CN108250281B - Sodium and hydrogen antiporter PbrNHX2 in pyrus betulaefolia and application thereof in improving salt tolerance of plants - Google Patents

Abstract

The invention provides sodium and hydrogen antiporter PbrNHX2 in pyrus betulaefolia and application thereof in improving the salt tolerance of plants, belonging to the technical field of genetic engineering. The invention provides a sodium and hydrogen antiporter protein in pyrus betulaefolia, a coding gene and a primer pair for cloning a cDNA sequence of the coding gene. The sodium and hydrogen reverse transport protein in the birch pear, the coding gene or the coding gene pair cloned by the primer pair are applied to improving the salt tolerance of plants. The tobacco transformation vector and the autumn pear transformation vector are constructed, salt treatment is carried out on the obtained positive plant seedlings, the electric conductivity of the positive plant seedlings is obviously reduced compared with that of the wild type, the chlorophyll content is obviously increased compared with that of the wild type, the survival rate is obviously improved compared with that of the wild type, and meanwhile, the positive plant seedlings have stronger ROS resistance compared with that of the wild type. The over-expressed coding gene can effectively enhance the active oxygen scavenging capacity of the transgenic plant, thereby improving the salt tolerance of the plant.

Description

Sodium and hydrogen antiporter PbrNHX2 in pyrus betulaefolia and application thereof in improving salt tolerance of plants

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a sodium and hydrogen antiporter PbrNHX2 in pyrus betulaefolia and application thereof in improving salt tolerance of plants.

Background

Pear is one of the main planted fruit trees in the world. The planting distribution of the pear trees in China is very wide, the main production area of the pears is distributed into three areas and four points, and the pear trees are planted from the northeast to the Guangxi and from the Yunnan to the Shandong. The layout and planning of the pear dominant production area greatly promote the development of the pear industry in China, but the pear dominant production area is easily influenced by various environmental factors such as drought, waterlogging, salt and alkali due to wide distribution. Therefore, whether to culture a new variety with excellent stress resistance becomes the most important factor for the development of the pear industry. Because of the existence of pear breeding: the conventional breeding method can not meet the variety requirements of modern pear planting due to the reasons of complex genetic background, long childhood period, self-incompatibility, uncertainty of breeding target and the like. Therefore, how to breed the novel stress-resistant pear variety for production in a short time becomes a difficult problem for breeders. With the rapid progress of biotechnology and tissue culture technology, plant breeding has new approaches, such as tissue culture technology breeding, genetic transformation breeding, somatic cell hybridization and the like. The characteristics of the plants are improved by utilizing the plant genetic engineering technology, so that the efficient and accurate cultivation of the new stress-resistant pear variety becomes possible.

The damage caused by salt stress to plants is initially manifested as osmotic stress and is present in the growth cycle of the plant; this is followed by toxic and nutrient element losses due to ionic imbalance; finally, oxidative stress causes changes in membrane permeability and physiological metabolic disorders and accumulation of toxic substances, which ultimately results in the retardation and the effects on plant growth and development, and even death (Flowers, 2004). The salt tolerance of the plants is improved mainly by the reconstruction of ion balance, osmotic adjustment and the elimination of active oxygen in cells.

According to the conventional studies, it has been found that NHX gene in plants and salt tolerance of plants are presentAre closely related. The NHX gene functions to encode Na⁺/H⁺The reverse transport protein can regulate and control Na in plants by changing the expression level of NHX family genes⁺/H⁺Synthesis of reverse transport protein, thus changing the salt tolerance of plants. Studies have shown that in higher plants, Na⁺/H⁺The functions of the antiporter are mainly: na (Na)⁺Efflux and restriction of Na⁺Transportation to the above ground (Shi et al, 2000; Shiet al, 2000). When the plant cells are stressed by salinity, the osmotic potential difference easily causes the water loss of the cells. Na located on plasma membrane and vacuolar membrane for maintaining ion balance in cells, reducing osmotic potential of cells, and making plants capable of normally absorbing water from external high-salt environment⁺/H⁺The antiporter will now be activated, Na⁺/H⁺Reverse transport protein is prepared by reacting Na⁺Transporting the Na in the cells by a reverse concentration gradient⁺Efflux or compartmentalization into the vacuole avoids water stress (Padan and Schuldiner, 1987). Except for Na⁺Efflux and compartmentalization, recent studies have shown that Na is localized to the vacuolar membrane⁺/H⁺The antiporter also has K⁺Compartmentalization into the vacuole. When the plant cell is stressed by salt, the expression level of NHX1 gene of the plant is up-regulated, thereby enhancing K⁺Compartmentalization into the vacuole by partitioning K⁺Compartmentalization into the vacuole, allowing the cell to produce K in the cytoplasm⁺Reduced signal, thereby activating high affinity K on plasma membrane⁺The transporter of (1). Activated high affinity K⁺The transporter can be specifically selected from low K⁺Environment will be more K⁺Transport into cells to maintain stable Na in cells⁺/H⁺. The research shows that the salt stress can effectively activate the NHX1 gene, thereby enhancing Na⁺/H⁺Expression of antiporters, increasing the salt tolerance of plants (Ohta et al, 2002; Jiang et al, 2010; Leidi et al, 2010). The pH value in the cell is mainly H generated by proton pump and metabolism⁺、OH^-Determinatively, the NHX protein passes through H intracellularly⁺The transportation of (a) is carried out,can change the ion concentration in the vacuole to adjust the pH value of the vacuole.

Na⁺/H⁺The first finding of antiporters was in mammals and in plants on the plasma membrane of barley (Ratner and Jacoby, 1976). From the present studies, it was found that Na is present in the middle of the membrane system of plants⁺/H⁺Antiporters are ubiquitous and have been studied more extensively in common species, such as AtNHX1 antiporter in Arabidopsis (Apse et al,1999), OsNHX1 antiporter in rice (Fukuda et al,1999), TtNHX1 antiporter in New Zealand (Lu Hui Gui, 2003), maize ZmNHX1 antiporter (Zorbet al, 2005), citrus cNHX1 antiporter (Portat et al, 2002), and others. Saier et al found that cation, proton antiporters can be divided into two families, CPA1 and CPA2 (Saier et al, 1999). With increasing Na⁺/H⁺Transporters are found in animals and plants and fungi and slowly constitute a family of transporters of NHX. This family can be divided into two groups, class1 and class2, according to their sequence homology, which is 20% to 30%, respectively. From the distribution of the transporter family of NHX, class1 family was shown to be present mainly in terrestrial plants, and its NHX subtype is present mainly on the vacuolar membrane, which is also one of its main properties. The class2 family is found in angiosperm spruce and bryophytes and gymnosperms, and is mainly found in various vesicles in plant cells. According to the existing research, Na is separated from the plant⁺/H⁺Antiporters, which are classified according to their localization, are localized to the plasma membrane of the cell, and are localized to the vacuolar membrane of the cell (Lu et al, 2005).

