Lpcdpk27 gene for regulating heat tolerance of perennial ryegrass and application thereof

Abstract

The application discloses a kind of LpCDPK27 genes for regulating perennial ryegrass heat tolerance and application thereof, the nucleotide sequence of LpCDPK27 gene is as shown in SEQ ID NO.1;Its corresponding amino acid sequence is as shown in SEQ ID NO.2;The nucleotide sequence of its corresponding promoter is as shown in SEQ ID NO.3.The application clones the cDNA sequence of calcium-dependent protein kinase gene LpCDPK27 from perennial ryegrass, and links the obtained LpCDPK27 gene with plant expression vector, constructs recombinant expression vector, and transforms perennial ryegrass callus with the constructed recombinant expression vector, and finally cultivates into transgenic plant.The results show that the gene overexpression can significantly improve the heat tolerance of transgenic plant.The application provides key gene resources and effective technical approach for cultivating new varieties of perennial ryegrass resistant to high temperature.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to a calcium-dependent protein kinase LpCDPK27 gene that regulates the heat tolerance of perennial ryegrass and its application. Background Technology

[0002] Abiotic stresses (such as drought, salinity, chilling, and heat) have a significant impact on plant growth and development, leading to reduced crop yields and affecting the normal growth and quality of forest trees, pastures, horticultural plants, and garden plants. Therefore, breeding new plant varieties with strong stress resistance has become one of the important goals of bio-breeding, and identifying and cloning stress-resistance-related genes is an important approach to breeding such new varieties.

[0003] Ca 2+ As a highly conserved second messenger in the eukaryotic signal transduction system, it plays a central role in plant stress responses. Plants precisely regulate intracellular calcium levels by activating specific calcium ion channels. 2+ The concentration of calcium in plant cells determines the specific calcium signal, which in turn triggers a series of adaptive physiological responses. There are four main classes of calcium-sensing proteins in plant cells: calcium-dependent protein kinases (CDPKs), calmodulin (CaM), calmodulin-like proteins (CML), and calmophosphatase B-like proteins (CBL). Among them, CDPKs possess unique molecular characteristics, simultaneously possessing calcium... 2+ The CDPK functions as both a sensor and an effector. It senses Ca through its N-terminal structural domain. 2+ By adjusting calcium concentrations and directly phosphorylating various substrates, including ion channels, transcription factors, and metabolic enzymes, through their C-terminal kinase domains, calcium signals are directly converted into physiological responses. This unique signal transduction mechanism does not rely on the participation of exogenous calmodulins, making CDPKs key regulatory elements in the plant calcium signal transduction network.

[0004] The functions of calcium-dependent protein kinases (CDPKs) in various plants have been widely reported, including model plants such as Arabidopsis thaliana and rice, as well as crops such as soybean, wheat, maize, grape, alfalfa, and pepper. These studies have shown that CDPKs play important roles in plant growth, development, and responses to abiotic stress. However, to date, very little research has been conducted on the role of CDPK family genes in heat stress tolerance in perennial ryegrass.

[0005] Perennial ryegrass (Lolium perenne), a widely cultivated cool-season turfgrass globally, possesses not only strong tolerance to trampling and disease and pest resistance, but also remarkable characteristics such as rapid establishment and long green period. These excellent properties make it one of the preferred grass species for turf establishment in temperate regions, playing an irreplaceable role in the construction of artificial grasslands and the improvement of natural pastures. However, its optimal growth temperature is 10-27 ℃. High temperatures adversely affect its growth, development, and physiological metabolism. When temperatures exceed 35 ℃, perennial ryegrass enters a dormant state, exhibiting yellowing and wilting leaves and seedling death. Therefore, in areas south of the Yangtze River, perennial ryegrass struggles to survive the summer and must be reseeded the following year, contradicting its "perennial" biological characteristics. This altered growth characteristic not only increases cultivation and management costs but also limits its widespread application in subtropical regions. Therefore, in-depth analysis of the molecular regulatory mechanisms of heat tolerance in perennial ryegrass is of significant theoretical and practical importance for improving its high-temperature adaptability and maintaining its perennial characteristics.

[0006] Studies have found that some CDPK family members participate in regulating plant growth and development or responding to stresses such as cold and salt in certain model plants. Plant responses to high-temperature stress involve protein homeostasis maintenance and heat shock signaling pathways, which are fundamentally different in molecular mechanism from the osmotic regulation pathways activated by cold or saline-alkali stress. Particularly for cool-season turfgrass like perennial ryegrass, its heat tolerance mechanism involves a unique heat shock response pathway, which is fundamentally different from the reported cold and salt tolerance mechanisms in model plants. Furthermore, due to the high species specificity and functional diversity of CDPK protein kinases, their homologous members often exhibit drastically different biological functions in different plants; therefore, their specific role in regulating heat tolerance in cool-season turfgrass remains highly uncertain. Whether endogenous CDPK family members in ryegrass can confer heat tolerance by stabilizing plasma membrane signaling and activating the antioxidant system lacks clear experimental evidence. Therefore, it is currently impossible to infer which specific CDPK member in perennial ryegrass plays a core role in responding to extreme high temperatures through sequence alignment or cross-species homology. Summary of the Invention

