Transcript of thioredoxin gene HbCXXS2 and its encoded protein and application

Abstract

The present invention discloses a transcript of a thioredoxin gene HbCXXS2 and its encoded protein and application, belongs to the field of genetic engineering technology, and is used to improve the resistance of rubber trees to oxidative stress from a molecular level. The present invention provides transcripts HbCXXS2.1 and HbCXXS2.2 of the thioredoxin gene HbCXXS2 and their encoded proteins, the nucleotide sequence of the transcript HbCXXS2.1 is shown in SEQ ID No.1, and the nucleotide sequence of the transcript HbCXXS2.2 is shown in SEQ ID No.3. The present invention separates and identifies genes that regulate oxidative stress resistance, enhances plant resistance to oxidative stress by genetic engineering means, and improves the ability of plants to resist various adverse stresses.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and more specifically to a transcript of a thioredoxin gene HbCXXS2 and its encoded protein and application. Background Art

[0002] Reactive oxygen species (ROS) are byproducts of plant aerobic metabolism, mainly including hydrogen peroxide (H2O2), superoxide anion (O2 - ), hydroxyl radical (OH - ) etc. Under normal environmental conditions, the generation and removal of ROS in plants is in a dynamic balance, and ROS will not accumulate in large quantities to cause toxicity to plants. However, under biotic or abiotic stress, the dynamic balance of the generation and removal of ROS in plants is broken, resulting in excessive accumulation of ROS, oxidative stress, oxidative damage to the structure and function of plant cells, hindering the normal growth and development of plants, and even leading to plant death. In order to adapt to adverse adverse environments, plants have formed a complex ROS balance regulation mechanism during the evolution process to improve their resistance to oxidative stress and avoid or reduce the oxidative damage to cells caused by the large accumulation of ROS.

[0003] Thioredoxin is an important small molecule redox regulatory protein that is ubiquitous in eukaryotic and prokaryotic organisms and plays an important role in maintaining redox balance in organisms. There are many members of the plant thioredoxin family. For example, there are at least 20 thioredoxin genes in the Arabidopsis genome. Based on amino acid sequence and structural characteristics, plant thioredoxins are divided into different types such as f, m, h, o, x, y and z. The vast majority of plant thioredoxins have a highly conserved WC(G / P)PC active center containing two cysteine ​​residues. In a very small number of h-type thioredoxins, the second cysteine ​​in the active center becomes serine, which is CXXS. At present, it is known that Arabidopsis contains two thioredoxin genes with CXXS active centers: AtCXXS1 and AtCXXS2. In addition, CXXS thioredoxin genes were also found in poplar and Medicago truncatula, but it is still unclear whether CXXS thioredoxin genes exist in rubber tree (Hevea brasiliensis Muell.Arg.).

[0004] Rubber tree is the main commercial source of important industrial raw materials and strategic materials natural rubber. my country's rubber tree planting area is about 17 million mu, distributed in Hainan, Yunnan and Guangdong. These areas belong to non-traditional rubber planting areas, are not the best areas for rubber tree planting, and are susceptible to the influence of abiotic stress such as low temperature and drought. In addition, the incidence of rubber tree cut surface drying up (tapping panel dryness, TPD, often referred to as dead skin in China) is also relatively high, resulting in serious yield and economic losses. Rubber tree cut surface drying up is a complex physiological syndrome, and the main symptom is that the cutting line is partially or completely not discharged after tapping. Studies have shown that redox homeostasis imbalance is one of the important reasons causing the rubber tree cut surface drying up, and the response of oxidative stress is regulated by multiple genes, and transcripts are one or more mature mRNAs that can be used for encoding proteins formed by transcription of a gene, and transcripts are related to biological function activity. Therefore, the present invention studies the transcript of thioredoxin gene HbCXXS2 and its application in regulating oxidative stress. Summary of the invention

[0005] The present invention provides a transcript of a thioredoxin gene HbCXXS2 and a protein encoded therein and an application thereof, which are used to improve the resistance of organisms to oxidative stress at a molecular level.

[0006] In the first aspect, the present invention provides a transcript of the thioredoxin gene HbCXXS2, wherein the transcript is transcript HbCXXS2.1 or transcript HbCXXS2.2, wherein the nucleotide sequence of the transcript HbCXXS2.1 is shown in SEQ ID No. 1, and the nucleotide sequence of the transcript HbCXXS2.2 is shown in SEQ ID No. 3. Transcript: One or more mature mRNAs that can encode proteins formed by transcription of a gene. In the present invention, the gene HbCXXS2 expresses two transcripts through different selective splicing methods: transcript HbCXXS2.1 and transcript HbCXXS2.2.

[0007] In a second aspect, the present invention provides thioredoxin HbCXXS2, which is encoded by the transcript of the gene HbCXXS2 described in the first aspect, and the amino acid sequence of the protein HbCXXS2 is shown in SEQ ID No.2 or SEQ ID No.4.

[0008] In the third aspect, the present invention provides a vector comprising the open reading frame sequence of the transcript HbCXXS2.1 or the open reading frame sequence of the transcript HbCXXS2.2 described in the first aspect; the nucleotide sequence of the open reading frame of the transcript HbCXXS2.1 is shown at positions 160 to 537 in SEQ ID No.1, and the nucleotide sequence of the open reading frame of the transcript HbCXXS2.2 is shown at positions 160 to 414 in SEQ ID No.3.

[0009] As a possible implementation method, the open reading frame sequence of the transcript HbCXXS2.1 or the open reading frame sequence of the transcript HbCXXS2.2 is inserted between the Nco I and BstEⅡ restriction sites of the pCAMBIA1301 plasmid to obtain a plant expression vector; or, the open reading frame sequence of the transcript HbCXXS2.1 or the open reading frame sequence of the transcript HbCXXS2.2 is inserted between the Kpn I and Xba I restriction sites of the pYES2 plasmid to obtain a yeast expression vector.

[0010] In a fourth aspect, the present invention provides use of the transcript of the gene HbCXXS2 in the first aspect, the protein HbCXXS2 in the second aspect, or the vector in the third aspect in improving the resistance of an organism to oxidative stress.

[0011] As a possible implementation manner, the transcript of the gene HbCXXS2 described in the first aspect or the protein HbCXXS2 described in the second aspect or the vector described in the third aspect is used to improve the survival rate and / or growth activity of yeast under oxidative stress.

[0012] As a possible implementation manner, the transcript of the gene HbCXXS2 described in the first aspect or the protein HbCXXS2 described in the second aspect or the vector described in the third aspect is used to promote the growth of tobacco under oxidative stress.

[0013] As a possible implementation manner, the transcript of the gene HbCXXS2 described in the first aspect or the protein HbCXXS2 described in the second aspect or the vector described in the third aspect is used to regulate the occurrence or recovery of cut surface drying of rubber trees.

[0014] As a possible implementation, the transcript of the gene HbCXXS2 described in the first aspect, the protein HbCXXS2 described in the second aspect, or the vector described in the third aspect is used to prepare a product for preventing or regulating the drying up of the cut surface of rubber trees.

[0015] In a fifth aspect, the present invention provides a method for cultivating a rubber tree variety with a reduced incidence of cut surface dry-up, which is achieved by up-regulating the expression of the transcript of the gene HbCXXS2 described in the first aspect.

[0016] The present invention separates and identifies the gene HbCXXS2 regulating oxidative stress resistance and its transcript, enhances the resistance of organisms to oxidative stress by genetic engineering means, improves the ability of organisms to resist various adverse external environments, and obtains organisms with the ability to resist oxidative stress.

[0017] The present invention separates and identifies the rubber tree gene HbCXXS2 and its transcripts which regulate cut surface dryness by adjusting antioxidant activity, improves the resistance of the rubber tree to oxidative stress by genetic engineering means, reduces the occurrence of cut surface dryness, and plays an important role in enhancing the adaptability of the rubber tree to adverse environments, increasing the yield of natural rubber, and cultivating rubber trees with improved oxidative stress resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The PCR amplification result of the rubber tree HbCXXS2 gene provided in the embodiment of the present invention.

[0020] Figure 2 The structure of the rubber tree HbCXXS2 gene and its two transcripts provided in the embodiments of the present invention.

[0021] Figure 3 The present invention provides a sequence alignment and conserved domain analysis of the proteins encoded by the two transcripts of the HbCXXS2 gene of the rubber tree, wherein the background portion is the conserved domain of thioredoxin and the underlined portion is the CXXS active motif.

