Application of cold response PaIBH1 protein and coding gene thereof in enhancing cold stress resistance of plants

Abstract

The invention discloses a cold response PaIBH1 protein and application of a coding gene thereof in enhancing cold stress resistance of plants. The cold response PaIBH1 protein is a protein PaIBH1 with an amino acid sequence as shown in SEQ ID NO: 2. Experiments prove that the gene for coding the protein PaIBH1, namely the gene PaIBH1, is overexpressed in wild type arabidopsis thaliana, so that the cold resistance of the arabidopsis thaliana can be improved; the improvement of the cold resistance shows that after cold treatment, the fresh weight of the overground part is increased, the wilting rate is reduced, the leaf membrane damage and electrolyte leakage are reduced, the photosynthetic ability is improved, the membrane lipid peroxidation damage is relieved, the active oxygen scavenging ability is enhanced and / or the active oxygen accumulation in the plant body is reduced. Therefore, the protein PaIBH1 can improve the cold resistance of the plants. The method has a very wide application prospect in the aspect of cold-resistant genetic improvement of crops or pasture.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology breeding technology, specifically the application of the cold-response PaIBH1 protein and its encoding gene in enhancing plant resistance to cold stress. Background Technology

[0002] Temperature is a core environmental factor affecting plant metabolism, geographical distribution, and growth and development. In recent years, global climate change has led to an increasing frequency and intensity of extreme low-temperature events, making sudden low-temperature stress one of the most common abiotic stresses in agricultural production. Low-temperature stress not only causes qualitative and quantitative losses in plant yield and quality by interfering with cell membrane stability and inhibiting photosynthetic organ function, but also has a profound impact on regional agricultural economies. Therefore, screening and cultivating crops with high cold resistance is crucial for my country's food security.

[0003] Over a long period of evolution, plants have developed multi-layered low-temperature stress response systems, forming stress response mechanisms through dynamic reconstruction of molecular networks and regulation of cellular homeostasis. When plants encounter low-temperature stress, they sense temperature changes through plasma membrane cold receptors, triggering Ca2+... 2+ Signaling cascades activate transcriptional regulatory hubs, driving the transcriptome, proteome, and metabolome to respond to changes in environmental temperature. In this process, antifreeze proteins protect cell structural integrity by inhibiting ice crystal nucleation and repairing damaged proteins. Simultaneously, the metabolome lowers the cell freezing point by accumulating osmotic regulators such as proline and betaine, while the lipidome dynamically increases the proportion of unsaturated fatty acids in membrane lipids to maintain membrane fluidity. Furthermore, antioxidant enzyme systems such as superoxide dismutase and peroxidase, along with the ascorbate-glutathione cycle, synergistically scavenge excess ROS, preventing membrane lipid peroxidation damage. Studying the self-adaptation and resistance mechanisms of plants to low-temperature stress, and clarifying their molecular mechanisms, can lay an important molecular foundation for discovering and utilizing cold-resistant plant resources, and is a hot research direction for alleviating plant growth and development stagnation and yield reduction caused by low temperatures.

[0004] pampas grass ( Pennisetum alopecuroides (L.) Spreng. Napier grass (Ponchocarpa) is an important plant in the genus *Ponchocarpa* of the Poaceae family, renowned for its high biomass, rich nutritional value, good palatability, and wide adaptability. Currently, my country has 11 species and 2 varieties of *Ponchocarpa*, mainly distributed in fields and hillsides in Northeast, North, East, Central South, and Southwest provinces. However, due to cold winters and frequent late spring frosts in northern China, Napier grass faces difficulties in overwintering, resulting in low regrowth rates and even large-scale failures to regrow. This impacts the stability of the Napier grass production system, increasing the risk of declining forage quality and yield losses, and seriously affecting the healthy development of the domestic forage industry.

[0005] Based on genetic engineering breeding technology, studying the mechanisms of plant responses to low-temperature stress, identifying cold-response-related genes under low-temperature stress conditions, and applying them to crop breeding are economical and effective measures to enhance crop stress resistance and breed cold-resistant varieties. However, to date, research on the cloning, functional identification, and expression regulation of cold-response genes at the gene level is still relatively limited. Therefore, identifying cold-resistance-related genes, elucidating cold-resistance mechanisms, and providing new ideas and technical support for the efficient breeding of transgenic cold-resistant crops or forage varieties have significant application prospects. Summary of the Invention

[0006] The purpose of this invention is to improve the cold resistance of plants.

[0007] This invention first protects the protein PaIBH1 derived from *Pennisetum purpureum*. Specifically, protein PaIBH1 can be as follows: a1), a2), a3), or a4). a1) The amino acid sequence is that of the protein shown in SEQ ID NO:2; a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO:2; a3) Proteins derived from Napier grass and associated with cold hardiness, obtained by substituting and / or deleting and / or adding one or more amino acid residues of the proteins shown in a1) or a2). a4) A protein derived from Napier grass and associated with cold hardiness, which has 90% or more homology with the amino acid sequence defined by SEQ ID NO:2.

[0008] Of these, SEQ ID NO:2 consists of 195 amino acid residues.

[0009] To facilitate the purification of the protein in a1), a tag as shown in Table 1 can be attached to the amino or carboxyl terminus of the protein shown in SEQ ID NO: 2.

