Protein PeuENH1 for improving plant salt tolerance, nucleotides and applications

Abstract

The present invention belongs to the field of genetic engineering and specifically provides a protein PeuENH1 for improving plant salt tolerance. The amino acid sequence of the protein PeuENH1 is shown in SEQ ID NO.2. The protein PeuENH1 can improve plant tolerance to salt stress, reduce plant sensitivity to salt stress, participate in plant response to salt stress, reduce reactive oxygen content, H2O2 content and malondialdehyde content in transgenic plants under salt stress, increase proline content in transgenic plants, enhance the antioxidant enzyme system, reduce oxidative damage to the membrane system by reactive oxygen, thereby improving plant tolerance to salt stress. The protein has important theoretical and practical significance for improving saline-alkali land environment, increasing saline-alkali land utilization efficiency and expanding saline-alkali land vegetation coverage area.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and particularly relates to a protein PeuENH1, nucleotides and applications thereof for improving the salt tolerance of plants. Background Art

[0002] Populus euphratica (Populus euphratica Oliver) is distributed in saline-alkali lands and semi-arid deserts from western my country, Central Asia, and North Africa (Browicz, 1977). As a natural barrier to desert expansion, it plays a vital role in regulating oasis climate, preventing wind and sand, and maintaining ecological balance (Mamat et al., 2018). Populus euphratica is resilient and can survive in a variety of extreme environments. It exhibits resistance to drought, high temperatures, and salinity, and is particularly adaptable to drought and salt stress (Ding et al., 2010). Studies have shown that Populus euphratica can tolerate stress environments up to 450 mM NaCl and 400 mM mannitol. It can even tolerate high concentrations of NaCl in its leaves and roots when placed in an environment with a NaCl concentration of 300 mM. + The effects of the PeDUB1 gene on drought and salt tolerance in poplars can last up to a month (Gu et al., 2004). Related patents have also been published in the prior art, such as invention patent CN116751767B, which discloses the use of the Populus euphratica PeDUB1 gene for improving plant drought and salt tolerance; and invention patent CN107858341B, which discloses the Populus euphratica PeMIPS1 gene and its applications.

[0003] During the research process, the inventors screened out a protein PeuENH1 from Populus euphratica that improves the salt tolerance of plants. The protein PeuENH1 can improve the tolerance of plants to salt stress, reduce the sensitivity of plants to salt stress, participate in the response of plants to salt stress, reduce the reactive oxygen content, H2O2 content and malondialdehyde content in transgenic plants under salt stress, increase the proline content in transgenic plants, enhance the antioxidant enzyme system, reduce the oxidative damage of reactive oxygen to the membrane system, thereby improving the tolerance of plants to salt stress. This has important theoretical and practical significance for improving the saline-alkali land environment, improving the utilization efficiency of saline-alkali land, and expanding the vegetation coverage area of ​​saline-alkali land.

[0004] References:

[0005] Browicz K.(1977)Chorology of Populus euphratica Olivier.ArboretumKornickie.22,5–27.

[0006] Ding MQ,Hou PC,Shen X.,Wang MJ,Deng SR,...&Chen SL(2010)Salt-induced expression of genes related to Na + / K + and ROS homeostasis inleaves of salt-resistant and salt-sensitive poplar species. Plant Mol Biol. 73, 251–269.

[0007] Gu RS,Liu QL,Pei D.&Jiang XN(2004)Understanding saline andosmotic tolerance of Populus euphratica suspended cells.Plant Cell TissueOrgan Cult.78,261–265.

[0008] Mamat Z.,Halik Keyimu M.,Keram A.&Nurmamat K.(2018)Variation of the floodplain forest ecosystem service value in the lower reaches of TarimRiver,China.Land Degrad Dev.29,47–57.

[0009] Rengasamy P. (2016) Soil Salinization. Oxford Research Encyclopedia of Environmental Science, New York. Summary of the Invention

[0010] The primary purpose of the present invention is to provide a protein PeuENH1 for improving plant salt tolerance, wherein the protein PeuENH1 is a protein as shown in S1), S2) or S3) below:

[0011] S1) protein PeuENH1 with the amino acid sequence shown in SEQ ID NO. 2;

[0012] S2) a protein having the same function as the amino acid sequence shown in SEQ ID NO. 2 after one or more amino acid residues are substituted and / or deleted and / or added;

[0013] S3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of S1) or S2).

