Potentilla serrata PsGSH1 gene and analysis and application of promoter of potentilla serrata PsGSH1 gene

Abstract

The invention relates to a potentilla serrata PsGSH1 gene and analysis and zinc resistance application of a promoter of the potentilla serrata PsGSH1 gene. The length of the nucleotide sequence of the gene is 1509bp, and the sequence is SEQ.NO.1. The length of the amino acid sequence coded by the gene is 502aa, and the sequence is SEQ.NO.2. The length of the nucleotide sequence of the promoter proPsGSH1 of the gene is 1541bp, and the sequence is SEQ.NO.3. The promoter proPsGSH1 of the gene has the nucleotide sequence. According to the invention, plant recombinant expression vectors GV1300-PsGSH1-GFP and pBI121-proPsGSH1-GUS are constructed, and are transferred into model plants, namely arabidopsis thaliana and tobacco, so that an overexpression strain is obtained. Through analysis and verification, it is proved that the promoter proPsGSH1 contains an anti-zinc cis-action element and has activation activity. Under zinc stress, it is verified that PsGSH1 has a zinc resistance function, the gene can effectively relieve damage of excessive zinc to plants, and a gene resource is provided for cultivation of zinc-resistant plants.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering and molecular biology, and specifically relates to the analysis of the PsGSH1 gene of Potentilla sericea and its promoter. The application of the gene is beneficial to improving the zinc tolerance of plants. Background Art

[0002] Potentilla sericea is an important garden ground cover herb and is widely used throughout the country, especially in the Northeast. Due to its unique rhizome structure, Potentilla sericea is cold-resistant, barren-resistant, and resistant to extensive management. It is often used as a forest ground cover to decorate the environment. It is also an excellent plant material for soil consolidation and slope protection to restore the ecology. Previous studies have shown that Potentilla sericea has the function of resisting heavy metals cadmium (Cd) and lead (Pb), but there is a lack of research on its resistance to high zinc environments. In addition, the unreasonable discharge of zinc-containing industrial "three wastes" such as zinc (Zn) mining, smelting, and galvanizing processing causes excessive accumulation of Zn in the soil. The Zn in the polluted soil is transmitted through plant enrichment-food chain, thereby threatening the health of humans and animals. In order to explore the enrichment mechanism of Zn in Potentilla sericea at the molecular biological level, and then cultivate new zinc-resistant varieties for the restoration of heavy metal contaminated soil, the inventors focused on the functional genes of the key enzyme pathways of zinc resistance in Potentilla sericea.

[0003] Glutathione (GSH), as an antioxidant in cells, plays a key role in protecting cells from oxidation and stress, and has become the focus of scientific researchers in recent years. In plants, GSH sulfhydryl groups form glutathione chelates with heavy metal ions. Relevant studies have shown that chelates can also be transported to vacuoles or plastids, cell membranes and other cells for detoxification. Glutathione synthetase 1 (GSH1), also known as glutamate-cysteine ​​ligase (GCL), is a biosynthetic rate-limiting enzyme at the front end of the GSH synthesis pathway. Overexpression of the GSH1 gene may promote the production of GSH and increase the synthesis of phytochelatin (PC). It can be seen that using molecular biology and genetic engineering technology to increase the expression of the GSH1 gene in plants to obtain overexpressed transgenic plants, thereby promoting the synthesis of a large amount of GSH, has great practical significance for the remediation of soil heavy metal zinc pollution. Summary of the invention

[0004] In view of this, an object of the present invention is to provide a glutathione synthetase gene PsGSH1 from Potentilla sericea and its encoded protein. The gene contains a 1509 bp coding sequence, encoding 502 amino acids, and the nucleotide sequence of the gene is shown in SEQ.NO.1, and the amino acid sequence is shown in SEQ.NO.2.

[0005] The present invention also provides a PsGSH1 gene promoter (proPsGSH1), which contains a 1541 bp coding sequence. The nucleotide sequence encoded by the promoter is shown in SEQ.NO.3.