[0010]

[0011] The protein in a3) above, wherein the substitution and / or deletion and / or addition of one or more amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0012] The proteins mentioned in a3) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0013] The gene encoding the protein in a3) above can be obtained by deleting one or more amino acid residues from the codons in the DNA sequence shown in SEQ ID NO: 1, and / or by performing a missense mutation of one or more base pairs, and / or by attaching the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0014] Nucleic acid molecules encoding any of the proteins PaIBH1 described above are also within the scope of protection of this invention.

[0015] The nucleic acid molecule encoding any of the aforementioned proteins PaIBH1 can be a DNA molecule as shown in b1), b2), b3), or b4): b1) The coding region is the DNA molecule shown in SEQ ID NO:1; b2) The nucleotide sequence of the DNA molecule is shown in SEQ ID NO:1; b3) A DNA molecule that has 90% or more homology with the nucleotide sequence defined in b1) or b2) and is derived from Napier grass and encodes PaIBH1, any of the proteins described above; b4) Hybridizes under stringent conditions to the nucleotide sequence defined in b1) or b2) a DNA molecule derived from Napier grass and encoding any of the proteins described above, PaIBH1.

[0016] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0017] Of these, SEQ ID NO: 1 consists of 588 nucleotides, and the nucleotides shown in SEQ ID NO: 1 encode the amino acid sequence shown in SEQ ID NO: 2.

[0018] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein PaIBH1 of this invention using known methods, such as directed evolution and point mutation. Any artificially modified nucleotides that have 90% or higher identity with the nucleotide sequence of the protein PaIBH1 isolated according to this invention, as long as they encode the protein PaIBH1, are derived from and equivalent to the nucleotide sequence of this invention.

[0019] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 90% or higher, or 95% or higher, identity with the nucleotide sequence of the protein PaIBH1, which encodes the amino acid sequence shown in SEQ ID NO: 2 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0020] The above-mentioned foxtail grass can specifically refer to *Lysimachia clethroides*.

[0021] Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic cell lines containing any of the nucleic acid molecules described above are also within the scope of protection of this invention.

[0022] The expression cassette includes a promoter, a nucleic acid molecule encoding the protein PaIBH1, and a terminator. The promoter may be a CaMV35S promoter; the terminator may be a NOS terminator.

[0023] The recombinant vector can be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding any of the proteins PaIBH1 described above into an expression vector.

[0024] The recombinant vector may specifically be the recombinant plasmid pBI121-GUS- PaIBH1 The recombinant plasmid pBI121-GUS- PaIBH1 Specifically, a recombinant plasmid can be obtained by inserting a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 1 from position 1 to 585 from the 5' end into the recognition site of the restriction endonuclease XbaI of the pBI121-GUS vector.

[0025] The recombinant microorganism can be obtained by introducing any of the above-described recombinant vectors into the starting microorganism.

[0026] The starting microorganism may be yeast, bacteria, algae, or fungi. The bacteria may be Gram-positive or Gram-negative. The Gram-negative bacteria may be Agrobacterium tumefaciens. The Agrobacterium tumefaciens may be Agrobacterium tumefaciens GV3101.

[0027] The transgenic plant cell lines do not include propagation material. The transgenic plant is understood to include not only the first-generation transgenic plant obtained by transforming the recipient plant with the nucleic acid molecule encoding any of the aforementioned proteins PaIBH1, but also its progeny. For transgenic plants, the gene can be propagated within the species, or it can be transferred into other varieties of the same species using conventional breeding techniques, particularly commercial varieties. The transgenic plant includes seeds, callus tissue, intact plants, and cells.

[0028] This invention also protects the application of any of the above-described proteins PaIBH1, any of the above-described nucleic acid molecules, or expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic cell lines containing any of the above-described nucleic acid molecules, which may be c1) or c2). c1) Improve the cold resistance of plants; c2) Cultivate transgenic plants with improved cold resistance.

[0029] In the above applications, the plant may be any one of the following c1) to c8): c1) dicotyledonous plants; c2) monocotyledonous plants; c3) grasses; c4) Napier grass; c5) Napier grass lichenifolia; c6) cruciferous plants; c7) Arabidopsis thaliana; c8) wild-type Arabidopsis thaliana Col-0.

[0030] In the above applications, the improvement of plant cold resistance can be manifested as increased fresh weight of aboveground parts, reduced wilting rate, reduced cell membrane damage and electrolyte leakage, improved photosynthetic capacity, reduced membrane lipid peroxidation damage, enhanced reactive oxygen species scavenging capacity, and / or reduced accumulation of reactive oxygen species in the plant after cold treatment.

[0031] The present invention also protects a method for cultivating transgenic plants, which may include the following steps: increasing the expression level and / or activity of any of the proteins PaIBH1 described above in a recipient plant to obtain a transgenic plant; the transgenic plant exhibits improved cold resistance compared to the recipient plant.

[0032] In the above method, the expression level and / or activity of any of the above-mentioned proteins PaIBH1 in the recipient plant can be increased by methods well known in the art, such as transgenic technology, multiple copies, alteration of promoters, and regulatory factors, to achieve the effect of increasing the expression level and / or activity of any of the above-mentioned proteins PaIBH1 in the recipient plant.

[0033] In the above method, increasing the expression level and / or activity of any of the aforementioned proteins PaIBH1 in the recipient plant can be achieved by introducing a nucleic acid molecule encoding any of the aforementioned proteins PaIBH1 into the recipient plant.

[0034] In the above method, the introduction of a nucleic acid molecule encoding any of the aforementioned proteins PaIBH1 into the recipient plant can be achieved by introducing a recombinant vector into the recipient plant; the recombinant vector is a recombinant plasmid obtained by inserting a nucleic acid molecule encoding any of the aforementioned proteins PaIBH1 into an expression vector.

