LpPR1L gene and application thereof in improving heat resistance of plants

Abstract

The invention discloses an LpPR1L gene and application thereof in improving heat resistance of plants, and belongs to the technical field of plant genetic engineering. The invention provides a perennial ryegrass disease-resistant related gene LpPR1L capable of improving the heat resistance of plants, and experiments find that the total chlorophyll content, the maximum photochemical efficiency, the electrolyte permeability and the recovery rate of transgenic arabidopsis thaliana overexpressed with the LpPR1L gene after high temperature stress are all superior to those of a wild type; therefore, overexpression of the LpPR1L gene can improve the heat resistance of transgenic arabidopsis thaliana. The invention provides an important target for improving the heat resistance of perennial ryegrass and relieving the problem that the perennial ryegrass is difficult to oversummering in actual production, and also provides a theoretical basis for cultivating heat-resistant high-quality plants.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant genetic engineering, in particular to LpPR1L gene and its application in improving plant heat tolerance. BACKGROUND

[0002] Temperature is an environmental factor that affects the seasonal growth and geographical distribution of plants. In the past 200 years, human activities have increased the emission of greenhouse gases, leading to global warming, which is expected to be 1.0℃ higher than the pre-industrial level. However, if the current growth rate continues, global warming may reach 4.0℃ by the end of this century. One negative effect of global warming is the decline in agricultural production. For every 1℃ increase in global average temperature, the global production of wheat, rice, corn, and soybeans will decrease by 6.0%, 3.2%, 7.4%, and 3.1%, respectively. In recent years, there has been significant progress in the study of molecular mechanisms of heat tolerance in crops. First, in the model plant Arabidopsis thaliana, a new temperature sensor, TWA1, was discovered, providing a powerful molecular tool for adjusting the heat stress response of crops through breeding and biotechnology. In wheat, genes such as TaMBF1 and TaGCN5 regulate the antioxidant system and glycolytic pathway, reducing the accumulation of reactive oxygen species and maintaining energy metabolism. In rice, the TT1 gene reduces the abundance of SCE1 protein by promoting its ubiquitination and degradation, thereby relieving its negative regulation of small heat shock proteins. The knockout strain of TT1 gene increased the yield of single plant by 15.1% and the yield of small area by 7.4% under high temperature stress in the field. In maize, the ZmMKK9-ZmMPK20-ZmRIN2 cascade regulates stomatal movement, reducing the stomatal opening of transgenic maize by 30% at 40℃, lowering the leaf temperature by 2-3℃, and reducing the yield loss by 15-20%. Therefore, studying the molecular mechanisms of plant response to high temperature stress is of great significance for increasing crop yield, maximizing agricultural production, and promoting world food security.

[0003] Perennial ryegrass (Lolium perenne L.) belongs to the family Poaceae and has the characteristics of fast growth, multiple tillering, tolerance to grazing and mowing, strong digestibility, and strong resistance to diseases and pests. It is an important pioneer grass species for lawn establishment and is widely planted in temperate regions of the world. However, as a typical cool-season forage and turfgrass, perennial ryegrass prefers warm and humid climate and is sensitive to high temperature, making it difficult to survive the summer.

[0004] High temperature stress is an important factor limiting the growth and development of many perennial grasses. At the same time, due to the cold season biological characteristics of perennial ryegrass, with the global warming, the occurrence of extreme high temperature, the growth and development of perennial ryegrass will be more affected, how to actively respond to the threat of global climate change to the survival of perennial ryegrass, explore how to improve the heat tolerance of perennial ryegrass, solve the limitation of its application as forage, turfgrass is particularly important. However, the research on heat tolerance of perennial ryegrass in China is relatively backward, in the face of high temperature stress, a highly complex regulatory network, the heat tolerance molecular mechanism of perennial ryegrass is mostly concentrated in the classic "HSF-HSP" high temperature response pathway. However, the research on the function of other heat tolerance related genes is seriously insufficient, which greatly restricts the process of heat tolerance molecular breeding of perennial ryegrass. Therefore, it is urgent to excavate the key genes of heat tolerance of perennial ryegrass and analyze their regulation and heat tolerance function, so as to speed up the process of heat tolerance molecular breeding of perennial ryegrass, and also provide reference for the research on heat tolerance of related cold season forage. SUMMARY

[0005] The purpose of the present application is to provide LpPR1L gene and its application in improving heat tolerance of plants, so as to solve the problems existing in the prior art. The LpPR1L gene can significantly improve the heat tolerance of plants, which is of great significance for improving the heat tolerance of perennial ryegrass and solving the problem of its difficulty in summering. At the same time, it provides a theoretical basis for breeding heat-resistant high-quality plants.