[0007] To overcome the shortcomings and deficiencies of existing technologies, such as the lack of stress-resistance genes in perennial ryegrass and the unclear function of the CDPKs family, this invention provides an LpCDPK27 gene that regulates the heat resistance of perennial ryegrass and its application.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] This invention provides the application of overexpression of the LpCDPK27 gene in improving the heat resistance of perennial ryegrass, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0010] Furthermore, the amino acid sequence of the protein encoded by the LpCDPK27 gene is shown in SEQ ID NO.2. This encoded protein contains 516 amino acids, has a molecular weight of approximately 57.8 kDa, an isoelectric point of 6.72, contains four EF-hand domains, and is located in the plasma membrane.

[0011] The present invention also provides a recombinant expression vector comprising the LpCDPK27 gene described above.

[0012] The present invention also provides a recombinant genetically engineered cell comprising the above-described recombinant expression vector.

[0013] The present invention also provides the application of the above-mentioned recombinant expression vector or recombinant genetically engineered cells in improving the heat resistance of perennial ryegrass.

[0014] The present invention also provides a method for cultivating heat-resistant perennial ryegrass by transferring the above-mentioned LpCDPK27 gene into perennial ryegrass.

[0015] This invention also provides a method for detecting the heat-resistance LpCDPK27 gene in plants, comprising the following steps:

[0016] Extracting plant genomic DNA;

[0017] The genomic DNA was amplified by PCR and then detected.

[0018] If the target band is present, the plant contains the heat-resistant LpCDPK27 gene.

[0019] Furthermore, the primer pair used for the PCR amplification includes an upstream primer as shown in SEQ ID NO.4 and a downstream primer as shown in SEQ ID NO.5.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) For the first time, it was revealed that the LpCDPK27 gene from perennial ryegrass has a significant heat resistance function, breaking through the limitation that existing CDPK heat resistance research is mostly focused on model plants or crops such as Arabidopsis thaliana and rice.

[0022] (2) The LpCDPK27 gene has been shown to be specifically induced by heat stress, and its overexpression can significantly enhance the survival ability of plants under high temperature, providing clear and reproducible evidence of heat tolerance phenotype.

[0023] (3) The leaves of transgenic plants showed lower cell membrane damage (such as significantly reduced relative conductivity), stronger photosynthetic system stability or higher antioxidant enzyme activity under heat stress, indicating that their heat resistance mechanism has a physiological basis.

[0024] (4) As an endogenous gene of perennial ryegrass, LpCDPK27 has a high genetic background matching degree when expressed in homologous or closely related grass species, avoiding developmental abnormalities or metabolic burden that may be caused by heterologous expression.

[0025] (5) The constructed overexpression vector system is suitable for cool-season turfgrass / pastures such as perennial ryegrass, which are difficult to genetically transform, providing an effective tool for improving heat tolerance for these important but poorly studied species.

[0026] (6) LpCDPK27 belongs to the calcium-dependent protein kinase family and also possesses Ca2+. 2+ Sensing and protein kinase activity were significantly upregulated under high temperature stress, and overexpression could effectively reduce the accumulation of reactive oxygen species in the plant and alleviate membrane lipid peroxidation, thereby enhancing the heat resistance of perennial ryegrass.

[0027] (7) It directly addresses the industry pain point that perennial ryegrass is prone to yellowing and degeneration under high summer temperatures, and provides an efficient and precise molecular breeding solution with clear application value and promotion potential. Attached Figure Description

[0028] Figure 1 Electrophoresis diagram of PCR amplification of the LpCDPK27 gene.

[0029] Figure 2 This is an electrophoresis image of the LpCDPK27 gene amplified by the overexpression vector constructed in this invention.

[0030] Figure 3 This is a schematic diagram of the overexpression vector constructed according to the present invention.

[0031] Figure 4 This is a diagram showing the subcellular localization results of the LpCDPK27 gene.

[0032] Figure 5 A bar chart showing the results of LpCDPK27 gene expression in different tissues of perennial ryegrass.

[0033] Figure 6 This image shows a comparison of phenotypes, gene expression levels, and electrolyte extravasation rates between LpCDPK27 transgenic perennial ryegrass plants and wild-type plants under high-temperature stress; among them, Figure 6 Figure A shows a phenotypic comparison between LpCDPK27 transgenic perennial ryegrass plants and wild-type plants under high temperature stress. Figure 6 Figure B in the diagram shows the comparison of gene expression levels between transgenic perennial ryegrass plants and wild-type plants. Figure 6 The figure in Figure C shows the comparison of electrolyte efflux rates between transgenic perennial ryegrass plants and wild-type plants. Detailed Implementation

[0034] The present invention will be further explained below with reference to the embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods.

