CN116640738B - Thioredoxin HbTRXy2 from rubber tree, related biological material and application thereof - Google Patents

Abstract

The invention discloses thioredoxin HbTRXy2 from rubber trees and related biological materials and application thereof, belonging to the technical field of biology. The y-type thioredoxin HbTRXy of the rubber tree provided by the invention is A1) or A2) as follows: a1 Amino acid sequence is protein shown as sequence 1 in a sequence table; a2 A protein derived from A1) and having the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the sequence 1. Experiments prove that the expression of HbTRXy coding genes in yeast can improve the resistance of recombinant yeast to oxidative stress, which shows that HbTRXy coding genes have the function of resisting oxidation and can be used for improving the oxidation resistance of plants or microorganisms.

Description

Thioredoxin HbTRXy2 from rubber tree and its related biomaterials and applications

Technical Field

The invention relates to the field of biotechnology, and in particular to thioredoxin derived from rubber trees and related biological materials and applications thereof.

Background Art

Natural rubber is an important industrial raw material and an indispensable strategic material. Rubber tree (Heveabrasiliensis Muell.Arg.) is the main source of natural rubber. my country is the world's largest consumer of natural rubber, with an annual consumption of about 5.5 million tons, but my country's annual production is only 800,000 tons, and the self-sufficiency rate is less than 15%. my country mainly grows rubber trees in some areas of Yunnan Province, Hainan Province and Guangdong Province. These areas belong to non-traditional rubber planting areas. Rubber trees are often subjected to abiotic stress such as low temperature, drought and typhoons during their growth. In addition, the occurrence of dead bark (or tapping panel dryness; the main symptom is that the tapping line does not discharge rubber partially or completely after tapping) of rubber trees in China's rubber plantations is also relatively serious. At present, the dead bark rate of my country's rubber plantations is generally above 30%, resulting in serious production and economic losses. Whether abiotic stress or dead bark is accompanied by the occurrence of oxidative stress, regulating and maintaining cellular redox balance is of great significance for rubber trees to resist abiotic stress and reduce the occurrence of dead bark.

Summary of the invention

The technical problem to be solved by the present invention is how to improve the resistance of organisms to oxidative stress and other abiotic stresses and/or determine the function and application of rubber tree thioredoxin HbTRXy2. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

In order to solve the above technical problems, the present invention provides the following technical solutions:

The object of the present invention is to provide an application of a protein, a substance for regulating the expression of a gene encoding the protein, or a substance for regulating the content of the protein, wherein the application is any of the following:

D1) Application in regulating biological oxidative stress resistance;

D2) Application in the preparation of products for regulating biological oxidative stress resistance;

D3) Application in breeding biological species with improved antioxidant capacity;

D4) Application in regulating the abiotic stress resistance of organisms;

D5) Use in the preparation of products for regulating the resistance of organisms to abiotic stress;

D6) Application in breeding biological varieties with improved resistance to abiotic stress;

The protein is named HbTRXy2, which is derived from the rubber tree (Hevea brasiliensis Muell.Arg.) of the Euphorbiaceae family, and is any of the following:

A1) the amino acid sequence is the protein of SEQ ID No. 1, consisting of 168 amino acids;

A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by substitution and/or deletion and/or addition of amino acid residues of the amino acid sequence shown in SEQ ID No.1;

A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and/or C-terminus of A1) or A2).

The tag proteins described herein include but are not limited to Poly-Arg tag proteins, which contain 5-6 amino acid residues (usually 5, with a sequence of RRRRR), or Poly-His tag proteins, which contain 2-10 amino acid residues (usually 6, with a sequence of HHHHHH), or FLAG tag proteins, which contain 8 amino acid residues (with a sequence of DYKDDDDK), or Strep-tag II tag proteins, which contain 8 amino acid residues (with a sequence of WSHPQFEK), or c-myc tag proteins, which contain 8 amino acid residues (with a sequence of WSHPQFEK).

The HbTRXy2 in A2) above can be synthesized artificially, or its coding gene can be synthesized first and then expressed biologically. The coding gene of HbTRXy2 in A2) above can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in SEQ ID No. 2 in the sequence table, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the aforementioned tag at its 5′ end and/or 3′ end.

In order to solve the above technical problems, the present invention also provides the use of the biomaterial related to the above HbTRXy2 protein in any of the following:

D1) Application in regulating biological oxidative stress resistance;

D2) Application in the preparation of products for regulating biological oxidative stress resistance;

D3) Application in breeding biological species with improved antioxidant capacity;

D4) Application in regulating the abiotic stress resistance of organisms;

D5) Use in the preparation of products for regulating the resistance of organisms to abiotic stress;

D6) Application in breeding biological varieties with improved resistance to abiotic stress;

The biological material is any one of the following B1) to B7):

B1) a nucleic acid molecule encoding the aforementioned protein;

B2) an expression cassette containing the nucleic acid molecule described in B1);

B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

B5) a transgenic plant cell line containing the nucleic acid molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2);

B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2);

B7) A transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2).

In the above biological material, the nucleic acid molecule described in B1) may be the gene of b1) or b2) or b3) or b4) as follows:

b1) the nucleotide sequence is the cDNA molecule or DNA molecule at positions 179 to 685 of SEQ ID No. 2 in the sequence list;

b2) the nucleotide sequence is a cDNA molecule or a DNA molecule of SEQ ID No. 2 in the sequence list;

b3) a cDNA molecule or genomic DNA molecule that has more than 90% identity with the nucleotide sequence defined in b1) or b2) and encodes HbTRXy2;

b4) a cDNA molecule or genomic DNA molecule that hybridizes with the nucleotide sequence defined in b1) or b2) under stringent conditions and encodes HbTRXy2.

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

Among them, SEQ ID No.2 consists of 852 nucleotides, and positions 179-685 are the coding region, encoding HbTRXy2 shown in SEQ ID No.1.

A person skilled in the art can easily mutate the nucleotide sequence encoding HbTRXy2 of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 90% or higher identity with the nucleotide sequence encoding HbTRXy2 isolated from the present invention are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode HbTRXy2 and have the function of HbTRXy2.

The term "identity" as used herein refers to sequence similarity with a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 90% or more, or 95% or more identity with the nucleotide sequence of the protein consisting of the amino acid sequence shown in HbTRXy2 of the present invention. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

The above stringent conditions are hybridization and washing at 68°C in a 2×SSC, 0.1% SDS solution for 5 min each time, and hybridization and washing at 68°C for 15 min each time in a 0.5×SSC, 0.1% SDS solution for 15 min each time; or hybridization and washing at 65°C in a 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution.

The above-mentioned 90% or more identity may be 90% or 95% or more identity.

In the above-mentioned biological material, the expression cassette (HbTRXy2 gene expression cassette) containing the nucleic acid molecule encoding HbTRXy2 described in B2) refers to a DNA capable of expressing HbTRXy2 in a host cell, and the DNA may include not only a promoter for initiating transcription of the HbTRXy2 gene, but also a terminator for terminating transcription of the HbTRXy2 gene. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present 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; the chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); the tomato proteinase inhibitor II promoter (PIN2) or the LAP promoter (both can be induced by methyl jasmonate); heat shock promoters; tetracycline-inducible promoters; seed-specific promoters, such as millet seed-specific promoter pF128, seed storage protein-specific promoters (e.g., promoters of phaseolin, 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, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators.

