Pennisetum americanum PgGP gene for regulating plant cold sensitivity and application thereof
By cloning and overexpressing the PgGP gene of Arabia, the cold sensitivity of Arabidopsis was regulated, and the problem of Arabia's sensitivity to low temperature stress was solved, and the cultivation expansion in high-latitude areas was achieved.
Patent Information
- Application Number
- CN202510625230.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-15
- Publication Date
- 2025-08-15
AI Technical Summary
American wolftail grass is sensitive to low temperature stress, limiting its promotion and cultivation in temperate and high-altitude cool areas. The existing technology lacks effective regulatory measures.
The PgGP gene of syrupus in Arabiana was cloned and overexpressed. By overexpressing the PgGP gene in Arabidopsis to positively regulate plant cold sensitivity, a recombinant vector was constructed and PgGP protein was expressed in the host bacteria, and its localization on the nucleus and membrane and expression pattern under cold stress were observed to verify its regulatory function.
Through the overexpression of the PgGP gene, the cold sensitivity of Arabidopsis is enhanced, and technical means to improve the cold sensitivity of the American wolftail grass and expand its distribution and planting range in high latitude areas.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, in particular to a Pennisetum gracilis PgGP gene for regulating plant cold sensitivity and application thereof. Background Art
[0002] Extreme weather disasters are occurring frequently around the world, and low temperatures have become one of the major environmental stresses affecting plant growth and agricultural production worldwide (Peixoto et al., 2022). Low temperatures severely hinder normal plant growth and development, restrict the geographical distribution of vegetation, and impact agricultural production. Plant adaptability and tolerance face even greater challenges. In a severe year, my country's grain production can decrease by approximately 10 billion tons (Ding et al., 2022). Low temperatures not only affect plant vegetative and reproductive growth but also limit yield and, in severe cases, even cause plant mortality, posing a severe challenge to agricultural development (Guo et al., 2022; Li et al., 2023).
[0003] Pennisetum glaucum L. is a multifunctional C4 plant originating in Africa. It is an annual, warm-season forage grass of the genus Pennisetum in the Poaceae family. Also known as royal millet and pearl chestnut, it is one of the six major cereal crops worldwide and is characterized by rapid growth, nutrient richness, high yield, and excellent quality (Tako et al., 2015). Its value lies primarily in its use as a food crop, forage crop, and energy forage. It is an important forage plant and coarse grain crop and exhibits exceptional resistance, maintaining high yields despite high temperatures, drought, and poor soil quality (Yan et al., 2023). However, it is sensitive to cold stress and lacks the ability to adapt to low temperatures (Wu et al., 2021), severely limiting its widespread cultivation in temperate regions and cool, high-altitude areas. Identifying cold-stress regulatory proteins in Pennisetum glaucum, improving its resistance, and cultivating new cold-tolerant Pennisetum varieties are of great theoretical significance.
[0004] Small GTPases are common proteins with GTPase activity in eukaryotic cells, typically with a molecular weight of 20–30 kDa (Peiner et al., 2018). Based on their sequence, structure, and function, small GTPases are divided into five subfamilies: Ras, Rho, Rab, Arf / Sar, and Ran (Barr et al., 2010). They function as "molecular switches" in cell signaling, regulating a variety of biological processes by switching between a GTP-bound active state and a GDP-bound inactive state, affecting nearly all cellular processes, including cell growth, differentiation, and cell motility (Assmann et al., 2002; Van et al., 2023; He et al., 2024). Small G proteins have been reported in model plants to participate in the regulation of plant cold stress. For example, the interaction between GPA1 and the copper-binding protein AtBCB may affect plant cold tolerance under cold stress (Zhang et al., 2013). In Arabidopsis, AtRAN1 enhances plant tolerance to cold stress by regulating nuclear function and signal transduction (Guo et al., 2021). Rice COLD1 encodes a G protein signaling regulator that is localized to the cell membrane and endoplasmic reticulum (ER). It interacts with the G protein α subunit to activate Ca 2+ The small GTPases that regulate cold stress in non-model plants are rarely identified. Summary of the Invention
[0005] The purpose of the present invention is to provide a Pennisetum gracilis PgGP gene for regulating plant cold sensitivity and its application to solve the problems existing in the above-mentioned prior art. The PgGP gene can positively regulate the cold sensitivity of Arabidopsis thaliana.
