Pennisetum purpureum PpDREB1D-like gene and application thereof in improving cold resistance and biomass of plants

Abstract

The invention discloses a pennisetum purpureum PpDREB1D-like gene and application thereof in improving cold resistance and biomass of plants, and relates to the technical field of plant genetic engineering. The PpDREB1D-like gene derived from the pennisetum purpureum is cloned and functionally identified for the first time, and experimental verification shows that the PpDREB1D-like gene can increase the cold resistance of plants, the growth state of the plants under cold stress is remarkably improved through overexpression of the PpDREB1D-like gene, the cold resistance of the plants is remarkably reduced through silence of the PpDREB1D-like gene, and the PpDREB1D-like gene can be used for improving the cold resistance of the plants. A molecular foundation is laid for popularization and planting of pasture and energy crops in cold regions, and the method has remarkable agricultural production and ecological value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genetic engineering, and particularly relates to a Pennisetum purpureum PpDREB1D-like gene and application thereof in improving plant cold resistance and biomass. BACKGROUND

[0002] Low temperature is one of the main abiotic stresses that limit plant distribution and agricultural productivity. To cope with this challenge, crop cold resistance research has become the focus of genetic improvement. Plants will initiate complex molecular responses under low temperature, including hormone signaling, oxidation-reduction, photosynthesis and transcription factor regulation pathways. Identifying key regulatory factors in these pathways is crucial for revealing the cold resistance mechanism of plants.

[0003] Pennisetum purpureum has soft texture, good palatability, and high dry matter content of up to 21.6%. The relatively high crude protein and moderate crude fat and nitrogen-free extract content make it suitable for feeding a variety of animals such as cattle, sheep, pigs, rabbits and fish. Pennisetum purpureum is a perennial clump large herbaceous plant of the genus Pennisetum in the family Poaceae, and has become one of the most important forage species and potential energy grasses in the tropical and subtropical regions of Asia, Africa and the Americas. As an excellent forage crop, it has a yield of up to 150 tons per hectare per year, can be harvested at least 4 times a year, and can resist high temperature, drought, low soil fertility and biological stress.

[0004] Pennisetum purpureum Schum. cv. Gui Min Yin is a perennial herb of the genus Pennisetum in the family Poaceae. It has strong drought resistance and disease resistance, and high sugar content, and is not only an excellent forage resource, but also an ecological pasture for greening barren hills and preventing soil erosion. At present, this variety has been widely applied in many places in southern China, but its cold tolerance is generally weak, which is the main factor limiting its production and efficient use. Therefore, in-depth study of its molecular response mechanism under low temperature stress is of great significance for expanding the ecological adaptation range, assisting the introduction of southern grasses to the north and the construction of efficient pasture production system, and provides a theoretical basis for the excavation and breeding of cold-tolerant germplasm. However, so far, there have been few studies on cold-resistant genes of Pennisetum purpureum, and its cold resistance mechanism is not clear.

[0005] DREB is an important transcription factor involved in the response to abiotic stress, and can be subdivided into six subfamilies in Arabidopsis. Different subfamilies have different responses to different stress signal transduction, and DREB1 / CBF is considered as the "main switch" in the process of responding to cold stress, and plays an important role in the freezing tolerance and low temperature acclimation of plants. DREB1 / CBFs activate the expression of low temperature stress related genes by binding to the DRE / CRT cis-acting elements in the promoter of downstream target genes, causing stress related physiological metabolism and functional structural response, thereby improving the cold resistance of plants. According to the potential of DREB1 / CBFs in cold resistance, it has broad application prospects to enhance the cold resistance of plants and improve the viability and yield of plants. However, so far, there are few studies on DREB1 / CBFs in elephant grass, especially on cold resistance. SUMMARY

[0006] The present application identifies a new cold stress induced PpDREB1-like gene from elephant grass through transcriptome analysis, and genetic studies show that the gene is a positive regulatory gene for cold resistance of elephant grass. The gene is of great significance for breeding low temperature resistant varieties, improving the overwintering of elephant grass, expanding the planting area, improving the quality of forage grass and environmental sustainable development.

