A PpDREB1D-like gene in elephant grass and its application in improving plant cold resistance and biomass.

Abstract

This invention discloses an elephant grass PpDREB1D-like gene and its application in improving plant cold resistance and biomass, belonging to the field of plant genetic engineering technology. This invention is the first to clone and functionally identify the elephant grass-derived PpDREB1D-like gene. Experimental verification revealed that the elephant grass PpDREB1D-like gene can increase the plant's cold resistance. Overexpression of the PpDREB1D-like gene significantly improved the plant's growth under cold stress, while silencing the elephant grass PpDREB1D-like gene significantly reduced its cold resistance. This lays the molecular foundation for the promotion and cultivation of forage and energy crops in cold regions, and has significant agricultural production and ecological value.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to an elephant grass PpDREB1D-like gene and its application in improving plant cold resistance and biomass. Background Technology

[0002] Low temperature is one of the major abiotic stresses limiting plant distribution and agricultural productivity. To address this challenge, research on crop cold resistance has become a key focus of genetic improvement. Plants initiate complex molecular responses under low temperatures, including pathways involving hormone signaling, redox mechanisms, photosynthesis, and transcription factor regulation. Identifying key regulatory factors in these pathways is crucial for elucidating the mechanisms of cold resistance in plants.

[0003] Elephant grass is soft, palatable, and boasts a high dry matter content of 21.6%. Its high crude protein, moderate crude fat, and nitrogen-free extract content make it suitable for feeding a variety of animals, including cattle, sheep, pigs, rabbits, and fish. Elephant grass is a perennial, clump-forming, large herbaceous plant belonging to the genus *Elephantgrass* of the Poaceae family. It has become one of the most important forage species and potential energy grasses in tropical and subtropical regions of Asia, Africa, and the Americas. As an excellent forage crop, it yields up to 150 tons per hectare annually, can be harvested at least four times a year, and is resistant to high temperatures, drought, low soil fertility, and biotic stress.

[0004] *Pennisetum purpureum* Schum.cv. Gui Min Yin, a perennial herbaceous plant belonging to the genus *Pennisetum* in the family Poaceae, possesses strong drought and disease resistance, and a high sugar content. It is not only an excellent forage resource but also an ecological forage grass for afforestation of barren hills and prevention of soil erosion. Currently, this variety has been widely promoted and applied in many parts of southern my country, but its generally weak cold tolerance is a major factor limiting its production, application, and efficient utilization. Therefore, in-depth research on its molecular response mechanism to low-temperature stress is of great significance for expanding its ecological adaptability, promoting the introduction of southern grasses to northern regions, and building a high-efficiency forage grass production system in my country. It also provides a theoretical basis for the discovery and breeding of cold-resistant germplasm. However, to date, research on cold-resistant genes in *Pennisetum purpureum* is limited, and its cold-resistance mechanism remains unclear.

[0005] DREBs are important transcription factors involved in abiotic stress responses, and in Arabidopsis thaliana, they can be further subdivided into six subfamilies. Different subfamilies exhibit varying responses to different stress signal transductions, with DREB1 / CBF considered a "master switch" in the cold stress response process, playing a crucial role in plant frost tolerance and low-temperature acclimatization. DREB1 / CBFs activate the expression of low-temperature stress-related genes by binding to the DRE / CRT cis-acting elements of downstream target gene promoters, evoking stress-related physiological, metabolic, and functional structural responses, thereby enhancing plant cold resistance. Based on the potential of DREB1 / CBFs in cold resistance, they have broad application prospects for enhancing plant cold resistance, improving plant viability and yield. However, to date, research on DREB1 / CBFs in elephant grass is limited, especially regarding their cold tolerance. Summary of the Invention

[0006] This invention identifies a novel, cold-stress-induced PpDREB1-like gene in elephant grass through transcriptome analysis. Genetic studies have shown that this gene is a positive regulator of the cold tolerance of elephant grass. This gene is of great significance for breeding cold-resistant varieties, improving the overwintering ability of elephant grass, expanding the planting area, improving the quality of forage and promoting environmental sustainability.

[0007] The technical solution adopted in this invention is as follows:

[0008] In a first aspect, the present invention provides an elephant grass PpDREB1D-like gene, the gene sequence of which is shown in SEQ ID NO:1 and the amino acid sequence encoded by it is shown in SEQ ID NO:2.

