Application of rubber tree thioredoxin or biomaterials related to thioredoxin

Abstract

This invention discloses the application of a rubber tree thioredoxin or related biomaterials, belonging to the field of biotechnology, for improving the resistance of rubber trees to low temperature and oxidative stress at the molecular level. The amino acid sequence of thioredoxin is shown in SEQ ID NO. 5. Experiments have shown that expression of the gene encoding this thioredoxin in yeast enhances the cold resistance and antioxidant capacity of recombinant yeast. Therefore, the gene encoding this thioredoxin can be used to improve the cold resistance and antioxidant capacity of plants or microorganisms.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of a rubber tree thioredoxin or biomaterials related to thioredoxin. Background Technology

[0002] Natural rubber is an important strategic resource and an indispensable industrial raw material. More than 98% of the world's commercially used natural rubber comes from rubber trees. Hevea brasiliensis (Muell. Arg.). Rubber trees frequently suffer from abiotic stresses such as low-temperature damage, seasonal drought, and typhoons during their growth. Abiotic stresses often lead to the production of large amounts of reactive oxygen species, resulting in oxidative stress and damaging plant cells. Therefore, identifying antioxidant and stress-resistance genes in rubber trees and using genetic engineering to improve their resistance to oxidative and abiotic stresses is of great significance for increasing the yield of natural rubber. Summary of the Invention

[0003] In view of the above-mentioned technical problems, the present invention provides an application of thioredoxin from rubber trees or biomaterials related to thioredoxin. The technical problems to be solved are not limited to the described technical topics, and those skilled in the art will clearly understand other technical topics not mentioned herein through the following description.

[0004] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0005] In a first aspect, the present invention provides an application of thioredoxin from rubber trees or biomaterials related to thioredoxin, wherein the application is any of the following:

[0006] (1) Application in improving the cold resistance of yeast;

[0007] (2) Application in the selection and breeding of microbial or plant varieties with improved cold resistance;

[0008] (3) Application in improving the antioxidant properties of yeast;

[0009] (4) Application in the selection and breeding of microbial or plant varieties with enhanced antioxidant properties;

[0010] The aforementioned thioredoxin is named HbTRXh2, and it originates from the rubber tree (Rubberia spp.) in the Euphorbiaceae family. Hevea brasiliensis Muell. Arg.), whose amino acid sequence is shown in SEQ ID NO.5, consists of 134 amino acids.

[0011] In a second aspect, the present invention provides the use of a biomaterial related to the above-mentioned HbTRXh2 protein in any of the following:

[0012] (1) Application in improving the cold resistance of yeast;

[0013] (2) Application in the selection and breeding of microbial or plant varieties with improved cold resistance.

[0014] (3) Application in improving the antioxidant properties of yeast;

[0015] (4) Application in the selection and breeding of microbial or plant varieties with enhanced antioxidant properties;

[0016] In a preferred embodiment of the present invention, the biomaterial is any one of B1) to B3) below:

[0017] B1) The nucleic acid molecule encoding the thioredoxin described in the text;

[0018] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0019] B3) A recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2).

[0020] In the above-mentioned biological materials, the nucleic acid molecule described in B1) can be a gene as shown in C1) or C2):

[0021] C1) cDNA or DNA molecules with nucleotide sequences as shown in positions 104-508 of SEQ ID NO.3;

[0022] C2) cDNA or DNA molecules with nucleotide sequences as shown in SEQ ID NO.3;

[0023] 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.

[0024] The sequence shown in SEQ ID NO.3 consists of 519 nucleotides, with positions 104 to 508 being the coding region, encoding HbTRXh2 as shown in SEQ ID NO.5.

[0025] Of the above-mentioned biological materials, B2) describes an expression cassette containing a nucleic acid molecule encoding HbTRXh2. HbTRXh2 Gene expression cassettes (GCs) are devices that can express gene information in host cells. HbTRXh2 The DNA, which can include not only the starter HbTRXh2 The promoter of gene transcription may also include a terminator. HbTRXh2The gene transcription terminator. Further, the expression cassette may also include an enhancer sequence. 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: constitutive promoter 35S of cauliflower mosaic virus; wound-inducible promoter from tomato, leucine aminopeptidase; chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato protease inhibitor II promoter (PIN2) or LAP promoter (both induced by methyl jasmonic acid); heat shock promoters; tetracycline-inducible promoters; seed-specific promoters, such as the millet seed-specific promoter pF128; and promoters specific to seed storage proteins (e.g., promoters of beta-glucan, napin, oleosin, and soybean beta-conglycin). They can be used alone or in combination with other plant promoters. 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 terminator.

[0026] Existing expression vectors can be used to construct structures containing the aforementioned... HbTRXh2 Recombinant vectors for gene expression cassettes. These plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microbombardment, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb (CAMBIA). The plant expression vectors may also contain the 3′ untranslated region of a foreign gene, i.e., a polyadenylated signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylated signal can guide the addition of polyadenylated acid to the 3′ end of the mRNA precursor, such as Agrobacterium crown gall tumor inducing (Ti) plasmid genes (e.g., carmine synthase genes). NosThe untranslated regions transcribed at the 3′ end of plant genes (such as soybean storage protein genes) have similar functions. When constructing plant expression vectors using the genes 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, but they 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. To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes encoding enzymes or luminescent compounds that can be expressed in plants (e.g., those encoding enzymes that produce color changes). GUS Genes, luciferase genes, etc.), antibiotic marker genes (such as those conferring resistance to kanamycin and related antibiotics). nptII Genes that confer resistance to the herbicide phosphinic acid bar Genes that confer resistance to the antibiotic hygromycin hph Genes, and the genes that confer resistance to methotrexate dhfr Genes that confer resistance to glyphosate EPSPS Genes such as herbicide-resistant marker genes or mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose can be used. From a safety perspective, transgenic plants can be directly selected by stress screening without adding any selective marker genes.

[0027] In the aforementioned biological materials, the vector is a plasmid, granule, bacteriophage, or viral vector. Preferably, the plasmid is the yeast expression vector pYES2.

