CN116640738A

CN116640738A - Thioredoxin HbTRXy2 from rubber tree, related biological material and application thereof

Info

Publication number: CN116640738A
Application number: CN202310632739.3A
Authority: CN
Inventors: 刘辉; 王真辉; 王帅; 何其光; 胡义钰; 袁坤; 冯成天
Original assignee: Rubber Research Institute Chinese Academy Tropical Agricultural Sciences
Current assignee: Rubber Research Institute Chinese Academy Tropical Agricultural Sciences
Priority date: 2023-05-31
Filing date: 2023-05-31
Publication date: 2023-08-25

Abstract

The invention discloses thioredoxin HbTRXy2 from rubber trees and related biological materials and application thereof, and belongs to the technical field of biology. The rubber tree y-type thioredoxin HbTRXy2 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 HbTRXy2 coding gene can be expressed in yeast to improve the resistance of recombinant yeast to oxidative stress, and the HbTRXy2 coding gene has an antioxidant function and can be used for improving the antioxidant property of plants or microorganisms.

Description

Thioredoxin HbTRXy2 from rubber tree, related biological material and application thereof

Technical Field

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

Background

Natural rubber is an important industrial raw material and an indispensable strategic material. Rubber tree (Hevea brasiliensis muell. Arg.) is a major source of natural rubber. China is the largest natural rubber consumption country in the world, the annual consumption is about 550 ten thousand tons, but the annual output of China is only 80 ten thousand tons, and the self-supporting rate is less than 15 percent. Rubber trees are planted in regions of Yunnan province, hainan province and Guangdong province in China, and the regions belong to non-traditional rubber planting regions, and the rubber trees are often subjected to abiotic stress such as low temperature, drought, typhoons and the like in the growth process. In addition, dead bark of rubber tree in rubber garden in China (or dry cut surface tapping panel dryness; the main symptoms are that the rubber is not discharged partially or completely after cutting). At present, the dead skin rate of the rubber garden in China is generally over 30 percent, which leads to serious yield and economic loss. Both abiotic and dead skin accompany the onset of oxidative stress, and regulating and maintaining cellular redox balance is of great importance for rubber trees to resist abiotic stress and to reduce dead skin.

Disclosure of Invention

The invention aims to solve the technical problems of improving the resistance of organisms to oxidative stress and other abiotic stresses and/or determining the functions and applications of thioredoxin HbTRXy2 of rubber trees. The technical problems to be solved are not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.

In order to solve the technical problems, the invention provides the following technical scheme:

the object of the present invention is to provide a protein, a substance regulating the expression of a gene encoding said protein or a substance regulating the content of said protein, said use being any of the following:

d1 Use in modulating resistance to biotic oxidative stress;

d2 The use of a polypeptide for the preparation of a product for modulating resistance to biotic oxidative stress;

d3 Application in breeding biological varieties with improved antioxidant capacity;

d4 Use in modulating abiotic stress resistance of an organism;

d5 The use of a polypeptide for the preparation of a product for modulating abiotic stress resistance of an organism;

d6 The application of the strain in breeding the biological varieties with improved abiotic stress resistance;

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

A1 Protein with the amino acid sequence of SEQ ID No.1, consisting of 168 amino acids;

a2 A protein which is obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence shown in SEQ ID No.1, has more than 80% of identity with the protein shown in A1) and has the same function;

a3 Fusion proteins having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of A1) or A2).

The tag proteins described herein include, but are not limited to, poly-Arg tag proteins containing 5-6 amino acid residues (typically 5, sequence RRRRR), or Poly-His tag proteins containing 2-10 amino acid residues (typically 6, sequence HHHHH), or FLAG tag proteins containing 8 amino acid residues (sequence DYKDDDK), or Strep-tag II tag proteins containing 8 amino acid residues (sequence WSHPQFEK), or c-myc tag proteins containing 8 amino acid residues (sequence WSHPQFEK).

HbTRXy2 in the A2) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing. The HbTRXy2 coding gene in A2) can be obtained by deleting one or more amino acid residues from the DNA sequence shown in SEQ ID No.2 of the sequence Listing, and/or performing missense mutation of one or more base pairs, and/or linking the coding sequence of the tag to the 5 'end and/or the 3' end thereof.

In order to solve the technical problems, the invention also provides the application of the biological material related to HbTRXy2 protein in any one of the following,

d1 Use in modulating resistance to biotic oxidative stress;

d4 Use in modulating abiotic stress resistance of an organism;

the biomaterial is any one of the following B1) to B7):

b1 Nucleic acid molecules encoding the aforementioned proteins;

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);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

b5 A transgenic plant cell line comprising the nucleic acid molecule of B1) or a transgenic plant cell line comprising the expression cassette of B2);

b6 A transgenic plant tissue comprising the nucleic acid molecule of B1) or a transgenic plant tissue comprising the expression cassette of B2);

B7 A transgenic plant organ comprising the nucleic acid molecule of B1) or a transgenic plant organ comprising the expression cassette of B2).

In the above biological material, the nucleic acid molecule of B1) may be a gene of B1) or B2) or B3) or B4) 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 A nucleotide sequence is a cDNA molecule or a DNA molecule of SEQ ID No.2 in a sequence table;

b3 A cDNA molecule or genomic DNA molecule having more than 90% identity to the nucleotide sequence defined in b 1) or b 2) and encoding HbTRXy 2;

b4 Under stringent conditions with the nucleotide sequence defined in b 1) or b 2), and a cDNA molecule or genomic DNA molecule encoding HbTRXy2.

Wherein 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, etc.

Wherein, SEQ ID No.2 is composed of 852 nucleotides, the 179 th to 685 th positions are coding regions, and HbTRXy2 shown in SEQ ID No.1 is coded.

The HbTRXy 2-encoding nucleotide sequence of the present invention can be easily mutated by a person skilled in the art using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 90% or more identity to the nucleotide sequence encoding HbTRXy2 isolated according to the present invention are all derived from and equivalent to the nucleotide sequence of the present invention as long as HbTRXy2 is encoded and HbTRXy2 functions.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 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 assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.

The stringent conditions are hybridization and washing the membrane 2 times at 68℃in a solution of 2 XSSC, 0.1% SDS for 5min each time, and hybridization and washing the membrane 2 times at 68℃in a solution of 0.5 XSSC, 0.1% SDS for 15min each time; alternatively, hybridization and washing of the membrane were performed at 65℃in a solution of 0.1 XSSPE (or 0.1 XSSC) and 0.1% SDS.

The identity of 90% or more may be 90% or 95% or more.

In the above biological material, the expression cassette (HbTRXy 2 gene expression cassette) described in B2) containing a nucleic acid molecule encoding HbTRXy2 means a DNA capable of expressing HbTRXy2 in a host cell, and the DNA may include not only a promoter for initiating transcription of HbTRXy2 gene but also a terminator for terminating transcription of HbTRXy2 gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, inducible promoters. Examples of promoters include, but are not limited to: a constitutive promoter of cauliflower mosaic virus 35S; a wound-inducible promoter from tomato, leucine aminopeptidase; a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester); tomato protease inhibitor II promoter (PIN 2) or LAP promoter (both inducible with methyl jasmonate); a heat shock promoter; a tetracycline-inducible promoter; seed-specific promoters, such as the millet seed-specific promoter pF128, seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin, and soybean beta-cone). They may be used alone or in combination with other plant promoters. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator.