[0006] To achieve the above object, the present invention provides the following solutions:
[0007] The present invention provides the use of the PgGP gene in any of the following:
[0008] (1) Application in regulating plant cold sensitivity;
[0009] (2) Application in the preparation of products for regulating plant cold sensitivity;
[0010] (3) Application in breeding of plants with high cold sensitivity;
[0011] (4) Application in the cultivation of highly cold-sensitive plants;
[0012] The nucleotide sequence of the PgGP gene is shown in SEQ ID NO.1.
[0013] The present invention also provides the use of the PgGP protein encoded by the PgGP gene in any of the following:
[0014] (1) Application in regulating plant cold sensitivity;
[0015] (2) Application in the preparation of products for regulating plant cold sensitivity;
[0016] (3) Application in breeding of plants with high cold sensitivity;
[0017] (4) Application in the cultivation of highly cold-sensitive plants;
[0018] The amino acid sequence of the PgGP protein is shown in SEQ ID NO.2.
[0019] The present invention also provides the use of a recombinant vector containing the PgGP gene in any of the following:
[0020] (1) Application in regulating plant cold sensitivity;
[0021] (2) Application in the preparation of products for regulating plant cold sensitivity;
[0022] (3) Application in breeding of plants with high cold sensitivity;
[0023] (4) Application in the cultivation of highly cold-sensitive plants;
[0024] The nucleotide sequence of the PgGP gene is shown in SEQ ID NO.1.
[0025] The present invention also provides the use of a host bacterium containing a recombinant vector in any of the following:
[0026] (1) Application in regulating plant cold sensitivity;
[0027] (2) Application in the preparation of products for regulating plant cold sensitivity;
[0028] (3) Application in breeding of plants with high cold sensitivity;
[0029] (4) Application in the cultivation of highly cold-sensitive plants;
[0030] The recombinant vector is a vector in which the PgGP gene is integrated. The nucleotide sequence of the PgGP gene is shown as SEQ ID No. 1.
[0031] Optionally, the regulation is positive regulation.
[0032] Optionally, the plant comprises Arabidopsis thaliana.
[0033] The present invention also provides a method for regulating plant cold sensitivity, comprising the steps of overexpressing a PgGP gene in a plant to increase the expression level of the PgGP gene to increase the cold sensitivity of the plant;
[0034] The nucleotide sequence of the PgGP gene is shown in SEQ ID No. 1.
[0035] Optionally, the plant comprises Arabidopsis thaliana.
[0036] The present invention also provides a method for cultivating highly cold-sensitive plants, comprising the steps of overexpressing a PgGP gene in a plant, increasing the expression level of the PgGP gene, and obtaining a highly cold-sensitive plant;
[0037] The nucleotide sequence of the PgGP gene is shown in SEQ ID No. 1.
[0038] Optionally, the plant comprises Arabidopsis thaliana.
[0039] The present invention discloses the following technical effects:
[0040] The present invention cloned a gene PgGP (GTPase) encoding a small G protein in the American grass Tifleaf3 variety. The gene is 660bp long and encodes 219 amino acids. It has been verified that the PgGP protein belongs to the Rab subfamily of small G proteins and is located in the cell nucleus and cell membrane. Analysis of the transcriptional expression pattern found that the expression of PgGP was induced by cold stress and showed obvious cold sensitivity in the brewer's yeast system. By treating overexpressed Arabidopsis with frost damage, it was found that PgGP can negatively regulate the cold tolerance of Arabidopsis, that is, positively regulate the cold sensitivity of Arabidopsis. In summary, the present invention cloned a gene PgGP that can regulate the cold sensitivity of plants and the PgGP protein it encodes in American grass. The acquisition of this gene has important production value for improving the cold sensitivity of American grass, expanding its distribution in high-latitude areas and promoting its planting. BRIEF DESCRIPTION OF THE DRAWINGS
[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0042] Figure 1 is the phylogenetic tree of PgGP and its orthologous proteins;
[0043] Figure 2The subcellular localization of PgGP; the scale bar is 10 μm;
[0044] Figure 3 is the expression pattern of PgGP under cold stress;
[0045] Figure 4 This is the preliminary functional validation analysis of PgGP; A: Heterologous expression analysis in Saccharomyces cerevisiae; pYES2 is the control; B: Phenotypes of overexpressing strains and wild-type plants after freezing (-10°C); the white line represents 0.5 cm; C: Survival statistics of overexpressing strains and wild-type plants after freezing treatment;
[0046] Figure 5 Functional analysis of PgGP overexpressing Arabidopsis; A: Phenotypes of overexpressing lines and wild-type plants treated with low temperature (4℃); scale bar is 2 cm; BC: Electrolyte permeability (EL) and relative water content (RWC) of leaves of overexpressing plants and wild-type plants under low temperature (4℃); D: Phenotypes of overexpressing lines and wild-type plants treated with frost damage (-8℃); scale bar is 2 cm; EG: Survival statistics of overexpressing lines and wild-type plants after frost damage (-8℃), electrolyte permeability (EL) and relative water content (RWC) of leaves of plants. DETAILED DESCRIPTION
[0047] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0048] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.
