Chilling tolerance related gene osgh29, protein encoded by the same and application thereof

Abstract

The application discloses a cold tolerance related gene OsGH29, a coding protein thereof and application thereof. The application solves the technical problem of how to regulate the cold tolerance of plants, especially how to improve the cold tolerance of plants. Specifically disclosed are the application of a protein, a substance for regulating the expression of a coding gene of the protein or a substance for regulating the activity or content of the protein in any one of the following: A1) application in regulating the cold tolerance of plants and / or application in preparing a product for regulating the cold tolerance of plants; A2) application in plant breeding and / or application in preparing a product for plant breeding; the protein is shown in sequence 2. The cold tolerance of plants can be improved by the method of the application, and a basis is provided for crop cold tolerance breeding.

Description

Technical Field

[0001] This invention relates to the cold-resistance-related gene OsGH29, its encoded protein, and its applications. Background Technology

[0002] Chilling injury, a common abiotic stress in rice production, seriously affects the stability and development of rice production. Therefore, solving the problem of chilling injury in rice is of great practical significance for global food security and promoting economic development in rice-producing areas. Studying the mechanisms of rice cold tolerance and breeding cold-resistant rice varieties are fundamental ways to solve the problem of low-temperature chilling injury in rice, while cloning and identifying cold-resistance-related genes in rice can lay a solid foundation for cold-resistance breeding. How to provide a gene that can be used to genetically modify cold-resistance-related genes to increase the cold tolerance of rice is a problem faced by researchers in this field. Summary of the Invention

[0003] The technical problem solved by this invention is how to regulate the cold tolerance of plants, especially rice.

[0004] To address the above problems, the present invention provides the following applications.

[0005] The use of a protein, a substance that regulates the expression of the gene encoding the protein, or a substance that regulates the activity or content of the protein in any of the following;

[0006] A1) Applications in regulating plant cold tolerance and / or applications in the preparation of products that regulate plant cold tolerance;

[0007] A2) Applications in plant breeding and / or applications in the preparation of plant breeding products;

[0008] The protein is any one of the following:

[0009] B1) The amino acid sequence of the protein is shown in sequence 2;

[0010] B2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in B1) and having the same function as the protein shown in B1).

[0011] B3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1) or B2).

[0012] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0013] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Perresidue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0014] In the aforementioned proteins, the 80% or more identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0015] Of the proteins described above, sequence 2 (SEQ ID No. 2) consists of 494 amino acid residues. It is named the OsGH29 protein. Its encoding gene is the OsGH29 gene.

[0016] Sequence 2 is as follows:

[0017] MMGRCASPLCALAAALIAAVVAATSSSLAEATATPPLPVLPVPTAAQLRWQRREVIMFFHFGMNTFTDSEWGTGREPPAAFRPAALDASQWMDAAAAAGASLVVLVAKHHDGFCLWPSAHTAH SVRASPWRGGRGDVVREFADAARARGLDIGIYLSPWDRHDKRYGREVAYNEYYLAQLHELLTGYGSVSEIWFDGAKGKNATNNMTYHFQEWFQTVRQLQSSINIFSDDGPDLRWVGDENGSAGST CWSTINRSKITIGEAGIEKYLNTGDPRGKDWVPPECDVSIRPGWFWHKNETAKPLPELLEVYYNSVGRNCVLLLNAPPNTTGLVDAADIARLREFRTAVTAIFGTDLAAGSAARASSERGGRFA AANVLDGRDDTYWAPAAAEAEDGGGYWIELRRPASAAARKFNVVRIQEHVAMGQRVERHEVYVDGGGAAVASGTTVGHKRLHRLGAPVAGRTVRVWLASRRGPPLLSAVGLHLDPFAAGGGTM.

[0018] In this application, the purpose of the breeding includes cultivating plants with high cold resistance. The purpose of the breeding also includes cultivating plants with low cold resistance.

[0019] The highly cold-resistant plants exhibit increased cold resistance compared to the target plants. The less cold-resistant plants exhibit decreased cold resistance compared to the target plants.

[0020] In the above text, the regulation can be increased, strengthened, or enhanced. The regulation can also be decreased, weakened, or reduced.

[0021] Substances that upregulate, enhance, or increase the expression of the gene encoding the protein, or substances that regulate the activity or content of the protein, can upregulate, enhance, or increase plant cold tolerance.

[0022] Substances that downregulate, weaken, or reduce the expression of the gene encoding the protein, or substances that regulate the activity or content of the protein, can downregulate, weaken, or reduce plant cold tolerance.

[0023] In the above text, the cold resistance mentioned can refer to improving the seedling survival rate. The seedling survival rate can refer to the budding stage seedling survival rate.

[0024] The cold tolerance mentioned above can also refer to enhanced seedling growth. Seedling growth can be measured by plant height and / or fresh weight of the crown per plant.

[0025] In the above applications, the protein is derived from rice.

[0026] The rice mentioned above may be the rice infiltration line SIL208 (Qin Jingjing, Identification and Functional Analysis of Cold-Tolerant QTL qSCT9 in Seedlings of Common Wild Rice in Dongxiang, Jiangxi, Master's Thesis, 2014, available to the public from China Agricultural University).