[0028] In the aforementioned biological materials, the microorganisms are yeast, bacteria, algae, or fungi, such as Escherichia coli. Preferably, the yeast is Saccharomyces cerevisiae strain INVScl.

[0029] The organisms mentioned in this article are microorganisms, such as yeast.

[0030] In one embodiment of the present invention, the HbTRXh2 encoding gene is obtained by means of a gene containing... HbTRXh2 Recombinant microorganisms were obtained by introducing a recombinant vector of the gene expression cassette into the *Saccharomyces cerevisiae* strain INVSScl. The recombinant vector was a vector of the yeast expression vector pYES2. Kpn I and Xba The sequence between the I recognition sites is replaced with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.4, resulting in a recombinant vector. This recombinant vector expresses HbTRXh2 as shown in SEQ ID NO.5. The recombinant microorganism expresses HbTRXh2 as shown in sequence 5.

[0031] In the above applications, the oxidative stress may be hydrogen peroxide-induced oxidative stress.

[0032] In a third aspect, the present invention provides a method for cultivating microbial or plant varieties with enhanced cold resistance, the method comprising increasing the expression level of the gene encoding the thioredoxin in a recipient organism to obtain a transgenic organism with higher cold resistance than the recipient organism.

[0033] In a fourth aspect, the present invention provides a method for cultivating microbial or plant varieties with enhanced antioxidant properties, the method comprising increasing the expression level of the gene encoding the thioredoxin in a recipient organism to obtain a transgenic organism with higher antioxidant properties than the recipient organism.

[0034] Furthermore, the increase in the expression level of the gene encoding the thioredoxin in the recipient organism is achieved by introducing the gene encoding the thioredoxin into the recipient organism.

[0035] In the above method, the gene encoding the protein can be the nucleic acid molecule described in B1).

[0036] In the above method, the recipient organism can be a microorganism or a plant. Preferably, the recipient organism is yeast, and the plant is a rubber tree.

[0037] This invention has discovered that, HbTRXh2 The gene is expressed in all tissues of the rubber tree, with the highest expression level in leaves during the stable stage. HbTRXh2 Gene expression was induced by low temperature, drought, salt, and oxidative stress. Further research was conducted using a yeast expression system to... HbTRXh2 Gene functional identification showed that overexpression in yeast HbTRXh2 Genes can enhance the cold resistance and antioxidant capacity of recombinant yeast. Therefore, this can be achieved by... HbTRXh2 Overexpression of genes in plants or microorganisms can enhance their cold resistance and antioxidant properties. Attached Figure Description

[0038] Figure 1 Sequence analysis of HbTRXh2 from rubber tree. The gray background indicates conserved thioredoxin domains; the underlined areas indicate conserved redox active sites.

[0039] Figure 2 Phylogenetic tree of rubber tree HbTRXh2 and other plant thioredoxins.

[0040] Figure 3 For rubber trees HbTRXh2 Gene expression in various tissues.

[0041] Figure 4 Under low temperature stress HbTRXh2Changes in gene expression. Different lowercase letters indicate significant differences between treatment time points. P <0.05).

[0042] Figure 5 For PEG-induced drought stress treatment HbTRXh2 Changes in gene expression. Different lowercase letters indicate significant differences between treatment time points. P <0.05).

[0043] Figure 6 Under salt stress treatment HbTRXh2 Changes in gene expression. Different lowercase letters indicate significant differences between treatment time points. P <0.05).

[0044] Figure 7 After hydrogen peroxide-induced oxidative stress treatment HbTRXh2 Changes in gene expression. Different lowercase letters indicate significant differences between treatment time points. P <0.05).

[0045] Figure 8 PCR detection was performed on pYES2-HbTRXh2 and pYES2-transformed yeast. "M" represents the DL2000 DNA marker (TaKaRa); "N1" and "N2" are negative controls without template; "P1" and "P2" are plasmid positive controls; "1, 2, 3" are single yeast clones transformed with pYES2; "4, 5, 6" are single yeast clones transformed with pYES2-HbTRXh2.

[0046] Figure 9 To induce recombinant yeast in 24h of culture HbTRXh2 Gene expression detection. "1, 2, 3" are cDNA samples from 3 yeast monoclonal cells transformed with pYES2 and induced for 24 h; "4, 5, 6" are cDNA samples from 3 yeast monoclonal cells transformed with pYES2-HbTRXh2 and induced for 24 h.

[0047] Figure 10 The survival difference between pYES2-HbTRXh2-transformed yeast and pYES2-transformed yeast after low temperature (-20℃) stress treatment is shown. "HbTRXh2" represents pYES2-HbTRXh2-transformed yeast; "pYES2" represents pYES2-transformed yeast.

[0048] Figure 11The survival difference between pYES2-HbTRXh2 transformed yeast and pYES2 transformed yeast after treatment with 20mM H2O2. Here, "HbTRXh2" represents pYES2-HbTRXh2 transformed yeast; and "pYES2" represents pYES2 transformed yeast. Detailed Implementation

[0049] 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.

[0050] 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.

[0051] In the quantitative experiments described below, all experiments were repeated three times. The results are expressed as the mean SD. The data were processed using SigmaPlot 12.0 statistical software. P <0.05 (different lowercase letters indicate significant differences).

[0052] Natural rubber is an important strategic resource and an indispensable industrial raw material. Over 98% of the world's commercially available natural rubber comes from rubber trees. During their growth, rubber trees frequently suffer from abiotic stresses such as low-temperature damage, seasonal drought, and typhoons. These abiotic stresses often lead to the production of large amounts of reactive oxygen species, resulting in oxidative stress and damaging plant cells. Therefore, identifying antioxidant and stress-resistance genes in rubber trees and using genetic engineering to improve their resistance to oxidative and abiotic stresses is of great significance for increasing natural rubber yield.

[0053] Furthermore, microorganisms possess significant advantages such as rapid growth and reproduction, low production costs, no secondary pollution, and convenient gene manipulation, playing an irreplaceable role in industrial production and the degradation of toxic and harmful environmental pollutants. However, in actual production and environmental remediation processes, microorganisms face numerous harsh environmental conditions, such as high temperature, low temperature, high osmotic pressure, and hypoxia. Therefore, improving the stress resistance of microorganisms can better leverage their functions and expand their application scope.

