Protein PeuENH1 for improving salt tolerance of plants, nucleotide and application

Abstract

The invention belongs to the field of gene engineering, and particularly provides a protein PeuENH1 capable of improving the salt tolerance of a plant, the amino acid sequence of the protein PeuENH1 is shown as SEQ ID NO.2, and the protein PeuENH1 can improve the salt stress tolerance of the plant, reduce the sensitivity of the plant to the salt stress, participate in the response of the plant to the salt stress and improve the salt tolerance of the plant. Under salt stress, the active oxygen content, the H2O2 content and the malondialdehyde content in the transgenic plant are reduced, the proline content in the transgenic plant is improved, an antioxidant enzyme system is enhanced, and oxidative damage of active oxygen to a membrane system is reduced, so that the tolerance of the plant to salt stress is improved, and the method has great significance in improving the saline-alkali soil environment and improving the saline-alkali soil utilization efficiency. The method has important theoretical and practical significance in expanding the vegetation coverage area of the saline-alkali soil.

Description

Technical Field

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

[0002] my country is the country with the largest arid and semi-arid areas in the world. At the same time, my country also has a large area of ​​saline-alkali land, which accounts for about 20% of the country's cultivated land. With the intensification of global climate change, seawater backflow and soil salinization have become increasingly serious problems. Soil salinization is one of the important environmental factors that restrict the growth and development of agricultural and forestry plants and lead to crop yield reduction, which seriously restricts the development of modern agriculture (Rengasamy, 2016). Exploring the tolerance mechanism of plants to salt stress and exploring excellent salt-resistant gene resources are of great significance for cultivating high-quality salt-tolerant crops and forest germplasm resources and ensuring food security and ecological security.

[0003] Populus euphratica Oliver is distributed in saline-alkali land and semi-arid desert areas in western my country, Central Asia and North Africa (Browicz, 1977). As a natural barrier to prevent desert expansion, it plays an important role in regulating oasis climate, preventing wind and sand, and maintaining ecological balance (Mamat et al., 2018). Populus euphratica has strong vitality and can still grow in various extreme environments. It has multiple stress resistance capabilities such as drought resistance, high temperature resistance and salt resistance, especially its adaptability to drought stress and salt stress is more prominent (Ding et al., 2010). Studies have shown that Populus euphratica can tolerate stress environments of up to 450mM NaCl and 400mM mannitol. Even when placed in an environment with a NaCl concentration of 300mM, it can tolerate high concentrations of Na in leaves and roots. + For as long as one month (Gu et al., 2004). Those skilled in the art have also applied for relevant patents, such as invention patent CN116751767B discloses the application of Populus euphratica PeDUB1 gene in improving plant drought tolerance and salt tolerance; invention patent CN107858341B discloses Populus euphratica PeMIPS1 gene and its application.

[0004] During the research process, the inventors screened out a protein PeuENH1 that improves the salt tolerance of plants from Populus euphratica. 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. 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.

[0005] References:

[0006] Browicz K.(1977)Chorology of Populus euphratica Olivier. Arboretum Kornickie.22,5–27.

[0007] 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.

[0008] 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.

[0009] Mamat Z., 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.

[0010] Rengasamy P. (2016) Soil Salinization. Oxford Research Encyclopedia of Environmental Science, New York. Summary of the invention

[0011] The primary purpose of the present invention is to provide a protein PeuENH1 for improving the salt tolerance of plants, wherein the protein PeuENH1 is a protein as shown in S1), S2) or S3) below:

[0012] S1) protein PeuENH1 with an amino acid sequence as shown in SEQ ID NO.2;

[0013] 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 replaced and / or deleted and / or added;

[0014] S3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of S1) or S2).