[0049] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.
[0050] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.
[0051] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0052] Example 1
[0053] 1. Material handling
[0054] Pennisetum seeds were evenly distributed in a Petri dish lined with filter paper, keeping the filter paper moist to encourage germination. After germination, the seeds were transferred to a hydroponic pot and cultured with 1 / 2 Hoagland's nutrient solution. Stress treatment was applied when the seedlings reached approximately 15-20 cm. Leaves were collected at 0, 1, 24, and 72 hours into the treatment period. All test materials were immediately frozen in liquid nitrogen and stored in a -80°C freezer.
[0055] 2. Analysis of PgGP expression patterns
[0056] The leaves collected after the above treatment were sent to the company for transcriptome sequencing, and the transcriptome data were analyzed to obtain the PgGP expression pattern diagram.
[0057] 3. Cloning of PgGP
[0058] A nested PCR cloning method was used, using Pennisetum americanum cDNA as a template. The first round of PCR amplification was performed using the 5'UTR upstream primer PgGP-F1 and the 3'UTR downstream primer PgGP-R1 (primer sequences are shown in Table 8). The CDS region-specific primers were then designed with the addition of protective bases and restriction sites (PgGP-CDS primer sequences are shown in Table 8). The recovered product from the first PCR reaction was used as a template for a second round of amplification (amplification system is shown in Table 2). After the PCR reaction, the fragment was analyzed by 1% agarose gel electrophoresis and subsequently purified and recovered. The recovered fragment was digested with EcoRI and HindIII endonucleases (enzyme digestion reaction system is shown in Table 3) and then ligated into the pENTR vector using T4 ligase to generate pEntry-PgGP (ligation reaction system is shown in Table 4). The plasmid was then purified and sent to a company for sequencing. The results were compared with the genomic reference sequence to confirm the correctness of the cloned gene sequence.
[0059] Table 1 First amplification system
[0060]
[0061] Reaction conditions: 98°C for 1 min, 98°C for 15 s, 66°C for 20 s, 72°C for 40 s, 30 cycles, 72°C for 3 min, and storage at 10°C.
[0062] Table 2 Second amplification system
[0063]
[0064] Reaction conditions: 98°C for 1 min, 98°C for 15 s, 69°C for 20 s, 72°C for 40 s, 30 cycles, 72°C for 3 min, and storage at 10°C.
[0065] Table 3 Enzyme digestion reaction system
[0066]
[0067]
[0068] Reaction conditions: 37°C, 2h.
[0069] Table 4 Ligation reaction system
[0070]
[0071] Reaction conditions: 16°C, 2-3h.
[0072] 4. Construction of expression vector
[0073] ① Construction of pEG103-PgGP vector: The correctly sequenced pEntry-PgGP plasmid was digested with Pvu I restriction endonuclease (see Table 5 for the enzyme digestion reaction system) and the product was recovered. The digestion recovery product was then recombined with the pEG103 expression vector using LR recombinase (see Table 6 for the recombination reaction system) to construct the pEG103-PgGP recombinant expression vector, which was then transformed into Agrobacterium GV3101.