[0035] In the following examples, the perennial ryegrass (Lolium perenne) variety 'Emerald' was used for gene cloning and phenotypic verification. Escherichia coli DH5α was purchased from Beijing Qingke Biotechnology Co., Ltd. The pCAMBIA1390 expression vector was preserved in the laboratory of Professor Zhang Lu's research group at the College of Landscape Architecture and Architecture, Zhejiang Agriculture and Forestry University.

[0036] The nucleotide sequence of the LpCDPK27 gene described in this invention is shown in SEQ ID NO.1; its corresponding amino acid sequence is shown in SEQ ID NO.2; and its corresponding promoter nucleotide sequence is shown in SEQ ID NO.3.

[0037] Example 1 Cloning of the LpCDPK27 gene

[0038] I. Preparation of cDNA template from perennial ryegrass: Place a small amount of perennial ryegrass (Lolium perenne) 'Emerald' seeds into a 1.5 mL sterile centrifuge tube, add 1 mL of 70% ethanol and treat for 30 s, then wash 3 times with sterile water. Add 10% sodium hypochlorite solution, invert for 10 min, and wash 3-5 times with sterile water. The seeds were then placed on 1 / 2 MS medium. After germination and rooting, they were transplanted into a mixed culture medium (vermiculite: nutrient soil = 1:1, V:V) and cultivated in a greenhouse at a temperature of 22 ℃ / 16 ℃ (day / night), a photoperiod of 16 h / 8 h (light / dark), a humidity of 60%, and a light intensity of 800 μmol·m⁻²·s⁻¹. The plants were regularly pruned and fertilized with an appropriate amount of compound fertilizer (N:P:K = 15:15:15, V:V:V) solution. After the materials grew uniformly, they were used for experimental treatment. Mature leaves of perennial ryegrass were taken, and total RNA was extracted using the RNA Prep PurePlant kit (Tiangen DP432). cDNA templates were obtained by reverse transcription using the PrimeScript RT reverse transcription kit (Takara, RR092 / 6210A) and stored at -20 ℃ for later use.

[0039] II. Design of LpCDPK27 gene primers

[0040] Based on the cDNA sequence of the LpCDPK27 gene from the perennial ryegrass genome data (NCBI, https: / / www.ncbi.nlm.nih.gov / ), specific primers for amplifying the open reading frame (ORF) of this gene were designed and synthesized by Beijing Qingke. The upstream primer for amplifying LpCDPK27, LpCDPK27-F, has the sequence SEQ ID NO.4: 5′-ATGGCAGAGAAAAAGAATTACAGC-3′; the downstream primer for amplifying LpCDPK27, LpCDPK27-R, has the sequence SEQ ID NO.5: 5′-TAGATTGCACCAGGTGCGTC-3′.

[0041] III. Amplification of the LpCDPK27 gene and recovery of the product

[0042] PCR amplification was performed using the template prepared in step one and the primers designed in step two:

[0043] PCR reaction system (50 μL): 1 μL high-fidelity DNA polymerase (Novazia P525), 25 μL 2× high-fidelity buffer, 5 μL dNTPs (10 mmol·L⁻¹), 1.5 μL upstream primer (10 μmol·L⁻¹), 1.5 μL downstream primer (10 μmol·L⁻¹), 1-2 μL reverse transcription first-strand cDNA, and ddH₂O to a final volume of 50 μL;

[0044] PCR reaction program: 94 ℃ for 3 min; 94 ℃ for 0.5 min, 55 ℃ for 0.5 min, 72 ℃ for 0.5 min, 35 cycles; 72 ℃ for 10 min.

[0045] The amplification product was detected by 1% agarose gel electrophoresis; a sequence of approximately 1551 bp was obtained, as shown in the figure. Figure 1 As shown, the obtained PCR products were recovered using a DNA gel recovery kit (OMEGA, D2500-02).

[0046] Example 2: Construction of the overexpression vector pCAMBIA1390-LpCDPK27

[0047] A schematic diagram of the carrier construction is shown below. Figure 2 As shown, based on the LpCDPK27 open reading frame sequence obtained in Example 1, as shown in SEQ ID NO.1, amplification primers with Kpn I and BamHI (pCAMBIA1390) restriction sites were designed:

[0048] An upstream primer LpCDPK27-1390-F was introduced to the Kpn I restriction site, the sequence of which is shown in SEQ ID NO.6:

[0049] 5′-TTACTTCTGCACTAGGTACCATGGGCGCCTGCCTCTCCTCCTCCTC-3′;

[0050] The downstream primer LpCDPK27-1390-R, which introduces the BamHI restriction site, has the sequence shown in SEQ ID NO.7:

[0051] 5′-GAATTCCCGGGGATCCTCACAAAGCCTGTGGATTTGGAAC-3′;

