Application of SlPsbW-LIKE gene in improving high temperature resistance and photosynthetic capacity of tomato

Abstract

The present invention relates to the field of plant biotechnology, and specifically to the application of the SlPsbW-LIKE gene in improving the high-temperature resistance and photosynthetic capacity of tomatoes. The present invention uses reverse genetics to first clarify the function of PsbW-LIKE in heat tolerance and photoprotection in tomatoes. It is very important for maintaining the stability of the photosystem II supercomplex. Therefore, increasing and supplementing the content of PsbW-LIKE under high-temperature stress is beneficial to improving the tolerance of plants such as tomatoes to high-temperature stress and is also beneficial to improving the photosynthetic capacity of plants in high-temperature environments. The heat tolerance mechanism of PsbW-LIKE discovered by the present invention is of great significance for improving the high-temperature tolerance and photosynthetic capacity of solanaceous crops such as tomatoes.

Description

Technical Field

[0001] The present invention relates to the field of plant biotechnology, and particularly to the application of the SlPsbW-LIKE gene in improving the high-temperature resistance and photosynthetic capacity of tomatoes. Background Art

[0002] Global climate change has made the problem of high-temperature heat damage very prominent, and high-temperature stress has gradually become a major environmental stress factor restricting crop distribution, growth and productivity. Some common vegetable crops such as tomatoes, cucumbers, peppers, etc. are extremely vulnerable to the influence of high-temperature stress, causing damage to the chloroplast photosystem in their bodies, reducing the activity of carbon assimilation-related enzymes and various metabolic disorders, and ultimately leading to a decline in crop yield and quality. Therefore, improving the heat resistance and photoprotection ability of tomato plants is of great significance for overcoming the difficulty of tomatoes surviving the summer.

[0003] Photosynthesis is the most important biochemical reaction on earth, providing a source of matter and energy for almost all organisms. However, under high-temperature environments, the photosynthetic rate of plants will be severely inhibited, causing disorders in the metabolism of matter and energy in the body. This is related to the influence of high temperature on the components of the photosystem. Photosystem II (PSII) is an important multi-component pigment-protein complex on the thylakoid membrane of chloroplasts, responsible for water splitting, oxygen release and the reduction of plastoquinone, and is particularly sensitive to high temperatures. The PSII complex includes reaction center proteins (D1, D2), inner peripheral light-harvesting core antenna proteins (CP43, CP47), oxygen-evolving complex proteins (OEC), light-harvesting complex II proteins, and 12 low-molecular-weight transmembrane proteins (PsbE, PsbF, PsbH, PsbI, PsbJ, PsbK, PsbL, PsbM, PsbTc, PsbW, PsbX and PsbZ). Under high-temperature stress, the PSII-LHCII superaggregate complex will first dissociate, followed by obvious degradation of the core protein D1, and its repair process is severely inhibited. Supplementing sufficient D1 protein can maintain the photosynthetic capacity of crops under high-temperature stress and increase crop yield. Therefore, maintaining the function of PSII under high-temperature stress is very important. Low-molecular-weight proteins are mostly the "bridges" connecting the major subunits and core proteins of PSII, and play a crucial role in the stability and activity of the PSII superaggregate complex.

[0004] PsbW is a typical low-molecular-weight protein in the chloroplast photosystem II (PSII) complex, with only one transmembrane helix. Its N-terminus is connected to the oxygen-evolving complex protein PsbO and the PSII reaction center protein D1 on the lumen side, and its C-terminus binds to the inner peripheral light-harvesting core antenna protein CP43 on the stroma side. In addition, the light-harvesting pigment protein complex LHCII also binds to the PsbW protein. This indicates that PsbW may play a very important "bridge" role in the PSII complex. However, inhibiting the expression of PsbW in Arabidopsis only causes a decrease in PSII core proteins and a decline in PSII function, without causing obvious growth phenotypes. But when it is under strong light stress, the psbw mutant with low levels of PSII supercomplexes has lower photosynthetic capacity, more serious degradation of the PSII complex, and a significant decrease in the content of D1 protein, and its growth and development are greatly affected. In addition, Western blot experiments show that when Arabidopsis is photoinhibited, the PsbW protein has a similar degradation rate and level to D1, which is more obvious than the degradation levels of core proteins such as D2 and CP43. This suggests that the structure and function of the PSII supercomplex may be more important under stress conditions, and PsbW is committed to maintaining the homeostasis of PSII, but it is easily damaged under stress conditions.