[0074] ② Construction of the pYES2-PgGP vector: The full-length CDS sequence of the PgGP gene (SEQ ID NO. 1) obtained in previous basic work was flanked by pYES2 homology arms. The pYES2 vector was double-digested with enzymes and the vector fragments were homologously recombined with the target gene (see Table 7 for the reaction system) to construct the pYES2-PgGP expression vector. After transformation into competent E. coli, single colonies were selected for colony PCR to confirm their correctness. Once confirmed, the plasmid was extracted and transformed into competent Saccharomyces cerevisiae culture (INVSCI).
[0075] Table 5 Enzyme digestion reaction system
[0076]
[0077] Reaction conditions: 37°C, 1h.
[0078] Table 6 Recombination reaction system
[0079]
[0080] Reaction conditions: 25°C, 1h.
[0081] Table 7 Reaction system
[0082]
[0083] Reaction conditions: 50°C, 30 min.
[0084] 5. Analysis of heterologous expression in Saccharomyces cerevisiae
[0085] The correct yeast monoclonal clone was selected and placed in SD-Ura liquid medium, sealed and cultured in a shaking incubator at 30°C for 2-3 days, and diluted with sterile water in a 10-fold concentration gradient (10 1 , 10 2 , 10 3 , 10 4 , 10 5 ), 5 μL of the diluted yeast solution was spotted on SD-Ura solid selective medium, and cultured at 30°C and 20°C for 3-4 days, respectively, and the yeast growth was observed and photographed.
[0086] 6. Subcellular localization
[0087] 2-3 Agrobacterium colonies carrying the pEG103-PgGP vector were selected and placed in a 2 mL centrifuge tube containing LB liquid medium containing 50 mg / mL Kan and 50 mg / mL Rif antibiotics, and cultured in a 28 ° C shaker for 1-2 days. Then 100 μL was transferred to a 50 mL centrifuge tube containing LB liquid medium containing 50 mg / mL Kan and 50 mg / mL Rif antibiotics, and cultured in a 28 ° C shaker until the OD 600 =0.8-1.0. Centrifuge 5 mL of bacterial solution at 4000 rpm for 10 min to collect the bacteria, discard the supernatant, and calculate the value of V bacterial solution × OD 600 =V infection fluid × 0.8, then add the corresponding volume of tobacco infection fluid (1 mL of 100 μM AS per 100 mL of tobacco infection fluid) to resuspend the cells. Finally, incubate the resuspension in the dark for 1-2 hours before injecting into tobacco plants. After 2 days of normal cultivation, observe GFP fluorescence using a Leica ultra-high-resolution confocal microscope (excitation wavelength 488 nm, scanning wavelength 505-530 nm).
[0088] 7. Obtaining Arabidopsis transgenic lines
[0089] Wild-type Arabidopsis thaliana was transformed by Agrobacterium-mediated inflorescence infection. The specific steps are as follows:
[0090] ① Select 2-3 Agrobacterium colonies carrying the pEG103-PgGP vector constructed above and place them into a 50 mL centrifuge tube containing LB liquid medium containing 50 mg / mL Kan and 50 mg / mL Rif antibiotics, and culture them in a shaking incubator at 28°C until the OD 600＝ 0.8-1.0.
[0091] ②Centrifuge at 5000r / min for 15 minutes to collect the bacterial solution and discard the supernatant. Add the previously prepared infection solution (containing 5% sucrose and 100μM Silwet L-77) to resuspend the bacteria and adjust the OD 600 Adjust to around 0.8.
[0092] ③ Before infection, cut off pollinated inflorescences to improve transformation efficiency. Immerse the inflorescences in the above bacterial solution for 50-60 seconds. After absorbing the excess solution with filter paper, place the plants flat on a tray. Under long-day conditions, keep them in the dark for 24 hours. The next day, place the plants vertically for normal culture.
[0093] ④ Repeat the above steps again after one week to improve the transformation efficiency and obtain more positive seedlings.
[0094] 8. Identification of transgenic Arabidopsis
[0095] T0 generation seeds of the infected Arabidopsis thaliana, harvested after seed maturity, were initially screened using antibiotics. T0 generation seeds were germinated on 1 / 2 MS medium containing 20 mg / L glufosinate (Basta) and cultured for approximately 10 days. Seedlings that grew normally were selected and transplanted to nutrient soil. They were cultured under long-day conditions for approximately three weeks, and genomic DNA from the transgenic Arabidopsis thaliana was extracted for PCR identification. Correctly identified transgenic positive lines were further cultured, and their T1 generation seeds were harvested. Lines with progeny segregation ratios close to 3:1 were selected for further sowing and culture, and T2 and T3 generation seeds were harvested. Subsequently, homozygous T3 generation lines (OE-3, OE-8, and OE-11) were used for functional verification.