[0014] A second object of the present invention is to provide a biomaterial, wherein the biomaterial is any one of the following A1) to A20):

[0015] A1) a nucleic acid molecule encoding protein PeuENH1, the nucleotide sequence of which is shown in SEQ ID NO. 1;

[0016] A2) an expression cassette containing the nucleic acid molecule described in A1);

[0017] A3) a recombinant vector containing the nucleic acid molecule described in A1);

[0018] A4) a recombinant vector containing the expression cassette described in A2);

[0019] A5) a recombinant microorganism containing the nucleic acid molecule described in A1);

[0020] A6) a recombinant microorganism containing the expression cassette described in A2);

[0021] A7) a recombinant microorganism containing the recombinant vector described in A3);

[0022] A8) a recombinant microorganism containing the recombinant vector described in A4);

[0023] A9) a transgenic plant cell line containing the nucleic acid molecule described in A1);

[0024] A10) a transgenic plant cell line containing the expression cassette described in A2);

[0025] A11) a transgenic plant cell line containing the recombinant vector described in A3);

[0026] A12) a transgenic plant cell line containing the recombinant vector described in A4);

[0027] A13) transgenic plant tissue containing the nucleic acid molecule described in A1);

[0028] A14) transgenic plant tissue containing the expression cassette described in A2);

[0029] A15) transgenic plant tissue containing the recombinant vector described in A3);

[0030] A16) transgenic plant tissue containing the recombinant vector described in A4);

[0031] A17) a transgenic plant organ containing the nucleic acid molecule described in A1);

[0032] A18) a transgenic plant organ containing the expression cassette described in A2);

[0033] A19) a transgenic plant organ containing the recombinant vector described in A3);

[0034] A20) A transgenic plant organ containing the recombinant vector described in A4).

[0035] Preferably, the expression vector is a binary Agrobacterium vector and / or a vector that can be used for plant microprojectile bombardment.

[0036] The third object of the present invention is to provide the use of protein PeuENH1 in any of the following (1)-(5):

[0037] (1) Regulating plant salt tolerance;

[0038] (2) Preparation of products for regulating plant salt tolerance;

[0039] (3) Improve plant salt tolerance;

[0040] (4) preparing products for improving plant salt tolerance;

[0041] (5) Plant breeding;

[0042] The protein PeuENH1 is as follows S1), S2) or S3):

[0043] S1) protein PeuENH1 with the amino acid sequence shown in SEQ ID NO. 2;

[0044] S2) a protein having the same function as the amino acid sequence shown in SEQ ID NO. 2 after one or more amino acid residues are substituted and / or deleted and / or added;

[0045] S3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of S1) or S2).

[0046] Preferably, the plants are monocots and dicots.

[0047] Preferably, the plants are crops, forages or trees.

[0048] Preferably, the purpose of the breeding is to breed salt-tolerant plants.

[0049] The fourth object of the present invention is to provide a method for improving plant salt tolerance, comprising: increasing the activity and / or content of the protein PeuENH1 that improves plant salt tolerance in a recipient plant, or promoting the expression of the gene encoding the protein PeuENH1 that improves plant salt tolerance, to obtain a target plant with improved salt tolerance compared to the recipient plant.

[0050] A fifth object of the present invention is to provide a method for cultivating transgenic plants, comprising increasing the activity and / or content of the protein PeuENH1 that improves plant salt tolerance in a recipient plant, or promoting the expression of the gene encoding the protein PeuENH1 that improves plant salt tolerance, to obtain a target plant with improved plant traits compared to the recipient plant.

[0051] Preferably, the transgenic plant is distinguished from the recipient plant in the following aspects (1)-(5):

[0052] (1) The reactive oxygen species content in transgenic plants under salt stress was lower than that in recipient plants;

[0053] (2) The H2O2 content in transgenic plants under salt stress was lower than that in recipient plants;

[0054] (3) The proline content of transgenic plants under salt stress was higher than that of recipient plants;

[0055] (4) The MDA content in transgenic plants under salt stress was lower than that in recipient plants;

[0056] (5) The antioxidant enzyme system of transgenic plants under salt stress is stronger than that of recipient plants.