[0015] The second object of the present invention is to provide a biomaterial, wherein the biomaterial is any one of the following A1) to A20):

[0016] A1) a nucleic acid molecule encoding protein PeuENH1, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0017] A2) an expression cassette containing the nucleic acid molecule described in A1);

[0018] A3) a recombinant vector containing the nucleic acid molecule described in A1);

[0019] A4) a recombinant vector containing the expression cassette described in A2);

[0020] A5) a recombinant microorganism containing the nucleic acid molecule described in A1);

[0021] A6) a recombinant microorganism containing the expression cassette described in A2);

[0022] A7) a recombinant microorganism containing the recombinant vector described in A3);

[0023] A8) a recombinant microorganism containing the recombinant vector described in A4);

[0024] A9) a transgenic plant cell line containing the nucleic acid molecule described in A1);

[0025] A10) a transgenic plant cell line containing the expression cassette described in A2);

[0026] A11) a transgenic plant cell line containing the recombinant vector described in A3);

[0027] A12) a transgenic plant cell line containing the recombinant vector described in A4);

[0028] A13) transgenic plant tissue containing the nucleic acid molecule described in A1);

[0029] A14) transgenic plant tissue containing the expression cassette described in A2);

[0030] A15) transgenic plant tissue containing the recombinant vector described in A3);

[0031] A16) transgenic plant tissue containing the recombinant vector described in A4);

[0032] A17) transgenic plant organs containing the nucleic acid molecule described in A1);

[0033] A18) a transgenic plant organ containing the expression cassette described in A2);

[0034] A19) a transgenic plant organ containing the recombinant vector described in A3);

[0035] A20) A transgenic plant organ containing the recombinant vector described in A4).

[0036] Preferably, the expression vector is a binary Agrobacterium vector and / or a vector that can be used for plant microprojectile bombardment.

[0037] The third object of the present invention is to provide a use of the protein PeuENH1 in any of the following (1)-(5):

[0038] (1) Regulate plant salt tolerance;

[0039] (2) Preparing products for regulating plant salt tolerance;

[0040] (3) Improve plant salt tolerance;

[0041] (4) preparing products for improving plant salt tolerance;

[0042] (5) Plant breeding;

[0043] The protein PeuENH1 is as follows S1), S2) or S3):

[0044] S1) protein PeuENH1 with an amino acid sequence as shown in SEQ ID NO.2;

[0045] 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 replaced and / or deleted and / or added;

[0046] S3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of S1) or S2).

[0047] Preferably, the plants are monocots and dicots.

[0048] Preferably, the plants are crops, forage grasses or trees.

[0049] Preferably, the purpose of the breeding is to breed salt-tolerant plants.

[0050] 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 for improving plant salt tolerance in a recipient plant, or promoting the expression of the gene encoding the protein PeuENH1 for improving plant salt tolerance, to obtain a target plant with improved salt tolerance compared with the recipient plant.

[0051] The 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.

[0052] Preferably, the transgenic plant is distinguished from the recipient plant in the following aspects (1)-(5):

[0053] (1) The reactive oxygen content of transgenic plants under salt stress was lower than that of recipient plants;

[0054] (2) the H2O2 content of transgenic plants under salt stress was lower than that of recipient plants;

[0055] (3) the proline content of transgenic plants under salt stress was higher than that of recipient plants;

[0056] (4) The MDA content of transgenic plants under salt stress was lower than that of recipient plants;

[0057] (5) Under salt stress, the antioxidant enzyme system of transgenic plants is stronger than that of recipient plants.

[0058] 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

[0059] It should be noted here 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:

[0060] Figure 1 Identification and phylogenetic tree of the PeuENH1 gene family in Populus euphratica.

[0061] 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; each ENH amino acid sequence shows the domain position in proportion; 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.

[0062] Figure 2 Expression pattern of PeuENH1 gene in different tissues of Populus euphratica under normal growth conditions.

[0063] Figure 3 Expression patterns of PeuENH1 gene in different tissues of Populus euphratica under NaCl treatment.

[0064] Figure 4 Schematic diagram of the Populus euphratica PeuENH1 gene overexpression vector 35S::PeuENH1.

