CN110256544B

CN110256544B - Application of NsNHX1 protein and related biological material thereof in cultivation of stress-tolerant poplar

Info

Publication number: CN110256544B
Application number: CN201910462501.4A
Authority: CN
Inventors: 林晓飞; 耿新; 张文波; 陈首业
Original assignee: Inner Mongolia University; Inner Mongolia Agricultural University
Current assignee: Inner Mongolia University; Inner Mongolia Agricultural University
Priority date: 2019-05-30
Filing date: 2019-05-30
Publication date: 2023-05-09
Anticipated expiration: 2039-05-30
Also published as: CN110256544A

Abstract

The invention discloses an application of an NsNHX1 protein and a related biological material thereof in cultivation of stress-tolerant poplar. The invention uses Siberian nitraria Na ⁺ /H ⁺ The inverse transporter gene NsNHX1 is used as a research object, and is transferred into aspen 84K to obtain the trans-NsNHX 1 aspen. Three transformants of N1, N2 and N3 were selected for stress-tolerant function identification. The saline-alkali resistance and the antioxidation capability of the trans-NSNHX 1 aspen under the stress are studied by taking the wild aspen as a control. As a result, the survival rate, biomass, leaf chlorophyll content and water content, plant height and oxidation resistance of the transformed NsNHX1 aspen are obviously higher than those of the wild aspen under the stress treatment condition. The result shows that the NsNHX1 can obviously improve the stress resistance and the oxidation resistance of transgenic poplar, and the NsNHX1 can be used as a salt-tolerant gene to cultivate stress-tolerant poplar varieties.

Description

Application of NsNHX1 protein and related biological material thereof in cultivation of stress-tolerant poplar

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an application of an NsNHX1 protein and a related biological material thereof in cultivation of stress-tolerant poplar.

Background

Plant roots are in direct contact with Na in soil ⁺ And therefore Na of root ⁺ Exocrine and aerial cell pair Na ⁺ Is an important mechanism for determining salt tolerance of plants. Na on vacuole film ⁺ /H ⁺ The antiport protein (NHX 1) willNa in cytoplasm ⁺ Reverse concentration gradient transport to vacuole compartmentalization; na on cytoplasmic membrane ⁺ /H ⁺ The antiport protein (SOS 1) can make Na in cytoplasm ⁺ Reverse transport of high Na to extracellular space ⁺ In the environment. Up to now, developed and applied Na ⁺ /H ⁺ The antiport protein gene is mainly from sweet soil plants such as arabidopsis, tomato and the like, and aims at woody plants, especially halophyte woody plants Na ⁺ /H ⁺ Development and research of the antiport protein gene are relatively deficient. It has been found that the expression level of Thesos 1 is about 8-10 times that of Arabidopsis AtSOs1, and that similar SOS signal pathways are found in sweet potato plant rice and halophyte Thellungiella, suggesting that higher plants may share a set of salt tolerance regulatory mechanisms. Thus, na utilizing halophytes was developed ⁺ /H ⁺ The antiport protein gene has very important significance.

The nitraria (Nirtaria L.) is a fallen leaf bush of the genus nitraria of the family Zygophyllicae, is a halophyte, is mainly distributed in the North to North of China in inner Mongolia, ningxia, gansu, qinghai, xinjiang and other places, and is one of the colonisation species of arid saline-alkali lands. The stems and leaves of the Nitraria plant have thicker cuticle, leaves are embedded by waxy skin and hair, the palisade tissue and vascular bundles are developed, and cell contents are filled with waxy crystals, so that the typical xerophyte structural characteristics lead the plant to play a role in resisting salt damage. The strong membrane protection capability and membrane repair capability under the condition of salt stress, and the high-efficiency intracellular ion compartmentalization and accumulation capability of osmotic adjusting substances endow the Nitraria plant with strong salt tolerance. There are mainly four species in the inner Mongolian region, siberian nitraria (N.silicapall), tangutta nitraria (N.tannagorus Bobr), paulownia (N.sphaerocarpa Maxim) and dentition She Baici (N.roborowskii Kom), wherein Siberian nitraria exhibits greater salt tolerance. Siberian nitraria has extremely important ecological value, and because the Siberian nitraria is subjected to triple stress of salt, alkali and drought in natural habitat, the Siberian nitraria has stronger salt and alkali resistance and drought resistance, and contains rich stress resistance gene resources. Therefore, the salt tolerance mechanism is researched, the salt tolerance related genes are identified and applied to the genetic improvement of the salt tolerance of pasture and agriculture and forestry crops, and the method has very important significance.