The recombinant vector containing the HbTRXy2 gene expression cassette can be constructed with an existing expression vector. The plant expression vector includes a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA company), etc. The plant expression vector can also include the 3' non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as the Agrobacterium crown gall induction (Ti) plasmid gene (such as the nopaline synthase gene Nos), the non-translated region transcribed at the 3' end of the plant gene (such as the soybean storage protein gene) all have similar functions. When using the gene construction plant expression vector of the present invention, enhancers can also be used, including translation enhancers or transcription enhancers. 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 the correct translation of the entire sequence. The source of the translation control signal and the start codon is extensive and can be natural or synthetic. The translation initiation region can come from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene that confers resistance to kanamycin and related antibiotics, bar gene that confers resistance to the herbicide phosphinothricin, hph gene that confers resistance to the antibiotic hygromycin, dhfr gene that confers resistance to methotrexate, EPSPS gene that confers resistance to glyphosate) or chemical resistance marker genes (such as herbicide resistance genes), mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker gene can be added, and transformed plants can be directly screened by adversity.

In the above biological material, the vector can be a plasmid, a cosmid, a phage or a virus vector. The plasmid can be a yeast expression vector pYES2.

In the above biological material, the microorganism can be yeast, bacteria, algae or fungi, such as Escherichia coli. The yeast can be Saccharomyces cerevisiae strain INVScl.

In the above biological materials, the transgenic plant cell line does not include propagation materials.

The organism described herein may be a microorganism, such as yeast.

In one embodiment of the present invention, the gene encoding HbTRXy2 is introduced into the Saccharomyces cerevisiae strain INVSC1 through a recombinant vector containing an HbTRXy2 gene expression cassette to obtain a recombinant microorganism. The recombinant vector is a recombinant vector obtained by replacing the sequence between the Kpn I and Xba I recognition sites of the yeast expression vector pYES2 with the DNA molecule shown in nucleotides 179-685 of SEQ ID No.2 in the sequence table. The recombinant vector expresses HbTRXy2 shown in SEQ ID No.1. The recombinant microorganism expresses HbTRXy2 shown in sequence 1.

In the above application, the oxidative stress may be oxidative stress induced by hydrogen peroxide. The abiotic stress may be one of low temperature, drought and salt stress.

Another object of the present invention is to provide a method for cultivating a transgenic organism with improved oxidative stress resistance, the method comprising increasing the expression level of the gene encoding the aforementioned protein in the target organism to obtain an antioxidant organism with higher oxidative stress resistance than the target organism.

Furthermore, the increasing the expression level of the gene encoding the protein in the target organism is achieved by introducing the gene encoding the aforementioned protein into the target organism.

In the above method, the nucleic acid molecule in B1) can be first modified as follows and then introduced into the recipient organism to achieve a better expression effect:

1) Modification and optimization according to actual needs to achieve efficient gene expression; for example, the codons of the HbTRXy2 encoding gene of the present invention can be changed to conform to the plant preference while maintaining the nucleotide sequence of the HbTRXy2 encoding gene of the present invention according to the codons preferred by the recipient plant; during the optimization process, it is best to maintain a certain GC content in the optimized coding sequence to best achieve high-level expression of the introduced gene in the plant, wherein the GC content can be 35%, more than 45%, more than 50% or more than about 60%;

2) Modifying the gene sequence adjacent to the initiation methionine to allow efficient initiation of translation; for example, using a sequence known to be effective in plants;

3) Connected to various plant-expressed promoters to facilitate their expression in plants; the promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters; the choice of promoter will vary with the temporal and spatial requirements of expression, and also depends on the target species; for example, a tissue or organ-specific expression promoter, depending on the stage of development at which the receptor is required; although it has been demonstrated that many promoters derived from dicotyledons can function in monocotyledons and vice versa, ideally, dicotyledon promoters are selected for expression in dicotyledons, and monocotyledon promoters are selected for expression in monocotyledons;

4) Connecting with a suitable transcription terminator can also improve the expression efficiency of the gene of the present invention; for example, tml derived from CaMV, E9 derived from rbcS; any available terminator known to work in plants can be connected with the gene of the present invention;

5) Introduction of enhancer sequences, such as intron sequences (e.g. from Adhl and bronzel) and viral leader sequences (e.g. from TMV, MCMV and AMV).

In the above method, the HbTRXy2 encoding gene can be the nucleic acid molecule described in B1).

In the above method, the recipient organism can be a microorganism or a plant. The target organism can be yeast, and the plant can be rubber tree.

In the above method, the oxidative stress may be oxidative stress induced by hydrogen peroxide.

The proteins and biological materials mentioned above also belong to the protection content of the present invention.

Experiments have shown that the HbTRXy2 gene of the present invention is expressed in various tissues of rubber trees, wherein the expression levels in the leaves of the color change period, the leaves of the light green period and the latex are higher. The expression of the HbTRXy2 gene is induced by oxidation, low temperature, drought and salt stress. The functional identification of the HbTRXy2 gene using a yeast expression system shows that overexpression of the HbTRXy2 gene in yeast can improve the resistance of recombinant yeast to the oxidative stress induced by hydrogen peroxide. The resistance to oxidative stress can be improved by overexpressing the HbTRXy2 gene in plants or microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the multiple sequence alignment results of HbTRXy2 from rubber tree and other plant y-type thioredoxins. The horizontal lines mark the conserved domains of thioredoxins, and the "*" marks the conserved redox active sites.

Figure 2 is a phylogenetic tree of rubber tree HbTRXy2 and other plant thioredoxins.

FIG. 3 shows the expression of HbTRXy2 gene in various tissues of rubber tree.

Figure 4 shows the effect of hydrogen peroxide (H ₂ O ₂ )-induced oxidative stress on the expression of HbTRXy2 gene in rubber trees, where different lowercase letters indicate significant differences among treatment time points (P<0.05).

Figure 5 shows the effect of methyl viologen (MV)-induced oxidative stress on the expression of HbTRXy2 gene in rubber trees. Different lowercase letters indicate significant differences among treatment time points (P<0.05).

Figure 6 shows the effect of low temperature stress on the expression of HbTRXy2 gene in rubber trees, where different lowercase letters indicate significant differences among treatment time points (P<0.05).

Figure 7 shows the effect of PEG-induced drought stress on the expression of HbTRXy2 gene in rubber trees, where different lowercase letters indicate significant differences among treatment time points (P<0.05).

Figure 8 shows the effect of salt stress on the expression of HbTRXy2 gene in rubber trees, where different lowercase letters indicate significant differences among treatment time points (P<0.05).

FIG. 9 shows the subcellular localization of HbTRXy2 in Hevea brasiliensis.

Figure 10 is the PCR positive detection of recombinant yeast INVSc1(pYES2-HbTRXy2) and INVSc1(pYES2). "M" represents DL2000 Plus DNAMarker (Vazyme); "N1" and "N2" are negative controls without template; "P1" and "P2" are plasmid positive controls; "1, 2, 3" are single clones of yeast transformed with pYES2-HbTRXy2; "4, 5, 6" are single clones of yeast transformed with pYES2.

Figure 11 is the expression detection of HbTRXy2 gene in recombinant yeast after 36h of induction culture. "M" represents DL2000Plus DNAMarker (Vazyme); "1, 2, 3" are cDNA samples of 3 monoclonal recombinant yeast INVSc1 (pYES2) after 36h of induction; "4, 5, 6" are cDNA samples of 3 monoclonal recombinant yeast INVSc1 (pYES2-HbTRXy2) after 36h of induction.