Apse et al, obtained by transferring the Arabidopsis AtNHX1 gene into Arabidopsis, showed a greatly improved salt tolerance in the line (Apse et al, 1999). Zhang et al, transfer of the AtNHX1 gene into tomato revealed that over-expressed tomato plants grew normally in 200mM NaCl (Zhang, 2001). Ohta et al transferred NHX gene in genus atriplex into rice, and also obtained transgenic rice plants with salt tolerance (Ohta et al, 2002). The plant over-expressing NHX gene can change the salt tolerance, He and the like of the plant, and in the plant over-expressing AtNHX1 gene of cotton, the cotton yield of the transgenic plant is improved under the NaCl treatment condition of 200mmol/L, and more and better cotton fibers can be produced (He et al, 2005). Some NHX genes can also be specifically expressed in flowers, such as TvNHX1 gene in morning glory, which is mainly specifically expressed in flowers, and can change flower color by adjusting the pH value of vacuole (Ohnishi et al, 2005). The NHX gene also has a certain yield increase effect, and the over-expression of the ZmNHX1 gene in the corn enhances the salt tolerance of plants and simultaneously improves the yield of the corn (Li et al, 2010). NHX genes in most species can be expressed under non-stress, partial NHX genes are expressed in tissues such as roots, stems and leaves and the like under salt stress, and the expression of the genes induced by ABA, dehydration, drought, KCl and cold stress is also reported (Rodriguez-Rosales et al, 2009). The research shows that the plant is separated from arabidopsis thaliana, tomato, rice, barley, soybean, common seepweed herb, salicornia europaea and other plants.

The birch-leaf pear is a stock widely applied in the pear industry, is extremely salt-resistant, and is an ideal material for researching the salt resistance of woody plants and cloning related salt-resistant genes. Therefore, cloning the salt-resistant related gene of the pyrus betulaefolia is the key and the basis of salt-resistant gene engineering.

Disclosure of Invention

In view of the above, the invention aims to provide a sodium and hydrogen antiporter PbrNHX2 in pyrus betulaefolia and an application thereof in improving the salt tolerance of plants, wherein PbrNHX2 has a function of regulating and controlling salt resistance, and effectively improves the salt tolerance of transgenic plants.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides sodium and hydrogen antiporter PbrNHX2 in pyrus betulaefolia, which has an amino acid sequence shown as SEQ ID NO.1 in a sequence table.

The invention provides a coding gene of sodium and hydrogen antiporter PbrNHX2 in pyrus betulaefolia, which has a nucleotide sequence shown as SEQ ID NO.2 in a sequence table.

The invention provides a primer pair for cloning a cDNA sequence of the coding gene, which comprises a forward primer and a reverse primer, wherein the forward primer has a nucleotide sequence shown as SEQ ID NO.3 in a sequence table; the reverse primer has a nucleotide sequence shown as SEQ ID NO.4 in the sequence table.

The invention provides application of sodium and hydrogen antiporter PbrNHX2, the coding gene or the primer pair in the pyrus betulaefolia in improving the salt tolerance of plants.

Preferably, the application comprises the following steps: and recombining and expressing the sodium and hydrogen antiporter PbrNHX2 or the coding gene cloned by the primer pair in plants to obtain the recombined plants with salt tolerance.

Preferably, the cloning procedure is a pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 90s, and extension at 72 ℃ for 90s, and 35 cycles of extension at 72 ℃ for 10min after the completion of the cycles.

Preferably, the plant comprises tobacco or autumn pears.

Preferably, the salt concentration of the salt tolerance of the plants is not higher than 1000 mmol/L.

Preferably, the salt-tolerant salt of the plant comprises a Na salt or a K salt.

The invention provides sodium and hydrogen antiporter PbrNHX2 in pyrus betulaefolia, which has an amino acid sequence shown as SEQ ID NO.1 in a sequence table. The functional complementation analysis experiment of the PbrNHX2 in the salt-sensitive yeast mutant AXT3 shows that the PbrNHX2 is recombined and expressed in the salt-sensitive yeast mutant AXT3, which shows that the PbrNHX2 can partially complement the salt tolerance of the salt-sensitive yeast mutant AXT 3; the salt-containing capacity in the yeast is simultaneously measured, and the result shows that: the salt content of the recombinant yeast is obviously lower than that of the yeast of a blank control group, and the recombinant yeast contains K⁺The amount is higher than that of the control group yeast, which indicates that PbrNHX2 has K absorption⁺Arrange Na⁺Thus indicating that the protein PbrNHX2 has the function of salt resistance.

The invention provides a coding gene of sodium and hydrogen antiporter PbrNHX2 in pyrus betulaefolia, which has a nucleotide sequence shown as SEQ ID NO.2 in a sequence table. The birch pear seedlings are sampled at corresponding time points under the treatment of 200mM NaCl solution, and the relative expression quantity of the coding gene provided by the invention is analyzed by adopting real-time quantitative PCR, and the result shows that the relative expression quantity of the coding gene is gradually increased along with the extension of a NaCl solution treatment experiment within 12h, which shows that the coding gene responds to salt stress treatment and has the function of regulating and controlling salt resistance.

The invention provides application of sodium and hydrogen antiporter PbrNHX2, the coding gene or the coding gene pair cloned by the primer pair in improving the salt tolerance of plants. By respectively constructing a tobacco transformation vector and a autumn pear transformation vector, respectively obtaining positive plant seedlings for salt treatment, the results show that: the conductivity of the positive plant seedling is obviously reduced compared with that of a Wild Type (WT), the chlorophyll content is obviously increased compared with that of the wild type, and the survival rate is increased by 116% -129% compared with that of the wild type, which indicates that the positive plant seedling has stronger ROS resistance than the wild type. Hydrogen peroxide (H) in transgenic tobacco₂O₂) And superoxide anion (O)^2-) The content activity of the compound is lower than that of a wild type, the active oxygen residue in a plant body is lower, and the cell damage is smaller. These results show that the over-expressed PbrNHX2 gene can effectively enhance the active oxygen scavenging ability of transgenic plants, thereby improving the salt tolerance of the plants.