The recombinant vector containing the HbTRXy2 gene expression cassette can be constructed by using the existing expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Co.), etc. The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal may direct the addition of polyadenylation to the 3 'end of the mRNA precursor and may function similarly to the 3' transcribed untranslated regions of Agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase gene Nos) and plant genes (e.g., soybean storage protein genes). When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. To facilitate identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to the herbicide phosphinothricin, hph gene conferring resistance to antibiotic hygromycin, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or chemical marker genes, etc. (such as herbicide resistance genes), mannose-6-phosphate isomerase gene providing mannose metabolization ability, etc. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

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

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi, such as E.coli. The yeast may be saccharomyces cerevisiae strain INVScl.

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

The organisms described herein may be microorganisms, such as yeasts.

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

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

It is another object of the present invention to provide a method for breeding a transgenic organism having increased oxidative stress resistance, which comprises increasing the expression level of a gene encoding the aforementioned protein in the organism of interest, to obtain an oxidative stress resistant organism having higher oxidative stress resistance than the organism of interest.

Further, the increase in the expression level of the gene encoding the protein in the target organism is achieved by introducing the gene encoding the protein into the target organism.

In the above method, the nucleic acid molecule of B1) may be modified as follows before being introduced into the recipient organism to achieve a better expression:

1) Modification and optimization are carried out according to actual needs so as to enable the genes to be expressed efficiently; for example, the codon of the HbTRXy2 encoding gene of the present invention can be changed to conform to plant preferences while maintaining the nucleotide sequence thereof according to the codon preferred by the recipient plant; during the optimization process, it is preferable 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 may 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, modifications are made using sequences known to be effective in plants;

3) Ligating to promoters expressed by various plants to facilitate expression thereof in plants; the promoter may include constitutive, inducible, chronologically regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space of expression requirements and will also depend on the target species; for example, a tissue or organ specific expression promoter, depending on the desired time period of development of the receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, it is desirable to select dicot promoters for expression in dicots and monocot promoters for expression in monocots;

4) The expression efficiency of the gene of the invention can be improved by connecting with a proper transcription terminator; e.g., tml derived from CaMV, E9 derived from rbcS; any available terminator known to function in plants may be ligated to the gene of the present invention;

5) Enhancer sequences such as intron sequences (e.g., derived from Adhl and bronzel) and viral leader sequences (e.g., derived from TMV, MCMV and AMV) are introduced.

In the above method, the HbTRXy2 encoding gene may be the nucleic acid molecule of B1).

In the above method, the recipient organism may be a microorganism or a plant. The organism of interest may specifically be yeast and the plant may specifically be rubber tree.

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

The proteins and biological materials described hereinbefore also belong to the protection of the present invention.

Experiments prove that HbTRXy2 gene of the invention is expressed in all tissues of rubber trees, wherein the expression amount of the HbTRXy2 gene in the color-changing period leaves, the light green period leaves and the latex is higher. The expression of HbTRXy2 gene is induced by oxidation, low temperature, drought and salt stress. Functional identification of HbTRXy2 gene using a yeast expression system shows that over-expression of HbTRXy2 gene in yeast increases resistance of recombinant yeast to hydrogen peroxide-induced oxidative stress. The resistance to oxidative stress can be increased by over-expressing HbTRXy2 gene in plants or microorganisms.

Drawings

FIG. 1 shows the result of multi-sequence alignment of HbTRXy2 of rubber tree with other plant y-type thioredoxin. Wherein the horizontal line is marked with thioredoxin conserved domain; the ". Times." are marked with conserved redox active sites.

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

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

FIG. 4 shows hydrogen peroxide (H) ₂ O ₂ ) Effect of induced oxidative stress treatment on HbTRXy2 gene expression in rubber trees. Wherein the different lowercase letters represent a significant difference between the processing time points (P<0.05)。

FIG. 5 is an effect of Methyl Viologen (MV) -induced oxidative stress treatment on HbTRXy2 gene expression in rubber trees. Wherein different lowercase letters indicate significant differences between treatment time points (P < 0.05).

FIG. 6 is an effect of low temperature stress treatment on HbTRXy2 gene expression of rubber tree. Wherein different lowercase letters indicate significant differences between treatment time points (P < 0.05).

FIG. 7 is the effect of PEG-induced drought stress treatment on the HbTRXy2 gene expression of rubber trees. Wherein different lowercase letters indicate significant differences between treatment time points (P < 0.05).

FIG. 8 is an effect of salt stress treatment on HbTRXy2 gene expression of rubber tree. Wherein different lowercase letters indicate significant differences between treatment time points (P < 0.05).

FIG. 9 shows subcellular localization of HbTRXy2 of rubber tree.

FIG. 10 shows PCR positive detection of recombinant yeasts INVSc1 (pYES 2-HbTRXy 2) and INVSc1 (pYES 2). Wherein "M" represents DL2000 Plus DNAMaroer (Vazyme); "N1" and "N2" are negative controls without template; "P1" and "P2" are plasmid positive controls; "1, 2, 3" is pYES2-HbTRXy2 transformed yeast monoclonal; "4, 5, 6" are pYES2 transformed yeast monoclonal.

FIG. 11 shows the detection of HbTRXy2 gene expression in recombinant yeast at 36h of induction culture. Wherein "M" represents DL2000 Plus DNAMaroer (Vazyme); "1, 2, 3" are cDNA samples at 36h of induction of 3 monoclonals of recombinant yeasts INVSc1 (pYES 2); "4, 5, 6" are cDNA samples at 36h of induction of 3 monoclonals of recombinant yeast INVSc1 (pYES 2-HbTRXy 2).

FIG. 12 is H ₂ O ₂ Growth (OD) of recombinant yeasts INVSc1 (pYES 2-HbTRXy 2) and INVSc1 (pYES 2) under induced oxidative stress ₆₀₀ Value) of the difference. Wherein "HbTRXy2" represents recombinant yeast INVSc1 (pYES 2-HbTRXy 2); "pYES2" is control recombinant yeast INVSc1 (pYES 2); "-CK" is H-free ₂ O ₂ Treated control group, "-H ₂ O ₂ "3 mM H ₂ O ₂ Treatment groups. * And represent that recombinant yeasts INVSc1 (pYES 2-HbTRXy 2) and INVSc1 (pYES 2) have significant differences at levels of 0.05 and 0.01, respectively.

FIG. 13 shows a high concentration H ₂ O ₂ Survival of recombinant yeasts INVSc1 (pYES 2-HbTRXy 2) and INVSc1 (pYES 2) 24h after treatment. Wherein "HbTRXy2" represents recombinant yeast INVSc1 (pYES 2-HbTRXy 2); "pYES2" is the control recombinant yeast INVSc1 (pYES 2).

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The quantitative experiments in the following examples were all performed in triplicate, 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 significant differences, and P < 0.01 (x) indicated very significant differences.