[0022] Figure 4 The expression of two transcripts provided in the embodiments of the present invention in various tissues of rubber trees.

[0023] Figure 5 The expression differences of the two transcripts provided in the embodiments of the present invention in the latex of healthy, TPD and TPD-restored plants, where different lowercase letters indicate significant differences among samples (P<0.05).

[0024] Figure 6 The expression changes of two transcripts under H2O2-induced oxidative stress treatment provided in the embodiments of the present invention, wherein different lowercase letters indicate significant differences between treatment time points (P<0.05).

[0025] Figure 7The expression changes of two transcripts under oxidative stress induced by methyl viologen (MV) provided in the examples of the present invention, wherein different lowercase letters indicate significant differences between treatment time points (P<0.05).

[0026] Figure 8 This is the PCR positive detection of the recombinant yeast provided in an embodiment of the present invention, wherein M is DL2000 DNA Marker (TaKaRa), N1 and N2 are negative controls without template, P1 is the pYES2 plasmid positive control, P2 is the pYES2-HbCXXS2.1 plasmid positive control, P3 is the pYES2-HbCXXS2.2 plasmid positive control, 1 to 3 are pYES2 transformed yeast monoclones, 4 to 6 are pYES2-HbCXXS2.1 transformed yeast monoclones, and 7 to 9 are pYES2-HbCXXS2.2 transformed yeast monoclones.

[0027] Fig. 9 The semi-quantitative RT-PCR provided in the embodiment of the present invention is used to detect the expression of the HbCXXS2 gene in the recombinant yeast, wherein M represents DL2000 DNA Marker (TaKaRa); 1 to 3 are cDNA samples of the recombinant yeast INVSc1 (pYES2) monoclonal induction for 24 hours; 4 to 6 are cDNA samples of the recombinant yeast INVSc1 (pYES2-HbCXXS2.1) monoclonal induction for 24 hours; 7 to 9 are cDNA samples of the recombinant yeast INVSc1 (pYES2-HbCXXS2.2) monoclonal induction for 24 hours.

[0028] Fig.10 The survival status of the recombinant yeast INVSc1(pYES2-HbCXXS2.1), INVSc1(pYES2-HbCXXS2.2) and INVSc1(pYES2) after 24h of H2O2 treatment provided in the embodiments of the present invention.

[0029] Fig.11 The growth rate (OD) of the recombinant yeast under H2O2-induced oxidative stress when cultured for 36 h is shown in the embodiment of the present invention. 600 Different lowercase letters indicate significant differences (P<0.05).

[0030] Fig.12PCR detection of transgenic regenerated Nicotiana benthamiana plants provided in an embodiment of the present invention, wherein M is DL2000 DNA Marker (TaKaRa); N is non-transgenic Nicotiana benthamiana, P1 and P2 are pCAMBIA1301-HbCXXS2.1 and pCAMBIA1301-HbCXXS2.2 plasmids, respectively; 1 to 7 are pCAMBIA1301-HbCXXS2.1 transgenic plants; 8 to 19 are pCAMBIA1301-HbCXXS2.2 transgenic plants.

[0031] Fig.13 The qRT-PCR provided in the embodiment of the present invention detects the expression levels of transcripts HbCXXS2.1 and HbCXXS2.2 in transgenic plants, wherein Wt is non-transgenic Nicotiana benthamiana.

[0032] Fig.14 The overexpression of HbCXXS2 transcripts HbCXXS2.1 or HbCXXS2.2 in Nicotiana benthamiana provided in the embodiments of the present invention both improves resistance to oxidative stress, wherein A represents the phenotypic difference of the plants after 4 weeks of cultivation, in A, Wt represents the Wt plants of 2 repeated experiments, HbCXXS2.2-11 represents the plants of the HbCXXS2.2-11 strain of 2 repeated experiments, and HbCXXS2.2-16 represents the plants of the HbCXXS2.2-11 strain of 2 repeated experiments. A represents plants of HbCXXS2.2-16 line with two repeated experiments, HbCXXS2.1-5 represents plants of HbCXXS2.1-5 line with two repeated experiments, HbCXXS2.1-7 represents plants of HbCXXS2.1-7 line with two repeated experiments, B is the fresh weight of the plant, C is the plant height; Wt is non-transgenic Nicotiana benthamiana; different lowercase letters indicate significant differences (P<0.05). DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0034] In order to improve the resistance of organisms to oxidative stress at the molecular level, the present invention provides a transcript of a thioredoxin gene HbCXXS2 and its encoded protein and application.

[0035] The present invention provides a transcript of a thioredoxin gene HbCXXS2, wherein the gene HbCXXS2 expresses two transcripts through different selective splicing modes: transcript HbCXXS2.1 and transcript HbCXXS2.2. The nucleotide sequence of transcript HbCXXS2.1 is shown in SEQ ID No.1, and the nucleotide sequence of transcript HbCXXS2.2 is shown in SEQ ID No.3.

[0036] The present invention separates and identifies genes regulating oxidative stress resistance and cut surface dryness, enhances the resistance of organisms to oxidative stress through genetic engineering means, improves the ability of organisms to resist various adverse external environments, and obtains organisms with enhanced anti-oxidative stress ability.

[0037] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0038] The quantitative tests in the examples of the present invention were all repeated three times, and the experimental results were expressed as average values. The data were processed using SigmaPlot 12.0 statistical software, and different lowercase letters indicated significant differences (P<0.05).

[0039] The rubber tree (Hevea brasiliensis Muell.Arg.) variety used in the embodiment of the present invention is Reyan 7-33-97: recorded in "Huang Huasun, Liang Maohuan, Wu Yuntong, Li Deshun, He Jinwei. Breeding of medium-scale promotion-level rubber tree excellent variety Reyan 7-33-97. Journal of Tropical Crops, 1994, 15 (2): 1-6", cultivated by Rubber Research Institute of Chinese Academy of Tropical Agricultural Sciences, obtained from Rubber Research Institute of Chinese Academy of Tropical Agricultural Sciences.

[0040] Example 1

[0041] This example provides the cloning and analysis of the rubber tree thioredoxin gene HbCXXS2.

[0042] 1. Cloning of two different transcripts of HbCXXS2 gene

[0043] The latex of rubber trees was subjected to transcriptome sequencing analysis, from which the sequence of the candidate HbCXXS2 gene was obtained. Based on the sequence, primers for amplifying the gene were designed using Primer Premier 5. The primers were synthesized by Hainan Nanshan Biotechnology Co., Ltd. The sequence of primer HbCXXS2-F is shown in SEQ ID No.6, and the sequence of primer HbCXXS2-R is shown in SEQ ID No.7;

[0044] SEQ ID No.6: 5′-CCAGCAGGGAACCTTTCATA-3′;

[0045] SEQ ID No. 7: 5′-GAGTTAAATGGTAAGGCCACAA-3′.

[0046] The total RNA of latex from rubber tree Reyan 7-33-97 was extracted using RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd.). The total RNA of latex was reverse transcribed using RevertAid 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 HbCXXS2-F / R. The PCR reaction system was: 5×TransStart FastPfuBuffer 5μL, 2.5mM dNTPs 2μL, TransStart FastPfu DNAPolymerase (2.5U / μL) (Beijing Quanshijin Biotechnology Co., Ltd.) 0.5μL, F1 / R1 primers (10μmol / L) 0.5μL each, latex cDNA 2μL, and ddH2O was added to a total volume of 25μL. The PCR reaction program was: 95°C for 3 min; 95°C for 20 s, 56°C for 20 s, 72°C for 1 min, 35 cycles; and 72°C for 10 min extension to obtain the PCR product.

[0047] The PCR products were detected by 1.2% agarose gel electrophoresis. Figure 1 The two bands of different sizes shown indicate that the HbCXXS2 gene expresses two transcripts of different sizes. The transcripts represented by the bands of different sizes are named HbCXXS2.1 and HbCXXS2.2. The two bands were cut out respectively, and the corresponding fragments were recovered using an agarose gel recovery kit (OMEGA). The two purified DNA fragments were connected to pEASY-Blunt Simple Cloning Vector (Beijing Quanshijin Biotechnology Co., Ltd.), and the connection products were heat-shocked and transformed into Escherichia coli Trans1-T1 Phage Resistant Chemically Competent Cell (Beijing Quanshijin Biotechnology Co., Ltd.). The positive single clones were identified by colony PCR and sent to Hainan Nanshan Biotechnology Co., Ltd. for sequencing.