[0054] Based on this, the present invention provides an application of rubber tree thioredoxin or biomaterials related to thioredoxin, specifically any of the following:

[0055] (1) Application in improving the cold resistance of yeast;

[0056] (2) Application in the selection and breeding of microbial or plant varieties with improved cold resistance;

[0057] (3) Application in improving the antioxidant properties of yeast;

[0058] (4) Application in the selection and breeding of microbial or plant varieties with enhanced antioxidant properties;

[0059] The amino acid sequence of the aforementioned thioredoxin, as shown in SEQ ID NO.5, consists of 134 amino acids. The cold resistance and antioxidant properties of plants or microorganisms can be enhanced by overexpressing the gene encoding thioredoxin.

[0060] The rubber tree involved in this invention ( Hevea brasiliensis Muell. Arg.) Reyan 7-33-97: recorded in "Huang Huasun, Liang Maohuan, Wu Yuntong, Li Deshun, He Jinwei. Breeding of excellent rubber tree variety Reyan 7-33-97 for medium-scale promotion. Journal of Tropical Crops, 1994, 15(2): 1-6", was bred by the Rubber Research Institute of the Chinese Academy of Tropical Agricultural Sciences. It can be obtained by the public from the Rubber Research Institute of the Chinese Academy of Tropical Agricultural Sciences. This biological material is only used to repeat the relevant experiments of this invention and cannot be used for other purposes.

[0061] YPD liquid culture medium: is a sterile liquid culture medium composed of solutes and solvents. The solutes and their concentrations are 1% (w / w) yeast extract, 2% (w / w) peptone and 2% (w / w) glucose, and the solvent is water.

[0062] SC-Ura liquid selective medium (a basal medium for sterile yeast lacking uric acid): 1L of SC-Ura liquid selective medium is obtained by adding 0.78g DO Supplement-Ura (Clontech), 6.7g amino-free yeast nitrogen source, and 20g glucose to deionized water, and then bringing the volume to 1L. 1L of SC-Ura solid selective medium is obtained by adding 20g agar powder to the above liquid medium.

[0063] SC-Ura liquid induction medium (SC-Ura liquid medium with 2% galactose as carbon source): 1L of liquid induction medium is obtained by adding 0.78g DO Supplement-Ura, 6.7g amino-free yeast nitrogen source, and 20g galactose to deionized water and then adjusting the volume to 1L.

[0064] A 40mM H2O2 aqueous solution is obtained by adding H2O2 to sterile distilled water, resulting in an aqueous solution with a H2O2 concentration of 40mM.

[0065] Example 1: Obtaining and sequence analyzing HbTRXh2, a thioredoxin from rubber trees, and its encoding gene.

[0066] A reference sequence containing the complete coding region was obtained through identification and bioinformatics analysis of the thioredoxin gene from rubber trees. Primers were designed and synthesized based on the reference sequence, and the specific primer sequences are as follows:

[0067] F1: 5′-GGGGTAAATTCGAAGCCCAG-3′, SEQ ID NO.1;

[0068] R1: 5′-GAAGGAAGGCATCAAGTGGC-3′, SEQ ID NO. 2.

[0069] Total RNA was extracted from leaves of the rubber tree Rhizophora 7-33-97 using a universal plant total RNA extraction kit (Beijing Biotech Biotechnology Co., Ltd.). First-strand cDNA was synthesized via reverse transcription using the RevertAid First Strand cDNA Synthesis Kit (ThermoScientific), yielding leaf cDNA. Using the leaf cDNA as a template, the target gene was amplified by PCR using the aforementioned primers F1 / R1.

[0070] The PCR reaction system consisted of: 2 μL cDNA template, 0.5 μL F1 / R1 primer (10 μM), 5 μL 5×TransStartFastPfu Buffer, 2 μL 2.5 mM dNTPs, 0.5 μL TransStartFastPfu DNA Polymerase (2.5 U / μL) (Beijing TransGen Biotech Co., Ltd.), and ddH2O added to a total volume of 25 μL.

[0071] The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 20 s, 55℃ annealing for 20 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ extension for 10 min.

[0072] The PCR products were detected by 1% agarose gel electrophoresis, and the results showed that a band matching the expected target (519 bp) was obtained. The band was excised and the fragment was recovered using an agarose gel extraction kit (OMEGA). The purified DNA fragment was then ligated into the pEASY-Blunt Simple Cloning Vector (Beijing TransGen Biotech Co., Ltd.), transformed into E. coli Trans1-T1 Phage Resistant Chemically Competent Cell (Beijing TransGen Biotech Co., Ltd.), and positive clones were selected and sent to Platinum Biotech (Hainan) Co., Ltd. for sequencing.

[0073] Sequencing results showed that the obtained PCR product had a nucleotide sequence of 519 bp, as shown in SEQ ID NO.3 of the sequence listing. The gene represented by this sequence was named... HbTRXh2 Gene. HbTRXh2 The coding sequence of the gene is shown as nucleotides 104-508 of SEQ ID NO.3 in the sequence listing (i.e., SEQ ID NO.4), and the protein it encodes is named HbTRXh2. The amino acid sequence of HbTRXh2 is shown as SEQ ID NO.5.

[0074] SEQ ID NO.3:

[0075] ggggtaaattcgaagcccaggcgaagctggctcttacggattgtagtttccaagagaatcttgctccgtagtttgagaaagagagagagagagagagagagcaATGGGAGGTTTATTCTCTGCTCTTAATGGAGACGCTGCAGCCGCCGGCGAAGATTCCTCATCGGCGGATCATTCGGCAGTCACAACTTTCCATTCCTCTGAGCGATGGCAGCTCCATTTCAATTCCATGAAAGAGTTATCCCAGCTGATGGTCGTAGATTTTGCTGCATCGTGGTGTGGACCTTGTAAGTTAATTGAACCACAAGTTAAAGCCTTGGCTGCCAAGTTCACTGATGTCCAGTTTGCCAAGATCGATGTGGATGAATTGCCTGATGTGGCCCAGGAATTTGGAGTTCAGGCTATGCCAACGTTTGTGTTGGTGAAGAAAGGGAAAGAAGTGGATAGGATTGTGGGAGCCAAGAAGGAAGAGCTTGAAAAGAAGATTCAGAAACACAGGGCCACTTGAtgccttccttc