[0035] The recombinant vector may specifically be the recombinant plasmid pBI121-GUS- PaIBH1 The recombinant plasmid pBI121-GUS- PaIBH1 Specifically, a recombinant plasmid can be obtained by inserting a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 1 from position 1 to 585 from the 5' end into the recognition site of the restriction endonuclease XbaI of the pBI121-GUS vector.

[0036] The transgenic plants mentioned in the examples may specifically be OE-3, OE-13, OE-20, OE-24, OE-28, OE-30, and OE-31, especially OE-24 and OE-28; in this case, the recipient plant is Arabidopsis thaliana, specifically wild-type Arabidopsis thaliana (…). Arabidopsis thaliana (Columbia-0 subtype).

[0037] The present invention also protects a plant breeding method comprising the following steps: increasing the expression level and / or activity of any of the above-described proteins PaIBH1 in plants, thereby improving the cold resistance of plants.

[0038] In any of the methods described above, the plant may be any one of the following c1) to c8): c1) dicotyledonous plant; c2) monocotyledonous plant; c3) grass; c4) Pennisetum alopecuroides; c5) Pennisetum rubrum; c6) Brassicaceae plant; c7) Arabidopsis thaliana; c8) wild-type Arabidopsis thaliana Col-0.

[0039] In any of the methods described above, the improvement of plant cold resistance can be manifested as an increase in the fresh weight of the aboveground parts, a decrease in the wilting rate, a decrease in cell membrane damage and electrolyte leakage, an increase in photosynthetic capacity, a reduction in membrane lipid peroxidation damage, an enhancement in the ability to scavenge reactive oxygen species, and / or a decrease in the accumulation of reactive oxygen species in the plant after cold treatment.

[0040] The aforementioned cold treatment can be performed at 0–-10°C (e.g., 0–-5°C, -5–-10°C, 0°C, -5°C, or -10°C). The treatment time can be 1–14 hours (e.g., 1–2 hours, 2–4 hours, 4–6 hours, 6–8 hours, 8–10 hours, 10–12 hours, 12–14 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, or 14 hours). Any of the aforementioned cell membrane damage and reduced electrolyte leakage can manifest as a decrease in conductivity. Conductivity can specifically refer to relative conductivity.

[0041] The improvement in photosynthetic capacity mentioned above can be manifested as an increase in chlorophyll content and / or an increase in chlorophyll fluorescence parameters.

[0042] The reduction of membrane lipid peroxidation damage mentioned above can be manifested as an increase in soluble sugar content, an increase in proline content, and / or a decrease in malondialdehyde content.

[0043] The enhanced reactive oxygen species scavenging capacity described above can be manifested as increased peroxidase activity, superoxide dismutase activity, and / or catalase activity.

[0044] The decrease in reactive oxygen species accumulation in any of the above-mentioned plants can manifest as a decrease in H2O2 content and / or O2 content. 2- The content decreased.

[0045] Experiments have shown that overexpression of the gene encoding the protein PaIBH1 in wild-type Arabidopsis thaliana, namely... PaIBH1 The gene PaIBH1 can improve the cold tolerance of Arabidopsis thaliana. This improved cold tolerance is manifested in increased aboveground fresh weight, reduced wilting rate, decreased leaf membrane damage and electrolyte leakage, increased photosynthetic capacity, reduced membrane lipid peroxidation damage, enhanced reactive oxygen species (ROS) scavenging capacity, and / or reduced ROS accumulation within the plant after cold treatment. Therefore, the protein PaIBH1 can enhance plant cold tolerance. This invention has broad application prospects in the genetic improvement of cold tolerance in crops and forage grasses. Attached Figure Description

[0046] Figure 1 The result is a 1% agarose gel electrophoresis of the PCR amplification product in step 3 of Example 1.

[0047] Figure 2 Under cold stress treatment PaIBH1 Relative expression levels of genes in the roots, stems, and leaves of *Pennisetum affine*.

[0048] Figure 3 The recombinant plasmid pBI121-GUS- PaIBH1 A structural diagram.

[0049] Figure 4 The result is a 1% agarose gel electrophoresis of the PCR amplification product from step one of Example 3.

[0050] Figure 5 For step two of Example 3, T1 is replaced. PaIBH1 Gene-positive seedlings PaIBH1 Gene relative expression level detection and GUS histochemical staining.

[0051] Figure 6 For step three (a) of Example 3, the transfer PaIBH1Phenotypic analysis of Arabidopsis thaliana under cold stress treatment.

[0052] Figure 7 For step three (ii) of Example 3 PaIBH1 Results of chlorophyll content and chlorophyll fluorescence parameters in Arabidopsis thaliana.

[0053] Figure 8 For step three (ii) of Example 3 PaIBH1 Results of detection of malondialdehyde, soluble sugar and proline content in Arabidopsis thaliana.

[0054] Figure 9 For step three (ii) of Example 3 PaIBH1 Results of DAB and NBT staining of Arabidopsis thaliana, as well as detection results of hydrogen peroxide content and superoxide anion content.

[0055] Figure 10 For step three (ii) of Example 3 PaIBH1 Results of detection of peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) activities in Arabidopsis thaliana. Detailed Implementation

[0056] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0058] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0059] In the following examples, SPSS 11.5 statistical software was used to process the data. The experimental results are expressed as mean ± standard deviation, and one-way ANOVA was used. P <0.05 (*) indicates a significant difference. P <0.01 (**) indicates a highly significant difference, and ns indicates no significant difference.