[0048] The sequencing results showed that the nucleotide sequence of the transcript HbCXXS2.1 was 753 bp long, and its nucleotide sequence was shown in SEQ ID No. 1; the open reading frame of HbCXXS2.1 was 378 bp long, and its sequence was shown in nucleotides 160 to 537 of SEQ ID No. 1, and the protein encoded by it was named HbCXXS2.1, and HbCXXS2.1 had 125 amino acids, and its amino acid sequence was shown in SEQ ID No. 2. The nucleotide sequence of the transcript HbCXXS2.2 was 630 bp long, and its nucleotide sequence was shown in SEQ ID No. 3; the open reading frame of HbCXXS2.2 was 255 bp long, and its sequence was shown in nucleotides 160 to 414 of SEQ ID No. 3, and the protein encoded by it was named HbCXXS2.2, and HbCXXS2.2 had 84 amino acids, and its amino acid sequence was shown in SEQ ID No. 4.

[0049] SEQ ID No.1:

[0050] CCAGCAGGGAACCTTTCATATTTTGCTAATCTGGTAGCAATGGAAGCATGAAAGAAATGGTAGTGTCTACTCTAGTCTAGAACAAGAATCAAAGCTCCAGGGAAGGAGGTAAAGAAGAGAAACCAAGACAAGAAGAAGAAGAAGAAGAAGAAAGGAGACGAGATGGAGACCCAAGAGCAGCAAGCTAAGTCACGAGTTGTGAAGGTGGATTCGGTGGAGTCTTGGGATTTCTATGTTACCCAAGCCAATAATCAAGGCTGCCCTATTGTTGTACACTTTACTGCTTCATGGTGTATCCCCTCTGTGGCCATGAACCCTTTTTTCGAGGAATTAGCCTCGGCTTATCCAGATGTCCTGTTTCTAGCTGTTGATGTCGATG AAGTCAAGGAGGTGGCTTCTAAACTGGAGGTAAAGGCCATGCCTACATTTGTGCTGATGAAGGATGGTGCACAAATTGACAGGCTGGTGGGTGCCAATCCAGAGGAGATAAAGAAAAGGATTGGTGGGTTTGCACAGTCCATTCGTGGCTGTAGCCTAGTTACTGTGTGTGTGTGTGTGTGTGTGTGA GAGAGAGAGAGAGAGAGAGTGGAGAGAGATCTTGTATATTATGAACAGGTAAAATATGTGGTTATGGTGGACGGTGGAGCAGCACTTTAAACAGAAACGATGTTGATGATTGTGGTTTTGTATATGTTGATTACTGTCGCAAATCATGTAGTTAGAAAGTGTTTTACTTTGTGGCCTTACCATTTAACTC

[0051] SEQ ID No.2:

[0052] METQEQQAKSRVVKVDSVESWDFYVTQANNQGCPIVVHFTASWCIPSVAMNPFFEELASAYPDVLFLAVDVDEVKEVASKLEVKAMPTFVLMKDGAQIDRLVGANPEEIKKRIGGFAQSIRVAVA

[0053] SEQ ID No.3:

[0054] CCAGCAGGGAACCTTTCATATTTTGCTAATCTGGTAGCAATGGAAGCATGAAAGAAATGGTAGTGTCTACTCTAGTCTAGAACAAGAATCAAAGCTCCAGGGAAGGAGGTAAAGAAGAGAAACCAAGACAAGAAGAAGAAGAAGAAGAAAGGAGACGAGATGGAGACCCAAGAGCAGCAAGCTAAGTCACGAGTTGTGAAGGTGGATTCGGTGGAGTCTTGGGATTTCTATGTTACCCAAGCCAATAATCAAGGCTGCCCTGAGGTGGCTTCTAAACTGGAGGTAAAGGCCATGCCTACATTTGTGCTGATGAAGGATGGTGCACAAATTGACAGGCTGGTGGGTGCCAATCCAGAGGAGATAAAGAAAAGGATTGGTGGGTTTGCACAGTCCATTCGTGTGGCTGTAGCCTAGTTACTGTGTGTGTGTGTGTGTGTGTGAGAGAGAGAGAGAGAGAGAGTGGAGAGAGATCTTGTATATTATGAACAGGTAAAATATGTGGTTATGGTGGACGGTGGAGCAGCACTTTAAACAGAAACGATGTTGATGATTGTGGTTTGTATATGTTGATTACTGTCGCAAATCATGTAGTTAGAAAGTGTTTTACTTTGTGGCCTTACCATTTAACTC

[0055] SEQ ID No.4:

[0056] METQEQQAKSRVVKVDSVESWDFYVTQANNQGCPEVASKLEVKAMP TFVLMKDGAQIDRLVGANPEEIKKRIGGFAQSIRVAVA

[0057] 2. HbCXXS2 gene structure analysis

[0058] DNA from the leaves of rubber tree Reyan 7-33-97 was extracted using a plant DNA extraction kit (OMEGA). The leaf DNA was used as a template and PCR amplification was performed using the aforementioned HbCXXS2-F / R primers. The amplified product was detected by agarose gel electrophoresis, recovered and purified, and then connected to pEASY-Blunt Simple Cloning Vector (Beijing Quanshijin Biotechnology Co., Ltd.). The connection product was heat-shocked and transformed into Escherichia coli Trans1-T1 Phage Resistant Chemically CompetentCell (Beijing Quanshijin Biotechnology Co., Ltd.). The positive single clone was identified by colony PCR and sent to Hainan Nanshan Biotechnology Co., Ltd. for sequencing.

[0059] The sequencing results showed that the genome sequence of HbCXXS2 from the rubber tree Reyan 7-33-97 was 3435 bp long, and its nucleotide sequence is shown in SEQ ID No. 5. The genome sequences of transcripts HbCXXS2.1 and HbCXXS2.2 were compared and analyzed with those of HbCXXS2, and it was determined that the transcript HbCXXS2.1 was derived from Figure 2 The sequence of Exon 1 is shown in nucleotides 1 to 261 of SEQ ID No. 5, the sequence of Exon 2 is shown in nucleotides 678 to 800 of SEQ ID No. 5, and the sequence of Exon 3 is shown in nucleotides 3067 to 3435 of SEQ ID No. 5. Figure 2 As shown, compared with the HbCXXS2.1 transcript, the HbCXXS2.2 transcript lacks Exon 2 and is composed only of Exons 1 and 3. This indicates that the HbCXXS2 gene pre-mRNA produces two transcripts, HbCXXS2.1 and HbCXXS2.2, through exon skipping splicing.

[0060] SEQ ID No.5:

[0061]

[0062] 3. HbCXXS2 protein sequence analysis

[0063] The molecular weight and theoretical isoelectric point of the proteins encoded by the two transcripts of HbCXXS2 were predicted using the online program Compute pI / Mw (https: / / web.expasy.org / compute_pi / ). The results showed that the molecular weight of HbCXXS2.1 protein was 13.82 kDa, and the theoretical isoelectric point was 4.82; the molecular weight of HbCXXS2.2 protein was 9.27 kDa, and the theoretical isoelectric point was 6.22. The amino acid sequences of HbCXXS2.1 and HbCXXS2.2 were analyzed for conserved domains using the online program SMART (http: / / smart.embl-heidelberg.de / ), and the following results were obtained: Figure 3 The results shown show that both HbCXXS2.1 and HbCXXS2.2 proteins contain a thioredoxin domain, but the difference is that the thioredoxin domain of HbCXXS2.1 contains a CXXS active motif, while the thioredoxin domain of HbCXXS2.2 lacks the CXXS active motif.

[0064] Example 2

[0065] This example provides an analysis of the expression characteristics of different transcripts of HbCXXS2.

[0066] 1. Tissue expression analysis of different transcripts of HbCXXS2

[0067] The bark, latex, new shoots, bronze leaves, light green leaves, discoloration leaves, stable leaves, senescent leaves, female flowers, male flowers and root tissue samples of Reyan 7-33-97 tissue culture seedlings (transplanted and cultured for 8 months) of healthy 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. PrimeScriptRTreagent Kit with gDNAEraser (Perfect Real Time, TaKaRa) was used to reverse transcribe each sample RNA into cDNA, and the specific implementation was referred to the kit instructions. The internal reference gene used the rubber tree HbUBC4 gene, and Primer Premier5 was used to design and synthesize specific primers for real-time fluorescence quantitative PCR (qRT-PCR) detection of HbUBC4 and transcripts HbCXXS2.1 and HbCXXS2.2 expression.

