A perennial ryegrass heat-tolerant gene lpHSP70-10

Abstract

The application discloses a perennial ryegrass heat-resistant gene LpHSP70-10, the gene is significantly up-regulated under high-temperature stress, and reaches a peak at 12 hours, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1; the gene LpHSP70-10 is knocked out through a gene editing technology, and a heat-resistant ryegrass variety is cultivated. Based on whole genome association analysis combined with transcriptome sequencing results, the application finds a gene LpHSP70-10 which is sensitive to heat stress. The heat-resistant gene LpHSP70-10 of the ryegrass provided by the application can regulate the expression of heat stress-related genes, and subsequent gene knockout can improve the heat resistance of the ryegrass, which is helpful for cultivating the ryegrass variety suitable for the growth in the relatively hot southern region, and simultaneously reduces the breeding workload, reduces the breeding scale, shortens the breeding period, improves the breeding efficiency, and accelerates the breeding of the heat-resistant ryegrass variety.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering for enhancing plant heat resistance, and relates to a heat resistance gene LpHSP70-10 in annual ryegrass, its encoded protein, amplification primers, overexpression vector, and applications of the gene LpHSP70-10. Background Technology

[0002] Perennial ryegrass (Lolium perenne L.) belongs to the genus Lolium in the family Poaceae. It is characterized by its well-developed fibrous root system, abundant tillering, rapid turf establishment, and strong resistance to diseases and pests, making it an important pioneer grass species for lawn establishment. Furthermore, perennial ryegrass exhibits strong regeneration ability and high yield after mowing, making it an important cultivated forage and green manure crop. As an excellent mixed forage grass for grasslands, a landscaping grass for turfgrass, and a species for ecological restoration and improvement, perennial ryegrass is widely planted in temperate regions worldwide.

[0003] However, as a typical cool-season forage and turfgrass, perennial ryegrass prefers a cool and humid climate and is sensitive to high temperatures, making it difficult to survive the summer in southern regions. High-temperature stress is a significant factor limiting the growth and development of perennial ryegrass, and with global warming and the occurrence of extreme high temperatures, its growth and development will be even more severely affected.

[0004] It is particularly important to actively address the threat posed by global warming to the survival of perennial ryegrass, explore ways to improve its heat resistance, and solve the limitations in its application as forage and turfgrass. Summary of the Invention

[0005] Therefore, the present invention aims to provide a heat-resistant gene LpHSP70-10 for perennial ryegrass.

[0006] The second objective of this invention is to provide a protein encoded by the gene LpHSP70-10.

[0007] A third objective of this invention is to provide a primer pair for cloning the ryegrass heat-resistant gene LpHS P70-10.

[0008] The fourth objective of this invention is to provide an overexpression vector containing the ryegrass heat-resistant gene LpHSP70-10.

[0009] The fifth objective of this invention is to provide a method for improving the heat resistance of perennial ryegrass.

[0010] The sixth objective of this invention is to provide a method for constructing transgenic Arabidopsis plants using the LpHSP70-10 gene.

[0011] The seventh objective of this invention is to provide a method for verifying the heat resistance of the Lp HSP70-10 gene through the aforementioned transgenic plant.

[0012] The eighth objective of this invention is to provide a use for the gene LpHSP70-10.

[0013] The ninth objective of this invention is to provide a breeding method for perennial ryegrass.

[0014] Through long-term exploration and experimentation, as well as numerous experiments and efforts, the inventors have continuously reformed and innovated to solve the above-mentioned technical problems. The technical solution provided by this invention is to provide a heat-resistant gene LpHSP70-10 for perennial ryegrass. The expression of the gene is significantly upregulated under high-temperature stress and reaches a peak at 12 hours. The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0015] The present invention also provides a protein encoded by the gene LpHSP70-10, the amino acid sequence of which is shown in SEQ ID NO.2.