Rubber tree (Hevea brasiliensis muell. Arg.) hot ground 7-33-97: the biological material is only used for repeated relevant experiments of the invention, but not used for other purposes, and is described in 'Huang Huasun, liang Maohuan, wu Yuntong, li Deshun and He Jinwei. Seed selection of excellent variety hot research 7-33-97 of middle-scale popularization rubber tree. Tropical crop school, 1994,15 (2): 1-6', which is cultivated by rubber institute of Tropical academy of agricultural sciences of China.

YPD liquid medium: the liquid culture medium consists of a solute and a solvent, wherein the solute and the concentration thereof are 1 percent (mass percent concentration) of yeast extract, 2 percent (mass percent concentration) of peptone and 2 percent (mass percent concentration) of glucose, and the solvent is water.

SC-Ura liquid selection Medium (sterile Yeast minimal Medium lacking Urapine): the 1L SC-Ura liquid selection medium was a medium obtained by adding 0.78g DO supply-Ura (Clontech), 6.7g amino-free yeast nitrogen source (YNB), 20g glucose to deionized water, and fixing the volume to 1L. The 1L SC-Ura solid selection medium was obtained by adding 20g of agar powder to the above liquid medium.

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

3mM H ₂ O ₂ SC-Ura liquid induction medium of (c): adding H into SC-Ura liquid induction culture medium ₂ O ₂ The H obtained ₂ O ₂ SC-Ura liquid induction medium with a concentration of 3 mM.

10mM and 20mM H ₂ O ₂ The aqueous solutions are prepared by adding H to sterile distilled water ₂ O ₂ The H obtained ₂ O ₂ The concentration of (C) is 10mM and 20mM respectively.

pCAMBIA1300-GFP empty plasmid: described in "Deng Zhi, zhao Manman, liu Hui, wang Yuekun, li Dejun. Molecular cloning, expression profiles and characterization ofa glutathione reductase in Hevea brilloiensis. Plant Physiology and Biochemistry.

2015, 96:53-63', which is available to the public from the national academy of tropical agriculture rubber research, is used only for repeated experiments related to the invention and is not used for other purposes.

Example 1, acquisition of thioredoxin HbTRXy2 of rubber tree and sequence analysis of Gene encoding the same

The sequence of an arabidopsis thioredoxin gene AtTRXy2 (NM_ 103481) is used as a probe, homologous comparison analysis is carried out on the rubber tree genome, TSA (Transcriptome ShotgunAssembly) and EST (Expressed Sequence Tags) databases, and a reference sequence containing a complete coding region is obtained through electronic splicing and bioinformatics analysis. Primers were designed and synthesized based on the reference sequence, and the specific primer sequences were as follows:

F1：5′-GGTGGCTTCTTTGGCTCTCT-3′；

R1：5′-GCAAAATTCCAGGAGTTCCA-3′。

Extracting latex total RNA of rubber tree by hot grinding 7-33-97 with RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit (Tiangen Biochemical technology Co., ltd.), reference to ReverteAid ^TM First Strand cDNA Synthesis Kit (Thermo Scientific) Instructions first strand cDNA was synthesized to give latex cDNA. The target gene was amplified by PCR using the latex cDNA as a template and the aforementioned primer F1/R1. The PCR reaction system is as follows: 2. Mu.L of cDNA template, 0.5. Mu. L F1/R1 primer (10. Mu. Mol/L), 5. Mu.LFastPfu Buffer，2μL 2.5mM dNTPs，0.5μL/>FastPfu DNAPolymerase (2.5U/. Mu.L) (Beijing full-scale gold biotechnology limited Law)Span), add ddH ₂ O to a total volume of 25. Mu.L. The reaction procedure is: pre-denaturation at 95℃for 3min; denaturation at 95℃for 20s, annealing at 55℃for 20s, extension at 72℃for 1min, setting 38 cycles in total; extending at 72℃for 10min.

The PCR products were subjected to 1% agarose gel electrophoresis, and the results showed that bands consistent with the expected targets were obtained. The band was excised and the fragment was recovered using an agarose gel recovery kit (OMEGA). The purified DNA fragment is then ligated to a vectorBlunt Simple Cloning Vector (Beijing full gold Biotechnology Co., ltd.) transformed E.coli Trans1-T1 Phage Resistant Chemically Competent Cell (Beijing full gold Biotechnology Co., ltd.) and positive clones were picked and sent to platinum Biotechnology (Shanghai) Co., ltd.) for sequencing.

Sequencing results show that the nucleotide sequence of the obtained PCR product is 852bp long, the nucleotide sequence is shown as SEQ ID No.2 in a sequence table, and the gene shown in the sequence is named HbTRXy2 gene. The coding sequence of HbTRXy2 gene is shown as 179 th-685 th nucleotide of SEQ ID No.2 (namely SEQ ID No. 3) in the sequence table, the coded protein is named HbTRXy2, and the amino acid sequence of HbTRXy2 is shown as SEQ ID No. 1.

SEQ ID No.1：

MAISSLSASTIPSLKTPHSQLSANLSCLSSLQFPAQLHRLQFGNRGISSPSRSRILPLLAAKKQTFSNLDELLANADKPVLVDFYAAWCGPCQLMSPILKEVSAILNDTIQVVKIDTEKYPSIADKYRIEALPTFIIFKDGKPYDRFEGAFSKDKFIQRIENSLQVKQ

SEQ ID No.2：

ggtggcttctttggctctctctcctctcttctttttcacagattttgtgttcttgcccagcgaagcatatttttggattttttgccctctcgattctatcctaagaataggcgttttgtggattctatttgtcggtagcagcagaagaagaagaaggagaagaagagtctagaaaactATGGCGATTTCTTCTCTCTCGGCTTCAACAATTCCTTCTTTGAAGACACCGCACTCGCAGTTGAGTGCTAATTTGAGTTGCTTGTCTTCGCTGCAGTTTCCAGCGCAGCTTCACAGGCTTCAGTTTGGGAACAGAGGGATTTCCTCTCCTTCTAGGTCTCGAATTTTGCCTCTGCTTGCAGCAAAGAAGCAAACATTTTCCAACTTGGATGAGTTATTGGCAAATGCTGACAAACCAGTCTTGGTTGACTTCTATGCAGCCTGGTGCGGTCCATGCCAGCTTATGAGTCCAATTCTTAAAGAGGTCAGTGCCATCCTGAATGACACAATCCAGGTGGTGAAAATCGATACTGAGAAGTACCCTAGCATTGCTGACAAATACAGAATAGAAGCATTGCCTACATTTATCATATTTAAGGATGGGAAACCTTATGATCGCTTCGAGGGTGCTTTCTCTAAAGATAAGTTCATTCAACGCATAGAAAATTCACTGCAAGTGAAGCAATAGttgaggttttacgaagaatgcaatatgaagacattacatatgaatcttactaggaatgccgcccattcttcccctcccagaatgttgtaatgactgttgttgtacctaaaaagagtgacaaacacaaattctaatggcatatttgtttggaactcctggaattttgc