Figure 12 shows the difference in growth (OD ₆₀₀ value) between recombinant yeast INVSc1(pYES2-HbTRXy2) and INVSc1(pYES2) under oxidative stress induced by H ₂ O _2. "HbTRXy2" represents recombinant yeast INVSc1(pYES2-HbTRXy2); "pYES2" is the control recombinant yeast INVSc1(pYES2); "-CK" is the control group without H ₂ O ₂ treatment, and "-H ₂ O ₂ " is the 3 mM H ₂ O ₂ treatment group. * and ** indicate that there are significant differences between recombinant yeast INVSc1(pYES2-HbTRXy2) and INVSc1(pYES2) at the 0.05 and 0.01 levels, respectively.

Figure 13 shows the survival of recombinant yeast INVSc1(pYES2-HbTRXy2) and INVSc1(pYES2) after 24h of high concentration H ₂ O ₂ treatment, where "HbTRXy2" represents recombinant yeast INVSc1(pYES2-HbTRXy2); "pYES2" is the control recombinant yeast INVSc1(pYES2).

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

The quantitative tests in the following examples were performed in triplicate, and the experimental results were expressed as mean ± SD. The data were processed using SigmaPlot 12.0 statistical software. P < 0.05 (* or different lowercase letters) indicated a significant difference, and P < 0.01 (*) indicated an extremely significant difference.

Rubber tree (Hevea brasiliensis Muell.Arg.) Reyan 7-33-97: recorded in "Huang Huasun, Liang Maohuan, Wu Yuntong, Li Deshun, He Jinwei. Breeding of medium-scale promotion-grade rubber tree excellent variety Reyan 7-33-97. Journal of Tropical Crops, 1994, 15(2): 1-6.", cultivated by the Rubber Research Institute of the Chinese Academy of Tropical Agricultural Sciences, the public can obtain it from the Rubber Research Institute of the Chinese Academy of Tropical Agricultural Sciences. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

YPD liquid culture medium: a sterile liquid culture medium composed of a solute and a solvent, wherein the solute and its concentration are 1% (mass percentage concentration) yeast extract, 2% (mass percentage concentration) peptone and 2% (mass percentage concentration) glucose, and the solvent is water.

SC-Ura liquid selective medium (a sterile yeast basic medium lacking urine): 1L SC-Ura liquid selective medium is a medium obtained by adding 0.78g DO Supplement-Ura (Clontech), 6.7g amino-free yeast nitrogen base (YNB), and 20g glucose to deionized water and fixing the volume to 1L. 1L SC-Ura solid selective medium is a medium obtained by adding 20g agar powder to the above liquid medium.

SC-Ura liquid induction medium (SC-Ura liquid medium with 2% galactose as carbon source): 1L liquid induction medium is prepared by adding 0.78g DO Supplement-Ura, 6.7g amino-free yeast nitrogen source (YNB), and 20g galactose to deionized water and fixing the volume to 1L.

SC-Ura liquid induction medium containing 3 mM H ₂ O ₂ : H ₂ O ₂ was added to the SC-Ura liquid induction medium to obtain a SC-Ura liquid induction medium having a H ₂ O ₂ concentration of 3 mM.

10 mM and 20 mM H ₂ O ₂ aqueous solutions are prepared by adding H ₂ O ₂ to sterile distilled water to obtain aqueous solutions with H ₂ O ₂ concentrations of 10 mM and 20 mM, respectively.

pCAMBIA1300-GFP empty vector plasmid: recorded in "Deng Zhi, Zhao Manman, Liu Hui, Wang Yuekun, Li Dejun. Molecular cloning, expression profiles and characterization of a glutathione reductase in Hevea brasiliensis. Plant Physiology and Biochemistry.

2015,96:53-63." The public can obtain it from the Rubber Research Institute of the Chinese Academy of Tropical Agricultural Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

Example 1. Acquisition and sequence analysis of rubber tree thioredoxin HbTRXy2 and its encoding gene

Using the Arabidopsis thioredoxin gene AtTRXy2 (NM_103481) sequence as a probe, homology comparison analysis was performed on the rubber tree genome, TSA (Transcriptome Shotgun Assembly) and EST (Expressed Sequence Tags) databases. A reference sequence containing the complete coding region was obtained through electronic splicing and bioinformatics analysis. Primers were designed and synthesized based on the reference sequence. The specific primer sequences are as follows:

F1: 5′-GGTGGCTTCTTTGGCTCTCT-3′;

R1: 5′-GCAAAATTCCAGGAGTTCCA-3′.

The total RNA of latex from Hevea brasiliensis Reyan 7-33-97 was extracted using RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd.), and the first-strand cDNA was synthesized according to the instructions of RevertAid ^TM First Strand cDNA Synthesis Kit (Thermo Scientific) to obtain latex cDNA. The target gene was amplified by PCR using the latex cDNA as a template and the aforementioned primers F1/R1. The PCR reaction system was: 2 μL cDNA template, 0.5 μL F1/R1 primers (10 μmol/L), 5 μL FastPfu Buffer, 2μL 2.5mM dNTPs, 0.5μL FastPfu DNA Polymerase (2.5U/μL) (Beijing Quanshijin Biotechnology Co., Ltd.), add ddH ₂ O to a total volume of 25 μL. The reaction program is: 95℃ pre-denaturation for 3min; 95℃ denaturation for 20s, 55℃ annealing for 20s, 72℃ extension for 1min, a total of 38 cycles; 72℃ extension for 10min.

The PCR product was tested by 1% agarose gel electrophoresis, and the results showed that a band consistent with the expected target was obtained. The band was cut out and the fragment was recovered using an agarose gel recovery kit (OMEGA). The purified DNA fragment was then ligated to the vector -Blunt Simple Cloning Vector (Beijing Quanshijin Biotechnology Co., Ltd.), transformed into Escherichia coli Trans1-T1 Phage Resistant Chemically Competent Cell (Beijing Quanshijin Biotechnology Co., Ltd.), picked positive clones, and sent to Boshang Biotechnology (Shanghai) Co., Ltd. for sequencing.

The sequencing results showed that the nucleotide sequence of the obtained PCR product was 852 bp long, and the nucleotide sequence was shown in SEQ ID No. 2 in the sequence table. The gene shown in the sequence was named HbTRXy2 gene. The coding sequence of HbTRXy2 gene was shown in nucleotides 179-685 of SEQ ID No. 2 in the sequence table (i.e., SEQ ID No. 3), and the protein encoded by it was named HbTRXy2. The amino acid sequence of HbTRXy2 was shown in SEQ ID No. 1.