[0005] During the long evolution of photosynthetic organisms, there has been genetic information exchange between the chloroplast genome and the nuclear genome. Some gene fragments encoding chloroplast photosynthetic proteins have also been integrated into different positions in the nuclear genome. Usually, these gene fragments are considered to be pseudogenes. However, we still don't know whether all of these nuclear photosynthetic gene fragments are non-functional. Summary of the Invention

[0006] Aiming at the above-mentioned prior art, the object of the present invention is to provide the application of the SlPsbW-LIKE gene in improving the high-temperature resistance and photosynthetic capacity of tomatoes.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, there is provided the application of the SlPsbW-LIKE protein in the following (1) or (2):

[0009] (1) Improving the tolerance of plants to high-temperature stress;

[0010] (2) Improving the photosynthetic capacity of plants in a high-temperature environment;

[0011] In the above application, the SlPsbW-LIKE protein is the protein shown in the following (A1) or (A2):

[0012] (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing;

[0013] (A2) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0014] Among them, the protein described in (A1) can be artificially synthesized, or its coding gene can be synthesized first and then obtained by biological expression.

[0015] In the above-mentioned protein, the protein tag refers to a polypeptide or protein that is fused and expressed together with the target protein by using in vitro DNA recombination technology for the purpose of facilitating the expression, detection, tracing, and / or purification of the target protein. Among them, in order to facilitate the purification of the protein in (A1), a tag can be linked to the amino terminus or carboxyl terminus of the protein in (A1). The tag can be Poly-Arg (usually 6 RRRRR), Poly-His (usually 6 HHHHHH), FLAG (DYKDDDDK), Strep-tagII (WSHPQFEK), or c-myc (EQKLISEEDL).

[0016] In the above application, preferably, the plant is tomato.

[0017] In the second aspect of the present invention, there is provided the use of the coding gene of the SlPsbW-LIKE protein in the following (1) or (2):

[0018] (1) Improving the tolerance of plants to high-temperature stress;

[0019] (2) Improving the photosynthetic ability of plants in a high-temperature environment.

[0020] In the above application, preferably, the nucleotide sequence of the coding gene of the SlPsbW-LIKE protein is as shown in SEQ ID NO.3.

[0021] In the third aspect of the present invention, there is provided the use of a recombinant expression vector, a transgenic cell line, or an engineered bacterium containing the coding gene of the SlPsbW-LIKE protein in the following (1) or (2):

[0022] (1) Improving the tolerance of plants to high-temperature stress;

[0023] (2) Improving the photosynthetic ability of plants in a high-temperature environment.

[0024] In the above application, the recombinant expression vector can be constructed using existing plant expression vectors. For example, pZP211-GFP, pMal-c2x-mbp, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derivative plant expression vectors.

[0025] The host cell of the engineered bacterium can be Escherichia coli, Agrobacterium, etc.

[0026] In the fourth aspect of the present invention, a method for improving the tolerance of tomatoes to high-temperature stress is provided, including: the step of overexpressing the SlPsbW-LIKE gene in tomatoes;

[0027] Or, the step of spraying the SlPsbW-LIKE protein onto tomato plants.

[0028] In the above method, overexpressing the SlPsbW-LIKE gene in tomatoes can be achieved by the method of exogenous transfer of the SlPsbW-LIKE gene; or upregulating the expression of the SlPsbW-LIKE gene in the tomato genome.

[0029] In the fifth aspect of the present invention, the use of the SlPsbW-LIKE gene or a recombinant expression vector, transgenic cell line or engineered bacterium containing the SlPsbW-LIKE gene in cultivating transgenic tomatoes is provided;

[0030] The nucleotide sequence of the SlPsbW-LIKE gene is as shown in SEQ ID NO.3.

[0031] In the above use, compared with wild-type tomatoes, the transgenic tomatoes cultivated have improved tolerance to high-temperature stress and photosynthetic ability in a high-temperature environment.

[0032] Advantages of the present invention:

[0033] The present invention uses reverse genetics to first clarify the function of PsbW-LIKE in heat tolerance and photoprotection in tomatoes. It is very important for maintaining the stability of the photosystem II supercomplex. Therefore, increasing or supplementing the content of PsbW-LIKE under high-temperature stress is beneficial to improving the tolerance of plants such as tomatoes to high-temperature stress and is also beneficial to improving the photosynthetic ability of plants in a high-temperature environment. The heat tolerance mechanism of PsbW-LIKE discovered by the present invention is of great significance for improving the high-temperature tolerance and photosynthetic ability of solanaceous crops such as tomatoes. Description of the Drawings