[0027] In the above text, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcriptional level of the gene; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0028] In the above-described uses, the substance regulating the expression of the protein-coding gene is any one of the following:

[0029] D1) Nucleic acid molecules encoding the above proteins;

[0030] D2), an expression cassette containing the nucleic acid molecules described in D1);

[0031] D3), ​​a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0032] D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3);

[0033] D5) A transgenic plant cell line containing the nucleic acid molecule described in D1), or a transgenic plant cell line containing the expression cassette described in D2), or a transgenic plant cell line containing the recombinant vector described in D3);

[0034] D6) Transgenic plant tissue containing the nucleic acid molecules described in D1), or transgenic plant tissue containing the expression cassette described in D2), or transgenic plant tissue containing the recombinant vector described in D3);

[0035] D7) A transgenic plant organ containing the nucleic acid molecule described in D1), or a transgenic plant organ containing the expression cassette described in D2), or a transgenic plant organ containing the recombinant vector described in D3).

[0036] In the nucleic acid molecule described in D1), those skilled in the art can easily mutate the nucleotide sequence encoding the protein OsGH29 of the present invention using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides that have 80% or more of the same nucleotide sequence as the protein OsGH29 isolated in the present invention, as long as they encode and function as protein OsGH29, are all derived from and equivalent to the nucleotide sequence of the present invention.

[0037] The aforementioned 80% or higher identity can be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0038] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Perresidue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, a search can be performed to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0039] In this document, the vectors described are known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specifically, it may be the pWMB110 vector;

[0040] In the aforementioned biological materials, the expression cassette described in D2) refers to DNA capable of expressing the gene in a host cell. This DNA may include not only promoters that initiate gene transcription but also terminators that terminate gene transcription. Furthermore, the expression cassette may also include enhancer sequences. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); chemically inducible promoters from tobacco, pathogenesis-related (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato protease inhibitor II promoter (PIN2) or LAP promoter (both can be induced by jasmonic acid methyl ester); heat shock promoter (US Patent 5,187,267); tetracycline inducible promoter (US Patent 5,057,422); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 200710099169.7)), seed storage protein-specific promoters (e.g., promoters of beta-conglycin, napin, oleosin and soybean beta-conglycin (Beachy et al. (1985) EMBOJ.4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited here are cited in full. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminators (see, for example: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627.

[0041] In D3) above, the recombinant vector can be a recombinant expression vector containing the gene expression cassette constructed using a plant expression vector. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, pMDC85, pCAMBIA1391-Xb, pTCK303 / JL1460, or Super1300. When constructing a recombinant expression vector using OsSOAR1, any enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin gene Ubiqutin promoter (pUbi), etc., which can be used alone or in combination with other plant promoters. Furthermore, when constructing a plant expression vector using the gene of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. As a specific embodiment, this application uses pTCK303 or Super1300 as the expression vector.

[0042] As a specific embodiment, the microbial strain in the recombinant microorganism may be Agrobacterium EHA105.

[0043] In the above-mentioned uses, the nucleic acid molecule described in D1) is a nucleotide sequence, which is the DNA molecule shown in Sequence 1.

[0044] In the above-mentioned uses, the plant is any one of the following:

[0045] G1) Monocotyledons;

[0046] G2) Gramineae plants;

[0047] G3) Plants of the genus Oryza;

[0048] G4) Rice.

[0049] Sequence 1 is as follows:

[0050]

[0051] To address the aforementioned problems, the present invention also provides a method for cultivating highly cold-resistant plants.

[0052] The method includes upregulating or enhancing or increasing the expression level of the gene encoding the aforementioned protein in the target plant, and / or, the activity and / or content of the protein to obtain a highly cold-resistant plant, wherein the cold resistance of the highly cold-resistant plant is higher than that of the target plant.

[0053] To address the aforementioned problems, this application also provides a method for cultivating plants with low cold tolerance.

[0054] The method includes downregulating or weakening or reducing the expression level of the gene encoding the aforementioned protein in the target plant, and / or, the activity and / or content of the protein to obtain a plant with low cold tolerance, wherein the cold tolerance of the plant with low cold tolerance is lower than that of the target plant.

[0055] To address the aforementioned problems, the present invention also provides a method for improving the cold tolerance of plants.

[0056] The method includes improving plant cold tolerance by upregulating or enhancing or increasing the expression of genes encoding the aforementioned proteins in plants, and / or the activity and / or content of the aforementioned proteins.

[0057] To address the aforementioned problems, this application also provides a method for reducing the cold tolerance of plants.

[0058] The method includes downregulating or weakening or reducing plant cold tolerance by downregulating or reducing the expression of genes encoding the aforementioned proteins in plants, and / or, the activity and / or content of the aforementioned proteins.

[0059] In this application, the plant may be rice. The rice may be the rice infiltration line SIL208 (Qin Jingjing, Identification and Functional Analysis of Cold-Tolerant QTL qSCT9 in Seedling Stage of Common Wild Rice in Dongxiang, Jiangxi Province, Master's Thesis, 2014, publicly available from China Agricultural University).

[0060] In the above method, upregulating or enhancing or increasing the expression of the gene encoding the protein in the plant includes introducing the above-mentioned nucleic acid molecule, expression cassette or recombinant vector into the target plant.

[0061] In the above text, the nucleic acid molecule may be the nucleic acid molecule described in Sequence 1.