SEQ ID No.3：

ATGGCGATTTCTTCTCTCTCGGCTTCAACAATTCCTTCTTTGAAGACACCGCACTCGCAGTTGAGTGCTAATTTGAGTTGCTTGTCTTCGCTGCAGTTTCCAGCGCAGCTTCACAGGCTTCAGTTTGGGAACAGAGGGATTTCCTCTCCTTCTAGGTCTCGAATTTTGCCTCTGCTTGCAGCAAAGAAGCAAACATTTTCCAACTTGGATGAGTTATTGGCAAATGCTGACAAACCAGTCTTGGTTGACTTCTATGCAGCCTGGTGCGGTCCATGCCAGCTTATGAGTCCAATTCTTAAAGAGGTCAGTGCCATCCTGAATGACACAATCCAGGTGGTGAAAATCGATACTGAGAAGTACCCTAGCATTGCTGACAAATACAGAATAGAAGCATTGCCTACATTTATCATATTTAAGGATGGGAAACCTTATGATCGCTTCGAGGGTGCTTTCTCTAAAGATAAGTTCATTCAACGCATAGAAAATTCACTGCAAGTGAAGCAATAG

The HbTRXy2 protein has a molecular weight of 18.70kD and a theoretical isoelectric point of 9.17. The amino acid sequence of HbTRXy2 was analyzed for conserved domains using ScanProsite (https:// prosite. Expanse/prosite. Html) and found to contain a Thioredoxin (Thioredoxin) conserved domain, located at amino acids 50-165, with highly conserved CGPC redox active sites at positions 89-92 (FIG. 1).

Multiple sequence comparison with other plant type-y thioredoxin shows that the thioredoxin conserved domain of different plant type-y thioredoxin is more conserved, but the sequence change of the protein N terminal is larger, wherein the difference between monocotyledonous plants and dicotyledonous plants is more obvious (figure 1). The selection of different types of plant thioredoxins together with HbTRXy2 protein was used to map the evolutionary tree using ClustalX and MEGA6.0 software, and the results indicated that these thioredoxins could be grouped into 7 classes: m, f, h, o, x, y and z. HbTRXy2 is grouped with other plant type y thioredoxins and therefore belongs to type y thioredoxins. HbTRXy2 was most closely related to cassava MeTRXy2 (XP_ 021600142.1) and castor RcTRXy2 (XP_ 002524295.1), with amino acid sequence identity of 87% and 84%, respectively (FIG. 2).

EXAMPLE 2 analysis of expression Pattern of rubber tree HbTRXy2 Gene

1. Tissue expression characterization of HbTRXy2 Gene

Collecting the root tissue samples of rubber tree hot ground into 7-33-97 (16 years old) latex, bark, female flower, male flower, young shoot, mature leaf, aged leaf, light green leaf, color-changing leaf, bronze leaf and tissue culture seedling transplanted and cultured for 8 months, and freezing with liquid nitrogen. And extracting total RNA of each tissue sample by using a general plant total RNA extraction kit (Beijing Baitaike biotechnology Co., ltd.) and referring to the kit instruction for specific extraction method. Using PrimeScript ^TM RT reagent Kit with gDNAEraser (Perfectreal Time) (TaKaRa) was reverse transcribed to give first strand cDNA, and the kit was used in the following methods. The cDNA of each tissue was used as a template, and the following primer pair was used for real-time fluorescent quantitative PCR (qRT-PCR) analysis. The HbUBC4 gene of rubber tree is used as an internal reference gene.

The primer sequences for detecting HbTRXy2 gene were as follows:

F2:5′-CTTTGAAGACACCGCACTCG-3′；

R2:5′-CCAAACTGAAGCCTGTGAAGC-3′。

the primer sequences for detecting HbUBC4 gene are as follows:

F3：5′-TCACCCTGAACCTGATAGCC-3′；

R3：5′-TTTCTTTGGTGACGCTGCAA-3′。

qRT-PCR was performed on a Bio-Rad CFX96 fluorescent quantitative PCR apparatus with 3 replicates per parallel assay. The reaction system: 10 mu LPremixEx Taq ^TM (2×) (TaKaRa), 1. Mu.L of each of the forward and reverse primers, 2. Mu.L of cDNA template, and ddH were added ₂ O to a total volume of 20. Mu.L. Reaction conditions: 95 ℃ for 5min;95 ℃ for 10s; 30s at 60 ℃; melting curve analysis was performed after 40 cycles to determine the specificity of the primers. The calculation of the relative expression amount of the gene adopts the formula 2 ^–ΔΔCt 。

As shown in FIG. 3, the results of the expression of HbTRXy2 gene in each tissue of rubber tree show that HbTRXy2 gene is expressed in rubber tree latex, bark, female flower, male flower, young shoot, mature leaf, senescent leaf, light green leaf, color-changing leaf, bronze leaf and root tissue. The first three tissues with higher expression level are leaf in color change period, leaf in light green period and latex in turn, and the tissues with relatively lower expression level are root and senescent leaf. The expression of HbTRXy2 gene increases and decreases with leaf development, and it is presumed that the gene may play a role in leaf development or leaf function.

2. Analysis of expression pattern of HbTRXy2 Gene under abiotic stress

Plant thioredoxin plays an important role in regulating redox balance, responding to abiotic stress and the like. To clarify the expression characteristics of HbTRXy2 gene under abiotic stress, applicants analyzed Methyl Viologen (MV) and hydrogen peroxide (H ₂ O ₂ ) Changes in HbTRXy2 gene expression under induced oxidative stress, low temperature, salt and PEG induced drought stress.

Methyl Viologen (MV) -induced oxidative stress treatment group: selecting healthy plants of rubber tree hot-ground 7-33-97 tissue culture seedlings which are transplanted and cultured for 8 months, and uniformly spraying 200 mu mol/L MV solution (the solute is Methyl Viologen (MV) and the solvent is sterile distilled water) on the front and back surfaces of all leaves of the plants. Plants 2 and 3 leaves were harvested at treatment times 0, 3, 6, 12, 24 and 48 hours, with 3 biological replicates per treatment time point, each replicate taken from 3 plants. And (5) freezing and storing the sample in liquid nitrogen after the sample is collected. Leaf total RNA was extracted, first-strand cDNA was synthesized by reverse transcription and HbTRXy2 gene expression was detected in each sample by qRT-PCR according to the method in step 1. Leaf samples treated for 0h (untreated) were used as controls.

Hydrogen peroxide (H) ₂ O ₂ ) Induced oxidative stress treatment group: selecting healthy plants of rubber tree cultivated for 8 months by transplanting and hot-grinding 7-33-97 tissue culture seedlings, and adding 20mmol/LH ₂ O ₂ The solution is uniformly sprayed on the front and back surfaces of all leaves of the plants. Plants 2 and 3 leaves were harvested at treatment times 0, 3, 6, 12, 24 and 48 hours, with 3 biological replicates per treatment time point, each replicate taken from 3 plants. And (5) freezing and storing the sample in liquid nitrogen after the sample is collected. The rest of the procedure was identical to that of Methyl Viologen (MV) -induced oxidative stress treatment group. To the point of Leaf samples treated for 0h (untreated) served as controls.

Low temperature stress treatment group: the healthy plants of the rubber tree cultivated for 8 months and subjected to hot grinding for 7-33-97 tissue culture seedlings are placed in a climatic chamber at 4 ℃ for low-temperature treatment, the illumination time is 16 hours, and the intensity is 600 mu mol/(m) ² S). The rest of the procedure was identical to that of Methyl Viologen (MV) -induced oxidative stress treatment group. Leaf samples treated for 0h (untreated) were used as controls.