[0007] The technical scheme adopted by the present application is as follows:

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

[0009] SEQ ID NO: 1: ATGGATTACTTGCACACCACTGGTACCGAGCCTCCGGGCGGCTCCACGACCACTGAGTCGGGGTCGGGTCGCCCGTCCCCGCCGAAGCGCCCCGCGGGGCGGACCAAGTTCCAGGAGACGCGGCACCCGGTGTTCCGCGGGGTGCGGCGTCGTGGCCGGGCGGGGCGGTGGGTGTGCGAGGTCCGCGTCCCCGGCAGCCGCGGGGACCGCCTCTGGGTCGGCACCTTCGACACCGCCGAGGCCGCCGCGCGCGCGCACGACGCCGCCATGCTCGCCCTCTGCGGCGCCGCCGCCACCCTCAACTTCCCGGACTCCGCCTGGCTGCTCGACGTCGCCGTCGCCCCGCGCGGCGCCCGTTCTGCCGCCGGCGCCCCCGAGCTGCCGCCGCTGCCCGACGTCCAACGCGCCGCCACCGATGCCGTCGCGGGGTTCCTCCGTCGCCACGGCGGCGACGCCACGGTAGCTACCCACCCCGCGGGCAATGCCGCTAATTCGGTGGCGTCCTCAGGAGGAACGGCGGCGACAGCGTCGGTGATGGACGACAATGCCGGCACGCTTGAGCTGGACGCGTTCGGCGGCATGGACGCCGGTTCCTACTACGCGAGCCTGGCGCAGGGGCTGCTCATCGACCCGCCGCCGCCGACCGCCGTGGAGTGCCCGGAGGAGGACGAGGACGACTGCGAAGCCGGCGAAGTCGAGCTGTGGAGCTAG.

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

[0011] Further, the PpDREB1D-like gene is derived from stems, leaf sheaths or roots of elephant grass.

[0012] Still further, the PpDREB1D-like gene is amplified by using the sequences as shown in SEQ ID NO. 3 and SEQ ID NO. 4 as primers.

[0013] SEQ ID NO: 3: CGAAGCCATGGATTACTTGAACA.

[0014] SEQ ID NO: 4: ACGAAACGGATCCTCTGTTCTACTC.

[0015] In a second aspect, the application discloses application of the PpDREB1D-like gene in preparing a product for improving cold resistance and biomass of plants.

[0016] Further, the low-temperature-resistant plant is Arabidopsis thaliana or elephant grass.

[0017] Still further, the process for improving cold resistance and biomass of plants by using the PpDREB1D-like gene comprises the following steps:

[0018] (1) connecting the PpDREB1D-like gene to an expression vector to obtain a recombinant vector;

[0019] (2) transforming the recombinant vector into a competent cell to obtain a recombinant cell;

[0020] (3) using the recombinant cell to dip a plant with improved cold resistance.

[0021] More preferably, the process for preparing the recombinant vector comprises the following steps:

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

[0023] (2) using the primers Homo-DREB1Df and Homo-DREB1Dr to perform an amplification reaction on the PpDREB1D-like gene to obtain PpDREB1D-like;

[0024] (3) performing enzyme cutting on the pCAMBIA1381-YFP vector to obtain a linearized vector;

[0025] (4) ligating PpDREB1D-like with a linearized vector.

[0026] More preferably, the competent cell is a competent strain of Agrobacterium tumefaciens strain GV3101.

[0027] In a third aspect, the present application provides a reagent for improving cold resistance and biomass of plants, wherein the reagent comprises the PpDREB1D-like gene of the first aspect, or a recombinant vector or a recombinant cell prepared using the PpDREB1D-like gene of the first aspect.

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

[0029] 1. The PpDREB1D-like gene from elephant grass is cloned and functionally identified for the first time, and it is found through experiments that the PpDREB1D-like gene can increase the cold resistance of plants, specifically as follows: the transgenic Arabidopsis thaliana overexpressing the gene has a better growth state than the control group under 8℃ low-temperature stress, the fresh weight of the plant is significantly increased, and the level of reactive oxygen species (ROS) in the leaves is reduced; silencing the gene in elephant grass leads to more severe wilting of the leaves of elephant grass under low-temperature stress, and the level of ROS is significantly increased, indicating that PpDREB1D-like improves the cold resistance of the plant by removing the ROS induced by low temperature. Therefore, overexpression of the PpDREB1D-like gene significantly improves the growth state of the plant under cold stress, and silencing the PpDREB1D-like gene in elephant grass significantly reduces its cold resistance.

[0030] 2. The PpDREB1D-like gene is verified to be a positive regulatory factor of cold stress through heterologous overexpression in the model plant Arabidopsis thaliana and gene silencing in elephant grass, which provides direct experimental evidence for its application in elephant grass and other crops.