[0076] SEQ ID NO.4:

[0077] ATGGGAGGTTTATTCTCTGCTCTTAATGGAGACGCTGCAGCCGCCGGCGAAGATTCCTCATCGGCGGATCATTCGGCAGTCACAACTTTCCATTCCTCTGAGCGATGGCAGCTCCATTTCAATTCCATGAAAGAGTTATCCCAGCTGATGGTCGTAGATTTTGCTGCATCGTGGTGTGGACCTTGTAAGTTAATTGAACCACAAGTTAAAGCCTTGGCTGCCAAGTTCACTGATGTCCAGTTTGCCAAGATCGATGTGGATGAATTGCCTGATGTGGCCCAGGAATTTGGAGTTCAGGCTATGCCAACGTTTGTGTTGGTGAAGAAAGGGAAAGAAGTGGATAGGATTGTGGGAGCCAAGAAGGAAGAGCTTGAAAAGAAGATTCAGAAACACAGGGCCACTTGA

[0078] SEQ ID NO.5:

[0079] MGGLFSALNGDAAAAGEDSSSADHSAVTTFHSSERWQLHFNSMKELSQLMVVDFAASWCGPCKLIEPQVKALAAKFTDVQFAKIDVDELPDVAQEFGVQAMPTFVLVKKGKEVDRIVGAKKEELEKKIQKHRAT

[0080] The predicted molecular weight of HbTRXh2 protein is 14.64 kDa, with a theoretical isoelectric point of 5.78. Conserved domain analysis of the HbTRXh2 amino acid sequence using CDD (http: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) revealed the presence of a conserved thioredoxin (TRX) domain located at amino acids 39–129, with positions 59–62 being highly conserved CGPC redox active sites. Figure 1 ).

[0081] Phylogenetic trees were constructed using ClustalW and MEGA11 software to group HbTRXh2 with different types of plant thioredoxins. The results showed that these thioredoxins clustered into seven classes: m, f, h, o, x, y, and z. HbTRXh2 clustered with type h thioredoxins, thus belonging to the type h thioredoxin group. HbTRXh2 clustered with cassava MeTRXh2 (XP_021593822.1) and Jatropha curcas JcTRXh2 (XP_012082815.1), indicating the closest phylogenetic relationship. Figure 2 ).

[0082] Example 2, Rubber Tree HbTRXh2 Gene expression characteristics analysis

[0083] 1. HbTRXh2 Analysis of tissue expression characteristics of genes

[0084] Latex, bark, new shoots, stable-stage leaves, senescent leaves, female flowers, male flowers, and root tissue samples were collected from rubber trees (Heat Research 7-33-97, 16 years old) and stored in liquid nitrogen. Total RNA was extracted from each tissue sample using a universal plant total RNA extraction kit (Beijing Biotech Biotechnology Co., Ltd.). Specific extraction methods were followed according to the kit instructions. First-strand cDNA was synthesized via reverse transcription using the PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa). Specific implementation methods were followed according to the kit instructions. Real-time quantitative PCR (qRT-PCR) analysis was performed using the cDNA from each tissue as a template, employing the primer pairs described below. HbUBC4 Genes are used as internal reference genes.

[0085] Used for detection HbTRXh2 The primer sequences for the gene are as follows:

[0086] F2: 5′-ATTTTGCTGCATCGTGGTGT-3′, SEQ ID NO.6;

[0087] R2: 5′-ACGTTGGCATAGCCTGAACT-3′, SEQ ID NO.7.

[0088] Used for detection HbUBC4 The primer sequences for the gene are as follows:

[0089] F3: 5′-TCACCCTGAACCTGATAGCC-3′, SEQ ID NO.8;

[0090] R3: 5′-TTTCTTTGGTGACGCTGCAA-3′, SEQ ID NO.9.

[0091] qRT-PCR was performed using a Bio-Rad CFX96 real-time PCR instrument, with each parallel experiment having three replicates. The reaction mixture consisted of 10 μL SYBR Premix Ex Taq (2×) (TaKaRa), 1 μL each of the forward and reverse primers, 2 μL cDNA template, and ddH2O added to a total volume of 20 μL. Reaction conditions were: 95℃ for 5 min; 95℃ for 10 s; 60℃ for 30 s; after 40 cycles, melting curve analysis was performed to determine primer specificity. The relative gene expression level was calculated using Formula 2. –ΔΔCt .

[0092] HbTRXh2 The expression results of genes in various tissues of rubber trees are as follows: Figure 3As shown, latex was detected in latex, bark, roots, mature leaves, senescent leaves, female flowers, male flowers, and new shoots. HbTRXh2 Gene expression varies in different tissues, with the expression levels from highest to lowest being: stable leaves, latex, male flowers, roots, senescent leaves, female flowers, bark, and new shoots. HbTRXh2 The expression level was highest in leaves during the stationary phase, significantly higher than in other tissues. P <0.05).

[0093] 2. Under abiotic stress HbTRXh2 Analysis of changes in gene expression

[0094] To clarify the effects of abiotic stress HbTRXh2 This invention analyzes gene expression changes under low temperature, PEG-induced drought, salt, and hydrogen peroxide (H2O2)-induced oxidative stress. HbTRXh2 Gene expression.

[0095] Low-temperature stress treatment group: Healthy rubber tree tissue culture seedlings (Heat Research 7-33-97) cultured for 8 months were placed in a 4℃ artificial climate chamber for low-temperature treatment. The treatment time was 16 hours, and the intensity was 600 μmol / (m²). 2 •s). Second and third leaves were collected from plants at 0, 3, 6, 12, 24, and 48 h after treatment, with three biological replicates at each time point, and three plants per replicate. Samples were cryopreserved in liquid nitrogen after collection. Total RNA was extracted from the leaves according to the method in step 1, and first-strand cDNA was synthesized by reverse transcription and detected by qRT-PCR. HbTRXh2 Gene expression in each sample. Leaf samples treated for 0 h (untreated) were used as controls.