[0016] The present invention also provides a primer pair for cloning the ryegrass heat-resistant gene LpHSP70-10, the base sequence of which is as follows:

[0017] Upstream primer F: 5'-ATGTCGGGCAAGGGCGAAGGTCCGG-3';

[0018] Downstream primer R: 5'-TTAGTCGACCTCCTCAATCTTGGGC-3'.

[0019] The present invention also provides an overexpression vector containing the ryegrass heat tolerance gene LpHSP70-10. The full-length ryegrass heat tolerance gene LpHSP70-10 is inserted into the vector, which contains the CaMV 35S promoter, to obtain the overexpression vector LpHSP70-10.

[0020] The present invention also provides a method for improving the heat resistance of perennial ryegrass by knocking out the gene LpHSP70-10 and analyzing the expression level of the gene by transcriptome sequencing and real-time quantitative PCR.

[0021] The present invention also provides a method for constructing Arabidopsis transgenic plants using the LpHSP70-10 gene, wherein transgenic plants are obtained through Agrobacterium-mediated transformation and screening, and their heat resistance is verified under high temperature conditions.

[0022] The present invention also provides a method for verifying the heat resistance of the gene LpHSP70-10 through the transgenic plants, by measuring indicators including chlorophyll fluorescence, electrical conductivity and chlorophyll content to evaluate the heat resistance of the transgenic plants.

[0023] The present invention also provides a use of the gene LpHSP70-10, knocking out the gene LpHS P70-10 to improve the heat resistance of plants, for the purpose of breeding new heat-resistant plant varieties.

[0024] The present invention also provides a breeding method for perennial ryegrass, which involves knocking out the LpHSP70-10 gene using gene editing technology to cultivate heat-resistant ryegrass varieties.

[0025] Compared with the prior art, one of the above technical solutions has the following advantages:

[0026] a) Based on genome-wide association analysis combined with transcriptome sequencing results, this invention discovered a gene, LpHSP70-10, that is sensitive to heat stress. The LpHSP70-10 heat-resistant gene in ryegrass provided by this invention can regulate the expression of heat stress-related genes. Subsequent gene knockout is planned to improve the heat resistance of ryegrass, which will help breed ryegrass varieties suitable for growth in hotter southern regions. Simultaneously, it will reduce breeding workload, shrink the breeding scale, shorten the breeding period, improve breeding efficiency, and accelerate the selection of heat-resistant ryegrass varieties.

[0027] (b) Due to the increasing frequency of extreme heat events caused by global climate change, the heat resistance enhancement technology provided by this invention helps ensure the stability of agricultural production and reduce losses caused by high temperatures. By knocking out the LpHSP70-10 gene, perennial ryegrass can better adapt to different ecological environments, especially in areas with higher temperatures, which contributes to the successful implementation of ecological restoration and greening projects.

[0028] c) With global warming, the impact of high-temperature stress on plant growth is becoming increasingly severe. The technical solution of this invention helps to improve the adaptability of plants to high-temperature environments, which is of great significance for protecting biodiversity and maintaining ecological balance. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0030] Figure 1This is a cloning sequence map encoding the ryegrass heat-resistant gene LpHSP70-10 of this invention.

[0031] Figure 2 This is a prediction diagram of the hydrophilicity / hydrophobicity of the protein encoded by the ryegrass heat-resistant gene LpHSP70-10 of this invention.

[0032] Figure 3 This is a predicted diagram of the secondary structure of the protein encoded by the ryegrass heat-resistant gene LpHSP70-10 of this invention.

[0033] Figure 4 This is a predicted tertiary structure diagram of the protein encoded by the ryegrass heat-resistant gene LpHSP70-10 of this invention.

[0034] Figure 5 This is a predicted map of the conserved structural domains of the protein encoded by the ryegrass heat-resistant gene LpHSP70-10 of this invention.

[0035] Figure 6 This is a phylogenetic tree analysis of the heat-resistant gene LpHSP70-10 in ryegrass of this invention with other species.