NaCl-induced salt stress treatment group: the healthy plants of the rubber tree cultured for 8 months and subjected to hot grinding for 7-33-97 tissue culture seedlings are taken out of the seedling bags, the culture medium is cleaned, and the roots are immersed in 400mmol/LNaCl solution (the solute is NaCl and the solvent is sterile distilled water). The rest of the procedure was identical to that of Methyl Viologen (MV) -induced oxidative stress treatment group. Leaf samples treated for 0h (untreated) were used as controls.

PEG6000 induced drought stress treatment group: and taking out the healthy plants of the rubber tree heat-ground 7-33-97 tissue culture seedlings which are cultured for 8 months from the seedling bags, cleaning the culture medium, and immersing the roots in 20% PEG6000 solution (the solute is PEG6000 and the solvent is sterile distilled water) by mass percent. The rest of the procedure was identical to that of Methyl Viologen (MV) -induced oxidative stress treatment group. Leaf samples treated for 0h (untreated) were used as controls.

The results show that MV and H ₂ O ₂ Under the induced oxidative stress, hbTRXy2 gene expression change modes are similar and all show a trend of rising and then falling; the expression levels reached the highest at 6h treatment, 5.94 and 4.35 times that of control (0 h), respectively, after which the expression levels were reduced but still significantly higher than that of control (0 h) (fig. 4 and 5). The low temperature treatment upregulated the expression of HbTRXy2 gene, and the expression level reached the highest at 3h treatment, followed by a fall back but still significantly higher than control (0 h) (FIG. 6). PEG-induced drought stress treatment also upregulated HbTRXy2 gene expression, which was relatively stable throughout the treatment period, approximately 2-fold that of the control (0 h) (fig. 7). Under salt stress treatment, hbTRXy2 gene expression tends to be increased and then decreased; the expression level reaches the highest level in the treatment for 6-12h, which is about 5.30 times that of the control (0 h); after 12 hours of treatment, hbTRXy2 gene expression gradually decreased, and 48 hours of treatment, the expression level was only1/5 of control (0 h) (FIG. 8). The results show that the expression of HbTRXy2 gene is regulated and controlled by abiotic stress such as oxidization, low temperature, drought, salt and the like.

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 Listing, a primer pair is designed, wherein the amplified coding region does not contain a stop codon (179 th to 682 th nucleotides at the 5 'end), and recognition sequences (shown by a underlined part in the primer below) and a protecting base of restriction enzymes Sal I and Kpn I are respectively introduced into the 5' end of the forward primer and the reverse primer, and the specific primer sequences are as follows:

F4：5′-ACGCGTCGACATGGCGATTTCTTCTCTCTCG-3′；

R4：5′-GCGGTACCTTGCTTCACTTGCAGTGAATT-3′。

the target gene was amplified by PCR using latex cDNA as a template. The PCR reaction system is as follows: 2. Mu.L cDNA template, 5. Mu.LFastpfu Buffer, 2. Mu.L of 2.5mM dNTPs, 0.5. Mu.L of each of the forward and reverse primers F4 and R4 (10. Mu. Mol/L), 0.5. Mu.L of->FastPfu DNAPolymerase (2.5U/L), add ddH ₂ O to a total volume of 25. Mu.L. The reaction procedure is: pre-denaturation at 95℃for 3min; denaturation at 95℃for 20s, annealing at 55℃for 20s, extension at 72℃for 45s, setting up 38 cycles altogether; extending at 72℃for 10min.

The PCR product is connected to the agarose gel after electrophoresis, recovery and purificationBlunt Simple Cloning Vector (Beijing full gold Biotechnology Co., ltd.) E.coli Trans1-T1 Phage Resistant Chemically Competent Cell was transformed, positive clones were picked and sent to platinum Biotechnology (Shanghai) Co., ltd.) for sequencing. The plasmid extraction kit (OMEGA) is used for extracting and sequencing the correct clone and subcellular localization vector pCAMBIA1300-GFP empty load The plasmid was digested simultaneously with the restriction enzymes Sal I and Kpn I (Thermo Scientific). Double enzyme digestion system: kpn I (10U/. Mu.L) 2. Mu.L, xba I (10U/. Mu.L) 2. Mu.L, 10 XBamHI Buffer 3. Mu.L, plasmid DNA 8. Mu.L, ddH ₂ O to 30. Mu.L. Water bath at 37 ℃ for 3h. And (3) carrying out 1.0% agarose gel electrophoresis on the enzyme-digested product, and then cutting the gel to recover a target band. The recovered target gene was backbone-ligated to the pCAMBIA1300-GFP vector using T4 DNA ligase (Thermo Scientific), ligation system: 6. Mu.L of target gene recovery product, 2. Mu.L of pCAMBIA1300-GFP vector backbone, 1. Mu. L T4 of DNA ligase, 1. Mu.L of 10 XBuffer; the ligation was carried out overnight at 16 ℃. The ligation product was transformed into E.coli DH5a competent cells, which were then plated on LB solid plates containing calicheamicin (100 mg/L) and incubated overnight at 37 ℃. Selecting monoclonal shake bacteria, carrying out positive screening on colonies by using a vector 35S promoter primer (5'-TGACGCACAATCCCACTATCC-3') and a gene specific reverse primer (R4), and further carrying out sequencing identification on the positive clones. Sequencing results show that the 179 th to 682 th nucleotide sequences in SEQ ID No.2 are contained between Sal I and Kpn I cleavage sites of the recombinant vector, which indicates that the constructed subcellular localization vector is correct. The recombinant vector was named pCAMBIA1300-HbTRXy2-GFP. pCAMBIA1300-HbTRXy2-GFP positive clone was shaken to extract plasmids using a plasmid extraction kit (OMEGA).

2. PEG-mediated transformation of tobacco protoplasts and observation of subcellular localization

1) Cutting 4 leaves of Bentonite grown for about 4 weeks into strips of 0.5-1mM, adding 10mL of enzymolysis solution (0.4M mannitol, 20mM KCl, 1% cellulose R10, 0.4%macerozyme R10, 10mM CaCl) ₂ 5mM beta-mercaptoethanol, 0.1% BSA and 20mM MES, pH 5.7, solvent sterile deionized water).

2) And (3) performing enzymolysis for 4 hours at 25 ℃ and 40rpm in the dark.

3) An equal volume of pre-chilled W5 solution (154 mM NaCl, 125mM CaCl2, 5mM KCl, 2mM MES, pH 5.7, sterile deionized water as solvent) was added, the enzymatic hydrolysate was gently shaken, passed through a 300 mesh nylon sieve, and the filtrate was collected in a 50mL round bottom centrifuge tube and centrifuged at 600rpm for 5min at 23 ℃.

4) Removing supernatant, adding 10mLW solution to the precipitate, suspending, and standing on ice for 30min.

5) Centrifugation at 600rpm for 5min at 4℃and removal of supernatant, re-suspension in 1mL MMG solution (0.4M mannitol, 15mM MgCl) ₂ 4mM MES, pH 5.7, solvent was sterile deionized water).

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

7) Adding an equal volume of PEG solution (40% PEG4000, 0.2M mannitol, 100mM CaCl) ₂ ) Gently sucking, beating and mixing, and standing at room temperature for 30min.

8) 440. Mu.LW 5 solution (room temperature) was added, mixed upside down, centrifuged at 23℃and 600rpm for 2min, and the supernatant was removed.