Disclosure of Invention

The invention aims to solve the technical problem of how to regulate and control plant stress tolerance.

In order to solve the technical problems, the invention firstly provides a novel application of the NsNHX1 protein.

The invention provides the use of an NsNHX1 protein in any one of the following 1) -10):

1) Regulating and controlling plant stress tolerance;

2) Regulating and controlling the growth and development of plants;

3) Regulating and controlling plant biomass;

4) Regulating plant height;

5) Regulating and controlling the chlorophyll content of the plants;

6) Regulating and controlling the water content of plants;

7) Regulating and controlling the antioxidant capacity of plants;

8) Regulating and controlling the activity of plant superoxide dismutase and/or peroxidase and/or catalase;

9) Regulating and controlling the proline content of plants;

10 Regulating and controlling the content of malondialdehyde in plants;

in the above application, the NsNHX1 protein is a protein as shown in a) or b) or c) or d) below:

a) The amino acid sequence is a protein shown in the sequence 2;

b) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein represented by the sequence 2;

c) The protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2;

d) A protein having 75% or more homology with the amino acid sequence shown in sequence 2 and having the same function.

In order to facilitate purification of the protein of a), a tag as shown in Table 1 may be attached to the amino-or carboxy-terminus of the protein shown in sequence 2 in the sequence listing.

TABLE 1 sequence of tags

Label (Label)	Residues	Sequence(s)
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein of c) above, wherein the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein in the c) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing.

The coding gene of the protein in c) can be obtained by deleting one or more amino acid residues from the DNA sequence shown in the 149 th to 1783 th positions of the sequence 1 and/or carrying out one or more base pair missense mutations and/or linking the coding sequences of the tags shown in the table 1 at the 5 'end and/or the 3' end.

In the above d), the "homology" includes an amino acid sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more homology with the amino acid sequence shown in the sequence 2 of the present invention.

In order to solve the technical problems, the invention also provides a novel application of the biological material related to the NsNHX1 protein.

The invention provides the use of a biological material associated with the NsNHX1 protein in any one of the following 1) -12):

1) Regulating and controlling plant stress tolerance;

2) Regulating and controlling the growth and development of plants;

3) Regulating and controlling plant biomass;

4) Regulating plant height;

5) Regulating and controlling the chlorophyll content of the plants;

6) Regulating and controlling the water content of plants;

7) Regulating and controlling the antioxidant capacity of plants;

9) Regulating and controlling the proline content of plants;

10 Regulating and controlling the content of malondialdehyde in plants;

11 Cultivating a transgenic plant with improved stress tolerance;

12 Plant breeding.

In the above application, the biomaterial is any one of the following A1) to a 12):

a1 Nucleic acid molecules encoding NsNHX1 protein;

a2 An expression cassette comprising A1) said nucleic acid molecule;

a3 A) a recombinant vector comprising the nucleic acid molecule of A1);

a4 A recombinant vector comprising the expression cassette of A2);

a5 A) a recombinant microorganism comprising the nucleic acid molecule of A1);

a6 A) a recombinant microorganism comprising the expression cassette of A2);

a7 A) a recombinant microorganism comprising the recombinant vector of A3);

a8 A) a recombinant microorganism comprising the recombinant vector of A4);

a9 A transgenic plant cell line comprising the nucleic acid molecule of A1);

A10 A transgenic plant cell line comprising the expression cassette of A2);

a11 A transgenic plant cell line comprising the recombinant vector of A3);

a12 A) a transgenic plant cell line comprising the recombinant vector of A4).

In the above application, the nucleic acid molecule of A1) is a gene as shown in the following 1) or 2) or 3):

1) The coding sequence is a cDNA molecule shown in 149 th to 1783 th positions of a sequence 1 or a genome DNA molecule shown in the sequence 1;

2) A cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) and encoding an NsNHX1 protein;

3) Hybridizing under stringent conditions to the nucleotide sequence defined in 1) or 2) and encoding a cDNA molecule or genomic DNA molecule of the NsNHX1 protein.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleotide sequence encoding the NsNHX1 protein of the present invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence encoding the NsNHX1 protein are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the NsNHX1 protein and have the same function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of a protein consisting of the amino acid sequence shown in the coding sequence 2 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.

