CN114752622B

CN114752622B - Application of polypeptide receptor PSKR1 gene in improving high-temperature stress resistance of tomato plants and/or tomato pollen

Info

Publication number: CN114752622B
Application number: CN202210482403.9A
Authority: CN
Inventors: 师恺; 王萍; 罗倩; 方瀚墨
Original assignee: Anqing Mayor's Triangle Future Industry Research Institute; Zhejiang University ZJU
Current assignee: Anqing Mayor's Triangle Future Industry Research Institute; Zhejiang University ZJU
Priority date: 2022-05-05
Filing date: 2022-05-05
Publication date: 2023-09-01
Anticipated expiration: 2042-05-05
Also published as: CN114752622A

Abstract

The invention discloses an application of a PSKR1 gene or a protein encoded by the PSKR1 gene in improving high-temperature stress resistance of tomato plants and/or tomato pollen, wherein the nucleotide sequence of a protein encoding region of the PSKR1 gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein is shown as SEQ ID NO. 2. The PSKR1 gene can interact with HsfA1a to regulate and control the resistance of tomato plants and/or tomato pollen to high-temperature stress, the PSKR1 gene overexpression vector is constructed by a transgenic technology, and the tissue culture technology is utilized to screen the obtained PSKR1 gene overexpression homozygous strain OE, wherein the PSKR1-1 and OE are obvious high-temperature stress resistance compared with a wild type control group, so that the over-expression PSKR1 gene can improve the resistance of tomato plants and/or tomato pollen to high-temperature stress, and important gene resources are provided for cultivating high-temperature resistant tomato varieties.

Description

Application of polypeptide receptor PSKR1 gene in improving high-temperature stress resistance of tomato plants and/or tomato pollen

Technical Field

The invention relates to the technical field of biology, in particular to application of a polypeptide receptor PSKR1 gene in improving high-temperature stress resistance of tomato plants and/or tomato pollen.

Background

Tomato (Solanum lycopersicum l.) is one of the most widely cultivated vegetables and commercial crops in the world, belonging to the genus solanum of the family solanaceae. The food has delicious taste, is rich in vitamins, mineral elements and carotenoid, has high nutrition and health care value, and is widely popular with consumers. Tomatoes are heat-loving and heat-labile crops, the optimal growth temperature is 20-25 ℃, but with global warming, the greenhouse effect is increased, the controllability of the cultivation environment is poor, and the like, the tomato crops in summer production usually suffer from high-temperature invasion. Tomatoes are very sensitive to high temperature, and the high temperature in the nutrition growth period can cause the phenomena of remarkable inhibition of photosynthesis, increase of water loss rate and the like; the high temperature in the reproductive growth period can cause pollen abortion, flower and fruit dropping and the like, and seriously affect the yield and quality of tomatoes. At various stages of crop growth and development, the pollen development process is most sensitive to high temperature stress, so that the high temperature response mechanism of pollen is a hot spot in current botanic research. The high temperature affects the growth of pollen tubes in the stigma by changing the balance of auxin and sugar in pollen and stigma cells, showing pollen germination failure and pollen tube end swelling, twisting and cracking, etc., eventually leading to failure of fertilization (Shi et al, "Pollen germination and in vivo fertilization in response to high-temperature during flowering in hybrid and inbred rice", plant Cell Environment,2018, 41:1287-1297). In addition, elevated temperatures can also promote premature programmed death of tapetum cells, which in turn affects pollen development, activity, and pollen scattering processes (De Storm et al, "The impact of environmental stress on male reproductive development in plants: biological processes and molecular mechanisms" Plant Cell Environment,2018, 37:1-18). The response and regulation mechanism of tomato plants and pollen thereof to high temperature are explored, so that the high temperature resistance of tomatoes in the nutrition and reproductive growth period is improved, the yield and quality of tomatoes are improved, and the annual balanced supply is ensured.

PSKR1 (Solyc 01g 008140) is a major receptor gene for plant polypeptide PSK, which is a novel peptide hormone that has been recently discovered to play a versatile role in plant growth and development and resistance. The mature plant polypeptide PSK on the cell surface can be recognized by a transmembrane receptor PSKR located on the cell membrane surface and transmits PSK signals into the cell. PSKR1 possesses a conserved extracellular LRR domain, transmembrane domain and intracellular kinase domain (Amano Y et al, "Tyrosine-sulfated glycopeptide involved in cellular proliferation and expansion in Arabidopsis." Proc Natl Acad Sci USA,2007, 104, 18333-18338). Previous researches show that PSKR1 gene is involved in regulating the growth and development of plants, biotic stress resistance and the like, and the regulation of the PSKR1 gene in abiotic resistance is rarely studied.