[0006] In order to achieve the above purpose, the present application provides the following scheme:

[0007] The present application provides the application of LpPR1L gene in any one of the following:

[0008] (1) in improving the heat tolerance of plants;

[0009] (2) in breeding heat-resistant plants;

[0010] The CDS sequence of the LpPR1L gene is shown as SEQ ID NO. 9.

[0011] The present application also provides the application of the protein encoded by LpPR1L gene in any one of the following:

[0012] (1) in improving the heat tolerance of plants;

[0013] (2) in breeding heat-resistant plants;

[0014] The CDS sequence of the LpPR1L gene is shown as SEQ ID NO. 9, and the amino acid sequence of the protein encoded by the LpPR1L gene is shown as SEQ ID NO. 10.

[0015] The application also provides application of the recombinant vector containing the LpPR1L gene in any of the following aspects:

[0016] (1) application in improving heat tolerance of plants;

[0017] (2) application in breeding heat-tolerant plants;

[0018] The recombinant vector is constructed by connecting the LpPR1L gene and an expression vector, and the CDS sequence of the LpPR1L gene is shown as SEQ ID NO. 9.

[0019] The application also provides application of the recombinant bacteria containing the LpPR1L gene in any of the following aspects:

[0020] (1) application in improving heat tolerance of plants;

[0021] (2) application in breeding heat-tolerant plants;

[0022] The recombinant bacteria are constructed by introducing the recombinant vector into host bacteria, and the recombinant vector is constructed by connecting the LpPR1L gene and an expression vector, and the CDS sequence of the LpPR1L gene is shown as SEQ ID NO. 9.

[0023] Preferably, the LpPR1L gene is overexpressed to improve heat tolerance of plants.

[0024] Preferably, the plants include Arabidopsis thaliana or perennial ryegrass.

[0025] The application also provides a method for improving heat tolerance of plants, which comprises the steps of overexpressing the LpPR1L gene in plants to improve heat tolerance of the plants.

[0026] The application also provides a method for breeding heat-tolerant plants, which comprises the steps of overexpressing the LpPR1L gene in plants to breed heat-tolerant transgenic plants.

[0027] Preferably, the plants include Arabidopsis thaliana or perennial ryegrass.

[0028] The application discloses the following technical effects:

[0029] The application identifies a disease resistance related gene LpPR1L in perennial ryegrass, the gene is 453 bp long and encodes 150 amino acids. The LpPR1L protein contains a typical CAP domain at the 15-150 amino acid site. By determining the total chlorophyll content, maximum photochemical efficiency, electrolyte permeability and recovery rate of transgenic Arabidopsis overexpressing LpPR1L gene after high temperature stress, it is found that the determination results of total chlorophyll content, maximum photochemical efficiency, electrolyte permeability and recovery rate in transgenic Arabidopsis overexpressing LpPR1L gene are better than those of wild type, which shows that overexpression of LpPR1L gene can improve the heat tolerance of transgenic Arabidopsis.

[0030] The obtaining of the disease resistance related gene LpPR1L of perennial ryegrass provides an important target for improving the heat tolerance of perennial ryegrass, relieving the problem of difficult summering in actual production, and provides a theoretical basis for breeding heat-tolerant high-quality plants. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0032] Figure 1 Expression of perennial ryegrass LpPR1L gene at different time points and in different tissue parts under high temperature; (A) Expression of LpPR1L in perennial ryegrass under high temperature treatment for 0, 4, 12 and 72 h; (B) Expression of LpPR1L in different tissue parts of perennial ryegrass, 1 st : the first fully expanded leaf, 2 nd : the second leaf, 3 rd : the third leaf, 4 th : the fourth leaf, 5 th : the fifth leaf, sheath: leaf sheath, stem: stem, crown: crown root, root: root;

[0033] Figure 2 LpPR1L protein multiple sequence alignment and phylogenetic tree construction and functional positioning map; (A) Construction and analysis of the phylogenetic tree of LpPR1L protein and other plant PR1 proteins; (B) Homology comparison of PR1 proteins in other plants and perennial ryegrass LpPR1L protein, the conserved region of the amino acid sequence is marked with a red underline;