[0009] SEQ ID NO:1:ATGGATTACTTGCACACCACTGGTACCGAGCCTCCGGGCGGCTCCACGACCACTGAGTCGGGGTCGGGTCGCCCGTCCCCGCCGAAGCGCCCCGCGGGGCGGACCAAGTTCCAGGAGACGCGGCACCCGGTGTTCCGCGGGGTGCGGCGTCGTGGCCGGGCGGGGCGGTGGGTGTGCGAGGTCCGCGTCCCCGGCAGCCGCGGGGACCGCCTCTGGGTCGGCACCTTCGACACCGCCGAGGCCGCCGCGCGCGCGCACGACGCCGCCATGCTCGCCCTCTGCGGCGCCGCCGCCACCCTCAACTTCCCGGACTCCGCCTGGCTGCTCGACGTCGCCGTCGCCCCGCGCGGCGCCCGTTCTGCCGCCGGCGCCCCCGAGCTGCCGCCGCTGCCCGACGTCCAACGCGCCGCCACCGATGCCGTCGCGGGGTTCCTCCGTCGCCACGGCGGCGACGCCACGGTAGCTACCCACCCCGCGGGCAATGCCGCTAATTCGGTGGCGTCCTCAGGAGGAACGGCGGCGACAGCGTCGGTGATGGACGACAATGCCGGCACGCTTGAGCTGGACGCGTTCGGCGGCATGGACGCCGGTTCCTACTACGCGAGCCTGGCGCAGGGGCTGCTCATCGACCCGCCGCCGCCGACCGCCGTGGAGTGCCCGGAGGAGGACGAGGACGACTGCGAAGCCGGCGAAGTCGAGCTGTGGAGCTAG。

[0010] SEQ ID NO:2:MDYLHTTGTEPPGGSTTTESGSGRPSPPKRPAGRTKFQETRHPVFRGVRRRGRAGRWVCEVRVPGSRGDRLWVGTFDTAEAAARAHDAAMLALCGAAATLNFPDSAWLLDVAVAPRGARSAAGAPELPPLPDVQRAATDAVAGFLRRHGGDATVATHPAGNAANSVASSGGTAATASVMDDNAGTLELDAFGGMDAGSYYASLAQGLLIDPPPPTAVECPEEDEDDCEAGEVELWS*。

[0011] Furthermore, the elephant grass PpDREB1D-like gene is derived from the elephant grass stem, leaf sheath, or root.

[0012] Furthermore, the elephant grass PpDREB1D-like gene can be amplified by using the sequences shown in SEQ ID NO.3 and SEQ ID NO.4 as primers.

[0013] SEQ ID NO:3:CGAAGCCATGGATTACTTGAACA.

[0014] SEQ ID NO: 4: ACGAAACGGATCCTTGTTCTACTC.

[0015] Secondly, this invention discloses the application of the elephant grass PpDREB1D-like gene described in the first aspect in the preparation of products that improve plant cold resistance and biomass.

[0016] Furthermore, the cold-resistant plant is Arabidopsis thaliana or elephant grass.

[0017] Furthermore, the process of using the elephant grass PpDREB1D-like gene to improve plant cold resistance and biomass includes the following steps:

[0018] (1) The elephant grass PpDREB1D-like gene was ligated into an expression vector to obtain a recombinant vector;

[0019] (2) The recombinant vector was transformed into competent cells to obtain recombinant cells;

[0020] (3) Use recombinant cells to inoculate plants whose low-temperature resistance needs to be improved.

[0021] More preferably, the preparation process of the recombinant vector includes the following steps:

[0022] (1) Design primers Homo-DREB1Df and Homo-DREB1Dr, whose sequences are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively;

[0023] (2) The PpDREB1D-like gene was amplified using primers Homo-DREB1Df and Homo-DREB1Dr to obtain PpDREB1D-like.

[0024] (3) The pCAMBIA1381-YFP vector was digested with enzymes to obtain a linearized vector;

[0025] (4) Connect PpDREB1D-like with the linearized vector.

[0026] More preferably, the competent cells are competent strains of Agrobacterium tumefaciens strain GV3101.

[0027] Thirdly, the present invention provides a reagent for improving the cold resistance and biomass of plants, the reagent comprising the elephant grass PpDREB1D-like gene described in the first aspect, or a recombinant vector or recombinant cell prepared using the elephant grass PpDREB1D-like gene described in the first aspect.

[0028] In summary, compared with the prior art, the present invention has the following advantages and benefits:

[0029] 1. This invention is the first to clone and functionally identify the elephant grass PpDREB1D-like gene. Experimental verification revealed that the elephant grass PpDREB1D-like gene can increase the plant's cold resistance. Specifically, transgenic Arabidopsis thaliana overexpressing this gene showed better growth than the control group under 8℃ low-temperature stress, with a significant increase in plant fresh weight and a decrease in reactive oxygen species (ROS) levels in leaves. Silencing this gene in elephant grass led to more severe wilting of leaves under low-temperature stress and a significant increase in ROS levels, indicating that PpDREB1D-like enhances the plant's cold resistance by scavenging low-temperature-induced leaf ROS. It is evident that overexpression of the PpDREB1D-like gene significantly improves the plant's growth under cold stress, while silencing the elephant grass PpDREB1D-like gene significantly reduces its cold resistance.

[0030] 2. This invention verifies that the PpDREB1D-like gene is a positive regulator of cold stress through heterologous overexpression in the model plant Arabidopsis thaliana and gene silencing in elephant grass, providing direct experimental evidence for its application in elephant grass and other crops.

[0031] 3. This invention provides a new source of genetically modified genes, laying the molecular foundation for the promotion and planting of forage and energy crops in cold regions, and has significant agricultural production and ecological value. Attached Figure Description

[0032] Figure 1 Gel electrophoresis image of PCR products of PpDREB1D-like gene;

[0033] Figure 2 Phylogenetic tree diagram of PpDREB1D-like genes;

[0034] Figure 3A comparison of PpDREB1D-like gene expression levels in different tissues;

[0035] Figure 4 A comparison of PpDREB1D-like gene expression under different cold stress durations;

[0036] Figure 5 This is a diagram of the PpDREB1D-like gene transcriptional activation activity experiment.