[0006] The present invention also provides biological materials containing the PsGSH1 and proPsGSH1, wherein the biological materials are expression vectors, Escherichia coli, Agrobacterium or transgenic cell lines.

[0007] Furthermore, the vectors are GV1300-PsGSH1-GFP and pBI121-proPsGSH1-GUS.

[0008] The present invention also provides the zinc resistance analysis of the cis-acting element of proPsGSH1 and the verification results of its activation activity.

[0009] The present invention also provides application of the PsGSH1 gene or biological materials containing the gene in plant zinc resistance.

[0010] The plants of the present invention include but are not limited to Arabidopsis thaliana and Potentilla sericea.

[0011] The present invention also provides the application of the PsGSH1 gene or biological materials containing the gene in the breeding of new plant varieties.

[0012] The purpose of breeding the new variety is to improve the zinc resistance of the plant. The PsGSH1 gene is involved in the zinc stress response of Potentilla sericea, and zinc stress induces the expression of the gene.

[0013] Preferably, the PsGSH1 gene is transferred into Arabidopsis plants to overexpress the PsGSH1 gene. More preferably, the PsGSH1 gene is transferred into Arabidopsis plants by Agrobacterium-mediated method and inflorescence infection method to obtain transgenic plants overexpressing the PsGSH1 gene. More preferably, the PsGSH1 gene is constructed into the plant expression vector GV1300, Agrobacterium is transformed, and then Arabidopsis inflorescence is infected to screen for overexpressing transgenic plants.

[0014] Compared with the prior art, the zinc-resistant gene PsGSH1 of the sericeous Potentilla provided by the present invention is a key glutathione synthetase gene with brand-new gene functionality. By constructing a sericeous Potentilla plant overexpression vector and combining it with the Agrobacterium-mediated method to carry out heterologous transformation of the model plant Arabidopsis thaliana, the effect of the zinc-resistant function of the PsGSH1 gene on the plant zinc stress response is verified, which provides a powerful tool for the study of key enzyme genes in the glutathione synthesis pathway of the sericeous Potentilla and provides valuable basis for the ecological restoration application of the sericeous Potentilla. BRIEF DESCRIPTION OF THE DRAWINGS

[0001] Figure 1The electrophoresis diagram of the PCR product of the 5′-end 400bp sequence clone of the PsGSH1 gene in Example 1; wherein lanes 1, 2, 3, 4, 5, and 6 are PCR products, and M is DL2000 Marker.

[0002] Figure 2 It is a schematic diagram of the comparison between the 400bp sequence at the 5′ end of the PsGSH1 gene and the transcriptome sequencing results in Example 1; wherein the region selected in the red box is the intron region at the 5′ end of the PsGSH1 gene.

[0003] Figure 3 This is the electrophoresis diagram of the two FPNI-PCR products of proPsGSH1 in Example 1; wherein, lanes 1-2 to 9-3 are the second and third round PCR products of the first FPNI-PCR, respectively, and lanes 1-5 to 9-6 are the second and third round PCR products of the second FPNI-PCR, respectively, M is DL2000 Marker, and the area selected by the red box is the gel recovery and sequencing fragment.

[0004] Figure 4 It is the full-length clone of proPsGSH1 in Example 1; wherein, lanes 1, 2, 3, and 4 are PCR products, and M is DL2000 Marker.

[0005] Figure 5 This is the cis-acting element predicted from the proPsGSH1 sequence in Example 2.

[0006] Figure 6 This is a visualization of the cis-acting elements of proPsGSH1 in Example 2.

[0007] Figure 7 This is a schematic diagram of the construction of the pBI121-proPsGSH1-GUS recombinant expression vector in Example 2.

[0008] Figure 8 The results of GUS staining after transient transformation of Nicotiana benthamiana with pBI121-proPsGSH1-GUS Agrobacterium in Example 2; - Buffer was used as negative control group, CK + The empty vector Agrobacterium was used as the positive control group, and the proPsGSH1:GUS promoter bacterial solution was used as the experimental group.