9) Protoplasts were gently resuspended in 1mLWI solution (0.5M mannitol, 20mM KCl, 4mM MES, pH 5.7) and transferred to 6 well plates.

10 25℃for 16 hours, and the pellet was prepared and observed with a ZEISS/LSM 800 laser confocal microscope. The excitation light wavelengths of GFP and chloroplast autofluorescence are 488nm and 552nm, and the fluorescence collection wavelengths are 500-530nm and 650-680nm respectively.

Subcellular localization observations as shown in FIG. 9, protoplasts transformed with pCAMBIA1300-GFP empty vector, showed green fluorescence in cytoplasm and nucleus, whereas no green fluorescence was detected in chloroplasts. The protoplast transformed with pCAMBIA1300-HbTRXy2-GFP showed green fluorescence in chloroplasts and their periplasms. The above results indicate that HbTRXy2 protein localizes to chloroplasts and cytoplasm.

Example 4 HbTRXy2 Gene can increase resistance of transgenic Yeast to oxidative stress

1. Construction of HbTRXy2 Gene Yeast expression vector

Primer pairs for amplifying a coding region (179 th to 685 th nucleotides at the 5 'end) are designed according to SEQ ID No.2 in a sequence table, and recognition sequences (shown by a underlined part in the following primer) and a protecting base of restriction endonucleases Kpn I and Xba I are respectively introduced into the 5' ends of a forward primer and a reverse primer, wherein the specific primer sequences are as follows:

F5：5′-GGGGTACCATGGCGATTTCTTCTCTCTCG-3′；

R5：5′-GCTCTAGACTATTGCTTCACTTGCAGTGA-3′。

Extracting total RNA of latex of rubber tree, reverse transcribing into cDNA, using the cDNA as template, amplifying target gene by PCR. The PCR reaction system is as follows: 2. Mu.L cDNA template, 5. Mu.LFastpfu Buffer, 2. Mu.L of 2.5mM dNTPs, 0.5. Mu.L of each of the forward and reverse primers F5 and R5 (10. Mu. Mol/L), 0.5. Mu.L of->FastPfu DNAPolymerase (2.5U/L), add ddH ₂ O to a total volume of 25. Mu.L. The reaction procedure is: pre-denaturation at 95℃for 3min; denaturation at 95℃for 20s, annealing at 55℃for 20s, extension at 72℃for 45s, setting up 38 cycles altogether; extending at 72℃for 10min.

The PCR product is connected to the agarose gel after electrophoresis, recovery and purificationBlunt Simple Cloning Vector (Beijing full gold Biotechnology Co., ltd.) E.coli Trans1-T1 Phage Resistant Chemically Competent Cell was transformed, positive clones were picked and sent to platinum Biotechnology (Shanghai) Co., ltd.) for sequencing. The correct clone was sequenced by extraction using a plasmid extraction kit (OMEGA Co.) and the empty vector plasmid of the yeast expression vector pYES2 (Invitrogen Co.) was digested with restriction enzymes Kpn I and Xba I (Thermo Scientific). Double enzyme digestion system: kpn I (10U/. Mu.L) 2. Mu.L, xba I (10U/. Mu.L) 1. Mu.L, 10 XTango Buffer 3. Mu.L, plasmid DNA 8. Mu.L, ddH ₂ O to 30. Mu.L. Water bath at 37 ℃ for 3h. And (3) carrying out 1.0% agarose gel electrophoresis on the enzyme-digested product, and then cutting the gel to recover a target band. By T ₄ The DNA ligase connects the recovered target gene with the pYES2 carrier framework, and the connection system is as follows: 6. Mu.L of target gene recovery product, 2. Mu.L of pYES2 vector backbone, 1. Mu. L T4 DNA ligase, 1. Mu.L of 10 XBuffer; the ligation was carried out overnight at 16 ℃. The ligation product was transformed into E.coli DH5a competent cells, which were then plated on LB solid plates containing ampicillin (100 mg/L) and incubated overnight at 37 ℃. Picking up monoclonal shake bacteria, adding base by using pYES2 carrier primer (F6: 5'-CCCGGATCGGACTACTAGC-3')The colony is positively screened by the specific reverse primer (R5), and the positive clone is further sequenced and identified. Obtaining HbTRXy2 gene yeast expression vector pYES2-HbTRXy2.pYES2-HbTRXy2 is a recombinant plasmid obtained by replacing a fragment between Kpn I and Xba I recognition sites of pYES2 with a DNA molecule (coding region sequence of HbTRXy2 gene) having nucleotide sequences of 179 th to 685 th nucleotides in SEQ ID No.2, keeping other nucleotide sequences of pYES2 unchanged. The amino acid sequence expressed by pYES2-HbTRXy2 is protein HbTRXy2 of SEQ ID No.1 in the sequence table. Coli containing pYES2-HbTRXy2 was designated DH5a (pYES 2-HbTRXy 2).

2. Construction of recombinant Yeast

Saccharomyces cerevisiae strain INVScl (Invitrogen) is an auxotroph of urinary amino acid (Ura) ^－ ) On SC-Ura medium, the yeast strain is hardly capable of growth and propagation. The yeast expression vector pYES2 contains URA3 gene, and the expression of the gene can enable the yeast transformant to normally grow on an SC-Ura culture medium. Thus, positive yeast transformants can be screened with SC-Ura medium. Extracting pYES2 and recombinant expression vector pYES2-HbTRXy2 plasmids, respectively converting the plasmids into competent cells of saccharomyces cerevisiae strain INVSc1 by a lithium acetate method, coating the transformed saccharomycetes liquid on an SC-Ura solid selection medium, and culturing the transformed saccharomycetes liquid in an inversion way for 2-3 days at 30 ℃. Yeast monoclonal plasmid DNA was picked and positive transformants were identified by PCR. The specific operation steps are as follows:

1) Yeast INVSc1 was selected and added to 10mLYPD liquid medium and shake cultured overnight at 30℃and 200 rpm.

2) Detection of OD of Yeast liquid ₆₀₀ Value, adding overnight cultured yeast solution into 50mLYPD liquid medium, diluting to OD ₆₀₀ The shaking culture was continued at 30℃and 200rpm for 2-4h at 0.4.

3) The cells were collected by centrifugation at 2500rpm at 4℃for 5min, and the supernatant was collected and resuspended in 40mL of l.times.TE buffer.

4) The cells were collected by centrifugation at 2500rpm for another 5 minutes at 4℃and the supernatant was discarded, and the cells were resuspended in 2mL l.times.LiAc/0.5X TE.

5) The obtained resuspended cells were aliquoted into 1.5mL centrifuge tubes, 100. Mu.L per tube.

6) The sub-packed yeast cells were incubated at room temperature for 10min.

7) To each transformation system (100. Mu.L), 1. Mu.g of the plasmid extracted by shaking DH5a (pYES 2-HbTRXy 2) prepared in step 1 was added, and 100. Mu.g of denatured salmon sperm DNA was mixed.

8) 700 mu L l XLiAc/40% PEG-3350/l XTE was added and mixed.

9) Incubate at 30℃for 30min.

10 88. Mu.L of DMSO was added, and after mixing, heat shock was performed at 42℃for 7min.