[0031] 3. The present application provides a new source of genetic improvement genes, which lays a molecular foundation for the popularization and planting of forage grasses and energy crops in cold regions, and has significant agricultural production and ecological value. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Fig. 1 is a gel electrophoresis diagram of the PCR product of the PpDREB1D-like gene;

[0033] Figure 2 Fig. 2 is a phylogenetic tree spectrum diagram of the PpDREB1D-like gene;

[0034] Figure 3Figure 6 is a graph showing the expression of PpDREB1D-like gene in different tissues;

[0035] Figure 4 Figure 7 is a graph showing the expression of PpDREB1D-like gene under different cold stress time;

[0036] Figure 5 Figure 8 is a graph showing the transcriptional activation activity of PpDREB1D-like gene;

[0037] Figure 6 Figure 9 is a graph showing the subcellular localization of PpDREB1D-like gene;

[0038] Figure 7 Figure 10 is a graph showing the detection results of DNA and RNA levels of PpDREB1D-like gene in transgenic Arabidopsis;

[0039] Figure 8 Figure 11 is a graph showing the growth state of different Arabidopsis under cold stress condition;

[0040] Figure 9 Figure 12 is a graph showing the aboveground fresh weight of different Arabidopsis under cold stress condition;

[0041] Figure 10 Figure 13 is a graph showing the leaf ROS staining of different Arabidopsis under different conditions;

[0042] Figure 11 Figure 14 is a graph showing the expression of PpDREB1D-like gene in different elephant grasses under cold stress condition;

[0043] Figure 12 Figure 15 is a graph showing the growth state of different elephant grasses under cold stress condition. DETAILED DESCRIPTION

[0044] The advantages and various effects of the present application will be more clearly presented by the following specific embodiments and examples. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present application, not to limit the present application.

[0045] Throughout the specification, unless otherwise specifically indicated, the terms used herein are understood to have the meanings 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 the present application belongs. If there is a contradiction, the present specification takes precedence.

[0046] Unless otherwise specifically indicated, various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by existing methods.

[0047] The application will be described in detail below with examples and experimental data.

[0048] Example 1

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

[0050] (1) Preparation of cDNA template: RNA extraction kit (Puluo Maige, item number: LS1040) was used to extract the RNA of "Gui Min Yin" elephant grass leaves, and then cDNA was obtained by reverse transcription. The specific steps are as follows:

[0051] S1, Experimental material: select 4-week-old elephant grass fresh stems about 50-100 mg, immediately placed in liquid nitrogen for quick freezing and transferred to-80℃ for storage for standby. Take the frozen tissue, grind it into powder in liquid nitrogen.

[0052] S2, RNA extraction: RNA extraction steps are extracted according to total RNA extraction kit (Puluo Maige, item number: LS1040).

[0053] S3, Reverse transcription pretreatment: add the following ingredients (calculated according to a single sample) in a RNase-free centrifuge tube, denaturation treatment: 65℃ heating for 5 minutes, quickly placed on ice for 2 minutes (destroying RNA secondary structure, improving reverse transcription efficiency).

[0054] Table 1 RNA dosage table

[0055]

[0056] S4, Reverse transcription: add 2 μL 5× gDNA wiper Mix to the pretreated RNA, mix gently. PCR instrument temperature control 42℃ incubation for 2 minutes, instant centrifugation. Add the following ingredients (total volume 20 μL) in the following order: 10 μL of the mixture from the previous step; 10× RT Mix 2 μL; HiScript III Enzyme Mix 2 μL; Oligo(dT)20VN primer (50 μM) 1 μL; Random hexamers primer (50 μM) 1 μL; RNase-Free ddH2O to 20 μL. Mix and dispense: mix gently by tapping the tube wall, avoid bubbles, dispense into pre-cooled centrifuge tubes on ice. Perform the following two-stage reaction and termination reaction:

[0057] First stage: transfer the reaction system to the PCR instrument, 37℃ incubation for 10 minutes (primer annealing and enzyme activation).

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

[0059] Termination reaction: 95°C heating for 5 minutes (inactivate reverse transcriptase).