Drawings

FIG. 1 is a schematic flow chart of the present invention;

FIG. 2 is a schematic diagram showing the expression of the gene encoding PbNHX2 under salt, dehydration and low temperature stress in example 2; wherein, FIG. 2-A shows that the coding gene is sampled at corresponding time points under the treatment of birch pear seedlings (not transgenic) in a 200mM NaCl solution, and the relative expression quantity of the coding gene is analyzed by adopting real-time quantitative PCR; FIG. 2-B is an expression pattern of dehydration of birch pear seedlings at room temperature at various time points; FIG. 2-C shows the relative expression level of the gene of the present invention analyzed by real-time quantitative PCR, wherein the birch pear seedlings were sampled at corresponding time points under 4 deg.C treatment;

FIG. 3 shows the subcellular localization of the gene encoding PbrNHX2 in example 3, in which FIG. 3-A shows the imaging of the GFP gene in dark field, positive control, overlay, bright field; FIG. 3-B is an image of the gene encoding PbrNHX2 in dark field, positive control, overlay, bright field;

FIG. 4 shows the results of the functional complementation analysis of PbrNHX2 in the salt-sensitive yeast mutant AXT3 in example 4;

FIG. 5 is the acquisition of tobacco plants transformed with PbrNHX2 in example 5, FIG. 5- (a) is the construction of vectors, FIG. 5- (b) is the whole flow chart of PbrNHX2 transformed tobacco, FIG. 5- (c) is the identification of transgenic plants by PCR, FIG. 5- (d) is the overexpression analysis of RT-PCR identified transgenic plants, and FIG. 5- (e) is the protein expression level of overexpressed plants by Western blotting analysis;

FIG. 6 is a schematic diagram of the transformation of autumn pear and plant with PbrNHX2 in example 6, wherein FIG. 6-A is a photograph after transformation; FIG. 6-B is a material grown on screening media for 30 days; FIG. 6-C is the induction of rooting by the regenerated shoots; FIG. 6-D is a photograph of transgenic plants grown in soil for 30 days; FIG. 6-E is a T0 generation transgenic plant after PCR identification of tobacco using gene specific primers, where M: marker, +: plasmid, -: wild type plant, 1-8: a transgenic line; FIG. 6-F is a real-time quantitative PCR analysis of the expression level of the coding gene of PbrNHX2 in different transgenic lines of autumn pear;

FIG. 7 shows the phenotypic and physiological index measurements of the gene line encoding PbrNHX2 transformed in example 7 before and after treatment with Wild Type (WT) sodium chloride, wherein FIG. 7-A shows the phenotype of 45-day-old tobacco plants before treatment with 300mM sodium chloride for 20 days; FIG. 7-B is the phenotype of 45 day old tobacco plants after 20 days of 300mM sodium chloride treatment; FIG. 7-C is a phenotype of 30 day old tobacco plants 4 days prior to treatment with 200mM salt solution; FIG. 7-D is a phenotype of 30-day-old tobacco plants treated with 200mM salt solution for 4 days; FIG. 7-E is a statistical result of survival rate of 30-day-old tobacco plants treated with 200mM salt solution for 4 days, FIG. 7-F is a result of conductivity measurement of 30-day-old tobacco plants treated with 200mM salt solution for 4 days, FIG. 7-G is a phenotype of 45-day-old tobacco plants treated with 300mM sodium chloride for 25 days, FIG. 7-H is a chlorophyll measurement of 45-day-old tobacco plants treated with 300mM sodium chloride for 25 days, and FIG. 7-I is a chlorophyll extraction of 30-day-old tobacco plants treated with 200mM salt solution for 4 days;

FIG. 8 shows the results of the phenotypic and physiological index measurements of PbrNHX2 transgenic autumn pear strains (OE2, OE6 and OE8) and wild type plants (WT) in example 7 after 15 days of pouring 500mM sodium chloride, wherein FIG. 8-A shows the phenotype after 15 days of pouring 500mM sodium chloride, FIG. 8-B shows the conductivity measurement after 15 days of pouring 500mM sodium chloride, and FIGS. 8-C-8-D show the chlorophyll measurement (FIG. 8-D) and chlorophyll extraction (FIG. 8-C) after 15 days of pouring 500mM sodium chloride;

FIG. 9 is histochemical staining analysis H of tobacco and autumn pear encoding gene transformed with PbrNHX2 in example 7₂O₂And O^2-Accumulation, FIGS. 9-A and 9-B show that 45-day-old tobacco plants were stained with Nitrotetrazolium (NBT) and Diaminobenzidine (DAB) for H and reactive oxygen histochemical staining of untransformed plants and two transgenic lines before and after 15d treatment with 300mM sodium chloride, respectively₂O₂(FIG. 9-A) and O^2-(FIG. 9-B) staining, FIG. 9-C shows cell death chromosomes after transgenic tobacco salt stress treatment, and FIGS. 9-D and 9-E show that 30-day-old Kazuki pear plants were stained with Nitrotetrazole (NBT) and Diaminobenzidine (DAB) for H + cells before and after 500mM sodium chloride treatment for 15D for untransformed plants and two transgenic lines₂O₂(FIG. 9-D) and O^2-(FIG. 9-E) staining was performed, and FIG. 9-F shows cell death chromosomes after salt stress treatment of transgenic autumn pears.

Detailed Description

The invention provides sodium and hydrogen antiporter PbrNHX2 in pyrus betulaefolia, which has an amino acid sequence shown as SEQ ID NO.1 in a sequence table. The PbrNHX2 codes 542 amino acids, has an isoelectric point of 8.49 and a molecular weight of 60.09 KDa. The PbrNHX2 has the function of absorbing K⁺Arrange Na⁺The function of the gene can increase the ROS resistance of the carrier plant, thereby indicating that the gene has the function of salt resistance. In the invention, the PbrNHX2 is obtained by recombinant expression of the coding gene of PbrNHX 2. The method of the recombinant expression is not particularly limited, and a recombinant expression method well known in the art may be used.

The invention provides a coding gene of sodium and hydrogen antiporter PbrNHX2 in pyrus betulaefolia, which has a nucleotide sequence shown as SEQ ID NO.2 in a sequence table. The source of the coding gene is preferably obtained by cloning.

The invention provides a primer pair for cloning a cDNA sequence of the coding gene, which comprises a forward primer and a reverse primer, wherein the forward primer has a nucleotide sequence shown as SEQ ID NO.3 in a sequence table; the reverse primer has a nucleotide sequence shown as SEQ ID NO.4 in the sequence table. The source of the primer pair is not particularly limited, and it may be synthesized by biosynthetic companies well known in the art. In the embodiment of the invention, the primer pair is synthesized by Nanjing Biotechnology Ltd.