[0096] 9. Analysis of cold-tolerant phenotypes in PgGP-overexpressing Arabidopsis
[0097] T2 transgenic Arabidopsis seeds were spread on 1 / 2MS solid culture medium containing 20 mg / L Basta. After the wild-type and overexpressing Arabidopsis seedlings grew 2-4 leaves, seedlings of the same size were selected and transferred to the same plate. Eight replicates were set up and placed in an incubator 5 days later. The seedlings were frozen and the survival rates of the wild-type and overexpressing plants were counted after 5-7 days of recovery.
[0098] Select robust and uniformly growing T3 transgenic and wild-type seedlings and transplant them into nutrient soil. When they grow to one month old, are of uniform size, and can cover the small black square soil surface, they are subjected to 4°C low temperature treatment and -8°C frost damage treatment in batches.
[0099] 4°C low temperature treatment: Wild-type Arabidopsis thaliana was used as a control and subjected to 4°C low temperature treatment. During the treatment, its phenotype was observed, photographed, and samples were taken.
[0100] -8℃ frost injury treatment: wild-type Arabidopsis was used as a control. After acclimation at 4℃ for 3 days, the plants were treated at -8℃ for 8 hours and then returned to normal conditions for recovery. After 5 days of recovery, photos were taken and samples were collected.
[0101] 10. Determination of physiological indicators
[0102] (1) Determination of electrolyte leakage rate (EL)
[0103] Take 0.1g of fresh plant leaves, wrap them in absorbent paper, and place them in a 50mL centrifuge tube filled with 30mL of deionized water, ensuring that the sample is fully immersed in the water. Set up five biological replicates, seal the tube, and place it on a shaker for 24 hours. After that, use a conductivity meter to measure the initial conductivity (C0). The centrifuge tube with the initial conductivity measurement is then sealed and placed in a boiling water bath for 20 minutes to fix the greenness. Remove the tube, cool it to room temperature, and place it on a shaker for 24 hours. The conductivity (C1) is then measured again. The conductivity is calculated using the formula: EL = C0 / C1 × 100%.
[0104] (2) Determination of relative water content (RWC)
[0105] Accurately weigh 0.1 g of plant leaf fresh weight (FW) using a 1 / 10,000 balance. Wrap the leaf in absorbent paper and immerse it in a 10 mL centrifuge tube filled with 10 mL of deionized water. Seal the tube and let it sit at room temperature for 24 hours. Then, remove the leaf and wipe dry with absorbent paper. Quickly weigh the saturated fresh weight (TW) of the leaf. Place the leaf in an envelope and dry it in an oven (65°C) until constant weight is reached. Finally, weigh the leaf dry weight (DW). The formula for calculating leaf relative water content is: RWC = (FW - DW) / (TW - DW) × 100%.
[0106] Table 8 Primer sequences
[0107]
[0108] 11. Results
[0109] 11.1 PgGP sequence
[0110] The CDS sequence of the PgGP gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded PgGP protein is shown in SEQ ID NO.2.
