomatal Movement

[0152] Arabidopsis thaliana epidermal strips grown for 3-4 weeks were used as experimental samples. Stomatal aperture of the leaf epidermal strips was used as the evaluation index. The epidermal strips were placed in stomatal buffer (50 mM KCl + 10 mM MES + 50 μM CaCl2, pH 6.15) and then exposed to light (150 μmol / L). -2 s -1 After 2 hours of treatment, the opening of stomata was observed and recorded under a microscope. The epidermal strips were then placed in the dark for another 2 hours. The results are as follows: Figure 14 As shown in Figure A, the stomatal aperture of KST1:LCOST1.5 was greater than that of the control after 2 hours of light exposure, but smaller than that of the control after 2 hours of darkness.

[0153] To further clarify the results of the above experiments, the inventors further verified the changes in stomatal conductance using a photosynthesis apparatus, as detailed below:

[0154] Using Arabidopsis thaliana plants grown for 4-5 weeks as samples, and stomatal conductance as the evaluation index, the stomata were subjected to alternating light and dark conditions, and the continuous changes in stomatal activity were recorded. The results are as follows: Figure 14 As shown in B and 14C, it can be seen that during the dark-to-light transition, the stomatal conductance of KST1:LCOST1.5 increases with light intensity (150 μmol m). -2 s -1 With increasing time, stomatal conductance gradually increased and gradually exceeded that of the control. Furthermore, the slope of KST1:LCOST1.5 was greater than that of the control, indicating that its stomatal opening speed was faster than the control. During the light-dark transition, the stomatal conductance of KST1:LCOST1.5 was lower than that of the control in darkness, but its closing speed was not significantly different from the control. These results indicate that LCOST1.5, after being specifically expressed on guard cells, can regulate stomatal movement.

[0155] To further verify that stomatal movement influenced by LCOST1.5 is caused by ABA signaling controlled by LCOST1.5, we performed qRT-PCR to detect the expression levels of ABA signaling marker genes in guard cells. Details are as follows:

[0156] Epidermal strips were harvested from 4-5 week old Arabidopsis thaliana plants. The strips were placed in stomatal buffer and treated under light and darkness for 2-3 hours each. The treated strips were then collected, and total RNA was extracted. qRT-PCR primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and purified by PAGE. The primer sequences are as follows:

[0157]

[0158] The results are as follows Figure 15 As shown, RD29A expression was significantly increased under dark conditions and decreased under light conditions, indicating that ABA signaling in guard cells is activated in the dark and blocked under light. Therefore, the stomatal movement changes regulated by LCOST1.5 are caused by LCOST1.5-regulated ABA signaling.

[0159] Example 6: LCOST 1.5 increases the biomass of cultivated tobacco.

[0160] 1) Genetic transformation of tobacco

[0161] The genetic transformation method for tobacco was the leaf disc method, in which the plasmid pCAMBIA-KST1-LCOST1.5 was obtained by transforming callus tissue with Agrobacterium. The specific method is as follows:

[0162] a. Cultivation of sterile tobacco seedlings: Take an appropriate amount of K326 tobacco seeds, disinfect them with 84 disinfectant for 25 minutes, rinse them 5-6 times with sterile water, add water and place the seeds in a 4℃ refrigerator for 2-3 days in the dark, then sprinkle the seeds on 1 / 2 MS solid medium (2.215g / L MS + 30g / L sucrose + 8g / L agar powder) and place them in an incubator at 25℃ with 16h light / 8h darkness for about 10 days.

[0163] b. Agrobacterium activation: First, take the bacterial culture out of the -80℃ freezer, streak it on LB medium (rifampin + kanamycin + gentamicin) and activate it for 2-3 days. Pick the clones on the plate and inoculate them into 2mL LB liquid (rifampin + kanamycin + gentamicin) medium and incubate overnight at 28℃.

[0164] c. Preparation of Agrobacterium infection medium: Centrifuge the overnight cultured Agrobacterium at 5000 rpm for 5 min at room temperature, discard the supernatant, resuspend the precipitate in infection medium (2.215 g / L MS + 30 g / L sucrose + 100 μM acetylsylgenone, pH 5.3), centrifuge again at 5000 rpm for 5 min at room temperature, discard the supernatant, resuspend the precipitate in infection medium, and adjust OD... 600 It is 0.01.

[0165] d. Infection and co-culture: Take out the cultivated sterile tobacco seedlings from the laminar flow hood, cut a wound in the seedling, and place the leaves in the Agrobacterium infection solution, so that the leaves are completely immersed in the liquid for about 10-15 minutes. Remove the infected leaves with autoclaved forceps and place them on sterile absorbent paper to absorb the excess liquid. Then place sterilized filter paper on the co-culture medium (4.43 g / L MS + 0.5 mg / L IAA + 2 mg / L 6-BA + 8 g / L agar powder, pH 5.3), and then place the leaves on the filter paper. Use forceps to press the wound part of the leaves tightly against the filter paper and incubate in the dark at 25°C for 3 days.