[0096] PEG6000-induced drought stress treatment group: Healthy rubber tree tissue culture seedlings (Heterotrophication 7-33-97) cultured for 8 months were removed from their seedling bags, washed to remove the cultivation substrate, and their roots were immersed in a 20% PEG6000 solution (PEG6000 as the solute and sterile distilled water as the solvent). The second and third leaves of the plants were collected at 0, 3, 6, 12, 24, and 48 hours after treatment. Three biological replicates were established for each treatment time point, with three plants per replicate. Samples were cryopreserved in liquid nitrogen after collection. Total RNA was extracted from the leaves according to the method described in step 1, and first-strand cDNA was synthesized by reverse transcription and detected by qRT-PCR. HbTRXh2 Gene expression in each sample. Leaf samples treated for 0 h (untreated) were used as controls.

[0097] NaCl-induced salt stress treatment group: Healthy rubber tree tissue culture seedlings (Heat Research 7-33-97) cultured for 8 months were removed from their seedling bags, washed to remove the cultivation substrate, and their roots were immersed in a 400 mM NaCl solution (solute: NaCl, solvent: sterile distilled water). The second and third leaves of the plants were collected at 0, 3, 6, 12, 24, and 48 h of treatment, with three biological replicates at each time point, and three plants per replicate. Samples were cryopreserved in liquid nitrogen after collection. Total RNA was extracted from the leaves according to the method described in step 1, and the first-strand cDNA was synthesized by reverse transcription and detected by qRT-PCR. HbTRXh2 Gene expression in each sample. Leaf samples treated for 0 h (untreated) were used as controls.

[0098] Hydrogen peroxide (H2O2)-induced oxidative stress treatment group: Healthy rubber tree seedlings of the Reyan 7-33-97 tissue culture variety, transplanted for 8 months, were selected. A 20 mM H2O2 solution was evenly sprayed onto both sides of all leaves. The second and third leaves were collected at 0, 3, 6, 12, 24, and 48 hours after treatment, with three biological replicates at each time point, and three plants per replicate. Samples were cryopreserved in liquid nitrogen after collection. Total RNA was extracted from the leaves according to the method described in step 1, and first-strand cDNA was synthesized by reverse transcription and detected by qRT-PCR. HbTRXh2 Gene expression in each sample. Leaf samples treated for 0 h (untreated) were used as controls.

[0099] The results showed that after 6 and 48 hours of low-temperature stress treatment, HbTRXh2 Gene expression was significantly higher than the control (0h), with the highest expression level at 48h, which was 2.2 times that of the control. Figure 4 ); during PEG6000-induced drought stress treatment at 12 and 48 h, HbTRXh2 Gene expression was significantly higher than the control (0h), with the highest expression level at 48h, which was 2.2 times that of the control. Figure 5 ); at 6, 12 and 48 h of salt stress treatment, HbTRXh2 Gene expression was significantly higher than the control (0h), with the highest expression level observed at 12h, which was 2.5 times that of the control. Figure 6 ); during H2O2-induced oxidative stress treatment for 6 and 48 h, HbTRXh2 Gene expression was significantly higher than the control (0h), with the highest expression level at 48h, which was 2.0 times that of the control. Figure 7 The above results indicate that... HbTRXh2 Gene expression is induced by low temperature, drought, salt, and oxidative stress.

[0100] Example 3: Expression in yeast HbTRXh2 Genes enhance cold resistance and antioxidant capacity

[0101] 1. HbTRXh2 Construction of yeast gene expression vector

[0102] Based on SEQ ID No. 3 in the sequence listing, primer pairs were designed to amplify the coding region (nucleotides 104 to 508 at the 5′ end). Restriction endonucleases were introduced at the 5′ ends of the forward and reverse primers, respectively. Kpn I and Xba The recognition sequence for I (the recognition sequence is shown in the underlined part of the primers below) is as follows:

[0103] F5: 5'- GGTACC ATGGGAGGTTTTTCTCTGCT-3′, SEQ ID NO.10;

[0104] R5: 5′- TCTAGA TCAAGTGGCCCTGTGTTTC-3', SEQ ID NO. 11.

[0105] Using the leaf cDNA as a template, the target gene was amplified by PCR. The PCR reaction system consisted of: 2 μL cDNA template, 5 μL 5×TransStart FastPfu Buffer, 2 μL 2.5 mM dNTPs, 0.5 μL each of forward and reverse primers F5 and R5 (10 μM), 0.5 μL TransStart FastPfu DNA Polymerase (2.5 U / L), and ddH2O added to a total volume of 25 μL. The reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 20 s, 55℃ annealing for 20 s, 72℃ extension for 30 s, for a total of 35 cycles; and 72℃ extension for 10 min.

[0106] PCR products were purified by agarose gel electrophoresis and ligated into pEASY-Blunt Simple Cloning Vector (Beijing TransGen Biotech Co., Ltd.). The ligation product was then transformed into *E. coli* Trans1-T1 Phage Resistant Chemically Competent Cell. Positive clones were selected and sent to PlatinumSure Biotechnology (Hainan) Co., Ltd. for sequencing. Plasmids that were correctly sequenced and the empty yeast expression vector pYES2 (Invitrogen) were extracted using a plasmid extraction kit (OMEGA). Restriction endonucleases were then used to extract the plasmids. Kpn I and Xba I (Thermo Scientific) performed double digestion.

[0107] Double enzyme digestion system: Kpn I (10 U / μL) 2μL, Xba1 μL of enzyme digestion solution (10 U / μL), 3 μL of 10×Tango Buffer, 8 μL of plasmid DNA, and ddH2O added to a final volume of 30 μL. Incubate at 37°C for 3 h. After electrophoresis of the digested products on a 1.0% agarose gel, the target band was excised and recovered.

[0108] The recovered target gene and the pYES2 vector backbone were ligated using T4 DNA ligase. The ligation system consisted of 6 μL of the recovered target gene product, 2 μL of the pYES2 vector backbone, 1 μL of T4 DNA ligase, and 1 μL of 10× Buffer. Ligation was carried out at 22°C for 3 h. The ligation product was transformed into E. coli DH5α competent cells and then plated on LB agar plates containing ampicillin (100 mg / L) and incubated overnight at 37°C.