The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

In the above application, the stringent conditions are hybridization and washing the membrane 2 times at 68℃in a solution of 2 XSSC, 0.1% SDS for 5min each time, and hybridization and washing the membrane 2 times at 68℃in a solution of 0.5 XSSC, 0.1% SDS for 15min each time; alternatively, hybridization and washing of the membrane were performed at 65℃in a solution of 0.1 XSSPE (or 0.1 XSSC) and 0.1% SDS.

In the above applications, the expression cassette containing the nucleic acid molecule encoding the NsNHX1 protein as described in A2) refers to a DNA capable of expressing the NsNHX1 protein in a host cell, and the DNA may include not only a promoter for initiating transcription of NsNHX1 but also a terminator for terminating transcription of NsNHX 1. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: a constitutive promoter; tissue, organ and development specific promoters and inducible promoters.

Recombinant vectors containing the NsNHX1 gene expression cassette can be constructed using existing expression vectors. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Co.), etc. The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal may direct the addition of polyadenylation to the 3 'end of the mRNA precursor and may function similarly to the 3' transcribed untranslated regions of Agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase gene Nos) and plant genes (e.g., soybean storage protein genes). When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. To facilitate identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to the herbicide phosphinothricin, hph gene conferring resistance to antibiotic hygromycin, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or chemical marker genes, etc. (such as herbicide resistance genes), mannose-6-phosphate isomerase gene providing mannose metabolization ability, etc. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

In the above applications, the vector may be a plasmid, cosmid, phage or viral vector. The plasmid may be pBI101-35:: gus-Hm.

In the above application, the microorganism may be a yeast, a bacterium, an alga or a fungus, such as Agrobacterium. The agrobacterium may be GV3101.

In the above applications, none of the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs include propagation material.

In the above application, the stress tolerance may be salt tolerance and/or alkali tolerance.

The saidRegulating plant stress tolerance to raise plant stress tolerance. The concrete steps are as follows: under salt or alkali stress, the transgenic NsNHX1 plants have increased survival, increased biomass or root biomass, increased chlorophyll content in leaves, increased water content in leaves, increased plant height, increased antioxidant capacity, increased superoxide dismutase and/or peroxidase and/or catalase activity in leaves, increased proline content in leaves, decreased malondialdehyde content in leaves, as compared to the recipient plants. The salt is specifically NaCl, and the NaCl concentration can be 50mM, 100mM or 150mM; the base may be NaHCO ₃ The method comprises the steps of carrying out a first treatment on the surface of the The NaHCO ₃ The concentration may be in particular 100mM or 200mM or 300mM.

The regulation and control of plant growth and development is to promote plant growth and development or promote plant root growth and development.

The regulation of plant biomass is to improve plant biomass; the biomass may be root biomass or plant biomass.

The plant height is regulated to improve the plant height.

The regulation of the chlorophyll content of the plant is to improve the chlorophyll content in the plant leaves.

The regulation of the water content of the plant is to increase the water content in the plant leaves. The water content is the relative water content.

The regulation of the plant oxidation resistance is to improve the plant oxidation resistance. The concrete steps are as follows: under salt stress conditions, superoxide dismutase and/or peroxidase and/or catalase activities in leaves of the transgenic NsNHX1 plant are increased, proline content is increased, and malondialdehyde content is reduced, as compared to the recipient plant.

The regulation of the activity of the plant superoxide dismutase and/or peroxidase and/or catalase is to increase the activity of the superoxide dismutase and/or peroxidase and/or catalase in the plant leaves.

The regulation of the proline content in the plant leaves is to increase the proline content in the plant leaves.

The control of the malondialdehyde content of the plants reduces the malondialdehyde content in the plant leaves.

In order to solve the technical problems, the invention finally provides a method for cultivating transgenic plants with improved stress tolerance.

The method for cultivating transgenic plants with improved stress tolerance comprises the steps of improving the expression quantity and/or activity of NsNHX1 protein in a receptor plant to obtain transgenic plants; the transgenic plant is stress tolerant higher than the recipient plant.

Further, the stress resistance is salt resistance and/or alkali resistance.