[0009] Fig. 9 This is the electrophoresis diagram of the PCR product of PsGSH1 gene cloning in Example 3; wherein lanes 1, 2, and 3 are PCR products, and M is DL2000 Marker.

[0010] Fig.10 Schematic diagram of the construction of the GV1300-PsGSH1-GFP recombinant expression vector in Example 3.

[0001] Fig.11 The results of PCR verification using PsGSH1-F / R using leaf DNA extracted from positive transgenic Arabidopsis in Example 4; OE-1 to 9 are 9 transgenic lines, and M is DL2000 Marker.

[0002] Fig.12 The seed germination rates of Arabidopsis thaliana strains under zinc stress in Example 5 are shown in the four figures from left to right, respectively, under stress treatments of 0, 1, 2, and 3 mM ZnSO 4 Seed germination of Arabidopsis thaliana strains at 37 °C. WT is the wild type, and OE-3 / 5 / 6 is the PsGSH1 overexpression strain.

[0003] Fig.13 The phenotypic differences of seedlings of each strain of Arabidopsis thaliana under zinc stress in Example 5; wherein, the four figures from left to right respectively show the growth status of each strain of Arabidopsis thaliana at 0, 7, 14, and 21 days of stress treatment, WT is the wild type, and OE3 / 5 / 6 is the strain overexpressing PsGSH1.

[0004] Fig.14 These are the physiological indicators of each strain of Arabidopsis thaliana under zinc stress in Example 5; wherein A is SOD activity, B is CAT activity, C is MDA content, and D is GSH content. DETAILED DESCRIPTION

[0005] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Chi Chunning et al. Plant Molecular Biology Research (Progress in Molecular Biology of Plant Heavy Metal Tolerance, 2016), or the conditions recommended by the kit manufacturer's instructions.

[0006] Example 1 Searching for the 5′ intron of the PsGSH1 gene of Potentilla sericea and cloning of proPsGSH1

[0001] Using Potentilla sericea with good growth and consistent cutting as the material, genomic DNA was extracted using the Plant DNA MaxiKit kit (D3488-01) from OMEGA. The nucleotide sequence of PsGSH1 was obtained from the transcriptome sequenced and analyzed by Beijing Biomike Biotechnology Company, and the first 400bp sequence was selected to search for the 5′ intron of the PsGSH1 gene. The primers PsGSH1-400bp-F / PsGSH1-400bp-R were designed using SnapGene 4.2.4 to amplify the 5′ end sequence of the PsGSH1 gene. 20μl of the PCR product was thoroughly mixed with 5μl of 6× Loading Buffer for agarose gel electrophoresis (200V, 400mA, 12min), and the strip with the correct band was cut ( Figure 1 ), the product was recovered and sequenced using the Gel Extraction Kit (D2500-01) from OMEGA (Jilin Kumei Biotechnology Co., Ltd.). The sequencing results were compared with the nucleotide sequence of PsGSH1 in the transcriptome and it was found that there was a partial intron region at the 266bp position ( Figure 2 ).

[0002] PsGSH1-400bp-F: 5'-CCAGTATGGTACTCCTTTCCAGATGAGTTC-3'

[0003] PsGSH1-400bp-R: 5'-TGTTATCACCTTCCATGACTTTCTCCCACTC-3'

[0004] 20μl system: 2× Rapid Taq Master Mix 10μl, 10μM upstream and downstream primers 1μl each, DNA template 2μl, ddH 2 O 6μl.

[0005] PCR reaction program: pre-denaturation at 95°C for 2 min; denaturation at 94°C for 10 s; annealing at 58°C for 30 s; extension at 72°C for 2 min, 35 cycles; 72°C for 5 min; storage at 4°C.