11 4 ℃, centrifugation lmin at 5000rpm, and discarding the supernatant.

12 The cells were resuspended in lmL.times.TE buffer, centrifuged at 5000rpm at 4℃for lmin, and the supernatant was discarded.

13 Bacteria were resuspended in 100. Mu. L l XTE buffer and plated on SC-Ura solid selection medium and incubated for 2-3 days at 30℃with inversion. InVSc1 (pYES 2-HbTRXy 2) single clones were picked 3 from the SC-Ura solid selection medium and placed in 3mL of SC-Ura liquid selection medium, respectively, and cultured with shaking at 30℃and 200rpm for 24 hours. Plasmid DNA was extracted using the yeast plasmid extraction kit (OMEGA). And (3) taking yeast plasmid DNA as a template, and adopting F6 and R5 primers to carry out bacterial liquid PCR positive detection. The positive clone was recombinant yeast containing pYES2-HbTRXy2, which was designated INVSc1 (pYES 2-HbTRXy 2).

The other steps were not changed except that pYES2-HbTRXy2 was replaced with pYES2 according to the above method. The PCR positive assay was performed using pYES2 vector primers F6 (5'-CCCGGATCGGACTACTAGC-3' and R6 (5'-ATTAAAGCCTTCGAGCGTCC-3'), and a recombinant yeast containing pYES2 was obtained and designated INVSc1 (pYES 2).

The agarose gel electrophoresis of the PCR products is shown in FIG. 10, and the recombinant yeast plasmids transferred with the pYES2 empty vector are amplified into bands (4, 5 and 6 in FIG. 10) with the sizes consistent with that of positive control (pYES 2 plasmid) and accord with the expected length of 402bp; the 3 pYES2-HbTRXy 2-transferred recombinant yeast plasmids amplified bands (1, 2, 3 in FIG. 10) that were identical in size to the positive control (pYES 2-HbTRXy2 plasmid) and were 577bp in expected length. The above results indicate that both pYES2-HbTRXy2 and pYES2 empty vector were successfully transferred into recombinant yeast.

3. Detection of HbTRXy2 Gene expression in recombinant Yeast

The INVSc1 (pYES 2-HbTRXy 2) and INVSc1 (pYES 2) positive clones obtained in step 2 were streaked onto SC-Ura solid selection medium and cultured upside down at 30℃for 2-3 days. Inoculating the monoclonal into 2mL SC-Ura liquid selection medium, shake culturing at 30deg.C and 200rpm for 24h, and measuring OD ₆₀₀ Values. Taking 1mL of cultured bacterial liquid, centrifuging at 4000r/min for 3min, discarding supernatant, re-suspending bacterial cells by using SC-Ura liquid induction culture medium, and adjusting OD ₆₀₀ When 10ml of each was subjected to induction culture at 30℃and 200rpm for 36 hours, 2ml of each was collected by low-speed centrifugation, and total RNA was extracted using a yeast total RNA rapid extraction kit (Shanghai Co., ltd.). Using PrimeScript ^TM RT reagent Kit with gDNAEraser (Perfectreal Time) (TaKaRa) first strand cDNA was synthesized, and the specific methods are described in the kit. Firstly, determining the addition amount of cDNA of each template by using reference gene Actin (GeneBank accession number: L00026.1) of Saccharomyces cerevisiae, wherein the sequence of an Actin primer is as follows:

F7：5′-AGTTGCCCCAGAAGAACACC-3′；

R7：5′-TACCGGCAGATTCCAAACCC-3′。

and then PCR amplification is carried out on the template by utilizing HbTRXy2 gene primers (F5 and R5). The PCR reaction system is as follows: template 1-3. Mu.L, buffer 2.5. Mu.L, dNTPs 2. Mu.L, forward and reverse primers each 0.5. Mu.L, easyTaq DNAPolymerase (Beijing full gold Biotechnology Co., ltd.) 0.5. Mu.L, and ddH ₂ O to a total volume of 25. Mu.L. The reaction procedure is: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 45s, 35 cycles were set up altogether; extending at 72℃for 10min.

As a result, as shown in FIG. 11, when cultured for 36 hours under induction, hbTRXy2 gene expression was detected in each clone of recombinant yeast INVSc1 (pYES 2-HbTRXy 2) (4, 5, 6 in FIG. 11), while HbTRXy2 gene expression was not detected in control yeast INVSc1 (pYES 2) (1, 2, 3 in FIG. 11). The above results indicate that the rubber tree HbTRXy2 gene was successfully transferred into recombinant yeast INVSc1 (pYES 2-HbTRXy 2) and induced to be expressed.

4. Oxidation resistance comparative analysis of recombinant Yeast INVSc1 (pYES 2-HbTRXy 2) and INVSc1 (pYES 2)

(1) Recombinant yeasts INVSc1 (pYES 2-HbTRXy 2) and INVSc1 (pYES 2) at H ₂ O ₂ Growth differential comparison in liquid induction medium

The INVSc1 (pYES 2-HbTRXy 2) and INVSc1 (pYES 2) positive clones obtained in step 2 were streaked onto SC-Ura solid selection medium and cultured upside down at 30℃for 2-3 days. INVSc1 (pYES 2-HbTRXy 2) and INVSc1 (pYES 2) were individually picked and inoculated into 5mL of SC-Ura liquid selection medium, followed by shaking culture at 30℃and 200rpm for 24 hours. 4000r/min, centrifuging for 3min, discarding supernatant, resuspending thallus with SC-Ura liquid induced culture medium, and adjusting OD ₆₀₀ 10mL of the resuspension broth was obtained and cultured at 30℃and 200rpm for 36 hours to induce the expression of HbTRXy2 gene, thereby obtaining INVSc1 (pYES 2-HbTRXy 2) induction culture broth and INVSc1 (pYES 2) induction culture broth, respectively.

pYES2-HbTRXy2 oxidative stress treatment group (i.e., hbTRXy2-H ₂ O ₂ ): taking 1mL of the above INVSc1 (pYES 2-HbTRXy 2) induction culture solution, centrifuging for 3min at 4000r/min, discarding supernatant, and adding 3mM H ₂ O ₂ The SC-Ura liquid induction culture medium of (C) is used for resuspension of thalli, and OD is regulated ₆₀₀ =0.2, resulting in an adjusted bacterial solution. The prepared bacterial liquid was cultured at 30℃and 200rpm in 10 mL. At the time of culturing for 12 hours, 24 hours and 36 hours, the OD of each culture broth was measured by using a spectrophotometer ₆₀₀ Values.

pYES2 oxidative stress treatment group (i.e., pYES 2-H) ₂ O ₂ ): substitution of INVSc1 (pYES 2) induction culture medium for INVSc1 (pYES 2-HbTRXy 2) induction culture medium, and the other procedures were the same as those of pYES2-HbTRXy2 oxidative stress treatment group (i.e., hbTRXy 2-H) ₂ O ₂ )。

pYES2-HbTRXy2 control group (i.e., hbTRXy 2-CK): replacement of 3mM H-containing medium with SC-Ura liquid induction ₂ O ₂ The other operations were identical to those of the pYES2-HbTRXy2 oxidative stress-treated group (i.e., pYES 2-H) ₂ O ₂ )。

pYES2 control group (i.e., pYES 2-CK): replacement of 3mM H-containing medium with SC-Ura liquid induction ₂ O ₂ The other operations are the same as those of the pYES2 oxidative stress treatment group (namely pYES 2-H) ₂ O ₂ )。