[0062] In the above method, the plant is any one of the following:

[0063] J1) Monocotyledons;

[0064] J2) Gramineae plants;

[0065] J3) Plants of the genus *Oryza*;

[0066] J4) Rice.

[0067] In this application, the rice may be the rice infiltration line SIL208 (Qin Jingjing, Identification and Functional Analysis of Cold-Tolerant QTL qSCT9 in Seedlings of Common Wild Rice in Dongxiang, Jiangxi, Master's Thesis, 2014, available to the public from China Agricultural University).

[0068] The aforementioned proteins or substances.

[0069] Beneficial effects

[0070] This experiment, combining QTL mapping and gene chip analysis, identified a glycosyl hydrolase member located within the QTL mapping region. This member was cold-induced and overexpressed, significantly enhancing early-stage cold tolerance in rice. This gene was named OsGH29. Obtaining the OsGH29 gene will be of great significance for rice production.

[0071] This invention cloned a gene, OsGH29, associated with early cold tolerance from wild rice in Dongxiang, Jiangxi Province. An OsGH29 RNAi interference vector (recombinant vector pTCK303-OsGH29) was constructed and transferred into Zhonghua 17. Self-pollination yielded T2 generation homozygous RNAi transgenic positive plants. Compared with Zhonghua 17, the T2 generation homozygous RNAi transgenic positive plants showed reduced OsGH29 expression, lower seedling survival rate under cold stress during the bud stage, weaker growth under cold stress during the seedling stage, and reduced cold tolerance.

[0072] An overexpression vector (recombinant vector Super1300-OsGH29) was constructed and transformed into Zhonghua 17. Self-pollination yielded T2 generation homozygous overexpression transgenic positive plants. Compared with Zhonghua 17, the T2 generation homozygous overexpression transgenic positive plants showed increased OsGH29 expression, increased survival rate under cold stress during the bud stage, stronger seedling growth than the control, and enhanced cold tolerance.

[0073] The gene OsGH29 and its encoded protein of this invention are related to early cold tolerance in rice, which helps to study the molecular mechanism of cold tolerance in plants, breed cold-resistant varieties, enhance their resistance to extreme weather, and thus improve crop yield and quality. Attached Figure Description

[0074] Figure 1 This is a diagram showing the expression of the OsGH29 gene under low temperature conditions. Figure 1 Image A shows the expression profile of the OsGH29 gene in the cold-tolerant line (IL) and Guichao 2 (GC2) under low-temperature treatment during the bud stage. Figure 1 Figure B shows the expression profile analysis of the OsGH29 gene in the cold-resistant line (IL) and Guichao 2 (GC2) under low temperature treatment during the seedling stage.

[0075] Figure 2 A graph showing the detection of cold tolerance in OsGH29 transgenic plants with RNAi interference; Figure 2 In Figure A, the relative expression levels of gene OsGH29 in the T2 generation transgenic plants (J1779, J1780) of Zhonghua 17 transgenic interference vector and Zhonghua 17 (ZH17) are compared. Figure 2 In section B, the cold tolerance during the budding stage of transgenic plants of Zhonghua 17 with the interference vector T2 generation is compared with that of Zhonghua 17. Figure 2 C represents a comparison of the cold tolerance of Zhonghua 17 seedlings with the T2 generation of the interference vector.

[0076] Figure 3 A graph showing the detection of cold tolerance in transgenic plants overexpressing OsGH29; Figure 3 In Figure A, the relative expression levels of gene OsGH29 in the T2 generation transgenic lines (J2098, J2150) of Zhonghua 17 transgenic vector are compared with those in Zhonghua 17. Figure 3 In section B, the cold tolerance during the budding stage was compared between the T2 generation transgenic line of Zhonghua 17 and the Zhonghua 17 transgenic line expressed by the expression vector. Figure 3 In the middle section (C), the cold tolerance of the seedling stage of Zhonghua 17 transgenic line transgenic line T2 overexpression vector is compared with that of Zhonghua 17. Detailed Implementation

[0077] 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.

[0078] 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.

[0079] The following examples used SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0080] The infiltrative line SIL208 of wild rice from Dongxiang, Jiangxi Province is described in the following literature: Qin Jingjing, Identification and functional analysis of cold-resistant QTL qSCT9 in seedling stage of common wild rice from Dongxiang, Jiangxi Province, Master's thesis, 2014, which can be obtained from China Agricultural University.

[0081] The vector pTCK303 is described in the following literature: Wang Z, Chen CG, Xu YY, et al. A practical vector for efficient knockdown of gene expression in rice (Oryza sativa L.). Plant Mol Biol Rep, 2004, 22:409-417. The vector pTCK303 is referred to as vectorpTCK303 in this literature and can be obtained from China Agricultural University.

[0082] The plant expression vector Super1300 is described in the following literature: Yang Q, Chen ZZ, Zhou XF, Yin HB, Li X, Xin XF, Hong XH, Zhu JK and Gong ZZ. Overexpression of SOS (Salt Overly Sensitive) Genes Increases Salt Tolerance in Transgenic Arabidopsis. Molecular Plant, 2009, 2:22-31. The plant expression vector Super1300 is named pCAMBIA 1300 in this literature and is available to the public from China Agricultural University.