[0006] According to the primer design principle of fusion primer nested PCR (FPNI-PCR), SnapGene 4.2.4 software was used to design 6 specific primers with the same direction to avoid the intron region. FPNI-PCR was divided into 3 rounds of PCR reactions. In the first round, the DNA of Potentilla sericea was used as a template, and the degenerate primers FP1-9 were used to perform thermal asymmetric PCR reactions with the proPsGSH1 specific primer proPsGSH1-SP1 respectively; in the second round, the PCR product of the first round was used as a template, and the nested specific primers FSP1 and proPsGSH1-SP2 were used for ordinary PCR reactions; in the third round, the PCR product of the second round was diluted 100 times as a template, and the nested specific primers FSP1 and proPsGSH1-SP3 were used for ordinary PCR reactions. Through the above 3 rounds of PCR reactions, the promoter fragments were gradually separated. The correct conditions for agarose gel electrophoresis were recovered ( Figure 3), connected to the pMD-18T vector, transformed into Escherichia coli DH5α, and sequenced the monoclonal bacterial solution. In the second FPNI-PCR, the specific primer design is based on the sequence amplified by the first FPNI-PCR as the reference sequence. According to the primer design principle, three specific primers with the same direction are designed, and then three rounds of PCR reactions are performed. When a promoter sequence of sufficient length is obtained, the spliced ​​sequence is used as the reference sequence to design the promoter full-length cloning primers proPsGSH1-F / proPsGSH1-R, which are connected to the pMD-18T vector (TaKaRa, according to the instructions), and finally sequenced to obtain the complete promoter sequence ( Figure 4 ). The nucleotide sequence of proPsGSH1 is shown in SEQ.NO.3.

[0007] The primers used are as follows:

[0008] proPsGSH1-SP1: 5'-TTACATCCAGAGGCAAGATAGC-3'

[0009] proPsGSH1-SP2: 5'-TTGTTTCTGCCCTGCAACATTG-3'

[0010] proPsGSH1-SP3: 5'-GGAGTACCATACTGGTTGTATACT-3'

[0011] proPsGSH1-SP4: 5'-GTGTTTACCACTGACAGCAAAACACG-3'

[0012] proPsGSH1-SP5: 5'-GCGGATTCAGGATTTTATAATGGGGTGT-3'

[0013] proPsGSH1-SP6: 5'-GCTGTAGCCCAACGTAATCTATGTGT-3'

[0014] proPsGSH1-F: 5'-CTGCATCCCACACAATGTTTATGCTCC-3'

[0015] proPsGSH1-R: 5'-CTGGGAAAGGAGTACCATACTGGTTGT-3'

[0016] FPNI-PCR first round 20μl system: 2× Rapid Taq Master Mix 10μl, 10μM SP1 / 4, FP1-9 primers 1μl each, DNA template 2μl, ddH 2O 6μl. Reaction program: 95℃90s; 94℃10s, 62℃30s, 72℃2min; 94℃10s, 25℃2min, 0.2℃ / s, 72℃2min; 94℃10s, 62℃30s, 72℃2min, 18 cycles; 72℃5min.

[0017] FPNI-PCR second round 20μl system: 2× Rapid Taq Master Mix 10μl, 10μM SP2 / 5, FSP1 primers 1μl each, first round PCR product 1μl, ddH 2 O 7μl. Reaction program: 95℃90s; 94℃10s, 62℃30s, 72℃2min, 30 cycles; 72℃5min.

[0018] FPNI-PCR third round 20μl system: 2× Rapid Taq Master Mix 10μl, 10μM SP3 / 6, FSP2 primers 1μl each, second round PCR product 1μl, ddH 2 O 7μl. Reaction program: 95℃90s; 94℃10s, 62℃30s, 72℃2min, 12 cycles; 72℃5min.

[0019] Example 2 Analysis of cis-acting elements of Potentilla sericea proPsGSH1 and verification of activation activity

[0020] The amplified proPsGSH1 sequence was analyzed using the PlantCARE online website ( Figure 5 ). Use Tbtools-Ⅱ software to visualize cis-elements ( Figure 6 ). It was found that proPsGSH1 not only contains TATA-box and CAAT-box core elements, but also contains stress response elements G-box, MYB, ARE, CTCGA-motif, TGACG-motif, and WUN-motif. Among them, G-box and MYB elements have been confirmed in relevant literature to have the function of regulating zinc stress after binding to transcription factors.