[0034] Figure 1:(A) Tobacco transient transformation experiment mediated by SlPsbW-LIKE-GFP. GFP: Green fluorescent protein; Chl.: Chlorophyll autofluorescence; ER: Endoplasmic reticulum-specific fluorescent probe staining; Bright field: White field; Merged: Merged image. (B) Identification of the PsbW protein levels in chloroplasts and endoplasmic reticulum of tomato PsbW-LIKE-GFP transgenic lines before and after heat treatment; D2: Chloroplast protein control; HSP21.5A: Endoplasmic reticulum protein control; LC: Protein loading reference. (C) Sequencing identification and analysis of the mutant lines psbw-1 and psbw-2 of PsbW, … represents the deleted sequence. (D) Identification of the PsbW protein levels in chloroplasts and endoplasmic reticulum of the psbw-like-2 mutant line before and after heat treatment; D2: Chloroplast protein control; HSP21.5A: Endoplasmic reticulum protein control; LC: Protein loading reference. (E) Heat tolerance phenotype analysis of psbw-like lines and wild type. (F) BN-PAGE analysis of the thylakoid membrane complex levels of psbw-like-2 lines and wild type before and after heat treatment; furthermore, Western blot was used to identify the changes in the protein levels of D1 and CP43 therein. (G, H) Detection of Fv / Fm and Pn in psbw-2 lines and wild type before and after heat treatment.

[0035] We discovered and cloned the PsbW-LIKE gene. Subcellular localization analysis showed that PsbW-LIKE was mainly distributed in the endoplasmic reticulum and chloroplasts (A, B). Two protein bands with different molecular weights were detected in the cytoplasmic (including endoplasmic reticulum) proteins, and the larger band was the same size as the PsbW-LIKE protein detected in the chloroplasts (B). We obtained the mutant (psbw-like) lines of PsbW-LIKE through the Crisper-Cas9 technology. Sequencing analysis showed that deletions occurred at different target sites in psbw-like-1 / 2, resulting in premature termination (C). Western blot experiments showed that the large molecular weight PsbW protein was still present in the chloroplasts of the psbw-like lines, while no PsbW protein was detected in the cytoplasm (including endoplasmic reticulum) (D). At the same time, we found that after heat treatment, the protein content of PsbW no longer increased (D). Comparative analysis of psbw-like and wild-type before and after heat treatment showed that under normal growth conditions, the psbw-like mutants did not show obvious phenotypes, but they were extremely intolerant to heat stress (E). Blue native polyacrylamide gel electrophoresis (BN-PAGE) experiments showed that the amount of PSII supercomplexes in the psbw-like mutants was significantly less than that in the wild-type before heat treatment, and this phenomenon was further amplified after heat stress (F). Correspondingly, the protein contents of D1 and CP43 also showed the same trend (F). In addition, physiological index measurements showed that the maximum photochemical efficiency (Fv / Fm) and photosynthetic rate (Pn) of the psbw-like lines were lower than those of the wild-type before heat treatment, and heat treatment also amplified these differences (G, H).

[0036] Figure 2 : (A) The upper part is a schematic diagram of the assembly of the CTPPsbW-LIKE-GFP expression vector; the lower part shows the fluorescence observation of the vector transferred into tobacco (Nt) and tomato (Sl) by Agrobacterium-mediated transformation. GFP: Green fluorescent protein; Chl.: Chlorophyll autofluorescence; Bright field: White field; Merged: Merged image. (B) The upper part shows the expression of the transferred gene identified by RT-qPCR in the WT (PsbW-L), WT (PsbW), and WT (GFP) lines. The lower part shows the analysis of their thylakoid membrane complexes and the insertion of the PsbW protein. (C) Heat tolerance analysis of the WT (PsbW-L), WT (PsbW), and WT (GFP) lines. (D) Analysis of the thylakoid membrane complex levels in the WT (PsbW-L), WT (PsbW), and WT (GFP) lines after heat treatment. (E) Detection of the Fv / Fm values in the WT (PsbW-L), WT (PsbW), and WT (GFP) lines before and after heat treatment.

[0037] Given the complexity of the mechanism by which PsbW-LIKE enters chloroplasts in tomatoes, we inserted a sequence encoding the chloroplast signal peptide of the small subunit of Rubisco, RbcS1, after the start codon at the N-terminus of PsbW-LIKE. Driven by the 35S promoter, it was highly expressed in tobacco chloroplasts (A), and was successfully transferred into tomato chloroplasts and assembled into the thylakoid membrane (A, B). We selected lines with the same expression level as the PsbW transgenic tomatoes driven by 35S (B), and compared their heat tolerance with that of the wild-type lines transfected with the GFP empty vector and the common wild-type lines. Phenotypic analysis showed that after high-temperature treatment, the transgenic tomato lines of CTPPsbW-LIKE and PsbW (WT(PsbW-L) and WT(PsbW)) were more heat-tolerant than the wild-type lines, but the heat tolerance of the WT(PsbW-L) line was stronger than that of WT(PsbW). BN-PAGE analysis showed that under normal growth conditions, the PSII supercomplex in the WT(PsbW-L) line and WT(PsbW) was slightly higher than that in the wild-type line (B). After high-temperature treatment, the levels of the PSII supercomplex and Fv / Fm in WT(PsbW) were higher than those in the wild-type line but lower than those in the WT(PsbW-L) line (D, E).