The family of heat shock transcription factors (Heat shock transcription factors, hsfs) protects the body from heat stress through transcriptional regulation. The HsfA1 subfamily gene is the main effective regulating factor responding to high temperature stress, the slHsfA1 gene is discovered in tomatoes for the first time, the high temperature resistance of tomato plants is high when the slHsfA1 gene is overexpressed, and the co-suppression transgenic plants are very sensitive to high temperature stress (Mishra et al, "In the complex family of heat stress transcription factors, hsfA1 has a unique role as master regulator of thermotolerance in tomato", genes and development,2002, 16:1555-1567). Trimer formation of tomato HsfA1a, hsfA2 and HsfB1 may enhance response to different stages of heat stress through interaction with molecular chaperones (Scharf et al, "The plant heat stress transcription fator (Hsf) family: structure, function and evolution", biophysica Acta,2012, 1819:104-119.). SlHsfA2 expression in tomato anthers is upregulated during pollen formation, thereby alleviating pollen sensitivity to heat stress (Frankostefanakis et al, "HsfA2 controls the activity of developmentally and stress-regulated heat stress protection mechanisms in tomato male reproductive tissues", plant Physiology,2016, 170:2461-1477.). Therefore, the plant HSF gene is an important transcription factor for regulating the expression of various stress response genes, and plays an important role in various abiotic stress resistances and plant growth and development.

In recent years, biotechnology development is gradually changed, and the establishment of DNA recombination technology, especially the application of gene overexpression technology, can exert the function of genes to the greatest extent, and reserve more resistant germplasm resources for solving the problems of environmental deterioration, resource shortage and the like faced by human beings.

The Chinese patent document with publication number of CN108841841A discloses cloning of a tomato transcription factor SlbZIP6 and application thereof in high temperature stress resistance, and results prove that the SlbZIP6 participates in a heat resistance regulation mechanism of tomatoes, and the heat resistance of the overexpressed SlbZIP6 tomatoes is poorer than that of wild tomatoes. The gene expression level is detected and used for screening heat-resistant breeding of tomatoes.

Disclosure of Invention

Experiments show that the polypeptide receptor PSKR1 gene can regulate and control the resistance of tomato plants and/or tomato pollen to high-temperature stress through interaction with a heat shock transcription factor HsfA1a, and can relieve plant leaf curl, photosynthetic damage, pollen abortion and the like caused by high temperature.

Based on the findings, the invention provides the application of the PSKR1 gene in improving the high-temperature stress resistance of tomato plants and/or tomato pollen, so as to provide a basis for cultivating high-temperature resistant tomato varieties.

The technical scheme adopted is as follows:

the application of the PSKR1 gene in improving the high-temperature stress resistance of tomato plants and/or tomato pollen is provided, and the nucleotide sequence of a protein coding region of the PSKR1 gene is shown as SEQ ID NO. 1.

The inventor finds through experiments that the PSKR1 gene can interact with a heat shock transcription factor HsfA1a, the heat shock transcription factor can enhance the resistance of plants and pollen to high-temperature stress, so that the PSKR1 gene can interact with HsfA1a to regulate and control the resistance of tomato plants and/or tomato pollen to high-temperature stress, and the sequence of a coding region of the heat shock transcription factor HsfA1a is shown as SEQ ID No. 5.

The PSKR1 gene is located on chromosome 1 of tomato, the length of a protein coding region is 2904bp, and the PSKR1 gene codes a transmembrane protein which consists of 967 amino acids.

The invention also provides application of the protein encoded by the PSKR1 gene in improving the high-temperature stress resistance of tomato plants and/or tomato pollen, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

The invention also provides a method for improving the high-temperature stress resistance of tomato plants and/or tomato pollen, wherein the PSKR1 gene is overexpressed in the tomato plants, and the nucleotide sequence of a protein coding region of the PSKR1 gene is shown as SEQ ID NO. 1.

The method specifically comprises the following steps:

(1) Constructing a vector for over-expressing PSKR1 genes;

(2) Constructing agrobacterium genetically engineered bacteria containing the vector for over-expressing PSKR1 gene in the step (1);

(3) And (3) transforming the agrobacterium genetic engineering bacteria in the step (2) into tomato tissues, and screening and culturing to obtain transgenic plants.

The PSKR1 gene overexpression vector is constructed by a transgenic technology, and a tissue culture technology is utilized to screen and obtain a PSKR1 gene overexpression homozygous strain; experiments show that the over-expression of the PSKR1 gene can improve the resistance of tomato plants and/or tomato pollen to high-temperature stress.

The vector is an expression vector pFGC1008-3HA with a 35S promoter.