[0109] Single-clone bacteria were picked and cultured. Positive screening was performed using pYES2 vector primers (F6: 5′-CCCGGATCGGACTACTAGC-3′, SEQ ID NO. 12) plus a gene-specific reverse primer (R5). Positive clones were further sequenced for identification. HbTRXh2 The gene is expressed in yeast using the vector pYES2-HbTRXh2. pYES2-HbTRXh2 uses nucleotides from positions 104 to 508 of SEQ ID No. 3. HbTRXh2 (The coding region sequence of the gene) replaces pYES2. Kpn I and Xba The recombinant plasmid was obtained by separating the fragment between the I recognition sites while keeping the other nucleotide sequences of pYES2 unchanged. The amino acid sequence expressed by pYES2-HbTRXh2 is the protein HbTRXh2, which is SEQ ID No. 5 in the sequence listing. The E. coli containing pYES2-HbTRXh2 is named DH5a(pYES2-HbTRXh2).

[0110] 2. Obtaining pYES2-HbTRXh2 and pYES2-converted yeast

[0111] Saccharomyces cerevisiae strain INVScl (Invitrogen) is a urea auxotroph (Ura - The yeast strain (pYES2) showed almost no growth or reproduction on SC-Ura medium. URA3The gene was identified, and its expression enabled the yeast transformants to grow normally on SC-Ura medium. Therefore, SC-Ura medium can be used to screen positive yeast transformants. The pYES2 and pYES2-HbTRXh2 plasmids were extracted and transformed into competent cells of *Saccharomyces cerevisiae* strain INVSC1 using the lithium acetate method. The transformed yeast culture was plated on SC-Ura solid selective medium and incubated at 30°C for 2-3 days. Single yeast clones were picked, plasmid DNA was extracted, and positive transformants were identified by PCR. The specific steps are as follows:

[0112] 1) Pick a single yeast INVSC1 clone and add it to 10 mL of YPD liquid medium. Shake overnight at 30°C and 200 rpm.

[0113] 2) Detection of OD in yeast culture 600 To determine the OD value, add overnight cultured yeast culture to 50 mL of YPD liquid medium and dilute to OD. 600 Set the pH to 0.4 and continue shaking at 30℃ and 200rpm for 2-4 hours.

[0114] 3) Centrifuge at 2500 rpm for 5 min at 4℃, discard the supernatant and collect the bacterial cells, then resuspend the bacterial cells in 40 mL of l×TE buffer.

[0115] 4) Centrifuge again at 2500 rpm for 5 min at 4℃, discard the supernatant and collect the bacterial cells, and resuspend the bacterial cells in 2 mL of l×LiAc / 0.5×TE.

[0116] 5) Dispense the obtained resuspended bacterial cells into 1.5mL centrifuge tubes, 100μL per tube.

[0117] 6) Incubate the dispensed yeast cells at room temperature for 10 minutes.

[0118] 7) In each transformation system (100 μL), add 1 μg of plasmid extracted from DH5a(pYES2-HbTRXh2) culture prepared in step 1 and 100 μg of denatured salmon sperm DNA, and mix well.

[0119] 8) Add 700 μL l×LiAc / 40% PEG-3350 / l×TE and mix well.

[0120] 9) Incubate at 30℃ for 30 minutes.

[0121] 10) Add 88 μL of DMSO, mix well, and heat shock at 42 °C for 7 min.

[0122] 11) Centrifuge at 4℃ and 5000rpm for 1min, then discard the supernatant.

[0123] 12) Resuspend the bacterial cells in 1 mL of 1×TE buffer, centrifuge at 4°C and 5000 rpm for 1 min, and discard the supernatant.

[0124] 13) Resuspend the bacterial cells in 100 μL of l×TE buffer and plate them onto SC-Ura solid selective medium. Incubate at 30°C with the medium inverted for 2–3 days. Pick three INVSC1(pYES2-HbTRXh2) monoclonal samples from the SC-Ura solid selective medium and place each sample in 3 mL of SC-Ura liquid selective medium. Incubate at 30°C with shaking at 200 rpm for 24 h. Extract plasmid DNA using a yeast plasmid extraction kit (OMEGA). Using the yeast plasmid DNA as a template, perform PCR detection of the bacterial culture using primers F6 and R5. The positive clone is a recombinant yeast containing pYES2-HbTRXh2, named INVSC1(pYES2-HbTRXh2).

[0125] Following the above method, pYES2-HbTRXh2 was replaced with pYES2, while all other steps remained unchanged. PCR positive detection was performed using pYES2 vector primers F6 (5′-CCCGGATCGGACTACTAGC-3′, SEQ ID NO.12) and R6 (5′-ATTAAAGCCTTCGAGCGTCC-3′, SEQ ID NO.13), yielding a recombinant yeast containing pYES2, which was named INVSC1(pYES2).

[0126] PCR products were subjected to agarose gel electrophoresis as follows: Figure 8 As shown, three recombinant yeast plasmids transformed with the empty pYES2 vector amplified and compared with the positive control (pYES2 plasmid). Figure 8 Bands of uniform size in P1) Figure 8 (1, 2, 3), and the length met the expected 402bp; three recombinant yeast plasmids transformed with pYES2-HbTRXh2 were amplified and compared with the positive control (pYES2-HbTRXh2 plasmid, Figure 8 Bands of uniform size in P2 (middle) Figure 8 The results show that pYES2-HbTRXh2 and the empty vector pYES2 were successfully transformed into recombinant yeast (4, 5, 6), and the length met the expected 467bp.