[0068] The sequence of the primer HbCXXS2.1-QF used to detect the transcript HbCXXS2.1 is shown in SEQ ID No.8, and the sequence of the primer HbCXXS2.1-QR is shown in SEQ ID No.9:

[0069] SEQ ID No.8: 5′-ATGGTGTATCCCCTCTGTGG-3′;

[0070] SEQ ID No. 9: 5′-TCGACATCAACAGCTAGAAACAG-3′.

[0071] The sequence of the primer HbCXXS2.2-QF used to detect the transcript HbCXXS2.2 is shown in SEQ ID No. 10, and the sequence of the primer HbCXXS2.2-QR is shown in SEQ ID No. 11:

[0072] SEQ ID No.10: 5′-CTGCCCTGAGGTGGCTTCTA-3′;

[0073] SEQ ID No. 11: 5′-CACGAATGGACTGTGCAAAC-3′.

[0074] The sequence of the primer HbUBC4-QF used to detect the HbUBC4 gene is shown in SEQ ID No. 12, and the sequence of the primer HbUBC4-QR is shown in SEQ ID No. 13:

[0075] SEQ ID No. 12: 5′-TACAAAGAGGTGCAGCGTGA-3′;

[0076] SEQ ID No. 13: 5′-ACTCCGCCCTCATAAGGAGT-3′.

[0077] qRT-PCR reaction system: SYBR Premix Ex Taq (2×) (TaKaRa) 5 μL, forward and reverse primers 0.4 μL each, cDNA template 1 μL, supplemented with ddH2O to a total volume of 10 μL. The detection was performed using a CFX96 fluorescence quantitative PCR instrument (Bio-Rad), and the reaction conditions were: 95°C for 30 s; 95°C for 5 s, 60°C for 30 s, and 40 cycles. The relative expression of the gene was calculated using formula 2 -ΔΔCt .

[0078] Expression of two transcripts of HbCXXS2 gene in various tissues of rubber tree Figure 4As shown in the results, HbCXXS2.1 and HbCXXS2.2 were expressed in the roots, bark, latex, new shoots, bronze leaves, light green leaves, color change leaves, stable leaves, senescent leaves, female flowers and male flowers of rubber trees. Among them, the expression levels of both transcripts were the highest in latex. In the same tissue, the expression level of HbCXXS2.1 was higher than that of HbCXXS2.2. The HbCXXS2 gene was highly expressed in latex, which suggested that it might be involved in the regulation of rubber production and discharge in rubber trees.

[0079] 2. Changes in HbCXXS2 expression during the occurrence and recovery of cut surface desiccation (TPD) in rubber trees

[0080] TPD symptoms were observed on rubber trees in rubber plantations. 90 rubber trees with a cut line and cut surface drying rate of 60% to 80% were selected and randomly divided into two groups: a TPD recovery treatment group (60 plants) and a TPD control group (30 plants). At the same time, 30 rubber trees with completely normal cut line rubber discharge were selected as a healthy control group. The plants in the TPD recovery treatment group were treated with a combination of dead skin recovery preparations (Dead Skin Condensation Gel and Dead Skin Condensation Aqueous Agent, developed by the Rubber Research Institute of the Chinese Academy of Tropical Agricultural Sciences, and can be obtained from the Rubber Research Institute of the Chinese Academy of Tropical Agricultural Sciences). The treatment method and cycle refer to the instructions of the Dead Skin Recovery Combination Preparation. The treatment lasted for 5 months, and the TPD symptoms of plants in each group were observed. 15 plants with a TPD length recovery rate of more than 80% were selected from the TPD recovery treatment group, 15 plants with a cut line length and cut surface drying rate close to that of the recovery group before treatment were selected from the TPD control group, and 15 plants with completely normal cut line rubber discharge were selected from the healthy control group. Latex samples of each plant were collected and frozen in liquid nitrogen. An equal amount of latex sample was taken from each strain, and 5 strains were mixed together as a biological replicate. According to the method in Example 1, total RNA of each sample was extracted, reverse transcribed to synthesize cDNA, and qRT-PCR was used to detect the expression levels of two transcripts of the HbCXXS2 gene in each sample.

[0081] Test results such as Figure 5 As shown by Figure 5 It can be seen that compared with the latex of healthy plants, the expression of HbCXXS2.1 and HbCXXS2.2 in the latex of TPD plants was significantly reduced; compared with the latex of TPD plants, the expression of HbCXXS2.1 in the latex of restored plants was significantly increased, and there was no significant difference with the expression in the latex of healthy plants. The expression of HbCXXS2.2 also increased significantly, but did not return to the normal level, and was still significantly lower than the latex of healthy plants. The above results show that the expression of HbCXXS2 gene is closely related to the occurrence and recovery of TPD in rubber trees. Improving the expression of HbCXXS2 gene in normal plants can reduce the occurrence of TPD, and promoting the expression of HbCXXS2 gene in the latex of TPD plants is conducive to the recovery of TPD.

[0082] 3. Expression changes of different transcripts of HbCXXS2 under oxidative stress

[0083] Expression analysis of different transcripts of HbCXXS2 under oxidative stress induced by hydrogen peroxide (H2O2): 54 healthy plants of rubber tree Reyan 7-33-97 tissue culture seedlings transplanted and cultured for 8 months were selected, and 20mmol / LH2O2 solution was evenly sprayed on the front and back of the leaves of the plants. The second and third leaves of the plants were collected at 0, 3, 6, 12, 24 and 48h after treatment, respectively. The leaves of every 3 plants were used as a biological replicate, and there were 3 biological replicates at each time point. After the samples were collected, they were frozen in liquid nitrogen. According to the method in Example 1, the total RNA of each sample was extracted, reverse transcribed to synthesize cDNA, and qRT-PCR was used to detect the expression levels of the two transcripts of the HbCXXS2 gene in each sample. The leaf samples treated for 0h were used as the control. The following is obtained: Figure 6 Results shown.

[0084] Expression analysis of different transcripts of HbCXXS2 under oxidative stress induced by methyl viologen (MV): 200 μmol / LMV solution (solute is MV, solvent is sterile distilled water) was used to replace 20 mmol / L H2O2 solution, and the rest of the operation was the same as that of the expression analysis of different transcripts of HbCXXS2 under oxidative stress induced by H2O2. Figure 7 Results shown.

[0085] Depend on Figure 6 It can be seen that under H2O2-induced oxidative stress, the expression change patterns of the two transcripts of HbCXXS2 were basically the same, both showing a trend of gradually decreasing first and then recovering; the expression level dropped to the lowest point at 12h of treatment, and then gradually recovered, but it was still significantly lower than the control (0h) at 48h of treatment. Figure 7 It can be seen that under the oxidative stress induced by MV, the expression change pattern of two transcripts of HbCXXS2 is basically the same, and the expression level significantly decreases when processing 3h, and then recovers; the difference is that when processing 6h, the expression level of HbCXXS2.2 returns to normal level, while the expression level of HbCXXS2.1 is still significantly lower than the control (0h). Same as H2O2 induced oxidative stress, the expression of two transcripts drops to the lowest point when MV processes 12h, and then gradually recovers, but is still significantly lower than the control (0h) when processing 48h. The above results show that the oxidative stress induced by H2O2 and MV significantly suppresses the expression of two transcripts of HbCXXS2. The result of the present embodiment shows that two transcripts of gene HbCXXS2 are significantly related to the oxidative stress of rubber tree, indicating that gene HbCXXS2 is significantly related to the oxidative stress of rubber tree.

[0086] Example 3

[0087] This example provides that expressing HbCXXS2 in yeast improves resistance to oxidative stress.

[0088] 1. Construction of yeast expression vectors for different transcripts of HbCXXS2

[0089] According to SEQ ID No. 1 and SEQ ID No. 3, a primer pair for amplifying the open reading frames of the two transcripts of HbCXXS2 was designed. The recognition sequences of restriction endonucleases Kpn I and Xba I (shown by the underlined parts in the following primers) and protective bases were introduced into the 5′ ends of the forward and reverse primers, respectively. The sequence of primer HbCXXS2-YF is shown in SEQ ID No. 14, and the sequence of primer HbCXXS2-YR is shown in SEQ ID No. 15:

[0090] SEQ ID No.14:5′-GG GGTACC ATGGAGACCCAAGAGCAGC-3′;

[0091] SEQ ID No.15: 5′-GC TCTAGA CTAGGCTACAGCCACACGAA-3′.