[0083] Example 1: Discovery of the early cold-resistance gene OsGH29 in rice

[0084] This experiment used common wild rice from Dongxiang, Jiangxi Province as the male parent and the indica rice variety "Guichao 2" (also known as Guichao No. 2), which had set a record for yield per mu (a Chinese unit of area, approximately 0.165 acres) in Yunnan Province for many years, as the female parent to construct a hybrid line of wild rice from Dongxiang with Guichao 2 as the background. The cold-resistant line SIL208 with stable cold tolerance was successfully screened. Subsequently, the F1 generation was constructed using SIL208 and Guichao 2. 2:3 In a population, a major-effect cold-resistant QTL (qSCT9) was located on chromosome 9 and confined to a 300 kb physical region. Gene chip expression profiling analysis revealed that after low-temperature treatment, the OsGH29 gene within this region was induced to express, and its expression level in the cold-resistant line was significantly higher than that of the recipient parent, Guichao 2. Figure 1 China A Figure 1 (B) is a cold-induced gene, so it was selected as a candidate gene for transgenic verification.

[0085] This gene was named OsGH29, and its gene sequence is shown in Sequence 3. Its CDS sequence is shown in Sequence 1. The protein sequence it encodes is shown in Sequence 2, and this protein was named OsGH29.

[0086] Sequence 3 is as follows:

[0087]

[0088] Example 2: Obtaining and Identifying OsGH29 Transgenic Rice

[0089] I. Construction of the RNAi-OsGH29 interference vector

[0090] Primers were designed based on the full-length cDNA sequence of OsGH29 (Sequence 2). Recognition sites for restriction endonucleases Spe I, Sac I, BamHI, and Kpn I, as well as protective bases, were introduced at both ends of the primers. The primer sequences are as follows:

[0091] 2R3F:5'- CGACTAGT GTTCTGGCACAAGAACGAGA-3' (The underlined bases are the recognition site and protective bases of the restriction endonuclease SpeI)

[0092] 2R3R:5'- CGGAGCTC TCACCACGTTGAACTTCCTC-3' (The underlined bases are the recognition site and protective bases of the restriction endonuclease SacI)

[0093] 2R4F:5'- CGGGATCC TCACCACGTTGAACTTCCTC-3' (The underlined bases are the recognition site and protective bases for the restriction endonuclease BamHI)

[0094] 2R4R:5'- CGGGTACC TCTGGCACAAGAACGAGAC-3' (The underlined bases are the recognition site and protective bases for the restriction endonuclease KpnI)

[0095] Total RNA was extracted from leaves of the introgression line SIL208 (Qin Jingjing, Identification and Functional Analysis of Cold-Tolerant QTLqSCT9 in Seedlings of Common Wild Rice in Dongxiang, Jiangxi Province, Master's Thesis, 2014, publicly available from China Agricultural University). Using this RNA as a template, cDNA was obtained by reverse transcription using SuperScript II reverse transcriptase (Invitrogen, Catno. 18064-014). This cDNA was then amplified using primers 2R3F and 2R3R to obtain fragment 1; fragment 2 was amplified using primers 2R4F and 2R4R.

[0096] The PCR product was digested using restriction enzyme sites introduced on the PCR primers. The RNAi empty vector pTCK303, with a length of 14621 bp, was recovered from the gel and yielded two fragments, 374 bp (fragment 1) and 380 bp (fragment 2). Fragment 1 and the empty vector were then digested with Spe I and Sac I, respectively, and the digested empty vector and fragment 1 were ligated using T4 ligase to obtain a vector carrying fragment 1. Fragment 2 and the vector carrying fragment 1 were then double-digested with BamHI and KpnI, respectively. The digested products were then ligated using T4 ligase and sequenced. The resulting RNAi vector containing fragments 1 and 2 of the rice OsGH29 gene was named RNAi-OsGH29.

[0097] The recombinant vector pTCK303-OsGH29 is obtained by replacing the small fragment between the restriction enzyme Spe I recognition site and the restriction enzyme Sac I recognition site in the pTCK303 vector with fragment 1 (sequence 4), and replacing the small fragment between the restriction enzyme BamH I recognition site and the restriction enzyme Kpn I recognition site in the pTCK303 vector with fragment 2 (sequence 5), while keeping the other sequences of the pTCK303 vector unchanged. The resulting recombinant vector is named recombinant vector pTCK303-OsGH29 (hereinafter also referred to as pTCK303-OsGH29).

[0098] The recombinant vector pTCK303-OsGH29 was transformed into Agrobacterium EHA105 to obtain the recombinant vector pTCK303-OsGH29 / Agrobacterium EHA105, abbreviated as Agrobacterium EHA105 / pTCK303-OsGH29. Agrobacterium EHA105 / pTCK303-OsGH29 is Agrobacterium EHA105 containing the recombinant vector pTCK303-OsGH29.

[0099] Sequence 4: GTTCTGGCACAAGAACGAGACGGCGAAGCCGCTGCCGGAGCTGCTCGAGGTATACTACAACTCGGTGGGCCGCAACTGCGTGCTGCTGCTGAACGCGCCGCCGAACACCACGGGCCTCGTCGACGCCGCCGACATCGCCAGGCTGCGCGAGTTCCGCACCGCCGTGACGGCCATCTTCGGCACCG ACCTCGCCGCGGGCAGCGGCGAGGGCGAGCAGCGAGCGCGGCGGCAGGTTCGCGGCGGCCAACGTGCTCGACGGCCGCGACGACACGTACTGGGCACCGGCCGCGGCGGAGGCGGAGGACGGCGGCGGGTACTGGATCGAGCTGCGGCGGCCGGCGTCGGCGGCGGCGAGGAAGTTCAACGTGGTGA.