Preferably, in the step (1), an upstream primer and a downstream primer are selected, PCR amplification is carried out by taking tomato cDNA as a template, and amplified products are connected to an expression vector pFGC1008-3HA after enzyme digestion and conversion to construct a vector for over-expressing PSKR1 genes.

The nucleotide sequence of the upstream primer is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4.

Preferably, the agrobacterium genetically engineered bacterium is an agrobacterium tumefaciens GV3101 strain.

Compared with the prior art, the invention has the beneficial effects that:

(1) The invention provides a cultivation method of high-temperature-resistant tomato germplasm by utilizing a transgenic technology, and a high-temperature-resistant transgenic homozygous plant is obtained.

(2) Compared with a control group, the cultured OE PSKR1-1 and OE PSKR1-2 transgenic tomatoes have obvious resistance to high-temperature stress, which shows that the over-expression PSKR1 gene can improve the resistance of tomato plants to high-temperature stress, and meanwhile, the pollen activity and pollen germination conditions after high-temperature treatment are observed, so that the over-expression PSKR1 gene can improve the resistance of tomato pollen to high-temperature stress.

Drawings

FIG. 1 is a Western blot verification HA tag protein map after PSKR1 over-expression plants in example 1 were successfully constructed. RBC is Rubisco and Ponc is ponceau stain for characterization of protein loading.

FIG. 2 is a phenotype diagram of wild type WT and PSKR1 gene over-expressed plant OE in example 2, PSKR1-1 at normal temperature and high temperature, PSII maximum photochemical efficiency and relative conductivity, wherein A is the phenotype diagram, B is the PSII maximum photochemical efficiency comparison diagram, and C is the relative conductivity comparison diagram; a, b represent a significant difference at the 5% level.

FIG. 3 is a phenotype diagram, PSII maximum photochemical efficiency and relative conductivity of a plant subjected to a plant polypeptide PSK exogenous treatment experimental group and a pure water treatment control group in example 3 under normal temperature and high temperature conditions, wherein A is the phenotype diagram, B is the PSII maximum photochemical efficiency comparison diagram, and C is the relative conductivity comparison diagram; a, b represent a significant difference at the 5% level.

FIG. 4 shows the pollen viability and pollen germination patterns of the wild type WT and overexpressed OE PSKR1-1 plants of example 4 under high temperature conditions, wherein A is the pollen viability FDA staining pattern, B is the pollen viability statistics pattern, C is the pollen in vitro germination aniline blue staining pattern, and D is the pollen in vitro germination rate statistics; a, b represent a significant difference at the 5% level.

FIG. 5 is a graph showing the results of yeast two-hybrid in example 5. Wherein pBD-PSKR1 intracellular +pAD represents that pGBKT7 plasmid and pGADT7 plasmid containing PSKR1 gene intracellular region are co-transformed into yeast competent cells; pBD-PSKR1 intracellular+pAD-HsfA 1a indicates that pGBKT7 plasmid containing the intracellular region of PSKR1 gene and pGADT7 plasmid containing HsfA1a are co-transformed into yeast competent cells; SD-Trp-Leu means a yeast solid medium lacking both Trp and Leu amino acids; SD-Trp-Leu-His-Ade represents a yeast solid medium lacking Trp, leu, his and Ade amino acids; if the yeast is grown on a yeast medium lacking amino acids, this indicates that there is an interaction between the two proteins.

Detailed Description

The invention is further elucidated below in connection with the drawings and the examples. It is to be understood that these examples are for illustration of the invention only and are not intended to limit the scope of the invention.

Example 1

Construction and identification of PSKR1 gene overexpression vector and tomato plant

The coding sequence of the PSKR1 gene (Solyc 01g 008140) is obtained according to a tomato genome database (https:// solgenomics. Net /), the nucleotide sequence of a protein coding region is shown as SEQ ID NO.1, and the amino acid sequence of a protein coded by the PSKR1 gene is shown as SEQ ID NO. 2. Referring to the Gateway handbook, the upstream Primer (SEQ ID NO. 3) was designed using Primer5 software: 5'> TTggcgcgccATGGTGATTTGGGAGTTTCT >3' and a downstream primer (SEQ ID NO. 4) 5'> CGGggtaccCCTCTCCTTTACACTTGCGATTG >3'.