[0127] 3. In recombinant yeast HbTRXh2 Detection of gene expression

[0128] The INVSC1(pYES2-HbTRXh2) and INVSC1(pYES2) positive clones obtained in step 2 were streaked onto SC-Ura solid selective medium and incubated upside down at 30°C for 2–3 days. Single clones were picked and inoculated into 2 mL of SC-Ura liquid selective medium and incubated at 30°C with shaking at 200 rpm for 24 h. Their OD values ​​were then measured. 600 Take 1 mL of the cultured bacterial suspension, centrifuge at 4000 rpm for 3 min, discard the supernatant, resuspend the bacterial cells in SC-Ura liquid induction medium, and adjust the OD value. 600 =0.2, take 10mL of each, and induce culture at 30℃ and 200rpm for 24h. Then, take 2mL of bacterial culture from each, collect the cells by low-speed centrifugation, and extract total RNA using a yeast total RNA rapid extraction kit (Sangon Biotech (Shanghai) Co., Ltd.). First-strand cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa), following the kit instructions. Firstly, the Saccharomyces cerevisiae internal reference gene was used. Actin (GeneBank accession number: L00026.1) Determine the amount of cDNA added for each template. Actin The primer sequences are as follows:

[0129] F7: 5′-AGTTGCCCCAGAAGAACACC-3′, SEQ ID NO.14;

[0130] R7: 5′-TACCGGCAGATTCCAAACCC-3′, SEQ ID NO. 15.

[0131] Reuse HbTRXh2 Gene primers (F5 and R5) were used to amplify the template by PCR. The PCR reaction system consisted of: 1-3 μL template, 2.5 μL buffer, 2 μL dNTPs, 0.5 μL each of forward and reverse primers, 0.5 μL EasyTaq DNA Polymerase (Beijing TransGen Biotech Co., Ltd.), and ddH2O added to a total volume of 25 μL. The reaction program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; and 72℃ extension for 10 min.

[0132] The results are as follows Figure 9 As shown, after 24 hours of induction culture, the recombinant yeast INVSC1(pYES2-HbTRXh2) was detected in all three clones. HbTRXh2 Gene expression ( Figure 9 (4, 5, 6) were detected in the control yeast INVSC1 (pYES2), while no detection was found in the control yeast. HbTRXh2Gene expression ( Figure 9 (1, 2, 3). The above results indicate that... HbTRXh2 The gene was successfully transferred into recombinant yeast INVSC1(pYES2-HbTRXh2) and expressed.

[0133] 4. Compare the cold resistance differences between recombinant yeast INVSC1(pYES2-HbTRXh2) and INVSC1(pYES2).

[0134] The INVSC1(pYES2-HbTRXh2) and INVSC1(pYES2) positive clones obtained in step 2 were streaked onto SC-Ura solid selective medium and incubated upside down at 30°C for 2-3 days. Single clones of INVSC1(pYES2-HbTRXh2) and INVSC1(pYES2) were picked and inoculated into 5 mL of SC-Ura liquid selective medium and cultured at 30°C with shaking at 200 rpm for 24 h. The cells were centrifuged at 4000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in SC-Ura liquid induction medium, with the OD adjusted. 600 =0.2, to obtain a resuspended bacterial solution. Take 10 mL of the resuspended bacterial solution and incubate at 30℃ and 200 rpm for 24 h to induce induction. HbTRXh2 Gene expression. Take 5 mL of induced culture of recombinant yeast INVSC1(pYES2-HbTRXh2) and control yeast INVSC1(pYES2), centrifuge at 4000 rpm for 5 min to collect the cells, resuspend the cells in sterile water, and adjust the OD. 600 Up to version 2.0.

[0135] Low temperature stress treatment group: OD was collected separately. 600 1 mL of INVSc1(pYES2-HbTRXh2) and INVSc1(pYES2) bacterial suspensions (2.0 g / L) were mixed with 1 mL of sterile distilled water. The mixed bacterial suspensions were placed in a -20°C freezer. Every 12 hours, the suspensions were removed, thawed, and then placed back in the -20°C freezer for further treatment. On days 5 and 7 of treatment, 1 mL of the treated bacterial suspensions were taken out and serially diluted 10-fold with sterile distilled water (10⁻¹⁰). -1 10 -2 10 -3 10 -4 Five μL of undiluted bacterial suspension and bacterial suspensions diluted at different ratios were spotted onto SC-Ura solid selective medium, with each treatment repeated three times. The culture was incubated upside down at 30°C for three days, and the growth differences between recombinant yeast INVSC1(pYES2-HbTRXh2) and INVSC1(pYES2) colonies were observed and compared.

[0136] Control group (no stress treatment): OD was collected separately. 6001 mL of INVSC1(pYES2-HbTRXh2) and INVSC1(pYES2) bacterial suspensions (2.0 g / mL) were added to 1 mL of sterile distilled water and mixed thoroughly. The mixed bacterial suspensions were then serially diluted 10-fold with sterile distilled water (10⁻¹⁰). -1 10 -2 10 -3 10 -4 Five μL of undiluted bacterial suspension and bacterial suspensions diluted at different ratios were spotted onto SC-Ura solid selective medium, with each treatment repeated three times. The culture was incubated upside down at 30°C for three days, and the growth differences between recombinant yeast INVSC1(pYES2-HbTRXh2) and INVSC1(pYES2) colonies were observed and compared.

[0137] The growth of the yeast in each group is as follows: Figure 10 As shown. In the control group, at the same dilution, there was no significant difference in the number of plaques between INVSc1(pYES2-HbTRXh2) and INVSc1(pYES2). In the 5-day low-temperature stress (-20℃) treatment group, undiluted and 10 -1 and 10 -2 When diluted bacterial suspensions were spotted onto plates, the number of plaques grown by INVSC1(pYES2-HbTRXh2) was significantly higher than that of INVSC1(pYES2). In the group treated with low-temperature stress (-20℃) for 7 days, 10 -2 After dilution, INVSc1(pYES2-HbTRXh2) still showed plaque growth, while INVSc1(pYES2) showed no plaque growth; using undiluted and 10 -1 When diluted bacterial suspensions were spotted onto plates, INVSC1(pYES2-HbTRXh2) produced significantly more plaques than INVSC1(pYES2). These results indicate that... HbTRXh2 The gene improved the survival rate of recombinant yeast after low-temperature stress (-20℃) treatment, and its expression... HbTRXh2 The gene enhanced the cold resistance of the recombinant yeast.

[0138] 5. Compare the antioxidant properties of recombinant yeast INVSC1(pYES2-HbTRXh2) and INVSC1(pYES2).