[0036] Figure 7 This is a graph showing the results of Arabidopsis thaliana heat stress experiments and phenotypic analysis. Figure 7 In the figure, A represents the control group (CK), and B represents the treatment group (33 / 38℃). The figures show the phenotypic distribution of Arabidopsis thaliana (WT, OE1, OE2, and OE3 lines) before heat treatment (day 0) and after 10 days of heat stress treatment. As can be seen from the figures, there were no significant differences in the phenotypic distribution of the four Arabidopsis thaliana lines in the control group. However, after heat stress, the phenotypic distribution of OE1, OE2, and OE3 lines was significantly worse than that of WT. This indicates that this gene is a negatively regulated gene.

[0037] Figure 8 These are the statistical results of Arabidopsis thaliana chlorophyll fluorescence; Normal is the control group, and Hot is the 33 / 38℃ heat treatment group.

[0038] Figure 9 These are the statistical results of the electrical conductivity of Arabidopsis thaliana under heat stress; Normal is the control group, and Hot is the group treated at 33 / 38℃.

[0039] Figure 10 These are the statistical results of chlorophyll content in Arabidopsis thaliana under heat stress; Normal represents the control group, and Hot represents the group treated at 33 / 38℃. Detailed Implementation

[0040] The following description, in conjunction with the accompanying drawings and a specific embodiment, will be provided.

[0041] This embodiment also describes the perennial ryegrass heat tolerance gene LpHSP70-10, the protein encoded by the gene LpHSP70-10, the primer pair for cloning the ryegrass heat tolerance gene LpHSP70-10, the overexpression vector containing the ryegrass heat tolerance gene LpHSP70-10, the method for improving the heat tolerance of perennial ryegrass, the method for constructing Arabidopsis transgenic plants using the gene LpHSP70-10, the method for verifying the heat tolerance of the gene LpHSP70-10 through the transgenic plants, the uses of the gene LpHSP70-10, and the breeding method for perennial ryegrass.

[0042] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These 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; and the materials and reagents used, unless otherwise specified, are commercially available.

[0043] In the process of realizing this invention, the inventors selected the "Gentleman" perennial ryegrass, a nationally approved variety with excellent traits in actual production, as the test material and planted it at the Wenjiang Campus of Sichuan Agricultural University.

[0044] Weigh 2g of seeds into each pot and sow them evenly in a plastic pot that is 20cm long, 15cm wide, and 10cm high. After 14 days of germination, water the pot with Hoagland nutrient solution (Hoagland Biotechnology Co., Ltd.).

[0045] The entire experiment was conducted in an incubator (RTOP series, Zhejiang Top Instrument Co., Ltd.). The normal growth environment in the incubator was: day / night temperature (25 / 15℃), relative humidity 70%, light duration 12h, and light intensity 500 μmol·(m²). 2 ·s) -1 .

[0046] After 45 days of germination culture, heat stress treatment was applied: the plants were placed in a growth chamber at 38 / 30℃ (day / night) with other conditions unchanged; leaf samples were taken at 0h, 6h, 12h, 24h and 48h of treatment, with 3 biological replicates for each. The samples were rapidly frozen in liquid nitrogen and stored at -80℃.

[0047] Total RNA was extracted from mature leaves. The plant total RNA extraction kit from Tiangen (Beijing) Biochemical Technology Co., Ltd. was used for RNA extraction, following the instructions provided. The extracted RNA plasmids were used for bioinformatics and functional analysis.

[0048] RNA was extracted and then reverse transcribed to synthesize cDNA.

[0049] The effectiveness of RNA-seq was verified using quantitative real-time PCR (qRT-PCR). The expression levels of 24 candidate genes in the HSP70 family of ryegrass were analyzed using qRT-PCR. All samples were prepared in triplicate (3 biological replicates and 2 technical replicates). The final results were analyzed based on 2... -ΔΔCt The method analyzes the relative expression levels of genes.