[0100] Sequence 5: TCACCACGTTGAACTTCCTCGCCGCCGCCGACGCCGGCCGCCGCAGCTCGATCCAGTACCCGCCGCCGTCCTCCGCTCCGCCGCGGCCGGTGCCCAGTACGTGTCGTCGCGGCCGTCGAGCACGTTGGCCGCCGCGAACCTGCCGCCGCGCTCGCTGCTCGCCCTCGCCGCGCTGCCCGCGGC GAGGTCGGTGCCGAAGATGGCCGTCACGGCGGTGCGGAACTCGCGCAGCCTGGCGATGTCGGCGGCGTCGACGAGGCCCGTGGTGTTCGGCGGCGCGTTCAGCAGCAGCACGCAGTTGCGGCCCACCGAGTTGTAGTATACCTCGAGCAGCTCCGGCAGCGGCTTCGCCGTCTCGTTCTTGTGCCAGA.

[0101] II. Construction of OsGH29 overexpression vector

[0102] Primers were designed based on the full-length cDNA sequence of OsGH29, and restriction endonuclease recognition sites for KpnI and XbaI, as well as protective bases, were introduced at both ends of the primers. The primer sequences are as follows:

[0103] GP9-2FN: 5' ATAGGTACCATGATGGGTCGGTGCGCGT-3' (The underlined bases are the recognition site and protective bases for the restriction endonuclease KpnI)

[0104] GP9-2RN: 5'- ATATCTAGA CATCGTGCCCCCGCCGG-3' (The underlined bases are the recognition site and protective bases of the restriction endonuclease Xba I)

[0105] Total RNA was extracted from leaves of the introgression line SIL208 (Qin Jingjing, Identification and Functional Analysis of Cold-Tolerant QTLqSCT9 in Seedlings of Common Wild Rice in Dongxiang, Jiangxi Province, Master's Thesis, 2014, publicly available from China Agricultural University) using TRIZOL reagent. Using this RNA as a template, cDNA was obtained by reverse transcription using SuperScript II reverse transcriptase (Invitrogen, Cat no. 18064-014). Using this cDNA as a template, the coding sequence of the rice OsGH29 gene (fragment 3, 1485 bp in length) was amplified using primers GP9-2FN and GP9-2RN. This 1485 bp DNA fragment was cloned into the multiple cloning site of the plant expression vector Super1300 between the KpnI and XbaI restriction sites, resulting in an overexpression vector containing the rice OsGH29 gene, named Super1300-OsGH29 (also known as the recombinant vector Super1300-OsGH29). The specific method is as follows.

[0106] The coding sequence (fragment 3) of the rice OsGH29 gene obtained above was double-digested with KpnI and XbaI enzymes to obtain the digested amplification product. The vector Super1300 was then double-digested with KpnI and XbaI enzymes to obtain the digested vector. The digested amplification product and the digested vector were ligated using DNA ligase. The ligation product was transformed into *E. coli* for screening and sequencing. The recombinant vector with correct sequencing was named recombinant vector Super1300-OsGH29.

[0107] The recombinant vector Super1300-OsGH29 is obtained by replacing the small fragment between the KpnI restriction enzyme recognition site and the XbaI restriction enzyme recognition site in the Super1300 vector with a DNA molecule (OsGH29 gene) whose nucleotide sequence is sequence 21-1502 of sequence 1, while keeping the other sequences of the Super1300 vector unchanged. This recombinant vector is named Super1300-OsGH29 (hereinafter also referred to as Super1300-OsGH29). Transforming the recombinant vector Super1300-OsGH29 into Agrobacterium EHA105 yields the recombinant vector Super1300-OsGH29 / Agrobacterium EHA105, also known as Agrobacterium EHA105 / Recombinant Vector Super1300-OsGH29 (Agrobacterium EHA105 / Super1300-OsGH29).

[0108] III. Obtaining OsGH29 transgenic rice

[0109] Preparation of mature embryo callus from Zhonghua 17 rice (japonica conventional rice, also known as Zhonghua 17, hereinafter referred to as ZH17):

[0110] (1) Select unopened rice seeds with no obvious fungal spots on the seed surface and place them in a 42℃ oven for 2 days to break dormancy.

[0111] (2) Remove the outer shell of the rice seeds with a threshing machine, and select seeds with normal endosperm and no bacterial spots on the surface and place them in a 100mL Erlenmeyer flask.

[0112] (3) Add 75% alcohol for 3 min, discard the 75% alcohol, then add 20% sodium hypochlorite solution and shake in a shaker at 37°C for 20 min.

[0113] (4) Pour out the sodium hypochlorite solution in the laminar flow hood, rinse the seeds 5-6 times with sterile deionized water until the water used to wash the seeds is relatively clear, and place them in a petri dish lined with 6 layers of sterile filter paper to air dry.

[0114] (5) Place the dried seeds on the NB basic culture medium with a spoon, and spread the seeds evenly on the NB culture medium with tweezers, about 30-50 seeds per dish.