Extracting RNA of tomato leaves, obtaining cDNA through reverse transcription, using cDNA as a template, utilizing an upstream primer and a downstream primer to amplify the full length of a PSKR1 gene coding region of the tomato, and connecting an amplified product to an expression vector pFGC1008-3HA with a 35S promoter after enzyme digestion and conversion, wherein the expression vector pFGC1008-3HA specifically comprises: 3HA tag proteins are added into the overexpression vector pFGC1008, and the HA tag proteins are plant selectable markers, so that identification and screening of transgenic tomato plants are facilitated. After the recombinant plasmid is successfully constructed, the recombinant plasmid is transformed into an agrobacterium tumefaciens GV3101 strain through electric shock, then transformed agrobacterium is used for infecting tomato cotyledons, tissue culture is carried out, OE: PSKR 1T 0 generation plants are obtained through screening, protein is extracted from tender leaf blades, western blot verification is carried out, T0 generation strains with target bands are used for continuous seed reproduction screening, and two homozygous T2 generation strains OE: PSKR1-1 and OE: PSKR1-2 are obtained (shown in figure 1).

Example 2

High temperature stress resistance research of PSKR1 gene over-expression plants

Placing tomato seedlings with the size of 3-4 weeks into a climatic incubator, and illuminating for 12 hoursDark, light intensity 200. Mu. Mol m ^-2 s ^-1 The temperature is set to be 45 ℃ high temperature treatment or 25 ℃ normal temperature treatment, and water is timely supplemented during the treatment, so that drought of seedlings is prevented. Two days later, wild type control plants WT were found to be more severe than PSKR1 gene over-expressed plants OE PSKR1-1 and OE PSKR1-2 wilting at high temperature, representative plants were selected for photographing, and tomato plants PSII maximum photochemical efficiency (Fv/Fm) and relative conductivity were determined.

Wherein, the method for measuring the maximum photochemical efficiency (Fv/Fm) of the plant PSII comprises the following steps: after tomato plants treated at different temperatures are placed in a dark environment to adapt to 30min, detection light is irradiated by using an Imaging-PAM modulation fluorescence Imaging system produced by Heinz-Walz company of Germany<0.5μmol m ^-2 s ^-1 ) Minimum fluorescence Fo was obtained, and saturated pulse light (4000. Mu. Mol m) ^-2 s ^-1 ) The maximum fluorescence Fm is obtained. The fluorescence parameter calculation method comprises the following steps: fv/fm= (Fm-Fo)/Fm.

Relative conductivity measurement: 0.3g of tomato leaf is weighed and dH is used ₂ O cleaning leaf, removing main vein, and cutting into 1cm ² Is placed in a 50mL centrifuge tube, 20mL dH is added ₂ O. The conductivity EL1 was measured after shaking at 200rpm for 2 hours at room temperature. The sample was cooled to room temperature in a 95℃water bath for 15min to determine the conductivity as EL2. Relative conductivity EL (%) =el1/el2×100.

The results show that PSKR1 gene overexpression can significantly improve the resistance of tomatoes to high temperature stress (A in FIG. 2). PSKR1 Gene over-expressed plants OE PSKR1-1 PSII maximum photochemical efficiency was significantly higher than that of the same condition control treated WT tomato plants (B in FIG. 2) after high temperature stress, while high temperature mediated relative conductivity improvement was effectively reduced (C in FIG. 2).

Example 3

Influence of exogenous treatment of plant polypeptide PSK on plant high-temperature stress resistance

0.85g PSK is slowly added into 100mL of water, and the mixture is stirred until the mixture is fully dissolved to obtain a mixed solution; to 100mL of the mixed solution, 9.9L of water and 2.5mL of organic silicon were added successively, and the mixture was mixed uniformly to prepare a diluted solution (preparation working solution) for use.

The preparation working solution is uniformly sprayed Shi Miaoling on the surfaces of five-leaf and one-heart tomato leaves until the two-face leaves are completely wet and the preparation working solution does not flow down in a water drop shape, and is sprayed for 1 time at 6 points a day for 3 days, and tomato plants sprayed with clear water are used as a control.

And (3) carrying out different temperature treatments on the tomato plants subjected to PSK exogenous spraying and control treatment. Tomato plants are placed in a climatic incubator with light intensity of 200 mu mol m ^-2 s ^-1 The temperature is set to 45 ℃ high temperature treatment or 25 ℃ normal temperature treatment. And water is timely supplied during the period, so that drought and water shortage of plants are prevented. After 10 hours of treatment at different temperatures, the tomato plants of the high temperature treatment group and the normal temperature treatment group were compared, a plant temperature response phenotype map was photographed, and the maximum photochemical efficiency (Fv/Fm) and the relative conductivity of the tomato plants PSII were determined.

The results show that the exogenous treatment of the plant polypeptide PSK can significantly improve the resistance of tomatoes to high-temperature stress (A in figure 3). PSK exogenously treated tomato plants subjected to high temperature stress have a PSII maximum photochemical efficiency (Fv/Fm) significantly higher than that of control treated tomato plants (B in FIG. 3); while exogenous treatment of PSK effectively reduces the high temperature-mediated increase in relative conductivity (C in fig. 3).