[0111] SEQ ID NO.1:
[0112] ATGGCAGGGGGTTACCGCACGGAGGAGGAGTACGACTACCTGTTCAAGGTGGTGCTGATCGGGGACAGCGGCGTCGGCAAGTCCAACCTCCTGTCGCGGTTCGCCAGGGACGAGTTCAGCCTCGAGACCAGGTCCACCATCGGCGTCGAGTTCGCCACCAAGACCGTCCAGGTCGATGACAAGCTCGTCAAGGCGCAGATCTGGGACACCGCCGGCCAGGAGAGGTACCGCGCCATCACGAGCGCATACTACCGCGGCGCGGTGGGCGCGCTGGTGGTGTACGACGTGACCCGCCGCGTGACGTTCGAGAACGCGGAGCGGTGGCTCCGGGAGCTGCGGGACCACACGGACGCCAACATCGTGGTCATGCTGGTGGGCAACAAGGCCGACCTGCGCCACCTCCGCGCCGTCTCGCCCGAGGACGCCGCCGCCTTCGCGGAGCGCCACGGCACCTTCTCAATGGAGACGTCGGCGCTGGACGCCACCAACGTGGAGCGCGCCTTCGCCGAGGTGCTCCGCCAGATCTACCACGTCGTCAGCCGGAACGCGCTCGACATCGGGGAGGACCCGGCCGCGCCGCCCAGGGGCAAGACCATCGACGTCGGCGCCTCCAAGGACGAGGTCTCGCCCGTGAACACGGGCGGGTGCTGCTCGGCTTGA。
[0113] SEQ ID NO.2:
[0114] MAGGYRTEEEYDYLFKVVLIGDSGVGKSNLLSRFARDEFSLETRSTIGVEFATKTVQVDDKLVKAQIWDTAGQERYRAITSAYYRGAVGALVVYDVTRRVTFENAERWLRELRDHTDANIVVMLVGNKADLRHLRAVSPEDAAAFAERHGTFSMETSALDATNVERAFAEVLRQIYHVVSRNALDIGEDPAAPPRGKTIDVGASKDEVSPVNTGGCCSA*。
[0115] 11.2 Phylogenetic Tree Analysis
[0116] The UPGMA phylogenetic tree of PgGP and its closest orthologous proteins in rice, maize, Brachypodium distachyon, perennial ryegrass and Arabidopsis thaliana was studied ( Figure 1 Its closest ortholog was found to be ZmRABA1f in maize (ZmRABA1f is a Ras-related protein RABA1f in maize). Its function has not yet been reported, indicating that PgGP is a gene of a new small G protein family whose function has not yet been studied.
[0117] 11.3 PgGP Subcellular Localization
[0118] To observe the subcellular localization of PgGP, the PgGP-GFP fusion protein was introduced into tobacco leaf cells by Agrobacterium-mediated infiltration. Confocal microscopy showed that the fluorescence signal generated by PgGP-GFP expression appeared simultaneously on the cell membrane and the nucleus ( Figure 2 ), the results showed that PgGP-GFP was co-localized in the nucleus and cell membrane.
[0119] 11.4 PgGP expression is induced by cold stress
[0120] Transcriptome data analysis revealed that the expression level of PgGP increased with the increase of cold stress treatment time, reaching the highest value at 72 h of cold stress treatment, and showed a significant difference in expression level compared with the expression level at 0 h ( Figure 3 The results showed that the expression level of PgGP was induced by cold stress.
[0121] 11.5 Preliminary Verification of PgGP Functionality
[0122] In order to preliminarily verify the function of PgGP in regulating plant cold tolerance, yeast carrying pYES2-PgGP and pYES2 expression vectors were gradiently diluted and spotted through heterologous expression in Saccharomyces cerevisiae, and grown at 30℃ and 20℃. Figure 4 It can be seen that under normal yeast growth conditions (30℃), the growth of the two yeasts is the same. Under low temperature conditions (20℃), the growth of yeast carrying pYES 2-PgGP is significantly worse than that of the control (yeast carrying only the pYES2 empty vector). Figure 4 Middle A). The results indicate that PgGP exhibits cold sensitivity under low temperature stress.
[0123] Overexpression and wild-type Arabidopsis seedlings grown on culture medium were subjected to freezing treatment below 0°C to -10°C, and then placed in normal temperature for recovery. The survival rate was observed and calculated. After freezing treatment, almost all leaves of Arabidopsis seedlings wilted and died. In the later recovery period, some leaves gradually returned to green. The survival rate of wild-type Arabidopsis was significantly higher than that of overexpression strains ( Figure 4 Middle B, Figure 4 Middle C). This preliminary suggests that PgGP can positively regulate the cold sensitivity of Arabidopsis.