[0092] The latex cDNA in Example 2 was used as a template for PCR amplification. The PCR reaction system was: 2 μL of cDNA template, 5 μL of 5×TransStartFastPfuBuffer, 2 μL of 2.5 mM dNTPs, 0.5 μL of each forward and reverse primer (10 μmol / L), 0.5 μL of TransStartFastPfuDNAPolymerase (2.5 U / L), and ddH2O was added to a total volume of 25 μL. The PCR reaction program was: 95°C for 3 min; 95°C for 20 s, 56°C for 20 s, 72°C for 30 s, 35 cycles; 72°C for 10 min.

[0093] The PCR products were detected by agarose gel electrophoresis, and the HbCXXS2.1 and HbCXXS2.2 target fragments were purified by gel excision and ligated with pEASY-Blunt Simple Cloning Vector (Beijing Quanshijin Biotechnology Co., Ltd.). The ligation products were transformed into Escherichia coli Trans1-T1 Phage Resistant Chemically Competent Cell. Single clones were picked and tested for colony PCR. Positive single clones were sent to Hainan Nanshan Biotechnology Co., Ltd. for sequencing. The plasmid extraction kit (OMEGA) was used to extract the correct single clones and yeast expression vector pYES2 (Invitrogen) plasmids, and double enzyme digestion was performed with restriction endonucleases Kpn I and Xba I (Thermo Scientific). Double enzyme digestion system: 2μL Kpn I (10U / μL), 8μL Xba I (10U / μL), 5μL 10×Kpn I Buffer, 10μL plasmid DNA, 25μL ddH2O. Incubate at 37℃ for 4h. Detect the digestion products by 1.2% agarose gel electrophoresis, cut the target fragment gel block, and recover the target band by GelExtractionKit (OMEGA). Use T4 DNA ligase (Thermo Scientific) to connect the recovered HbCXXS2.1 and HbCXXS2.2 fragments to the pYES2 vector backbone, respectively. The connection system is: 5.5μL of HbCXXS2.1 / HbCXXS2.2 recovery product, 2.5μL of pYES2 vector backbone recovery product, 1μL of 10×Buffer, and 1μL of T4 DNA ligase. Connect overnight at 22℃. Transform the ligation product into E. coli DH5α competent cells, spread on LB solid (containing 100mg / L ampicillin) plates, and culture overnight at 37℃. Single clones were picked and identified by colony PCR using pYES2 vector primers (T7: 5′-CCCGGATCGGACTACTAGC-3′) plus HbCXXS2-YR primers, and positive single clone plasmids were extracted to obtain yeast expression vectors pYES2-HbCXXS2.1 and pYES2-HbCXXS2.2 of different transcripts of the HbCXXS2 gene.pYES2-HbCXXS2.1 is a recombinant plasmid obtained by replacing the fragment between the Kpn I and Xba I recognition sites of pYES2 with the nucleotide sequence of positions 160 to 537 (open reading frame of HbCXXS2.1) in SEQ ID No.1, while keeping the other nucleotide sequences of pYES2 unchanged; pYES2-HbCXXS2.2 is a recombinant plasmid obtained by replacing the fragment between the Kpn I and Xba I recognition sites of pYES2 with the nucleotide sequence of positions 160 to 414 (open reading frame of HbCXXS2.2) in SEQ ID No.3, while keeping the other nucleotide sequences of pYES2 unchanged. pYES2-HbCXXS2.1 expresses the protein HbCXXS2.1 whose amino acid sequence is SEQ ID No.2 in the sequence list. pYES2-HbCXXS2.2 expresses the protein HbCXXS2.2 whose amino acid sequence is SEQ ID No.4 in the sequence list.

[0094] 2. Obtaining recombinant yeast

[0095] Streak the Saccharomyces cerevisiae strain INVScl (Invitrogen) stored at -80℃ on YPD solid medium and culture at 30℃ for 2-3 days. Pick a single clone and transfer it to 10mL YPD liquid medium and culture it at 30℃ and 200rpm for 12h. Measure the OD value of the bacterial solution. 600 value, pipette an appropriate amount of bacterial solution into 50mL YPD liquid culture medium and adjust the OD 600=0.4, culture at 30℃ and 200rpm for 4h. Centrifuge at 2500rpm for 5min, discard the supernatant, and resuspend the cells with 40mL1×TE buffer. Centrifuge at 2500rpm for 5min, discard the supernatant, resuspend the cells with 2mL 1×LiAc / 0.5×TE, and incubate at room temperature for 10min to obtain yeast competent cells. Divide the yeast competent cells into 1.5mL centrifuge tubes, 100μL per tube. Take 3 tubes of yeast competent cells, add 1μgpYES2-HbCXXS2.1 / pYES2-HbCXXS2.2 / pYES2 plasmid and 100μg denatured salmon sperm DNA respectively, and mix well. Add 700μL 1×LiAc / 40%PEG-3350 / 1×TE to each tube, mix well, and incubate at 30℃ for 30min. Add 88 μL of DMSO to each tube, mix well, and heat shock at 42°C for 7 minutes. Centrifuge at room temperature and 5000 rpm for 2 minutes, and discard the supernatant. Resuspend the cells with 1 mL of 1×TE buffer, centrifuge at room temperature and 5000 rpm for 1 minute, and discard the supernatant. Resuspend the cells with 100 μL of 1×TE buffer and apply them on SC-Ura solid selection medium [1L: 0.78g DO Supplement-Ura (Clontech), 6.7g amino-free yeast nitrogen base (YNB), 20g glucose, 20g agar powder], and culture them upside down at 30°C for 2 to 3 days. Pick 3 single clones grown by each plasmid transformation and place them in 5 mL of SC-Ura liquid selection medium (1L: 0.78g DO Supplement-Ura (Clontech), 6.7g YNB, 20g glucose), and culture them at 30°C and 200 rpm for 2 days. The plasmid DNA of each single clone was extracted using a yeast plasmid extraction kit (OMEGA). Using yeast plasmid DNA as a template, PCR detection was performed on the pYES2-HbCXXS2.1 / pYES2-HbCXXS2.2 transformed yeast plasmid using T7 and HbCXXS2-YR primers, and PCR detection was performed on the pYES2 transformed yeast plasmid using T7 and pYES2-R: 5′-ATTAAAGCCTTCGAGCGTCC-3′ (SEQ ID No. 24) primers.

[0096] The results of agarose gel electrophoresis of PCR products were as follows Figure 8 As shown, Figure 8 The three recombinant yeast plasmids transformed with the pYES2 empty vector shown in 1, 2, and 3 were amplified and compared with the positive control (pYES2 plasmid, Figure 8 The pYES2 empty vector was successfully transferred into Saccharomyces cerevisiae INVScl, and the recombinant yeast transformed with the pYES2 empty vector was named INVSc1 (pYES2). Figure 8The three recombinant yeast plasmids transformed with pYES2-HbCXXS2.1 shown in 4, 5, and 6 were amplified with the positive control (pYES2-HbCXXS2.1 plasmid, Figure 8 The pYES2-HbCXXS2.1 vector was successfully transferred into Saccharomyces cerevisiae INVScl. The recombinant yeast transformed with the pYES2-HbCXXS2.1 vector was named INVSc1 (pYES2-HbCXXS2.1). Figure 8 The three recombinant yeast plasmids transformed with pYES2-HbCXXS2.2 shown in Figures 7, 8, and 9 were amplified with the positive control (pYES2-HbCXXS2.2 plasmid, Figure 8 The pYES2-HbCXXS2.2 vector was successfully transformed into Saccharomyces cerevisiae INVScl. The recombinant yeast transformed with the pYES2-HbCXXS2.2 vector was named INVSc1 (pYES2-HbCXXS2.2).

[0097] 3. Detection of HbCXXS2 gene expression in recombinant yeast

[0098] The positive clones of recombinant yeast INVSc1(pYES2-HbCXXS2.1), INVSc1(pYES2-HbCXXS2.2) and INVSc1(pYES2) were streaked onto SC-Ura solid selection medium and inverted at 30°C for 2-3 days. A single clone was inoculated into 2 mL SC-Ura liquid selection medium and cultured at 30°C and 200 rpm for 24 h. The OD was measured. 600 Take an appropriate amount of bacterial solution and centrifuge at room temperature at 4000 rpm for 3 min. Discard the supernatant and resuspend the cells in 5 mL SC-Ura liquid induction medium [1 L: 0.78 g DO Supplement-Ura (Clontech), 6.7 g YNB, 20 g galactose] to adjust the OD 600About 0.2. Culture at 30°C and 200rpm for 36h, take 2mL of bacterial solution, centrifuge at room temperature and 4000rpm for 3min. Discard the supernatant and collect the bacteria. According to the instructions of the kit, use the yeast total RNA rapid extraction kit (Shanghai Biotechnology Co., Ltd.) to extract the total RNA of recombinant yeast, and use PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa) to reverse transcribe the total RNA into cDNA. Using the Saccharomyces cerevisiae Actin (GeneBank accession number: L00026.1) gene as the internal reference, semi-quantitative RT-PCR was used to detect the expression of HbCXXS2.1 or HbCXXS2.2 in recombinant yeast.