Example 4

High temperature stress resistance research of PSKR1 gene over-expression plant pollen

About 10d of tomato flowering is the meiotic phase of microsporocytes, which is considered the most sensitive developmental stage to high temperature stress. And (3) the wild type plants with inflorescences and PSKR1 gene over-expression plants are subjected to stress treatment at the high temperature of 39 ℃ for 3 hours and then are placed under normal culture conditions again for growth, and the pollen viability and pollen germination are measured until flowering.

The pollen viability detection method comprises the following steps: the activity of tomato pollen was detected by using fluorescein diacetate solution (Fluorescein diacetate, FDA) staining method. At 2mg mL ^-1 The FDA acetone solution of (C) was used as a mother solution and stored at 4℃in the dark, and a 100-fold diluted solution of 0.5M sucrose solution was used as a working solution. Collecting the flowers of tomato just blooming in the morning, uniformly dispersing pollen in anther on a glass slide dripped with FDA working solution, keeping moisture at 28deg.C for 1 hr, and coveringSlide, observed under fluorescence microscope (Leica) and photographed for statistics.

The method for detecting the germination rate of pollen in vitro comprises the following steps: in vitro germination of pollen, aniline blue (Sigma-aldrich solution) is adopted to dye a growing pollen tube, 0.1% aniline blue solution is used as working solution, flowers just bloomed in the morning of tomatoes are taken, pollen in anthers is evenly scattered on a glass slide dripped with a semi-solid culture medium for in vitro germination of pollen, after light-shielding and moisture-preserving culture for 1h at 28 ℃,20 mu L of 0.1% aniline blue solution is dripped for 5min, a cover slip is covered, and observation and photographing statistics are carried out under a fluorescence microscope (Leica).

As shown in A-D of FIG. 4, the pollen viability and pollen germination rate of the high temperature PSKR1 gene over-expressed plants OE, PSKR1-1 were higher than those of the wild-type WT.

Example 5

The intracellular fragment of the protein coding region of the PSKR1 gene (SEQ ID NO. 6) was amplified and recombined into pGBKT7 vector, hsfA1a fragment and pGADT7 vector, respectively, using control tomato cDNA as template. The pGBKT7 and pGADT7 strain described above were co-transformed into the yeast AH109 strain.

The specific operation steps are as follows: AH109 single colonies on YPAD medium were picked up into 3mL YPAD, shake-cultured at 250rpm for 30min for 8h, and about 200. Mu.L of the bacterial liquid was transferred to a 50mL centrifuge tube containing 20mL YPAD, and cultured overnight under the same conditions. When the OD600 value reached between 0.6 and 0.8, the supernatant was discarded by centrifugation at 4000rpm for 5 min. The supernatant was discarded by re-suspension with TE/LiAc, centrifugation at 4000rpm for 5 min. Re-suspending with TE/LiAc to obtain yeast competence. The recombinant pGBKT7 and pGADT7, salmon sperm DNA, PEG/LiAc were mixed with yeast competence, allowed to stand at 30℃for 30min, and 60. Mu.L of dimethyl sulfoxide was added. Placing in a water bath at 42deg.C for 15min on ice for 5min, centrifuging to remove supernatant, and adding 100 μl of sterile ddH ₂ O, smearing on an auxotroph solid medium SD-Trp-Leu, and culturing at 30 ℃ for 3-4 days. The monoclonal yeast successfully transferred into the two recombinant plasmids is selected and cultured by using SD-Trp-Leu liquid culture medium until the OD600 value reaches between 0.6 and 0.8, centrifuged by using 4000rpm for 5min, and the supernatant is discarded. By aseptic ddH ₂ O was resuspended until the OD600 reached 0.3 and diluted 10-fold and 100-fold, respectively. Absorbing 10 mu L of bacterial liquid, respectively dripping the bacterial liquid into SD-Trp-Leu and SD-Trp-Leu-His-Ade for immobilizationThe results were observed after 3-4 days of culture in the body culture medium at 30 ℃.

As shown in FIG. 5, yeast colonies grew well on both SD-Trp-Leu and SD-Trp-Leu-His-Ade solid media, demonstrating that the PSKR1 gene was able to interact with the heat shock transcription factor HsfA1 a.

While the foregoing embodiments have been described in detail in connection with the embodiments of the invention, it should be understood that the foregoing embodiments are merely illustrative of the invention and are not intended to limit the invention, and any modifications, additions, substitutions and the like made within the principles of the invention are intended to be included within the scope of the invention.