[0037] Figure 6 This is a diagram of the subcellular localization experiment of the PpDREB1D-like gene.

[0038] Figure 7 The image shows the results of DNA and RNA level detection of the PpDREB1D-like gene in transgenic Arabidopsis thaliana.

[0039] Figure 8 A comparison of the growth states of different Arabidopsis species under cold stress conditions;

[0040] Figure 9 A statistical chart showing the aboveground fresh weight of different Arabidopsis species under cold stress conditions;

[0041] Figure 10 ROS staining images of leaves from different Arabidopsis species under different conditions;

[0042] Figure 11 A diagram illustrating the expression levels of the PpDREB1D-like gene in different elephant grasses under cold stress conditions.

[0043] Figure 12 This is a comparison of the growth states of different elephant grasses under cold stress. Detailed Implementation

[0044] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0045] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0046] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0047] The present application will now be described in detail with reference to embodiments and experimental data.

[0048] Example 1

[0049] In this embodiment, the PpDREB1D-like gene was extracted, and the specific experimental steps are as follows:

[0050] (1) cDNA template preparation: RNA was extracted from the leaves of 'Gui Min Yin' elephant grass using an RNA extraction kit (Promega, catalog number: LS1040), and then cDNA was obtained by reverse transcription. The specific steps are as follows:

[0051] S1. Experimental Materials: Approximately 50-100 mg of fresh stems from 4-week-old elephant grass were selected, immediately flash-frozen in liquid nitrogen, and then transferred to -80℃ for storage. The frozen tissue was then thoroughly ground into powder in liquid nitrogen.

[0052] S2. RNA extraction: RNA extraction was performed according to the procedure of the total RNA extraction kit (Promega, catalog number: LS1040).

[0053] S3. Reverse transcription pretreatment: Add the following components (calculated per sample) to an RNase-free centrifuge tube according to Table 1. Denaturation treatment: Heat at 65°C for 5 minutes, then quickly place on ice for 2 minutes (to destroy the secondary structure of RNA and improve reverse transcription efficiency).

[0054] Table 1 RNA Dosage Table

[0055]

[0056] S4. Reverse Transcription: Add 2 μL of 5× gDNA wiper Mix to the pretreated RNA and gently tap to mix. Incubate at 42℃ for 2 minutes using a PCR instrument, then briefly centrifuge. Add the following components in order (total volume 20 μL): 10 μL of the mixture from the previous step; 2 μL of 10× RT Mix; 2 μL of HiScript III Enzyme Mix; 1 μL of Oligo(dT)20VN primer (50 μM); 1 μL of Random hexamers primer (50 μM); and bring the RNase-Free ddH2O to 20 μL. Mix and aliquot: Gently tap the tube walls to mix, avoiding air bubbles, and aliquot into pre-chilled centrifuge tubes on ice. Perform the following two stages of the reaction and terminate the reaction:

[0057] First stage: Transfer the reaction system to a PCR instrument and incubate at 37°C for 10 minutes (primer annealing and enzyme activation).

[0058] Second stage: Adjust the temperature to 50℃ and incubate for 45 minutes (cDNA synthesis).

[0059] Terminate the reaction: Heat at 95°C for 5 minutes (to inactivate reverse transcriptase).

[0060] (2) Primer design: The full-length CDS sequence of the PpDREB1D-like homolog (Pgi04A00024830) was extracted from the genome of the closely related giant Napier grass (a hybrid of elephant grass and elephant grass) and used as a template to design specific primers:

[0061] PpDREB1D-like-f:CGAAGCCATGGATTACTTGAACA (SEQ ID NO.3)

[0062] PpDREB1D-like-r: ACGAAACGGATCCTCTTGTTCTACTC (SEQ ID NO. 4).

[0063] (3) Amplification and extraction of the PpDREB1D-like gene: Using the cDNA obtained in step (1) as a template, the PpDREB1D-like fragment was amplified using the amplification primers PpDREB1D-like-f and PpDREB1D-like-r with the high-fidelity enzyme 2 × Phanta FlashMaster Mix (Dye Plus) from Vazyme. After electrophoresis, the correctly sized band was excised and the DNA fragment was recovered using an agarose gel DNA recovery kit (Tiangen, DP219-03). The electrophoresis diagram is shown below. Figure 1 As shown, the PCR product was purified and then ligated into a T vector for sequencing (T-PpDREB1D-like), yielding the full-length CDS sequence of PpDREB1D-like (SEQ ID NO. 1). Alignment analysis of the sequencing results showed that the nucleic acid sequence identity between PpDREB1D-like and Pgi04A00024830 was as high as 95.38%, indicating successful cloning of the homologous gene of Pgi04A00024830 from elephant grass. Blast analysis confirmed that this gene is a homologous gene of rice OsDREB1D, hence the name PpDREB1D-like.