[0038] Figure 3 Protein expression under different induction conditions (A) and heat tolerance investigation of tomato plants under different treatments (B);

[0039] The above CTPPsbW-LIKE sequence was constructed into the prokaryotic expression vector pMal-c2x-mbp vector by homologous recombination, and the constructed vector was transferred into BL21 strains. The strain was induced to culture in a shaker at 37 °C with 0.2 mM isopropyl thiogalactoside (IPTG). The induced CTP-PsbW-LIKE-mbp protein is shown by the yellow arrow in Figure A. The bacterial solution was ultrasonically disrupted to separate the supernatant protein. The solution was evenly sprayed on the leaves of tomato plants (the supernatant protein obtained from the empty mbp vector and water were used as two control groups), and then placed in a high-temperature light incubator at 42 °C for treatment. The results showed that the heat tolerance of the tomato plants sprayed with the crude extract of CTP-PsbW-LIKE-mbp was significantly stronger than that of the two control plants (B). Specific embodiments

[0040] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0041] As mentioned above, global climate change has made the problem of high-temperature heat damage very prominent, and high-temperature stress severely limits the yield and quality of vegetable crops such as tomatoes. Photosynthesis is the driving force for plant growth, and high temperature seriously affects the progress of photosynthesis in plants. Therefore, improving the heat tolerance of vegetable crops such as tomatoes from the perspective of photosynthesis is of great significance for overcoming the difficulties of tomatoes and other vegetable crops in surviving the summer.

[0042] Based on this, the present invention has conducted in-depth research on the proteins that affect the high-temperature resistance and photosynthetic ability of tomatoes. The PsbW protein is a typical low-molecular-weight protein in the chloroplast photosystem II (PSII) complex, with only one transmembrane helix, and its amino acid sequence is shown as follows:

[0043] MASSITACTTTSLAAREALLHKASALSRPSVLGLPSMRKNVGRVKCSMEREDSKSGICGSLVAAT LSFSPTAMALVDERMSTEGTGLPFGLSNNLLGWILFGVFGLVWSLYTVYTSSLDEDEDSAMSL. (SEQ ID NO.1)

[0044] The PsbW protein has only been reported in Arabidopsis thaliana so far. It is speculated that the PsbW protein is committed to maintaining the stability of PSII, but it is extremely vulnerable to damage under stress conditions.

[0045] The PsbW-LIKE protein is a protein similar to PsbW, and its amino acid sequence is shown as follows:

[0046] MAAAVAASMSSPVAMALVDERMSTEGTGLPFGLSNNLLGWILLGVFGLIWSLYTVYTSGLDEDEESGMSL. (SEQ ID NO.2)

[0047] Compared with the PsbW protein, the PsbW-LIKE protein has a large deletion of amino acids at the N-terminus and 5 amino acid differences at the C-terminus. At present, the coding gene of the PsbW-LIKE protein is generally recognized as a "pseudogene", and there is no report on the function of the PsbW-LIKE protein.

[0048] The present invention has conducted in-depth research on the function of the PsbW-LIKE protein for the first time. The present invention has cloned the PsbW-LIKE gene, and its nucleotide sequence is shown in SEQ ID NO.3, specifically as follows:

[0049] ATGGCAGCAGCAGTAGCAGCAAGCATGTCAAGCCCAGTAGCAATGGCTTTGGTTGATGAAAGAATGAGCACTGAAGGAACAGGGCTACCATTTGGACTCAGCAACAATCTTCTTGGTTGGATCCTTTTAGGTGTATTTGGTTTGATTTGGTCTCTCTACACTGTTTACACTTCTGGACTTGATGAAGATGAAGAATCTGGAATGTCTCTTTAA。(SEQ IDNO.3)

[0050] Subcellular localization analysis showed that PsbW-LIKE was mainly distributed in the endoplasmic reticulum and chloroplasts. The results indicated that the PsbW-LIKE gene was different from the traditional "pseudogene" in that PsbW-LIKE had very important functions and a unique mechanism of action.

[0051] To further analyze the function of PsbW-LIKE, the present invention obtained mutant lines of the tomato PsbW-LIKE gene using the Crisper Cas9 technology. These mutants were significantly intolerant to high temperature, had low photosynthetic capacity under high temperature stress, and severe degradation of the photosystem II supercomplex. At the same time, in the present invention, using a plant expression vector, tomato overexpression lines of SlPsbW-LIKE driven by the 35S promoter and tomato overexpression lines of SlPsbW-LIKE with a chloroplast signal peptide (CTP) driven by the 35S promoter were obtained respectively. Phenotypic analysis showed that the heat tolerance of these two overexpression lines was stronger than that of the wild-type control plants.