[0001] The vector map information of the pBI121-GUS empty plasmid and the nucleotide sequence information of proPsGSH1 preserved by the laboratory of the College of Landscape Architecture of Northeast Forestry University were used to determine the restriction sites of proPsGSH1, HindⅢ (AAGCTT) and BamHI (GGATCC). Using proPsGSH1-18T as a template, the homology arm specific primers proPsGSH1-HindIII-F / proPsGSH1-BamHI-R were designed. The pBI121-GUS vector was double-digested with HindⅢ and BamHI, and connected according to the instructions of the Novizan C112 homologous recombination kit. The recombinant product was transformed into Escherichia coli competent DH5α (Weidi Biotechnology, according to the instructions), and the plasmid was extracted after sequence determination to obtain the pBI121-proPsGSH1-GUS recombinant expression vector ( Figure 7 ). The plasmid was extracted from the correctly sequenced bacterial solution and then transformed into Agrobacterium GV3101 (pSoup-p19) (Weidi Biotechnology, follow the instructions). According to the method of Liang Tongyao for transient transformation of Nicotiana benthamiana (Establishment of Agrobacterium-mediated transient expression system in tobacco, Salvia miltiorrhiza, and Prunella vulgaris, 2015), pBI121-proPsGSH1-GUS Agrobacterium was injected and cultured for 36 hours for staining. The GUSstain Kit from Coolbo was used for staining. The staining results showed that proPsGSH1 has activation activity in tobacco ( Figure 8 ), providing a basis for further study of PsGSH1 gene.

[0002] proPsGSH1-HindIII-F: 5'-GACCATGATTACGCCAAGCTTTTATGCTCCAGTTGATTCTTCGAG-3'

[0003] proPsGSH1-BamHI-R: 5'-AAGGGACTGACCACCCGGGGATCCACTGGTTGTATACTACAACTTCTTTTCTTG-3'

[0004] Example 3 Cloning of the PsGSH1 gene from Potentilla sericea and construction of the GV1300-PsGSH1-GFP vector

[0005] Potentilla sericea with good growth and consistent cutting was used as the material. Total RNA was extracted using the RNA Plant Kit from Kangwei Company. Takara PrimeScript TMcDNA was obtained using the RT reagent Kit. The nucleotide sequence of PsGSH1 was obtained from the data returned by Beijing Biomike Biotechnology Co., Ltd. after sequencing and analyzing the transcriptome. The open reading frame (ORF) sequence position was determined using NCBI (https: / / www.ncbi.nlm.nih.gov / orffinder / ). The gene accession number in the GenBank database is PQ810936, and the query coverage (query coverage) with the Arabidopsis thaliana glutathione synthetase (glutamate-cysteine ​​ligase) gene (NP_001190808.1) is predicted to be 95%. Primers PsGSH1-F / PsGSH1-R were designed using SnapGene 4.2.4 to amplify the PsGSH1 gene ORF. The PCR product was subjected to agarose gel electrophoresis and the correct band was cut out ( Fig. 9 ), and the product was recovered using the Gel Extraction Kit (D2500-01) from OMEGA.

[0006] PsGSH1-F: 5'-CCAGTATGGTACTCCTTTCCCAGATGAGTTC-3'

[0007] PsGSH1-R: 5'-CTGAAGTTTCCCACCTCAGTATAGTAGCTCTT-3'

[0008] 50μl PCR reaction system: 10×Taq Buffer 5μl, 2mM dNTPs 5μl, 25mM MgSO 4 3μl, 10μM upstream and downstream primers 1.5μl each, cDNA template 2μl, KOD-Plus-Neo 2μl, ddH 2 O 30 μl.