[0052] Using the LpCDPK27 gene PCR product recovered in Example 1 as a template, amplification was performed using primers (SEQ ID NO. 6 and NO. 7) with homologous arms containing Kpn I (TaKaRa) and BamHI (TaKaRa) restriction sites. The PCR reaction system and procedure for this step were consistent with those in Example 1. After amplification of the homologous arms of the PCR-recovered fragment, it was used for vector construction: the pCAMBIA1390 expression vector was double-digested with restriction endonucleases Kpn I and BamHI, respectively. The target fragment amplified from the homologous arms of the recovered LpCDPK27 gene and the large fragment of the digested vector were ligated using ClonExpress II recombination reaction solution at a molar ratio of 1:2. The reaction system was as follows: 4 μL of 5×CE II Buffer, 2 μL of Exnase II, 0.06 pmol of the recovered target fragment, water added to 20 μL, ligation was performed at 37 °C for 30 min, and the ligation product was obtained after cooling on ice.

[0053] Preparation of competent *E. coli* cells: *E. coli* DH5α bacterial culture stored at -80 °C was inoculated onto LB solid medium and cultured overnight at 37 °C. A single colony of *E. coli* DH5α was picked and inoculated into 1 mL of LB liquid medium and cultured on a shaker at 37 °C with shaking at 200 rpm for approximately 6 hours. The culture was then inoculated into 200 mL of LB liquid medium and cultured on a shaker at 37 °C with shaking at 200 rpm until OD (Organic Dose) was reached. 550 =0.6; collect bacterial cells by centrifugation at 2500 rpm for 10 min, wash with pre-cooled 50% glycerol (glycerol:LB liquid medium), centrifuge to collect bacteria; repeat washing with pre-cooled 10% glycerol (glycerol:LB liquid medium), centrifuge to collect bacteria, finally aliquot the bacteria and store at -80 ℃ for later use;

[0054] Heat shock transformation of E. coli with ligation product: The ligation product was added to 100 μL of DH5α competent cells and placed on ice for 30 min; after heat shock at 42 ℃ for 45 s, it was placed on ice for 2 min; then the cells were transferred to 1 mL of LB liquid medium and cultured on a shaker at 37 ℃ with shaking at 200 rpm for 1 h. 100 μL of the bacterial culture was spread on LB solid medium (containing 200 μg / mL Kan) and incubated upside down overnight at 37 ℃.

[0055] Screening, purification, and sequencing of recombinant plasmid pCAMBIA1390-LpCDPK27: White colonies were picked for colony PCR detection. PCR-positive colonies were picked and inoculated into 2 mL of LB liquid medium containing 200 μg / mL kanamycin, and cultured overnight at 37 ℃ with shaking at 200 rpm. 0.5 mL of the bacterial culture was retained for later use, and 1.5 mL of the bacterial culture was used to extract the plasmid using a plasmid DNA purification kit (Qiagen), and stored at 4 ℃ for later use. The purified recombinant plasmid was named pCAMBIA1390-LpCDPK27.

[0056] PCR detection, using pCAMBIA1390-LpCDPK27 plasmid as a template, and following the PCR reaction system and procedure described in Example 1, amplified the correctly sized fragment using primer pairs SEQ ID NO. 6 and SEQ ID NO. 7. The band size was approximately 1551 bp, indicating that the recombinant vector contains the LpCDPK27 gene. PCR electrophoresis results are shown below. Figure 2 As shown in the figure. Further sequencing of the insert fragment showed that the sequence of the insert fragment was completely identical to the sequence of the coding region of LpCDPK27, and the restriction enzyme sites at both ends of the insert fragment were also completely correct, thus proving that the recombinant expression vector pCAMBIA1390-LpCDPK27 was successfully constructed. The expression cassette diagram of this recombinant expression vector is shown in the figure. Figure 3 As shown.

[0057] Example 3 Subcellular localization of LpCDPK27

[0058] Based on the amplified CDS sequence of LpCDPK27, specific primers with the stop codon (TAG) removed were designed. The terminal sequence of the original LpCDPK27 CDS sequence (SEQ ID NO.1) was SEQ ID NO.8: 5'-GACGCACCTGGTGCAATCTAG-3'; during vector construction, the three letters TAG at the end were removed through primer design, resulting in the terminal sequence of the amplified target fragment being SEQ ID NO.9: 5'-GACGCACCTGGTGCAATC-3'. This fragment was then constructed into the pCAMBIA1305-GFP vector and transiently expressed in Nicotiana benthamiana, as detailed below:

[0059] Introduce the upstream primer for the Spe I restriction site, SEQ ID NO.10:

[0060] 5′-AGGACAGCCCAGATCACTAGTATGGGGCGCCTGCCTCTCCTCCTCCTCCT-3′;

[0061] A downstream primer with an Xma I restriction site was introduced, SEQ ID NO.11:

[0062] 5′-GCTCACCATGGATCCCCCGGGCAAAGCCTGTGGATTTGGAACTCCT-3′;

[0063] (1) Take a newly activated Agrobacterium monoclonal carrying LpCDPK27-GFP and inoculate it into YEP medium containing 35 mg / L rifampicin (Rif) and 50 mg / L kanamycin (Kan). Incubate on a shaker (28 ℃) at 200 rpm for 12 hours.

