CN118834913A - Application of alpha-TTP expression circular vector in improving rice high light stress resistance - Google Patents

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

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

Technical Field

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

In nature, plants are inevitably exposed to high light radiation. Under the action of strong light, chlorophyll (Chl) is activated and converted to the single excited state ¹ Chl, which is converted to the triple excited state ³ Chl inside the system. The triple excited state ³ Chl exists longer than the single excited state ¹ Chl, and can also react with molecular oxygen to produce singlet oxygen ¹ O _2. Singlet oxygen ¹ O ₂ is the most destructive to the photosystem. It can cause damage to lipids, key ^pigment cofactors, and D1 protein subunits in PSⅡ, and ultimately lead to the inactivation of the PSⅡ reaction center, severely inhibiting photosynthesis. Therefore, how to protect the plant's photosystem so that it can efficiently operate photosynthesis under high light stress is an urgent problem to be solved.

In plants, tocopherol is located on membrane structures such as chloroplast membranes, thylakoid membranes, and plastid globule membranes. Studies have shown that the aromatic ring head of tocopherol 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 inside of the membrane. α-Tocopherol forms a charge transfer excitation complex by providing an electron to singlet oxygen ¹ O ₂ , and finally forms free α-tocopherol and molecular oxygen, resulting in the inactivation of singlet oxygen ¹ O ₂ , thereby protecting the membrane lipids and photosystems.

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

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.

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

The circular vector claimed in the present invention comprises 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 gene encoding α-tocopherol transfer protein (α-TTP), 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 the 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.

Furthermore, the expression cassette 1 is composed, from the 5' end to the 3' end, of 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.

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.

Furthermore, the expression cassette 3 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 PIP5KIA3 protein, and the termination sequence capable of terminating gene transcription in plants.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

(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;

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

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:

(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.

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

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.

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.

In the above-mentioned related aspects, the high light refers to light having a light intensity greater than the light intensity suitable for the plant. In an implementation case of the present invention, the light intensity of the high light is 1200 μmol·m ^-2 s ^-1 .

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.

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

(C1) monocots;

(C2) Gramineae;

(C3) Oryza plants;

(C4) Rice.

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

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 the α-Toc content in the thylakoid membrane is increased in a directional manner, thereby improving the ability to remove ¹ O ₂ and enhancing the light protection ability of plants. 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, so that they are specifically positioned on the outside of the thylakoid membrane; at the same time, through the co-expression of 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, a gene circuit is designed, and different high light-inducible promoters, leader peptides, and terminators are matched. The modules are assembled by multi-gene expression cassette technology and then genetically transformed. The modules are induced and controllably expressed in rice chloroplasts to maintain the effective operation of photosynthesis under high light stress of rice, thereby increasing the dry weight of the aboveground part of rice and the yield per plant. The present invention has great application potential in the fields of breeding plant varieties resistant to high light, improving the efficiency of light energy utilization of plants, and increasing crop yields.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the changes in expression of some candidate genes screened by the present invention before and after high light (1200 μmol·m ^-2 s ^-1 ) treatment. A shows the changes in expression of ELIP2 gene before and after high light treatment; B shows the changes in expression of APX2 gene before and after high light treatment; C shows the changes in expression of ELIP1 gene before and after high light treatment; and D shows the changes in expression of SEP1 gene before and after high light treatment. The expression of ELIP1 and ELIP2 genes continued to increase with the increase in the duration of high light treatment.

Figure 2 shows the transient transformation experiment of α-TTP, PI4KB, PIP5KIA3 and GFP fusion protein expression vector in rice protoplasts, which shows that the fluorescence signals of the three fusion proteins all appear in chloroplasts.

Figure 3 shows 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.

Figure 4 shows the results of rice transformation and positive plant identification using the recombinant expression vector. A shows that the expression of PI4KB and PIP5KIA3 genes in positive rice plants was significantly upregulated after 2 hours of high light treatment (1200 μmol·m ^-2 s ^-1 ) compared with 0 hours; B shows that the aboveground dry weight of the transgenic lines was significantly increased compared with the wild type; C shows that the yield per plant of the transgenic lines was significantly increased compared with the wild type. 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

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.