[0050] In transcriptomic data, the expression of LpHSP70-10 significantly increased under high-temperature stress, and the expression level of LpHSP70-10 peaked at 12 h in qRT-PCR analysis. This suggests that it may play an important role in the later stages of high-temperature stress. The nucleotide sequence of LpHSP70-10 is shown in Sequence Listing Seq ID NO.1, and the protein it encodes is shown in Sequence Listing Seq ID NO.2.

[0051] Phylogenetic analysis results of LpHSP70-10 with other species are as follows: Figure 6 As shown.

[0052] Cloning and Bioinformatics Analysis of the Ryegrass Heat Tolerance Gene LpHSP70-10

[0053] RNA plasmid integrity was assessed using 1% agarose gel electrophoresis, and RNA concentration and purity were determined using a micro spectrophotometer. Reverse transcription was performed using ABM's 5X All-In-One RT MasterMix with AccuRT; the procedure was described in the included instruction manual.

[0054] Primers were designed using the full-length CDS sequence of ryegrass reference genome as a template:

[0055] Upstream primer F: 5'-ATGTCGGGCAAGGGCGAAGGTCCGG-3', as shown in Seq ID NO.3 of the sequence listing;

[0056] Downstream primer R: 5'-TTAGTCGACCTCCTCAATCTTGGGC-3', as shown in Seq ID NO.4 of the sequence listing.

[0057] cDNA was used as a template for amplification. The amplification was performed using the PrimeSTAR Max DNA Polymerase kit from TaKaRa. The procedure was described in the included instruction manual. The PCR amplification system is shown in Table 1.

[0058] Table 1 PCR amplification system

[0059]

[0060] The reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 1 min; 55℃ annealing for 30 s; 72℃ extension for 1 min; 35 cycles; and a final run at 72℃ for 5 min. The PCR products were detected by 0.8% agarose gel electrophoresis.

[0061] In this embodiment, the LpHSP70-10 gene can be cloned with just one pair of primers and one amplification.

[0062] The gel was excised under UV light, and the target fragment was recovered and purified using the TaKaRa Mini BEST Agarose Gel DNA Extraction Kit (see the included instruction manual for details). An "A" tail was added to the 3' end of the target DNA fragment using the TaKaRa DNAA-Tailing Kit. After completion, 4 μl of the above DNA solution was added to 1 μl of pMD18-T vector and 5 μl of Solution (containing ligase), and the mixture was incubated at 16°C for 30 min. After the reaction, the above solution was added to 100 μl of DH5α competent cells, incubated on ice for 30 min, heated at 42°C for 45 s, and then placed on ice for 1 min. The transformed competent cells were then added to 890 μl of SOC medium and incubated at 37°C for 60 min. The cells were then plated on LB medium containing ampicillin (Amp) and incubated upside down overnight. Single colonies were selected for culture, and bacterial PCR was used to verify successful insertion of the target fragment. The target band was amplified by bacterial PCR and subjected to paired-end sequencing using primer M13 to verify successful cloning.