[0057] The beneficial effects of the present invention are as follows: the present invention provides a protein PeuENH1 for improving the salt tolerance of plants, whose amino acid sequence is shown in SEQ ID NO.2; the protein PeuENH1 can improve the tolerance of plants to salt stress, reduce the sensitivity of plants to salt stress, participate in the response of plants to salt stress, reduce the active oxygen content, H2O2 content and malondialdehyde content in transgenic plants under salt stress, increase the proline content in transgenic plants, enhance the antioxidant enzyme system, reduce the oxidative damage of active oxygen to the membrane system, thereby improving the tolerance of plants to salt stress, and has important theoretical and practical significance for improving the saline-alkali land environment, improving the utilization efficiency of saline-alkali land, and expanding the vegetation coverage area of ​​saline-alkali land. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] It should be noted that the accompanying drawings are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the accompanying drawings:

[0059] Figure 1Identification and phylogenetic tree of the PeuENH1 gene family in Populus euphratica.

[0060] Note: RaxML was used to construct an ML phylogenetic tree based on the amino acid sequences of plant ENHs. Plant ENHs were clustered into four subfamilies (I-IV), represented by different background colors. The domain positions of each ENH amino acid sequence are shown proportionally. Peu, Populus euphratica; Ppr, Populus grayus; Ptr, Populus trichocarpa; Pav, Populus xinjiangensis; At, Arabidopsis thaliana; Vv, Vitis vinifera; Am, Cinnamomum camphora; Pp, Physcomitrella patens; Sm, Selaginella chinensis; Os, Rice; Zm, Zea mays; Gb, Ginkgo biloba; Pa, Norway spruce.

[0061] Figure 2 Expression patterns of the PeuENH1 gene in different tissues of Populus euphratica under normal growth conditions.

[0062] Figure 3 Expression patterns of PeuENH1 gene in different tissues of Populus euphratica under NaCl treatment.

[0063] Figure 4 Schematic diagram of the Populus euphratica PeuENH1 gene overexpression vector 35S::PeuENH1.

[0064] Figure 5 Overexpression of the Populus euphratica PeuENH1 gene improves the tolerance of transgenic Arabidopsis thaliana to salt stress.

[0065] Note: A: Root growth phenotypes of wild-type (WT), mutant (enh1), and overexpressing (OE2, OE6, and OE9) Arabidopsis plants treated with 0, 50, 100, and 150 mM NaCl. B: Root length statistics of wild-type (WT), mutant (enh1), and overexpressing (OE2, OE6, and OE9) Arabidopsis plants treated with 0, 50, 100, and 150 mM NaCl as shown in Figure A. At least 30 seedlings were measured for each line, replicated three times. * indicates P < 0.05.

[0066] Figure 6 Overexpression of the Populus euphratica PeuENH1 gene improves salt stress tolerance in transgenic Arabidopsis by reducing reactive oxygen species (ROS) levels. Note: A: Phenotypic comparison of wild-type (WT), mutant (enh1), and overexpressing (OE2, OE6, and OE9) Arabidopsis plants treated with 200 mM NaCl in soil; B: Hydrogen peroxide (H2O2) content (B), superoxide dismutase (SOD) activity (C), proline (PRO) content (D), and malondialdehyde (MDA) content (E) in wild-type (WT), mutant (enh1), and overexpressing (OE2, OE6, and OE9) Arabidopsis plants after 21 days of 200 mM NaCl treatment in soil. DETAILED DESCRIPTION

[0067] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with examples and drawings. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and are not intended to limit the present invention.

[0068] In the following examples of the present invention, the experimental materials used are Populus euphratica Oliver and Arabidopsis thaliana.

[0069] Arabidopsis thaliana is a commonly used model plant in this field, so Arabidopsis thaliana is generally used first in plant verification experiments in this field. However, it should be noted that the experimental results verified with Arabidopsis thaliana also have the same effect in other plants.