The invention provides application of sodium and hydrogen antiporter PbrNHX2, the coding gene or the coding gene pair cloned by the primer pair in improving the salt tolerance of plants.

In the present invention, the application preferably comprises the steps of: the sodium and hydrogen antiporter PbrNHX2 or the coding gene cloned by the primer pair in the birch pear are recombined and expressed in plants, and the obtained recombined plants have salt tolerance (see figure 1).

In the present invention, the cloning procedure is preferably pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 90s, and extension at 72 ℃ for 90s, and 35 cycles of extension at 72 ℃ for 10min after the completion of the cycles. The coding gene is preferably transformed into plants in the form of a pMV-PbrNHX2 recombinant vector. The construction method of the pMV-PbrNHX2 recombinant vector is not particularly limited in the present invention, and any construction method known in the art may be employed.

The method for the recombinant expression of the coding gene or the coding gene cloned by the primer pair in the plant preferably adopts an agrobacterium-mediated genetic transformation method. The method for said agrobacterium-mediated genetic transformation is not particularly limited in the present invention, and conventional methods well known in the art may be used. The plant preferably comprises tobacco or autumn pears.

In the present invention, before the salt tolerance of the recombinant plant is tested, the screening of the positive transgenic plant is preferably included. The screening method of the positive transgenic plant is preferably carried out by adopting a PCR amplification method. During screening, a forward primer of the primer for PCR amplification has a nucleotide sequence shown as SEQ ID NO.3 in a sequence table; the primer reverse primer for PCR amplification has a nucleotide sequence shown as SEQ ID NO.4 in a sequence table. The reaction procedure for the PCR amplification is as follows:

step (ii) of	94℃	58℃	Extension temperature		4℃	Number of cycles
							Step
1	3min				1
						Step 2	30s	30s	55s(58℃)		35
			90s(60℃)		1
						Step 3			10min(72℃)
Step 4				30min

The PCR reaction system for PCR amplification is as follows:

after the PCR amplification is finished, if the plant line to be detected can amplify a fragment (1626bp) with an expected size, the result shows that the plant line to be detected is a positive transgenic line.

In the invention, the obtained positive transgenic plant is subjected to salt treatment, and the phenotype, physiological index and active oxygen of the positive transgenic plant obtained after the salt treatment are measured to verify the salt tolerance function of the positive transgenic plant. The determination method of the active oxygen is preferably to use DAB and NBT histochemical staining method to perform plant leaf H₂O₂And O^2-Accumulation was analyzed (by the shade and extent of staining) and visually observed and photographed. The active oxygen includes hydrogen peroxide (H) respectively₂O₂) And superoxide anion (O)^2-). By measuring the change of the active oxygen content, the following results are found: before salt stress treatment, the positive transgenic line and a control group have no obvious difference in color, after 30d of salt stress, the leaves dyed by DAB have brown leaf areas obviously larger than those of the transgenic lines and are darker in color, and the leaves dyed by NBT have blue color and larger area than those of the transgenic lines, which shows that the positive transgenic line has lower ROS (H) than the wild type under the salt stress₂O₂And O^2-) Accumulation, thereby ensuring less cell damage.

In the present invention, the conductivity measurement and the chlorophyll extraction and measurement method are not particularly limited, and those well known in the art may be used. Analyzing the measurement results of the conductivity and the chlorophyll, and finding that: the conductivity of the positive plant seedling is obviously reduced compared with that of a Wild Type (WT), the chlorophyll content is obviously increased compared with that of the wild type, and the survival rate is increased by 116% -129% compared with that of the wild type, which indicates that the positive plant seedling has stronger ROS resistance than the wild type.

In the invention, the salt concentration of the plant salt tolerance is preferably not higher than 1000 mmol/L. The salt-tolerant salt of the plant preferably comprises Na salt or K salt. The plant preferably comprises tobacco or autumn pears.

The sodium, potassium and hydrogen antiporter PbrNHX2 in Pyrus pyrifolia and its application in improving the salt tolerance of plants provided by the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.

Example 1

Cloning of full-length cDNA of birchleaf pear PbNHX2 gene

In the earlier stage of the subject group, an efficient yeast expression system is adopted, a sodium-hydrogen antiporter protein PbrNHX2 is screened from the birch, a primer is designed according to the sequence of a PbrNHX2 gene and a primer premier 5.0, and the full length of the birch is amplified by an RT-PCR method. The detailed steps are as follows: mu.g of birch-leaf pear RNA was treated with 1U of DNase I at 37 ℃ for 30min and immediately placed on ice, and 1. mu.l of 50mM EDTA was added and treated at 65 ℃ for 10min and immediately placed on ice. First strand cDNA synthesis was performed according to the manual of the TOYOBO reverse transcription kit. The resulting first strand cDNA was used for amplification of the PbrNHX2 gene. PCR was performed as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 90s, and extension at 72 ℃ for 90s, and 35 cycles of extension at 72 ℃ for 10min after the completion of the cycles. After the amplification is finished, a PCR product with a single band is generated, and after electrophoresis of 1% agarose gel, a specific target band is recovered by using a gel recovery kit according to the extraction steps of the instructions.

The recovered and purified solution was ligated to pMD19-T vector at a molar ratio of gene to vector of 3:1 in a ligation system in a total reaction volume of 10. mu.l, 5. mu.l buffer, 4.5. mu.l of PCR-purified product, 0.5. mu.l vector, overnight at 16 ℃ and transformed into E.coli competent DH5 α by heat shock method, and PCR-verified and sequenced with the target gene sequence primers (done by Shanghai Yiwei Jiji Co.).

Bioinformatics analysis of cDNA sequence showed that the full length of PbNHX2 gene was 1626bp, including coding reading frame coding 542 amino acids, isoelectric point was 8.49, and predicted molecular weight was 60.09 KDa. BLASTX analysis of the sequence with known (all published literature and database) plant sequences with high homology. Amino acid sequence comparison shows that the deduced amino acid sequence of PbrNHX2 gene is highly homologous with the reported populus euphratica PeNHX2, Begonia fuciformis MzNHX1, apple MdNHHX 2 and MdHX 1, maize ZmNHX2, Arabidopsis thaliana AtNHX1 and AtNHX2 sequences. ExPASy analysis indicated that the encoded amino acid PbrNHX2 has a tonoplast localization signal. According to the evolutionary tree constructed by amino acid multiple sequence alignment, the pear PbrNHX2 family protein has the closest relationship with apple MdHX 2, and MdHX 1 times has the furthest evolutionary distance with Arabidopsis AtNHX5 and AtNHX 6.