[0065] Figure 5 Overexpression of the Populus euphratica PeuENH1 gene improves the tolerance of transgenic Arabidopsis thaliana to salt stress.

[0066] Note: A: Root growth phenotypes of wild type (WT), mutant (enh1) and overexpression (OE2, OE6 and OE9) Arabidopsis under 0, 50, 100 and 150 mM NaCl treatment; B: Root length statistics of wild type (WT), mutant (enh1) and overexpression (OE2, OE6 and OE9) Arabidopsis under 0, 50, 100 and 150 NaCl treatment in Figure A. At least 30 seedlings were measured for each line, repeated 3 times, * indicates P < 0.05.

[0067] Figure 6 Overexpression of the PeuENH1 gene in Populus euphratica improves the tolerance of transgenic Arabidopsis to salt stress by reducing the content of reactive oxygen species. Note: A: Phenotypic comparison of wild-type (WT), mutant (enh1) and overexpressed (OE2, OE6 and OE9) Arabidopsis treated with 200 mM NaCl in soil; BE: Hydrogen peroxide H2O2 content (B), superoxide dismutase SOD activity (C), proline PRO content (D) and malondialdehyde MDA content (E) of wild-type (WT), mutant (enh1) and overexpressed (OE2, OE6 and OE9) Arabidopsis treated with 200 mM NaCl in soil for 21 days. DETAILED DESCRIPTION

[0068] 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 embodiments and drawings. The exemplary embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0069] In the following examples of the present invention, the experimental materials used are Populus euphratica Oliver and Arabidopsis thaliana.

[0070] 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.

[0071] Example 1 Identification and phylogenetic analysis of the PeuENH1 gene family in Populus euphratica

[0072] We first used the AtENH1 protein sequence of Arabidopsis thaliana to identify the homologous ENHs sequences of 13 species, including Populus xinjiangensis, Populus grisea, Populus trichocarpa, Arabidopsis thaliana, grape, rice, maize, Norway spruce, Ginkgo biloba, Cinnamomum camphora, Physcomitrella patens and Selaginella chinensis, by the Blastp method. Among them, 6 ENHs sequences were identified in Populus euphratica, Populus trichocarpa, Populus grisea and Physcomitrella patens, 5 in Populus xinjiangensis and Norway spruce, 4 in Arabidopsis thaliana, rice, grape, Ginkgo biloba, Cinnamomum camphora, 8 in maize and 9 in Selaginella chinensis. The ML phylogenetic tree was constructed using the RAxML software using a total of 71 ENHs amino acid sequences identified from 13 species. The results showed that the ENH sequences of all species were clustered into four subfamilies (I-IV), among which Populus euphratica PeuENH1, PeuENH4, PeuENH2 and PeuENH3, PeuENH5 and PeuENH6 were clustered in subfamilies I, II, III and IV, respectively ( Figure 1 shown).

[0073] Example 2 Cloning of Populus euphratica PeuENH1 gene sequence

[0074] The total RNA of different tissues (root, xylem, phloem and leaves) of Populus euphratica was extracted using 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 sucked into a 1.5 ml centrifuge tube. The tube was placed at room temperature for 10 min. The supernatant was taken into a new 1.5 ml centrifuge tube at 4 °C and 12000 r / min for 10 min. 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 stand at room temperature for 2-3 minutes; centrifuge at 4°C, 12000 r / min 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 stand at room temperature for 10 minutes; centrifuge at 4°C, 12000 r / min for 10 minutes to precipitate RNA, and discard the supernatant; add 75% ethanol to wash the precipitate 1-2 times, and centrifuge at 4°C, 12000 r / min for 2 minutes; dry the precipitate, dissolve in 50 μL DEPC water, and store at -80°C for later use.