[0070] Example 1 Identification and phylogenetic analysis of the Populus euphratica PeuENH1 gene family

[0071] We first used the AtENH1 protein sequence of Arabidopsis thaliana to identify ENHs homologous sequences from 13 plant species, including Populus sinica, Populus grisea, Populus trichocarpa, Arabidopsis thaliana, grape, rice, maize, Norway spruce, Ginkgo biloba, Cinnamomum camphora, Physcomitrella patens, and Selaginella chinensis, using the Blastp method. Six ENHs sequences were identified from Populus euphratica, Populus trichocarpa, Populus grisea, and Physcomitrella patens, five from Populus sinica and Norway spruce, four from Arabidopsis thaliana, rice, grape, Ginkgo biloba, Cinnamomum camphora, eight from maize, and nine from Selaginella chinensis. A phylogenetic tree was constructed using the RAxML software using a total of 71 ENHs amino acid sequences identified from the 13 species. The results showed that the ENH sequences of all species clustered into four subfamilies (I-IV), with Populus euphratica PeuENH1, PeuENH4, PeuENH2 and PeuENH3, and PeuENH5 and PeuENH6 clustering in subfamilies I, II, III, and IV, respectively. Figure 1 shown).

[0072] Example 2 Cloning of Populus euphratica PeuENH1 gene sequence

[0073] The total RNA of different tissues (roots, xylem, phloem and leaves) of Populus euphratica was extracted using the RNA extraction and separation reagent Trizol. The specific method was as follows: 0.1 g of fresh Xinjiang Populus tissue was weighed and immediately ground into powder in liquid nitrogen. Then 1 ml of Trizol reagent was added, mixed thoroughly, and the mixture was aspirated into a 1.5 ml centrifuge tube and placed at room temperature for 10 min. The supernatant was collected and transferred to a new 1.5 ml centrifuge tube. 1 ml of the supernatant was used for each tube. Add 0.2 ml of chloroform to Trizol, shake vigorously for 15 seconds, and let it stand at room temperature for 2-3 minutes; centrifuge at 12000 r / min at 4°C for 15 minutes, take the supernatant to a new 1.5 ml centrifuge tube, and discard the middle and lower layers; add an equal volume of isopropanol, mix well, and let it stand at room temperature for 10 minutes; centrifuge at 12000 r / min at 4°C for 10 minutes to precipitate RNA, and discard the supernatant; add 75% ethanol to wash the precipitate 1-2 times, and centrifuge at 12000 r / min at 4°C for 2 minutes; dry the precipitate, dissolve it in 50 μL of DEPC water, and store it at -80°C until use.

[0074] The full-length sequence of the PeuENH1 gene was obtained from the Populus euphratica genome, and then the following full-length sequence amplification primers were designed using Primer5.0 software:

[0075] Upstream primer F: 5'-ATGTCATCAGCTGTAGTTGCAGG-3'

[0076] Downstream primer R: 5'-TCACATCATACCAACCCCAGCC-3'

[0077] The PeuENH1 gene sequence was obtained by reverse transcription and PCR amplification according to the user manual of the Plant RT-PCR Kit 2.01 (TaKaRa, Japan). 1 μg of total RNA (approximately 1-2 μl) was mixed with the various reverse transcription reagents in the kit (MgCl₂ 4 μl; 10× RNA PCR Buffer 2 μl; RNase Inhibitor 0.5 μl; RNase-free Water 8.5 μl; dNTP Mixture 2 μl; Reverse Transcriptase 1 μl; Oligo dT-Adapter 1 μl). After mixing, the reverse transcription reaction was completed by incubating at 42°C for 30 minutes, 99°C for 5 minutes, and 4°C for 5 minutes. One μl of the reverse transcription product was aspirated and used as a template for the PCR reaction: 94°C for 5 minutes, followed by the amplification program: 94°C for 50 seconds, 53°C for 50 seconds for 30 minutes, and 72°C for 1 minute. After 38 cycles, the reaction was continued at 72°C for 10 minutes. Through the above process, the CDS sequence of the PeuENH1 gene was amplified, and the amplified PeuENH1 CDS sequence was 801 bases in total from the start codon to the stop codon, the nucleotide sequence of which was shown in SEQ ID No. 1, and the amino acid sequence was shown in SEQ ID No. 2.