[0099] The sequence of the primer Actin-RF of Saccharomyces cerevisiae Actin is shown in SEQ ID No.16, and the sequence of the primer Actin-RR is shown in SEQ ID No.17:

[0100] SEQ ID No. 16: 5′-AGTTGCCCCAGAAGAACACC-3′;

[0101] SEQ ID No. 17: 5′-TACCGGCAGATTCCAAACCC-3′.

[0102] The sequence of the primer HbCXXS2-RF for detecting the expression of HbCXXS2.1 and HbCXXS2.2 is shown in SEQ ID No. 18, and the sequence of the primer HbCXXS2-RR is shown in SEQ ID No. 19:

[0103] SEQ ID No. 18: 5′-ACCCAAGAGCAGCAAGCTAA-3′;

[0104] SEQ ID No. 19: 5′-CACGAATGGACTGTGCAAAC-3′.

[0105] The PCR reaction system was: 2.5 μL 10×EasyTaq Buffer, 0.5 μL EasyTaq DNA Polymerase (Beijing Quanshijin Biotechnology Co., Ltd.), 2 μL dNTPs (2.5 mM), 0.5 μL each of forward and reverse primers (10 μM), 1-3 μL cDNA template, supplemented with ddH2O to a total volume of 25 μL. The PCR reaction program was: 94°C for 3 min; 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 10 min.

[0106] The semi-quantitative RT-PCR test results are as follows Fig. 9 As shown, at 36 h of induction culture, three recombinant yeast INVSc1 (pYES2-HbCXXS2.1) clones ( Fig. 9 4, 5, 6) were detected in HbCXXS2.1 expression, and three recombinant yeast INVSc1 (pYES2-HbCXXS2.2) clones ( Fig. 9 7, 8, 9), while HbCXXS2.1 and HbCXXS2.2 were not detected in the control yeast INVSc1(pYES2) ( Fig. 9 The above results indicate that both transcripts of HbCXXS2 gene of rubber tree were successfully transferred into recombinant yeast and induced to express.

[0107] 4. Expression of the HbCXXS2 gene in yeast improves its resistance to oxidative stress

[0108] (1) Survival differences of recombinant yeast after oxidative stress treatment

[0109] Streak the positive clones of recombinant yeast INVSc1(pYES2-HbCXXS2.1), INVSc1(pYES2-HbCXXS2.2) and INVSc1(pYES2) on SC-Ura solid selection medium and invert and culture at 30℃ for 2-3 days. Pick out each recombinant yeast single clone and inoculate it into 5mL SC-Ura liquid selection medium and culture it at 30℃ and 200rpm for 24h. Take an appropriate amount of bacterial solution, centrifuge it at room temperature and 4000rpm for 3min, discard the supernatant, resuspend the bacteria in 10mL SC-Ura liquid induction medium, and adjust the OD to 0. 600 =0.2. The culture was shaken at 30°C and 200 rpm for 36 h to induce HbCXXS2 gene expression. The OD of each induced culture solution was measured. 600 Take appropriate amount of INVSc1(pYES2-HbCXXS2.1), INVSc1(pYES2-HbCXXS2.2) and INVSc1(pYES2) induction culture medium, centrifuge at room temperature and 4000rpm for 3min, and discard the supernatant. Resuspend the cells with sterile water and adjust the OD 600 =2.0. OD 600 =2.0 of INVSc1(pYES2-HbCXXS2.1), INVSc1(pYES2-HbCXXS2.2) and INVSc1(pYES2) bacterial solutions.

[0110] INVSc1(pYES2-HbCXXS2.1)10mM H2O2 treated group: OD 600= 2.0 INVSc1 (pYES2-HbCXXS2.1) bacterial solution 0.5 mL in a centrifuge tube, add 0.5 mL 20 mM H2O2 solution, and obtain OD 600 =1.0, 10mM H2O2 treated INVSc1 (pYES2-HbCXXS2.1) bacterial solution. Treat at 30°C, 160rpm for 24h. Use sterile distilled water to dilute the treated bacterial solution in 5-fold concentration gradients (5 -1 , 5 -2 , 5 -3 , 5 -4 , 5 -5 5 μL of undiluted bacterial solution and bacterial solution diluted at different dilution multiples were spotted on SC-Ura solid selective medium, and each treatment was repeated 3 times. Inverted culture was carried out at 30℃ for 3 days.

[0111] INVSc1(pYES2-HbCXXS2.2)10mM H2O2 treated group: INVSc1(pYES2-HbCXXS2.2) replaced INVSc1(pYES2-HbCXXS2.1), and the rest of the operations were the same as those of INVSc1(pYES2-HbCXXS2.1)10mM H2O2 treated group.

[0112] INVSc1(pYES2)10mM H2O2 treated group: INVSc1(pYES2) was used to replace INVSc1(pYES2-HbCXXS2.1), and the rest of the operations were the same as those of the INVSc1(pYES2-HbCXXS2.1)10mM H2O2 treated group.

[0113] INVSc1(pYES2-HbCXXS2.1)15mM H2O2 treated group: OD 600 = 2.0 INVSc1 (pYES2-HbCXXS2.1) bacterial solution 0.5 mL in a centrifuge tube, add 0.5 mL 30 mM H2O2 solution, and obtain OD 600 =1.0, 15mM H2O2 treated INVSc1 (pYES2-HbCXXS2.1) bacterial solution. Treat at 30°C, 160rpm for 24h. Use sterile distilled water to dilute the treated bacterial solution in 5-fold concentration gradients (5 -1 , 5 -2 , 5 -3 , 5 -4 , 5 -5 5 μL of undiluted bacterial solution and bacterial solution diluted at different dilution multiples were spotted on SC-Ura solid selective medium, and each treatment was repeated 3 times. Inverted culture was carried out at 30℃ for 3 days.

[0114] INVSc1(pYES2-HbCXXS2.2)15mM H2O2 treated group: INVSc1(pYES2-HbCXXS2.2) replaced INVSc1(pYES2-HbCXXS2.1), and the rest of the operations were the same as those of INVSc1(pYES2-HbCXXS2.1)15mM H2O2 treated group.

[0115] INVSc1(pYES2)15mM H2O2 treated group: INVSc1(pYES2) was used to replace INVSc1(pYES2-HbCXXS2.1), and the rest of the operations were the same as those of the INVSc1(pYES2-HbCXXS2.1)15mM H2O2 treated group.

[0116] INVSc1(pYES2-HbCXXS2.1) control group: take OD 600 = 2.0 INVSc1 (pYES2-HbCXXS2.1) bacterial solution 0.5 mL in a centrifuge tube, add 0.5 mL sterile distilled water, and obtain OD 600 =1.0 INVSc1 (pYES2-HbCXXS2.1) bacterial solution. Treat at 30°C, 160 rpm for 24 h. Use sterile distilled water to dilute the treated bacterial solution in 5-fold gradients (5 -1 , 5 -2 , 5 -3 , 5 -4 , 5 -5 5 μL of undiluted bacterial solution and bacterial solution diluted at different dilution multiples were spotted on SC-Ura solid selective medium, and each treatment was repeated 3 times. Inverted culture was carried out at 30℃ for 3 days.

[0117] INVSc1(pYES2-HbCXXS2.2) control group: INVSc1(pYES2-HbCXXS2.2) was used to replace INVSc1(pYES2-HbCXXS2.1), and the rest of the operations were the same as those of the INVSc1(pYES2-HbCXXS2.1) control group.

[0118] INVSc1(pYES2) control group: INVSc1(pYES2) was used to replace INVSc1(pYES2-HbCXXS2.1), and the rest of the operations were the same as those of the INVSc1(pYES2-HbCXXS2.1) control group.