[0052] The above results indicated that PsbW-LIKE could maintain the stability of the photosystem II supercomplex in tomatoes under high temperature stress, thereby maintaining the photosynthetic capacity of tomatoes. Further research found that increasing the expression of PsbW-LIKE in tomatoes or spraying the PsbW-LIKE protein with a chloroplast signal peptide in vitro could improve the high temperature resistance and photosynthetic capacity of tomatoes. Thus, the present invention provided a feasible strategy for improving the heat tolerance of tomato vegetable crops from the perspective of photosynthesis.

[0053] Based on the above research findings, the protection scope of the present invention also includes: the functions of DNA fragments homologous to the PsbW-LIKE gene, as long as the proteins encoded by them are functionally equivalent to the protein shown in SEQ ID NO.2. The "functionally equivalent to the protein shown in SEQ ID NO.2" referred to herein means that the protein encoded by the target DNA fragment is the same as or similar to the protein shown in SEQ ID NO.2 of the present invention in terms of biological functions and physiological and biochemical characteristics. The typical biological function of the protein shown in SEQ ID NO.2 is to improve the high-temperature resistance and photosynthetic ability of tomatoes.

[0054] These DNA fragments homologous to the PsbW-LIKE gene include allelic genes, homologous genes, mutant genes, and derivative genes corresponding to the nucleotide sequence (SEQ ID NO.3) of the present invention; the proteins encoded by them are similar to the protein shown in SEQ ID NO.2 of the present invention, or there are one, several, or dozens of amino acid substitutions, deletions, or insertions, which all belong to the content of the present invention.

[0055] Another object of the present invention is to provide a method for regulating the heat tolerance and photoprotective ability of plants by changing the expression level of the SlPsbW-LIKE gene in plants, and this regulation can be to reduce or increase the expression level of SlPsbW-LIKE.

[0056] The SlPsbW-LIKE gene in the present invention can be used in the field of plant genetic engineering. Since the increase or deletion of this gene can lead to the up-regulation or down-regulation of the heat tolerance and photoprotective ability of tomato plants. Therefore, heat-resistant breeding of vegetable crops such as tomatoes can be carried out by increasing the expression level of the SlPsbW-LIKE gene in plants to improve the heat tolerance of plants. In this way, the current situation of difficult over-summering of vegetable crops such as tomatoes can be improved.

[0057] The SlPsbW-LIKE protein in the present invention can be used in the development of plant heat-resistant amino acid fertilizers. By inducing the expression of the SlPsbW-LIKE protein described in the present invention or the SlPsbW-LIKE protein with a chloroplast signal peptide in Escherichia coli, the aqueous solution rich in the SlPsbW-LIKE protein or the SlPsbW-LIKE protein with a chloroplast signal peptide obtained is sprayed on tomato plants. The heat tolerance of this plant is significantly stronger than that of the control tomato plants, and it has better photosynthetic ability. The specific application can be to obtain the SlPsbW-LIKE protein or the SlPsbW-LIKE protein with a chloroplast signal peptide, and mix it with other macro- or trace elements that are beneficial to plant growth, and use this as a fertilizer to spray vegetable crops such as tomatoes to improve their heat tolerance and photoprotective ability under high-temperature stress.

[0058] To enable those skilled in the art to more clearly understand the technical solution of this application, the technical solution of this application will be described in detail below in combination with specific embodiments.

[0059] The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels. The experimental methods without specific conditions are carried out according to conventional test methods or the operation manuals recommended by the suppliers.

[0060] Example 1: Functional identification of tomato PsbW-LIKE

[0061] 1. Experimental method:

[0062] 1.1. Obtaining of PsbW-LIKE mutant lines

[0063] The coding sequence of PsbW-LIKE (numbered Solyc09g065910 in the https: / / phytozome.jgi.doe.gov / pz / portal.html database) was obtained through BLAST analysis. The target sequences of Crisper Cas9 designed through https: / / crispr.cos.uni-heidelberg.de are as follows:

[0064] AGCCCAGTAGCAATGGCTT; (SEQ ID NO.4)

[0065] AATGAGCACTGAAGGAACA. (SEQ ID NO.5)

[0066] And they were constructed onto the pHSE401 expression vector (this expression vector is described in the journal article "A CRISPR / Cas9 toolkit for multiplex genome editing in plants", and the public can obtain it from the applicant for repeating this experiment). The above vector was transformed into the Agrobacterium strain LBA4404 by the freeze-thaw method, and then transferred into tomato (Micro-TOM) by the Agrobacterium-mediated leaf disc method to obtain the mutant lines of PsbW-LIKE. The obtained lines were identified by DNA sequencing and Western blot experiments.