[0064] (4) Evolutionary analysis: The homologous gene sequences of DREB1 from rice and giant reed were downloaded and analyzed to construct a phylogenetic tree. The results are as follows: Figure 2As shown, PpDREB1D-like genes cluster in one branch with rice OsDREB1D. Furthermore, in this subfamily of giant elephant grass, besides the ortholog of OsDREB1D (Pgi01B00034760), a new gene, Pgi04A00024830, has evolved, indicating that this gene may play an important role in elephant grass species. Therefore, we conducted an in-depth study of the PpDREB1D-like gene in hybrid elephant grass in subsequent experiments.

[0065] Example 2

[0066] This embodiment investigated the expression levels of PpDREB1D-like in different tissues of elephant grass. Referring to the RNA extraction and transcription steps in Example 1, we also extracted and transcribed RNA from fresh roots, leaves, and leaf sheaths of 4-week-old elephant grass. The amount of RNA used during transcription is shown in Table 2.

[0067] Table 2 RNA Usage Table

[0068]

[0069] RT-qPCR was also performed after transcription, and the reaction system is shown in Table 3:

[0070] Table 3 RT-qPCR system sample loading table

[0071]

[0072] Among them, qPpDREB1D-like-f and qPpDREB1D-like-r are primers, and their sequence information is as follows.

[0073] qPpDREB1D-like-f: TCAACTTCCCGGACTCCGCCTG (SEQ ID NO.5)

[0074] qPpDREB1D-like-r: GCTGTCGCCGCCGTTCCTCCT (SEQ ID NO.6)

[0075] SYBR reagent is Novizan RT-qPCR quantitative reagent.

[0076] PCR reaction program: 95℃ for 3 min; 95℃ for 10 s, 60℃ for 30 s, 72℃ for 15 s, 40 cycles; fluorescence was collected every 0.5℃ from 65℃ to 95℃. Results showed the expression of PpDREB1D-like in different tissues as follows: Figure 3As shown in the results, the PpDREB1D-like gene was detected and expressed in all tissues. The expression level in the stem and leaf sheath was significantly higher than in other tissues. This indicates that the gene plays an important function in the stem and leaf sheath.

[0077] Example 3

[0078] This embodiment investigated the expression levels of PpDREB1D-like cells under different cold stress durations. The specific process is as follows:

[0079] S1. Sample processing and RNA extraction

[0080] Stem segments of *Elephantgrass 'Guiminyin'* with lateral buds were isolated and immersed in 1 / 2 Hoagland nutrient solution for germination. After approximately 3-4 days, stem segments with uniform germination were selected and transplanted into pots containing nutrient soil. These were then placed in a greenhouse (25°C, 16-hour light / 8-hour dark cycle) for 20 days of growth. Seedlings with uniform growth were selected and treated in a 4°C incubator for 0, 6, 12, 24, and 48 hours. Leaf tissues of *Elephantgrass* were collected at each time point and immediately frozen in liquid nitrogen. RNA extraction and reverse transcription were performed using the same methods as in the cDNA template preparation section.

[0081] S2, RT-qPCR

[0082] RT-qPCR was used to detect the expression levels of PpDREB1D-like proteins at different time points (method as described in the tissue differential analysis section), and the results are as follows: Figure 4 As shown, PpDREB1D-like was induced by cold stress, with the highest expression level at 24 h, indicating that PpDREB1D-like in elephant grass plays a key role in cold stress adaptation.

[0083] Example 4

[0084] This embodiment investigated the transcriptional activation activity of PpDREB1D-like structures, and the investigation process is as follows:

[0085] (I) Experimental Materials

[0086] (1) Strains and vectors

[0087] Yeast strain: Y2H gold (with HIS3 reporter gene). Vector: pGBKT7, positive control vector pGBKT7-53.

[0088] (2) Reagents: SD / -Trp medium, SD / -Trp / -His. Molecular cloning reagents: EcoRI restriction endonuclease, plasmid extraction kit, gel extraction kit, yeast transformation kit.

[0089] (II) Experimental Procedure

[0090] Construction of S1 and BD-PpDREB1D-like expression vectors

[0091] (1) Primer design and target gene amplification

[0092] Based on the CDS sequence (SEQ ID NO.1) of the PpDREB1D-like gene and the multiple cloning site of the pGBKT7 vector, specific primers with homologous arms were designed. The primer sequences are as follows (lowercase letters represent homologous arm sequences): BD-DREB1D-F: 5'-GGCCATGGAGGCCGAATTCATGGATTACTTGCA-3' (SEQ ID NO.7); BD-DREB1D-R: 5'-CGGATCCCCGGGAATTCTAGCTCCACAGCTCG-3' (SEQ ID NO.8)

[0093] The PCR amplification reaction system is as follows:

[0094] Table 4. PCR amplification system for PpDREB1D-like gene

[0095]

[0096] PCR program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; 72℃ final extension for 5 min; store at 4℃.

[0097] The product was verified by 1% agarose gel electrophoresis to confirm the size of the target fragment, and then purified and recovered.