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.

Example 1: Screening of high light-inducible promoters

The experimental material used in this application is Arabidopsis thaliana (Col-0) grown for three weeks. Leaf samples with good growth status and placed under high light conditions of LED light source (1200μmol·m ^-2 s ^-1 ) for 0h, 0.5h, 1h, 2h, 3h, 6h, 12h, and 24h in the same period were taken to extract RNA.

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

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.

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

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

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.

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

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

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.

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.

⑨ Add 50 μL of DEPC-treated ddH ₂ O and store in a -80°C refrigerator.

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.

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:

qRT-ELIP2-F: 5’-GCGATCCTATCAAGGAAGATCC-3’ (SEQ ID No. 10);

qRT-ELIP2-R: 5’-ACTCACCTTAGGCTTGCTAAC-3’ (SEQ ID No. 11);

qRT-ELIP1-F: 5’-ATTATCCGGTGGGAGTGAGA-3’ (SEQ ID No. 12);

qRT-ELIP1-R: 5’-TTCATAGGAGGAGGAGGAGATG-3’ (SEQ ID No. 13);

qRT-SEP1-F: 5’-GCTTCTGGTTCTCCTCCTTG-3’ (SEQ ID No. 14);

qRT-SEP1-R: 5’-CCACTGCTTCCTTCTGTACTT-3’ (SEQ ID No. 15);

qRT-APX2-F: 5’-GGAAGCTCCGTGGTCTTATT-3’ (SEQ ID No. 16);

qRT-APX2-R: 5’-CTCCTGTCTTCGTCTTCACATC-3’ (SEQ ID No. 17);

qRT-Actin2-F: 5’-GACCTTTAACTCTCCCGCTATG-3’ (SEQ ID No. 18);

qRT-Actin2-R: 5’-GAGACACACCATCACCAGAAT-3’ (SEQ ID No. 19).

Table 1. qPCR reaction system

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.

By calculating the quantitative relative expression of qRT-PCR, the expression pattern of Arabidopsis high-light response genes before and after high-light treatment can be determined. The results are shown in Figure 1, and it can be seen that the expression levels of some candidate genes screened by the present invention before and after high-light treatment change, among which the expression levels of ELIP1 and ELIP2 genes continue to increase with the increase of high-light treatment duration, and the increase is large. Therefore, the present invention selects the region 2kb upstream of the transcription start site of ELIP1 and ELIP2 genes as the promoter sequence of the gene (hereinafter referred to as ELIP1 promoter and ELIP2 promoter. Among them, the sequence of ELIP2 promoter is shown in the 1st to 2000th positions of SEQ ID No.2, and the sequence of ELIP1 promoter is shown in the 1st to 2085th positions of SEQ ID No.3), for the subsequent construction of recombinant expression vectors.

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

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 the Rubisco small subunit gene RBCS1 before the α-TTP sequence, and constructs a vector expressing the fusion protein of α-TTP and GFP with the chloroplast signal peptide driven by the 35S promoter. The specific operation is as follows:

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) of 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 is available to 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) between the Eco RI and Xba I restriction sites. 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.

The structural description of the pM999-rCTP-αTTP-GFP vector is as follows: The recombinant plasmid is obtained by replacing the small fragment between the restriction sites Eco R I and Xba I of the pM999 vector (carrying the GFP gene) with the rCTP-αTTP fragment. The rCTP-αTTP fragment contains the rCTP coding sequence shown in positions 678-842 of SEQ ID No.1 and the coding gene of α-TTP shown in positions 843-1679 of SEQ ID No.1 from the 5' end to the 3' end.

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:

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.

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.

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 for 10 min at 100 g with an acceleration and deceleration of 2, and pre-cool the MMG (formula: 4 mM MES, 600 mM mannitol, 15 mM MgCl ₂ ) solution.

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 CaCl ₂ , 5mM KCl) solution, mix gently, and then centrifuge at 100g speed, acceleration and deceleration acceleration of 2, for 10 minutes.

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.

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.