SEQ ID No.1:

MAISSLSASTIPSLKTPHSQLSANLSCLSSLQFPAQLHRLQFGNRGISSPSRSRILPLLAAKKQTFSNLDELLANADKPVLVDFYAAWCGPCQLMSPILKEVSAILNDTIQVVKIDTEKYPSIADKYRIEALPTFIIFKDGKPYDRFEGAFSKDKFIQRIENSLQVKQ

SEQ ID No.2:

ggtggcttctttggctctctctcctctcttctttttcacagattttgtgttcttgcccagcgaagcatatttttggattttttgccctctcgattctatcctaagaataggcgttttgtggattctatttgtcggtagcagcagcagagaagaagaaggagaagaagagtctagaaaactATGGCGATTTCTTCTCTCTCGGCTTCAACAATTCC TTCTTTGAAGACACCGCACTCGCAGTTGAGTGCTAATTTGAGTGCTTGTCTTCGCTGCAGTTTCCAGCGCAGCTTCACAGGCTTCAGTTTGGGAACAGAGGGATTTCCTCTCCTTCTAGGTCTCGAATTTTGCCTCTGCTTGCAGCAAAGAAGCAAACATTTTCCAACTTGGATGAGTTATTGGCAAATGCTGACAAACCAGTCTTGGTTGA CTTCTATGCAGCCTGGTGCGGTCCATGCCAGCTTATGAGTCCAATTCTTAAAGAGGTCAGTGCCATCCTGAATGACACAATCCAGGTGGTGAAAATCGATACTGAGAAGTACCCTAGCATTGCTGACAAATACAGAATAGAAGCATTGCCTACATTTATCATATTTAAGGATGGGAAACCTTATGATCGCTTCGAGGGTGCTTTCTCTAAAGATAAGTTCATTCAACGCATAGAAAATTCACTGCAAGTGAAGCAATAGtt gaggttttacgaagaatgcaatatgaagacattacatatgaatcttactaggaatgccgcccattcttcccctcccagaatgttgtaatgactgttgttgtacctaaaaagagtgacaaacacaaattctaatggcatatttgtttggaactcctggaattttgc

SEQ ID No.3:

ATGGCGATTTCTTCTCTCTCGGCTTCAACAATTCCTTCTTTGAAGACACCGCACTCGCAGTTGAGTGCTAATTTGAGTTGCTTGTCTTCGCTGCAGTTTCCAGCGCAGCTTCACAGGCTTCAGTTTTGGGAACAGAGGGATTTCCTCTCCTTCTAGGTCTCGAATTTTGCCTCTGCTTGCAGCAAAGAAGCAAACATTTTCCAACTTGGATGAGTTATTGGCAAATGCTGACAAACCAGTCTTGGTTGACTTCT ATGCAGCCTGGTGCGGTCCATGCCAGCTTATGAGTCCAATTCTTAAAGAGGTCAGTGCCATCCTGAATGACACAATCCAGGTGGTGAAAATCGATACTGAGAAGTACCCTAGCATTGCTGACAAATACAGAATAGAAGCATTGCCTACATTTATCATATTTAAGGATGGGAAACCTTATGATCGCTTCGAGGGTGCTTTCTCTAAAGATAAGTTCATTCAACGCATAGAAAATTCACTGCAAGTGAAGCAATAG

It is predicted that the molecular weight of HbTRXy2 protein is 18.70 kD and the theoretical isoelectric point is 9.17. ScanProsite (https://prosite.expasy.org/prosite.html) was used to analyze the conserved domain of the amino acid sequence of HbTRXy2, and it was found that the protein contains a thioredoxin conserved domain located at amino acids 50-165, of which 89-92 is a highly conserved CGPC redox active site (Figure 1).

Multiple sequence alignment with other plant y-type thioredoxins revealed that the thioredoxin conserved domains of different plant y-type thioredoxins were relatively conservative, but the N-terminal sequences of the proteins varied greatly, with the differences between monocots and dicots being more obvious (Figure 1). Different types of plant thioredoxins were selected together with HbTRXy2 proteins to draw an evolutionary tree using ClustalX and MEGA6.0 software. The results showed that these thioredoxins could be clustered into 7 categories: m, f, h, o, x, y and z. HbTRXy2 was clustered in a group with other plant y-type thioredoxins, so it belongs to the y-type thioredoxin. HbTRXy2 is most closely related to cassava MeTRXy2 (XP_021600142.1) and castor RcTRXy2 (XP_002524295.1), with amino acid sequence identities of 87% and 84%, respectively (Figure 2).

Example 2, expression pattern analysis of the HbTRXy2 gene in rubber tree

1. Analysis of tissue expression characteristics of HbTRXy2 gene

The latex, bark, female flowers, male flowers, new shoots, mature leaves, senescent leaves, light green leaves, discolored leaves, bronze leaves and root tissue samples of the tissue culture seedlings transplanted and cultured for 8 months of rubber tree Reyan 7-33-97 (16 years old) were collected and frozen in liquid nitrogen. The total RNA of each tissue sample was extracted using a universal plant total RNA extraction kit (Beijing Baitai Biotechnology Co., Ltd.), and the specific extraction method was referred to the kit instructions. PrimeScript ^TM RT reagent Kit with gDNAEraser (PerfectReal Time) (TaKaRa) was used to reverse transcribe and synthesize the first-chain cDNA, and the specific implementation method was referred to the kit instructions. Using the cDNA of each tissue as a template, the following primer pairs were used for real-time fluorescence quantitative PCR (qRT-PCR) analysis. The rubber tree HbUBC4 gene was used as an internal reference gene.

The primer sequences used to detect the HbTRXy2 gene are as follows:

F2:5′-CTTTGAAGACACCGCACTCG-3′;

R2: 5′-CCAAACTGAAGCCTGTGAAGC-3′.

The primer sequences used to detect the HbUBC4 gene are as follows:

F3: 5′-TCACCCTGAACCTGATAGCC-3′;

R3: 5′-TTTCTTTGGTGACGCTGCAA-3′.

qRT-PCR was performed on a Bio-Rad CFX96 fluorescence quantitative PCR instrument, with 3 replicates per parallel experiment. Reaction system: 10 μL PremixEx Taq ^TM (2×) (TaKaRa), 1 μL each of forward and reverse primers, 2 μL cDNA template, supplemented with ddH ₂ O to a total volume of 20 μL. Reaction conditions: 95°C for 5 min; 95°C for 10 s; 60°C for 30 s; melting curve analysis was performed after 40 cycles to determine the specificity of the primers. The relative expression of the gene was calculated using the formula 2 ^–ΔΔCt .

The expression results of HbTRXy2 gene in various tissues of rubber tree are shown in Figure 3. HbTRXy2 gene is expressed in rubber tree latex, bark, female flowers, male flowers, new shoots, mature leaves, senescent leaves, light green leaves, color change leaves, bronze leaves and root tissues. The top three tissues with higher expression levels are color change leaves, light green leaves and latex, respectively, and the tissues with relatively lower expression levels are roots and senescent leaves. The expression of HbTRXy2 gene increases first and then decreases with leaf development, and it is speculated that this gene may play a role in leaf development or leaf function.

2. Analysis of the expression pattern of HbTRXy2 gene under abiotic stress

Plant thioredoxin plays an important role in regulating redox balance and responding to abiotic stress. In order to clarify the expression characteristics of the HbTRXy2 gene under abiotic stress, the applicant analyzed the expression changes of the HbTRXy2 gene under oxidative stress induced by methyl viologen (MV) and hydrogen peroxide (H ₂ O ₂ ) and drought stress induced by low temperature, salt and PEG.

Methyl viologen (MV)-induced oxidative stress treatment group: Select healthy plants of rubber tree Reyan 7-33-97 tissue culture seedlings that have been transplanted and cultured for 8 months, and spray 200μmol/L MV solution (the solute is methyl viologen (MV), and the solvent is sterile distilled water) evenly on the front and back of all leaves of the plant. The second and third leaves of the plant were collected at 0, 3, 6, 12, 24 and 48h of treatment. Three biological replicates were set for each treatment time point, and each replicate was taken from 3 plants. After sample collection, it was frozen in liquid nitrogen. According to the method in step 1, the total RNA of the leaves was extracted, the first-chain cDNA was synthesized by reverse transcription, and the expression of the HbTRXy2 gene in each sample was detected by qRT-PCR. The leaf sample treated for 0h (untreated) was used as the control.