[0060] (2) Primer design: The full-length CDS sequence of PpDREB1D-like homologous gene (Pgi04A00024830) was extracted from the closely related giant reed (Erianthus arundinaceus and its hybrid) genome as a template, and specific primers were designed:

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

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

[0063] (3) Amplification and extraction of PpDREB1D-like gene: The cDNA obtained in step (1) was used as a template, and the Vazyme high-fidelity enzyme 2 × Phanta Flash Master Mix (Dye Plus) was used to amplify the PpDREB1D-like fragment using the amplification primers PpDREB1D-like-f and PpDREB1D-like-r. After electrophoresis, the correct size band was cut off and the DNA fragment was recovered using an agarose gel DNA recovery kit (Tiangen, DP219-03). The electrophoresis map is shown in Figure 1 , the PCR product was purified, then the product was ligated to a T vector for sequencing (T-PpDREB1D-like), and the PpDREB1D-like full-length CDS sequence (SEQ ID NO. 1) was obtained. The alignment analysis of the sequencing results showed that the nucleic acid sequence consistency of PpDREB1D-like and Pgi04A00024830 was as high as 95.38%, indicating that the homologous gene of Pgi04A00024830 in elephant grass was successfully cloned. The Blast result showed that the gene is the homologous gene of rice OsDREB1D, and therefore named PpDREB1D-like.

[0064] (4) Evolution analysis: The sequences of rice and giant reed DREB1 homologous genes were downloaded for analysis, and a phylogenetic evolution tree was constructed, and the results are shown in Figure 2PpDREB1D-like and rice OsDREB1D are clustered in one branch. In addition, Pgi04A00024830, a new gene in addition to the orthologous gene (Pgi01B00034760) of OsDREB1D, is evolved in P. giganteum, indicating that the gene may play an important role in Pennisetum species. Therefore, we further studied the PpDREB1D-like gene in the hybrid P. giganteum in the subsequent experiments.

[0065] Example 2

[0066] In this example, the expression amount of PpDREB1D-like in different tissues of P. giganteum was studied. Referring to the RNA extraction and transcription steps in Example 1, we also extracted RNA from the fresh root, leaf and leaf sheath tissues of 4-week-old P. giganteum and transcribed the RNA. In the transcription process, the RNA dosage is shown in Table 2:

[0067] Table 2 RNA dosage table

[0068]

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

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

[0071]

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

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

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

[0075] The SYBR reagent is a Roche RT-qPCR quantitative reagent.

[0076] The PCR reaction program is as follows: 95℃ for 3 min; 95℃ for 10 s, 60℃ for 30 s, 72℃ for 15 s, 40 cycles; 65-95℃, collect fluorescence once every 0.5℃. The expression of PpDREB1D-like in different tissues is shown in Figure 3As shown in the results, it is shown that PpDREB1D-like gene is detected in all tissues. Among them, the expression amount in stems and leaf sheaths is significantly higher than that in other tissues. It can be seen that the gene plays an important function in stems and leaf sheaths.

[0077] Example 3

[0078] This example is directed to the expression amount of PpDREB1D-like under different cold stress times, and the specific process is as follows:

[0079] S1, sample processing and RNA extraction

[0080] The stems of "Guiminyin" elephant grass with lateral buds were immersed in 1 / 2 Hoagland nutrient solution for germination. After about 3-4 days, the germinated stems were selected and transplanted into flowerpots containing nutrient soil, and then transferred to a greenhouse (25°C, 16 hours light / 8 hours dark cycle) for growth for 20 days. The uniform seedlings were selected and treated in a 4°C incubator for 0, 6, 12, 24 and 48h. At each time point, the leaf tissue of elephant grass was collected and immediately frozen in liquid nitrogen. The RNA extraction and reverse transcription method were the same as the cDNA template preparation part.

[0081] S2, RT-qPCR

[0082] The expression level of PpDREB1D-like at different time points was detected by RT-qPCR (the method was the same as the tissue difference analysis part), and the results were as shown in Figure 4 It can be seen that PpDREB1D-like is induced to express under cold stress, and the expression amount is the highest at 24h, indicating that PpDREB1D-like of elephant grass plays a key role in the cold stress adaptation process.