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.

8. Mix the protoplasts, add 200μl of protoplasts and 220μl of 40% PEG4000 solution (formula: 200 mM mannitol, 400g/L PEG4000, 100mM CaCl ₂ ) 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.

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).

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.

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:

Hind III and Eco R I restriction endonucleases were used to cut off the 35S promoter on the pM999 vector (carrying the GFP gene), and then the pEASY -Basic Seamless Cloning and Assembly Kit (CU201-03) from Quanshijin Company was used to introduce the ELIP2 and ELIP1 high light-inducible promoters between the Hind III and Eco R I restriction sites of the pM999 vector (carrying the GFP gene) using the homologous recombinase 2×Basic Assembly Mix to replace the 35S promoter carried by the vector itself. 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 Eco R I and Xba I restriction sites of the pM999 vector (carrying the GFP gene). Subsequently, based on the sequencing results, expression vectors with correct gene element connection direction and correct sequence were screened and named pELIP2::pM999-ALB3 _1-250aa -PI4KB-GFP and pELIP1:: pM999-ALB3 _1-250aa -PIP5KIA3-GFP according to their different structural compositions.

The structure description of pELIP2::pM999-ALB3 _1-250aa -PI4KB-GFP vector is as follows: the 35S promoter between the restriction sites Hind III and Eco RI of the pM999 vector (carrying the GFP gene) is replaced with the pELIP2 promoter (positions 1-2000 of SEQ ID No.2), and the small fragment between Eco RI and Xba I is replaced with the ALB3 _1-250aa -PI4KB fragment to obtain the recombinant plasmid. Among them, the ALB3 _1-250aa -PI4KB fragment contains the ALB3 _1-250aa coding gene shown in positions 2001-2750 of SEQ ID No.2, the coding sequence of the (GGGGS)3 connecting peptide shown in positions 2751-27980 of SEQ ID No.2, and the PI4KB coding gene shown in positions 2799-5204 of SEQ ID No.2 from the 5' end to the 3' end.

Structural description of pELIP1:: pM999-ALB3 _1-250aa -PIP5KIA3-GFP vector: The 35S promoter between the restriction sites Hind III and Eco R I of the pM999 vector (carrying the GFP gene) was replaced with the pELIP1 promoter (positions 1-2085 of SEQ ID No. 3), and the small fragment between Eco R I and Xba I was replaced with the ALB3 _1-250aa -PIP5KIA3 fragment to obtain the recombinant plasmid. Among them, the ALB3 _1-250aa -PIP5KIA3 fragment is, from the 5' end to the 3' end, the ALB3 _1-250aa coding gene shown at positions 2086-2835 of SEQ ID No.3, the coding sequence of the (GGGGS)3 connecting peptide shown at positions 2836-2884 of SEQ ID No.3, and the PIP5KIA3 coding gene shown at positions 2885-4386 of SEQ ID No.3.

The pELIP2::pM999-ALB3 _1-250aa -PI4KB-GFP vector and the pELIP1:: pM999-ALB3 _1-250aa -PIP5KIA3-GFP vector were transformed into rice protoplasts, respectively. After culturing in the dark for 14 h, sections were prepared and the fluorescence expression was observed using a laser confocal microscope. The specific operations were the same as above.

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

Example 3: Construction of recombinant expression vector

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 the research group of Liu Yaoguang of South China Agricultural University through the 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).

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;

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;

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.

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.

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 odd-numbered round target gene expression frame (expression frame 1, expression frame 3) is connected to donor I (pYL322d1), and the even-numbered round target gene expression frame (expression frame 2) is connected to donor II (pYL322d2). The constructed donor vector and the recipient vector are co-transformed into Escherichia coli NS3529 that can express the Cre enzyme. After the pYL322d1-expression frame 1 donor vector and the recipient empty pYLTAC380H are recombined, it will continue to recombine with the pYL322d2-expression frame 2 donor vector as the recipient of the even-numbered round. After three rounds of recombination, the three expression cassettes are integrated into the pYLTAC380H expression vector in turn. In addition, in order to verify whether the target gene is completely connected to the receptor vector and to verify the integrity of the vector backbone, we use the nucleic acid restriction endonuclease Not I to perform 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 sites will be introduced on both sides of the vector, so that an electrophoresis band of the same size as the target gene expression frame can be digested. The specific operation is as follows:

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

(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;

(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);

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

(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.

