Application of circular vector expressing α-TTP in improving rice resistance to high light stress

Abstract

The present invention discloses the application of a circular vector expressing α-TTP in improving the resistance of rice to high light stress. The present invention relates to the field of biotechnology. The present invention provides a circular vector, comprising expression cassette 1 (a plant constitutive promoter, a signal peptide coding sequence for locating plant chloroplasts, a coding gene for α-tocopherol transfer protein, and a plant termination sequence), expression cassette 2 (a plant high light inducible promoter, a signal peptide coding sequence for locating plant thylakoid membranes, a coding gene for PI4KB protein, and a plant termination sequence) and expression cassette 3 (a plant high light inducible promoter, a signal peptide coding sequence for locating plant thylakoid membranes, a coding gene for PIP5KIA3 protein, and a plant termination sequence). The present invention has great application potential in the fields of breeding plant high light resistant varieties, improving the light energy utilization efficiency of plants, and increasing crop yields.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of a circular vector expressing an alpha-tocopherol transport module TTP in improving the high light stress resistance of rice. Background Art

[0002] In nature, plants are inevitably exposed to high light radiation. Under the action of strong light, chlorophyll (Chl) is activated and converted to a single excited state. 1 Chl, single excited state 1 Chl is converted to a triple excited state within the system 3 Chl. Triple excited state 3 Chl single excited state 1 Chl exists longer and can also react with molecular oxygen to produce singlet oxygen 1 O2, singlet oxygen 1 O2 has the greatest destructive effect on the photosystem, which can damage lipids, key pigment cofactors, and D1 protein subunits in PSⅡ, ultimately leading to the inactivation of the PSⅡ reaction center and severely inhibiting photosynthesis. Therefore, how to protect the photosystem of plants so that they can efficiently perform photosynthesis under high light stress is an urgent problem to be solved.

[0003] In plants, tocopherols are localized on membrane structures such as chloroplast membranes, thylakoid membranes, and plastid globule membranes. Studies have shown that the aromatic ring head of tocopherols is located on the outside of the hydrophilic layer of the lipid bimolecule of the biological membrane, while the phytyl tail is located on the hydrophobic interior of the membrane. α-Tocopherols can be used to generate a singlet oxygen by donating an electron. 1 O2, forming a charge transfer excitation complex, and finally forming free α-tocopherol and molecular oxygen, leading to singlet oxygen 1 O2 inactivates and thus protects membrane lipids and photosystems.

[0004] α-Tocopherol transfer protein (α-TTP) is the only protein known so far that specifically recognizes α-tocopherol (α-Toc), the most abundant and biologically active form of vitamin E in higher animals. α-TTP is highly expressed in the liver, where it selects α-Toc from vitamin E forms taken up by plasma lipoproteins and promotes its secretion into circulating lipoproteins. Therefore, α-TTP is a major determinant of plasma α-Toc concentrations. In hepatocytes, α-TTP catalyzes the transport of α-Toc from endocytic compartments to the plasma membrane by targeting phosphatidylinositol phosphates (PIPs) such as PI(4,5)P2. However, to date, no proteins with tocopherol-binding activity have been found in plants. Summary of the invention

[0005] The purpose of the present invention is to provide an application of a circular vector expressing an alpha-tocopherol transport module TTP in improving the ability of rice to resist high light stress.

[0006] In a first aspect, the present invention claims a ring-shaped carrier.

[0007] The circular vector claimed in the present invention comprises the following three expression cassettes:

[0008] Expression cassette 1: From the 5' end to the 3' end, it includes a constitutive promoter capable of initiating gene transcription in plants, a signal peptide coding sequence capable of localizing to plant chloroplasts, a gene encoding α-tocopherol transfer protein (α-TTP), and a termination sequence capable of terminating gene transcription in plants.

[0009] Expression cassette 2: From the 5' end to the 3' end, it includes a high light-inducible promoter capable of initiating gene transcription in plants, a signal peptide coding sequence capable of localizing plant thylakoid membranes, a coding gene for PI4KB protein, and a termination sequence capable of terminating gene transcription in plants.

[0010] Expression cassette 3: From the 5' end to the 3' end, it includes a high light-inducible promoter capable of initiating gene transcription in plants, a signal peptide coding sequence capable of localizing plant thylakoid membranes, a gene encoding PIP5KIA3 protein, and a termination sequence capable of terminating gene transcription in plants.

[0011] Furthermore, the expression cassette 1 is composed of, from the 5' end to the 3' end, the constitutive promoter capable of initiating gene transcription in plants, the signal peptide coding sequence capable of localizing plant chloroplasts, the coding gene of α-tocopherol transfer protein (α-TTP), and the termination sequence capable of terminating gene transcription in plants.

[0012] Furthermore, the expression cassette 2 is composed, from the 5' end to the 3' end, of the high light-inducible promoter capable of initiating gene transcription in plants, the signal peptide coding sequence capable of localizing the plant thylakoid membrane, the coding sequence of the (GGGGS)3 connecting peptide, the coding gene of the PI4KB protein, and the termination sequence capable of terminating gene transcription in plants.

[0013] Furthermore, the expression cassette 3 is composed of, from the 5' end to the 3' end, the high light-inducible promoter capable of initiating gene transcription in plants, the signal peptide coding sequence capable of localizing the plant thylakoid membrane, the coding sequence of the (GGGGS)3 connecting peptide, the coding gene of the PIP5KIA3 protein, and the termination sequence capable of terminating gene transcription in plants.

[0014] Furthermore, in the expression cassette 1, the constitutive promoter may be a 35S promoter; the signal peptide may be an rCTP signal peptide; and the termination sequence may be a Tnos terminator.

[0015] Furthermore, in the expression cassette 2, the high light inducible promoter may be the ELIP2 promoter; the signal peptide may be the first 250 amino acids of the Arabidopsis thaliana thylakoid membrane protein ALB3; and the termination sequence may be the Tags terminator.

[0016] Furthermore, in the expression cassette 3, the high light inducible promoter may be the ELIP1 promoter; the signal peptide may be the first 250 amino acids of the Arabidopsis thaliana thylakoid membrane protein ALB3; and the termination sequence may be the Tmas terminator.