[0078] Example 3 Expression patterns of the Populus euphratica PeuENH1 gene in different tissues under salt treatment

[0079] Using the RNA extraction method in Example 2, total RNA was extracted from different tissues of Populus euphratica: roots, leaves, xylem, and phloem, and reverse transcribed into cDNA. Then, Primer 5.0 software was used to design the following real-time quantitative qRT-PCR primers:

[0080] Upstream primer F: 5'-AAGTCTGGCGGTGGTGTAGTC-3'

[0081] Downstream primer R: 5'-TGCTTGGATGGCAGTTTTAGT-3'

[0082] The specific method is: according to the kit PrimeScript TMFollow the user manual of the RT reagent kit with gDNAEraser (Perfect Real Time) (TaKaRa, Japan). Mix 1 μg of total RNA (approximately 1-2 μl) with the various reverse transcription reagents in the kit (5× gDNAEraser Buffer 2.0 μl, gDNAEraser 1.0 μl, Total RNA 1 μg, RNase-free dH2O up to 10 μl). After mixing, incubate at 42°C for 2 minutes, then at 4°C for 5 minutes to complete the first reverse transcription reaction. Pipette 10 μl of the first reverse transcription product and mix with the remaining reagents (PrimeScript RT Enzyme Mix I 1.0 μl, RT Primer Mix 1.0 μl, 5× PrimeScript Buffer 2 4.0 μl, RNase-free dH2O 4.0 μl). After mixing, incubate at 37°C for 15 minutes, then at 85°C for 5 seconds, and finally at 4°C to complete the qRT reverse transcription reaction.

[0083] The reverse transcription product was used as a template according to the kit TB Premix Ex Taq TM qRT-PCR was performed using Taq II (Tli RNaseH Plus). The specific method was as follows: 10 μl of TB Green Premix Ex Taq II (Tli RNaseH Plus) (2X), 1.0 μl of PCR Forward Primer, 1.0 μl of PCR Reverse Primer, 1.0 μl of reverse transcription product, and 7.0 μl of sterile water. After mixing, the qRT-PCR reaction was completed at 95°C for 30 seconds for 1 cycle for Segment 1; at 95°C for 5 seconds, 60°C for 30 seconds, and 40 cycles for Segment 2; and at 95°C for 1 minute, 55°C for 30 seconds, and 95°C for 30 seconds, and 1 cycle for Segment 3.

[0084] The results of expression pattern analysis were as follows Figure 2 As shown in Figure 2, under normal growth conditions, the PeuENH1 gene of Populus euphratica is mainly expressed in leaves and phloem, with the lowest expression level in roots. Figure 3 As shown in the figure, after 48 hours of treatment with 200 mM NaCl, the expression level of the PeuENH1 gene in Populus euphratica leaves and roots increased significantly, while its expression level in stems decreased significantly. The results indicate that the expression level of the PeuENH1 gene in Populus euphratica is significantly induced by salt stress and participates in regulating the plant's response to salt stress.

[0085] Example 4 Overexpression of the Populus euphratica PeuENH1 gene improves Arabidopsis thaliana's tolerance to salt stress

[0086] The method for constructing an expression vector containing the gene PeuENH1 comprises the following steps:

[0087] (1) The gene fragment was connected to the expression vector by homologous recombination, transformed into Escherichia coli DH5α competent cells, and positive clones were initially obtained by screening with 50 mg / L kanamycin;

[0088] (2) Single clones were further selected for RCR amplification and sequencing verification, and finally a positive strain of the expression vector 35S::PeuENH1 fused with the PeuENH1 gene was obtained; Figure 4 As shown;

[0089] (3) extracting the successfully connected plasmid from the positive strain obtained in step (2), transforming it into competent Agrobacterium GV3101, and preliminarily screening the positive clone strain with 50 mg / L kanamycin, 50 mg / L rifampicin, and 50 mg / L gentamicin;

[0090] (4) The positive monoclonal strain obtained in step (3) was selected, amplified by RCR, and verified by sequencing to finally obtain the successfully transformed Agrobacterium GV3101 positive strain, which was used to infect Arabidopsis thaliana.

[0091] (5) The Populus euphratica PeuENH1 gene was transferred into Arabidopsis thaliana by Agrobacterium-mediated floral invasion. After progeny screening, homozygous Arabidopsis thaliana overexpressing the Populus euphratica PeuENH1 gene was obtained. Based on the expression level of the PeuENH1 gene in homozygous Arabidopsis thaliana, we selected three overexpression Arabidopsis thaliana lines, OE2, OE6, and OE9, with high, medium, and low expression levels, respectively, for salt stress experiments.