[0063] The full-length LpHSP70-10 gene fragment of ryegrass is 1947 bp, and its sequence is shown in SEQ ID NO.1. The cloning sequence map encoding LpHSP70-10 is shown below. Figure 1 As shown in the figure. The protein encoded by LpHSP70-10 contains 648 amino acids, and its sequence is shown in SEQ ID NO.2. The protein has a molecular weight of 71.16 kD, a pI of 5.03, a lipophilic index of 82.65, an average hydrophilicity coefficient of -0.422, and an instability coefficient of 34.25, suggesting that it is a stable hydrophilic protein. The hydrophilicity / hydrophobicity prediction diagram of the protein encoded by LpHSP70-10 is shown in the figure. Figure 2 As shown, the conservative structural domain prediction is as follows: Figure 5 As shown. Subcellular prediction indicates the encoded protein is located in the cytoplasm, and transmembrane region analysis shows it lacks a transmembrane helical region. For the predicted secondary structure of the LpHSP70-10 protein, see [link to relevant documentation]. Figure 3The results showed that the protein contained 41.82% α-helices, 18.06% extended strands, 7.25% β-turn structures, and 32.87% random coils. Tertiary structure modeling of the protein encoded by the LpHSP70-10 gene was performed using the SWISS-MODEL online software; see [link to documentation]. Figure 4 The selected template modeling ranged from 5 to 615 amino acids, with a coverage of 0.93. The sequence identity with LpHSP70-10 was 63.89%, and the GMQE value was 0.79, indicating good quality. The QMEAN4 value was 0.77 ± 0.05, indicating a high degree of matching between the LpHSP70-10 encoded protein and the template protein. The prediction results are usable.

[0064] LpHSP70-10 Functional analysis of genes transferred into Arabidopsis thaliana

[0065] S1. Construction of overexpression vector

[0066] Using pCAMBIA1301-35SN as a vector, two restriction enzyme sites, XbaI and SalI, were selected to design... LpHSP70-10 Using specific primers and the aforementioned RNA plasmid as a template, the target gene was amplified using a high-fidelity enzyme, and the product was purified and recovered. Then, pCAMBIA1301-35SN was linearized using XbaI and SalI restriction endonucleases (NEB), and the product was purified and recovered. Using the ClonExpress II One Step Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd.), the purified and recovered target fragment was ligated to the linearized pCAMBIA1301-35SN. The ligation products were transformed into Trelief™ 5α Chemically Competent Cell competent cells, plated on LB agar solid medium containing kanamycin (Kan, 50 mg / L), and positive single colonies were selected for PCR and sequencing verification using universal primer M13. Plasmids were extracted from the correct bacterial cultures to obtain the overexpression vector pCambia1301-35S- LpHSP70-10 .

[0067] Constructing primer designs for overexpression of Arabidopsis thaliana plants:

[0068] LpHSP70-10(1301)F:

[0069] 5′-aagctttatcgataccgtcgacATGTCGGGCAAGGGCGAA-3′

[0070] LpHSP70-10(1301)R:

[0071] 5′-gggggatccactagttctagaTTAGTCGACCTC CTCAATCTTGG-3′

[0072] M13F:

[0073] 5′-cgccagggttttcccagtcacgac-3′

[0074] M13R:

[0075] 5′-agcggataacaatttcacacagga-3′

[0076] 2. Transformation of Agrobacterium with overexpression vector

[0077] The recombinant plasmid pCambia1301-35S-LpHSP70-10 from S1 was transferred into GV3101 Chemically Competent Cell (Shanghai Weidi Biotechnology Co., Ltd.) according to the manufacturer's instructions. The plasmid was then cultured in antibiotic-free LB liquid medium for 4 hours. After centrifugation and resuspending, the plasmid was spread onto LB solid medium containing Kan (50 mg / L) and rifampin (50 mg / L) and incubated upside down at 28°C for 2-3 days. Single colonies were selected and cultured in liquid LB medium containing Kan and rifampin at 28°C on a shaker at 200 rpm for 24 hours. Colony PCR was then performed; positive single colonies were identified as host cells. The host cells were stored at -80°C with glycerol.

[0078] S3. Transformation and Functional Verification of Arabidopsis thaliana

[0079] Arabidopsis thaliana cultivation: Wild-type seeds were sown on 1 / 2 MS medium. After vernalization for two days, they were placed in a plant growth chamber with 14 hours of light per day and 40-60% humidity for about two weeks. Then, they were transplanted into flowerpots (9cm×9cm×11cm), with 4 seedlings per pot. After sowing, the seeds were watered and covered with plastic wrap, and then cultured in a plant growth chamber.