[0034] Figure 3 Subcellular localization map of tobacco; scale: 10um;

[0035] Figure 4 Figure 8 is a diagram of positive identification of Arabidopsis thaliana overexpressing LpPR1L transgene; wherein OE-1, OE-2 and OE-3 are overexpression lines, and WT is wild type plant;

[0036] Figure 5 Figure 9 is a diagram of phenotype analysis of wild type and transgenic Arabidopsis thaliana under high temperature stress; (A) high temperature stress phenotype of wild type and transgenic Arabidopsis thaliana; (B) maximum photochemical efficiency; (C) electrolyte permeability; (D) total chlorophyll content; (E) recovery rate; wherein OE-1, OE-2 and OE-3 are overexpression lines, Col-0 is wild type plant, and prll is mutant plant; scale bar: 3.5 cm. DETAILED DESCRIPTION

[0037] Various exemplary embodiments of the present application will now be described in detail with reference to the drawings. The detailed description is made with reference to the drawings, but the present application is not limited to the embodiments described herein. Rather, the present application should be understood to encompass any number of variations within the scope of the embodiments, which will become apparent to those of ordinary skill in the art, and it is therefore intended that such variations include in the scope of the present application. The following detailed description includes specific details for the purpose of providing a thorough understanding of the present application. However, it will be apparent to those skilled in the art that the present application can be practiced without these specific details. In some instances, well-known structures and functions have not been described in detail in order to avoid obscuring the concept of the present application.

[0038] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, for a range of values of a parameter, unless otherwise indicated, each intervening value by each intervening value, as well as any other stated or intervening value in that stated range is encompassed. In addition, any combination of the above ranges, as well as any other stated or intervening value in that stated range, is encompassed. All values within the ranges stated and / or described herein have their full range of equivalents in some embodiments, the stated ranges can be carried out or achieved by any means within the scope of this application. Accordingly, the numerical parameters are reported herein with a certain degree of precision. However, it must be readily recognized by those skilled in the art that optimal properties can be achieved and / or desired results can be achieved with modifications to reflect insignificant deviations.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In the case of conflict between the present specification and any document incorporated by reference, the present specification will control.

[0040] Various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those of ordinary skill in the art from consideration of the specification and practice of the application. The specification and examples given are exemplary only. It is to be understood that the application is not to be limited by the specific details described herein.

[0041] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.

[0042] The inventors found that LpPR1L is up-regulated by long-term high temperature stress in the high temperature transcriptome data of perennial ryegrass, indicating that LpPR1L is involved in the response of perennial ryegrass to high temperature stress, and therefore LpPR1L was selected for the research of the present application. The following embodiments are further described in detail.

[0043] Example 1 Expression of LpPR1L gene under high temperature stress

[0044] 1, Material treatment of perennial ryegrass

[0045] The perennial ryegrass (Bneua Vista) samples were provided by the Zhang Xinqian research group of Sichuan Agricultural University, and were subjected to high temperature stress treatment, specifically 16h light / 8h darkness, with the high temperature stress set conditions being 38℃ during the day and 33℃ at night.

[0046] 2, Analysis of relative expression amount of LpPR1L under high temperature stress

[0047] Total RNA was extracted from the leaves collected after treatment using a plant RNA extraction kit. The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit. The relative expression amount of LpPR1L at different time points under high temperature stress was determined using fluorescent quantitative polymerase chain reaction. LpeIF4A was used as an internal reference gene. The primers used in the fluorescent quantitative polymerase chain reaction process are as follows:

[0048] qLpPR1L-F: 5'-GCCCAAGACTACCTTTCACCC-3' (SEQ ID NO. 1);

[0049] qLpPR1L-R: 5'-CGGATCCCCAGAAGATGTTC-3' (SEQ ID NO. 2);

[0050] LpeIF4A-F: 5'-AACTCAACTTGAAGTGTTGGAGTG-3' (SEQ ID NO. 3);

[0051] LpeIF4A-R: 5'-AGATCTGGTCCTGGAAAGAATATG-3' (SEQ ID NO. 4).