[0075] The full-length sequence of the PeuENH1 gene was obtained based on the Populus euphratica genome, and then the following full-length sequence amplification primers were designed using Primer5.0 software:

[0076] Upstream primer F: 5'-ATGTCATCAGCTGTAGTTGCAGG-3'

[0077] Downstream primer R: 5'-TCACATCATACCAACCCCAGCC-3'

[0078] The PeuENH1 gene sequence was obtained by reverse transcription and PCR amplification. The specific method was as follows: According to the user manual of the kit Plant RT-PCR Kit 2.01 (TaKaRa, Japan). 1 μg of total RNA (about 1-2 μl) was mixed with various reverse transcription reagents of the Kit (MgCl2 4 μl; 10× RNA PCR Buffer 2 μl; RNase Inhibitor 0.5 μl; RNase free Water 8.5 μl; dNTPMixture 2 μl; Reverse Transcriptase 1 μl; Oligo dT-Adaptor 1 μl). After mixing, the reverse transcription reaction was completed at 42°C for 30 min; 99°C for 5 min; and 4°C for 5 min. 1 μl of the reverse transcription product was taken and used as a template for PCR reaction: 94°C for 5 min, then the amplification program was entered, 94°C for 50 s, 53°C for 50 s for 30 min, 72°C for 1 min, and after 38 cycles, 72°C for 10 min. Through the above process, the CDS sequence of the PeuENH1 gene was amplified, and the amplified PeuENH1 CDS sequence had a total length of 801 bases 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 of which was shown in SEQ ID No.2.

[0079] Example 3 Expression patterns of the PeuENH1 gene in different tissues of Populus euphratica under salt treatment

[0080] Using the RNA extraction method in Example 2, total RNA from different tissues of Populus euphratica: roots, leaves, xylem and phloem was extracted, reverse transcribed into cDNA, and then the following real-time quantitative qRT-PCR primers were designed using Primer5.0 software:

[0081] Upstream primer F: 5'-AAGTCTGGCGGTGGTGTAGTC-3'

[0082] Downstream primer R: 5'-TGCTTGGATGGCAGTTTTAGT-3'

[0083] The specific method is: according to the kit PrimeScript TMRT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Japan) user manual. 1μg total RNA (about 1-2μl) was mixed with various reverse transcription reagents of the Kit (5×gDNA Eraser Buffer 2.0μl, gDNA Eraser 1.0μl, Total RNA 1μg, RNase Free dH2O up to 10μl). After mixing, incubate at 42℃ for 2min; 4℃ for 5min to complete the first step of reverse transcription reaction. Pipette 10μl of the first step reverse transcription product and mix it with the remaining reagents (PrimeScript RT Enzyme Mix I 1.0μl, RTPrimer Mix 1.0μl, 5×PrimeScript Buffer 24.0μl, RNase Free dH2O 4.0μl). After mixing, incubate at 37℃ for 15min; 85℃ for 5sec; 4℃ to complete the qRT reverse transcription reaction.

[0084] The reverse transcription product was used as a template according to the kit TB Premix Ex Taq TM II (Tli RNaseHPlus) for qRT-PCR reaction. The specific method is: TB Green Premix Ex Taq II (Tli RNaseH Plus) (2X) 10μl, PCR Forward Primer 1.0μl, PCR Reverse Primer 1.0μl, reverse transcription product 1.0μl, sterile water 7.0μl. After mixing, Segment 1, 95℃30sec, 1Cycle; Segment 2, 95℃5sec, 60℃30sec, 40Cycle; Segment 3, 95℃1min, 55℃30sec, 95℃30sec, 1Cycle, complete the qRT-PCR reaction.

[0085] 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 leaves and roots of Populus euphratica significantly increased, and the expression level in stems significantly decreased. The results showed that the expression level of the PeuENH1 gene in Populus euphratica was significantly induced by salt stress and participated in regulating the response of plants to salt stress.