[0139] The INVSC1(pYES2-HbTRXh2) and INVSC1(pYES2) positive clones obtained in step 2 were streaked onto SC-Ura solid selective medium and incubated upside down at 30°C for 2-3 days. Single clones of INVSC1(pYES2-HbTRXh2) and INVSC1(pYES2) were picked and inoculated into 5 mL of SC-Ura liquid selective medium and cultured at 30°C with shaking at 200 rpm for 24 h. The cells were centrifuged at 4000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in SC-Ura liquid induction medium, with the OD adjusted. 600 =0.2, to obtain a resuspended bacterial solution. Take 10 mL of the resuspended bacterial solution and incubate at 30℃ and 200 rpm for 24 h to induce induction. HbTRXh2 Gene expression. Take 5 mL of induced culture of recombinant yeast INVSC1(pYES2-HbTRXh2) and control yeast INVSC1(pYES2), centrifuge at 4000 rpm for 5 min to collect the cells, resuspend the cells in sterile water, and adjust the OD. 600 Up to version 2.0.

[0140] Oxidative stress treatment group: OD was taken 600 1 mL of bacterial suspension with a concentration of 2.0 μM was added to 1 mL of 40 mM H₂O₂ solution and mixed well to obtain a 20 mM H₂O₂ treatment group bacterial suspension. The treatment group bacterial suspension was placed in a shaker at 30℃ and 160 rpm for treatment. At 10 h and 20 h of treatment, 1 mL of each solution was taken and serially diluted 10-fold with sterile distilled water (10...). -1 10 -2 10 -3 10 -4 Five μL of undiluted bacterial suspension and bacterial suspensions diluted at different ratios were spotted onto SC-Ura solid selective medium, with each treatment repeated three times. The culture was incubated upside down at 30°C for three days, and the growth differences between recombinant yeast INVSC1(pYES2-HbTRXh2) and INVSC1(pYES2) colonies were observed and compared.

[0141] Control group (no stress treatment): OD was collected. 600 Add 1 mL of 2.0 μL bacterial culture to 1 mL of sterile distilled water and mix well. Then, perform a 10-fold serial dilution of the mixed bacterial culture with sterile distilled water (10...). -1 10 -2 10 -3 10 -4Five μL of undiluted bacterial suspension and bacterial suspensions diluted at different ratios were spotted onto SC-Ura solid selective medium, with each treatment repeated three times. The culture was incubated upside down at 30°C for three days, and the growth differences between recombinant yeast INVSC1(pYES2-HbTRXh2) and INVSC1(pYES2) colonies were observed and compared.

[0142] The growth of the yeast in each group is as follows: Figure 11 As shown. In the control group, at the same dilution, there was no significant difference in the number of plaques between INVSc1(pYES2-HbTRXh2) and INVSc1(pYES2). In the group treated with 20mM H2O2 for 10h, the number of plaques was significantly different. -3 After dilution, INVSc1(pYES2-HbTRXh2) still showed plaque growth, while INVSc1(pYES2) showed no plaque growth; using 10 -1 and 10 -2 When diluted bacterial suspensions were spotted onto the plate, the number of plaques grown by INVSC1(pYES2-HbTRXh2) was significantly higher than that of INVSC1(pYES2). In the 20mH2O2 treatment group for 20h, undiluted and 10... -1 and 10 -2 When diluted bacterial suspensions were spotted onto plates, the number of plaques grown by INVSC1(pYES2-HbTRXh2) was significantly higher than that of INVSC1(pYES2). These results indicate that... HbTRXh2 The gene improved the survival rate of recombinant yeast after high-concentration H2O2-induced oxidative stress treatment, and its expression... HbTRXh2 The gene enhanced the antioxidant capacity of the recombinant yeast.

[0143] 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. Use of a Hevea brasiliensis thioredoxin or a biological material related to thioredoxin, characterized in that, The application is any one of the following: (1) application in improving the cold resistance of yeast; (2) application in breeding yeast strains with improved cold resistance; (3) application in improving the oxidation resistance of yeast; (4) application in breeding yeast strains with improved oxidation resistance; The amino acid sequence of the sulfiredoxin is shown in SEQ ID NO. 5; The biological material is any one of the following: (1) a nucleic acid molecule encoding the sulfiredoxin; (2) an expression cassette containing the nucleic acid molecule in (1); (3) a recombinant vector containing the nucleic acid molecule in (1); (4) a recombinant vector containing the expression cassette in (2).

2. Use according to claim 1, characterized in that, The nucleic acid molecule is a cDNA molecule or a DNA molecule with a nucleotide sequence shown in SEQ ID NO. 3, or a cDNA molecule or a DNA molecule with a nucleotide sequence shown in positions 104-508 of SEQ ID NO.

3.

3. A method of breeding a yeast strain with improved cold tolerance, characterized in that, The method comprises increasing the expression amount of the gene encoding the sulfiredoxin in claim 1 in a recipient yeast to obtain a transgenic yeast with higher cold resistance than the recipient yeast; and the increase of the expression amount of the gene encoding the sulfiredoxin in the recipient yeast is achieved by introducing the gene encoding the sulfiredoxin into the recipient yeast.

4. A method of breeding a yeast strain with improved antioxidant properties, characterized in that, The method comprises increasing the expression amount of the gene encoding the sulfiredoxin in claim 1 in a recipient yeast to obtain a transgenic yeast with higher oxidation resistance than the recipient yeast; and the increase of the expression amount of the gene encoding the sulfiredoxin in the recipient yeast is achieved by introducing the gene encoding the sulfiredoxin into the recipient yeast.

5. A recombinant yeast having improved cold or oxidative resistance, wherein the yeast is a Saccharomyces cerevisiae strain having a mutation in the YGLO 18 gene. It is obtained by introducing the gene encoding the sulfiredoxin in claim 1 into a Saccharomyces cerevisiae strain through a recombinant vector containing a gene expression cassette.

6. The recombinant yeast of claim 5, wherein, The recombinant vector is obtained by replacing the sequence between the recognition sites of I and I of the yeast expression vector pYES2 Kpn I and Xba I and I recognition sites with a DNA molecule having a nucleotide sequence as shown in SEQ ID NO. 4.

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