[0119] The growth of the above-mentioned recombinant yeast INVSc1(pYES2-HbCXXS2.1), INVSc1(pYES2-HbCXXS2.2) and INVSc1(pYES2) is shown in Fig.10 As shown by Fig.10It can be seen that in the control group (no oxidative stress treatment), at the same dilution multiple, there was no significant difference in the number of plaques (CFU) of INVSc1(pYES2-HbCXXS2.1), INVSc1(pYES2-HbCXXS2.2) and INVSc1(pYES2). -3 After dilution, INVSc1(pYES2) had no plaque growth, while INVSc1(pYES2-HbCXXS2.1) and INVSc1(pYES2-HbCXXS2.2) had plaque growth, and the number of plaques of INVSc1(pYES2-HbCXXS2.1) was greater than that of INVSc1(pYES2-HbCXXS2.2); -4 After dilution, only INVSc1(pYES2-HbCXXS2.1) showed plaque growth, while INVSc1(pYES2-HbCXXS2.2) and INVSc1(pYES2) showed no plaque growth. -2 After dilution, only one plaque grew in INVSc1(pYES2), while many plaques still grew in INVSc1(pYES2-HbCXXS2.1) and INVSc1(pYES2-HbCXXS2.2), and the number of plaques in INVSc1(pYES2-HbCXXS2.1) was greater than that in INVSc1(pYES2-HbCXXS2.2); -3 After dilution, INVSc1(pYES2) had no plaque growth, while INVSc1(pYES2-HbCXXS2.1) and INVSc1(pYES2-HbCXXS2.2) still had plaque growth, and the number of plaques of INVSc1(pYES2-HbCXXS2.1) was greater than that of INVSc1(pYES2-HbCXXS2.2). The above results showed that expressing the transcripts HbCXXS2.1 or HbCXXS2.2 of the HbCXXS2 gene in yeast increased the survival rate of the recombinant yeast after oxidative stress induced by high concentration H2O2, enhanced the resistance of the recombinant yeast to oxidative stress, and the antioxidant activity of the recombinant yeast expressing the transcript HbCXXS2.1 was better than that of the recombinant yeast expressing the transcript HbCXXS2.2.

[0120] (2) Growth differences of recombinant yeast under oxidative stress

[0121] Streak the positive clones of recombinant yeast INVSc1(pYES2-HbCXXS2.1), INVSc1(pYES2-HbCXXS2.2) and INVSc1(pYES2) on SC-Ura solid selection medium and invert and culture at 30℃ for 2-3 days. Pick out each recombinant yeast single clone and inoculate it into 5mL SC-Ura liquid selection medium and culture it at 30℃ and 200rpm for 24h. Take an appropriate amount of bacterial solution, centrifuge it at room temperature and 4000rpm for 3min, discard the supernatant, resuspend the bacteria in 10mL SC-Ura liquid induction medium, and adjust the OD to 0. 600 =0.2. The culture was shaken at 30°C and 200 rpm for 36 h to induce HbCXXS2 gene expression. The OD of each induced culture solution was measured. 600 Take appropriate amount of INVSc1(pYES2-HbCXXS2.1), INVSc1(pYES2-HbCXXS2.2) and INVSc1(pYES2) induction culture medium, centrifuge at room temperature and 4000rpm for 3min, and discard the supernatant. Resuspend the cells with SC-Ura liquid induction medium and adjust the OD 600 =0.4.

[0122] INVSc1(pYES2-HbCXXS2.1)2.5mM H2O2 treatment group: take the above OD 600 =0.4 INVSc1 (pYES2-HbCXXS2.1) bacterial solution 2.5 mL in a 50 mL sterile centrifuge tube, add 2.5 mL SC-Ura liquid induction medium containing 5 mM H2O2, and obtain OD 600 =0.2, 2.5mM H2O2 treated INVSc1 (pYES2-HbCXXS2.1) bacterial solution. Each treatment was repeated 3 times. The culture was shaken at 30°C and 200 rpm. After 36 hours of culture, the OD of each culture solution was measured by a spectrophotometer. 600 value.

[0123] INVSc1(pYES2-HbCXXS2.2)2.5mM H2O2 treated group: INVSc1(pYES2-HbCXXS2.2) replaced INVSc1(pYES2-HbCXXS2.1), and the rest of the operations were the same as those of INVSc1(pYES2-HbCXXS2.1)2.5mM H2O2 treated group.

[0124] INVSc1(pYES2)2.5mM H2O2 treated group: INVSc1(pYES2) was used to replace INVSc1(pYES2-HbCXXS2.1), and the rest of the operations were the same as those of the INVSc1(pYES2-HbCXXS2.1)2.5mM H2O2 treated group.

[0125] INVSc1(pYES2-HbCXXS2.1)3mM H2O2 treatment group: take the above OD 600 =0.4 INVSc1 (pYES2-HbCXXS2.1) bacterial solution 2.5 mL in a 50 mL sterile centrifuge tube, add 6 mM H2O2 containing SC-Ura liquid induction medium 2.5 mL, and obtain OD 600 =0.2, 3mM H2O2 treated INVSc1 (pYES2-HbCXXS2.1) bacterial solution. Each treatment was repeated 3 times. The culture was shaken at 30°C and 200 rpm. After 36 hours of culture, the OD of each culture solution was measured by a spectrophotometer. 600 value.

[0126] INVSc1(pYES2-HbCXXS2.2)3mM H2O2 treated group: INVSc1(pYES2-HbCXXS2.2) replaced INVSc1(pYES2-HbCXXS2.1), and the rest of the operations were the same as those of INVSc1(pYES2-HbCXXS2.1)3mM H2O2 treated group.

[0127] INVSc1(pYES2)3mM H2O2 treated group: INVSc1(pYES2) was used to replace INVSc1(pYES2-HbCXXS2.1), and the rest of the operations were the same as those of the INVSc1(pYES2-HbCXXS2.1)3mM H2O2 treated group.

[0128] INVSc1(pYES2-HbCXXS2.1) control group: take the above OD 600 =0.4 INVSc1 (pYES2-HbCXXS2.1) 2.5 mL of bacterial solution was placed in a 50 mL sterile centrifuge tube, and 2.5 mL of SC-Ura liquid induction medium was added to obtain an OD 600 = 0.2, INVSc1 (pYES2-HbCXXS2.1) culture solution without H2O2 treatment. Each treatment was repeated 3 times. The culture was shaken at 30°C and 200 rpm. After 36 h of culture, the OD of each culture solution was measured by a spectrophotometer. 600 value.

[0129] INVSc1(pYES2-HbCXXS2.2) control group: INVSc1(pYES2-HbCXXS2.2) was used to replace INVSc1(pYES2-HbCXXS2.1), and the rest of the operations were the same as those of the INVSc1(pYES2-HbCXXS2.1) control group.

[0130] INVSc1(pYES2) control group: INVSc1(pYES2) was used to replace INVSc1(pYES2-HbCXXS2.1), and the rest of the operations were the same as those of the INVSc1(pYES2-HbCXXS2.1) control group.

[0131] After 36h of culture, the OD of yeast culture in each group 600 Value Fig.11 In the control group, the OD values ​​of INVSc1(pYES2-HbCXXS2.1) and INVSc1(pYES2-HbCXXS2.2) were 600 The value was slightly lower than that of INVSc1(pYES2), but the difference was not significant. H2O2 treatment significantly inhibited the growth of yeast, and the higher the H2O2 concentration, the more severe the inhibition. In the 2.5 and 3 mM H2O2 treatment groups, the OD 600 The values ​​were significantly higher than those of INVSc1(pYES2), and the OD 600 The value was significantly higher than that of INVSc1(pYES2-HbCXXS2.2). The above results show that expressing the transcripts HbCXXS2.1 or HbCXXS2.2 of the HbCXXS2 gene in yeast enhances the resistance of recombinant yeast to oxidative stress and improves the growth activity of recombinant yeast under H2O2-induced oxidative stress treatment, and the antioxidant activity of recombinant yeast expressing transcript HbCXXS2.1 is better than that of recombinant yeast expressing transcript HbCXXS2.2.

[0132] Example 4

[0133] This example provides that the transcript of gene HbCXXS2 improves the resistance of tobacco to oxidative stress.

[0134] 1. Construction of plant overexpression vectors of different HbCXXS2 transcripts and genetic transformation of tobacco

[0135] According to SEQ ID No. 1 and SEQ ID No. 3, a primer pair for amplifying the open reading frames of the two transcripts of HbCXXS2 was designed, and the recognition sequences of restriction endonucleases Nco I and BstEII (shown by the underlined parts in the following primers) and protective bases were introduced into the 5′ ends of the forward and reverse primers, respectively. The sequence of primer HbCXXS2-OF is shown in SEQ ID No. 20, and the sequence of primer HbCXXS2-OR is shown in SEQ ID No. 21:

[0136] SEQ ID No.20: 5′-TA CCATGGATGGAGACCCAAGAGCAGC-3′;

[0137] SEQ ID No.21: 5′-GT GGTTACC CTAGGCTACAGCCACACGAA-3′.