[0098] (2) Enzyme digestion of linearized vector

[0099] The pGBKT7 vector was digested with enzymes in the following 30 μL solution:

[0100] Table 5. PGBKT7 Enzyme Digestion System for Vector

[0101]

[0102] The linearized pGBKT7 vector was obtained by incubating at 37°C for 1 hour in a PCR instrument and then purifying and recovering it.

[0103] (3) Connect PpDREB1D-like to pGBKT7 vector

[0104] Mixed connection systems as shown in the table below:

[0105] Table 6. Linkage system of PpDREB1D-like and linearized carrier

[0106]

[0107] The culture was incubated at 50℃ for 30 min, and then the product was transformed into E. coli DH5α competent cells, plated on LB solid medium containing 50 μg / mL kanamycin, and incubated at 37℃ for 12-16 h. Single colonies were picked, and sequencing confirmed the correct construction of the BD-PpDREB1D-like fusion vector. High-purity plasmids were extracted using the Tiangen plasmid extraction kit for yeast transformation.

[0108] S2, yeast conversion

[0109] Y2H Gold yeast competent cells were removed from a -80℃ freezer, thawed on ice, and then the following three plasmids were transformed into the yeast competent cells, setting up three treatment groups:

[0110] Experimental group: BD-PpDREB1D-like fusion vector (1 μg); Positive control group: pGBKT7-53 vector (1 μg); Negative control group: pGBKT7 vector (1 μg)

[0111] The yeast conversion system is as follows:

[0112] Table 7 Yeast Conversion System

[0113]

[0114] After gently mixing, incubate at 30°C for 30 min, then heat shock at 42°C for 15 min, centrifuge at 5000 rpm for 5 min, discard the supernatant, and resuspend the bacterial cells in 100 μL of sterile water.

[0115] S3, Transcription activation activity assay

[0116] Take 20 μL of each group of resuspended bacterial solution, spread them evenly on SD / -Trp solid medium (for screening yeast that has been successfully transformed into plasmid) and SD / -Trp / -His solid medium (for screening yeast with transcriptional activation activity), and incubate them upside down at 30℃ for 5 days to observe yeast growth.

[0117] The results are as follows Figure 5 As shown, all groups were able to grow on SD / -Trp, indicating that the plasmids had been successfully transformed. Yeast transformed with the empty pGBKT7 vector could not grow on SD / -Trp / -His. However, yeast transformed with pGBKT7-p53 (positive control group) and pGBKT7-PpDREB1D-like (experimental group) were able to grow on SD / -Trp / -His medium, indicating that PpDREB1D-like has transcriptional activation activity and activates HIS3 expression.

[0118] Example 5

[0119] This embodiment performed subcellular localization testing on the PpDREB1D-like gene, and the specific process is as follows:

[0120] (a) Tobacco cultivation

[0121] Sow tobacco seeds in pots filled with nutrient soil, water thoroughly, and allow them to grow to 5 leaves (about 4-5 weeks) under conditions of 14 hours of light / 10 hours of darkness, temperature of 25°C, and relative humidity of 60%-70%.

[0122] (II) Construction of PpDREB1D-like-YFP plant expression vector

[0123] S1, Primer Design

[0124] Based on the CDS sequence of the PpDREB1D-like gene, primers with vector homologous arms were designed:

[0125] Homo-DREB1Df (SEQ ID NO.9):

[0126] GGCATGGACGAGCTGTACAAGATGGATTACTTGCACA

[0127] Homo-DREB1Dr (SEQ ID NO.10):

[0128] ACGATCGGGGAAAATTCGAGCTCCTAGCTCCACAGCTCGACT

[0129] S2. PCR amplification and recovery of the PpDREB1D-like gene sequence

[0130] The PCR system was the same as in Table 3, but the primers were replaced with those listed above. The PCR program was: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 1 min, for 30 cycles; stored at 4℃. The PCR products were purified by 1% agarose gel electrophoresis and then recovered from the gel.

[0131] S3 and pCAMBIA1381-YFP vector were digested with enzymes, and the digestion system is shown in Table 8.

[0132] Table 8 Enzyme digestion system

[0133]

[0134] Enzyme digestion procedure: 37℃ for 30 min; 65℃ for 20 min; store at -20℃.

[0135] S4. The amplified PpDREB1D-like product with homologous arms was ligated to the linearized vector, and the system is shown in Table 9:

[0136] Table 9. Connection System of PpDREB1D-like with Linearized Carrier

[0137]

[0138] The PCR instrument was set at 50℃ for 30 minutes, and then stored at 4℃.

[0139] S5. Transform the ligation product into competent E. coli cells.

[0140] Thaw 50 μL of competent E. coli cells on an ice box, add all the ligation product to the competent cells, and gently tap to mix. Incubate the competent cells in an ice box for 30 min. Then heat shock in a 42°C water bath for 30 s, followed by an ice bath for 2 min. Add 500 μL of LB liquid medium and incubate at 37°C with shaking for 1 h. Remove the competent cells and centrifuge at 12000 rpm for 1 min to collect the cells. Pick a single colony into a sterile centrifuge tube and add LB liquid medium (containing 50 mg / L kan). Place the centrifuge tube in a 37°C shaker and incubate at 200 rpm for 12 h. Discard the supernatant and excess LB medium. Mix the cells at the bottom by pipetting and aspirating, then spread onto LB solid medium (containing kan, 50 mg / L). Incubate the plate at 37°C overnight (approximately 15 h) until single colonies grow.