Sequence listing

<110> Anqing city long triangle future industry research institute

University of Zhejiang

Application of <120> polypeptide receptor PSKR1 gene in improving high-temperature stress resistance of tomato plants and/or tomato pollen

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Met Gly Val Leu Gln Val Cys Val Ile Phe Leu Phe Leu Gly Ile Cys

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Leu Gln Ala Gln Ser Gln Asn Leu Gln Asn Leu Ile Cys Asn Pro Lys

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Asp Leu Lys Ala Leu Glu Gly Phe Val Lys Ser Leu Glu Thr Val Ile

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Asp Phe Trp Asp Leu Gly Asn Ser Thr Asn Cys Cys Asn Leu Val Gly

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Val Thr Cys Asp Ser Gly Arg Val Val Lys Leu Glu Leu Gly Lys Arg

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Arg Leu Asn Gly Lys Leu Ser Glu Ser Leu Gly Asn Leu Asp Glu Leu

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Arg Thr Leu Asn Leu Ser His Asn Phe Phe Lys Gly Pro Val Pro Phe

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Thr Leu Leu His Leu Ser Lys Leu Glu Val Leu Asp Leu Ser Asn Asn

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Asp Phe Phe Gly Leu Phe Pro Ser Ser Met Asn Leu Pro Leu Leu Gln

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Val Phe Asn Ile Ser Asp Asn Ser Phe Gly Gly Pro Val Pro Leu Gly

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Ile Cys Glu Asn Ser Thr Arg Val Ser Val Ile Lys Met Gly Val Asn

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Tyr Phe Asn Gly Ser Leu Pro Val Gly Ile Gly Asn Cys Gly Ser Leu

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Lys Leu Phe Cys Val Gly Ser Asn Leu Leu Ser Gly Ser Leu Pro Asp

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Glu Leu Phe Lys Leu Ser Arg Leu Thr Val Leu Ser Leu Gln Glu Asn

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Arg Phe Ser Gly Gln Leu Ser Ser Gln Ile Gly Asn Leu Ser Ser Leu

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Val His Leu Asp Ile Cys Ser Asn Gly Phe Ser Gly Asn Ile Pro Asp

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Val Phe Asp Arg Leu Gly Lys Leu Thr Tyr Leu Ser Ala His Ser Asn