[0064] (2) When the bacterial solution OD 600 When the bacterial concentration is 0.6-0.8, centrifuge at 5000 rpm for 10 min and collect the bacterial pellet.

[0065] (3) Resuspend the bacterial culture in working solution (10 mM MgCl2, 10 mM MES, 200 μM acetylsuccine) and adjust to OD. 600= 0.1; The preparation of Agrobacterium tumefaciens suspension carrying the plasma membrane localization marker vector mCherry was carried out according to the aforementioned method for preparing Agrobacterium tumefaciens suspension carrying LpCDPK27; Experimental group combination: Agrobacterium tumefaciens suspension carrying the fusion expression vector pCAMBIA1305-LpCDPK27-GFP was mixed with Agrobacterium tumefaciens suspension carrying the plasma membrane localization marker mCherry at a 1:1 volume ratio; Control group combination: Agrobacterium tumefaciens suspension carrying the empty vector pCAMBIA1305-GFP was mixed with Agrobacterium tumefaciens suspension carrying the plasma membrane localization marker mCherry at a 1:1 volume ratio.

[0066] (4) Gently inject the substance into the underside of leaves of 1-month-old Nicotiana benthamiana using a syringe without a needle. Place the plants in a growth chamber and grow at 25 ℃ for 48-72 h. Observe and photograph the transformed leaves using a laser confocal scanning microscope (Zeiss LSM800, Germany).

[0067] The results are as follows Figure 4 The results showed that the fluorescence signal of LpCDPK27-GFP was observable in the cell membrane and overlapped with the fluorescence of the cell membrane-localized mCherry-labeled signal; the GFP fluorescence of the control vector was visible throughout the cell. This indicates that LpCDPK27 is localized in the cell membrane.

[0068] Example 4: High-Temperature Induced Expression Analysis of LpCDPK27

[0069] I. Obtaining cDNA templates from perennial ryegrass under high temperature conditions

[0070] Perennial ryegrass seedlings grown under natural greenhouse light for 30 days were selected and placed in an artificial climate chamber for high-temperature treatment (38 ℃). Different tissue parts (newly unfolded leaves, mature leaves, senescent leaves, leaf sheaths, roots, rhizomes, and stems) were cut and placed in 2 mL centrifuge tubes, then rapidly frozen in liquid nitrogen and stored at -80 ℃ for later use. The samples stored at -80 ℃ were then removed, ground with liquid nitrogen, and total RNA was extracted from the leaves using TRE-Trizol reagent (TaKaRa). Reverse transcription was performed using the Prime Script RT reagent Kit with gDNA Erase (TaKaRa) to prepare cDNA templates.

[0071] II. Design of Specific Detection Primers

[0072] Based on the cDNA sequence of the LpCDPK27 gene in perennial ryegrass, primers were designed and their specificity evaluated using Primer 5 software in conjunction with NCBI's Primer-BLAST online tool. The following primers were determined for real-time quantitative PCR:

[0073] LpCDPK27 gene upstream primer (LpCDPK27-qPCR F), SEQ ID NO.12:

[0074] 5′-AACAGCCGCTATAGTGAGAAAG-3′;

[0075] Downstream primer of LpCDPK27 gene (LpCDPK27-qPCR R), SEQ ID NO.13:

[0076] 5′-GCTTCATATCTCGGTGGACTAAC-3′;

[0077] Reference gene LpelF4A upstream primer (LpelF4A-qPCR F), SEQ ID NO.14:

[0078] 5′-GCACCCTGTTCTTTCTTACCGAG-3′;

[0079] Reference gene LpelF4A downstream primer (LpelF4A-qPCR R), SEQ ID NO.15:

[0080] 5′-AGTAAGGTCACGTCCAGCAAGG-3′.

[0081] III. Real-time quantitative PCR detection of LpCDPK27 gene expression

[0082] The cDNA prepared in step one was diluted 30-fold and used as a qRT-PCR template. The reaction system was 10 μL: 5 μL ChamQ Universal SYBR qPCR Master Mix (2×), 0.2 μL each of forward and reverse primers (10 μM), 4 μL cDNA template, and 0.6 μL ddH2O.

[0083] PCR reaction program: 95 ℃ for 30 s; followed by 94 ℃ for 10 s, 60 ℃ for 60 s, for a total of 40 cycles; melting curve was set at 95 ℃ for 15 s, 60 ℃ for 30 s, and 95 ℃ for 15 s.

[0084] A negative control (NTC) without cDNA was set up, with three technical replicates for each sample; the internal reference gene LpeIHF4A was used as a reference. Two [samples were analyzed]. -ΔΔCT The relative expression levels were calculated, and melting curve analysis was performed after the reaction to verify the amplification specificity.