[0017] More specifically, the sequence of the rCTP signal peptide is shown in SEQ ID No.4; the sequence of the α-tocopherol transfer protein is shown in SEQ ID No.5; the sequence of the first 250 amino acids of the Arabidopsis thaliana thylakoid membrane protein ALB3 is shown in SEQ ID No.6; the sequence of the PI4KB protein is shown in SEQ ID No.7; the sequence of the PIP5KIA3 protein is shown in SEQ ID No.8; the amino acid sequence of the (GGGGS)3 connecting peptide is shown in SEQ ID No.9.

[0018] In the expression cassette 1, the sequence of the 35S promoter is shown in positions 1-677 of SEQ ID No.1; the coding sequence of the rCTP signal peptide is shown in positions 678-842 of SEQ ID No.1; the sequence of the coding gene of the α-tocopherol transfer protein is shown in positions 843-1679 of SEQ ID No.1; and the sequence of the Tnos terminator is shown in positions 1680-1934 of SEQ ID No.1.

[0019] In the expression cassette 2, the sequence of the ELIP2 promoter is shown as positions 1-2000 of SEQ ID No.2; the coding sequence of the first 250 amino acids of the Arabidopsis thaliana thylakoid membrane protein ALB3 is shown as positions 2001-2750 of SEQ ID No.2; the coding sequence of the (GGGGS)3 connecting peptide is shown as positions 2751-2798 of SEQ ID No.2; the sequence of the gene encoding the PI4KB protein is shown as positions 2799-5204 of SEQ ID No.2; and the sequence of the Tags terminator is shown as positions 5205-5610 of SEQ ID No.2.

[0020] In the expression cassette 3, the sequence of the ELIP1 promoter is shown as positions 1-2085 of SEQ ID No.3; the coding sequence of the first 250 amino acids of the Arabidopsis thaliana thylakoid membrane protein ALB3 is shown as positions 2086-2835 of SEQ ID No.3; the coding sequence of the (GGGGS)3 connecting peptide is shown as positions 2836-2884 of SEQ ID No.3; the sequence of the gene encoding the PIP5KIA3 protein is shown as positions 2885-4386 of SEQ ID No.3; and the sequence of the Tmas terminator is shown as positions 4387-4639 of SEQ ID No.3.

[0021] In an embodiment of the present invention, the sequence of the expression cassette 1 is shown as SEQ ID No.1; the sequence of the expression cassette 2 is shown as SEQ ID No.2; and the sequence of the expression cassette 3 is shown as SEQ ID No.3.

[0022] In one embodiment of the present invention, the circular vector is obtained by integrating the expression cassette 1, the expression cassette 2 and the expression cassette 3 into the pYLTAC380H expression vector of the TransGene Stacking II vector system. On the circular vector, the fragment located between the left border (LB) of the insertion sequence T-DNA and the right border (RB) of the insertion sequence T-DNA is, from the 5' end to the 3' end, the following: hygromycin resistance gene expression frame, loxP site, NotI restriction site, the aforementioned expression frame 3, NotI restriction site, NotI restriction site, the aforementioned expression frame 2, NotI restriction site, NotI restriction site, the aforementioned expression frame 1, NotI restriction site.

[0023] In a second aspect, the present invention claims protection for any of the following biological materials:

[0024] (A1) a set of expression cassettes, consisting of the expression cassette 1, the expression cassette 2 and the expression cassette 3 in the first aspect above;

[0025] (A2) A recombinant bacterium containing the circular vector described in the first aspect or the expression cassette described in (A1) above.

[0026] In a third aspect, the present invention claims the use of the annular carrier described in the first aspect or the biomaterial described in the second aspect in any of the following:

[0027] (B1) Improve the ability of plants to resist high light stress;

[0028] (B2) Improve plant yield under high light stress;

[0029] (B3) Increase the aboveground dry weight of plants under high light stress;

[0030] (B4) Increase plant yield under high light stress.

[0031] In a fourth aspect, the present invention claims protection for any of the following methods:

[0032] Method I: A method for improving the ability of plants to resist high light stress, comprising the following steps: introducing the circular vector described in the first aspect above into a recipient plant to obtain a transgenic plant; the transgenic plant has improved resistance to high light stress compared with the recipient plant.

[0033] Method II: A method for increasing plant yield under high light stress, comprising the following steps: introducing the circular vector described in the first aspect above into a recipient plant to obtain a transgenic plant; the yield of the transgenic plant under high light stress is increased compared with the recipient plant.

[0034] In the above-mentioned related aspects, the high light refers to light intensity greater than the suitable light intensity for the plant. In one embodiment of the present invention, the high light intensity is 1200 μmol·m -2 s -1 .

[0035] In the above-mentioned related aspects, the increasing of plant yield may be embodied as: increasing the dry weight of the above-ground part of the plant, and / or increasing the yield per plant.

[0036] In the present invention, the plant may be any of the following:

[0037] (C1) monocots;

[0038] (C2) Gramineae;

[0039] (C3) Oryza plants;

[0040] (C4) Rice.

[0041] In a specific implementation case of the present invention, the rice is the rice variety Zhonghua 11.

[0042] The present invention establishes a new α-tocopherol transport pathway from the inner membrane to the thylakoid membrane. This branch is activated under high light stress and increases the α-Toc content in the thylakoid membrane in a directional manner, thereby improving the clearance of 1O2 capacity, enhance the plant light protection ability. For the two kinase proteins, phosphatidylinositol 4-kinase (PI4KB) and phosphatidylinositol 4-monophosphate 5-kinase (PIP5KIA3K), which are necessary for the synthesis of phosphatidylinositol (4,5)-bisphosphate [PI(4,5) P2], different high-light-inducible promoters are selected and fused with the first 250 amino acids of the thylakoid membrane protein ALB3 to specifically locate them on the outside of the thylakoid membrane; at the same time, by co-expressing the chloroplast transit peptide and α-tocopherol transfer protein, the expression and localization of an α-tocopherol transfer protein (α-TTP) in rice chloroplasts are regulated. Based on the above α-tocopherol transport pathway-related genes, gene circuits are designed, with different high-light-inducible promoters, leader peptides, and terminators. The above modules are assembled by multi-gene expression cassette technology and then genetically transformed. It is induced and controllably expressed in rice chloroplasts to maintain the effective operation of photosynthesis under high light stress of rice, thereby promoting the increase of dry weight of the aboveground part of rice and yield per plant. The invention has great application potential in the fields of breeding plant varieties resistant to high light, improving the light energy utilization efficiency of plants, increasing crop yields, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Some of the candidate genes selected by the present invention are high light (1200 μmol·m -2 s -1 ) before and after treatment. A is the expression change of ELIP2 gene before and after high light treatment; B is the expression change of APX2 gene before and after high light treatment; C is the expression change of ELIP1 gene before and after high light treatment; D is the expression change of SEP1 gene before and after high light treatment. Among them, the expression levels of ELIP1 and ELIP2 genes continued to increase with the increase of high light treatment time.