[0060] 'Liqiu' Napier Grass, with the improved variety number S-BV-PA-007-2021, was approved by the Grass Variety Approval Committee of the National Forestry and Grassland Administration in October 2021. In the following examples, 'Liqiu' Napier Grass is referred to as Napier Grass.

[0061] Example 1 PaIBH1 Cloning of genes 1. Extract total RNA from Napier grass leaves; use the total RNA from Napier grass leaves as a template for reverse transcription to obtain cDNA from Napier grass leaves.

[0062] 2. After completing step 1, using cDNA from Napier grass leaves as a template, PCR amplification was performed using primer pair consisting of primer P1: 5'-ATGGCGTTCAAGCGTGACTT-3' (SEQ ID NO: 3) and primer P2: 5'-CTACAATTCTCCCATGCTTTGAG-3' (SEQ ID NO: 4) to obtain PCR amplification products.

[0063] Reaction conditions: 94℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 58℃ annealing for 15 s, 68℃ extension for 30 s, 35 cycles; 68℃ extension for 10 min.

[0064] 3. After completing step 2, perform PCR amplification products by 1% agarose gel electrophoresis.

[0065] Test results are shown Figure 1 (Marker is DNA Marker, 1, 2 and 3 are all PCR amplification products). The results show that step 2 yielded a 588bp PCR amplification product.

[0066] 4. After completing step 2, sequence the PCR amplification products obtained in step 2.

[0067] Sequencing results showed that the nucleotide sequence of the PCR amplification product obtained in step 2 is shown in SEQ ID NO: 1. This sequence is named... PaIBH1 Gene. PaIBH1 The gene encodes the protein PaIBH1, and the amino acid sequence of the protein PaIBH1 is shown in SEQ ID NO: 2.

[0068] Example 2: Cold stress treatment PaIBH1 Analysis of gene expression patterns in Napier grass 1. Take the roots, stems, and leaves of *Pennisetum purpureus* after being treated at -5℃ (i.e., cold stress) for 0 h (as a blank control), 2 h, 4 h, 6 h, or 8 h, respectively, and quickly place them in liquid nitrogen to obtain the test samples.

[0069] 2. Extract total RNA from the sample obtained in step 1. The integrity of the total RNA in the sample was assessed by 1.2% (w / v) agarose gel electrophoresis, and the RNA concentration was determined using a Quawell Q5000 micro-volume nucleic acid and protein analyzer.

[0070] Total RNA from test samples with good RNA integrity, an RNA 260nm / 280nm ratio between 1.9 and 2.1, and a 260nm / 230nm ratio greater than 2.0 was used for the next step of analysis.

[0071] 3. After completing step 2, collect the total RNA from the sample to be tested using PrimeScript. TM The RT reagent kit with gDNA Eraser (TaKaRa) was used for reverse transcription to obtain cDNA from the sample to be tested.

[0072] 4. After completing step 3, using the cDNA template of the sample to be tested, employ SYBR... ® The Premix Ex Taq II kit (TaKaRa) was used for real-time quantitative qRT-PCR on a Bio-Rad CFX96 instrument to detect... PaIBH1 Relative gene expression levels (in terms of) Actin (The gene was used as an internal reference gene). All experiments were performed in triplicate.

[0073] Detection PaIBH1 The primers for the gene are primer P3: 5'-CCATGAGCGTCGAGGAGAG-3' (SEQ ID NO: 5) and primer P4: 5'-TCCTCGTCATCCTCTTAGCG-3' (SEQ ID NO: 6).

[0074] Detection Actin The primers for the gene are primer P5: 5'-CTGAGCGGGAAATTGTGAGG-3' (SEQ ID NO: 7) and primer P6: 5'-CATGGATGGCTGGAAGAGGA-3' (SEQ ID NO: 8).

[0075] Test results are shown Figure 2 (CK was the blank control). The results showed that low-temperature treatment... PaIBH1 The gene was induced to be upregulated in roots, stems, and leaves, and PaIBH1 Gene expression patterns differ significantly across different tissue sites. In root and stem tissues... PaIBH1 The relative gene expression level gradually increased with the extension of low-temperature treatment time, reaching peak values ​​at 6 h and 4 h, respectively, while the leaf expression level peaked at 8 h. This indicates that... PaIBH1The gene is significantly induced and regulated by cold stress, and it actively participates in the low-temperature stress response of Napier grass.

[0076] Example 3, Transfer PaIBH1 Obtaining and identifying the cold resistance of Arabidopsis thaliana gene I. Recombinant plasmid pBI121-GUS- PaIBH1 Construction 1. Extract total RNA from Napier grass leaves; use the total RNA from Napier grass leaves as a template for reverse transcription to obtain cDNA from Napier grass leaves.

[0077] 2. After completing step 1, using cDNA from Napier grass leaves as a template, PCR amplification was performed using primer pair consisting of primer P1: 5'-ATGGCGTTCAAGCGTGACTT-3' (SEQ ID NO: 3) and primer P2: 5'-CTACAATTCTCCCATGCTTTGAG-3' (SEQ ID NO: 4) to obtain PCR amplification product 1.

[0078] 3. After completing step 2, using PCR amplification product 1 as a template, and employing primer P7: 5'-AGAACACGGGGGAC TCTAG A ATGGCGTTCAAGCGTGACTT-3' (SEQ ID NO: 9; underlined is the recognition site of restriction endonuclease XbaI) and primer P8: 5'-CCACCCGGGGATCC TCTAGA The primer pair consisting of CAATTCTCCCATGCTTTGAG-3' (SEQ ID NO: 10; the underline is the recognition site of restriction endonuclease XbaI) and high-fidelity PrimeSTAR®Max DNA Polymerase were used for PCR amplification to obtain PCR amplification product 2.