[0052] The RT-qPCR reaction system is shown in the following table:

[0053] Table 1 RT-qPCR reaction system

[0054]

[0055] The RT-qPCR reaction program is shown in the following table:

[0056] Table 2 RT-qPCR reaction procedure

[0057]

[0058] The results are shown in Table 3, wherein A shows the expression level of LpPR1L in perennial ryegrass under high temperature treatment for 72h, and B shows the expression level of LpPR1L in different tissue parts of perennial ryegrass. Figure 1 The results are shown in Table 3, wherein A shows the expression level of LpPR1L in perennial ryegrass under high temperature treatment for 72h, and B shows the expression level of LpPR1L in different tissue parts of perennial ryegrass.

[0059] 3. Analysis of the relative expression amount of LpPR1L in different tissue parts of perennial ryegrass

[0060] Total RNA was extracted from different tissue parts of perennial ryegrass by using a plant RNA extraction kit. The extracted RNA was reverse transcribed into cDNA by using a reverse transcription kit. The relative expression amount of LpPR1L at different time points under high temperature stress was determined by using a fluorescent quantitative polymerase chain reaction. LpeIF4A was used as an internal reference gene. The fluorescent quantitative polymerase chain reaction was the same as described in “2. Analysis of the relative expression amount of LpPR1L under high temperature stress”.

[0061] The results are shown in Table 3, wherein A shows the expression level of LpPR1L in perennial ryegrass under high temperature treatment for 72h, and B shows the expression level of LpPR1L in different tissue parts of perennial ryegrass. Figure 1 The results are shown in Table 3, wherein A shows the expression level of LpPR1L in perennial ryegrass under high temperature treatment for 72h, and B shows the expression level of LpPR1L in different tissue parts of perennial ryegrass.

[0062] Example 2. Identification of LpPR1L

[0063] 1. Cloning of LpPR1L

[0064] PCR was performed by using perennial ryegrass cDNA as a template, using 5'UTR upstream primer F1 (LpPR1L-F) and 3'UTR downstream primer R1 (LpPR1L-R) for first round PCR amplification. CDS region-specific primers (LpPR1L-CDS-F and LpPR1L-CDS-R) were designed again, and the PCR recovery product obtained in the first round was used as a template for second round amplification.

[0065] The primers required for PCR amplification are as follows:

[0066] LpPR1L-F: 5'-AATTTTGCTGGCCCTAGCC-3' (SEQ ID NO. 5);

[0067] LpPR1L-R: 5'-GTGGGGAACGAGGGTCTAT-3' (SEQ ID NO. 6);

[0068] LpPR1L-CDS-F: 5'-ctagacccggggaattcATGGCAGCCGCCATGGT-3' (SEQ ID NO. 7);

[0069] LpPR1L-CDS-R: 5'-tcgacactagtaagcttGTATGGTTTCTGTCCAACG-3' (SEQ ID NO. 8),

[0070] wherein the lowercase letters in the sequences of SEQ ID NO. 7 and SEQ ID NO. 8 represent enzyme cutting site amplification sequences.

[0071] The PCR reaction system is shown in the following table:

[0072] Table 3 PCR reaction system

[0073]

[0074] The PCR reaction program is shown in the following table:

[0075] Table 4 PCR reaction program

[0076]

[0077] The CDS sequence fragment obtained by amplification was subjected to enzyme cutting, and then was connected to the pENTRY vector by T4 ligase to obtain a recombinant vector pEntry-LpPR1L, which was sent to a company for sequencing, and the results were compared with the perennial ryegrass genome reference sequence.

[0078] The enzyme cutting reaction system is as follows:

[0079] Table 5 Enzyme cutting reaction system

[0080]

[0081] The reaction condition is 37°C for 1h.

[0082] The LpPR1L gene is 453bp long and encodes 150 amino acids. The LpPR1L protein contains a typical CAP domain at the 15-150 amino acid site.