[0044] Figure 2 The transient transformation experiment of the expression vectors for α-TTP, PI4KB, PIP5KIA3 and GFP fusion proteins in rice protoplasts showed that the fluorescence signals of the three fusion proteins all appeared in chloroplasts.

[0045] Figure 3 The identification results of the recombinant expression vector and transgenic positive seedlings. A is the result of agarose gel electrophoresis enzyme digestion detection of the recombinant expression vector. The three complete gene expression frames all appear at the correct band position (indicated by the arrows), and the recombinant expression vector is successfully constructed; B is the identification result of the transgenic positive seedlings. Lanes 1-23 are positive seedlings that have been successfully transformed, and lane 24 is the wild-type negative control.

[0046] Figure 4 The results of rice transformation with recombinant expression vector and positive plant identification. A is the positive rice plant under high light treatment (1200 μmol·m-2 s -1 ) After 2 hours, the expression levels of PI4KB and PIP5KIA3 genes were significantly upregulated compared with 0 hours; B shows that compared with the wild type, the aboveground dry weight of the transgenic line increased significantly; C shows that compared with the wild type, the yield per plant of the transgenic line increased significantly. In the figure, HL 2h means high light treatment for 2 hours; * means P<0.05 compared with the wild type; ** means P<0.01 compared with the wild type. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0048] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0049] Example 1: Screening of high light-inducible promoters

[0050] The experimental materials used in this application are Arabidopsis thaliana (Col-0) grown for three weeks. The plants with good growth status and at the same period were placed under high light conditions of LED light source (1200 μmol·m -2 s -1 ) RNA was extracted from leaf samples treated for 0h, 0.5h, 1h, 2h, 3h, 6h, 12h, and 24h.

[0051] The RNA extraction method used in this experiment is the Trizol one-step method. The specific steps are as follows:

[0052] ① Add two small steel beads treated at 180℃ to a 1.5mL centrifuge tube treated with DEPC, take 50mg-100mg of Arabidopsis leaf tissue, freeze it with liquid nitrogen, and quickly crush it with a sample grinder.

[0053] ② Add 1 ml of Trizol and place at room temperature for 5 minutes, shaking gently to completely dissolve the cells.

[0054] ③Add 200 μL of chloroform, mix by inversion for 15-30 seconds, leave at room temperature for 10-15 minutes, and centrifuge at 4°C and 12,000 g for 15-20 minutes.

[0055] ④ Transfer the aqueous phase to a new centrifuge tube that has been pre-treated with DEPC, add 0.5 ml of isopropanol, shake well to mix, and place at -20°C for 10 min.

[0056] ⑤ Centrifuge at 4°C and 12,000 g for 15-20 minutes. The gel-like precipitate is RNA.

[0057] ⑥Discard the supernatant and add 500 μL of anhydrous ethanol to remove DNA.

[0058] ⑦ Wash with 500 μL of 7% (volume percentage) ethanol (prepared with DEPC-treated water), shake to mix, and centrifuge at 4°C and 12,000 g for 5 min.

[0059] ⑧Discard the supernatant, centrifuge briefly, drain the liquid in the centrifuge tube, and place it at room temperature for 5-10 minutes until it is dry.

[0060] ⑨Add 50 μL of DEPC-treated ddH2O and store in a -80℃ refrigerator.

[0061] The reverse transcription kit TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix provided by Quanshijin Biotechnology Co., Ltd. was used to reverse transcribe RNA into cDNA and store it at -80°C. The cDNA reverse transcribed from RNA is a double-stranded stable structure, which is a sequence with no introns but only exons. The cDNA library specifically reflects the coding genes of proteins expressed in a certain tissue or cell at a specific developmental stage. Therefore, the cDNA library has tissue or cell specificity, and it is relatively easy to screen and clone genes specifically expressed by cells.

[0062] qRT-PCR primers were designed based on the gene sequences in the Arabidopsis reference genome and compared on the BLAST-Search website to determine the specificity of the primers, and the GC content of the designed primer sequences was controlled at 40%-60%. The cDNAs of the 8 leaf samples before and after high light treatment were used as templates for subsequent qRT-PCR of the screened genes and the Actin reference gene. The PerfectStart® Green qPCR SuperMix real-time fluorescence quantitative PCR kit provided by Quanshijin Biotechnology Co., Ltd. was used for qRT-PCR. The qPCR reaction system is shown in Table 1, and the primer sequences are as follows:

[0063] qRT-ELIP2-F: 5'-GCGATCCTATCAAGGAAGATCC-3' (SEQ ID No. 10);

[0064] qRT-ELIP2-R: 5'-ACTCACCTTAGGCTTGCTAAC-3' (SEQ ID No. 11);

[0065] qRT-ELIP1-F: 5'-ATTATCCGGTGGGAGTGAGA-3' (SEQ ID No. 12);

[0066] qRT-ELIP1-R: 5'-TTCATAGGAGGAGGAGGAGATG-3' (SEQ ID No. 13);

[0067] qRT-SEP1-F: 5'-GCTTCTGGTTCTCCTCCTTG-3' (SEQ ID No. 14);

[0068] qRT-SEP1-R: 5'-CCACTGCTTCCTTCTGTACTT-3' (SEQ ID No. 15);

[0069] qRT-APX2-F: 5'-GGAAGCTCCGTGGTCTTATT-3' (SEQ ID No. 16);

[0070] qRT-APX2-R: 5'-CTCCTGTCTTCGTCTTCACATC-3' (SEQ ID No. 17);

[0071] qRT-Actin2-F: 5'-GACCTTTAACTCTCCCGCTATG-3' (SEQ ID No. 18);

[0072] qRT-Actin2-R: 5'-GAGACACACCATCACCAGAAT-3' (SEQ ID No. 19).