[0079] 4. After completing step 3, recover the PCR amplification product 2 using a standard agarose gel DNA recovery kit (purchased from Tiangen Biotech Co., Ltd., catalog number DP209).

[0080] 5. After completing step 4, digest the pBI121-GUS vector (purchased from Changsha Youbao Biotechnology Co., Ltd., catalog number VT1388) with the restriction endonuclease XbaI and recover the vector backbone.

[0081] 6. The PCR amplification product 2 recovered in step 4 and the vector backbone recovered in step 5 were seamlessly ligated using the In-Fusion HD Cloning kit (purchased from TaKaRa, catalog number 639649) to obtain the recombinant plasmid pBI121-GUS- PaIBH1 .

[0082] Recombinant plasmid pBI121-GUS- PaIBH1 See the structural diagram. Figure 3 .

[0083] 7. The recombinant plasmid pBI121-GUS- obtained in step 6... PaIBH1 Transformed Escherichia coli competent cells (purchased from Beijing TransGen Biotech Co., Ltd., catalog number CD501-02), normally growing Escherichia coli colonies were picked and inoculated into LB liquid medium (purchased from Beijing Coollab Technology Co., Ltd., catalog number PM0010), and incubated overnight on a shaker at 37°C.

[0084] 8. After completing step 7, use primers P1 and P2 to perform PCR amplification on the overnight cultured E. coli solution from step 7. The PCR amplification product size is 588 bp (see...). Figure 4 The sample was then sent to Beijing Sangon Biotech for sequencing. The results showed that the nucleotide sequence of the PCR amplification product was as shown in SEQ ID NO: 1.

[0085] The above results indicate that the recombinant plasmid pBI121-GUS- PaIBH1 A recombinant plasmid was obtained by inserting a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 1 from position 1 to 585 from the 5' end into the recognition site of the restriction endonuclease XbaI of the pBI121-GUS vector.

[0086] II. Transfer PaIBH1 Obtaining the gene from Arabidopsis thaliana Wild-type Arabidopsis thaliana ( Arabidopsis thaliana (Columbia-0 subtype) was purchased from Beijing Huayueyang Company, product number NRR00220. In the following text, wild-type Arabidopsis thaliana (… Arabidopsis thaliana (Columbia-0 subtype) is also known as wild-type Arabidopsis thaliana.

[0087] 1. The recombinant plasmid pBI121-GUS- PaIBH1 Agrobacterium tumefaciens GV3101 (purchased from Beijing Huayueyang Co., Ltd., catalog number NRR01020) was introduced to obtain recombinant Agrobacterium, named GV3101 / pBI121-GUS- PaIBH1 .

[0088] 2. The Arabidopsis thaliana inflorescence dipping transformation method was adopted (described in the following literature: Clough, SJ, and Bent, AF. Floraldip: as simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. (1998) 16, 735-743.), and GV3101 / pBI121-GUS- PaIBH1 Transplanted into wild-type Arabidopsis thaliana, resulting in T0 generation transgenic strains. PaIBH1 Genetically modified Arabidopsis thaliana seeds.

[0089] 3. Transfer the T0 obtained in step 2. PaIBH1 Arabidopsis thaliana seeds were sown on MS solid medium containing 50 mg / L kanamycin. Arabidopsis thaliana seedlings that grew normally (resistant seedlings) were identified as T1 generation transgenic plants. PaIBH1 Gene-positive seedlings.

[0090] A total of T1 generation transfers were obtained PaIBH1 Seven gene-positive seedlings were named OE-3, OE-13, OE-20, OE-24, OE-28, OE-30 and OE-31, respectively.

[0091] 4. Total RNA was extracted from the leaves of the plants to be tested (wild-type Arabidopsis, OE-3, OE-13, OE-20, OE-24, OE-28, OE-30, or OE-31), and then PrimeScript was used. TM Reverse transcription was performed using the RT reagent kit with gDNA Eraser (TaKaRa) to obtain cDNA from the leaves of the plants to be tested. Using the cDNA from the leaves as a template, SYBR Green was employed. ® The Premix Ex Taq II kit (TaKaRa) was used for real-time quantitative qRT-PCR on a Bio-Rad CFX96 instrument to detect... PaIBH1 Relative gene expression levels (in terms of) AtActin (The gene was used as an internal reference gene). All experiments included three biological replicates.

[0092] Detection PaIBH1 The primers for the gene are primer P3 and primer P4.

[0093] Detection AtActinThe primers for the gene are primer P9: 5'-AGTCCACCCTTCATCTTGTTCTC-3' (SEQ ID NO: 11) and primer P10: 5'-GTCAGCCAAAGTTCTTCCATCT-3' (SEQ ID NO: 12).

[0094] Test results are shown Figure 5 Middle A (WT is wild-type Arabidopsis thaliana).

[0095] 5. Take leaves from the plants to be tested (wild-type Arabidopsis thaliana, OE-3, OE-13, OE-20, OE-24, OE-28, OE-30 or OE-31) and perform GUS histochemical staining.

[0096] Test results are shown Figure 5 Middle B (WT is wild-type Arabidopsis thaliana).