(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.

(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;

(8) Extract plasmid and determine plasmid concentration;

(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.

(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;

(11) After successful sequencing, the positive plasmid was digested with restriction endonuclease Not I to verify the integrity of the vector backbone.

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.

Figure 3 A 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 appeared 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.

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

(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:

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.

(2) Agrobacterium-mediated genetic transformation of rice:

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.

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.

(3) Positive identification of transgenic seedlings:

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 carries a hygromycin selection tag, and PCR amplification was performed using the hyg501-J-F and hyg501-J-R primer combinations. The primer combination sequences used are as follows:

hyg501-J-F: 5’-GAGCATATACGCCCGGAGTC-3’ (SEQ ID No. 20);

hyg501-J-R: 5’-CAAGACCCTGCCTGAAACCGA-3’ (SEQ ID No. 21).

The identification results of transgenic positive seedlings are shown in Figure 3B. The appearance of the correct band position (around 500 bp) in agarose gel electrophoresis is considered to be successful in DNA level verification. Lanes 1-23 are positive seedlings with successful transformation, and lane 24 is a wild-type negative control.

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

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, extract RNA from leaf samples at 0 hours and 2 hours after high light treatment (1200 μmol·m ^-2 s ^-1 ) in good growth state at the same time, and use the Trizol one-step method (extraction method refers to Example 1), and then use the reverse transcription kit TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription kit provided by Quanshijin Biotechnology Co., Ltd. (see the instructions for steps) to reverse transcribe RNA into cDNA and store it at -80°C.

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:

Primers used to amplify the α-TTP gene:

qRT-α-TTP-F: 5’-GCACGGGAACAACTACAAATC-3’ (SEQ ID No. 22);

qRT-α-TTP-R: 5’-TGTCCACTCCTGACAAATATCC-3’ (SEQ ID No. 23).

Primers used to amplify the PI4KB gene:

qRT-PI4KB-F: 5’-ACGAGGACGTGGACTTCTAT-3’ (SEQ ID No. 24);

qRT-PI4KB-R: 5’-GGTGGACTATGTAGGGCTTAATG-3’ (SEQ ID No. 25).

Primers used to amplify the PIP5KIA3 gene:

qRT-PIP5KIA3-F: 5’-CGGCCCGATGATTACTTGTAT-3’ (SEQ ID No. 26);

qRT-PIP5KIA3-R: 5’-CGTCGCTGGACACATAGAATAG-3’ (SEQ ID No. 27).

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

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.

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

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.

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 (10)

1. The annular carrier, its characterized in that: the circular vector comprises the following three expression cassettes:

Expression cassette 1: the plant chloroplast-localizing gene comprises a constitutive promoter capable of promoting gene transcription in plants, a signal peptide coding sequence capable of localizing plant chloroplasts, an alpha-tocopherol transfer protein coding gene and a termination sequence capable of terminating gene transcription in plants from the 5 'end to the 3' end in sequence;

expression cassette 2: the kit sequentially comprises a highlight inducible promoter capable of promoting gene transcription in plants, a signal peptide coding sequence capable of positioning plant thylakoid membranes, a coding gene of PI4KB protein and a termination sequence capable of terminating gene transcription in plants from the 5 'end to the 3' end;

Expression cassette 3: the polypeptide comprises a high light induction type promoter capable of promoting gene transcription in plants, a signal peptide coding sequence capable of locating plant thylakoid membranes, a PIP5KIA3 protein coding gene and a termination sequence capable of terminating gene transcription in plants from the 5 'end to the 3' end.