[0073] Table 1. qPCR reaction system

[0074]

[0075] The amplification program was as follows: pre-denaturation at 94°C for 30 s; denaturation at 94°C for 5 s, extension at 60°C for 30 s, for 45 cycles.

[0076] By calculating the relative expression of qRT-PCR, the expression pattern of Arabidopsis high-light response genes before and after high-light treatment can be determined. Figure 1 As shown in FIG. 1 , it can be seen that the expression levels of some candidate genes screened by the present invention change before and after high light treatment, among which ELIP1, ELIP2 The gene expression level continued to increase with the increase of high light treatment time, and the increase was large. ELIP1, ELIP2 The region 2 kb upstream of the gene transcription start site is used as the gene promoter sequence (hereinafter referred to as ELIP1 Promoter and ELIP2 Promoter. Among them, ELIP2 The sequence of the promoter is shown in positions 1-2000 of SEQ ID No. 2, ELIP1 The sequence of the promoter is shown in positions 1-2085 of SEQ ID No. 3) and is used for the subsequent construction of a recombinant expression vector.

[0077] Example 2: Identification of gene expression patterns related to the α-tocopherol transport pathway

[0078] In order to introduce the α-tocopherol transport-related core protein into the chloroplast, the present invention attempts to add the chloroplast signal peptide sequence of the Arabidopsis thaliana encoding Rubisco small subunit gene RBCS1 before the α-TTP sequence to construct an expression 35S The promoter-driven vector of the fusion protein of α-TTP and GFP with chloroplast signal peptide. The specific operation is as follows:

[0079] The chloroplast signal peptide sequence (rCTP) and α-TTP sequence of the Rubisco small subunit gene RBCS1 were seamlessly connected to the pM999 vector preserved in the laboratory using the pEASY-Basic Seamless Cloning and Assembly Kit (CU201-03) from Quanshijin Company and the homologous recombinase 2×Basic Assembly Mix. The vector is recorded in the article “Yanlong Guan et al. Gene refashioning through innovative shifting of reading frames in mosses. NATURE COMMUNICATIONS, (2018) 9:1555”, which can be obtained by the public from the applicant and can only be used to repeat the experiments of the present invention and cannot be used for other purposes. The pM999 vector carries the GFP gene) Eco RI and XB I restriction site, and subsequently, based on the sequencing results, an expression vector with the correct gene element connection direction and correct sequence was screened and named pM999-rCTP-αTTP-GFP.

[0080] pM999-rCTP-αTTP-GFP vector structure description: pM999 vector (carrying GFP gene) restriction site Eco RI and XBThe recombinant plasmid is obtained by replacing the small fragment between I with the rCTP-αTTP fragment. The rCTP-αTTP fragment contains the rCTP coding sequence shown in the 678th to 842th positions of SEQ ID No.1 and the coding gene of α-TTP shown in the 843th to 1679th positions of SEQ ID No.1 from the 5' end to the 3' end.

[0081] The pM999-rCTP-αTTP-GFP vector transient expression vector was transformed into rice protoplasts, and the slices were prepared after dark culture for 13-16 hours, and the fluorescence expression was observed using a laser confocal microscope. The specific operation is as follows:

[0082] 1. Prepare enzymatic solution with the following formula: 1.5% (w / v) cellulase R10, 0.4% (w / v) cleavage enzyme R10, 20mM MES, 600mM mannitol, 20mM KCl. Prepare it for immediate use and prepare 0.6M mannitol at the same time.

[0083] 2. Pour the enzymatic solution into a clean culture dish of suitable size. Take out the yellowing seedlings of Zhonghua 11 rice that have been cultured in the dark for 10-15 days, immerse the leaf sheaths in 0.6M mannitol, and quickly cut the leaf sheaths into small segments less than 1mm with a sharp blade. The cut leaf sheaths are immediately transferred to the prepared enzymatic solution. Generally, the leaf sheaths of 50 yellowing seedlings can be enzymatically hydrolyzed with 10ml of enzymatic solution, and at least 5 plasmid combinations can be transformed. More transformations can expand the corresponding reaction system. After the leaf sheaths are cut, soak them in the enzymatic solution and vacuum for 10 minutes to make most of the leaf sheaths sink to the bottom of the enzymatic solution. Wrap the enzymatic solution with tin foil to avoid light, place it on a shaker at 28℃ and 50rpm, and enzymatically hydrolyze for 5 hours. After the enzymatic solution is completed, take a drop of enzymatic solution for microscopic examination. If the cells are round and shiny, they are healthy and continue to do the next step; if the cells are flat and black, discard them.

[0084] 3. Mix the enzymatic solution, filter it through a 150-mesh sieve into a new culture dish, and then transfer it to a 10 ml round-bottom centrifuge tube. Centrifuge at 100 g with an acceleration and deceleration of 2 for 10 min, and pre-cool MMG (formula: 4 mM MES, 600 mM mannitol, 15 mM MgCl2) solution.

[0085] 4. Use a 1ml pipette to remove the supernatant. You can see a lot of yellow-green precipitates at the bottom of the tube, which indicates healthy cells. If there is little precipitate or the precipitate is very white, the cell quality is poor. Slowly add 4ml W5 (formula: 2mM MES, 150mM NaCl, 125mM CaCl2, 5mM KCl) solution, mix gently, and then centrifuge at 100g speed, acceleration and deceleration acceleration of 2, for 10 minutes.

[0086] 5. Use a 1ml pipette to aspirate the supernatant, slowly add 4ml of pre-cooled MMG solution, and centrifuge for 10min at 4℃, 100g, with acceleration and deceleration of 2.

[0087] 6. Use a 1ml pipette to remove the supernatant, slowly add 2ml pre-cooled MMG solution, gently mix, and centrifuge for 10min at 4℃, 100g speed, acceleration and deceleration acceleration of 2. Calculate the required volume of protoplasts: 200μl×the number of carriers, and leave 500μl. Use a pipette to remove the supernatant, mix the protoplasts with the required volume of MMG solution, and place on ice for 30min.