[0166] e. Selection and Rooting Culture: After co-culture, the leaves were transferred to a selection medium (4.43 g / L MS + 0.5 mg / L IAA + 2 mg / L 6-BA + 100 mg / L termethin + 50 mg / L hygromycin, pH 5.8) and placed in a 25°C incubator with a light intensity of 150 μmol / L. -2 s -1 The plants were cultured continuously under 16 hours of light and 8 hours of darkness, with the culture medium changed weekly, until resistant shoots emerged from the callus tissue that differentiated from the leaves. Once the shoots had grown, they were cut off and inserted into rooting medium (2.215 g / L MS + 30 g / L sucrose + 100 mg / L termethin + 50 mg / L hygromycin, pH 5.8) for rooting culture. After roots had grown, they were transferred to individual tissue culture bottles to continue growing. After hardening off for 3-5 days, they were transplanted into soil for further culture.

[0167] 2) Identification of genetically modified tobacco

[0168] a. After the tobacco plants were transplanted to the soil for 1-2 weeks, the leaves were removed, and laser confocal microscopy was used to observe whether LCOST1.5 was specifically expressed in guard cells. The results were as follows: Figure 16 As shown in Figure A, LCOST1.5 is indeed specifically expressed in guard cells.

[0169] b. To determine the expression level, RT-PCR was used to analyze the expression level of LCOST1.5 in positive seedlings. The tobacco housekeeping gene ACTIN was used as an internal control gene. Primers for ACTIN were synthesized by Shanghai Sangon Biotech Co., Ltd., and purified by PAGE. The primer sequences are as follows:

[0170]

[0171] The results are as follows Figure 16 As shown in Figure B, no LCOST1.5 band was observed in the negative control K326, and the expression levels of the KST1:LCOST1.5 lines were higher than those of the control, indicating that these two lines are independent transgenic lines that overexpress LCOST1.5.

[0172] 3) Determination of leaf area of ​​KST1:LCOST1.5 transgenic tobacco

[0173] Leaf area was measured in tobacco plants after 4 weeks. The measurement method was the same as in "Example 5". The results are as follows: Figure 16 As shown in C and 16D, the leaf area of ​​the transgenic tobacco was significantly larger than that of the control.

[0174] 4) Determination of fresh and dry weight of KST1:LCOST1.5 transgenic tobacco

[0175] Fresh weight of Arabidopsis thaliana was measured at 4 weeks of age. The measurement method was the same as in "Example 5". The results are as follows: Figure 16 As shown in E and 16F, the fresh weight and dry weight of the two transgenic lines were significantly higher than those of the control. These results indicate that LCOST1.5 expression in tobacco guard cells significantly increases tobacco biomass.

[0176] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A fusion protein, characterized in that, The fusion protein includes at least a protein kinase OST1 in the ABA signal transduction component, and an optogenetic element cpLOV2 linked to the protein kinase OST1; wherein the protein kinase OST1 is located at the N-terminus of the fusion protein, and the cpLOV2 is located at the C-terminus of the fusion protein.

2. The fusion protein as described in claim 1, characterized in that, The fusion protein is any one of the following (a1)-(a3): (a1) The amino acid sequence shown in any one of SEQ ID NO. 1-5; (a2) A protein derived from (a1) by substitution and / or deletion and / or addition of one or more amino acid residues and having the same function; (a3) Other genes encode proteins that have at least 80% (including 80%, 85%, 90%, 95%, 99%) the same amino acid sequence composition as shown in (a1) and have the same or similar activity as the fusion protein shown in (a1).

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule can encode the fusion protein as described in claim 1 or 2.

4. The nucleic acid molecule as described in claim 3, characterized in that, The nucleic acid molecule has any of the nucleotide sequences described in (b1)-(b4): (b1) A nucleotide sequence as shown in any of SEQ ID NO. 6-10; (b2) A sequence formed by replacing, deleting or inserting one or more nucleotides as shown in (b1); (b3) has at least 80% (including 80%, 85%, 90%, 95%, 99%) identity with the nucleotide sequence defined in (b1) or (b2) and encodes the same or similar functional protein. (b4) A nucleotide sequence complementary to any of the nucleotide sequences described in (b1)-(b3).

5. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid molecule described in claim 3 or 4.

6. A host cell, characterized in that, The host cell contains the nucleic acid molecule of claim 3 or 4, the recombinant expression vector of claim 5, or is capable of expressing the fusion protein of claim 1 or 2; Furthermore, the host cell includes bacterial cells, fungal cells, or plant cells.

7. The use of the fusion protein of claim 1 or 2, the nucleic acid molecule of claim 3 or 4, the recombinant expression vector of claim 5, and / or the host cell of claim 6 in any one or more of the following: (1) Regulate the plant’s endogenous ABA signaling pathway; (2) Regulating plant stomatal movement; (3) Increase plant biomass.

8. The application as described in claim 7, characterized in that, The plant is a monocotyledonous or dicotyledonous plant; further, the plant is a crop, including food crops and cash crops.

9. A method for increasing plant biomass, characterized in that, The method includes: transferring the nucleic acid molecule of claim 3 or 4 into the plant for expression; Furthermore, the nucleic acid molecules are expressed in plant guard cells.

10. The method as described in claim 9, characterized in that, The increase in plant biomass is specifically manifested in at least the following ways: increasing the leaf area of ​​the plant, and increasing the fresh weight and dry weight of the plant line.

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