Hydrogen peroxide (H ₂ O ₂ )-induced oxidative stress treatment group: Select healthy plants of rubber tree Reyan 7-33-97 tissue culture seedlings that have been transplanted and cultured for 8 months, and spray 20mmol/LH ₂ O ₂ solution evenly on the front and back of all leaves of the plants. The second and third leaves of the plants were collected at 0, 3, 6, 12, 24 and 48h of treatment. Three biological replicates were set for each treatment time point, and each replicate was taken from three plants. After the samples were collected, they were frozen in liquid nitrogen. The rest of the operations were the same as those of the methyl viologen (MV)-induced oxidative stress treatment group. The leaf samples treated at 0h (untreated) were used as the control.

Low temperature stress treatment group: Healthy plants of Hevea brasiliensis Reyan 7-33-97 tissue culture seedlings cultured for 8 months were placed in a 4℃ artificial climate chamber for low temperature treatment. The light duration during treatment was 16h and the intensity was 600μmol/(m ² ·s). The rest of the operations were the same as those of the oxidative stress treatment group induced by methyl viologen (MV). Leaf samples treated for 0h (untreated) were used as controls.

NaCl-induced salt stress treatment group: The healthy plants of Hevea brasiliensis Reyan 7-33-97 tissue culture seedlings that had been cultured for 8 months were taken out of the seedling bags, the culture medium was washed, and the roots were immersed in 400mmol/L NaCl solution (solute: NaCl, solvent: sterile distilled water). The rest of the operations were the same as those of the oxidative stress treatment group induced by methyl viologen (MV). The leaf samples treated for 0h (untreated) were used as controls.

PEG6000-induced drought stress treatment group: The healthy plants of Hevea brasiliensis Reyan 7-33-97 tissue culture seedlings that had been cultured for 8 months were taken out of the seedling bags, the culture medium was washed, and the roots were immersed in a 20% PEG6000 solution (solute: PEG6000, solvent: sterile distilled water) by mass. The rest of the operations were the same as those of the oxidative stress treatment group induced by methyl viologen (MV). The leaf samples treated for 0 h (untreated) were used as controls.

The results showed that the expression patterns of HbTRXy2 gene under MV and H ₂ O ₂ induced oxidative stress were similar, both showing a trend of first increasing and then decreasing; the expression level reached the highest level at 6 h of treatment, which was 5.94 times and 4.35 times of the control (0 h), respectively, and then decreased, but still significantly higher than the control (0 h) (Figure 4 and Figure 5). Low temperature treatment upregulated the expression of HbTRXy2 gene, and the expression level reached the highest level at 3 h of treatment, and then decreased, but still significantly higher than the control (0 h) (Figure 6). PEG-induced drought stress treatment also upregulated the expression of HbTRXy2 gene. During the entire treatment period, the expression of HbTRXy2 gene was relatively stable, which was about 2 times of the control (0 h) (Figure 7). Under salt stress, the expression of HbTRXy2 gene showed a trend of increasing first and then decreasing; the expression reached the highest level at 6-12h of treatment, which was about 5.30 times that of the control (0h); after 12h of treatment, the expression of HbTRXy2 gene gradually decreased, and at 48h of treatment, the expression level was only 1/5 of the control (0h) (Figure 8). The above results show that the expression of HbTRXy2 gene is regulated by abiotic stresses such as oxidation, low temperature, drought and salt.

Example 3: Subcellular localization analysis of HbTRXy2 protein

1. Construction of HbTRXy2 gene subcellular localization vector

According to SEQ ID No. 2 in the sequence table, a primer pair was designed to amplify the coding region without a stop codon (nucleotides 179 to 682 at the 5′ end). The recognition sequences of restriction endonucleases Sal I and Kpn I (shown by the underlined portions in the following primers) and protective bases were introduced into the 5′ ends of the forward and reverse primers, respectively. The specific primer sequences are as follows:

F4: 5′-ACGC GTCGAC ATGGCGATTTCTTCTCTCTCG-3′;

R4: 5′-GC GGTACC TTGCTTCACTTGCAGTGAATT-3′.

Using latex cDNA as template, the target gene was amplified by PCR. The PCR reaction system was: 2 μL cDNA template, 5 μL FastPfu Buffer, 2 μL 2.5 mM dNTPs, 0.5 μL each of forward and reverse primers F4 and R4 (10 μmol/L), 0.5 μL Fast Pfu DNA Polymerase (2.5U/L), add ddH ₂ O to a total volume of 25 μL. The reaction program is: 95°C pre-denaturation for 3 min; 95°C denaturation for 20 s, 55°C annealing for 20 s, 72°C extension for 45 s, a total of 38 cycles; 72°C extension for 10 min.

The PCR product was purified by agarose gel electrophoresis and then ligated to -Blunt Simple Cloning Vector (Beijing Quanshijin Biotechnology Co., Ltd.), transformed into Escherichia coli Trans1-T1 Phage Resistant Chemically Competent Cell, picked positive clones, and sent to Boshang Biotechnology (Shanghai) Co., Ltd. for sequencing. The plasmid extraction kit (OMEGA) was used to extract the sequencing correct clone and subcellular localization vector pCAMBIA1300-GFP empty vector plasmid, and double enzyme digestion was performed with restriction endonucleases Sal I and Kpn I (Thermo Scientific). Double enzyme digestion system: KpnII (10U/μL) 2μL, Xba I (10U/μL) 2μL, 10×BamHI Buffer 3μL, plasmid DNA 8μL, supplemented with ddH ₂ O to 30μL. 37℃ water bath for 3h. After the enzyme digestion product was subjected to 1.0% agarose gel electrophoresis, the gel was cut to recover the target band. T4 DNA ligase (Thermo Scientific) was used to connect the recovered target gene and the pCAMBIA1300-GFP vector backbone. The connection system was as follows: 6 μL target gene recovery product, 2 μL pCAMBIA1300-GFP vector backbone, 1 μL T4 DNA ligase, 1 μL 10×Buffer; connection was carried out at 16°C overnight. The ligation product was transformed into E. coli DH5a competent cells, and then spread on LB solid plates containing kanamycin (100 mg/L) and cultured at 37°C overnight. Single clones were picked and the colonies were positively screened using the vector 35S promoter primer (5′-TGACGCACAATCCCACTATCC-3′) plus the gene-specific reverse primer (R4). The positive clones were further sequenced and identified. The sequencing results showed that the nucleotide sequence from 179 to 682 in SEQ ID No. 2 was contained between the Sal I and Kpn I restriction sites of the recombinant vector, indicating that the constructed subcellular localization vector was correct. The recombinant vector was named pCAMBIA1300-HbTRXy2-GFP. The pCAMBIA1300-HbTRXy2-GFP positive clone was inoculated and the plasmid was extracted using a plasmid extraction kit (OMEGA).

2. PEG-mediated tobacco protoplast transformation and subcellular localization observation

1) Take 4 leaves of Nicotiana benthamiana grown for about 4 weeks, cut them into strips of 0.5-1 mm, and put them into 10 mL of enzymatic solution (0.4 M mannitol, 20 mM KCl, 1% cellulase R10, 0.4% macerozyme R10, 10 mM CaCl ₂ , 5 mM β-mercaptoethanol, 0.1% BSA and 20 mM MES, pH 5.7, the solvent is sterile deionized water).

2) Enzymatic hydrolysis at 25°C, 40 rpm, in the dark for 4 h.

3) Add an equal volume of pre-cooled W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES, pH 5.7, solvent is sterile deionized water), gently shake the enzymatic hydrolysate, pass it through a 300-mesh nylon sieve, collect the filtrate in a 50 mL round-bottom centrifuge tube, and centrifuge it at 23°C, 600 rpm for 5 min.