[0088] 7. Preparation during the ice bath: dilute the plasmid to be transformed into 20μl, the amount of plasmid is 2-10μg, and add it to a 2ml round-bottom centrifuge tube. Change the centrifuge rotor to a 2ml small rotor and set the temperature to room temperature.

[0089] 8. Mix the protoplasts, add 200μl of protoplasts and 220μl of 40% PEG4000 solution (formula: 200 mM mannitol, 400g / L PEG4000, 100mM CaCl2) to a 2ml centrifuge tube (to which 20μl of plasmid has been added), and immediately mix by gently inverting upside down and timing. Leave at room temperature for 20 min.

[0090] 9. Add 1 ml of W5 solution to a 2 ml centrifuge tube, mix well, and centrifuge for 10 min at room temperature at 100 g with an acceleration and deceleration of 2. Carefully aspirate the supernatant with a 1 ml pipette, add 1.5 ml of W5, and incubate at 28°C in the dark for 14 h (not less than 8 h or more than 24 h).

[0091] 10. Before observation, centrifuge at room temperature, 100g, acceleration and deceleration of 2 for 10 minutes. Aspirate the upper liquid, leaving only 200μl cells at the bottom. Mix gently and observe the fluorescence expression using a laser confocal microscope.

[0092] In addition, the present invention also attempts to combine the two kinases PI4KB and PIP5KIA3 required for the synthesis of phosphatidylinositol phosphate PI(4,5)P2 with the first 250 amino acid sequences of Arabidopsis thaliana thylakoid membrane protein ALB3, and constructs vectors of fusion proteins of PI4KB, PIP5KIA3 and GFP with thylakoid membrane localization signal peptides driven by ELIP2 and ELIP1 high light inducible promoters, respectively. The specific operations are as follows:

[0093] use Hind III and Eco RI restriction endonuclease cuts off the pM999 vector (carrying the GFP gene) 35SPromoter, then use Quanshijin Company pEASY -Basic Seamless Cloning and Assembly Kit (CU201-03) uses homologous recombinase 2×Basic Assembly Mix to introduce ELIP2 and ELIP1 high light-inducible promoters into the pM999 vector (carrying the GFP gene) Hind III and Eco The 35S promoter carried by the vector was replaced between the RI restriction sites. Then the nucleotide sequence of the first 250 amino acids of the Arabidopsis thaliana thylakoid membrane protein ALB3 and the PI4KB / PIP5KIA3 sequence were seamlessly connected to the pM999 vector (carrying the GFP gene). Eco RI and XB I restriction site, and then based on the sequencing results, the expression vector with the correct gene element connection direction and correct sequence was screened and named pELIP2::pM999-ALB3 according to the different structural compositions. 1-250aa -PI4KB-GFP, pELIP1::pM999-ALB3 1-250aa -PIP5KIA3-GFP.

[0094] pELIP2::pM999-ALB3 1-250aa -PI4KB-GFP vector structure description: pM999 vector (carrying GFP gene) restriction site Hind III and Eco Between RI 35S The promoter was replaced with the pELIP2 promoter (positions 1-2000 of SEQ ID No. 2), and Eco R I and XB The small fragment between I is replaced by ALB3 1-250aa -PI4KB fragments to obtain the recombinant plasmid. 1-250aa -PI4KB fragment from 5' to 3' end is ALB3 shown in 2001-2750 of SEQ ID No.2 1-250aa the coding gene of , the coding sequence of the (GGGGS)3 connecting peptide shown at positions 2751-27980 of SEQ ID No.2, and the coding gene of PI4KB shown at positions 2799-5204 of SEQ ID No.2.

[0095] pELIP1::pM999-ALB3 1-250aa - Description of the structure of PIP5KIA3-GFP vector: The restriction site of pM999 vector (carrying GFP gene) Hind III and Eco Between RI 35S The promoter was replaced with the pELIP1 promoter (positions 1-2085 of SEQ ID No. 3), and Eco RI and XB The small fragment between I is replaced by ALB3 1-250aa -PIP5KIA3 fragments to obtain the recombinant plasmid. 1-250aa -PIP5KIA3 fragments are ALB3 shown in positions 2086-2835 of SEQ ID No. 3 from 5' to 3' end. 1-250aa the coding gene of , the coding sequence of the (GGGGS)3 connecting peptide shown at positions 2836-2884 of SEQ ID No.3, and the coding gene of PIP5KIA3 shown at positions 2885-4386 of SEQ ID No.3.

[0096] pELIP2::pM999-ALB3 1-250aa -PI4KB-GFP vector and pELIP1::pM999-ALB3 1-250aa -PIP5KIA3-GFP vectors were used to transform rice protoplasts, and the samples were prepared after culturing in the dark for 14 h. The fluorescence expression was observed using a laser confocal microscope. The specific operation was the same as above.

[0097] Rice protoplast transient transformation experiments showed that the fluorescence of the three fusion proteins appeared in chloroplasts, which proved that the fusion proteins related to the α-tocopherol transport pathway can be expressed in rice chloroplasts. Figure 2 .

[0098] Example 3: Construction of recombinant expression vector

[0099] In this example, the following three gene expression cassettes were integrated into the pYLTAC380H expression vector of the TransGene Stacking II vector system developed by Liu Yaoguang's research group at South China Agricultural University through a multi-gene expression cassette assembly vector system and Cre-loxP recombination technology. The system includes donor vectors pYL322d1, pYL322d2 and expression vector pYLTAC380H (these three vectors are recorded in the article "Qinlong Zhu et al. Development of "Purple Endosperm Rice" by Engineering Anthocyanin Biosynthesis in the Endosperm with a High-Efficiency Transgene Stacking System. Molecular Plant, DOI: 10.1016 / j.molp.2017.05.008", which can be obtained by the public from the applicant and can only be used to repeat the experiments of the present invention and cannot be used for other purposes).