[0086] Example 4 Overexpression of Populus euphratica PeuENH1 gene improves Arabidopsis thaliana tolerance to salt stress

[0087] The method for constructing an expression vector containing the gene PeuENH1 comprises the following steps:

[0088] (1) connecting the gene fragment to the expression vector by homologous recombination, transforming into Escherichia coli DH5α competent cells, and preliminarily obtaining positive clones by screening with 50 mg / L kanamycin;

[0089] (2) further selecting single clones for RCR amplification and sequencing verification, and finally obtaining positive strains of the expression vector 35S::PeuENH1 fused with the PeuENH1 gene; Figure 4 As shown;

[0090] (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;

[0091] (4) The positive monoclonal strain obtained in step (3) was selected, amplified by RCR, and verified by sequencing, and finally the successfully transformed Agrobacterium GV3101 positive strain was obtained, which was used to infect Arabidopsis thaliana.

[0092] (5) The Populus euphratica PeuENH1 gene was transferred into Arabidopsis thaliana by Agrobacterium-mediated floral invasion. After progeny screening, we obtained Arabidopsis thaliana overexpressing the PeuENH1 gene in homozygous form. 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.

[0093] In order to clarify the function of ENH1 under salt stress, we purchased the Arabidopsis ENH1 gene mutant enh1 (SALK_018190) and selected the homozygous enh1 mutant as the subsequent experimental material by the three-primer method. We purified the seeds of OE2, OE6, OE9, WT and enh1 at 4°C in the dark for 2 days. After purification, the sterilized seeds were planted on plates containing MS medium. After growing for 3 days, seedlings with the same growth status were selected and transferred to MS medium containing 0mM, 50mM, 100mM and 150mM NaCl for salt stress treatment.

[0094] 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 enh1 mutant was significantly inhibited, while the root growth of overexpression lines OE2, OE6 and OE9 was better, followed by WT Arabidopsis ( Figure 5 A, 5B). Under 150 mM NaCl treatment, excessively high salt concentrations 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 lower than that of the WT, and the root length of the OE-overexpressing Arabidopsis was significantly higher than that of the WT ( Figure 5 B); Under 50 mM 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 conclusion, overexpression of the PeuENH1 gene in Populus euphratica enhances the tolerance of Arabidopsis thaliana to salt stress, the enh1 mutant enhances the sensitivity of Arabidopsis thaliana to salt stress, and the PeuENH1 gene in Populus euphratica is involved in the response of plants to salt stress.

[0095] Example 5 Overexpression of Populus euphratica PeuENH1 reduces the content of reactive oxygen species (ROS) in Arabidopsis thaliana under salt stress

[0096] To further verify the function of PeuENH1 in Populus euphratica under salt stress, we subjected WT, enh1, OE2, OE6 and OE9 Arabidopsis to salt treatment under soil culture conditions and observed the growth phenotype. First, we transplanted wild-type WT, mutant enh1 and overexpressing OE Arabidopsis seedlings grown on MS medium for 7 days into pots, and after growing for 3 weeks under normal conditions, irrigated with 200mM NaCl for 21 days, and 0mM NaCl was used as a control.

[0097] The results showed that under 0 mM 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, the growth of Arabidopsis thaliana with OE overexpression was significantly better than that of WT and enh1 mutant plants, with the leaves partially turning purple and new leaves growing in the center of the plant ( Figure 6 A); WT Arabidopsis thaliana grew second, 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 6 A); 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). The above results indicate that PeuENH1 in Populus euphratica is involved in regulating the response of plants to salt stress. Overexpression enhances the tolerance of Arabidopsis to salt stress. The enh1 mutant enhances the sensitivity of Arabidopsis to salt stress. The results of salt treatment under soil culture conditions are consistent with those in MS medium.

[0098] Reactive oxygen species (ROS) induced by plants under stress can damage plant cell membranes and endomembrane systems, seriously affecting normal cell metabolism and even causing cell apoptosis. The antioxidant enzyme system can remove excess ROS in plants under stress, maintain normal cell metabolism, and reduce the toxic effects of ROS on plants. The hydrogen peroxide H2O2 content and antioxidant enzyme activity were detected in 3-week-old WT, enh1 mutant and OE-overexpressing Arabidopsis thaliana treated with 200 mM NaCl for 21 days.