[0067] 1.2. Subcellular localization analysis of PsbW-LIKE

[0068] The full-length coding sequence of PsbW-LIKE (excluding the terminator) was constructed into the pZP211-GFP plant expression vector and transformed into the Agrobacterium strain GV3101 by the freeze-thaw method for transient transformation of tobacco experiments. The specific transient transformation operation was referred to Zhuang et al., (2020). Two days after transformation, the tobacco plants were placed in a high-temperature incubator for cultivation, and then a part of the lower epidermis of the leaves was taken for GFP fluorescence observation. Another part was used to extract chloroplast proteins and cytoplasmic (including endoplasmic reticulum) proteins to detect the localization of the PsbW-LIKE-GFP fusion protein in plant cells.

[0069] 1.3 Analysis of high-temperature resistance of PsbW-LIKE mutant lines

[0070] The obtained PsbW-LIKE mutant lines and wild-type lines were subjected to a high-temperature treatment at 40 °C to observe the phenotypes. Mild blue native polyacrylamide gel electrophoresis (BN-PAGE) experiments were performed on the plant leaves before and after the treatment to analyze the damage degree of each photosynthetic membrane complex and the degradation degree of the core proteins; in addition, the photosynthetic rates and the maximum photochemical efficiency of photosystem II (Fv / Fm) of each line before and after the high-temperature treatment were measured. The measurement methods were referred to “A chloroplast-targeted DnaJ protein contributes to maintenance of photosystem II under chilling stress”. The net photosynthetic rate (Pn) was measured by an open-gas portable photosynthesis system (CIRAS-3, PP Systems, Herts, UK), and Fv / Fm was measured by an FMS-2 pulse-activated modulation fluorometer (Hansatech, Cambridge, UK).

[0071] 2. Planting method: Tomatoes (Micro-TOM) and tobacco (Nicotiana benthamiana) were both planted in the greenhouse. The sunshine time was 16 hours, the dark culture time was 8 hours, the culture temperature was normal temperature 25 °C, and the air humidity was 60 - 70%; the E-41L2 type Percival incubator was used for the high-temperature treatment of tomatoes and tobacco.

[0072] The greenhouse is located in the State Key Laboratory of Crop Biology, Shandong Agricultural University.

[0073] 3. Experimental results

[0074] The results are shown in Figure 1, Figure A shows that PsbW-LIKE is mainly distributed in the endoplasmic reticulum and chloroplasts. Figure B confirmed the results of Figure A again and showed that two protein bands with different molecular weights were detected in the cytoplasmic (including endoplasmic reticulum) proteins, and the large band was the same size as the PsbW-LIKE protein detected in the chloroplasts. Figure C shows that two mutant lines (psbw-like-1 / 2) of PsbW-LIKE were obtained by Crisper-Cas9 technology. The sequencing identification results showed that deletions occurred at different target sites in psbw-like-1 / 2, resulting in premature termination; Figure D shows the protein level identification of psbw-like-1 / 2. This experiment showed that a large molecular weight PsbW protein was still present in the chloroplasts of the psbw-like lines, and its content gradually decreased with the progress of high temperature treatment. No PsbW protein was detected in the cytoplasm (including endoplasmic reticulum). Figure E shows a comparative analysis of psbw-likes and wild-type tomatoes before and after high temperature treatment, showing that under normal growth conditions, the psbw-likes mutants did not show obvious phenotypes, but they were extremely intolerant to high temperature stress. Figure F shows a blue native polyacrylamide gel electrophoresis (BN-PAGE) experiment, and the results showed that the amount of PSII supercomplex in the psbw-like mutants was significantly less than that of the wild type before high temperature treatment, and this phenomenon was further amplified after high temperature stress. Correspondingly, the contents of D1 and CP43 proteins also showed the same trend. Figures G and H respectively show that the maximum photochemical efficiency (Fv / Fm) and photosynthetic rate (Pn) of the PSII in the psbw-like lines were lower than those of the wild type before high temperature treatment, and high temperature treatment also amplified these differences.

[0075] The above experimental results indicate that the deletion of the PsbW-LIKE gene leads to the dissociation of the PSII supercomplex in tomatoes, thereby reducing the photosynthetic capacity and high temperature resistance of the plant under high temperature stress.