[0100] ① Expression frame 1: 35S promoter fragment, r-CTP signal peptide (amino acid sequence as shown in SEQ ID No.4) coding sequence, α-TTP (amino acid sequence as shown in SEQ ID No.5) coding gene, Tnos terminator fragment;

[0101] ② Expression frame 2: ELIP2 promoter fragment, the coding sequence of the first 250 amino acids of Arabidopsis thaliana thylakoid membrane protein ALB3 (as shown in SEQ ID No.6), the coding sequence of (GGGGS)3 connecting peptide (amino acid sequence as shown in SEQ ID No.9), the coding gene of phosphatidylinositol kinase PI4KB (amino acid sequence as shown in SEQ ID No.7), and the Tags terminator fragment;

[0102] ③ Expression frame 3: ELIP1 promoter fragment, the coding sequence of the first 250 amino acids of Arabidopsis thaliana thylakoid membrane protein ALB3 (as shown in SEQ ID No.6), the coding sequence of (GGGGS)3 connecting peptide (amino acid sequence as shown in SEQ ID No.9), the coding gene of phosphatidylinositol kinase PIP5KIA3 (amino acid sequence as shown in SEQ ID No.8), and the Tmas terminator fragment.

[0103] Wherein, the sequence of the expression frame 1 is shown in SEQ ID No.1. The 1st to 677th positions of SEQ ID No.1 are the 35S promoter fragment, the 678th to 842th positions are the r-CTP signal peptide, the 843th to 1679th positions are the coding gene of α-TTP, and the 1680th to 1934th positions are the Tnos terminator fragment. The sequence of the expression frame 2 is shown in SEQ ID No.2. The 1st to 2000th positions of SEQ ID No.2 are the ELIP2 promoter fragment, the 2001st to 2750th positions are the coding sequence of the first 250 amino acids of the Arabidopsis thaliana thylakoid membrane protein ALB3, the 2751st to 2798th positions are the coding sequence of the (GGGGS) 3 connecting peptide, the 2799th to 5204th positions are the coding gene of the phosphatidylinositol kinase PI4KB, and the 5205th to 5610th positions are the Tags terminator fragment. The sequence of the expression frame 3 is shown in SEQ ID No.3. Positions 1-2085 of SEQ ID No.3 are the ELIP1 promoter fragment, positions 2086-2835 are the coding sequence of the first 250 amino acids of the Arabidopsis thaliana thylakoid membrane protein ALB3, positions 2836-2884 are the coding sequence of the (GGGGS)3 connecting peptide, positions 2885-4386 are the coding gene of the phosphatidylinositol kinase PIP5KIA3, and positions 4387-4639 are the Tmas terminator fragment.

[0104] The specific operation is as follows: First, the corresponding promoter, signal peptide, gene, and terminator are connected to the donor vector by homologous recombination. The target gene expression frame (expression frame 1, expression frame 3) in the odd-numbered round is connected to donor I (pYL322d1), and the target gene expression frame (expression frame 2) in the even-numbered round is connected to donor II (pYL322d2). The constructed donor vector and the acceptor vector are co-transformed into Escherichia coli NS3529 that can express the Cre enzyme. After the pYL322d1-expression frame 1 donor vector and the acceptor empty pYLTAC380H recombine, it will continue to recombine with the pYL322d2-expression frame 2 donor vector as the acceptor in the even-numbered round. After three rounds of recombination, the three expression frames are integrated into the pYLTAC380H expression vector in turn. In addition, in order to verify whether the target gene is completely connected to the acceptor vector and to verify the integrity of the vector backbone, we use nucleic acid restriction endonucleases Not I performed enzyme digestion verification on the constructed vector. In this system, each time a complete gene expression frame is connected, a pair of Not I restriction site, so that an electrophoresis band of the same size as the target gene expression frame can be cut out. The specific operation is as follows:

[0105] (1) Take out an appropriate number of NS3529 competent cells from the -80°C freezer and thaw them on ice;

[0106] (2) Add 1 μL of receptor plasmid and 1 μL of donor plasmid (receptor: 20 ng / μL; donor: 5 ng / μL) to 40 μL of competent cells and mix gently;

[0107] (3) Use a micropipette to transfer the entire bacterial DNA suspension to the gap between the pillars of the electroporation chamber of the instrument (0.1 cm gap);

[0108] (4) Using the electroporation instrument Gene Pulser (BIO RAD), release 2.5KV electric pulses to the cells in the electroporation chamber;

[0109] (5) Transfer the cells to a 1.5 mL centrifuge tube containing 1 mL of LB medium, place on a shaker, and culture at 37°C and 220 rpm for 2.5 h.

[0110] (6) Collect the cells at room temperature, 6,000 rpm, for 1 min, discard the supernatant, resuspend the cell pellet with the remaining culture medium, spread on LB solid medium containing double resistance to kanamycin and chloramphenicol or ampicillin, and culture at 37°C overnight.

[0111] (7) Wash off all plaques on the electroporated LB solid medium with a dual-resistance liquid LB medium containing kanamycin and chloramphenicol or ampicillin resistance, add the plaques to a 15 mL centrifuge tube containing liquid LB, place on a shaker, and culture at 37°C and 220 rpm for 3 hours;

[0112] (8) Extract plasmid and determine plasmid concentration;

[0113] (9) Use restriction endonuclease I-SceI to digest and remove some non-target plasmids. 100 ng of plasmid was digested with 2.5 units (0.5 µl) of I-SceI at 37°C for 2 h. After the excision, the enzyme was inactivated at 65°C for 10 min. The digestion product was transformed into E. coli competent DH5α.

[0114] (10) Send the positive bacterial spots to Sangon Biotechnology Co., Ltd. for sequencing to verify whether the target gene is linked to the vector and whether the loxP site sequence is intact;

[0115] (11) After successful sequencing, use restriction endonucleases Not I was used to digest the positive plasmid to verify the integrity of the vector backbone.

[0116] The final recombinant plasmid verified by sequencing was named 380H-TVN expression vector. On the 380H-TVN expression vector, the fragment located between the left border (LB) of the insertion sequence T-DNA and the right border (RB) of the insertion sequence T-DNA from the 5' end to the 3' end is: hygromycin resistance gene expression frame, loxP site, NotI restriction site, the aforementioned expression frame 3 (SEQ ID No.3), NotI restriction site, NotI restriction site, the aforementioned expression frame 2 (SEQ ID No.2), NotI restriction site, NotI restriction site, the aforementioned expression frame 1 (SEQ ID No.1), NotI restriction site. Among them, the hygromycin resistance gene expression frame, loxP site, and NotI restriction site are originally present on the pYLTAC380H vector. The 380H-TVN vector introduces the aforementioned expression frame 1 (SEQ ID No.1), expression frame 2 (SEQ ID No.2), expression frame 3 (SEQ ID No.3) and more NotI restriction sites on the basis of pYLTAC380H.