260 265 270

Arg Phe Phe Gly Asn Ile Pro Thr Ser Leu Ala Asn Ser Gly Thr Val

275 280 285

Ser Ser Leu Ser Leu Arg Asn Asn Ser Leu Gly Gly Ile Ile Glu Leu

290 295 300

Asn Cys Ser Ala Met Val Ser Leu Val Ser Leu Asp Leu Ala Thr Asn

305 310 315 320

Gly Phe Arg Gly Leu Val Pro Asp Tyr Leu Pro Thr Cys Gln Arg Leu

325 330 335

Gln Thr Ile Asn Leu Ala Arg Asn Ser Phe Thr Gly Gln Leu Pro Glu

340 345 350

Ser Phe Lys Asn Phe His Ser Leu Ser Ser Leu Ser Val Ser Asn Asn

355 360 365

Ser Met His Asn Ile Asp Ala Ala Leu Arg Ile Leu Gln His Cys Lys

370 375 380

Asn Leu Ser Thr Leu Val Leu Thr Leu Asn Phe Arg Asp Glu Glu Leu

385 390 395 400

Pro Thr Asp Ser Ser Leu Gln Phe Ser Glu Leu Lys Ala Leu Ile Ile

405 410 415

Ala Asn Cys Arg Leu Thr Gly Val Val Pro Gln Trp Leu Arg Asn Ser

420 425 430

Ser Lys Leu Gln Leu Leu Asp Leu Ser Trp Asn Arg Leu Ser Gly Thr

435 440 445

Leu Pro Pro Trp Ile Gly Asp Phe Gln Phe Leu Phe Tyr Leu Asp Phe

450 455 460

Ser Asn Asn Ser Phe Thr Gly Glu Ile Pro Lys Glu Ile Thr Arg Leu

465 470 475 480

Lys Ser Leu Ile Ser Ala Ile Leu Pro Glu Phe Gly Asn Leu Lys Arg

485 490 495

Leu His Val Leu Asp Leu Lys Ser Asn Asn Leu Ser Gly Thr Ile Pro

500 505 510

Ser Ser Leu Ser Gly Met Ala Ser Val Glu Asn Leu Asp Leu Ser His

515 520 525

Asn Asn Leu Ile Gly Ser Ile Pro Ser Ser Leu Val Gln Cys Ser Phe

530 535 540

Met Ser Lys Phe Ser Val Ala Tyr Asn Lys Leu Ser Gly Glu Ile Pro

545 550 555 560

Thr Gly Gly Gln Phe Pro Thr Phe Pro Thr Ser Ser Phe Glu Gly Asn

565 570 575

Gln Gly Leu Cys Gly Glu His Gly Ser Thr Cys Arg Asn Ala Ser Gln

580 585 590

Val Pro Arg Asp Ser Val Ala Lys Gly Lys Arg Arg Lys Gly Thr Val

595 600 605

Ile Gly Met Gly Ile Gly Ile Gly Leu Gly Thr Ile Phe Leu Leu Ala

610 615 620

Leu Met Tyr Leu Ile Val Val Arg Ala Ser Ser Arg Lys Val Val Asp

625 630 635 640

Gln Glu Lys Glu Leu Asp Ala Ser Asn Arg Glu Leu Glu Asp Leu Gly

645 650 655

Ser Ser Leu Val Ile Phe Phe His Asn Lys Glu Asn Thr Lys Glu Met

660 665 670

Cys Leu Asp Asp Leu Leu Lys Cys Thr Asp Asn Phe Asp Gln Ser Asn

675 680 685

Ile Val Gly Cys Gly Gly Phe Gly Leu Val Tyr Lys Ala Ile Leu Arg

690 695 700

Asp Gly Arg Lys Val Ala Ile Lys Arg Leu Ser Gly Asp Tyr Gly Gln

705 710 715 720

Met Glu Arg Glu Phe Gln Ala Glu Val Glu Ser Leu Ser Arg Ala Gln

725 730 735

His Pro Asn Leu Val His Leu Gln Gly Tyr Cys Lys Tyr Arg Thr Asp

740 745 750

Arg Leu Leu Ile Tyr Ser Tyr Met Glu Asn Gly Ser Leu Asp Tyr Trp

755 760 765

Leu His Glu Lys Val Asp Gly Pro Ala Leu Leu Asp Trp Asp Leu Arg

770 775 780

Leu Gln Ile Ala Gln Gly Ala Ala Arg Gly Leu Ala Tyr Leu His Leu

785 790 795 800

Ala Cys Glu Pro His Ile Leu His Arg Asp Ile Lys Ser Ser Asn Ile

805 810 815

Leu Leu Asp Glu Asn Phe Glu Ala His Leu Ala Asp Phe Gly Leu Ala

820 825 830

Arg Ile Ile Arg Pro Tyr Asp Thr His Val Thr Thr Asp Val Val Gly

835 840 845

Thr Leu Gly Tyr Ile Pro Pro Glu Tyr Gly Gln Ala Ser Val Ala Thr

850 855 860

Tyr Lys Gly Asp Val Tyr Ser Phe Gly Val Val Leu Leu Glu Leu Leu

865 870 875 880

Thr Cys Lys Arg Pro Met Asp Pro Cys Lys Pro Arg Ala Ser Arg Asp

885 890 895

Leu Ile Ser Trp Val Ile Gln Met Lys Lys Gln Lys Arg Glu Thr Glu

900 905 910

Val Phe Asp Pro Leu Ile Tyr Asp Lys Gln His Ala Lys Glu Met Leu

915 920 925

Leu Val Leu Glu Ile Ala Cys Leu Cys Leu His Glu Ser Pro Lys Ile

930 935 940

Arg Pro Ser Ser Gln Gln Leu Val Thr Trp Leu Asp Asn Ile Asn Thr

945 950 955 960

Pro Pro Asp Val His Val Phe

965

<210> 3

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

ttggcgcgcc atggtgattt gggagtttct 30

<210> 4

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

cggggtaccc ctctccttta cacttgcgat tg 32

<210> 5

<211> 1584

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

atggagccga attcttatgg cagcgggaag gctgctgtcg gtgacggagt aggagcgcca 60

atgttgcaga cggcgccggc gccggcgccg atacccagtg cgaatgcgcc gccgccgttt 120

ctggtgaaga cgtatgatat ggtggatgat ccgagtacgg ataagatcgt gtcgtggagt 180

cctacgaata atagttttgt ggtttgggat cctccggagt ttgctaaaga cctacttccg 240