Example 2

qRT-PCR analysis of PbrNHX2 Gene and subcellular localization of PbrNHX2 Gene under different stress conditions

In order to analyze the response pattern of PbrNHX2 gene in Du pear to low temperature, high salt and dehydration, the expression pattern of PbrNHX2 gene was analyzed using Real-timePCR technique. RNA was extracted by CTAB method, and first strand DNA was synthesized according to the manual of TOYOBO reverse transcription kit. In a 20. mu.l reaction: mu.l of 2 Xmix, 0.1. mu.l of cDNA, 5. mu.l of primers (ubiqutin as internal reference primers (SEQ ID NO.5 and SEQ ID NO.6), length 208), 4.9. mu.l of water. The procedure for quantitative PCR is shown in table 1:

TABLE 1 quantitative PCR procedure

The results are shown in FIG. 2. FIG. 2 is a schematic diagram of the expression of the PbNHX2 encoding gene of the invention under salt, dehydration and low temperature stress, wherein FIG. 2-A is a sample of the encoding gene at a corresponding time point under the treatment of Du pear seedlings (not transgenic) with 200mM NaCl, and the relative expression of the gene of the invention is analyzed by real-time quantitative PCR; FIG. 2-B is an expression pattern of dehydration of birch pear seedlings at room temperature at various time points; FIG. 2-C shows the relative expression of the coding gene in the seedlings of Pyrus betulaefolia after the corresponding time point sampling and real-time quantitative PCR analysis at 4 ℃. As can be seen from FIG. 2-A, the expression level of the coding gene showed an increasing tendency within 12 hours as the treatment time was prolonged, and the expression level began to decrease after 12 hours. This shows that the coding gene is affected by salt stress, so that the salt-resistant mechanism is responded, and the salt-resistant function of the pyrus betulaefolia is realized.

Example 3

Subcellular localization of the Gene encoding PbrNHX2

A nucleotide sequence of a coding gene of PbrNHX2 and a pJIT166-GFP vector map are respectively added with SalI and BamHI restriction sites before and after the gene sequence, a target gene extraction plasmid with a correct sequencing result is used as a template, primers (SEQ ID NO.7 and SEQ ID NO.8) added with the restriction sites are used for amplification, the used PCR program comprises the steps of pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 1min, extension at 72 ℃ for 1min for 30s, 35 cycles, extension at 72 ℃ for 10min, removal of a termination codon TAG from the gene 3', fusion of the gene and GFP, recovery of a target band by using a gel kit after electrophoresis of the PCR product by 1% agarose gel, recovery of the purified amplification fragment and cloning into a pMD19-T vector, transformation into Escherichia coli competent DH5 α, detection of the transformed gene by PCR, identification of positive bacteria liquid, extraction of the bacteria liquid with correct PCR identification, sequencing results named as a correct bacteria liquid and a pJIT166-GFP plasmid, recovery of the plasmid of the two plasmid DNA, restriction enzymes, ligation of the PbrNHX 2-GFP, recovery of the plasmid, digestion products by a PbrNHX vector, ligation of the plasmid, digestion of the plasmid, recovery of the plasmid 4, and the plasmid 35-GFP, and the plasmid DNA, and ligation of the plasmid.

Reagents and methods for transformation using protoplasts, the reagents used: arabidopsis protoplast enzymatic hydrolysate (prepared one day and night in advance and stored at 4 ℃): 0.4M mannitol; 1% cellulase; 0.1% of an eductase; 5mM MES; 0.1% pectinase.

The method comprises the following steps: heating in 55 deg.C water bath for 10min to inactivate various proteases and enhance solubility of cellulase etc., cooling to room temperature, and adding the following two reagents: 0.15% BSA and 8mM CaCl₂(ii) a And (3) MaMg solution: 0.4M mannitol; 0.1% MgCl₂(ii) a 4mM MES; w5 solution (ready for use, attention was paid to the addition of the carboxymethyl antibiotic during culture): 154mM NaCl; 125mM CaCl₂(ii) a 5mM KCl; 2mM MES; PEG solution (40%, v/v): 4g PEG 4000(sigma-Fluka, #81240) was dissolved in 4mL water; 0.72868g mannitol into 3mL water, 1mL 1M CaCl₂If not dissolved, the solution can be made soluble at 65 ℃. Mixing the two, after completely mixing, fixing the volume to 10mL, and finally adjusting the pH value to 7.5-8.0 by using KOH.

The results are shown in FIG. 3. FIG. 3 is the subcellular localization of the gene encoding PbrNHX2, where FIG. 3-A is the image of the GFP gene (control) in the bright field (right panel) and dark field (left panel), and the image (middle) is the image after the two are superimposed; FIG. 3-B, the PbrNHX2 gene was imaged in bright field (right panel) and dark field (left panel), and the images were superimposed on each other (middle panel). According to the cell location map, the protein PbrNHX2 can be located at the vacuolar membrane.

Example 4

Yeast strain AXT3(ean1:: HIS3:: ena4, nha1:: LEU2, nhx1:: TRP1) (Na)⁺Endogenous genes of transporter are knocked out) to study the anaplerotic effect of NHX genes on salt tolerance. The expression vector used was pYES2 (Invitrigen). Empty pYES2 vector and the vector carrying PbrNHX2 gene were transformed into the mutant strain by chemical transformation and then screened using SC/-Ura solid medium, respectively. Selection of Positive transformants in liquid SC/-Ura Medium, Yeast transformants with the transformation pYES2, pYES 2:PbrNHX 2 and pYES 2:PbrNHX 2 in logarithmic growth phase were adjusted to OD by double distilled water₆₀₀After 1.0, 3. mu.L of yeast stock solution and 10 dilutions were taken¹、10²And 10³Duplicate yeast cultures were spotted onto SC/-Ura selection media containing concentrations of 0, 90, 120, 150mM NaCl, respectively, to observe the growth of different transformants. The results show that: the growth condition of the yeast transformant transformed with the PbrNHX2 coding gene is obviously better than that of the yeast transformant transformed with an empty pYES2 vector, which shows that the coding gene of PbrNHX2 can partially supplement the salt tolerance of the salt-sensitive yeast mutant AXT 3. The salt-containing capability of the compounds in yeast is also tested, and the results show that the salt content of the yeast with the coding gene transferred PbrNHX2 is obviously lower than that of the yeast with the empty pYES2 vector, and the yeast with the K content is⁺The amount is higher than that of the empty vector, indicating that the gene has K uptake⁺Arrange Na⁺Thus proving that the gene encoding PbrNHX2 has a salt-resistant function (see FIG. 4).