[0124] 11.6PgGP positively regulates cold sensitivity in transgenic Arabidopsis plants
[0125] The overexpression and wild-type Arabidopsis plants grown in soil were subjected to a low-temperature treatment at 4°C, and the ion permeability and relative water content were measured. The results showed that after 10 days of low-temperature treatment, the leaves of the overexpression Arabidopsis began to turn purple, while the wild-type Arabidopsis remained relatively green. After 14 days of low-temperature treatment, almost all the leaves of the overexpression Arabidopsis turned purple and began to turn yellow, while only some of the leaves of the wild-type Arabidopsis turned purple ( Figure 5 After the physiological indexes were measured, it was found that there was no significant difference in ion permeability and relative water content between wild type and overexpressed Arabidopsis before low temperature treatment. After low temperature treatment, the ion permeability of PgGP overexpressed Arabidopsis was significantly higher than that of wild type, and the relative water content was significantly lower than that of wild type ( Figure 5 Middle B, Figure 5 (C) This indicates that the cell membrane of PgGP-overexpressing Arabidopsis strains is more severely damaged after low temperature stress, and overexpression of PgGP can enhance the cold sensitivity of Arabidopsis.
[0126] The overexpression and wild-type Arabidopsis plants grown in soil were acclimated at 4°C and then subjected to -8°C freezing treatment. They were then returned to normal conditions for recovery. The survival rate was observed and calculated, and the ion permeability and relative water content were measured. The results showed that after freezing treatment, all the leaves of Arabidopsis thaliana wilted. In the late recovery period, almost all the leaves of the overexpression Arabidopsis turned yellow, with only a small part returning to green. In contrast, most of the leaves of the wild-type Arabidopsis returned to green. The survival rate of the overexpression Arabidopsis was significantly lower than that of the wild-type ( Figure 5 Middle D, Figure 5 Middle E). After freezing treatment, the ion permeability of the overexpressing Arabidopsis was significantly higher than that of the wild type, and the relative water content was significantly lower than that of the wild type ( Figure 5 Middle F, Figure 5 Middle G). The results indicate that overexpression of PgGP can enhance the cold sensitivity of Arabidopsis.
[0127] By enhancing the cold sensitivity of plants, plants with high cold sensitivity can be cultivated, which can provide technical support for subsequent research and exploration of genes related to cold sensitivity resistance.
[0128] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.
Claims
1. Use of the PgGP gene in any of the following: (1) Application in regulating plant cold sensitivity; (2) Application in the preparation of products for regulating plant cold sensitivity; (3) Application in breeding of plants with high cold sensitivity; (4) Application in the cultivation of highly cold-sensitive plants; The nucleotide sequence of the PgGP gene is shown in SEQ ID NO.
1.
2. Use of the PgGP protein encoded by the PgGP gene in any of the following: (1) Application in regulating plant cold sensitivity; (2) Application in the preparation of products for regulating plant cold sensitivity; (3) Application in breeding of plants with high cold sensitivity; (4) Application in the cultivation of highly cold-sensitive plants; The amino acid sequence of the PgGP protein is shown in SEQ ID NO.
2.
3. Use of a recombinant vector containing the PgGP gene in any of the following: (1) Application in regulating plant cold sensitivity; (2) Application in the preparation of products for regulating plant cold sensitivity; (3) Application in breeding of plants with high cold sensitivity; (4) Application in the cultivation of highly cold-sensitive plants; The nucleotide sequence of the PgGP gene is shown in SEQ ID NO.
1.
4. Use of host bacteria containing recombinant vectors in any of the following: (1) Application in regulating plant cold sensitivity; (2) Application in the preparation of products for regulating plant cold sensitivity; (3) Application in breeding of plants with high cold sensitivity; (4) Application in the cultivation of highly cold-sensitive plants; The recombinant vector is a vector in which the PgGP gene is integrated. The nucleotide sequence of the PgGP gene is shown as SEQ ID No.
1.
5. The use according to any one of claims 1 to 4, characterized in that The regulation is positive regulation.
6. The use according to any one of claims 1 to 4, characterized in that The plants include Arabidopsis thaliana.
7. A method for regulating plant cold sensitivity, characterized in that: The method comprises the steps of overexpressing the PgGP gene in the plant to increase the expression level of the PgGP gene, thereby increasing the cold sensitivity of the plant; The nucleotide sequence of the PgGP gene is shown in SEQ ID No.
1.
8. The method according to claim 7, wherein The plants include Arabidopsis thaliana.
9. A method for cultivating plants with high cold sensitivity, characterized in that: The method comprises the steps of overexpressing the PgGP gene in the plant, increasing the expression level of the PgGP gene, and obtaining a plant with high cold sensitivity; The nucleotide sequence of the PgGP gene is shown in SEQ ID No.
1.
10. The method according to claim 9, wherein The plants include Arabidopsis thaliana.