2. The toroidal support of claim 1, wherein: in the expression cassette 1, the constitutive promoter is a 35S promoter; and/or, the signal peptide is an rCTP signal peptide; and/or, the termination sequence is a Tnos terminator; and/or

In the expression cassette 2, the highlight inducible promoter is ELIP promoter; and/or, the signal peptide is the first 250 amino acids of an arabidopsis thaliana capsule membrane protein ALB 3; and/or, the termination sequence is a Tags terminator; and/or

In the expression cassette 3, the highlight inducible promoter is ELIP promoter; and/or, the signal peptide is the first 250 amino acids of an arabidopsis thaliana capsule membrane protein ALB 3; and/or, the termination sequence is Tmas terminator.

3. The toroidal support of claim 2, wherein: the sequence of the rCTP signal peptide is shown as SEQ ID No. 4; and/or

The sequence of the alpha-tocopherol transfer protein is shown in SEQ ID No. 5; and/or

The sequence of the first 250 amino acids of the arabidopsis thaliana thylakoid membrane protein ALB3 is shown as SEQ ID No. 6; and/or

The sequence of the PI4KB protein is shown in SEQ ID No. 7; and/or

The sequence of the PIP5KIA3 protein is shown as SEQ ID No. 8.

4. A toroidal support as claimed in claim 3, wherein: in the expression cassette 1, the sequence of the 35S promoter is shown in positions 1-677 of SEQ ID No. 1; and/or, the coding sequence of the rCTP signal peptide is shown in the 678-842 th position of SEQ ID No. 1; and/or, the sequence of the coding gene of the alpha-tocopherol transfer protein is shown in 843-1679 of SEQ ID No. 1; and/or, the sequence of the Tnos terminator is shown in 1680-1934 positions of SEQ ID No. 1; and/or

In the expression cassette 2, the sequence of the ELIP promoter is shown in positions 1-2000 of SEQ ID No. 2; and/or the coding sequence of the first 250 amino acids of the arabidopsis thaliana capsule membrane protein ALB3 is shown in 2001-2750 of SEQ ID No. 2; and/or the sequence of the encoding gene of the PI4KB protein is shown in the 2799-5204 positions of SEQ ID No. 2; and/or the sequence of the Tags terminator is shown in 5205-5610 bits of SEQ ID No. 2; and/or

In the expression cassette 3, the sequence of the ELIP promoter is shown in positions 1-2085 of SEQ ID No. 3; and/or the coding sequence of the first 250 amino acids of the arabidopsis thaliana capsule membrane protein ALB3 is shown in positions 2086-2835 of SEQ ID No. 3; and/or the sequence of the coding gene of the PIP5KIA3 protein is shown in 2885-4386 positions of SEQ ID No. 3; and/or, the Tmas terminator has a sequence shown in 4387-4639 positions of SEQ ID No. 3.

5. The toroidal support of any one of claims 1 to 4, wherein: the sequence of the expression cassette 1 is shown as SEQ ID No. 1; and/or

The sequence of the expression cassette 2 is shown as SEQ ID No. 2; and/or

The sequence of the expression cassette 3 is shown as SEQ ID No. 3.

6. The complete set of expression cassettes is characterized in that: the kit of expression cassettes consists of the expression cassette 1, the expression cassette 2 and the expression cassette 3 according to any one of claims 1 to 5.

7. Use of a circular vector according to any one of claims 1 to 5 or a kit of expression cassettes according to claim 6 in any one of the following:

(B1) Improving the capability of the plant for resisting the high light stress;

(B2) Improving plant yield under high light stress;

(B3) Increasing the dry weight of the aerial parts of the plants under high light stress;

(B4) And the yield of a single plant of the plant is improved under high light stress.

8. The use according to claim 7, characterized in that: the intensity of the high light is greater than the appropriate illumination intensity of the plant.

9. Use according to claim 7 or 8, characterized in that: the plant is any one of the following:

(C1) Monocotyledonous plants;

(C2) A grass plant;

(C3) A plant of the genus oryza;

(C4) And (3) rice.

10. A method for improving the capability of plants to resist high light stress, which is characterized in that: the method comprises the following steps: introducing the circular vector of any one of claims 1-5 into a recipient plant to obtain a transgenic plant; the transgenic plants have increased resistance to high light stress as compared to the recipient plants.