[0083] Example 4

[0084] This example studies the transcriptional activation activity of PpDREB1D-like, and the research process is as follows:

[0085] (I) Experimental materials

[0086] (1) Strains and vectors

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

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

[0089] (II) Experimental steps

[0090] S1, Construction of BD-PpDREB1D-like expression vector

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

[0092] According to the CDS sequence (SEQ ID NO. 1) of PpDREB1D-like gene and the multiple cloning site of pGBKT7 vector, a specific primer with homologous arm was designed, and the primer sequence was as follows (the lowercase letters were 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 was as follows:

[0094] Table 4 PCR amplification system table of PpDREB1D-like gene

[0095]

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

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

[0098] (2) Linearization of the vector

[0099] The pGBKT7 vector was digested, and the digestion system (30 μL) was as follows:

[0100] Table 5 Digestion system table of vector PGBKT7

[0101]

[0102] The linearized pGBKT7 vector was purified and recovered by incubation at 37°C in a PCR instrument for 1 h.

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

[0104] The linking system was mixed according to the following table:

[0105] Table 6 Linking system table of PpDREB1D-like and linearized vector

[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] The embodiment is aimed at PpDREB1D-like gene subcellular localization test, the specific process is as follows:

[0120] (I) Planting tobacco

[0121] Tobacco seeds are sowed in pots containing nutrient soil, and water is poured, and grown to 5 leaves (about 4-5 weeks) under the conditions of 14 h light / 10 h darkness, temperature 25°C, and relative humidity 60%-70%.

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

[0123] S1, primer design

[0124] According to the CDS sequence of PpDREB1D-like gene, a primer with vector homologous arm is designed:

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

[0126] GGCATGGACGAGCTGTACAAGATGGATTACTTGCACA

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

[0128] ACGATCGGGGAAATTCGAGCTCCTAGCTCCACAGCTCGACT

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

[0130] The PCR system is the same as Table 3, and the primer is replaced with the above primer; the PCR program is 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 1 min, 30 cycles; 4°C storage. The PCR product is electrophoresed on 1% agarose gel and purified by gel recovery.

[0131] S3, pCAMBIA1381-YFP vector digestion, the enzyme digestion system is shown in Table 8.

[0132] Table 8 Enzyme digestion system

[0133]

[0134] The enzyme digestion program is 37°C for 30 min; 65°C for 20 min; -20°C storage.

[0135] S4, the PpDREB1D-like product with homologous arm obtained by amplification is connected with the linearized vector, and the system is shown in Table 9:

[0136] Table 9 PpDREB1D-like and linearization vector connection system table

[0137]

[0138] PCR instrument control temperature 50℃ 30 min, then 4℃ preservation.

[0139] S5, connection product transformation E. coli competent cell

[0140] Take 50 μL E. coli competent cell on ice box melt, add all the connection product to the competent cell, shake mix; the competent cell is inserted into the ice box and incubated for 30 min; then 42℃ water bath pot heat shock 30 s, ice bath 2 min; add 500 μL LB liquid medium, 37℃ shaking culture 1h; the competent cell is taken out, 12000 rpm centrifugal 1 min to collect bacterial body; pick single colony in sterile centrifuge tube, add LB liquid medium (containing 50 mg / L kan); the centrifuge tube is placed in 37℃ shaking bed, and the culture is cultured at 200 rpm for 12 h; the supernatant LB culture medium is discarded, and the bottom bacterial body is blown and mixed, then coated with LB solid culture medium (containing kan, 50 mg / L), and the plate is placed in 37℃ culture for overnight culture (about 15 h), until single colony grows.

[0141] S6, bacteria liquid PCR identification positive clone

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

[0143] Table 10 E. coli positive identification PCR system table

[0144]

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

[0146] The bacteria liquid of positive band is selected and sent to Shengong Biotechnology Company for sequencing, and the sequencing result comparison analysis determines that the PpDREB1D-like-YFP vector is successfully connected and has no mutation.

[0147] S7, tobacco transient transformation

[0148] The PpDREB1D-like-YFP vector was extracted, transformed into Agrobacterium competent cells, and a single colony of Agrobacterium transformed with the expression plasmid was selected and inoculated into 2 mL of LB liquid medium containing the corresponding antibiotic and cultured overnight at 28°C with shaking at 200 rpm. 1 mL of the overnight culture of Agrobacterium was transferred to 20 mL of LB liquid medium containing the corresponding antibiotic and cultured at 28°C with shaking at 200 rpm until the Agrobacterium grew to the logarithmic phase (OD 600 = 0.6-0.8). The bacteria were collected by centrifugation at room temperature at 5,000 rpm for 10 min, resuspended in infiltration solution (containing 10 mM MgCl2, 10 mM MES, 150 μM acetosyringone, pH = 5.6) to an OD 600 = 1.0. The bacteria were allowed to stand at room temperature for 2 h, and then tobacco leaves were injected. The injected plants were placed in the dark for 12 h, and then normally cultured in the greenhouse for 48 h. The yellow fluorescence signal of the region of the tobacco leaf injected with Agrobacterium was photographed using a laser confocal microscope. The results of the subcellular localization test of the PpDREB1D-like gene are shown in Figure 6 . The results show that the gene may be subcellularly localized mainly in the nucleus and expressed in a small amount in the cell membrane.