The transgenic plant has stress tolerance higher than that of the recipient plant, and is specifically expressed in: the survival rate of the transgenic plant is higher than that of the acceptor plant under the stress of salt or alkali; and/or the biomass or root biomass of the transgenic plant is higher than that of the recipient plant; and/or, chlorophyll content in leaves of the transgenic plant is higher than that of the recipient plant; the water content (relative water content) in the leaves of the transgenic plant is greater than that of the recipient plant; the transgenic plant has a higher plant height than the recipient plant; and/or the transgenic plant has a higher antioxidant capacity than the recipient plant; and/or the superoxide dismutase and/or peroxidase and/or catalase activity in the transgenic plant leaf is higher than in the recipient plant; and/or, the proline content in leaves of the transgenic plant is higher than that of the recipient plant; and/or the malondialdehyde content in leaves of the transgenic plant is lower than in the recipient plant. The salt is specifically NaCl, and the NaCl concentration can be 50mM, 100mM or 150mM; the base may be NaHCO ₃ The method comprises the steps of carrying out a first treatment on the surface of the The NaHCO ₃ The concentration may be in particular 100mM or 200mM or 300mM.

Further, the method for increasing the expression level and/or activity of the NsNHX1 protein in the recipient plant is to overexpress the NsNHX1 protein in the recipient plant;

the over-expression method is to introduce the coding gene of the NsNHX1 protein into a receptor plant. The coding gene of the NsNHX1 protein can be specifically obtained by transforming plant cells or tissues by using Ti plasmid, ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, agrobacterium-mediated and other conventional biological methods, and culturing the transformed plant tissues into plants.

The nucleotide sequence of the coding gene of the NsNHX1 protein is a DNA molecule shown in 149-1783 of the sequence 1.

In a specific embodiment of the invention, the coding gene of the NsNHX1 protein is introduced into a recipient plant through a recombinant vector pBI101-NsNHX1, wherein the recombinant vector pBI101-NsNHX1 is obtained by replacing a fragment between XbaI and SacI cleavage sites of a pBI101-35:: gus-Hm vector with a DNA fragment shown in 149 th to 1783 th of the sequence 1, and keeping other sequences of the pBI101-35:: gus-Hm vector unchanged.

In the above method, the transgenic plant is understood to include not only the first generation transgenic plant obtained by transforming the NsNHX1 gene into a recipient plant, but also the progeny thereof. For transgenic plants, the gene may be propagated in that species, and may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, calli, whole plants and cells.

In the above method or application, the plant is a monocot or dicot. Further, the monocot plant may be a poplar; still further, the poplar may be aspen. In one embodiment of the present invention, the Populus tomentosa may be Populus tomentosa 84K (Populus alba× Populus glandulose).

The invention uses Siberian nitraria Na ⁺ /H ⁺ The inverse transporter gene NsNHX1 was used as a subject, and transferred into Populus alba X Populus glandulose to obtain NsNHX 1-transferred Populus alba. Three transformants of N1, N2 and N3 were selected for stress-tolerant function identification. The saline-alkali resistance and the antioxidation capability of the trans-NSNHX 1 aspen under the stress are studied by taking the wild aspen as a control. As a result, the survival rate, biomass, leaf chlorophyll content and water content, plant height and oxidation resistance of the transformed NSNHX1 aspen are obviously higher than those of the wild aspen under the stress condition. The result shows that the NsNHX1 can obviously improve the stress resistance and the oxidation resistance of transgenic poplar, and the NsNHX1 can be applied to cultivation of stress-resistant poplar varieties.

Drawings

FIG. 1 shows the amplification of the NsNHX1 ORF.

FIG. 2 is a double restriction identification of recombinant vector pBI101-NsNHX 1.

FIG. 3 is a genetic transformation of populus tomentosa. (A) cutting and preculturing of the explants; (B) dip dyeing; (C) differentiation culture; (D) screening; (E) rooting culture; and (F) transplanting.

FIG. 4 is a PCR identification of NsNHX 1-transformed aspen. M:200bp DNA Ladder; +: recombinant plasmid pBI101-NsNHX1; -: wild aspen; n1, N2, N3: transferring NsNHX1 aspen.

FIG. 5 is an RT-PCR analysis of the transformed NsNHX1 aspen. WT: wild aspen; n1, N2, N3: transferring NsNHX1 aspen.