[0115] (6) The rice was cultured in the dark in a constant temperature incubator at 28℃ for 7 days until pale yellow callus tissue grew. The induced callus tissue was peeled off and placed on NB basic medium to recover for 2 days to obtain mature embryo callus tissue.

[0116] NB medium:

[0117] NB basal medium (pH 5.85–6.0)

[0118]

[0119] Adjust the pH to 5.85–6.0 with KOH, and dispense into 1L bottles.

[0120]

[0121] Mature embryo callus tissues of Zhonghua 17 rice prepared above were individually infected with recombinant vectors pTCK303-OsGH29 / Agrobacterium EHA105 and Super1300-OsGH29 / Agrobacterium EHA105, respectively. Three rounds of selection were performed using NB medium containing 50 mg / L hygromycin, with each round lasting 20 days, to obtain transgenic plants. The transgenic plants were identified by PCR using hygromycin primer sequences and target gene fragment sequences. Twenty T0 generation RNAi transgenic positive plants (obtained by infection with recombinant vector pTCK303-OsGH29 / Agrobacterium EHA105) were named J1-J20, and fifteen T0 generation overexpression transgenic positive plants (obtained by infection with Super1300-OsGH29 / Agrobacterium EHA105) were named O1-O15.

[0122] IV. PCR and Phenotypic Identification of Transgenic Rice

[0123] Materials Preparation: Two RNAi transgenic positive plants obtained above were continuously self-crossed to obtain seeds of T1 generation homozygous RNAi transgenic positive plants (T2 generation homozygous RNAi transgenic positive lines). Seeds of the T1 generation homozygous RNAi transgenic positive lines named J1779 (hereinafter referred to as J1779 line seeds) and J1780 (hereinafter referred to as J1780 line seeds) were used for the following experiments.

[0124] Two overexpressing transgenic positive plants obtained above were continuously self-crossed to obtain seeds of T1 generation homozygous overexpressing transgenic positive plants (T2 generation homozygous overexpressing transgenic positive lines). Seeds of the T1 generation homozygous overexpressing transgenic positive lines named J2098 (hereinafter referred to as J2098 line seeds) and J2150 (hereinafter referred to as J2150 line seeds) were used for the following experiments.

[0125] Cold tolerance test of RNAi transgenic organisms:

[0126] Plump seeds of J1779, J1780, and control ZH17 were placed in a 42℃ oven for 2 days to break dormancy. The dormancy-broken seeds were then disinfected by soaking in a 20% sodium hypochlorite solution for 30 minutes, followed by rinsing several times with deionized water until no odor remained. The seeds were then placed in a 37℃ incubator for 2 days to promote germination. Once the seeds showed signs of sprouting, they were transferred to disposable plastic petri dishes (lined with two layers of sterile filter paper and containing 15-20 mL of sterile deionized water) and placed in a light incubator (temperature 28℃, humidity 70%, light conditions set to 16h light / 8h dark) until the sprouts reached approximately 5mm in length. Seedlings of J1779, J1780, and ZH17 were obtained. Cold tolerance tests during the sprouting period were then performed on the seedlings of J1779, J1780, and ZH17, using the following method:

[0127] Thirty seedlings each from the J1779 and J1780 lines and the ZH17 line were placed at 2℃ for 10 days, followed by 28℃ for 10 days. The growth and survival rate (survival rate) of the T2 generation homozygous RNAi transgenic positive seedlings and the ZH17 seedlings were then recorded, and their average values ​​were calculated. Results are as follows: Figure 2 As shown in Figure B, ZH17 is Zhonghua 17 rice, J1779 is a seed plant of the J1779 series, and J1780 is a seed plant of the J1780 series. Figure 2 In section B, from left to right, are the growth status diagrams of ZH17, J1779, and J1780 after being placed in a cold environment at 2℃ for 10 days and then placed in a cold environment at 28℃ for 10 days, and the bar chart of the average survival rate of ZH17, J1779, and J1780 after cold treatment.

[0128] Plump seeds from the J1779, J1780, and control ZH17 lines were dried in a 42℃ oven for 2 days to break dormancy. The dormancy-broken seeds were then disinfected by soaking in a 20% sodium hypochlorite solution for 30 minutes. After disinfection, the seeds were rinsed several times with deionized water and placed in a 37℃ incubator for 2 days. Once the seeds showed signs of sprouting, they were placed in 96-well PCR plates (bottom removed) and cultured in a 28℃ artificial climate chamber (14 hours light, 10 hours dark) with deionized water, changing the water every two days. When the plants reached the two-leaf-one-heart stage (12 days after transplanting), full nutrient solution was applied. Seedlings of the J1779, J1780, and ZH17 lines were obtained. The seedlings of these three lines were then tested for cold tolerance during the seedling stage using the following method:

[0129] Ten seedlings each of the J1779 and J1780 lines and ten seedlings of ZH17 were collected and placed at 4℃ for 2 days, followed by 7 days at 28℃. The growth, plant height, and fresh weight of the crown per plant were then recorded for the T2 generation homozygous RNAi transgenic positive seedlings (J1779 and J1780 lines) and ZH17 seedlings, and the average values ​​were calculated. The results are as follows: Figure 2 As shown in C, Figure 2 The left side of image C shows the seedling growth status of ZH17, J1779, and J1780 seedlings before and after cold treatment. The upper two images show the seedling growth status before the cold tolerance test, while the lower two images show the seedling growth status after 2 days at 4℃ and then 7 days at 28℃. Figure 2 The right side of the middle section (C) shows the average plant height and average fresh weight of the crown of seedlings ZH17, J1779, and J1780 after seedling cold tolerance testing.