[0149] Example 6

[0150] In this example, the genetic transformation ability of the PpDREB1D-like gene was studied using Arabidopsis thaliana as an example. 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 liquid medium containing antibiotics (rifampicin 50 mg / L, kanamycin 50 mg / L) and cultured overnight (about 12 h) at 28°C with shaking. 2 ml of the cultured Agrobacterium was transferred to 100 mL of LB liquid medium containing antibiotics and cultured at 28°C with shaking for about 8 hours. The Agrobacterium was transferred to a 50 mL centrifuge tube and centrifuged at room temperature at 6000 rpm / min for 10 minutes. Then the supernatant was poured out, the precipitate was resuspended (OD 600 = 0.8) with infiltration solution (5% sucrose solution, with the addition of 0.01% Silwet L-77), and the Agrobacterium suspension was transferred to an open sterile culture dish.

[0153] S2. Infection

[0154] Select healthy Arabidopsis plants, cut off the open flowers, pods, immerse the inflorescence in a container containing Agrobacterium suspension for 3 min. After the infection, the Arabidopsis is placed in a dark box, and water is sprinkled to keep it moist. Dark culture for 12 hours. The Arabidopsis plants are transferred to a normal culture under light conditions at 22-25°C until flowering and seed collection.

[0155] S3, screening of transgenic Arabidopsis

[0156] (1) Arabidopsis seed disinfection

[0157] Sterilize the centrifuge tube, gun head, and culture dish. Put the seeds into a 1.5 mL centrifuge tube, add 1 mL of sterile water, and store in a 4°C refrigerator for 3 days (to promote germination synchronicity). Discard the sterile water, add 1 mL of 75% ethanol, shake for 1 minute, and immediately discard the ethanol (to avoid damage to the seeds caused by residual alcohol). Add 1 mL of 10% sodium hypochlorite (containing 0.1% Triton X-100 to enhance permeability), shake for 10 minutes, and discard the sodium hypochlorite. Wash with sterile water 8 times, and discard the water after each wash.

[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 culture dish (thickness about 0.5 cm). Cool and solidify. Use a sterile gun head to suck the seed suspension and evenly sow it on the surface of the culture medium. Seal the culture dish with a sealing film.

[0160] (3) Germination and transplanting

[0161] Transfer the culture dish to a light incubator: 22±1°C, 16 h light / 8 h dark. Observe the germination and root growth after 7 days of culture. When the seedlings grow to 2 true leaves, transplant them into soil, seal with plastic wrap to keep moist, and transfer them to a greenhouse for further culture to obtain transgenic plants.

[0162] S4, evaluation of cold resistance of Arabidopsis overexpressing PpDREB1D-like gene

[0163] (1) Seed disinfection and sowing of Arabidopsis overexpressing PpDREB1D-like gene and Arabidopsis transformed with empty vector: the same as the previous step. Transplant the germinated Arabidopsis into pots containing nutrient soil and continue to culture for one week.

[0164] (2) After one week, transfer the Arabidopsis transformed with YFP empty vector and the Arabidopsis overexpressing PpDREB1D-like gene to 8°C cold stress conditions for culture. Compare the PpDREB1D-like gene expression and phenotypic changes of Arabidopsis, and the results are shown in Figures 7-10 .

[0165] Figure 7 Figure 7 shows the identification of DNA and RNA levels of Arabidopsis thaliana overexpressing PpDREB1D-like gene under low temperature condition of 8℃. The results show that the PpDREB1D-like gene was detected in the genome of two independent Arabidopsis thaliana lines; semi-quantitative results show that the expression of PpDREB1D-like gene transcript was detected in two overexpressing Arabidopsis thaliana lines under cold treatment, while the empty control group (YFP) had no expression.

[0166] Figure 8 Figure 8 shows the growth status of Arabidopsis thaliana under low temperature condition of 8℃, and the growth status of overexpressing Arabidopsis thaliana is stronger than that of the control group.