[0141] S6. PCR identification of positive clones using bacterial culture.

[0142] The bacterial culture was amplified using 35S-f and GFP-r primers, and the amplification system is shown in Table 10.

[0143] Table 10 PCR System for Escherichia coli Positive Identification

[0144]

[0145] Program: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 2 min, 30 cycles; store at 4℃.

[0146] The bacterial culture with positive bands was selected and sent to Sangon Biotech for sequencing. The sequencing results were compared and analyzed to confirm that the PpDREB1D-like-YFP vector was successfully ligated and without mutations.

[0147] S7, Instantaneous Tobacco Conversion

[0148] The PpDREB1D-like-YFP vector was extracted and transformed into Agrobacterium competent cells. Single clones of Agrobacterium expressing the plasmid were picked and cultured overnight at 28°C and 200 rpm in 2 mL of LB broth containing the appropriate antibiotic. 1 mL of the overnight Agrobacterium culture was transferred to 20 mL of LB broth containing the appropriate antibiotic and cultured at 28°C and 200 rpm until the logarithmic growth phase (OD50) of Agrobacterium. 600 = 0.6-0.8). Collect bacterial cells by centrifugation at 5,000 rpm for 10 min at room temperature. Resuspend the Agrobacterium cells in staining buffer (containing 10 mM MgCl2, 10 mM MES, 150 μM acetylsylgenone, pH = 5.6) to OD. 600 = 1.0. After standing at room temperature for 2 hours, tobacco leaves were injected. Injected plants were placed in darkness for 12 hours, then cultured normally in a greenhouse for 48 hours. The yellow fluorescence signal in the injected Agrobacterium region of the tobacco leaves was captured using a laser confocal microscope. The subcellular localization test results of the PpDREB1D-like gene are as follows: Figure 6 As shown in the figure. The results indicate that this gene may be subcellularly localized, primarily expressed in the cell nucleus, with limited expression in the cell membrane.

[0149] Example 6

[0150] This embodiment uses Arabidopsis thaliana as an example to study the genetic transformation ability of the PpDREB1D-like gene. The specific process is as follows:

[0151] S1, Agrobacterium culture

[0152] Two days before transformation, Agrobacterium containing the PpDREB1D-like-YFP vector was inoculated into 2 mL of LB broth containing antibiotics (rifampin 50 mg / L, kanamycin 50 mg / L) and cultured overnight (approximately 12 h) at 28°C with shaking. The 2 mL cultured Agrobacterium was then transferred to 100 mL of LB broth containing antibiotics and cultured at 28°C with shaking for approximately 8 hours. The Agrobacterium was then transferred to a 50 mL centrifuge tube and centrifuged at 6000 rpm for 10 minutes at room temperature. The supernatant was then discarded, and the precipitate was resuspended in staining buffer (5% sucrose solution with 0.01% Silwet L-77) (OD). 600 =0.8), and the Agrobacterium suspension was transferred to an open sterile petri dish.

[0153] S2, Infection

[0154] Select robust Arabidopsis thaliana plants, remove open flowers and pods, and immerse the inflorescences in a container containing Agrobacterium suspension for 3 minutes. After immersion, place the Arabidopsis thaliana in a dark chamber, sprinkle with water to maintain humidity, and incubate in the dark for 12 hours. Transfer the Arabidopsis thaliana plants to a light condition of 22-25℃ for normal cultivation until flowering and fruiting, then harvest mature seeds.

[0155] S3, Screening of transgenic Arabidopsis thaliana

[0156] (1) Disinfection of Arabidopsis thaliana seeds

[0157] Autoclave centrifuge tubes, pipette tips, petri dishes, and other consumables. Place an appropriate amount of seeds into a 1.5 mL centrifuge tube, add 1 mL of sterile water, and vernalize at 4°C for 3 days (to promote synchronous germination). Discard the sterile water, add 1 mL of 75% ethanol, shake for 1 minute, and immediately discard the ethanol (to avoid alcohol residue damaging the seeds). Add 1 mL of 10% sodium hypochlorite (containing 0.1% Triton X-100 to enhance permeability), shake to sterilize for 10 minutes. Discard the sodium hypochlorite, rinse 8 times with sterile water, and absorb all water after each rinse.

[0158] (2) Sowing

[0159] Prepare and sterilize the culture medium (0.8% agar + 1 / 2 MS medium, pH 5.7); add 30 mg / L hygromycin to the melted culture medium and pour it into a petri dish (about 0.5 cm thick), and let it cool and solidify; use a sterile pipette tip to draw up the seed suspension and sow it evenly on the surface of the culture medium; seal the petri dish with sealing film.

[0160] (3) Germination and transplanting

[0161] Transfer the petri dishes to a light incubator: 22±1℃, 16 h light / 8 h darkness. Observe germination and root development after 7 days of cultivation. When the seedlings have grown two true leaves, transplant them into soil, seal with plastic wrap to retain moisture, and transfer to a greenhouse for further cultivation to obtain transgenic plants.