[0092] To clarify the function of ENH1 under salt stress, we purchased the Arabidopsis thaliana ENH1 mutant enh1 (SALK_018190) and selected homozygous enh1 mutants using a three-primer method for subsequent experimental material. Seeds of OE2, OE6, OE9, WT, and enh1 were purified for 2 days in the dark at 4°C. After purification, the sterilized seeds were seeded on plates containing MS medium. After 3 days of growth, seedlings with consistent growth status were transferred to MS medium containing 0 mM, 50 mM, 100 mM, and 150 mM NaCl for salt stress treatment.

[0093] After 10 days of treatment, under normal growth conditions (0 mM NaCl), the growth of WT, enh1, OE2, OE6 and OE9 seedlings was basically the same, and there was no significant change in root length statistics ( Figure 5A, 5B). Under 50mM and 100mM NaCl treatment, the root growth of the enh1 mutant was significantly inhibited, while the root growth of the overexpression lines OE2, OE6, and OE9 was better, followed by the WT Arabidopsis ( Figure 5 A, 5B). Under 150 mM NaCl treatment, excessively high salt concentration significantly inhibited the root growth of WT, enh1, OE2, OE6, and OE9 Arabidopsis ( Figure 5 A, 5B). Root length statistics showed that under 100 mM NaCl treatment, the root length of the enh1 mutant was significantly shorter than that of the WT, while the root length of the OE-overexpressing Arabidopsis was significantly longer than that of the WT ( Figure 5 B); Under 50mM NaCl treatment, the root length of the enh1 mutant was shorter than that of the WT, and the root length of the OE-overexpressing Arabidopsis was longer than that of the WT, but the statistical results were not significant ( Figure 5 B); Under 150 mM NaCl treatment, the root length of WT, enh1 mutant and OE overexpressing Arabidopsis was significantly inhibited, and there was no significant difference in root length statistics ( Figure 5 B). In summary, overexpression of the Populus euphratica PeuENH1 gene enhances Arabidopsis tolerance to salt stress, and the enh1 mutant increases Arabidopsis sensitivity to salt stress. This suggests that the Populus euphratica PeuENH1 gene is involved in the plant response to salt stress.

[0094] Example 5 Overexpression of Populus euphratica PeuENH1 reduces reactive oxygen species (ROS) content in Arabidopsis thaliana under salt stress

[0095] To further validate the function of PeuENH1 in Populus euphratica under salt stress, we subjected WT, enh1, OE2, OE6, and OE9 Arabidopsis plants to salt treatment in soil culture and observed their growth phenotypes. First, we transplanted 7-day-old wild-type WT, enh1 mutant, and OE-overexpressing Arabidopsis seedlings grown on MS medium into pots. After 3 weeks of normal growth, they were watered with 200 mM NaCl for 21 days, with 0 mM NaCl serving as a control.

[0096] The results showed that under 0mM NaCl treatment, the growth status of WT, enh1 and OE overexpressing Arabidopsis was consistent and grew well, with no significant changes ( Figure 6 A); Under 200 mM NaCl treatment, OE-overexpressing Arabidopsis plants grew significantly better than WT and enh1 mutant plants, with some leaves turning purple and new leaves growing in the center of the plant ( Figure 6 A); WT Arabidopsis thaliana grew second best, with some leaves turning yellow and wilting and drying up. The leaves growing in the center of the plant were smaller and white, and the plant growth rate slowed down ( Figure 6A); enh1 mutant plants had the worst growth, with growth significantly inhibited. Most leaves wilted, dried up, and turned white, and almost no new leaves grew in the center of the plant ( Figure 6 A). These results indicate that PeuENH1 in Populus euphratica is involved in regulating plant responses to salt stress. Overexpression of PeuENH1 enhances salt tolerance in Arabidopsis, and the enh1 mutant increases the sensitivity of Arabidopsis to salt stress. The results of salt treatment under soil culture conditions were consistent with those in MS medium.

[0097] Reactive oxygen species (ROS) produced by plants under stress can damage cell membranes and endomembrane systems, severely impairing normal cellular metabolism and even causing apoptosis. Antioxidant enzyme systems can eliminate excess ROS under stress, maintaining normal cellular metabolism and mitigating the toxic effects of ROS on plants. Three-week-old WT, enh1 mutant, and OE-overexpressing Arabidopsis seedlings were treated with 200 mM NaCl for 21 days and then assayed for hydrogen peroxide (H2O2) content and antioxidant enzyme activity.