[0167] Figure 9 Figure 9 shows the fresh weight of Arabidopsis thaliana under low temperature treatment condition of 8℃, and the results show that under cold stress, the fresh weight of Arabidopsis thaliana overexpressing PpDREB1D-like gene is significantly increased compared with the control group.

[0168] Figure 10 Figure 10 shows the identification of active oxygen level in the leaves of Arabidopsis thaliana overexpressing PpDREB1D-like gene and the control group under low temperature stress. The results show that the NBT and DAB coloring degree of Arabidopsis thaliana overexpressing PpDREB1D-like gene is weaker than that of the control group, indicating that the active oxygen level in the leaves is lower than that of the control group.

[0169] Example 7

[0170] This example studies the growth effect of the target grass instantaneously silenced PpDREB1D-like gene of the grass under low temperature stress, and the specific process is as follows:

[0171] I. Materials and reagents

[0172] Young grass seed nodes, BSMV-VIGS system three-plasmid vector (pCaBS-α, pCaBS-β, pCaBS-γbLIC), Agrobacterium strain GV3101 (pSoup).

[0173] II. Silencing PpDREB1D-like gene of the grass

[0174] S1. Construction of recombinant silencing vector

[0175] 1) Gene fragment design

[0176] According to the nucleic acid sequence of the sequenced PpDREB1D-like gene, multiple sequence alignment of the homologous genes in the genome was performed. A specific region of about 300 bp (SEQ ID NO. 11) was selected as a specific silencing fragment of PpDREB1D-like gene.

[0177] SEQ ID NO. 11: CACCCTCAACTTCCCGGACTCCGCCTGGCTGCTCGACGTCGCCGTCGCCCCGCGCGGCGCCCGTTCTGCCGCCGGCGCCCCCGAGCTGCCGCCGCTGCCCGACGTCCAGCGCGCCGCCACCGAGGCCGTCGCGGGGTTCCTCCGTCGCCACGGCGGGGACGCGACGGCAGCGACGCAGCCCGCGCGCAATGCCGCTAATTCGGTGGCGTCCTCAGGAGGAACGGCGGCGACAGCGTCGGTGATGGACGACAATGCCGGCACGCTTGAGCTGGACGCGTTCGGCGGCATGGACGCCGGTTCCTACT.

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

[0179] Specific primers pCaBS-PpDREB1D-F: 5'-AAGGAAGTTTAAATCACCCTCAACTTCCCGGA-3' (SEQ ID NO. 12), pCaBS-PpDREB1D-R: 5'-AACCACCACCACCGTAGTAGGAACCGGCGTCCAT-3' (SEQ ID NO. 13) were designed to amplify PpDREB1D-like specific fragment from T-PpDREB1D-like plasmid using high fidelity enzyme PCR. The PCR product was purified by gel recovery. The purified fragment was inserted into pCaBS-γbLIC linearized vector according to LIC cloning method to construct recombinant plasmid pCaBS-γbLIC-PpDREB1D-like. The recombinant plasmid was transformed into E. coli DH5α, and positive clones were screened and verified by sequencing.

[0180] S2 Preparation of infection solution

[0181] 1) The correct recombinant plasmid pCaBS-γbLIC-PpDREB1D-like was transformed into Agrobacterium GV3101 competent cells with helper plasmids pCaBS-α and pCaBS-β, respectively. The cells were plated on LB solid plates containing kanamycin (50 mg / L) and rifampicin (25 mg / L) and incubated at 28°C for 2-3 days. Single colonies were selected.

[0182] 2) Pick single colonies of Agrobacterium containing pCaBS-a, pCaBS-b and pCaBS-g bLIC-PpDREB1D-like, respectively, and shake-cultured in LB liquid medium containing the corresponding antibiotics to OD 600 ≈ 1.5. Centrifuge at 6000 rpm for 10 minutes to collect the bacterial cells, resuspend the bacterial cells with the infection liquid, and adjust the OD 600 to 1.0. After standing at room temperature for 2 hours, use for infection.

[0183] S3 Infection of elephant grass young shoots

[0184] (1) Mix the three Agrobacterium bacterial solutions (GV3101 / pCaBS-a, GV3101 / pCaBS-b, GV3101 / pCaBS-g bLIC-PpDREB1D-like) at a volume ratio of 1:1:1 to obtain the treatment group mixed bacterial solution.

[0185] (2) Negative control: Replace GV3101 / pCaBS-g bLIC-PpDREB1D-like with GV3101 / pCaBS-g bLIC (empty vector), and mix in the same way at a volume ratio of 1:1:1.