[0076] Example 2: Increasing the content of PsbW-LIKE in vivo improves the high temperature resistance of tomato plants

[0077] 1. Experimental method:

[0078] 1.1 Obtaining two overexpression lines of PsbW-LIKE

[0079] The full-length coding sequence of PsbW-LIKE (shown in SEQ ID NO.3) and the full-length coding sequence of PsbW-LIKE with the gene fragment (CTP) of the chloroplast signal peptide of tomato-encoded Rubisco small subunit RbcS1 at the N-terminus (shown in SEQ ID NO.6) were inserted into the plant expression vector pZP211-GFP (provided by Shandong Agricultural University) by homologous recombination ligation method, respectively, to obtain two plant expression vectors, PsbW-LIKE-GFP and CTP-PsbW-LIKE-GFP. Among them, CTP-PsbW-LIKE-GFP was first confirmed whether it could enter the chloroplast through CTP by tobacco transient transformation experiment. The above vectors and the pZP211-GFP empty vector were transformed into the Agrobacterium strain LBA4404 by the freeze-thaw method, and then transferred into tomato (Micro-TOM) by the Agrobacterium-mediated leaf disc method to obtain two overexpression lines of PsbW-LIKE (WT(PsbW-L), WT(C-PsbW-L)) and the empty vector control line (WT(GFP)). The obtained lines were identified by Qrt-PCR, fluorescence observation and BN-PAGE combined with Western blot analysis.

[0080] Given the complexity of the mechanism of PsbW-LIKE entering the chloroplast in tomato, we inserted the sequence encoding the chloroplast signal peptide of Rubisco small subunit RbcS1 at the N-terminus of PsbW-LIKE, a common mechanism for entering the chloroplast, which is beneficial for it to enter the chloroplast more efficiently and play its role. The full-length coding sequence of PsbW-LIKE with the gene fragment (CTP) of the chloroplast signal peptide of tomato-encoded Rubisco small subunit RbcS1 at the N-terminus is as follows:

[0081] ATGGCTTCCTCAGTTCTTTCCTCAGCAGCAGTTGCCACCCGCAGCAATGTTGCTCAAGCTAACATGGTTGCACCTTTCACTGGTCTTAAGTCAGCTGCCTCGTTCCCTGTTTCAAGGAAGCAAAACCTTGACATCACTTCCATTGCCAGCAACGGCGGAAGAGTGCAAGCAGCAGCAGTAGCAGCAAGCATGTCAAGCCCAGTAGCAATGGCTTTGGTTGATGAAAGAATGAGCACTGAAGGAACAGGGCTACCATTTGGACTCAGCAACAATCTTCTTGGTTGGATCCTTTTAGGTGTATTTGGTTTGATTTGGTCTCTCTACACTGTTTACACTTCTGGACTTGATGAAGATGAAGAATCTGGAATGTCTCTTTAA. (Shown as SEQ ID NO.6)

[0082] 1.2 Analysis of high temperature resistance of PsbW-LIKE mutant lines

[0083] The obtained WT (PsbW-L), WT (C-PsbW-L), and WT (GFP) lines with consistent expression levels and the wild-type line were placed under a high temperature treatment of 42 °C, and their phenotypes were observed. Mild blue native polyacrylamide gel electrophoresis (BN-PAGE) experiments were performed on the plant leaves before and after the treatment to analyze the damage degree of each photosynthetic membrane complex and the degradation degree of the core proteins; in addition, the maximum photochemical efficiency (Fv / Fm) of photosystem II of each line before and after the high temperature treatment was measured.

[0084] 2. Planting method: Tomatoes (Micro-TOM) and tobacco (Nicotiana benthamiana) were both planted in the greenhouse with a 16-hour sunshine time, 8-hour dark culture, a normal temperature of 25 °C, and an air humidity of 60 - 70%; an E-41L2 type Percival incubator was used for the high temperature treatment (42 °C) of tomatoes and tobacco. The greenhouse is located in the State Key Laboratory of Crop Biology, Shandong Agricultural University.

[0085] 3. Experimental results

[0086] The results are shown in Figure 2, Panel A shows that CTP-PsbW-LIKE-GFP is highly expressed in tobacco leaves and tomatoes under the drive of the 35S promoter. It was found that this chloroplast signal peptide could successfully import the PsbW-LIKE-GFP fusion protein into the chloroplast for high-level expression. The upper part of Panel B shows the WT(PsbW-L), WT(C-PsbW-L), and WT(GFP) lines with basically the same GFP overexpression levels selected by Qrt-PCR. The lower part shows that the PsbW-LIKE-GFP fusion proteins in the WT(PsbW-L) and WT(C-PsbW-L) lines were successfully integrated into the thylakoid membranes of tomato chloroplasts. And the photosystem II supercomplexes in the WT(PsbW-L) and WT(C-PsbW-L) lines were slightly more abundant than those in the WT(GFP). Panel C shows that under normal growth conditions, the psbw-likes mutant did not show obvious phenotypes, but both the WT(PsbW-L) and WT(C-PsbW-L) lines had stronger high-temperature resistance than the WT(GFP) line. Panel D is a blue native polyacrylamide gel electrophoresis (BN-PAGE) experiment, and the results show that the levels of the PSII supercomplexes in the WT(PsbW-L) and WT(C-PsbW-L) lines were significantly higher than those in the WT(GFP) line after high-temperature treatment. Panel E shows that after high-temperature treatment, the Fv / Fm levels in the WT(PsbW-L) and WT(C-PsbW-L) were higher than those in the WT(GFP) line.