[0097] The results showed that, compared with wild-type Arabidopsis thaliana, 7 T1 generation transgenic strains... PaIBH1 Gene-positive seedlings (i.e., OE-3, OE-13, OE-20, OE-24, OE-28, OE-30, and OE-31) PaIBH1 The relative expression levels of all genes were significantly increased, and GUS histochemical staining showed a blue color; particularly in OE-24 and OE-28. PaIBH1 The gene has the highest relative expression level, and the GUS histochemical staining is the darkest, which is used for subsequent experiments.

[0098] T1 transfer of OE-24 and OE-28 PaIBH1 Seeds received from gene-positive seedlings are OE-24 and OE-28 T2 generation transgenic seeds. PaIBH1 Genetically modified Arabidopsis thaliana seeds.

[0099] 6. Replace OE-24 and OE-28 T2 PaIBH1 The seeds of the gene-modified Arabidopsis thaliana were sown on MS solid medium containing 50 mg / L kanamycin for screening. The seedlings that were resistant were identified as homozygous lines, namely OE-24 homozygous line and OE-28 homozygous line.

[0100] The seeds received from the OE-24 homozygous and OE-28 homozygous lines were respectively OE-24 T3 generation homozygous transgenic strains. PaIBH1 Arabidopsis thaliana seeds and OE-28 T3 generation homozygous transgenic genes PaIBH1 Genetically modified Arabidopsis thaliana seeds.

[0101] III. Transfer PaIBH1 Cold resistance identification of Arabidopsis thaliana (a) Turn PaIBH1 Phenotypic analysis of Arabidopsis thaliana under cold stress All experiments were performed in triplicate and the average value was taken.

[0102] 1. Treat 4-week-old plants (wild-type Arabidopsis thaliana, OE-24 or OE-28) with low temperature or room temperature.

[0103] The low-temperature treatment was as follows: First, pre-cooled at 4℃ for 24 h, then lowered to -5℃ at a rate of 1℃ / h and maintained for 12 h, and finally restored to culture at 22℃ with alternating light and dark (16 h of light treatment and 8 h of dark treatment) for 72 h.

[0104] The specific treatment at room temperature was as follows: cultured in a 22℃ artificial culture room with alternating light and dark conditions (16 h of light treatment followed by 8 h of dark treatment). The treatment time at room temperature was the same as that at low temperature.

[0105] 2. After completing step 1, observe the phenotype of the plants to be tested, and count the fresh weight of the aboveground parts and the wilting rate of the plants to be tested.

[0106] Phenotypic observation results are shown in Figure 6 (A) (Control refers to room temperature treatment, Cold stress refers to low temperature treatment). Results showed that after low temperature treatment, wild-type Arabidopsis plants exhibited severe leaf wilting, while transformed... PaIBH1 The wilting of leaves in the genetically modified Arabidopsis thaliana (OE-24 and OE-28) plants was less severe compared to that in wild-type Arabidopsis thaliana plants.

[0107] The statistical results of the aboveground fresh weight of the tested plants are shown in the figure. Figure 6 (B) The results showed that after treatment at room temperature, wild-type Arabidopsis and the transformed [various species]... PaIBH1 There was no significant difference in the fresh weight of the aboveground parts of the transgenic Arabidopsis thaliana (OE-24 and OE-28); after low-temperature treatment, compared with wild-type Arabidopsis thaliana, the transgenic... PaIBH1 The aboveground fresh weight of the gene-modified Arabidopsis (OE-24 and OE-28) was significantly increased.

[0108] The statistical results of the wilting rate of the tested plants are shown in the figure. Figure 6 (C). The results showed that, compared with wild-type Arabidopsis, the modified... PaIBH1 The wilting rate of the gene-modified Arabidopsis thaliana (OE-24 and OE-28) was significantly reduced.

[0109] 3. After completing step 1, take a glass tube, place a 1g leaf of the plant to be tested and 10 mL of deionized water inside, and shake at 25℃ and 180 rpm for 30 min. Measure the initial conductivity (C1) using a conductivity meter (DDBJ-350). Then boil the glass tube for 30 minutes, cool it to 25℃, and measure the final conductivity (C2). Take deionized water at 25℃ and measure its conductivity (C0) as a blank control. Finally, calculate the relative conductivity of the leaves of the plant to be tested, i.e., the electrolyte leakage rate REL. REL = (C1 - C0) / (C2 - C0) × 100%.

[0110] Relative conductivity reflects cell membrane damage and electrolyte leakage; a higher value indicates more severe damage. Measurement results for relative conductivity are shown in [link to measurement]. Figure 6 (D). The results showed that after treatment at room temperature, wild-type Arabidopsis and transgenic Arabidopsis... PaIBH1 There was no significant difference in the relative electrical conductivity between the transgenic Arabidopsis thaliana (OE-24 and OE-28); after low-temperature treatment, compared with wild-type Arabidopsis thaliana, the transgenic Arabidopsis thaliana showed significantly higher electrical conductivity. PaIBH1 The relative electrical conductivity of the gene-derived Arabidopsis thaliana (OE-24 and OE-28) was significantly reduced.

[0111] Therefore, it can be seen that... PaIBH1 Genetically modified Arabidopsis thaliana (OE-24 and OE-28) showed significantly better electrolyte leakage and cell membrane damage than wild-type Arabidopsis thaliana, i.e., transgenic Arabidopsis thaliana... PaIBH1 The genetically modified Arabidopsis thaliana exhibits greater resistance to cold stress, i.e., cold tolerance.