[0085] The results of real-time quantitative PCR are as follows Figure 5The results showed that LpCDPK27 was expressed at different levels in different tissues of perennial ryegrass (newly unfolded leaves, mature leaves, senescent leaves, leaf sheaths, roots, rhizomes, and stems) under high-temperature treatment, indicating that high temperature induces the expression of the LpCDPK27 gene.

[0086] Example 5: Transgenic perennial ryegrass plants and their molecular detection

[0087] I. Genetically modified perennial ryegrass plants

[0088] 1. The recombinant expression vector pCAMBIA1390-LpCDPK27 was introduced into Agrobacterium tumefaciens EHA105.

[0089] Streak EHA105 bacterial culture on YEP+Kan+Rif plates and incubate at 28 ℃ for 48 h. Pick a single colony and inoculate it into 50 mL of liquid LB medium, incubating overnight at 28 ℃. Take 0.5 mL of the bacterial culture and inoculate it into 500 mL of liquid YEP medium, incubating at 28 ℃ for 8 h until OD reaches zero. 600 = 0.6, cool in an ice bath for 10 min; pour into a sterile 200 mL centrifuge tube, equilibrate, centrifuge at 4 ℃ and 4000 rpm for 10 min, collect the cells, discard the culture medium, and invert on a sterile paper towel to drain; add 50 mL of pre-chilled 10% glycerol (glycerol to YEP volume ratio of 1:9), gently shake on ice to suspend, centrifuge at 4 ℃ and 4000 rpm for 15 min, collect the cells; repeat washing once; add 2 mL of pre-chilled 10% glycerol to suspend the cells, aliquot into 25 μL / tube, flash freeze in liquid nitrogen, and store at -80 ℃.

[0090] The competent EHA105 cells were thawed on ice and transformed using the traditional heat shock method: In a clean bench, 2 μL of pCAMBIA1390-LpCDPK27 plasmid (20 ng / μL) was added to 50 μL of thawed competent cells, the mixture was gently tapped to mix, and the cells were incubated on ice for 5 min, then heat-shocked at 37 ℃ for 5 min, and then cooled on ice for 2-5 min; 1 mL of pre-warmed YEP liquid medium was then added, and the cells were gently shaken at 28 ℃ and 180-200 rpm for 2-4 h to recover; 0.3 mL of the bacterial culture was spread on YEP plates (containing 50 mg / L Kan and 35 mg / L Rif), and incubated upside down at 28 ℃ for 48 h, and single colonies were picked for subsequent identification.

[0091] 2. Identification of Agrobacterium-positive colonies containing the recombinant expression vector pCAMBIA1390-LpCDPK27

[0092] Single colonies were picked from the plate for colony PCR detection and labeled. The primers used for detection were SEQ ID NO.6 and SEQ ID NO.7 as described in Example 2. The PCR reaction system (20 μL) contained: 10 μL of 2×Taq Master Mix, 0.5 μL each of forward and reverse primers (10 μmol·L⁻¹), 1 μL of sterile aqueous suspension of a small amount of bacterial cells as template, and 8 μL of ddH₂O. The PCR reaction program was as follows: 95 ℃ pre-denaturation for 5 min; followed by 30 cycles of 95 ℃ for 30 s, 58 ℃ for 30 s, and 72 ℃ for 90 s; and finally 72 ℃ for 10 min. Further PCR-positive colonies were picked and cultured in 3 mL YEP liquid medium (containing 35 mg / L Rif and 50 mg / L Kan) at 28 ℃ with shaking for 40 h. 2 mL of bacterial culture was taken and plasmid was extracted using the alkaline lysis method. The expression vector pCAMBIA1390-LpCDPK27 was digested with restriction endonucleases Kpn I and BamHI to confirm that the positive clones contained the plant expression vector pCAMBIA1390-LpCDPK27. 0.8 mL of Agrobacterium culture was taken, and 0.2 mL of 50% glycerol (glycerol: YEP liquid medium) was added. After mixing, the culture was stored at -80 ℃ for preservation.

[0093] 3. Obtaining transgenic perennial ryegrass plants

[0094] Genetic transformation and identification of perennial ryegrass

[0095] Agrobacterium-mediated transformation of perennial ryegrass using pCAMBIA1390-LpCDPK27. The overexpression vector was constructed as pCAMBIA1390-LpCDPK27; the Agrobacterium tumefaciens strain was EHA105. The plasmid was transformed into EHA105 using the heat shock method. Activated positive clones were used for subsequent transformations, and the cells were activated overnight in YEP liquid medium to OD. 600 = 0.8-1.2 spare.

[0096] Callus preparation: Take perennial ryegrass seeds (after sterilization) and cut them in half lengthwise to expose the endosperm. Inoculate the cut side down into the induction medium and change the medium every 2 weeks. After induction for about 1 month, select well differentiated callus tissues and transfer them to the subculture medium.