[0087] The above experimental results indicate that increasing the expression of the PsbW-LIKE gene in tomatoes can enhance the stability of the PSII supercomplex under high-temperature stress and improve the high-temperature resistance of tomato plants.

[0088] Example 3: Spraying the crude extract of CTP-PsbW-LIKE to improve the high-temperature resistance of tomato plants

[0089] 1. Experimental method:

[0090] 1.1 Prokaryotic induction expression of CTP-PsbW-LIKE

[0091] CTP-PsbW-LIKE (shown in SEQ ID NO.6) was inserted into the prokaryotic induction expression vector pMal-c2x-mbp (provided by Shandong Agricultural University) by homologous recombination ligation. The vector and the pMal-c2x-mbp empty vector were transformed into Escherichia coli competent BL21. They were induced and cultured for 6 - 9 hours at 37°C in a constant temperature shaker with 1 mM IPTG. Then they were ultrasonically disrupted in a low-temperature environment, and the supernatant was collected for protein gel electrophoresis to detect whether the target protein was obtained.

[0092] 1.2 Detection of the effect of spraying the crude extract of CTP-PsbW-LIKE on the high-temperature resistance of tomatoes

[0093] The obtained crude extract of CTP-PsbW-LIKE-mbp (induced at 37 °C for 4 h), the crude extract of the mbp empty vector, and water were respectively sprayed on the wild-type tomato lines, and high-temperature treatment was carried out on them, and the phenotypes were observed. CK was the blank control (not treated).

[0094] 2. Planting method: Wild-type tomatoes (Micro-TOM) were planted in a greenhouse with a 16-hour sunshine time, 8-hour dark culture, a normal temperature of 25 °C, and an air humidity of 60-70%; the E-41L2 type Percival incubator was used for the high-temperature treatment of tomatoes. The greenhouse was located in the State Key Laboratory of Crop Biology, Shandong Agricultural University.

[0095] 3. Experimental results

[0096] The results are shown in Figure 3 , Figure A shows that CTP-PsbW-LIKE-mbp was successfully induced to express, and when the induction temperature was 37 °C, the induction effect was better. Figure B shows that the heat tolerance of tomato plants sprayed with the crude extract of CTP-PsbW-LIKE was significantly stronger than that of the two control plants.

[0097] The above experimental results show that spraying the crude extract of CTP-PsbW-LIKE can enhance the high-temperature resistance of tomato plants under high-temperature stress.

[0098] The above is only the preferred embodiment of the present application and is not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of overexpressing SlPsbW-LIKE protein in the following (1) or (2): (1) Improving the tolerance of plants to high temperature stress; (2) Improving the photosynthetic capacity of plants in high temperature environment; The SlPsbW-LIKE protein is the protein shown in the following (A1) or (A2): (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing; (A2) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1); The plant is tomato.

2. Use of the coding gene of overexpressing SlPsbW-LIKE protein in the following (1) or (2): (1) Improving the tolerance of tomato to high temperature stress; (2) Improving the photosynthetic capacity of tomato in high temperature environment; The nucleotide sequence of the coding gene of SlPsbW-LIKE protein is shown in SEQ ID NO.

3.

3. Use of a recombinant expression vector, transgenic cell line or engineering bacterium containing the coding gene of SlPsbW-LIKE protein in the following (1) or (2): (1) Improving the tolerance of tomato to high temperature stress; (2) Improving the photosynthetic capacity of tomato in high temperature environment; The nucleotide sequence of the coding gene of SlPsbW-LIKE protein is shown in SEQ ID NO.

3.

4. A method for improving the tolerance of tomato to high temperature stress, comprising: The step of overexpressing the SlPsbW-LIKE gene in tomato; the nucleotide sequence of the SlPsbW-LIKE gene is shown in SEQ ID NO.3; Or, the step of spraying SlPsbW-LIKE protein onto tomato plants, and the amino acid sequence of SlPsbW-LIKE protein is shown in SEQ ID NO.

2.

5. The method according to claim 4, characterized in that By exogenous transfer of the SlPsbW-LIKE gene or up-regulating the expression of the SlPsbW-LIKE gene in the tomato genome, the SlPsbW-LIKE gene in tomato is overexpressed.

6. Use of the SlPsbW-LIKE gene or a recombinant expression vector, transgenic cell line or engineering bacterium containing the SlPsbW-LIKE gene in cultivating transgenic tomatoes; The nucleotide sequence of the SlPsbW-LIKE gene is shown in SEQ ID NO.

3.

7. The application according to claim 6, characterized in that Compared with wild-type tomatoes, the transgenic tomatoes cultivated have improved tolerance to high temperature stress and photosynthetic capacity in high temperature environment.

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