FIG. 6 is a comparison of growth of wild type aspen and three trans-NSNHX 1 aspen strains under salt stress conditions. (A) Plants were grown in P5 medium containing 0mM, 50mM, 100mM, 150mM and 200mM NaCl for 2 weeks; (B) comparison of root growth; (C) Plants were grown in P5 medium containing 0mM, 50mM, 100mM, 150mM and 200mM NaCl for 2 weeks; (D) Biomass of roots of wild aspen and trans-NsNHX 1 aspen lines; (E) Survival rates of wild aspen and ns nhx 1-transformed aspen. WT: wild aspen; n1, N2, N3: transferring NsNHX1 aspen. Error bars represent Standard Deviation (SD) from three independent biological replicates; the different lowercase letters of the label represent the difference in significance between each set of samples (P < 0.05), respectively

FIG. 7 shows propagation of populus tomentosa. (a) cutting seedlings into a culture medium; (B) transplanting the seedlings into the soil; (C) covering a preservative film; (D) preparing seedlings subjected to salt stress.

FIG. 8 is a stress tolerance analysis of NsNHX 1-transformed aspen. (a) stress treatment; (B) transferring chlorophyll content in the leaves of the NsNHX1 aspen; error bars represent Standard Deviation (SD) from three independent biological replicates; the different lowercase letters of the label represent the significant differences (P < 0.05) between each set of samples, respectively. WT: wild aspen; n1, N2, N3: transferring NsNHX1 aspen.

FIG. 9 is a phenotypic analysis of wild type aspen and trans-NSNHX 1 aspen under NaCl stress. (A) Influence of salt stress on wild aspen and transformed NsNHX1 aspen whole plant (scale bar=10 cm); (B) Influence of salt stress on wild aspen and transformed NsNHX1 aspen leaves.

FIG. 10 is a stress tolerance analysis of NsNHX 1-transformed aspen. (a) biomass; (B) plant height; (C) leaf chlorophyll content; (D) relative blade moisture content; (E) SOD activity; (F) POD activity; (G) CAT activity; (H) proline content; (I) MDA content. WT: wild aspen; n1, N2, N3: transferring NsNHX1 aspen. Error bars represent Standard Deviation (SD) from three independent biological replicates; the different lowercase letters of the label represent the significant differences (P < 0.05) between each set of samples, respectively.

Detailed Description

The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

Cultivation conditions of the plant material Populus tomentosa 84K (Populus alba. Times. Populus glandulose) sterile tissue culture seedlings referred to in the following examples: the temperature is 24+/-2 ℃, and the illumination intensity is 200 mu mol.m ^-2 ·s ^-1 The illumination period is 16h illumination/8 h darkness.

The pBI101-35 of the following examples is described in the literature "WANG L, MA Y K, LIN N, et al isolation and characterization of a tonoplast Na" Gus-Hm vector ⁺ /H ⁺ antiporter from the halophyte Nitraria sibirica[J]BIOLOGIA PLANTARUM,2016,60 (1): 113-122,2016", which is publicly available from the applicant, is used only for repeated experiments related to the invention and is not used for other purposes.

The medium formulations referred to in the examples below were as follows:

preculture medium P1: MS (4.33 g/L) +6-BA (0.5 mg/L) +NAA (0.05 mg/L) +Sucrose (30 g/L) +Agar (0.7%).

Co-culture medium P2: MS (4.33 g/L) +6-BA (1.0 mg/L) +NAA (0.1 mg/L) +Sucross (30 g/L) +Agar (0.7%) +AS (200. Mu. Mol/mL).

Differentiation medium P3: MS (4.33 g/L) +6-BA (1.0 mg/L) +NAA (0.1 mg/L) +Sucross (30 g/L) +agar (0.7%) +Cef (300 mg/L).

Screening medium P4: MS (4.33 g/L) +6-BA (1.0 mg/L) +NAA (0.1 mg/L) +Sucross (30 g/L) +Agar (0.7%) +Cef (200 mg/L) +Kan (50 mg/L) or Gent (40 mg/L).

Rooting medium P5:1/2MS (2.17 g/L) +IBA (0.4 mg/L) +Sucross (30 g/L) +Agar (0.7%).

Both the Hoagland medium (Hoagland nutrient solution) and the MS medium are products of Phytotechnology Laboratories (https:// phytotechnab. Com).

Siberian nitraria Na in the following examples ⁺ /H ⁺ The sequence of the antiport protein gene (called NsNHX1 gene for short) is shown as a sequence 1, and comprises an open reading frame of 1635bp (149 th to 1783 th positions of the sequence 1), and Siberian nitraria Na encoded by the NsNHX1 gene ⁺ /H ⁺ The amino acid sequence of the antiport protein (abbreviated as NsNHX1 protein) is shown as a sequence 2.