[0097] The culture media used for transformation include the following types, and the formulations of each culture medium are as follows (per 1L):

[0098] Induction medium (pH 5.85±0.05): 4 g N6 medium, 1 g acid-hydrolyzed casein, 1 g proline, 30 g maltose, 7 mL 2,4-dichlorophenoxyacetic acid, 0.05 mL 6-benzylaminopurine, 6 g agar;

[0099] Subculture medium (pH 5.85±0.05): 4 g N6 medium, 1 g acid-hydrolyzed casein, 1 g proline, 30 g maltose, 5 mL 2,4-dichlorophenoxyacetic acid, 0.1 mL 6-benzylaminopurine, 6 g agar;

[0100] Co-culture medium (pH 5.7±0.05): 4 g N6 medium, 1 g acid-hydrolyzed casein, 1 g proline, 60 g maltose, 5 mL 2,4-dichlorophenoxyacetic acid, 0.1 mL 6-benzylaminopurine, 6 g agar.

[0101] Selective medium (pH 5.85±0.05): 4 g N6 medium, 1 g acid-hydrolyzed casein, 1 g proline, 30 g maltose, 5 mL 2,4-dichlorophenoxyacetic acid, 0.1 mL 6-benzylaminopurine, 6 g agar, plus 50 mg / L hygromycin and termethin;

[0102] Differentiation medium (pH 5.7±0.05): 2.2 g MS medium, 30 g maltose, 0.5 mL 6-benzylaminopurine, 6 g agar, 25 mg / L hygromycin;

[0103] Rooting medium (pH 5.7±0.05): 2.2 g MS medium, 30 g maltose, 5 g agar, 150 mg / L termethin and 25 mg / L hygromycin;

[0104] Tissue infection solution (pH 5.4±0.05): 4 g N6 medium, 1 g proline, 30 g glucose, 5 mL 2,4-dichlorophenoxyacetic acid, 0.1 mL 6-benzylaminopurine.

[0105] Preparation of Agrobacterium tumefaciens culture: EHA105 containing pCAMBIA1390-LpCDPK27 was cultured in YEP liquid medium for 12 hours until OD. 600 = 0.8-1.2, centrifuge, discard the supernatant, resuspend in tissue infection solution and wash twice, adjust the bacterial culture to OD. 600 =0.6-0.8, stand at 28 ℃ for 1-2 h, then ice bath for 20 min.

[0106] Callus cold shock: Select vigorous callus pieces with a diameter of about 5 mm and place them in a sterile bottle for 20 min on ice.

[0107] Infection and co-culture: The cold-shocked callus block was mixed with the bacterial suspension and vacuum-treated for 15 min at a vacuum degree of -0.08 MPa, followed by shaking at 28 ℃ and 100 rpm for 10 min; after removing the excess bacterial solution on the surface, it was transferred to the co-culture medium and incubated in the dark at 25 ℃ for 3 days.

[0108] Sterilization and resistance screening: Callus blocks co-cultured for 3 days were thoroughly rinsed 3-4 times with sterile water containing 200 mg / L termethin and the surface moisture was dried. They were then transferred to selective medium (containing hygromycin) and cultured in the dark at 25 ℃. Subculture was performed every 14 days for 4-6 weeks until stable resistance was obtained from transformed callus.

[0109] Differentiation, regeneration, and rooting: The resistant callus was transferred to differentiation medium (MS + 0.5 mL / L 6-BA, containing 25 mg / L hygromycin) and cultured under light for about 2 weeks to induce shoot differentiation; the regenerated shoots were transferred to rooting medium (MS, containing 25 mg / L hygromycin and 150 mg / L termethin) and cultured for about 4 weeks to obtain plants.

[0110] Transplanting and acclimatization: After the roots have grown strong, transplant them into the substrate and manage them in the greenhouse according to routine procedures. Once the growth has stabilized, they can be used for molecular identification and phenotypic determination.

[0111] II. PCR detection of genetically modified perennial ryegrass

[0112] 1. Sample and genomic DNA extraction

[0113] Regenerated perennial ryegrass plants under natural greenhouse light for about 30-40 days were selected for testing. Fresh leaves (about 50-100 mg) were cut and genomic DNA was extracted using the conventional CTAB method or a plant genomic DNA extraction kit. After the quality was confirmed by 1% agarose gel electrophoresis and spectrophotometry, the samples were stored at -20 ℃ for later use.

[0114] 2. Detection primers and design basis

[0115] To improve the specificity for exogenous copies, two sets of primers were used for parallel detection:

[0116] Primer set A (covering the exogenous open reading frame (ORF), the amplified product is approximately 1551 bp in length and is used to detect whether the exogenous CDS is present in its entirety):

[0117] Upstream primer SEQ ID NO.4: 5′-ATGGCAGAGAAAAAGAATTACAGC-3′;

[0118] Downstream primer SEQ ID NO.5: 5′-TAGATTGCACCAGGTGCGTC-3′.