[0162] S4. Evaluation of cold resistance in Arabidopsis thaliana overexpressing the PpDREB1D-like gene

[0163] (1) The disinfection and sowing methods for the seeds of Arabidopsis thaliana in the empty vector control group and Arabidopsis thaliana overexpressing PpDREB1D-like gene were the same as the previous step. The germinated Arabidopsis thaliana were transplanted into flower pots filled with nutrient soil and continued to be cultivated for one week.

[0164] (2) One week later, Arabidopsis thaliana transfected with the empty YFP vector and Arabidopsis thaliana overexpressing the PpDREB1D-like gene were transferred to a cold stress condition of 8℃ for culture. The expression of the PpDREB1D-like gene and phenotypic changes in Arabidopsis thaliana were compared. The results are as follows: Figure 7-10 As shown.

[0165] Figure 7 This study demonstrates the identification of DNA and RNA levels in Arabidopsis thaliana overexpressing the PpDREB1D-like gene under 8°C low-temperature conditions. Results showed that the PpDREB1D-like gene was detected in the genomes of two independent Arabidopsis thaliana lines. Semi-quantitative results indicated that PpDREB1D-like gene transcripts were expressed in both overexpressing Arabidopsis thaliana lines under cold treatment conditions, while no expression was observed in the empty vector control group (YFP).

[0166] Figure 8 The growth status of Arabidopsis thaliana under a low temperature of 8℃ was shown, and the overexpressed Arabidopsis thaliana showed stronger growth than the control group.

[0167] Figure 9 The results show that the fresh weight of Arabidopsis thaliana under 8℃ low temperature treatment conditions was significantly increased compared with the control group under cold stress.

[0168] Figure 10 This study illustrates the identification of reactive oxygen species (ROS) levels in leaves of Arabidopsis thaliana overexpressing the PpDREB1D-like gene and the control group under low-temperature stress. The results showed that the NBT and DAB staining gradations in the leaves of the PpDREB1D-like gene-overexpressing Arabidopsis thaliana line were weaker than those in the control group, indicating that the ROS level in the leaves was lower in the PpDREB1D-like gene line than in the control group.

[0169] Example 7

[0170] This embodiment investigated the effects of transiently silenced PpDREB1D-like gene in elephant grass on growth under low-temperature stress. The specific process is as follows:

[0171] I. Materials and Reagents

[0172] The seed nodes of *Eriocaulon buergerianum* during the seedling stage, the three plasmid vectors of the BSMV-VIGS system (pCaBS-α, pCaBS-β, pCaBS-γbLIC), and *Agrobacterium* strain GV3101 (pSoup).

[0173] II. Silent Elephant Grass PpDREB1D-like Gene

[0174] S1. Construction of recombinant silencing vector

[0175] 1) Gene fragment design

[0176] Based on the sequenced nucleic acid sequence of the PpDREB1D-like gene, multiple sequence alignment was performed on genomic homologous genes. A specific region of approximately 300 bp (SEQ ID NO. 11) was selected as the specific silencing fragment for the PpDREB1D-like gene.

[0177] SEQ ID NO.11: CACCCTCAACTTCCCGGACTCCGCCTGGCTGCTCGACGTCGCCGTCGCCCCGCGCGGCGCCCGTTCTGCCGCCGGCGCCCCCGAGCTGCCGCCGCTGCCCGACGTCCAGCGCGCCGCCACCGAGGCCGTCGCGGGGTTCCTCCGTCGCCA CGGCGGGGACGCGACGGCAGCGACGCAGCCCGCGCGCAATGCCGCTAATTCGGTGGCGTCCTCAGGAGGAACGGCGGCGACAGCGTCGGTGATGGACGACAATGCCGGCACGCTTGAGCTGGACGCGTTCGGCGGCATGGACGCCGGTTCCTACT.

[0178] 2) Construction of pCaBS-γbLIC-PpDREB1D-like silencing vector

[0179] Specific primers pCaBS-PpDREB1D-F: 5'-AAGGAAGTTTAAATCACCCTCAACTTCCCGGA-3' (SEQ ID NO.12) and pCaBS-PpDREB1D-R: 5'-AACCACCACCACCGTAGTAGGAACCGGCGTCCAT-3' (SEQ ID NO.13) were designed. Using the T-PpDREB1D-like plasmid as a template, the PpDREB1D-like specific fragment was amplified by high-fidelity enzyme PCR. The PCR product was purified by gel extraction. The purified fragment was inserted into the pCaBS-γbLIC linearized vector according to the LIC cloning method to construct the recombinant plasmid pCaBS-γbLIC-PpDREB1D-like. The recombinant plasmid was transformed into E. coli DH5α for positive clone screening and sequencing verification.

[0180] S2 Infection Solution Preparation

[0181] 1) The verified recombinant plasmid pCaBS-γbLIC-PpDREB1D-like, along with the helper plasmids pCaBS-α and pCaBS-β, were transformed into Agrobacterium GV3101 competent cells. The cells were plated on LB agar plates containing kanamycin (50 mg / L) and rifampin (25 mg / L), and incubated upside down at 28°C for 2-3 days. Single colonies were then picked.