4) Remove the supernatant, resuspend the precipitate in 10 mL of W5 solution, and place on ice for 30 min.

5) Centrifuge at 4°C, 600 rpm for 5 min, remove the supernatant, and resuspend in 1 mL MMG solution (0.4 M mannitol, 15 mM MgCl ₂ , 4 mM MES, pH 5.7, the solvent is sterile deionized water).

6) Add 10 μL of the target plasmid to a 2 ml centrifuge tube, add 100 μL of the protoplasts from step 5), and mix gently.

7) Add an equal volume of PEG solution (40% PEG4000, 0.2M mannitol, 100mM CaCl ₂ ), mix by gentle pipetting, and leave at room temperature for 30 minutes.

8) Add 440 μL of W5 solution (room temperature), mix by inversion, centrifuge at 23°C, 600 rpm for 2 min, and remove the supernatant.

9) Add 1 mL of WI solution (0.5 M mannitol, 20 mM KCl, 4 mM MES, pH 5.7) to gently resuspend the protoplasts and transfer to a 6-well culture plate.

10) Cultivate at 25°C under weak light for 16 h, prepare slides, and observe using a ZEISS/LSM 800 laser confocal microscope. The excitation wavelengths for GFP and chloroplast autofluorescence are 488 nm and 552 nm, and the fluorescence collection wavelengths are 500-530 nm and 650-680 nm, respectively.

The results of subcellular localization observation are shown in Figure 9. In the protoplasts transformed with the empty vector pCAMBIA1300-GFP, green fluorescence was observed in the cytoplasm and nucleus, but no green fluorescence was detected in the chloroplasts. In the protoplasts transformed with pCAMBIA1300-HbTRXy2-GFP, green fluorescence was observed in the chloroplasts and the surrounding cytoplasm. The above results indicate that the HbTRXy2 protein is localized in the chloroplasts and cytoplasm.

Example 4: HbTRXy2 gene can improve the resistance of transgenic yeast to oxidative stress

1. Construction of yeast expression vector of HbTRXy2 gene

According to SEQ ID No. 2 in the sequence table, a primer pair for amplifying the coding region (nucleotides 179 to 685 at the 5′ end) was designed. The recognition sequences of restriction endonucleases Kpn I and Xba I (shown by the underlined portions in the following primers) and protective bases were introduced into the 5′ ends of the forward and reverse primers, respectively. The specific primer sequences are as follows:

F5: 5′-GG GGTACC ATGGCGATTTCTTCTCTCTCG-3′;

R5: 5′- GCTCTAGACTATTGCTTCACTTGCAGTGA -3′.

The total RNA of rubber tree latex was extracted and reverse transcribed into cDNA. The target gene was amplified by PCR using the cDNA as a template. The PCR reaction system was: 2 μL cDNA template, 5 μL FastPfu Buffer, 2 μL 2.5 mM dNTPs, 0.5 μL each of forward and reverse primers F5 and R5 (10 μmol/L), 0.5 μL Fast Pfu DNA Polymerase (2.5U/L), add ddH ₂ O to a total volume of 25 μL. The reaction program is: 95°C pre-denaturation for 3 min; 95°C denaturation for 20 s, 55°C annealing for 20 s, 72°C extension for 45 s, a total of 38 cycles; 72°C extension for 10 min.

The PCR product was purified by agarose gel electrophoresis and then ligated to -Blunt Simple Cloning Vector (Beijing Quanshijin Biotechnology Co., Ltd.), transformed into Escherichia coli Trans1-T1 Phage Resistant Chemically Competent Cell, picked positive clones, and sent to Boshang Biotechnology (Shanghai) Co., Ltd. for sequencing. The plasmid extraction kit (OMEGA) was used to extract the correct clones for sequencing and the empty vector plasmid of the yeast expression vector pYES2 (Invitrogen), and double enzyme digestion was performed with restriction endonucleases Kpn I and Xba I (Thermo Scientific). Double enzyme digestion system: Kpn I (10U/μL) 2μL, Xba I (10U/μL) 1μL, 10×Tango Buffer 3μL, plasmid DNA 8μL, supplemented with ddH ₂ O to 30μL. 37℃ water bath for 3h. After the enzyme digestion product was subjected to 1.0% agarose gel electrophoresis, the gel was cut to recover the target band. _T4 DNA ligase was used to connect the recovered target gene and pYES2 vector backbone. The connection system was as follows: 6 μL target gene recovery product, 2 μL pYES2 vector backbone, 1 μL T4 DNA ligase, 1 μL 10×Buffer; connection was carried out at 16°C overnight. The ligation product was transformed into Escherichia coli DH5a competent cells, and then spread on LB solid plates containing ampicillin (100 mg/L) and cultured at 37°C overnight. Single clones were picked and positive colonies were screened using pYES2 vector primers (F6: 5′-CCCGGATCGGACTACTAGC-3′) plus gene-specific reverse primers (R5). Positive clones were further sequenced and identified. The HbTRXy2 gene yeast expression vector pYES2-HbTRXy2 was obtained. pYES2-HbTRXy2 is a recombinant plasmid obtained by replacing the fragment between the Kpn I and Xba I recognition sites of pYES2 with a DNA molecule having a nucleotide sequence of nucleotides 179 to 685 in SEQ ID No.2 (coding region sequence of HbTRXy2 gene), while keeping the other nucleotide sequences of pYES2 unchanged. pYES2-HbTRXy2 expresses the protein HbTRXy2 having an amino acid sequence of SEQ ID No.1 in the sequence list. The Escherichia coli containing pYES2-HbTRXy2 is named DH5a (pYES2-HbTRXy2).

2. Construction of recombinant yeast

The Saccharomyces cerevisiae strain INVScl (Invitrogen) is a strain with uridine auxotrophy ( ^Ura- ). On SC-Ura medium, this yeast strain can hardly grow and reproduce. The yeast expression vector pYES2 contains the URA3 gene, and the expression of this gene can enable yeast transformants to grow normally on SC-Ura medium. Therefore, SC-Ura medium can be used to screen positive yeast transformants. Extract pYES2 and the recombinant expression vector pYES2-HbTRXy2 plasmids, and transform them into the competent cells of the Saccharomyces cerevisiae strain INVSc1 by the lithium acetate method. Apply the transformed yeast liquid on the SC-Ura solid selection medium and invert and culture at 30°C for 2-3 days. Pick the yeast monoclonal plasmid DNA and identify the positive transformants by PCR. The specific operation steps are as follows:

1) Pick a yeast INVSc1 single clone and add it to 10 mL YPD liquid culture medium, and shake and culture it at 30°C and 200 rpm overnight.

2) Detect the _OD600 value of the yeast solution, add the yeast solution cultured overnight to 50 mL YPD liquid medium, dilute to _OD600 of 0.4, and continue shaking at 30°C and 200 rpm for 2-4 hours.

3) Centrifuge at 2500 rpm for 5 min at 4°C, discard the supernatant to collect the cells, and resuspend the cells in 40 mL of 1×TE buffer.

4) Centrifuge again at 2500 rpm at 4°C for 5 min, discard the supernatant to collect the cells, and resuspend the cells in 2 mL of 1×LiAc/0.5×TE.

5) The resuspended cells were divided into 1.5 mL centrifuge tubes, 100 μL per tube.

6) Incubate the aliquoted yeast cells at room temperature for 10 minutes.