Example 5

Genetic transformation of tobacco

1. Construction of plant transformation vectors

According to the multiple cloning site of pMV vector (in the plant binary transformation vector pBI121 for cutting GUS gene) and coding region sequence of PbrNHX2 gene, using primer primier5.0 software to design upstream and downstream PCR primers for amplifying whole coding region of gene (said primer pair is the primer pair for amplifying PbrNHX2 gene cDNA sequence), using clone of PbrNHX2 gene as template to make PCR amplification, annealing temp. is 58 deg.C, PCR reaction system and amplification program are identical to that of PbrNHX2 gene clone, after amplification making double digestion, its total volume is 20. mu.l, in which the purified product of PCR is 10. mu.l, 10 XG 2. mu.l, KpnI and PbI are respectively 1. mu.l, double distilled water is 6. mu.l, and after making double digestion, they are gel-purified and recovered, and its double digestion reaction volume is 20. mu.l, in which the screened pMV vector is 10X 8. times, 10. mu.l, the recombinant vector containing plasmid is obtained by adding into the PCR digestion system, and the plasmid containing plasmid of PbrNHV DNA, and its fragment is recovered, after the PCR amplification is added into the PCR system, and the plasmid containing plasmid of Kpn 5. mu.5. mu.l, DNA, the plasmid containing plasmid, DNA, the plasmid, DNA, its plasmid is recovered, its plasmid is added into the plasmid, its plasmid is added into the plasmid, its plasmid is added into plasmid, its plasmid is added into plasmid, its plasmid is added into.

2. The agrobacterium-mediated tobacco genetic transformation procedure was as follows:

(1) and (3) agrobacterium culture: taking the agrobacterium tumefaciens bacterial liquid stored in an ultra-low temperature refrigerator, streaking on a flat plate added with LB with 50mg/L kanamycin, scraping streaked bacterial plaque, adding the streaked bacterial plaque into a liquid MS basic culture medium, rotating for min at 28 ℃, and carrying out shake culture, wherein the bacterial liquid is subjected to dip dyeing when the concentration of the bacterial liquid reaches OD (0.3-0.8).

(2) Dip dyeing: taking non-transgenic tobacco leaf, cutting into 0.5 × 0.5cm, soaking in the prepared Agrobacterium tumefaciens bacterial solution for 8-10min, and oscillating continuously.

(3) Co-culturing: taking the impregnated tobacco leaves, sucking the bacterial liquid on the tobacco leaves by sterile filter paper, then inoculating the tobacco leaves on a co-culture medium (the back of the leaves faces downwards), and culturing the tobacco leaves for 3 days in dark at 25 ℃.

(4) Screening and culturing: the tobacco leaves after 3d of co-culture are washed once by using a 500mg/L cefuroxime axetil solution, washed 3-5 times by using sterile water and then transferred into a screening culture medium added with 100mg/L kanamycin and 500mg/L cefuroxime axetil.

(5) Rooting culture: when the adventitious bud on the screening culture medium grows to about 1cm, cutting off and transferring the adventitious bud to a Cef rooting culture medium added with 100mg/LKm and 500 mg/L.

(6) Transferring the tobacco seedlings into soil culture: after the transformed seedling grows to full of culture bottles after rooting, taking out the transformed seedling from the rooting culture medium, washing the culture medium on the transformed seedling by using tap water, and planting the transformed seedling in sterilized nutrient soil. The culture medium used for tobacco transformed shoots is shown in Table 2.

TABLE 2 culture medium recipe for tobacco transformation seedlings

3. Screening of transgenic Positive seedlings

Obtaining tobacco with the PbrNHX2 gene according to the method, extracting DNA from each tobacco plant, and designing primer gene inner primers to perform PCR amplification to identify positive seedlings.

3.1 tobacco leaf DNA extraction

(1) Putting a proper amount of young tobacco leaves into a 1.5mL centrifuge tube, adding liquid nitrogen, and fully grinding into powder; then 700. mu.l of a 65 ℃ preheated DNA extraction buffer cetyltriethylammonium bromide (abbreviated as CTAB, formulation: 100mM Tris-HCl (pH8.0), 1.5M NaCl, 50mM EDTA (pH8.0) solution, 1% polyvinylpyrrolidone, 2% (by volume) CTAB, and 65 ℃ water bath were added and sufficiently dissolved for use, and 1 to 4% (by volume) mercaptoethanol was added and mixed well after preheating in 65 ℃ water bath before use.

(2) Bathing at 65 deg.C for 60-90min, taking out at 15min interval, slightly turning upside down, and mixing; centrifuging at 10000g for 10min at normal temperature; collecting supernatant (directly poured into a centrifuge tube), adding 600 μ l chloroform isoamyl alcohol (chloroform to isoamyl alcohol volume ratio is 24: 1), and mixing and extracting for 3 min.

(3) Centrifuging at 10000g for 15 min; taking 450 μ l of supernatant (taking care not to suck protein layer, causing protein contamination), adding 900 μ l-20 deg.C precooled absolute ethanol, 34 μ l 5M NaCl, mixing by inversion, and freezing at-20 deg.C for 30 min.

(4)10000g, centrifuging for 10 min; after the supernatant is discarded, the mixture is washed for 3 times by 1mL of 75% ethanol, centrifuged for one minute in an empty tube, placed on a superclean bench to be blown for half an hour until DNA is colorless and transparent, added with a proper amount of double distilled water, placed in an incubator at 37 ℃ to be dissolved for 40min, and subjected to gel detection.

3.2 Positive transgenic plant detection

PCR amplification is carried out by using primer gene specific primers. The reaction procedures and systems are shown in tables 3 and 4, respectively. PCR is carried out by adopting 35S gene right-side inner primers (SEQ ID NO.9 and SEQ ID NO.10), and the selected transgenic lines have fragments with expected sizes, which indicates that the transgenic lines are positive transgenic lines.

TABLE 3 PCR reaction procedure

Step (ii) of	94℃	58℃	72℃	4℃	Number of cycles
						Step
1	3min				1
						Step 2	30s	30s	55s		35
Step 3			90s		1
						Step 4			10min
Step
	5				30min

TABLE 4 PCR reaction System

Reaction components	Dosage (mu l)
		Template DNA	1
PCRBuffer	2
		dNTDMix(2.5mmol/L)	1.6
Right forward primer	1
		Left reverse primer	1
TaqDNA polymerase (5U)	0.2
		Ribozyme-free water	13.2

Pbrhhxx 2 was expressed in tobacco by agrobacterium-mediated transformation. Through molecular genetic analysis, the transgenic tobacco which obtains a single copy homozygous insertion and stably expresses PbrNHX2 from T1-T2 generations is identified, so that the phenotypic character can be stably inherited. The insertion site analysis proves that the phenotypic change of the transgenic material transformed with PbrNHX2 is not caused by the influence of other genes caused by transgenic operation. Therefore, the transgenic material provides material guarantee for the research of the project. The vector construction is shown in figure 5- (a), the whole flow of the transgenic tobacco is shown in figure 5- (b), the transgenic plant is identified in figure 5- (c) by using RT-PCR (35S primer + gene downstream primer), and TG17 and TG20 are further identified as two overexpression lines by using RT-PCR and Western blotting (figure 5- (d) and figure 5- (e)).