[0119] Primer set B (used to amplify the LpCDPK27 coding sequence to construct the recombinant plasmid pCAMBIA1390, and for positive verification of this recombinant plasmid):

[0120] The upstream primer is shown in SEQ ID NO. 6; the downstream primer is shown in SEQ ID NO. 7.

[0121] 3. PCR reaction system (25 μL system)

[0122] 2×Taq Master Mix 12.5 μL; forward and reverse primers (10 μM) 0.5 μL each (primer set A or primer set B); template gDNA 1.0 μL (approximately 50-100 ng); ddH2O to bring the total volume to 25 μL.

[0123] Genomic DNA from the perennial ryegrass plant 'Emerald' WT (wild type) was used as a negative control; pCAMBIA1390-LpCDPK27 plasmid was used as a positive control.

[0124] 4. PCR procedure

[0125] 95 ℃ for 3 min; then 95 ℃ for 10 s, 58-60 ℃ for 30 s, 72 ℃ for 60-90 s, 35 cycles; 72 ℃ for 10 min.

[0126] 5. Electrophoresis and Judgment

[0127] Take 5 μL of the amplification product and perform electrophoresis on a 1% agarose gel to detect the bands:

[0128] A PCR positive result is defined as a single, distinct band of approximately 1551 bp amplified using primer set A or primer set B; this specific band should not be present in WT.

[0129] Example 6: Identification of heat tolerance in transgenic perennial ryegrass

[0130] The heat tolerance phenotypes of transgenic overexpression lines (OE1, OE2) and wild-type (WT) materials of perennial ryegrass grown in a plant incubator for 40 days were compared.

[0131] Perennial ryegrass cuttings were propagated in plastic pots filled with a mixture of peat moss, vermiculite, and perlite in a 3:1:1 ratio (V:V:V). They were then cultured for approximately 40 days in a long-day incubator at 24°C (16 h light, 8 h dark). Plants with uniform growth were then subjected to high-temperature stress treatment at 42°C and 60% relative humidity for 21 consecutive days, during which time the substrate was kept moist by watering with 1 / 2 Hoagland nutrient solution every 3 days. The control group was kept at room temperature under the same conditions.

[0132] Observe and record the changes in plant appearance before and after treatment. For example... Figure 6 As shown in A, before treatment, WT, OE1, and OE2 plants showed good growth with no significant differences. After 21 days of stress at 42 ℃, the leaves of WT plants showed obvious chlorosis, curling, and drying, while OE1 and OE2 plants maintained good green color and upright posture.

[0133] Total RNA was extracted from transgenic ryegrass leaves and reverse transcribed into cDNA. The relative expression level of LpCDPK27 was detected using qRT-PCR. The qRT-PCR reaction procedure and system were as described in Example 4, with LpGAPDH as the internal reference gene. -ΔΔCT ) method to calculate expression level ( Figure 6 (B in the text). Subsequently, leaves from wild-type and transgenic ryegrass plants before and after high-temperature treatment (42 °C) were collected, and their relative electrolyte leakage rate (REC) was measured to assess the degree of cell membrane damage. Figure 6 (C in the text). The results showed that perennial ryegrass plants overexpressing LpCDPK27 exhibited stronger heat resistance under high temperature stress, with significantly less leaf damage than the wild type, and were able to maintain higher physiological activity and tissue integrity.

[0134] In summary, the LpCDPK27 protein identified in this invention is specifically located on the cytoplasmic membrane. This subcellular localization allows it to preferentially sense and transduce transient calcium signaling impulses induced by high temperatures, thereby establishing a regulatory pathway from environmental perception to phenotypic resistance at the molecular level. This specific regulatory function is difficult to deduce directly from existing studies based on sequence homology, and its significant resistance advantage under extreme high-temperature stress of 42 °C confirms the innovative application value of this gene in improving the stress resistance of cool-season forage grasses.

[0135] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of overexpression of the LpCDPK27 gene in improving the heat tolerance of perennial ryegrass, characterized in that, The nucleotide sequence of the LpCDPK27 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. A recombinant expression vector comprising the LpCDPK27 gene as described in claim 1.

3. A recombinant genetically engineered cell, characterized in that, It includes the recombinant expression vector as described in claim 2.

4. The application of the recombinant expression vector according to claim 2 or the recombinant genetically engineered cell according to claim 3 in improving the heat resistance of perennial ryegrass.

5. A method for cultivating heat-resistant perennial ryegrass, characterized in that, The recombinant expression vector of claim 2 or the recombinant genetically engineered cell of claim 3 is genetically transformed into perennial ryegrass.

6. A method for detecting the LpCDPK27 gene, which regulates heat tolerance, in perennial ryegrass, characterized in that... Includes the following steps: Extracting plant genomic DNA; The genomic DNA was amplified by PCR and then detected. If the target band is present, the plant contains the LpCDPK27 gene that regulates heat tolerance.

7. The method according to claim 6, characterized in that, The primer pairs used in the PCR amplification include the upstream primer shown in SEQ ID NO.4 and the downstream primer shown in SEQ ID NO.5.

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