Example 1 obtaining of transgenic NsNHX1 aspen and stress tolerance analysis thereof

1. Obtaining of trans-NsNHX 1 aspen

1. Construction of recombinant vectors

(1) Amplification of target Gene

According to Siberian nitraria Na ⁺ /H ⁺ A pair of primers (NHX-pBI-F: 5' -GC) was designed for the sequence of the antiport protein gene (abbreviated as NsNHX 1) (Gene sequence accession number: AB 859847) TCTAGAATGGATCAATTAAGTT-3',5' end includes an XbaI recognition sequence; NHX-pBI-R:5' -CGAGCTCTCACTGCCATTGGGGGAT-3',5' end comprises SacI recognition sequence), using RNA of Siberian nitraria Nitraria sibirica Pall as a template, adopting NHX-pBI-F and NHX-pBI-R primers, and amplifying the NsNHX1 ORF region by using TaKaRa RNA PCR Kit (AMV) Ver.3.0 kit to obtain a PCR product. The reaction conditions are as follows: 94 ℃ for 3min;94℃for 30s,52℃for 30s,72℃for 1min,35cycles; and at 72℃for 5min. The result of electrophoresis of the PCR products is shown in FIG. 1.

(2) Ligation of the target Gene and expression vector

Gus-Hm and the PCR products are respectively subjected to double digestion by using XbaI and SacI, and then are connected by using T4 ligase to obtain the recombinant vector pBI101-NsNHX1. And sequencing and enzyme digestion identification are carried out on the recombinant vector pBI101-NsNHX1.

Sequencing results showed that: the recombinant vector pBI101-NsNHX1 is obtained by replacing a fragment between XbaI and SacI cleavage sites of a pBI 101-35:Gus-Hm vector with DNA fragments shown in 149-1783 of a sequence 1, and keeping other sequences of the pBI 101-35:Gus-Hm vector unchanged.

The results of the cleavage assay of the recombinant vector pBI101-NsNHX1 are shown in FIG. 2. The recombinant vector pBI101-NsNHX1 identified by double digestion and sequencing is used for genetic transformation of poplar.

2. Genetic transformation of populus tomentosa and screening and identification of transgenic plants

The recombinant vector pBI101-NsNHX1 is transformed into the aspen by a leaf disc method mediated by an agrobacterium strain GV3101 to obtain the transgenic aspen. The genetic transformation flow of populus tomentosa is shown in figure 3, and the specific steps are as follows:

(1) Selecting the sterile tissue culture seedling leaf of Populus tomentosa 84K (Populus alba× Populus glandulose) with vigorous growth, vertically arranging the leaf main She Maiqie-4 cutters, spreading the leaf back face upwards in a P1 culture medium, and pre-culturing for 4 days.

(2) The recombinant vector pBI101-NsNHX1 was introduced into GV3101 Agrobacterium to give GV3101 Agrobacterium containing pBI101-NsNHX 1.

(3) Picking single colony of GV3101 agrobacterium containing pBI101-NsNHX1 activated by coating plate, adding into LB liquid medium containing 20mg/L kanamycin, 50mg/L rifampicin and 200 μmol/L acetosyringone, and shaking culturing at 28deg.C for about 8 hr until OD ₆₀₀ 0.6-0.8% Silwet L-77 was added as a dip.

(4) Soaking the pre-cultured leaf in the soaking solution for 10min, taking out the leaf, sucking the bacterial solution on the leaf surface with filter paper, spreading the leaf back upwards in P2 culture medium, and dark culturing at 24 deg.c for 2-3 days.

(5) After the agrobacterium colonies appear on the P2 medium, the medium containing 300mg/L ceftiofur sodium and 200mg/L sterilized ddH of timentin ₂ O washes the leaves and absorbs water, and transfers the leaves to P3 medium for continuous culture, after about two weeks, the leaves develop differentiated adventitious buds.

(6) When the adventitious bud length is 1-2cm, cutting off and transferring the adventitious bud length into a P4 culture medium containing 50mg/L kanamycin for screening, and after three weeks, selecting resistant seedlings for PCR identification.