[0112] (II) Transfer PaIBH1 Analysis of physiological and biochemical indicators of Arabidopsis thaliana under cold stress All experiments were performed in triplicate and the average value was taken.

[0113] 1. Four-week-old plants (wild-type Arabidopsis thaliana, OE-24, or OE-28) were subjected to low-temperature treatment or room-temperature treatment. The chlorophyll content of the leaves of the plants was then determined by ethanol extraction, and the chlorophyll fluorescence parameter (Fv / Fm) was measured using a plant efficiency analyzer (HandyPEA+, PP SYSTEMS, UK).

[0114] The low-temperature treatment was as follows: First, pre-cooled at 4℃ for 24 h, then lowered to -5℃ at a rate of 1℃ / h and maintained for 12 h, and finally restored to culture at 22℃ with alternating light and dark (16 h of light treatment and 8 h of dark treatment) for 72 h.

[0115] The specific room temperature treatment was as follows: culturing at 22℃ with alternating light and dark conditions (16 h of light treatment followed by 8 h of dark treatment). The room temperature treatment time was the same as the low temperature treatment time.

[0116] Test results are shown Figure 7The results showed that after treatment at room temperature, wild-type Arabidopsis and the transformed [various species]... PaIBH1 There were no significant differences in chlorophyll content and chlorophyll fluorescence parameters between the transgenic Arabidopsis thaliana (OE-24 and OE-28); after low-temperature treatment, compared with wild-type Arabidopsis thaliana, the transgenic Arabidopsis thaliana showed... PaIBH1 The chlorophyll content and chlorophyll fluorescence parameters of Arabidopsis thaliana genes OE-24 and OE-28 were significantly increased. This indicates that overexpression of the protein PaIBH1 in Arabidopsis thaliana can improve photosynthetic capacity under cold stress.

[0117] 2. Four-week-old test plants (wild-type Arabidopsis thaliana, OE-24, or OE-28) were subjected to low-temperature treatment or room-temperature treatment. Subsequently, the malondialdehyde (MDA), soluble sugar (SS), and proline (Pro) content in the leaves of the test plants were detected using a kit from Suzhou Keming Biotechnology Co., Ltd. The degree of plant membrane lipid peroxidation can be reflected by key physiological indicators such as MDA and Pro.

[0118] The low-temperature treatment is as follows: First, pre-cool at 4℃ for 24 h, then lower to -5℃ at a rate of 1℃ / h and maintain for 12 h.

[0119] The specific room temperature treatment was as follows: culturing at 22℃ with alternating light and dark conditions (16 h of light treatment followed by 8 h of dark treatment). The room temperature treatment time was the same as the low temperature treatment time.

[0120] Test results are shown Figure 8 The results showed that after treatment at room temperature, wild-type Arabidopsis and the transformed [various species]... PaIBH1 There were no significant differences in MDA, SS, and Pro contents between the transgenic Arabidopsis thaliana (OE-24 and OE-28); after low-temperature treatment, compared with wild-type Arabidopsis thaliana, the transgenic Arabidopsis thaliana showed higher levels of MDA, SS, and Pro. PaIBH1 The contents of SS and Pro in Arabidopsis thaliana (OE-24 and OE-28) were significantly increased, while the contents of MDA were significantly decreased. This indicates that overexpression of the protein PaIBH1 in Arabidopsis thaliana can alleviate membrane lipid peroxidation damage caused by low-temperature stress.

[0121] 3. Four-week-old plants (wild-type Arabidopsis thaliana, OE-24, or OE-28) were subjected to low-temperature or room-temperature treatment. Leaves were then collected and soaked in diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining solutions for 12 h, respectively. Afterward, the leaves were transferred to 10 mL of anhydrous ethanol for decolorization. Once decolorization was complete, the leaves were rinsed three times with distilled water. The leaf color was observed to determine the accumulation of hydrogen peroxide and superoxide anions, and photographs were taken.

[0122] The low-temperature treatment is as follows: First, pre-cool at 4℃ for 24 hours, then lower the temperature to -5℃ at a rate of 1℃ / h and maintain it for 0h, 2h, 4h or 6h.

[0123] The specific room temperature treatment was as follows: culturing at 22℃ with alternating light and dark conditions (16 h of light treatment followed by 8 h of dark treatment). The room temperature treatment time was the same as the low temperature treatment time.

[0124] Histochemical staining (DAB and NBT staining) is used to detect the accumulation of reactive oxygen species in cells, and can visually display the levels of H2O2 and O2 in leaves. 2- The accumulation status. DAB and NBT staining results are shown in [the table / reference needed]. Figure 9 (A) and (B). The results show that after treatment at room temperature, the conversion... PaIBH1 The staining of gene-modified Arabidopsis thaliana (OE-24 and OE-28) was significantly reduced compared to wild-type Arabidopsis thaliana; after low-temperature treatment, the staining of wild-type Arabidopsis thaliana and the gene-modified Arabidopsis thaliana was significantly reduced. PaIBH1 Leaves of both gene-modified Arabidopsis (OE-24 and OE-28) showed increased staining, with wild-type Arabidopsis exhibiting extensive brown (H2O2) and dark blue (O2O2) staining. 2- ) spots, and turn PaIBH1 The number of staining spots in Arabidopsis thaliana (OE-24 and OE-28) was significantly lower than that in wild-type Arabidopsis thaliana. This indicates that overexpression of the protein PaIBH1 in Arabidopsis thaliana can reduce the content of reactive oxygen species.