[0083] The CDS sequence (SEQ ID NO. 9) of the LpPR1L gene obtained by amplification is as follows:

[0084] ATGGCAGCCGCCATGGTTAATCCTTCCCAGGCGCAGAACTCGGCCCAAGACTACCTTTCACCCCACAACTCTGTCCGCGCCGCCGTCGGCGTTGGCGCGGTGAGCTGGAGCACAAGGCTGCAGTCGTACGCCCAGACCTACGCCAACCAGAGGATCGGCGACTGCAAGCTTCAGCACTCCGGCGGGCCCTATGGGGAGAACATCTTCTGGGGATCCGGTTCGGGCTGGAAGGCGGCGGACGCGGTGAACTTGTGGGCCGGCGAGAAGAGCGACTACGACTACGGCTCCAACAGCTGCGCGGCGGGGAAGCAGTGCGGGCACTACACACAGATTGTGTGGCGCGCGACGACGAGCATCGGCTGCGCTCGCGTGGTTTGCAACAACAACGCGGGCGTATTCATCATCTGCAGCTACGATCCCCCGGGCAATTTCGTTGGACAGAAACCATACTAA.

[0085] The amino acid sequence of the obtained LpPR1L gene (SEQ ID NO. 10) is as follows:

[0086] MAAAMVNPSQAQNSAQDYLSPHNSVRAAVGVGAVSWSTRLQSYAQTYANQRIGDCKLQHSGGPYGENIFWGSGSGWKAADAVNLWAGEKSDYDYGSNSCAAGKQCGHYTQIVWRATTSIGCARVVCNNNAGVFIICSYDPPGNFVGQKPY.

[0087] 2. Construction of recombinant expression vector and recombinant bacteria

[0088] The recombinant vector pEntry-LpPR1L was transformed into E. coli competent DH5a for replication and proliferation by heat shock method. The competent cells were taken out from the -80°C refrigerator and placed on ice to melt. The recombinant vector plasmid was added to the competent cell suspension, and the contents were mixed by gently rotating the centrifuge tube. The centrifuge tube was placed in a 42°C water bath for 90s, and then quickly transferred to ice for 3min. 700μL of antibiotic-free liquid LB medium was added to the centrifuge tube, and after mixing, it was placed in a 37°C shaking incubator for 1h. The transformed competent cells were centrifuged at low speed, and part of the supernatant was discarded. 100-150μL of culture medium was retained, and the bacterial cells were suspended by gently blowing with a pipette. The entire suspension was added to LB solid agar medium containing the corresponding antibiotic, and the cells were evenly spread using a sterile spreader. The plate was placed at room temperature until the liquid was absorbed. The plate was inverted and incubated at 37°C for 12-16h to obtain single colonies. The single colonies were picked and placed in 5mL of liquid LB medium containing the corresponding antibiotic, and incubated at 37°C for 12-16h. The plasmid was extracted and digested with PvuI restriction enzyme. Then, the LR recombination method was used to recombine with the pEarlyGate103 expression vector to form a new recombinant expression vector pEarlyGate103-LpPR1L. The plasmid was extracted using the same method as above, and the expression vector was transferred into Agrobacterium competent GV3101. 5μL of pEarlyGate103-LpPR1L vector plasmid was added to 100μL of Agrobacterium competent cells, mixed well, and incubated in ice water for 5min. Then, it was quickly frozen in liquid nitrogen for 5min, and then incubated in a 37°C water bath for 5min. 700μL of antibiotic-free liquid LB medium was added, and the mixture was incubated at 28°C for 4-5h. Part of the supernatant was discarded, and 100-150μL of culture medium was retained. The entire suspension was added to LB solid agar medium containing the corresponding antibiotic, and the cells were evenly spread using a sterile spreader. The plate was incubated at 28°C for 48h. After the bacterial colonies grew, three single colonies were picked, and the positive detection confirmed the successful transformation. The expanded bacterial liquid could be used for subsequent genetic transformation.

[0089] 3. Multiple sequence alignment and bioinformatics analysis

[0090] The precise LpPR1L sequence was obtained by PCR technology. At the same time, the homologous protein sequences of other species were downloaded from the National Center for Biotechnology Information (NCBI), and Jalview software was used to analyze the amino acid sequences under default parameters. MEGA11 software was used for phylogenetic tree analysis, and the results are shown in Figure 2 A and B.