[0186] (3) Completely immerse the elephant grass young shoots in the mixed bacterial solution, and use a vacuum pump to treat at a vacuum pressure of 1.0 bar for 60 minutes. After infection, transplant the plants into nutrient soil, and cultivate under conditions of day and night temperatures of 24 / 22°C, light and dark cycles of 16 / 8 h for 20 days.

[0187] (4) Sample at 20 days after infection, use the elephant grass Actin gene as an internal reference, and use PpDREB1D-like specific primers to detect the PpDREB1D-like gene expression level by qRT-PCR. Select the silenced strain and the control group to be treated at 8 degrees for 20 days. The results are shown in Figures 11-12 .

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

[0189] Figure 12The growth state of PpDREB1D-like silenced Miscanthus under cold stress is shown. PpDREB1D-like gene silenced plants show more sensitive phenotype to cold stress, showing wilting of leaves, and the ROS level in leaves is significantly increased, indicating that PpDREB1D-like gene improves the cold tolerance of Miscanthus by removing the ROS level induced by low temperature.

[0190] Finally, it should be noted that the terms "comprising", "including", or any other variant thereof are intended to cover a non-exclusive inclusion, such that processes, methods, articles, or apparatuses that comprise a list of elements are not limited to those elements, but can include other elements not expressly listed or inherent to such processes, methods, articles, or apparatuses.

[0191] Although the preferred embodiments of the present application have been described, those skilled in the art who have the benefit of the present disclosure will appreciate that additional modifications and changes can be made without departing from the scope of the present application. Accordingly, it is intended that all such modifications and changes be included within the scope of the claims.

[0192] The above-described embodiments only express the specific implementation of the present application, which is described in a more specific and detailed manner, but should not be understood as a limitation to the protection scope of the present application. It should be noted that for those skilled in the art, without departing from the concept of the technical scheme of the present application, a number of modifications and improvements can be made, which are all within the protection scope of the present application.

Claims

1. A Puccinellia peisoni PpDREBlD-like gene, characterized in that, The sequence of the PpDREB1D-like gene is shown as SEQ ID NO: 1, and the amino acid sequence encoded by the PpDREB1D-like gene is shown as SEQ ID NO.

2.

2. The PpDREBlD-like gene of Pennisetum purpureum according to claim 1, characterized in that, The PpDREB1D-like gene of the elephant grass is derived from the stem, leaf sheath or root of the elephant grass.

3. The PpDREBlD-like gene of Pennisetum purpureum according to claim 2, characterized in that, The PpDREB1D-like gene of the elephant grass can be obtained by using the sequences shown as SEQ ID NO. 3 and SEQ ID NO. 4 as primers for amplification reaction.

4. Use of the PpDREB1D-like gene of the elephant grass in the preparation of a product for improving the cold resistance and biomass of a plant.

5. The use according to claim 4, wherein the compound is ###0002### The plant is Arabidopsis thaliana or elephant grass.

6. Use according to claim 5, wherein The process for improving the cold resistance and biomass of a plant by using the PpDREB1D-like gene of the elephant grass comprises the following steps: (1) linking the PpDREB1D-like gene of the elephant grass to an expression vector to obtain a recombinant vector; (2) transforming the recombinant vector into a competent cell to obtain a recombinant cell; (3) using the recombinant cell to infiltrate a plant whose cold resistance is to be improved.

7. Use according to claim 6, wherein The process for preparing the recombinant vector comprises the following steps: (1) designing primers Homo-DREB1Df and Homo-DREB1Dr, the sequences of which are shown as SEQ ID NO. 9 and SEQ ID NO. 10, respectively; (2) using the primers Homo-DREB1Df and Homo-DREB1Dr to perform amplification reaction on the PpDREB1D-like gene to obtain PpDREB1D-like with vector homologous sequences; (3) performing enzyme digestion on the pCAMBIA1381-YFP vector to obtain a linearized vector; (4) linking the PpDREB1D-like gene to the linearized vector.

8. The use according to claim 6, wherein The competent cell is a competent strain of Agrobacterium tumefaciens strain GV3101.

9. An agent for improving cold tolerance and biomass of a plant, characterized by comprising a polynucleotide encoding a protein having an amino acid sequence of SEQ ID NO:

1. The recombinant vector or the recombinant cell prepared by using the PpDREB1D-like gene of the elephant grass.

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