Example 6

Overexpression analysis of transgenic autumn pear plants

Transgenic autumn pear plants were constructed according to the method of example 5. Extracting RNA of 8 transgenic positive seedlings transplanted to live, detecting the complete structure of the seedlings by glue running, determining the concentration of the RNA by using nanodrop (the concentration is all 600ng/ul at 200-. The nucleotide sequence of the Tublin primer is as follows:

tublin forward primer: 5'-TGGGCTTTGCTCCTCTTAC-3' (SEQ ID NO. 11);

tublin reverse primer: 5'-CCTTCGTGCTCATCTTACC-3' (SEQ ID NO. 12).

The brightness of the bands amplified by Tublinn is consistent, which indicates that the concentration of the reverse transcription cDNA is the same, then the target band is amplified by using a PbrNHX2 specific primer as a template, and the nucleotide sequence of the PbrNHX2 primer is as follows:

a forward primer: 5'-ATGGCTGTTGCACATTTGAGCATGATG-3' (SEQ ID NO. 3);

reverse primer: 5'-TTGCCACTGAACGTTGTTGTCCCGTTC-3' (SEQ ID NO. 4).

According to the brightness of a target band amplified by the PbrNHX2 specific primer, the expression quantity of the PbrNHX2 gene in positive transgenic pears can be judged, 2,6 and 8 with high brightness are selected, namely three over-expression strains with high expression quantity are named as OE2, OE6 and OE8 are used as independent transgenic strains, and then the individual transgenic strains are respectively propagated.

The results are shown in FIG. 6. FIG. 6 is a schematic diagram of the transformation of autumn pear with PbrNHX2 and the regeneration process of the plant, wherein FIG. 6-A is the photograph after transformation; FIG. 6-B is a material grown on screening media for 30 days; FIG. 6-C is the induction of rooting by the regenerated shoots; FIG. 6-D is a photograph of transgenic plants grown in soil for 30 days; FIG. 6-E is a T0 generation transgenic plant after PCR identification of tobacco using gene specific primers, where M: marker, +: plasmid, -: wild type plant, 1-8: a transgenic line; FIG. 6-F shows the real-time quantitative PCR analysis of the expression level of PbrNHX2 encoding gene in different transgenic lines of autumn pear. FIG. 6 illustrates overexpression of the PbrNHX2 gene in pear-transformed PbrNHX2 gene plants.

Example 7

PbrNHX2 transgenic resistant plant resistance identification

1. Salt resistance analysis of transgenic tobacco plants

To identify whether PbrNHX2 transgenic tobacco is associated with salt stress resistance, control and transgenic lines were subjected to short-term salt stress and long-term salt stress. The tobacco seeds and Wild Type (WT) seeds of the PbrNHX2 transgenic line (TG17, TG20) received from the same batch are respectively sown on an MS screening culture medium and a commonly used MS non-resistant culture medium after sterilization treatment, and the seeds are transplanted into soil for culture after about 3 days after germination. And (3) performing salt treatment on transgenic plants with different seedling ages, observing the treated phenotypes, counting the survival rate, and measuring the conductivity, chlorophyll and the like.

FIG. 7 shows the phenotype and physiological index measurements of the gene line encoding PbrNHX2 and before and after Wild Type (WT) sodium chloride treatment, where FIG. 7-A shows the phenotype of 45 day old tobacco plants before 20 days of 300mM sodium chloride treatment; FIG. 7-B is the phenotype of 45 day old tobacco plants after 20 days of 300mM sodium chloride treatment; FIG. 7-C is a phenotype of 30 day old tobacco plants 4 days prior to treatment with 200mM salt solution; FIG. 7-D is a phenotype of 30-day-old tobacco plants treated with 200mM salt solution for 4 days; FIG. 7-E is a statistical result of survival rate of 30-day-old tobacco plants treated with 200mM salt solution for 4 days, FIG. 7-F is a result of conductivity measurement of 30-day-old tobacco plants treated with 200mM salt solution for 4 days, FIG. 7-G is a phenotype of 45-day-old tobacco plants treated with 300mM sodium chloride for 25 days, FIG. 7-H is a chlorophyll measurement of 45-day-old tobacco plants treated with 300mM sodium chloride for 25 days, and FIG. 7-I is a chlorophyll extraction of 30-day-old tobacco plants treated with 200mM salt solution for 4 days. The measurement of the indexes is an important measurement index for evaluating the salt resistance of the transgenic plant, and the PbrNHX2 gene can be obtained in figure 7 to enhance the salt resistance of the transgenic plant.

2. Salt resistance analysis of transgenic autumn pears

To identify whether PbrNHX2 transgenic autumn pears were associated with salt stress resistance, control and transgenic lines were subjected to salt stress. The phenotypic and physiological indices of PbrNHX2 transgenic autumn pear lines (OE2, OE6 and OE8) and wild type plants (WT) were assessed 15 days after 500mM sodium chloride.

The results are shown in FIG. 8. FIG. 8 shows the results of the phenotypic and physiological index measurements of PbrNHX2 transgenic autumn pear strains (OE2, OE6 and OE8) and wild type plants (WT) in example 7 after 15 days of pouring 500mM sodium chloride, wherein FIG. 8-A shows the phenotype after 15 days of pouring 500mM sodium chloride, FIG. 8-B shows the conductivity measurement after 15 days of pouring 500mM sodium chloride, and FIGS. 8-C and 8-D show the chlorophyll measurement (FIG. 8-D) and chlorophyll extraction (FIG. 8-C) after 15 days of pouring 500mM sodium chloride.

3. Histochemical stain analysis H₂O₂And O^2-Accumulation of

In the transgenic lines (tobacco/autumn pears), the lower conductivity and high survival rate indicate that they may have a stronger ability to resist ROS than WT. It is necessary to identify the amount of ROS accumulated in the plant. Staining of plant leaves with DAB and NBT histochemical staining for detection of hydrogen peroxide (H)₂O₂) And superoxide anion (O)^2-) The content of (a).