[0091] 4. Subcellular localization of LpPR1L

[0092] Three pEG103-LpPR1L Agrobacterium single colonies were picked into 2 mL centrifuge tubes containing 50 mg / L Kan and 50 mg / L Rif antibiotics, and incubated at 28°C for 2-3 days. Then 100 μL was taken into 50 mL centrifuge tubes containing 50 mg / L Kan and 50 mg / L Rif antibiotics, and incubated at 28°C until OD 600 = 0.8-1.0. According to V 菌液 x OD 600 = 0.8, the corresponding volume of bacterial solution was centrifuged at 6000 rpm for 2 min to collect the bacteria, the supernatant was discarded, and the bacterial pellet was resuspended with 1 mL of tobacco infection solution (1 mL of 100 μM acetosyringone was added to 100 mL of tobacco infection solution). Finally, the resuspension was injected into tobacco after being treated at 25°C in the dark for 2-3 h. After the tobacco was normally cultured for 2 days, the eGFP fluorescence was observed using a Leica super-resolution confocal microscope (the excitation wavelength was 488 nm, and the scanning wavelength was 505-530 nm).

[0093] The results showed that LpPR1L was located in the nucleus, as Figure 3 shown.

[0094] Example 3 Phenotype analysis of LpPR1L overexpression transgenic plants under high temperature stress

[0095] 1. Obtaining of Arabidopsis thaliana transgenic lines

[0096] The Agrobacterium carrying the pEarlyGate103-LpPR1L vector prepared above was cultured to OD 600 = 0.6, the bacterial solution was collected and resuspended, and then the Arabidopsis inflorescences were immersed in the resuspended Agrobacterium for 45-60 s. After the infection was completed, the plants were treated in the dark for 12 h and then transferred to normal culture conditions. After the Arabidopsis seeds matured, positive Arabidopsis was screened on 1 / 2 MS + 20 mg / L glyphosate (Basta) medium.

[0097] 2. Identification of transgenic Arabidopsis

[0098] The received T0 generation Arabidopsis seeds were plated on 1 / 2 MS solid medium containing 20 mg / L Basta, and after they grew for about 2 weeks, the green and healthy seedlings were transferred to nutrient soil for further culture. When the Arabidopsis leaves grew to be slightly larger, total RNA was extracted from the transgenic Arabidopsis leaves for RT-qPCR overexpression fold determination, and the results are shown in Figure 4 The gene expression level in the LpPR1L overexpression transgenic Arabidopsis was significantly higher than that in the wild type Arabidopsis. Meanwhile, the obtained transgenic positive lines were further cultured, and the T1 generation seeds were collected. After screening, the progeny segregation ratio was close to 3:1, and the seeds were collected. The T3 generation homozygous lines were used for functional verification.

[0099] 3. Analysis of heat tolerance phenotype of transgenic Arabidopsis overexpressing LpPR1L

[0100] 3.1. Experimental methods

[0101] T3 generation transgenic Arabidopsis seeds were spread on 1 / 2 MS (pH = 5.8) solid medium, and after the Arabidopsis grew for about 2 weeks, they were transferred to nutrient medium for culture. After about 4 weeks of continuous culture, transgenic Arabidopsis with similar growth vigor were selected, and wild-type Arabidopsis was used as a control for high-temperature stress treatment (42°C / 37°C). The phenotype was observed during the treatment process, and the physiological indicators were determined.

[0102] The determination of physiological indicators includes the following operations.

[0103] (1) Determination of maximum photochemical efficiency (Fv / Fm):

[0104] The related parameters were measured using a LI-6400 photosynthetic instrument, and the leaves of Arabidopsis were measured at each time point under high-temperature stress.

[0105] (2) Determination of electrolyte leakage rate (EL):

[0106] 0.1 g of fresh plant leaves were wrapped with absorbent paper and placed in a 50 mL centrifuge tube containing 30 mL of deionized water, so that the sample was fully immersed in water. Five biological replicates were set, sealed and placed in a shaker for 24 h, and then the initial conductivity value C0 was measured using a conductivity meter. Then, the centrifuge tube with the measured initial conductivity value was sealed and placed in a boiling water bath for 20 min for fixation. After cooling to room temperature, it was placed in a shaker for 24 h, and the conductivity C1 was measured again. The conductivity calculation formula is: EL = C0 / C1 x 100%.

[0107] (2) Determination of total chlorophyll (Chl):

[0108] Fresh leaves were cut into small pieces (0.1 g) and placed in a 15 mL centrifuge tube, 5 mL of dimethyl sulfoxide was added to fully immerse the leaves in the liquid, and the tube was covered. It was placed in the dark at room temperature. When the leaves turned completely white, the extract was directly taken for measurement. The absorbance of the chlorophyll extract at 663 nm and 645 nm, and 470 nm wavelengths was measured using a spectrophotometer, and the total chlorophyll content was calculated using the Arnon formula.