[0117] Figure 3 A in the middle is the result of agarose gel electrophoresis enzyme digestion detection of the constructed 380H-TVN expression vector. Among them, three complete gene expression frames all appear in the correct band positions (indicated by arrows), with sizes of about 6kb, 4.9kb, and 2kb respectively. As shown in the figure, the recombinant expression vector was successfully constructed.

[0118] Example 4: Transformation of rice with recombinant expression vector and identification of positive plants

[0119] (1) The 380H-TVN expression vector constructed according to the method of Example 3 was transformed into Agrobacterium competent cells EHA105 using the heat shock transformation method, the steps are as follows:

[0120] Thaw a 50μL EHA105 competent medium on ice, add 5μL recombinant plasmid to the competent medium, blow and mix, then place on ice and let stand for 5 minutes. Quickly freeze in liquid nitrogen for 5 minutes. Heat shock in a 37℃ water bath for 5 minutes and then slowly place on ice and let stand for 5 minutes. Incubate at 30℃, 200r / min for 3h. Centrifuge at 5000r / min for 5min, discard the supernatant, resuspend the remaining bacteria and apply them to YEB solid culture medium containing 25μg / ml kanamycin and 25μg / ml rifampicin. After the plate is blown dry, place it in a 30℃ constant temperature incubator and invert for 36-48h. Pick 10-20 plaques of appropriate size, culture them in YEB liquid medium containing 25 μg / ml kanamycin and 25 μg / ml rifampicin at 30°C and 200 rpm for 12-16 h, and store the positive bacterial solution (50% glycerol: bacterial solution = 1:1) in a -80°C refrigerator.

[0121] (2) Agrobacterium-mediated genetic transformation of rice:

[0122] Agrobacterium contains Ti plasmid. The injured tissues of plants produce sugars and phenolic substances that attract Agrobacterium tumefaciens to concentrate on the injured tissues. It transfers the T-DNA on its Ti plasmid and integrates it into the DNA of the plant, thus completing the plant infection process.

[0123] The original wild-type material used in this example is the japonica rice variety "Zhonghua 11" (abbreviated as ZH11). The positive seedlings after rice genetic transformation were provided by Boyuan Biotechnology Co., Ltd.

[0124] (3) Positive identification of transgenic seedlings:

[0125] Leaf tissues of T0 transgenic rice plants were taken, DNA was extracted by SDS method, and the DNA was used as template. The pYLTAC380H expression vector has a hygromycin selection tag, and PCR amplification was performed using the hyg501-JF and hyg501-JR primer combinations. The primer combination sequences used are as follows:

[0126] hyg501-JF: 5'-GAGCATATACGCCCGGAGTC-3' (SEQ ID No. 20);

[0127] hyg501-JR: 5'-CAAGACCCTGCCTGAAACCGA-3' (SEQ ID No. 21).

[0128] The results of identification of transgenic positive seedlings are as follows Figure 3 As shown in B, the DNA level verification was successful when the correct band position (around 500 bp) appeared during agarose gel electrophoresis. Lanes 1-23 are positive seedlings with successful transformation, and lane 24 is a wild-type negative control.

[0129] Example 5: Expression of target gene in rice transformed with recombinant expression vector and detection of agronomic traits

[0130] In order to detect whether the expression of PI4KB and PIP5KIA3 genes transferred into rice positive seedlings is induced by high light, this example uses the rice positive seedlings successfully identified in Example 4 as experimental materials, and selects the high light treatment (1200 μmol·m -2 s -1) were taken from leaf samples at 0 and 2 hours after the incubation. The RNA was extracted using the Trizol one-step method (see Example 1 for the extraction method). The RNA was then reverse transcribed into cDNA using the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription kit provided by Quanshijin Biotechnology Co., Ltd. (see the instructions for the steps) and stored at -80°C.

[0131] Design target gene qRT-PCR primers based on the gene sequence in the reference genome and compare the sequences on the BLAST-Search website to determine the specificity of the primers. Use the cDNA of leaf samples before and after high light treatment as templates for subsequent qRT-PCR of the target gene and the Actin internal reference gene. Use the PerfectStart® Green qPCR SuperMix real-time fluorescence quantitative PCR kit provided by Quanshijin Biotechnology Co., Ltd. for qRT-PCR (refer to Example 1 for steps). By calculating the quantitative relative expression amount of qRT-PCR, the expression pattern of the successfully transformed exogenous gene before and after high light treatment can be determined. The primer sequences are as follows:

[0132] Primers used to amplify the α-TTP gene:

[0133] qRT-α-TTP-F: 5'-GCACGGGAACAACTACAAATC-3' (SEQ ID No. 22);

[0134] qRT-α-TTP-R: 5'-TGTCCACTCCTGACAAATATCC-3' (SEQ ID No. 23).

[0135] Primers used to amplify the PI4KB gene:

[0136] qRT-PI4KB-F: 5'-ACGAGGACGTGGACTTCTAT-3' (SEQ ID No. 24);

[0137] qRT-PI4KB-R: 5'-GGTGGACTATGTAGGGCTTAATG-3' (SEQ ID No. 25).

[0138] Primers used to amplify the PIP5KIA3 gene:

[0139] qRT-PIP5KIA3-F: 5'-CGGCCCGATGATTACTTGTAT-3' (SEQ ID No. 26);

[0140] qRT-PIP5KIA3-R: 5'-CGTCGCTGGACACATAGAATAG-3' (SEQ ID No. 27).

[0141] The results are as follows Figure 4 As shown in A, six positive rice plants (numbered 2-1, 2-5, 2-7, 2-8, 2-11, and 2-12) under high light treatment (1200 μmol·m -2 s -1 ), 2 hours after high light induction, the expression levels of PI4KB and PIP5KIA3 genes in the leaves were significantly increased compared with those at 0 hours, indicating that the expression levels of the target genes in the transgenic plants were significantly upregulated after high light induction, indicating that the expression of PI4KB and PIP5KIA3 genes was regulated by high light adversity.