The results are shown in FIG. 9. FIG. 9 shows histochemical staining analysis H of tobacco transformed with PbrNHX2 gene and autumn pear₂O₂And O^2-Accumulation, FIGS. 9-A and 9-B show that 45-day-old tobacco plants were subjected to reactive oxygen histochemical staining with nitrotetrazolium after and after 300mM sodium chloride treatment for 15 days for untransformed plants and two transgenic lines(NBT) and Diaminobenzidine (DAB) separately for H₂O₂(FIG. 9-A) and O^2-(FIG. 9-B) staining, FIG. 9-C shows cell death chromosomes after transgenic tobacco salt stress treatment, and FIGS. 9-D and 9-E show that 30-day-old Kazuki pear plants were stained with Nitrotetrazole (NBT) and Diaminobenzidine (DAB) for H + cells before and after 500mM sodium chloride treatment for 15D for untransformed plants and two transgenic lines₂O₂(FIG. 9-D) and O^2-(FIG. 9-E) staining was performed, and FIG. 9-F shows cell death chromosomes after salt stress treatment of transgenic autumn pears. As in fig. 9-a and 9-B), there was no significant difference in color between the transgenic lines and the control before salt stress treatment. As shown in FIGS. 9-A and 9-B, after salt stress for 30d, in the leaves stained with DAB, the leaves of wild type were significantly larger in size and darker in color than those of the transgenic lines, and in the leaves stained with NBT, the wild type was darker in color and larger in size than those of the transgenic lines. These evidence indicate that transgenic lines have lower ROS (H) under salt stress than wild type₂O₂And O^2-) And (4) accumulating. Similar results were obtained in transgenic autumn pear plants (as shown in 9-D and FIG. 9-E).

4. Comprehensive analysis shows that the function of the gene is identified after the gene is transferred into tobacco and autumn pears, and the salt resistance of a transgenic over-expression strain is greatly improved compared with that of a control wild type. Hydrogen peroxide (H) in transgenic tobacco₂O₂) And superoxide anion (O)^2-) The content activity of the compound is lower than that of a wild type, the active oxygen residue in a plant body is lower, and the cell damage is smaller. These results show that the over-expressed PbrNHX2 gene can effectively enhance the active oxygen scavenging ability of transgenic plants, thereby improving the salt tolerance of the plants.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Nanjing university of agriculture

<120> sodium and hydrogen antiporter PbrNHX2 in pyrus betulaefolia and application thereof in improving salt tolerance of plants

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Trp Val Asn Glu Ser Ile Thr Ala Leu Leu Ile Gly Leu Cys Thr Gly

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Val Val Ile Leu Leu Ile Ser Arg Gly Arg Ser Ser His Leu Leu Val

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Phe Ser Glu Asp Leu Phe Phe Ile Tyr Leu Leu Pro Pro Ile Ile Phe

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Asn Ala Gly Phe Gln Val Lys Lys Lys Gln Phe Phe Val Asn Phe Met

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Thr Ile Val Met Phe Gly Ala Ile Gly Thr Leu Val Ser Cys Thr Ile

115 120 125

Ile Ser Leu Gly Ala Thr Gln Phe Phe Lys Lys Leu Asp Ile Gly Thr

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Leu Glu Leu Gly Asp Phe Leu Ala Ile Gly Ala Ile Phe Ala Ala Thr

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Asp Ser Val Cys Thr Leu Gln Val Leu Asn Gln Asp Glu Thr Pro Leu

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Leu Tyr Ser Leu Val Phe Gly Glu Gly Val Val Asn Asp Ala Thr Ser

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Val Val Leu Phe Asn Ala Ile Gln Ser Phe Asp Leu Thr His Ile Asp

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Ser Ser Ile Ala Leu His Phe Met Gly Asn Phe Leu Tyr Leu Phe Phe

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Ala Ser Thr Met Leu Gly Val Phe Ala Gly Leu Leu Ser Ala Tyr Ile

225 230 235 240

Ile Lys Lys Leu Tyr Phe Gly Ser His Ser Thr Asp Arg Glu Val Ala

245 250 255

Leu Met Met Leu Met Ala Tyr Leu Ser Tyr Ile Leu Ala Glu Leu Phe

260 265 270

Tyr Leu Ser Gly Ile Leu Thr Val Phe Phe Cys Gly Ile Val Met Ser

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His Tyr Thr Trp His Asn Val Thr Glu Ser Ser Arg Thr Thr Lys His

290 295 300

Ala Phe Ala Thr Leu Ser Phe Val Ala Val Glu Thr Phe Ile Phe Leu

305 310 315 320

Tyr Val Gly Met Asp Ala Leu Asp Ile Glu Lys Trp Arg Phe Val Ser

325 330 335

Asp Ser Pro Gly Thr Ser Val Ala Val Ser Ser Ile Leu Leu Gly Leu

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Val Met Leu Gly Arg Ala Ala Phe Val Phe Pro Leu Ser Phe Leu Ser

355 360 365

Asn Leu Thr Lys Lys Asn Gln Arg Asp Lys Ile Ser Leu Arg Gln Gln

370 375 380

Val Ile Ile Trp Trp Ala Gly Leu Met Arg Gly Ala Val Ser Met Ala

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Leu Ala Tyr Asn Gln Phe Thr Arg Ser Gly His Thr Gln Leu Arg Ala

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Asn Ala Ile Met Ile Thr Ser Thr Ile Thr Val Val Leu Val Ser Thr

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Val Val Phe Gly Leu Met Thr Lys Pro Leu Ile Arg Phe Leu Leu Pro

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Pro Lys Ser Val Ile Val Pro Leu Leu Gly Gln Asp Ser Val Asp Asp

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atggctgttg cacatttgag catgatgatc tcaaagttac aaaatgtatc cacttcggac 60

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cttgttggcc aagatattcg acggccggcc agcttacgcg atcttctgac aactccaacg 1500

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Claims (4)

1. The application of the coding gene of sodium and hydrogen antiporter PbrNHX2 in the pyrus betulaefolia or the coding gene of sodium and hydrogen antiporter PbrNHX2 in the pyrus betulaefolia in improving the salt tolerance of plants is characterized in that the amino acid sequence of the sodium and hydrogen antiporter PbrNHX2 in the pyrus betulaefolia is shown in SEQ ID NO. 1;

the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.

2. The application according to claim 1, characterized in that it comprises the following steps: the sodium and hydrogen antiporter PbrNHX2 in the birch pear as claimed in claim 1 is expressed in plant to obtain recombinant plant with salt tolerance.

3. Use according to claim 1 or 2, wherein the plant comprises tobacco or autumn pears.

4. Use according to claim 1, wherein the salt comprises a Na salt or a K salt.

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