aagtacttta agcataataa cttctccagc tttgttcggc agctgaatac ttatggtttt 300

agaaaggttg atccagaccg ctgggaattt gctaatgagg gattcttaag aggtcagaag 360

cacctgctta aaagtataag tcgacgtaaa cctgctcatg gacatgctca acaacagcag 420

cagccacatg gaaatgctca acaacagatg cagccacctg gacacagtgc atccgttggg 480

gcttgtgtcg aggttgggaa atttgggctt gaagaagagg ttgagaggct gaaaagggac 540

aagaacgtgc ttatgcaaga gctggttagg ctcagacaac agcagcaagc cactgacaat 600

cagttgcaag ggatggtgca gcgccttcaa ggtatggagc tgcgacaaca acaaatgatg 660

tcgttcctgg caaaagctgt caacaggcct ggattcttgg cacagtttgt tcagcagcaa 720

aatgagagta ataagcgaat agctgaaggc agcaagaaac gaaggattaa gcaggatatt 780

gaatcacagg atccatccgt tactcctgcg gatggacaga ttgttaagta ccaacctggg 840

ataaatgagg cagcaaaggc aatgttgagg gagctttcaa aactagattc atctcctaga 900

ttagataact tcagcaacag tcctgaaagt ttcctgattg gtgatggttc accacagtct 960

aatgcatctt caggtcgtgt ttcgggagtc actcttcagg aggtcccacc aacttctggg 1020

aagcccttgc tgaatacagc ttcagcaatt gcaggtcaaa gtttgttgcc agccacttct 1080

gagatgcagt catctcatct tggcacatgt tccgaaatca tcaacaatca attgtcaaac 1140

ataatcccgt tggtaggagg tgaagatttg catcctggtt cactttctgc atccgatatg 1200

attatgcctg agttatcaca gttgcaagga attttgcctg aaaacaatac agatgtgatt 1260

ggatgtgatt ctttcatgga tactagtgca gttgagggaa aagtgggact agatattatt 1320

ggtagttgtt tgtctcctgg tgctgatatt gactggcaga gtggtttgct ggatgaaata 1380

gaagagtttc ctagtgtggg tgaccctttc tgggaaaagt ttctccaaag cccttgttcc 1440

cctgatgctg caatggatga tgatatttca aacacaagtg aaaccaaacc acaaataaat 1500

ggatgggata aaactcagaa catggaacat cttactgaac aaatgggggc tactaatatc 1560

aaacaacaaa aacatatgat ctaa 1584

<210> 6

<211> 893

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

cgggcaagca gtcgaaaagt agttgatcag gaaaaggagc tggatgcttc taacagggaa 60

ctggaggact tgggctcaag tctggtcata tttttccata acaaggagaa cactaaagag 120

atgtgtcttg atgacctttt gaaatgtact gacaactttg atcaatcaaa tattgttgga 180

tgtggaggct tcggcttggt ctacaaggcc atccttcgtg atggtaggaa agttgccatc 240

aagcggcttt caggtgacta cgggcaaatg gagcgagaat tccaagccga agttgaatca 300

ctttcaagag ctcagcatcc gaatctggtt catcttcaag gatattgcaa gtacagaact 360

gaccggcttc taatttattc ctacatggag aatggaagtt tggattattg gctgcacgag 420

aaagttgacg gacctgcttt attggactgg gatctgaggc ttcaaattgc tcaaggggct 480

gcaagaggac tagcgtactt gcacctagcg tgcgagcctc atatcttgca ccgagatata 540

aagtctagta acattcttct tgacgaaaat ttcgaagctc acttagctga tttcggtctt 600

gcaaggatta ttcggcccta cgacactcat gtgaccactg atgttgtcgg aacattaggc 660

tatatacctc ctgaatatgg ccaagcctcc gtagctacct ataaagggga cgtttatagc 720

tttggtgtgg ttcttttgga gcttctaaca tgcaaaagac cgatggatcc gtgcaagcct 780

agagcaagcc gagatttaat ctcttgggtg atccaaatga agaaacagaa gagggaaact 840

gaagtctttg atcctctgat atatgacaag cagcacgcaa aggaaatgtt att 893

Claims

Application of PSKR1 gene or coded protein thereof in improving high-temperature stress resistance of tomato plants and/or tomato pollen; the nucleotide sequence of the protein coding region of the PSKR1 gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.
2. A method for improving the high-temperature stress resistance of tomato plants and/or tomato pollen is characterized in that PSKR1 genes are overexpressed in tomato plants, and the nucleotide sequence of a protein coding region of the PSKR1 genes is shown as SEQ ID NO. 1.
3. The method according to claim 2, characterized in that it comprises in particular the following steps:

(1) Constructing a vector for over-expressing PSKR1 genes;

(2) Constructing agrobacterium genetically engineered bacteria containing the vector for over-expressing the PSKR1 gene in the step (1);

(3) And (3) transforming the agrobacterium genetic engineering bacteria in the step (2) into tomato tissues, and screening and culturing to obtain transgenic plants.
4. The method of claim 3, wherein the vector is a pFGC1008-3HA vector.
5. A method according to claim 3, wherein the agrobacterium genetically engineered bacterium is agrobacterium tumefaciens GV3101 strain.