[0130] Expression level detection:

[0131] Plump seeds of the J1779 and J1780 series, as well as control ZH17, were placed in a 42℃ oven for 2 days to break dormancy. The dormancy-broken seeds were then immersed in a 20% sodium hypochlorite solution for 30 minutes for disinfection, followed by rinsing several times with deionized water until no odor remained. The seeds were then placed in a 37℃ incubator for 2 days to promote germination. After the seeds sprout, transfer them to disposable plastic petri dishes with a diameter of 90 mm (lined with two layers of sterile filter paper and 15-20 mL of sterile deionized water), and place them in a light incubator (temperature 28℃, humidity 70%, light conditions set to 16 h light / 8 h dark) to cultivate until the two-leaf-one-heart stage. Take the aboveground parts of the seedlings, extract RNA, and reverse transcribe it into cDNA. Using the UBI2 gene of rice as an internal control (amplification primers UBI2F / UBI2R), detect the expression level of the OsGH29 gene in the two-leaf-one-heart stage seedlings of J1779 and J1780 lines (amplification primers Q9-2F / Q9-2R). PCR amplification: The reaction system is 10 μl, including: 4 μl cDNA template, 5 μl SYBR greenⅠ mix (1000×; SR4110-100 μl), and 1 μl 4 μM primers. The amplification program was as follows: 95℃ for 10 min; 95℃ for 10 s, 58℃ for 30 s, 72℃ for 45 s, 39 cycles; 72℃ for 5 min; melting curves were plotted at 0.3℃ intervals from 65℃ to 95℃. The results are as follows. Figure 2 As shown in Figure A, the expression level of the OsGH29 gene was significantly reduced in seedlings of the 1779 and J1780 series at the two-leaf-one-heart stage.

[0132] The results showed that when the OsGH29 interference vector was transferred into Zhonghua 17, the expression level of OsGH29 was reduced in T2 generation homozygous RNAi transgenic positive plants compared with Zhonghua 17, the survival rate of seedlings at the bud stage was decreased, the seedling growth was weakened, and the cold tolerance of rice was reduced. Figure 2 China A Figure 2 B, Figure 2 (C) Figure 2 In the text, ZH17 refers to the rice variety ZH17, J1779 refers to the seed seedling of the J1779 series, and J1780 refers to the seed seedling of the J1780 series.

[0133] Cold tolerance test of transgenic overexpression:

[0134] Plump seeds of the J2098 and J2150 series, as well as control ZH17, were placed in a 42℃ oven for 2 days to break dormancy. The dormancy-broken seeds were then disinfected by immersing in a 20% sodium hypochlorite solution for 30 minutes, followed by rinsing several times with deionized water until no odor remained. The seeds were then placed in a 37℃ incubator for 2 days to promote germination. Once the seeds showed signs of sprouting, they were transferred to disposable plastic petri dishes (lined with two layers of sterile filter paper and containing 15-20 mL of sterile deionized water) and placed in a light incubator (temperature 28℃, humidity 70%, light conditions set to 16h light / 8h dark) until the sprouts reached approximately 5mm in length. Cold tolerance during the sprouting period was then tested using the following method:

[0135] Thirty seedlings each of the J2098 and J2150 lines and the ZH17 line were placed at 2℃ for 10 days, followed by 28℃ for 10 days. The growth and survival rate of the T2 generation homozygous overexpressing transgenic positive seedlings and the ZH17 seedlings were then recorded, and the average values ​​were calculated. Results are as follows: Figure 3 As shown in Figure B, ZH17 is Zhonghua 17 rice, J2098 is a seed plant of the J2098 series, and J2150 is a seed plant of the J2150 series. Figure 3 In section B, from left to right, are the growth status diagrams of ZH17, J2098, and J2150 after being placed in a cold environment at 2℃ for 10 days and then placed in a 28℃ environment for 10 days, as well as the bar chart of the average survival rate of ZH17, J2098, and J2150 after cold treatment.

[0136] Plump seeds of J2098, J2150, and control ZH17 were dried in a 42℃ oven for 2 days to break dormancy. The dormancy-broken seeds were then disinfected by soaking in a 20% sodium hypochlorite solution for 30 minutes. After disinfection, the seeds were rinsed several times with deionized water and placed in a 37℃ incubator for 2 days. Once the seeds showed signs of sprouting, they were placed in 96-well PCR plates (bottom removed) and cultured in a 28℃ artificial climate chamber (14 hours light, 10 hours dark) with deionized water, changing the water every two days. The plants were cultured at 28℃ until they reached the two-leaf-one-heart stage (12 days after transplanting), at which point they were watered with a complete nutrient solution. Seedlings of J2098, J2150, and ZH17 were then tested for cold tolerance during the seedling stage using the following method:

[0137] Twenty seedlings each of the J2098, J2150, and ZH17 lines were collected and placed at 4℃ for 2 days, followed by 7 days at 28℃. The growth, plant height, and fresh weight of the canopy per plant of the T2 generation homozygous overexpressing transgenic positive seedlings (J2098 and J2150 lines) and ZH17 seedlings were recorded, and their average values ​​were calculated. Results are as follows: Figure 3 As shown in C, Figure 3 The left side of image C shows the seedling growth status of ZH17, J2098, and J2150 seedlings before and after cold treatment. The upper two images show the seedling growth status before the cold tolerance test, while the lower two images show the seedling growth status after 2 days at 4℃ and then 7 days at 28℃. Figure 2 The right side of the middle section (C) shows the average plant height and average fresh weight of the single plant crown after cold tolerance testing of seedlings of ZH17, J2098, and J2150.