7) Add 1 μg of the plasmid extracted from DH5a (pYES2-HbTRXy2) prepared in step 1 and 100 μg of denatured salmon sperm DNA to each transformation system (100 μL) and mix well.

8) Add 700 μL 1×LiAc/40% PEG-3350/1×TE and mix well.

9) Incubate at 30°C for 30 min.

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

11) Centrifuge at 4°C, 5000 rpm for 1 min and discard the supernatant.

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

13) Resuspend the bacteria with 100 μL l×TE buffer and apply it to SC-Ura solid selection medium, and invert and culture at 30°C for 2-3 days. Pick 3 INVSc1 (pYES2-HbTRXy2) monoclones from the SC-Ura solid selection medium, place them in 3 mL SC-Ura liquid selection medium, and shake and culture them at 30°C and 200 rpm for 24 hours. Use the yeast plasmid extraction kit (OMEGA) to extract plasmid DNA. Use F6 and R5 primers to perform positive PCR detection of bacterial liquid using yeast plasmid DNA as a template. The positive clone is a recombinant yeast containing pYES2-HbTRXy2, which is named INVSc1 (pYES2-HbTRXy2).

According to the above method, pYES2-HbTRXy2 was replaced by pYES2, and the other steps remained unchanged. The pYES2 vector primers F6 (5′-CCCGGATCGGACTACTAGC-3′) and R6 (5′-ATTAAAGCCTTCGAGCGTCC-3′) were used for PCR positive detection to obtain the recombinant yeast containing pYES2, which was named INVSc1 (pYES2).

Agarose gel electrophoresis of PCR products is shown in Figure 10. The three recombinant yeast plasmids transformed with the pYES2 empty vector amplified bands of the same size as the positive control (pYES2 plasmid) (4, 5, 6 in Figure 10), and the expected length was 402 bp; the three recombinant yeast plasmids transformed with pYES2-HbTRXy2 amplified bands of the same size as the positive control (pYES2-HbTRXy2 plasmid) (1, 2, 3 in Figure 10), and the expected length was 577 bp. The above results show that both pYES2-HbTRXy2 and pYES2 empty vectors were successfully transformed into recombinant yeast.

3. Detection of HbTRXy2 gene expression in recombinant yeast

The positive clones of INVSc1(pYES2-HbTRXy2) and INVSc1(pYES2) obtained in step 2 were streaked on SC-Ura solid selection medium and inverted at 30°C for 2-3 days. A single clone was picked and inoculated into 2mL SC-Ura liquid selection medium, shaken and cultured at 30°C and 200rpm for 24h, and its OD ₆₀₀ value was measured. 1mL of cultured bacterial solution was taken, centrifuged at 4000r/min for 3min, the supernatant was discarded, and the bacteria were resuspended in SC-Ura liquid induction medium, and OD ₆₀₀ = 0.2. 10mL was taken from each, and induced and cultured at 30°C and 200rpm for 36h. 2ml of bacterial solution was taken from each, and the bacteria were collected by low-speed centrifugation. The total RNA was extracted using a yeast total RNA rapid extraction kit (Shanghai Sangon Biotech Co., Ltd.). PrimeScript ^TM RT reagent Kit with gDNAEraser (PerfectReal Time) (TaKaRa) was used to synthesize the first-strand cDNA. The specific implementation method was referred to the kit instructions. First, the amount of each template cDNA was determined using the Saccharomyces cerevisiae internal reference gene Actin (GeneBank accession number: L00026.1). The Actin primer sequence is as follows:

F7: 5′-AGTTGCCCCAGAAGAACACC-3′;

R7: 5′-TACCGGCAGATTCCAAACCC-3′.

Then, the template was amplified by PCR using the HbTRXy2 gene primers (F5 and R5). The PCR reaction system was: template 1-3μL, buffer 2.5μL, dNTPs 2μL, forward and reverse primers 0.5μL each, EasyTaq DNA Polymerase (Beijing Quanshijin Biotechnology Co., Ltd.) 0.5μL, and ddH ₂ O was added to a total volume of 25μL. The reaction procedure was: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30s, 58℃ annealing for 30s, 72℃ extension for 45s, a total of 35 cycles; 72℃ extension for 10min.

The results are shown in Figure 11. At 36 hours of induction culture, HbTRXy2 gene expression was detected in each clone of recombinant yeast INVSc1 (pYES2-HbTRXy2) (4, 5, 6 in Figure 11), while HbTRXy2 gene expression was not detected in the control yeast INVSc1 (pYES2) (1, 2, 3 in Figure 11). The above results show that the rubber tree HbTRXy2 gene was successfully transferred into the recombinant yeast INVSc1 (pYES2-HbTRXy2) and induced to express.

4. Comparative analysis of the antioxidant activity of recombinant yeast INVSc1(pYES2-HbTRXy2) and INVSc1(pYES2)

(1) Comparison of growth differences between recombinant yeast INVSc1(pYES2-HbTRXy2) and INVSc1(pYES2) in H ₂ O ₂ liquid induction medium

The positive clones of INVSc1(pYES2-HbTRXy2) and INVSc1(pYES2) obtained in step 2 were streaked on SC-Ura solid selection medium and inverted at 30°C for 2-3 days. INVSc1(pYES2-HbTRXy2) and INVSc1(pYES2) were picked and inoculated into 5mL SC-Ura liquid selection medium and cultured at 30°C and 200rpm for 24h. Centrifuge at 4000r/min for 3min, discard the supernatant, resuspend the bacteria with SC-Ura liquid induction medium, and adjust OD ₆₀₀ = 0.2 to obtain a resuspended bacterial solution. Take 10mL of the resuspended bacterial solution and culture at 30°C and 200rpm for 36h to induce the expression of the HbTRXy2 gene, and obtain INVSc1(pYES2-HbTRXy2) induction culture solution and INVSc1(pYES2) induction culture solution, respectively.

pYES2-HbTRXy2 oxidative stress treatment group (i.e. HbTRXy2-H ₂ O ₂ ): Take 1 mL of the aforementioned INVSc1 (pYES2-HbTRXy2) induction culture medium, centrifuge at 4000 r/min for 3 min, discard the supernatant, resuspend the bacteria in SC-Ura liquid induction medium containing 3 mM H ₂ O ₂ , and adjust OD ₆₀₀ = 0.2 to obtain an adjusted bacterial solution. Take 10 mL of the adjusted bacterial solution and culture at 30°C and 200 rpm. At 12 h, 24 h and 36 h of culture, measure the OD ₆₀₀ value of each culture solution using a spectrophotometer.

pYES2 oxidative stress treatment group (ie, pYES2-H ₂ O ₂ ): the INVSc1 (pYES2) induction culture medium was used to replace the INVSc1 (pYES2-HbTRXy2) induction culture medium, and the rest of the operations were the same as those of the pYES2-HbTRXy2 oxidative stress treatment group (ie, HbTRXy2-H ₂ O ₂ ).

pYES2-HbTRXy2 control group (ie, HbTRXy2-CK): SC-Ura liquid induction medium containing 3 mM H ₂ O ₂ was replaced with SC-Ura liquid induction medium, and other operations were the same as those of the pYES2-HbTRXy2 oxidative stress treatment group (ie, pYES2-H ₂ O ₂ ).

pYES2 control group (ie, pYES2-CK): SC-Ura liquid induction medium containing 3 mM H ₂ O ₂ was replaced with SC-Ura liquid induction medium, and other operations were the same as those of the pYES2 oxidative stress treatment group (ie, pYES2-H ₂ O ₂ ).