As a result, as shown in FIG. 12, there was no oxidative stress, and the OD of the pYES2-HbTRXy2 control group (i.e., hbTRXy 2-CK) and the pYES2 control group (i.e., pYES 2-CK) were increased at the time of culturing for 12h, 24h and 36h ₆₀₀ There is no significant difference between the values, which indicates that HbTRXy2 gene transfer into yeast has no obvious effect on the growth of yeast. H ₂ O ₂ pYES2-HbTRXy2 oxidative stress treatment group (i.e. HbTRXy 2-H) under induced oxidative stress ₂ O ₂ ) And pYES2 oxidative stress treatment group (i.e., pYES 2-H) ₂ O ₂ ) Is significantly inhibited. However, at each treatment time point, pYES2-HbTRXy2 oxidative stress treatment group (i.e., hbTRXy2-H ₂ O ₂ ) OD of (d) ₆₀₀ The values were all significantly higher than in the pYES2 oxidative stress treated group (i.e.pYES 2-H ₂ O ₂ ) Indicating that pYES2-HbTRXy2 oxidative stress treatment group (i.e. HbTRXy2-H ₂ O ₂ ) The bacterial liquid concentration of (2) is obviously higher than that of the pYES2 oxidative stress treatment group (namely pYES 2-H) ₂ O ₂ ) As a result, it was found that the recombinant yeast expressing HbTRXy2 gene had an increased resistance to oxidative stress.

(2) High concentration H ₂ O ₂ Survival differences between recombinant yeasts INVSc1 (pYES 2-HbTRXy 2) and INVSc1 (pYES 2) after stress treatment

The INVSc1 (pYES 2-HbTRXy 2) and INVSc1 (pYES 2) positive clones obtained in step 2 were streaked onto SC-Ura solid selection medium and cultured upside down at 30℃for 2-3 days. INVSc1 (pYES 2-HbTRXy 2) and INVSc1 (pYES 2) were individually picked and inoculated into 5mL of SC-Ura liquid selection medium, followed by shaking culture at 30℃and 200rpm for 24 hours. 4000r/min, centrifuging for 3min, discarding supernatant, resuspending the thallus with SC-Ura liquid induction culture medium, adjusting OD600 = 0.2 to obtain a resuspension bacterial liquid, taking 10mL of the resuspension bacterial liquid, culturing at 30 ℃ and 200rpm for 36h to induce HbTRXy2 gene expression, and respectively obtaining INVSc1 (pYES 2-HbTRXy 2) induction culture liquid or INVSc1 (pYES 2) induction culture liquid.

HbTRXy210mM H ₂ O ₂ Treatment group: taking 2mL of INVSc1 (pYES 2-HbTRXy 2) induction culture solution, centrifuging for 3min at 4000r/min, discarding supernatant, and suspending the strain in 10mM H ₂ O ₂ Aqueous solution, adjust OD ₆₀₀ =1.0, resulting in an adjusted weightSuspension bacteria liquid. 1mL of the adjusted resuspended bacterial liquid was taken out in a 10mL centrifuge tube, and treated at 30℃and 160rpm for 24 hours to obtain a treated bacterial liquid. The treated bacterial liquid was subjected to 5-fold gradient dilution with sterile distilled water (5 ^-1 、5 ^-2 、5 ^-3 、5 ^-4 、5 ^-5 ). The undiluted bacterial solutions and bacterial solutions with different dilution factors are respectively spotted on SC-Ura solid selection medium in 5 mu L, and each treatment is repeated for 3 times. Inverted culture at 30℃for 3 days, and observation of the difference in growth of the comparative colonies.

pYES210mM H ₂ O ₂ Treatment group: INVSc1 (pYES 2) was used instead of INVSc1 (pYES 2-HbTRXy 2), and the rest of the procedure was identical to HbTRXy210mM H ₂ O ₂ Treatment groups.

HbTRXy220mM H ₂ O ₂ Treatment group: at 20mM H ₂ O ₂ Instead of 10mM H ₂ O ₂ The other operations are the same as HbTRXy210mM H ₂ O ₂ Treatment groups.

pYES220mM H ₂ O ₂ Treatment group: at 20mM H ₂ O ₂ Instead of 10mM H ₂ O ₂ The remaining procedure was as for pYES210mM H ₂ O ₂ Treatment groups.

HbTRXy2 control: replacement of 10mM H with sterile distilled water ₂ O ₂ The other operations are the same as HbTRXy210mM H ₂ O ₂ Treatment groups.

pYES2 control group: replacement of 10mM H with sterile distilled water ₂ O ₂ The remaining procedure was as for pYES210mM H ₂ O ₂ Treatment groups.

The growth of the above-described INVSc1 (pYES 2-HbTRXy 2) and INVSc1 (pYES 2) recombinant yeasts in each group is shown in FIG. 13. There was no significant difference in the number of plaques (CFU) of INVSc1 (pYES 2-HbTRXy 2) and INVSc1 (pYES 2) at the same dilution of the control group (without oxidative stress). 10mM H ₂ O ₂ Treatment group, 5 ^-1 And 5 ^-2 After dilution, INVSc1 (pYES 2-HbTRXy 2) still had plaque growth, while INVSc1 (pYES 2) had no plaque growth; the number of plaques grown by INVSc1 (pYES 2-HbTRXy 2) was significantly greater than that of INVSc1 (pYES 2) using undiluted bacterial liquid spot plates. 20mM H ₂ O ₂ Treatment group, 5 ^-1 After dilution, INVSc1 (pYES2-HbTRXy 2) still had plaque growth, whereas INVSc1 (pYES 2) had no plaque growth; the number of plaques grown by INVSc1 (pYES 2-HbTRXy 2) was also significantly greater than that of INVSc1 (pYES 2) using undiluted bacterial liquid spot plates. The results show that the HbTRXy2 gene can improve the high-concentration H of the recombinant yeast ₂ O ₂ The survival rate of the induced oxidative stress after treatment, and the HbTRXy2 gene is expressed to improve the resistance of the recombinant yeast to the oxidative stress.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims

1. Use of a protein, a substance regulating the expression of a gene encoding said protein or a substance regulating the content of said protein, characterized in that said use is any of the following:

d1 Use in modulating resistance to biotic oxidative stress;

d4 Use in modulating abiotic stress resistance of an organism;

the protein is any one of the following:

a1 A protein having 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 modulating resistance to biotic oxidative stress;

d4 Use in modulating abiotic stress resistance of an organism;

the biomaterial is any one of the following B1) to B7):

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);

4. The use according to claim 3, wherein the nucleic acid molecule of B1) is the gene of B1) or B2) or B3) or B4) as follows:

b3 A cDNA molecule or DNA molecule having more than 90% identity to the nucleotide sequence defined in b 1) or b 2) and encoding HbTRXy 2;

b4 Under stringent conditions with the nucleotide sequence defined in b 1) or b 2) and encoding a cDNA molecule or a DNA molecule of HbTRXy 2.

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

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

7. A protein as claimed in claim 1 or claim 2.

8. The biomaterial of claim 3 or 4.