[0109] (4) Determination of recovery rate:

[0110] The stressed seedlings were placed under normal culture conditions, and the recovery of the seedlings was observed. If the leaves of the seedlings turned green and new rosette leaves grew after stress, it was recorded as a recovered individual, and the recovery rate of different lines was calculated based on the number of recovered individuals in each square pot. Each square pot was recorded as a biological replicate.

[0111] 3.2 Experimental Results

[0112] like Figure 4 As shown, Arabidopsis thaliana was subjected to high-temperature stress treatment, followed by recovery at normal temperature, and the recovery rate was observed. Upon reaching normal temperature, almost all Arabidopsis seedlings died in the early recovery stage, while the leaves gradually returned to green in the later recovery stage. Furthermore, the recovery rate of Arabidopsis thaliana overexpressing LpPR1L was significantly higher than that of the wild type. Meanwhile, the mutant Arabidopsis thaliana exhibited the opposite phenotype to the overexpressed variety, with a significantly lower recovery rate than the wild type. This indicates that LpPR1L improves the heat tolerance of Arabidopsis thaliana. Figure 5 A, E).

[0113] Simultaneously, the maximum photochemical efficiency, ion permeability, and total chlorophyll content were measured. Figure 5 (B-D). The results showed that after high-temperature treatment, almost all leaves of wild-type Arabidopsis thaliana turned white, while some leaves of the overexpressing Arabidopsis thaliana remained relatively green. The maximum photochemical efficiency of the overexpressing Arabidopsis thaliana was superior to that of the wild type, while the mutant Arabidopsis thaliana showed the opposite, with a significantly lower maximum photochemical efficiency than the wild type. Before high-temperature treatment, there were no significant differences in ion permeability and relative water content between the wild type and the overexpressing Arabidopsis thaliana. However, after treatment, the ion permeability of the LpPR1L-overexpressing Arabidopsis thaliana was significantly lower than that of the wild type, while the maximum photochemical efficiency and total chlorophyll content were significantly higher. This indicates that the LpPR1L-overexpressing Arabidopsis thaliana experienced less cell membrane damage after high-temperature stress, and LpPR1L enhanced the heat resistance of Arabidopsis thaliana.

[0114] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of the LpPR1L gene in any of the following: (1) Application in improving the heat resistance of plants; (2) Application in cultivating heat-resistant plants; The CDS sequence of the LpPR1L gene is shown in SEQ ID NO.

9.

2. The application of the protein encoded by the LpPR1L gene in any of the following: (1) Application in improving the heat resistance of plants; (2) Application in cultivating heat-resistant plants; The CDS sequence of the LpPR1L gene is shown in SEQ ID NO.9, and the amino acid sequence of the protein encoded by the LpPR1L gene is shown in SEQ ID NO.

10.

3. Application of recombinant vectors containing the LpPR1L gene in any of the following: (1) Application in improving the heat resistance of plants; (2) Application in cultivating heat-resistant plants; The recombinant vector was constructed by connecting the LpPR1L gene and an expression vector, and the CDS sequence of the LpPR1L gene is shown in SEQ ID NO.

9.

4. Application of recombinant bacteria containing the LpPR1L gene in any of the following: (1) Application in improving the heat resistance of plants; (2) Application in cultivating heat-resistant plants; The recombinant bacteria were constructed by introducing a recombinant vector into a host bacterium. The recombinant vector was constructed by connecting the LpPR1L gene and an expression vector. The CDS sequence of the LpPR1L gene is shown in SEQ ID NO.

9.

5. The application as described in any one of claims 1-4, characterized in that, Overexpression of the LpPR1L gene improves heat tolerance in plants.

6. The application as described in any one of claims 1-4, characterized in that, The plants mentioned include Arabidopsis thaliana or perennial ryegrass.

7. A method for improving the heat resistance of plants, characterized in that, The method includes the step of overexpressing the LpPR1L gene in plants to improve plant heat tolerance; wherein the CDS sequence of the LpPR1L gene is shown in SEQ ID NO.

9.

8. A method for cultivating heat-resistant plants, characterized in that, This includes the steps of overexpressing the LpPR1L gene in plants to cultivate heat-resistant transgenic plants.

9. The method as described in claim 7 or 8, characterized in that, The plants mentioned include Arabidopsis thaliana or perennial ryegrass.

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