[0138] PCR amplification was performed with reference to the reaction system and procedure in Example 3. The target fragments of HbCXXS2.1 and HbCXXS2.2 were recovered and purified, and connected to pEASY-Blunt Simple Cloning Vector (Beijing Quanshijin Biotechnology Co., Ltd.). The ligation products were transformed into Escherichia coli Trans1-T1 Phage Resistant Chemically Competent Cell. Single clones were picked and positive colony PCR was performed. The positive single clones were sent to Hainan Nanshan Biotechnology Co., Ltd. for sequencing. The correct single clones and plant overexpression vector pCAMBIA1301 plasmids were extracted and double digested with restriction endonucleases Nco I and BstEII (Thermo Scientific). The digestion products were detected by agarose gel electrophoresis, and the target bands were recovered. T4 DNA ligase (Thermo Scientific) was used to connect the recovered HbCXXS2.1 and HbCXXS2.2 fragments to the pCAMBIA1301 vector backbone. The ligation product was transformed into competent E. coli DH5α cells, spread on LB solid (containing 100 mg / L ampicillin) plates, and cultured at 37°C overnight. Single clones were picked and identified by colony PCR using CaMV35S promoter primer (35S: 5′-TGACGCACAATCCCACTATCC-3′) plus HbCXXS2-OR primer. Plant overexpression vectors pCAMBIA1301-HbCXXS2.1 and pCAMBIA1301-HbCXXS2.2 of different transcripts of the HbCXXS2 gene were obtained. pCAMBIA1301-HbCXXS2.1 and pCAMBIA1301-HbCXXS2.2 plasmids were extracted and transformed into Agrobacterium EHA105 competent cells (Coolaber Company). The two plant overexpression vectors were transformed into Nicotiana benthamiana (obtained from Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences) using Agrobacterium-mediated tobacco leaf disc transformation.

[0139] 2. Molecular detection of genetically modified tobacco

[0140] The plant DNA extraction kit (OMEGA) was used to extract the leaf DNA of the transgenic tobacco regenerated plants. Using the leaf DNA as a template, the aforementioned CaMV35S promoter primer plus the HbCXXS2-OR primer was used for PCR amplification. PCR amplification and agarose gel electrophoresis detection were performed with reference to the reaction system and procedure in Example 3. The total RNA of each positive transgenic plant leaf 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 manual. The total RNA of each sample was reverse transcribed into cDNA using PrimeScript RTreagentKitwithgDNAEraser (Perfect Real Time, TaKaRa), and the specific implementation was referred to the kit manual. The expression of HbCXXS2.1 and HbCXXS2.2 genes in the corresponding transgenic plants was detected with reference to the qRT-PCR reaction system and conditions of Example 2. The qRT-PCR primers for detecting transcripts HbCXXS2.1 and HbCXXS2.2 were the same as those in Example 2, and the internal reference gene was Nicotiana benthamiana EF1a (AY206004.1). The sequence of primer EF1a-QF was shown in SEQ ID No. 22, and the sequence of primer EF1a-QR was shown in SEQ ID No. 23:

[0141] SEQ ID No. 22: 5′-TACCACCCCCAAGTACTCCA-3′;

[0142] SEQ ID No. 23: 5′-ATATTGTCCCTCGAAACC-3′.

[0143] Agarose gel electrophoresis results Fig.12 shown. Fig.12 The results showed that 7 transgenic plants with HbCXXS2.1 gene were obtained, and 12 transgenic plants with HbCXXS2.2 gene were obtained. The expression levels of the corresponding genes in the corresponding plants were detected, and the following results were obtained: Fig.13 The results shown show that 6 out of 7 HbCXXS2.1 gene-positive plants (2-7) were detected with higher HbCXXS2.1 expression, and 8 out of 12 HbCXXS2.2 gene-positive plants (10, 11, 13, 15-19) were detected with higher HbCXXS2.2 expression.

[0144] 3. Identification of antioxidant activity of transgenic strains

[0145] The seeds of homozygous T3 transgenic lines (HbCXXS2.1-5, -7 and HbCXXS2.2-11, -16) and wild-type (Wt) tobacco with high expression were sterilized with 75% alcohol for 30s, 30% sodium hypochlorite for 10min, and washed with sterile water. They were then inoculated on solid MS medium and cultured in an artificial climate chamber for 14 days. Seedlings with similar growth were selected and transplanted into solid MS medium containing 0 (control group) or 4mM H2O2 (H2O2 treatment group). After 4 weeks of culture in an artificial climate chamber, the fresh weight and plant height of the plants were measured. Twelve plants were measured for each line to compare the differences between the transgenic lines and the wild-type plants.

[0146] After 4 weeks of culture, Fig.14 The results shown. Fig.14 It can be seen that in the control group, there was no significant difference in the phenotypes of HbCXXS2.1 and HbCXXS2.2 transgenic lines and Wt plants, and the plant size, plant height and fresh weight were basically similar, indicating that overexpression of HbCXXS2.1 and HbCXXS2.2 in tobacco had no significant effect on the growth and development of plants under normal conditions. Oxidative stress treatment induced by 4mmol / L H2O2 significantly inhibited the growth of transgenic lines and Wt tobacco seedlings, but the inhibitory effect on transgenic HbCXXS2.1 and HbCXXS2.2 gene lines was significantly less than that on Wt. After 4 weeks of oxidative stress culture, there was no significant difference between transgenic HbCXXS2.1 and HbCXXS2.2 gene lines, but the plants of transgenic HbCXXS2.1 and HbCXXS2.2 gene lines were significantly larger than Wt. The average fresh weight of HbCXXS2.1-5, HbCXXS2.1-7, HbCXXS2.2-11, and HbCXXS2.2-16 strains were 93, 83, 85, and 89 mg, respectively, which were significantly heavier than Wt (58 mg). The average plant height of HbCXXS2.1-5, HbCXXS2.1-7, HbCXXS2.2-11, and HbCXXS2.2-16 strains was 1.63, 1.58, 1.57, and 1.58 cm, respectively, which were significantly higher than Wt (1.17 cm). The above results showed that overexpression of HbCXXS2.1 and HbCXXS2.2 in Nicotiana benthamiana promoted the growth of transgenic plants under H2O2-induced oxidative stress and improved their resistance to oxidative stress.

[0147] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0148] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A use of a transcript of a thioredoxin gene HbCXXS2 in improving the resistance of an organism to oxidative stress, characterized in that: The transcript of the thioredoxin gene HbCXXS2 is transcript HbCXXS2.1 or transcript HbCXXS2.2, the nucleotide sequence of the transcript HbCXXS2.1 is shown in SEQ ID No.1, and the nucleotide sequence of the transcript HbCXXS2.2 is shown in SEQ ID No.3; The use of improving the resistance of organisms to oxidative stress includes improving the survival rate and / or growth activity of yeast under oxidative stress.

2. The use according to claim 1, characterized in that: The application of improving the resistance of organisms to oxidative stress also includes promoting the growth of tobacco under oxidative stress.

3. The use according to claim 1, characterized in that: The amino acid sequence of thioredoxin encoded by the nucleotide sequence of the transcript HbCXXS2.1 is shown in SEQ ID No.2, and the amino acid sequence of thioredoxin encoded by the nucleotide sequence of the transcript HbCXXS2.2 is shown in SEQ ID No.

4.

4. The use according to claim 1, characterized in that: The vector was constructed using the open reading frame sequence of transcript HbCXXS2.1 or transcript HbCXXS2.2; The nucleotide sequence of the open reading frame of the transcript HbCXXS2.1 is shown at positions 160 to 537 in SEQ ID No.1, and the nucleotide sequence of the open reading frame of the transcript HbCXXS2.2 is shown at positions 160 to 414 in SEQ ID No.

3.

5. The use according to claim 4, characterized in that: The open reading frame sequence of the transcript HbCXXS2.1 or the open reading frame sequence of the transcript HbCXXS2.2 is inserted between the Nco I and BstEⅡ restriction sites of the pCAMBIA1301 vector plasmid to obtain a plant expression vector; or the open reading frame sequence of the transcript HbCXXS2.1 or the open reading frame sequence of the transcript HbCXXS2.2 is inserted between the Kpn I and Xba I restriction sites of the pYES2 plasmid to obtain a yeast expression vector.

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