[0125] Leaves were collected from the test plants after 6 h of low-temperature treatment (i.e., decreasing to -5℃ at a rate of 1℃ / h and maintaining for 6 h) or treatment at room temperature for the same period. The content of hydrogen peroxide (H2O2) and superoxide anion (O2) in the leaves were measured. 2- The content of [unspecified substance], peroxidase activity (POD), superoxide dismutase activity (SOD), and catalase activity (CAT) were measured. t -The test analyzed the significance of differences between different treatments. Hydrogen peroxide (H2O2) content, superoxide anion (O2) content... 2- The test results for the content of ) are shown in the following figures. Figure 9 (C) and (D). The results show that after room temperature treatment and low temperature treatment, the conversion... PaIBH1 H2O2 content and O2 content in gene-modified Arabidopsis thaliana (OE-24 and OE-28) 2- The contents were all significantly lower than those of wild-type Arabidopsis. This indicates that the H2O2 content is related to O2 content. 2- The trend of content change is consistent with the results of histochemical staining.

[0126] Oxidative damage caused by reactive oxygen species (ROS) in plants can be cleared by various antioxidant enzymes (SOD, POD, CAT). The results of peroxidase activity (POD), superoxide dismutase activity (SOD), and catalase activity (CAT) are shown below. Figure 10 The results showed that after treatment at room temperature, the SOD and POD activities in wild-type Arabidopsis leaves were significantly lower than those in the transgenic variety. PaIBH1Genetically modified Arabidopsis thaliana (OE-24 and OE-28) (except for the POD activity of OE-24); after low-temperature treatment, compared with wild-type Arabidopsis thaliana, the genetically modified Arabidopsis thaliana... PaIBH1 The SOD, POD, and CAT activities of the Arabidopsis genes (OE-24 and OE-28) were significantly increased (except for the CAT activity of OE-28). This indicates that the protein PaIBH1 enhances the cold resistance of plants by increasing the activity of antioxidant enzymes, thereby inhibiting excessive ROS accumulation and mitigating membrane lipid peroxidation damage.

[0127] Therefore, PaIBH1 Genes can enhance plant cold resistance. This can be achieved through overexpression. PaIBH1 The gene can also be used to develop cold-resistant varieties of plants including cabbage, rice, wheat, soybeans, and corn.

[0128] The above results fully demonstrate that PaIBH1 Genes can serve as candidate genes for the genetic improvement of cold resistance in crops or forage grasses, and have broad application prospects.

[0129] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Protein PaIBH1, which may be a1), a2), a3), or a4 as follows: a1) The amino acid sequence is that of the protein shown in SEQ ID NO:2; a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO:2; a3) Proteins derived from Napier grass and associated with cold hardiness, obtained by substituting and / or deleting and / or adding one or more amino acid residues of the proteins shown in a1) or a2). a4) A protein derived from Napier grass and associated with cold hardiness, which has 90% or more homology with the amino acid sequence defined by SEQ ID NO:

2.

2. A nucleic acid molecule encoding the protein PaIBH1 of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that: The nucleic acid molecule is the DNA molecule shown in b1), b2), b3), or b4): b1) The coding region is the DNA molecule shown in SEQ ID NO:1; b2) The nucleotide sequence of the DNA molecule is shown in SEQ ID NO:1; b3) Having 90% or more homology with the nucleotide sequence defined in b1) or b2), and being a DNA molecule derived from Napier grass and encoding the protein PaIBH1 of claim 1; b4) Hybridizes under stringent conditions to the nucleotide sequence defined in b1) or b2) a DNA molecule derived from Napier grass and encoding the protein PaIBH1 of claim 1.

4. An expression cassette, recombinant vector, recombinant microorganism, or transgenic cell line containing the nucleic acid molecule described in claim 2 or 3.

5. The application of the protein PaIBH1 of claim 1, the nucleic acid molecule of claim 2 or 3, or an expression cassette, recombinant vector, recombinant microorganism, or transgenic cell line containing the nucleic acid molecule of claim 2 or 3, is c1) or c2). c1) Improve the cold resistance of plants; c2) Cultivate transgenic plants with improved cold resistance.

6. A method for cultivating transgenic plants, comprising the following steps: increasing the expression level and / or activity of the protein PaIBH1 of claim 1 in a recipient plant to obtain a transgenic plant; the transgenic plant exhibits improved cold resistance compared to the recipient plant.

7. The method according to claim 6, characterized in that: The improvement in the expression level and / or activity of the protein PaIBH1 of claim 1 in the recipient plant is achieved by introducing a nucleic acid molecule encoding the protein PaIBH1 into the recipient plant.

8. A plant breeding method comprising the following steps: increasing the expression level and / or activity of the protein PaIBH1 of claim 1 in a plant, thereby improving the plant's cold resistance.

9. The application according to claim 5 or the method according to any one of claims 6 to 8, characterized in that: The plant is any one of the following c1) to c8): c1) dicotyledonous plants; c2) monocotyledonous plants; c3) grasses; c4) Napier grass; c5) Napier grass lichenifolia; c6) cruciferous plants; c7) Arabidopsis thaliana; c8) wild-type Arabidopsis thaliana Col-0.

10. The application according to claim 5 or the method according to any one of claims 6 to 8, characterized in that: The improvement in plant cold resistance is manifested in the following ways after cold treatment: increased fresh weight of aboveground parts, reduced wilting rate, reduced cell membrane damage and electrolyte leakage, improved photosynthetic capacity, reduced membrane lipid peroxidation damage, enhanced reactive oxygen species scavenging capacity, and / or reduced accumulation of reactive oxygen species in the plant.

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