[0099] The results showed that H2O2 content was significantly increased in the enh1 mutant and significantly decreased in Arabidopsis overexpressing OE ( Figure 6 B); The activity of the antioxidant enzyme superoxide dismutase (SOD) was significantly reduced in the enh1 mutant and significantly increased in OE-overexpressing Arabidopsis ( 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, and the PRO content in the OE-overexpressing Arabidopsis was higher than that in the WT and enh1, especially in OE9, where the PRO content 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 increased significantly, while the MDA content in the OE-overexpressing Arabidopsis was significantly reduced ( 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 tolerance of plants to salt stress.

[0100] In summary, the present invention provides a gene PeuENH1 for improving the salt resistance of plants, 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, which 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 breeding crops, trees and other plants with stronger salt resistance, improving the saline-alkali land environment, 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), S2) or S3) below: S1) protein PeuENH1 with an amino acid sequence as shown in SEQ ID NO.2; 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 replaced and / or deleted and / or added; S3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of S1) or S2).

2. A biomaterial, characterized in that: The biological material is any one of the following A1) to A20): 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); A9) a transgenic plant cell line containing the nucleic acid molecule described in A1); A10) a transgenic plant cell line containing the expression cassette described in A2); A11) a transgenic plant cell line containing the recombinant vector described in A3); A12) a transgenic plant cell line containing the recombinant vector described in A4); A13) transgenic plant tissue containing the nucleic acid molecule described in A1); A14) transgenic plant tissue containing the expression cassette described in A2); A15) transgenic plant tissue containing the recombinant vector described in A3); A16) transgenic plant tissue containing the recombinant vector described in A4); A17) transgenic plant organs containing the nucleic acid molecule described in A1); A18) a transgenic plant organ containing the expression cassette described in A2); A19) a transgenic plant organ containing the recombinant vector described in A3); A20) A transgenic plant organ containing the recombinant vector described in A4).

3. The biomaterial according to claim 2, characterized in that The expression vector is a binary Agrobacterium vector and / or a vector that can be used for plant microprojectile bombardment.

4. Application of protein PeuENH1 in any of the following (1)-(5): (1) Regulate plant salt tolerance; (2) Preparing products for regulating plant salt tolerance; (3) Improve plant salt tolerance; (4) preparing products for improving plant salt tolerance; (5) Plant breeding; The protein PeuENH1 is as follows S1), S2) or S3): S1) protein PeuENH1 with an amino acid sequence as shown in SEQ ID NO.2; 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 replaced and / or deleted and / or added; S3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of S1) or S2).

5. The use according to claim 4, characterized in that The plants are crops, forage grass or trees.

6. The use according to claim 4, characterized in that The purpose of the breeding is to breed salt-tolerant plants.

7. A method for improving plant salt tolerance, comprising: The activity and / or content of the protein PeuENH1 for improving plant salt tolerance as claimed in claim 1 is increased in a recipient plant, or the expression of the gene encoding the protein PeuENH1 for improving plant salt tolerance as claimed in claim 1 is promoted, so as to obtain a target plant with improved salt tolerance compared with the recipient plant.

8. A method for cultivating transgenic plants, characterized in that: The method comprises increasing the activity and / or content of the protein PeuENH1 for improving the salt tolerance of plants as claimed in claim 1 in a recipient plant, or promoting the expression of the gene encoding the protein PeuENH1 for improving the salt tolerance of plants as claimed in claim 1, thereby obtaining a target plant with improved plant traits compared with the recipient plant.

9. The method according to claim 8, characterized in that The transgenic plant is different from the recipient plant in any of the following aspects (1) to (5): (1) The reactive oxygen 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) Under salt stress, the antioxidant enzyme system of transgenic plants is stronger than that of recipient plants.

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