[0080] Remove the top: Water every two days after transplanting, and apply Hoagland nutrient solution every week. Cut off the flower buds when Arabidopsis thaliana first blooms.

[0081] Preparation of infiltration solution: Resuspend Agrobacterium in 5% sucrose solution to OD = 0.8. Before infiltration, add surfactant silwet-77 to a concentration of 0.02% (200 μL / L), mix well, and let stand at room temperature for 1 hour;

[0082] Infection: After removing the pods and flowers of Arabidopsis thaliana during the peak flowering period, immerse the above-ground parts of the plant in a suspension of host cells for 20-30 seconds;

[0083] Dark culture: After infection, the plants were bagged and cultured in the dark for 48 hours;

[0084] Post-infection cultivation: Watering method is the same as before infection;

[0085] Seed collection: Collect seeds when the siliques split open naturally;

[0086] Transgenic seed screening: Seeds obtained after inoculation on 1 / 2 MS medium containing 50 mg / L hygromycin were cultured normally for 7-10 days after vernalization. Transgenic seeds were determined based on their growth status: seeds successfully inoculated with recombinant plasmids grew normally on the resistant medium, while non-transgenic seeds did not grow normally.

[0087] Transplantation of transgenic plants and positive test: After the transgenic seeds germinate on plates for 2 weeks, the positive plants are transferred to soil for further cultivation. When the plants are growing well, leaves of the positive plants are taken for DNA extraction and PCR verification is performed using primers for the target gene sequence.

[0088] Germination heat stress experiments were conducted on T3 generation transgenic plant seeds, with heat stress conditions of 33 / 38℃ for 10 days.

[0089] Experimental results are as follows Figure 7 As shown, Figure 7 In the figures, A represents the phenotypic pattern of Arabidopsis thaliana before heat treatment, and B represents the phenotypic pattern of Arabidopsis thaliana after 10 days of heat treatment. A is the control group, and B is the treatment group (treated at 33 / 38℃ for 10 days). WT represents untransgenic wild-type Arabidopsis thaliana plants, and OE1, OE2, and OE3 represent transgenic Arabidopsis thaliana plants. The results indicate that Arabidopsis thaliana transgenic with the LpHSP70-10 gene exhibits significantly lower heat tolerance under heat stress than WT.

[0090] When the T3 generation transgenic plants reached the two-pair-leaf stage, some Arabidopsis transgenic plants (OE1, OE2, OE3) and non-transgenic wild-type Arabidopsis (WT) plants were cultured in soil. After about 3 weeks of normal growth, samples were taken from each plant before treatment to measure chlorophyll fluorescence, electrical conductivity, and chlorophyll content. After sampling, some Arabidopsis transgenic plants (OE1, OE2, and OE3) and non-transgenic wild-type Arabidopsis (WT) plants were placed in a high-temperature incubator (heat treatment at 33 / 38℃ for 10 days) to simulate heat stress. Sampling and phenotypic photography were completed after about 9-10 days. See [link to relevant documentation]. Figure 7 , 8 In the control treatments (9 and 10), WT, OE1, OE2, and OE3 plants all grew normally. Under heat stress, the growth of WT, OE1, OE2, and OE3 plants was inhibited, but WT plants showed better growth than the three OE lines. The results indicate that Arabidopsis thaliana transgenic with the LpHSP70-10 gene exhibits significantly greater heat tolerance than weakly wild-type Arabidopsis thaliana.

[0091] Based on the results of this embodiment, subsequent experiments showed that knocking out the LpHSP70-10 gene using gene editing technology could be used to cultivate heat-resistant perennial ryegrass varieties, in order to breed heat-resistant ryegrass varieties.

[0092] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of the heat-sensitive gene LpHSP70-10 in perennial ryegrass, characterized in that, The thermosensitivity of Arabidopsis thaliana was improved by overexpressing the gene LpHSP70-10; the nucleotide sequence of the gene LpHSP70-10 is shown in SEQ ID NO.1.

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