[0009] PCR reaction program: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 15 s; annealing at 62°C for 30 s; extension at 68°C for 2 min, 40 cycles; 72°C for 1 min; storage at 4°C.

[0010] 4 μl of the purified DNA product was mixed with Beijing Quanshijin Biotechnology Co., Ltd. -Blunt Zero Cloning Vector plant vector 1μl was mixed and reacted in a 25℃ water bath for 15min to obtain PsGSH1-Blunt recombinant plasmid, which was transformed into Escherichia coli DH5α and the monoclonal bacterial solution was sequenced.

[0011] The vector map information of the GV1300-GPF empty plasmid and the nucleotide sequence information of the PsGSH1 gene preserved in the laboratory of the College of Landscape Architecture of Northeast Forestry University were used to determine the restriction sites of PsGSH1, SalⅠ(GTCGAC) and BamHI(GGATCC). Using PsGSH1-Blunt as a template, the homology arm specific primers PsGSH1-SalⅠ-F / PsGSH1-BamHI-R were designed. The GV1300-GPF vector was double-digested with SalⅠ and BamHI, and connected according to the instructions of the Novizan C117 homologous recombination kit. The recombinant product was transformed into Escherichia coli competent DH5α (Weidi Biotechnology, according to the instructions), and the plasmid was extracted after sequence determination to obtain the GV1300-PsGSH1-GFP recombinant expression vector ( Fig.10 ).

[0012] PsGSH1-SalⅠ-F:5'-TTGATACATATGCCCGTCGACATGGTACTCCTTTCCCAGATGAGT-3'

[0013] PsGSH1-BamHI-R:5'-GCCCTTGCTCACCATGGATCCGTATAGTAGCTCTTCAAAAACAGGGTCT-3'

[0014] Example 4 Plant expression vector GV1300-PsGSH1-GFP transformation of Arabidopsis

[0015] (1) Infection of Arabidopsis thaliana

[0016] First, the recombinant vector GV1300-PsGSH1-GFP plasmid obtained in Example 3 was transformed into Agrobacterium competent cells GV3101 (Weidi Biotechnology, according to the instructions). -1 , centrifuge at 4500rpm for 10min to collect the bacteria. Prepare the resuspension solution: add 10g sucrose, 0.1g MES, 4μl 0.5mM 6-BA, 80μl Silwet-77, and 20μl 40mM AS per 200ml. Resuspend the precipitated bacteria with the resuspension solution so that the OD600 of the resuspended infection solution reaches 0.8μg·mL -1 Soak the aerial part of the flowering Arabidopsis in the infection solution for 7 minutes, cover the plant with plastic wrap to maintain humidity, and culture it in the dark for 48 hours. Repeat the same method of infection once a week to ensure the success rate of infection.

[0017] (2) Screening homozygous Arabidopsis

[0018] In a sterile oven, Arabidopsis thaliana T0 seeds were sterilized and evenly sown on the screening medium (containing 50 mg·L - 1 Kana), put it in an artificial culture room until it grows four complete, healthy, green cotyledons, and then move it into the soil for further cultivation. After maturity, separate the strains and bag the seeds. The same method as above was used to screen Arabidopsis T2 and T3 seeds according to the strains.

[0019] (3) PCR identification of positive transgenic plants

[0020] The RNA and DNA of the leaves of positive transgenic Arabidopsis were extracted, and the RNA was reverse transcribed into cDNA, and PCR verification was performed using PsGSH1-F and PsGSH1-R. The results showed that the positive transgenic plants all contained the PsGSH1 gene ( Fig.11 ), indicating that PsGSH1 was successfully transferred into Arabidopsis thaliana.

[0021] Example 5 Analysis of zinc resistance in Arabidopsis thaliana overexpressing PsGSH1

[0022] (1) Analysis of zinc resistance in Arabidopsis seeds overexpressing PsGSH1

[0023] T3 seeds of wild-type and transgenic Arabidopsis lines (OE-3, OE-5, and OE-6) were sown in a medium containing 0, 1, 2, or 3 mM ZnSO. 4 The results showed that the germination rates of the strains under no stress were relatively small, all above 90%. As the concentration of Zn treatment gradually increased, the germination rate of the WT strain dropped below 5%; in contrast, the germination rate of the overexpression strain dropped to a minimum of 13% ( Fig.12 ).