[0142] Transgenic rice plants that had been screened and verified by hygromycin and tested for expression in high light treatment and wild-type ZH11 rice (10 homozygous T2 plants of transgenic line 2-1 and 10 wild-type plants) were planted at the field experimental base of Shandong Agricultural University, with conventional soil and fertilizer management. When the rice was yellow and the seeds were ripe, the dry weight, yield per plant and other traits were investigated.

[0143] The results showed that compared with the wild type, the aboveground dry weight of the transgenic lines increased significantly ( Figure 4 Middle B), the yield per plant increased significantly ( Figure 4 Middle C).

[0144] In summary, the α-tocopherol transfer protein disclosed in the present invention can improve the high light resistance of plants by regulating the transport of α-tocopherol. It has great application potential in the fields of breeding plant varieties resistant to high light, improving the light energy utilization efficiency of plants, and increasing crop yields.

[0145] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A ring-shaped carrier, characterized in that: The circular vector contains the following three expression cassettes: Expression cassette 1: from the 5' end to the 3' end, it includes a constitutive promoter capable of initiating gene transcription in plants, a signal peptide coding sequence capable of localizing to plant chloroplasts, a coding gene for α-tocopherol transfer protein, and a termination sequence capable of terminating gene transcription in plants; Expression cassette 2: from the 5' end to the 3' end, it includes a high light-inducible promoter capable of initiating gene transcription in plants, a signal peptide coding sequence capable of localizing the plant thylakoid membrane, a coding gene for PI4KB protein, and a termination sequence capable of terminating gene transcription in plants; Expression cassette 3: from the 5' end to the 3' end, it includes a high light-inducible promoter capable of initiating gene transcription in plants, a signal peptide coding sequence capable of localizing the plant thylakoid membrane, a gene encoding the PIP5KIA3 protein, and a termination sequence capable of terminating gene transcription in plants; the sequence of the PIP5KIA3 protein is shown in SEQ ID No. 8; In the expression cassette 1, the constitutive promoter is a 35S promoter; the signal peptide is an rCTP signal peptide; and the termination sequence is a Tnos terminator; In the expression cassette 2, the high light inducible promoter is the ELIP2 promoter; the signal peptide is the first 250 amino acids of the Arabidopsis thaliana thylakoid membrane protein ALB3; the termination sequence is the Tags terminator; In the expression cassette 3, the high light inducible promoter is the ELIP1 promoter; the signal peptide is the first 250 amino acids of the Arabidopsis thaliana thylakoid membrane protein ALB3; and the termination sequence is the Tmas terminator.

2. The annular carrier according to claim 1, characterized in that: The sequence of the rCTP signal peptide is shown as SEQ ID No.

4.

3. The annular carrier according to claim 1, characterized in that: The sequence of the α-tocopherol transfer protein is shown as SEQ ID No.

5.

4. The annular carrier according to claim 1, characterized in that: The sequence of the first 250 amino acids of the Arabidopsis thaliana thylakoid membrane protein ALB3 is shown in SEQ ID No.

6.

5. The annular carrier according to claim 1, characterized in that: The sequence of the PI4KB protein is shown in SEQ ID No.

7.

6. The annular carrier according to claim 1, characterized in that: In the expression cassette 1, the sequence of the 35S promoter is shown as positions 1-677 of SEQ ID No.1; and / or, the coding sequence of the rCTP signal peptide is shown as positions 678-842 of SEQ ID No.1; and / or, the sequence of the coding gene of the α-tocopherol transfer protein is shown as positions 843-1679 of SEQ ID No.1; and / or, the sequence of the Tnos terminator is shown as positions 1680-1934 of SEQ ID No.

1.

7. The annular carrier according to claim 1, characterized in that: In the expression cassette 2, the sequence of the ELIP2 promoter is shown as positions 1-2000 of SEQ ID No.2; and / or, the coding sequence of the first 250 amino acids of the Arabidopsis thaliana thylakoid membrane protein ALB3 is shown as positions 2001-2750 of SEQ ID No.2; and / or, the sequence of the gene encoding the PI4KB protein is shown as positions 2799-5204 of SEQ ID No.2; and / or, the sequence of the Tags terminator is shown as positions 5205-5610 of SEQ ID No.

2.

8. The annular carrier according to claim 1, characterized in that: In the expression cassette 3, the sequence of the ELIP1 promoter is shown as positions 1-2085 of SEQ ID No.3; and / or, the coding sequence of the first 250 amino acids of the Arabidopsis thaliana thylakoid membrane protein ALB3 is shown as positions 2086-2835 of SEQ ID No.3; and / or, the sequence of the gene encoding the PIP5KIA3 protein is shown as positions 2885-4386 of SEQ ID No.3; and / or, the sequence of the Tmas terminator is shown as positions 4387-4639 of SEQ ID No.

3.

9. The annular carrier according to any one of claims 1 to 8, characterized in that: The sequence of the expression cassette 1 is shown as SEQ ID No.

1.

10. The annular carrier according to any one of claims 1 to 8, characterized in that: The sequence of the expression cassette 2 is shown as SEQ ID No.

2.

11. The annular carrier according to any one of claims 1 to 8, characterized in that: The sequence of the expression cassette 3 is shown as SEQ ID No.

3.

12. A complete set of expression cassettes, characterized in that: The set of expression cassettes consists of the expression cassette 1, the expression cassette 2 and the expression cassette 3 in any one of claims 1-11.

13. Use of the circular vector according to any one of claims 1 to 11 or the set of expression cassettes according to claim 12 in any of the following: (B1) Improve the ability of plants to resist high light stress; (B2) Improve plant yield under high light stress; (B3) Increase the aboveground dry weight of plants under high light stress; (B4) Increase plant yield under high light stress.

14. The use according to claim 13, characterized in that: The intensity of the high light is greater than the suitable light intensity for the plants.

15. The use according to claim 13 or 14, characterized in that: The plant is a monocotyledonous plant.

16. The use according to claim 13 or 14, characterized in that: The plant is a grass plant.

17. The use according to claim 13 or 14, characterized in that: The plant is a rice plant.

18. The use according to claim 13 or 14, characterized in that: The plant is rice.

19. A method for improving the ability of plants to resist high light stress, characterized in that: The method comprises the following steps: introducing the circular vector described in any one of claims 1 to 11 into a recipient plant to obtain a transgenic plant; and the transgenic plant has improved high light stress resistance compared with the recipient plant.

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