[0098] The results showed that H2O2 content was significantly increased in the enh1 mutant and significantly decreased in OE-overexpressing Arabidopsis ( Figure 6 B); The activity of the antioxidant enzyme superoxide dismutase (SOD) was significantly reduced in the enh1 mutant and significantly increased in Arabidopsis thaliana overexpressing OE ( Figure 6 C). Proline (PRO) is an important indicator of plant stress resistance. After salt treatment, the PRO content in the enh1 mutant was significantly lower than that in the WT. In Arabidopsis overexpressing OE, the PRO content was higher than that in the WT and enh1. In particular, the PRO content in OE9 was significantly increased ( Figure 6 D). Malondialdehyde (MDA) is an important indicator for evaluating the degree of cell membrane damage. After salt treatment, the MDA content in the enh1 mutant was significantly increased, while the MDA content in Arabidopsis thaliana with OE overexpression was significantly decreased ( Figure 6 E). In summary, under salt stress, overexpression of the PeuENH1 gene reduces the content of reactive oxygen species in plants, enhances the antioxidant enzyme system, and reduces the oxidative damage of reactive oxygen species to the membrane system, thereby improving the plant's tolerance to salt stress.

[0099] In summary, the present invention provides a gene PeuENH1 for improving plant salt tolerance, the nucleotide sequence of which is shown in SEQ ID No. 1 and the amino acid sequence of which is shown in SEQ ID No. 2. The gene can significantly improve the tolerance of transgenic plants to salt stress. The discovery of the Populus euphratica PeuENH1 gene has important theoretical and practical significance for cultivating crops, trees, and other plants with stronger salt tolerance, improving saline-alkali land environments, and expanding the vegetation coverage area of ​​saline-alkali land.

Claims

1. A protein PeuENH1 for improving plant salt tolerance, characterized in that: The protein PeuENH1 is the protein shown in S1): S1) The amino acid sequence of protein PeuENH1 is shown in SEQ ID NO.

2.

2. A biomaterial, characterized in that The biological material is any one of the following A1) to A8): A1) a nucleic acid molecule encoding protein PeuENH1, the nucleotide sequence of which is shown in SEQ ID NO. 1; A2) an expression cassette containing the nucleic acid molecule described in A1); A3) a recombinant vector containing the nucleic acid molecule described in A1); A4) a recombinant vector containing the expression cassette described in A2); A5) a recombinant microorganism containing the nucleic acid molecule described in A1); A6) a recombinant microorganism containing the expression cassette described in A2); A7) a recombinant microorganism containing the recombinant vector described in A3); A8) A recombinant microorganism containing the recombinant vector described in A4).

3. The biomaterial according to claim 2, wherein The vector is a binary Agrobacterium vector or a vector used for plant microprojectile bombardment.

4. Application of protein PeuENH1 in any of the following (1)-(3): (1) Improve plant salt tolerance; (2) Preparation of products to improve plant salt tolerance; (3) Plant breeding, where the purpose of the breeding is to breed salt-tolerant plants; The protein PeuENH1 is as follows (S1): S1) protein PeuENH1 with the amino acid sequence shown in SEQ ID NO. 2; The plants are Arabidopsis thaliana and Populus euphratica.

5. A method for improving salt tolerance of plants, comprising: The content of the protein PeuENH1 for improving plant salt tolerance according to claim 1 is increased in a recipient plant to obtain a target plant with improved salt tolerance compared with the recipient plant, wherein the recipient plant is Arabidopsis thaliana or Populus euphratica.

6. A method for cultivating transgenic plants, characterized in that: The method comprises increasing the content of the protein PeuENH1 for improving plant salt tolerance as claimed in claim 1 in a receptor plant to obtain a target plant with improved plant traits compared with the receptor plant, wherein the receptor plant is Arabidopsis thaliana or Populus euphratica.

7. The method according to claim 6, wherein The transgenic plant is distinguished from the recipient plant by any of the following (1) to (5): (1) The reactive oxygen species content of transgenic plants under salt stress was lower than that of recipient plants; (2) The H2O2 content of transgenic plants under salt stress was lower than that of recipient plants; (3) The proline content of transgenic plants under salt stress was higher than that of recipient plants; (4) The MDA content of transgenic plants under salt stress was lower than that of recipient plants; (5) The antioxidant enzyme system of transgenic plants under salt stress is stronger than that of recipient plants.

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