(7) Transferring the positive transgenic NsNHX1 white poplar plants identified by tissue PCR into a P5 culture medium containing 100mg/L ceftiofur sodium for rooting culture, transplanting the transgenic plants after rooting into nutrient soil, culturing in a greenhouse and detecting stress tolerance.

3. PCR identification of trans-NSNHX 1 aspen

TransDirect Plant Tissue PCR Kit from Whole gold Inc. was used, primer 35S-F:5'-AGGAAGGTGGCTCCTACAAATG-3' and NHX1-pBI-R:5'-CGAGCTCTCACTGCCATTGGGGGA-3' PCR identification of populus tomentosa resistant plants was performed.

The reaction conditions are as follows: 94 ℃ for 5min;94℃for 30s,55℃for 30s,72℃for 1min for 30s,35cycles;72 ℃ for 5min; forever at 12 ℃.

The PCR products were detected by gel electrophoresis, and found that three resistant plants N1, N2, N3 and the recombinant vector pBI101-NsNHX1 all amplified a specific band of NsNHX1 of about 1.9kb (252 bp 35S promoter sequence +1635bp NsNHX ORF sequence +6bp cleavage sequence), whereas wild type aspen did not have the band, indicating that the exogenous gene NsNHX1 had been successfully transferred into aspen.

4. RT-PCR analysis of trans-NSNHX 1 aspen

And transferring the plant identified as the transformed NsNHX1 white poplar through tissue PCR identification into a P5 culture medium containing 100mg/L ceftiofur sodium for rooting culture, and carrying out RT-PCR detection after the plant leaves grow up. Extracting RNA of positive strain with TaKaRa MiniBEST Plant RNA Extraction Kit kit (procedure Protocol-I for procedure of description, see below), using

First-Strand cDNA Synthesis SuperMix Kit Synthesis of cDNA first strand, NHX-pBI-F and NHX1-pBI-R primers detected the expression of the NsNHX1 gene. The aspen action gene is taken as an internal reference, and the primer is Actin (Populus) -F:5'-AAACTGTAATGGTCCTCCCTCCG-3', actin (Populus) -R:5'-GCATCATCACAATCACTCTCCGA-3', reaction conditions: 94 ℃ for 3min;94℃for 30s,63℃for 30s,72℃for 20s,30cycles; and at 72℃for 5min.

The results are shown in FIG. 5. The results show that: the NsNHX1 expression products can be amplified in all three transformed NsNHX1 aspen lines N1, N2 and N3. The NsNHX1 gene is stably expressed in the NsNHX 1-transformed populus tomentosa.

2. Stress tolerance detection of NsNHX 1-transformed populus tomentosa

1. Stress tolerance detection of trans-NsNHX 1 aspen under aseptic culture condition

Selecting vigorous wild aspen and trans-NSNHX 1 aspen sterile tissue culture seedlings, and cutting 5-6 cm cutting (retaining top buds and three leaves). 20 strains of wild aspen and three transformed NsNHX1 aspen strains N1, N2 and N3 were selected and respectively cut into P5 culture medium containing NaCl (0, 50, 100, 150 and 200 mM) with different concentrations for salt stress treatment. After two weeks of treatment, the viability of the plants and the biomass of the roots (fresh quantity of roots) were determined.

The results are shown in FIG. 6A. The results show that: the phenotypes of the wild-type aspen and the three transformed NsNHX1 aspen lines were not significantly different at 0mM, 50mM and 100mM NaCl (fig. 6A), the survival rates were all 100%, but the biomass of the roots of the three transformed NsNHX1 aspen lines was significantly higher than that of the wild-type aspen (fig. 6B, D); under the condition of 150mM NaCl, the phenomena of yellowing, necrosis, growth inhibition and the like occur in the wild type populus tomentosa and the three trans-NSNHX 1 populus tomentosa strains, but the survival rate (N1-83.33%, N2-50% and N3-66.67%) of the three trans-NSNHX 1 populus tomentosa strains is obviously higher than that of the wild type populus tomentosa (25%) (FIG. 6E); all wild type aspen and three transgenic NsNHX1 aspen lines died after two weeks of salt stress at 200mM NaCl, indicating that transgenic aspen overexpressing NsNHX1 were able to survive in medium with a NaCl concentration of no more than 150mM (fig. 6A). On the other hand, root development of the transgenic Chinese white poplar with the over-expressed NsNHX1 is better than that of wild Chinese white poplar, and biomass of roots of three transgenic NsNHX1 Chinese white poplar lines is obviously higher than that of the wild Chinese white poplar under normal growth and salt stress conditions (figure 6B, C, D). The above results indicate that overexpression of NsNHX1 not only improves salt tolerance of transgenic poplar, but also promotes root growth (fig. 6).