[0024] (2) Analysis of zinc resistance in Arabidopsis seedlings overexpressing PsGSH1

[0025] The wild-type and transgenic Arabidopsis thaliana seedlings were transplanted into soil for cultivation and irrigated every 3 days with 50 ml of 800 mg·L -1 ZnSO 4 Solution, three biological replicates were set for each strain. Photographs were taken at 0, 7, 14, and 21 days after treatment to record the growth status of the plants. On the 14th day after treatment, the leaves were sampled for three biological replicates to determine the physiological indicators of zinc resistance. The GSH and malondialdehyde (MDA) contents were determined using the kits (GSH-G0206F, MDA-G0109F) of Suzhou Grace Biotechnology Co., Ltd.; the superoxide dismutase (SOD) activity was determined by referring to the nitroblue tetrazolium (NBT) method; and the catalase (CAT) activity was determined by referring to the ultraviolet spectrophotometer absorption method. The results showed that after Zn stress treatment, the transgenic strains grew better than the wild type, as reflected by greener leaves and lower mortality ( Fig.13 ), and the antioxidant enzymes SOD and CAT activities and the non-enzymatic antioxidant GSH content in the transgenic lines were significantly higher than those in the wild type ( Fig.14 AB, D). Further analysis results showed that the MDA content, an indicator of oxidative stress, in the transgenic lines was significantly lower than that in the wild type ( Fig.14 C). This indicates that the zinc resistance of transgenic Arabidopsis is stronger than that of wild type, and the PsGSH1 gene can improve the zinc resistance of plants through enzymatic and non-enzymatic systems.

[0026] Although the above description has generally described the PsGSH1 gene and its application and provided a specific experimental operation scheme, it is obvious to those skilled in the art that some modifications or improvements can be made to it based on the present invention. Therefore, modifications or improvements made without departing from the main technical route of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A PsGSH1 gene of Potentilla sericea, characterized in that: Its nucleotide sequence is shown in SEQ.NO.

1. The amino acid sequence of the encoded protein is shown in SEQ.NO.

2.

2. A proPsGSH1 sequence based on the PsGSH1 gene promoter according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ.NO.

3.

3. An expression vector, characterized in that: The vector comprises the Potentilla sericea PsGSH1 gene of claim 1.

4. An expression vector, characterized in that: The vector comprises the promoter proPsGSH1 sequence according to claim 2.

5. A cell, characterized in that The cell is a cell comprising the expression vector according to claim 3 after transformation of a host cell, and the host cell is an Escherichia coli or an Agrobacterium cell.

6. A cell, characterized in that The cell is a cell comprising the expression vector according to claim 4 after transformation of a host cell, and the host cell is an Escherichia coli or an Agrobacterium cell.

7. Use of the promoter sequence of claim 2, the expression vector of claim 4, and the cell of claim 6 in analyzing promoter cis-acting elements and verifying activation activity.

8. The use according to claim 7, characterized in that: proPsGSH was transferred into tobacco plants by Agrobacterium-mediated method to obtain transgenic tobacco plants transiently expressing proPsGSH1.

9. Use of the gene according to claim 1, the expression vector according to claim 3, and the cell according to claim 5 in preparing zinc-resistant transgenic plants and plant breeding, wherein the plant is Arabidopsis thaliana or Potentilla sericea, and the purpose of the breeding is to improve the zinc resistance of the plant.

10. The use according to claim 9, characterized in that: The PsGSH1 gene of Potentilla sericea was transferred into Arabidopsis thaliana plants by Agrobacterium-mediated method to obtain transgenic Arabidopsis thaliana overexpressing the PsGSH1 gene.

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