[0182] 2) Pick single colonies of Agrobacterium containing pCaBS-α, pCaBS-β, and pCaBS-γbLIC-PpDREB1D-like, respectively, and culture them with shaking in LB liquid medium containing the corresponding antibiotics until OD. 600 ≈ 1.5. Collect bacterial cells by centrifugation at 6000 rpm for 10 minutes, resuspend the cells in the infection solution, and set the OD value accordingly. 600 Adjust to 1.0. After standing at room temperature for 2 hours, it can be used for infection.

[0183] S3 Infects Elephant Grass Sprouts

[0184] (1) The three Agrobacterium bacterial solutions (GV3101 / pCaBS-α, GV3101 / pCaBS-β, GV3101 / pCaBS-γbLIC-PpDREB1D-like) were mixed in a volume ratio of 1:1:1 to obtain the mixed bacterial solution of the treatment group.

[0185] (2) Negative control: Replace GV3101 / pCaBS-γbLIC-PpDREB1D-like with GV3101 / pCaBS-γbLIC (empty vector), and mix them at the same ratio of 1:1:1.

[0186] (3) Completely immerse the elephant grass seedlings in the mixed bacterial solution and treat them for 60 minutes under a vacuum pressure of 1.0 bar using a vacuum pump. Transplant the infected plants into nutrient soil and cultivate them for 20 days under the conditions of day / night temperature of 24 / 22℃ and light / dark cycle of 16 / 8 h.

[0187] (4) Samples were taken 20 days after infection. Using the elephant grass Actin gene as an internal control, qRT-PCR was performed using PpDREB1D-like specific primers to detect the expression level of the PpDREB1D-like gene. Silent lines and control groups were simultaneously treated at 8 degrees Celsius for 20 days. Results are as follows... Figure 11-12 As shown.

[0188] Figure 11 RT-qPCR was used to identify the expression of the PpDREB1D-like gene in elephant grass. The results showed that, compared with the control group, the mRNA expression level of the PpDREB1D-like gene in the leaves of the silenced plants was significantly reduced, proving that the BSMV-VIGS system was successfully introduced and silenced the PpDREB1D-like gene.

[0189] Figure 12The comparison shows the growth status of PpDREB1D-like silent elephant grass under cold stress. Plants with the PpDREB1D-like gene silenced exhibit a more sensitive phenotype to cold stress, characterized by wilting leaves and a significant increase in ROS levels in the leaves. This indicates that the PpDREB1D-like gene improves the cold resistance of elephant grass by clearing low-temperature-induced ROS levels.

[0190] Finally, it should be noted that the terms “comprising,” “including,” or any other variations are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0191] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0192] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. Pennisetum PpDREB1D-like The use of the gene in the preparation of a product for increasing cold tolerance and biomass of a plant, characterized in that, The PpDREB1D-like The sequence of the gene is shown as SEQ ID NO: 1 and the encoded amino acid sequence is shown as SEQ ID NO. 2; The plant is Arabidopsis thaliana or elephant grass, the elephant grass PpDREB1D-like Genes are overexpressed in plants to increase plant cold tolerance and biomass.

2. Use according to claim 1, wherein The elephant grass PpDREB1D-like The gene is derived from the stem, leaf sheath or root of elephant grass.

3. The use according to claim 1, wherein The elephant grass PpDREB1D-like The gene can be obtained by amplification reaction using the sequence shown in SEQ ID NO. 3, SEQ ID NO. 4 as primers PpDREB1D-like Gene.

4. The use according to claim 1, wherein The use of said elephant grass PpDREB1D-like The process for increasing cold tolerance and biomass of plants comprising the steps of: (1) the elephant grass PpDREB1D-like gene is linked to an expression vector to obtain a recombinant vector; (2) Transform the recombinant vector into the competent cells to obtain the recombinant cells; (3) Use the recombinant cells to soak the plants whose cold resistance is to be improved.

5. The use according to claim 4, wherein the compound is ###0002### The preparation process of the recombinant vector comprises the following steps: (1) Design primers Homo- DREB1Df and Homo- DREB1Dr , the sequences of which are shown in SEQ ID NO. 9, SEQ ID NO. 10, respectively; (2) Using primers Homo- DREB1Df and Homo- DREB1Dr right PpDREB1D-like The gene undergoes an amplification reaction to obtain a vector-homogeneous sequence. PpDREB1D-like ; (3) Perform enzyme cutting on the pCAMBIA1381-YFP vector to obtain a linearized vector; (4) PpDREB1D-like Connected to the linearized carrier.

6. Use according to claim 4, characterized in that, The competent cells are Agrobacterium tumefaciens Strain GV3101 competent strain.

7. A composition comprising guinea grass PpDREB1D-like Use of a recombinant vector or a recombinant cell containing a gene for increasing cold tolerance and biomass of a plant, characterized in that, The PpDREB1D-like The sequence of the gene is shown as SEQ ID NO: 1 and the encoded amino acid sequence is shown as SEQ ID NO. 2; The plant is Arabidopsis thaliana or elephant grass, the elephant grass PpDREB1D-like Genes are overexpressed in plants to increase plant cold tolerance and biomass.

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