[0138] Expression level detection:

[0139] Plump seeds of the J2098 and J2150 series, as well as control ZH17, were placed in a 42℃ oven for 2 days to break dormancy. The dormancy-broken seeds were then immersed in a 20% sodium hypochlorite solution for 30 minutes for disinfection, followed by rinsing several times with deionized water until no odor remained. The seeds were then placed in a 37℃ incubator for 2 days to promote germination. After the seeds sprout, transfer them to disposable plastic petri dishes with a diameter of 90 mm (lined with two layers of sterile filter paper and 15-20 mL of sterile deionized water), and place them in a light incubator (temperature 28℃, humidity 70%, light conditions set to 16 h light / 8 h dark) to cultivate until the two-leaf-one-heart stage. Take the aboveground parts of the seedlings, extract RNA, and reverse transcribe it into cDNA. Using the UBI2 gene of rice as an internal control (amplification primers UBI2F / UBI2R), detect the expression level of the OsGH29 gene in the two-leaf-one-heart stage seedlings of J2098 and J2150 lines (amplification primers Q9-2F / Q9-2R). PCR amplification: The reaction system is 10 μl, including: 4 μl cDNA template, 5 μl SYBR greenⅠ mix (1000×; SR4110-100 μl), and 1 μl 4 μM primers. The amplification program was as follows: 95℃ for 10 min; 95℃ for 10 s, 58℃ for 30 s, 72℃ for 45 s, 39 cycles; 72℃ for 5 min; melting curves were plotted at 0.3℃ intervals from 65℃ to 95℃. The results are as follows. Figure 3 As shown in Figure A, the expression level of the OsGH29 gene was significantly increased in the two-leaf-one-heart stage seedlings of the J2098 and J2150 series.

[0140] The results showed that when the OsGH29 overexpression vector was transferred into Zhonghua 17, the T2 generation homozygous overexpression transgenic positive plants showed increased OsGH29 expression, increased seedling survival rate, better seedling growth, and enhanced cold tolerance compared to Zhonghua 17. Figure 3 China A Figure 3 B, Figure 3 (C) Figure 3 In the text, ZH17 refers to the rice variety ZH17, J2098 refers to the seed seedling of the J2098 series, and J2150 refers to the seed seedling of the J2150 series.

[0141] The present invention has been described in detail above. For those skilled in the art, 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. Although specific embodiments have been given, 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. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The use of a protein or a substance that upregulates, enhances, or increases the expression of the gene encoding the protein in any of the following: A1) Applications in enhancing or improving plant cold tolerance and / or applications in the preparation of products that enhance or improve plant cold tolerance; A2) Applications in plant breeding and / or in the preparation of plant breeding products, wherein the purpose of the breeding includes the cultivation of highly cold-resistant plants; The protein is any one of the following: B1) The amino acid sequence of the protein is shown in sequence 2; B2) The fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1); The substance that upregulates, enhances, or increases the expression of the gene encoding the protein is any one of the following: D1) The nucleic acid molecule encoding the protein; D2), an expression cassette containing the nucleic acid molecules described in D1); D3), ​​a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3); D5) A transgenic plant cell line containing the nucleic acid molecule described in D1), or a transgenic plant cell line containing the expression cassette described in D2), or a transgenic plant cell line containing the recombinant vector described in D3); D6) Transgenic plant tissue containing the nucleic acid molecules described in D1), or transgenic plant tissue containing the expression cassette described in D2), or transgenic plant tissue containing the recombinant vector described in D3); D7) Transgenic plant organs containing the nucleic acid molecules described in D1), or transgenic plant organs containing the expression cassette described in D2), or transgenic plant organs containing the recombinant vector described in D3); The plant in question is rice.

2. Use according to claim 1, characterized in that, The nucleic acid molecule described in D1) is a DNA molecule with the nucleotide sequence shown in Sequence 1.

3. A method for cultivating highly cold-resistant plants, characterized in that, This includes obtaining a highly cold-resistant plant by upregulating, enhancing, or increasing the expression level of the gene encoding the protein described in claim 1 or 2 in the target plant, wherein the highly cold-resistant plant has a higher cold resistance than the target plant; the plant is rice.

4. A method for improving cold tolerance in plants, characterized by, This includes improving plant cold tolerance by upregulating or enhancing or increasing the expression of genes encoding the proteins described in claim 1 or 2 in the plant; the plant being rice.

5. The method of claim 3 or 4, wherein, The upregulation, enhancement, or increase of the expression of the gene encoding the protein of claim 1 or 2 in the plant comprises introducing the nucleic acid molecule, expression cassette, or recombinant vector of claim 1 into the target plant.

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