The results are shown in Figure 12. Without oxidative stress, there was no significant difference in the OD ₆₀₀ values between the pYES2-HbTRXy2 control group (i.e., HbTRXy2-CK) and the pYES2 control group (i.e., pYES2-CK) at 12h, 24h, and 36h of culture, indicating that the transfer of the HbTRXy2 gene into yeast had no significant effect on the growth of yeast. Under _{H 2} O ₂ -induced oxidative stress, the yeast growth of the pYES2-HbTRXy2 oxidative stress treatment group (i.e., HbTRXy2-H ₂ O ₂ ) and the pYES2 oxidative stress treatment group (i.e., pYES2-H ₂ O ₂ ) was significantly inhibited. However, at each treatment time point, the OD ₆₀₀ values of the pYES2-HbTRXy2 oxidative stress treatment group (i.e., HbTRXy2-H ₂ O ₂ ) were significantly higher than those of the pYES2 oxidative stress treatment group (i.e., pYES2-H ₂ O ₂ ), indicating that the bacterial liquid concentration of the pYES2-HbTRXy2 oxidative stress treatment group (i.e., HbTRXy2-H ₂ O ₂ ) was significantly higher than that of the pYES2 oxidative stress treatment group (i.e., pYES2-H ₂ O ₂ ). This shows that the recombinant yeast expressing the HbTRXy2 gene has enhanced resistance to oxidative stress.

(2) Survival differences between recombinant yeast INVSc1(pYES2-HbTRXy2) and INVSc1(pYES2) after high concentration H ₂ O ₂ stress

The positive clones of INVSc1(pYES2-HbTRXy2) and INVSc1(pYES2) obtained in step 2 were streaked on SC-Ura solid selection medium and inverted at 30°C for 2-3 days. INVSc1(pYES2-HbTRXy2) and INVSc1(pYES2) were picked and inoculated into 5mL SC-Ura liquid selection medium respectively, and cultured at 30°C and 200rpm for 24h. Centrifuge at 4000r/min for 3min, discard the supernatant, resuspend the bacteria with SC-Ura liquid induction medium, and adjust OD600=0.2 to obtain a resuspended bacterial solution. Take 10mL of the resuspended bacterial solution and culture at 30°C and 200rpm for 36h to induce the expression of HbTRXy2 gene, and obtain INVSc1(pYES2-HbTRXy2) induction culture solution or INVSc1(pYES2) induction culture solution respectively.

HbTRXy2 10mM H ₂ O ₂ treatment group: Take 2mL INVSc1 (pYES2-HbTRXy2) induction culture medium, centrifuge at 4000r/min for 3min, discard the supernatant, resuspend the bacteria in 10mM H ₂ O ₂ aqueous solution, adjust OD ₆₀₀ = 1.0, and obtain the adjusted resuspended bacterial solution. Take 1mL of the adjusted resuspended bacterial solution in a 10mL centrifuge tube, treat at 30℃ and 160rpm for 24h to obtain the treated bacterial solution. Use sterile distilled water to dilute the treated bacterial solution 5 times ( ^5-1 , ^5-2 , ^5-3 , ^5-4 , ^5-5 ). Take 5μL of the undiluted bacterial solution and the bacterial solution with different dilution multiples and spot them on SC-Ura solid selection medium, and repeat 3 times for each treatment. Incubate at 30℃ for 3 days, and observe and compare the growth differences of the colonies.

pYES2 10 mM H ₂ O ₂ treated group: INVSc1 (pYES2) was used instead of INVSc1 (pYES2-HbTRXy2), and the rest of the operations were the same as those of the HbTRXy2 10 mM H ₂ O ₂ treated group.

HbTRXy220mM H ₂ O ₂ treatment group: 10mM H ₂ O ₂ was replaced by 20mM H ₂ O ₂ , and the rest of the operations were the same as those of the HbTRXy210mM _{H 2} O ₂ treatment group.

pYES220mM H ₂ O ₂ treatment group: 10mM H ₂ O ₂ was replaced by 20mM H ₂ O ₂ , and the rest of the operations were the same as those of the pYES210mM H ₂ O ₂ treatment group.

HbTRXy2 control group: 10 mM H ₂ O ₂ was replaced by sterile distilled water, and the rest of the operations were the same as those in the HbTRXy2 10 mM H ₂ O ₂ treatment group.

pYES2 control group: 10 mM H ₂ O ₂ was replaced by sterile distilled water, and other operations were the same as those of the pYES2 10 mM H ₂ O ₂ treatment group.

The growth of the recombinant yeast INVSc1(pYES2-HbTRXy2) and INVSc1(pYES2) in the above groups is shown in Figure 13. In the control group (without oxidative stress), there was no significant difference in the number of plaques (CFU) between INVSc1(pYES2-HbTRXy2) and INVSc1(pYES2) at the same dilution multiple. In the 10mM H ₂ O ₂ treatment group, after ^5-1 and ^5-2 dilutions, INVSc1(pYES2-HbTRXy2 still had plaque growth, while INVSc1(pYES2) had no plaque growth; when the undiluted bacterial solution was used to spot the plate, the number of plaques grown by INVSc1(pYES2-HbTRXy2 was significantly more than that of INVSc1(pYES2). In the 20mM H ₂ O ₂ treatment group, after ^5-1 dilution, INVSc1(pYES2-HbTRXy2) still had plaque growth, while INVSc1(pYES2) had no plaque growth; when the undiluted bacterial solution was used to spot the plate, the number of plaques grown by INVSc1(pYES2-HbTRXy2) was also significantly higher than that of INVSc1(pYES2). The above results indicate that the HbTRXy2 gene transfection increased the survival rate of the recombinant yeast after the oxidative stress treatment induced by high concentration of H ₂ O ₂ , and the expression of the HbTRXy2 gene increased the resistance of the recombinant yeast to oxidative stress.

The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims (6)

1. Use of a protein, characterized in that the use is any of the following:

D1 Use in increasing oxidative stress resistance of yeast;

d2 Use of a yeast for increasing oxidative stress resistance;

d3 Application in breeding yeast varieties with improved antioxidant capacity;

the protein is a protein with an amino acid sequence of SEQ ID No. 1.

2. The use according to claim 1, wherein the protein is derived from rubber tree.

3. The use of a biological material associated with a protein as described in claim 1 or 2 in any of the following,

D1 Use in increasing oxidative stress resistance of yeast;

d2 Use of a yeast for increasing oxidative stress resistance;

d3 Application in breeding yeast varieties with improved antioxidant capacity;

the biomaterial is any one of the following B1) to B3):

b1 A nucleic acid molecule encoding a protein as claimed in claim 1 or 2;

B2 An expression cassette comprising the nucleic acid molecule of B1);

B3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2).

4. The use according to claim 3, wherein the nucleic acid molecule of B1) is a gene of B1) or B2) as follows:

b1 Nucleotide sequence is cDNA molecule or DNA molecule of 179 th-685 th positions of SEQ ID No.2 in sequence table;

b2 Nucleotide sequence is cDNA molecule or DNA molecule of SEQ ID No.2 in the sequence table.

5. A method for breeding a transgenic yeast having improved oxidative stress resistance, which comprises increasing the expression level of a gene encoding the protein according to claim 1 or 2 in a target yeast to obtain an oxidative stress resistant yeast having higher oxidative stress resistance than the target yeast.

6. The method according to claim 5, wherein the increase in the expression level of the gene encoding the protein in the target yeast is achieved by introducing the gene encoding the protein in claim 1 or 2 into the target yeast.