2. Stress tolerance detection of trans-NSNHX 1 aspen transplanted into soil

Selecting vigorous wild aspen and trans-NSNHX 1 aspen sterile tissue culture seedlings, cutting 5-6 cm cutting slips, reserving top buds and three leaves, and cutting in a P5 culture medium for rooting culture. Culturing in an illumination incubator for two weeks, transferring to an illumination culture rack, continuously culturing for 3 days (at the moment, the plant grows out of the root), taking out the plant from the culture medium, cleaning the residual culture medium of the root, and wiping off the residual culture medium. Then transplanting the plant into soil (the ratio of peat soil to vermiculite is 5:1), hardening, irrigating Hoagland nutrient solution, covering preservative film for preserving moisture, and spraying the Hoagland nutrient solution on the leaves once a day in the morning and evening. And removing the preservative film after one week to obtain the plant with basically consistent growth vigor.

(1) Leaf disk method for detecting saline-alkali resistance and oxidation resistance of NsNHX 1-transformed aspen

Transplanting wild aspen and trans-NSNHX 1 aspen sterile tissue culture seedlings subjected to cuttage culture for three weeks into peat soil, selecting robust and stretched leaves at the same position after two months of greenhouse culture, and beating the leaves into leaf discs with the diameter of 1cm by using a puncher while avoiding main veins. The leaf discs were placed in NaCl solutions (0 mM, 50mM, 100mM and 150 mM), naHCO, respectively, containing different concentrations ₃ Solutions (0 mM, 100mM, 200mM and 300 mM) and H ₂ O ₂ The solutions (0%, 1.0%, 1.5% and 2.0%) were placed in a 16h light/8 h dark environment and incubated at 24℃for 72h, after which the chlorophyll content in the leaves was determined using the plant chlorophyll content kit from Suzhou Ming Biotech Co.

The results are shown in fig. 8, which shows that: there was no significant difference between leaf discs of wild type aspen and ns nhx 1-transformed aspen at 0mM and 50mM NaCl; however, most of leaf discs of wild type populus tomentosa are caused by high salt solution under the conditions of 100mM and 150mM NaClIs a strong osmotic action to shrink the cell water loss, resulting in softening of the leaf disc. Meanwhile, the chlorophyll content of leaf discs of the three trans-NSNHX 1 aspen strains is higher than that of wild aspen at 100mM and 150mM NaCl. The leaf disc of the transgenic populus tomentosa with the over-expression of NsNHX1 has stronger salt tolerance. NaHCO at 100mM, 200mM and 300mM ₃ Although the chlorophyll content of the wild aspen and the transformed NsNHX1 aspen leaf discs were not significantly different; however, leaf discs of wild aspen showed more severe damage, indicating that leaf discs of NsNHX 1-transformed aspen had greater alkali resistance. At 1.0% and 1.5% H ₂ O ₂ The transformed NsNHX1 aspen lines N2 and N3 exhibited a stronger oxidation resistance than the wild type and N1: the leaf discs whiten less in quantity and have a higher chlorophyll content. At 2.0% H ₂ O ₂ Under the conditions of (1), leaf discs of both wild aspen and transformed NsNHX1 aspen were severely damaged. The above results show that the overexpression of the NsNHX1 gene can improve the salt tolerance, alkali resistance and oxidation resistance of transgenic populus tomentosa to a certain extent.

(2) Salt tolerance and phenotypic analysis of ns nhx1 transformed aspen

Selecting pretreated seedlings of wild aspen and three transformed NsNHX1 aspen strains, dividing the pretreated seedlings into two groups, and irrigating one group with Hoagland nutrient solution to serve as a control; the other group was irrigated with a holland nutrient solution containing NaCl and subjected to salt stress treatment: watering Hoagland nutrient solution containing 25mM NaCl every two days for the first week; the second week, the concentration of NaCl was increased by 25mM every two days, gradually to 150mM; and in the third week, the mixture is irrigated with Hoagland nutrient solution containing 150mM NaCl once every two days, and stress is continued for one week. Each plant was irrigated with the same volume of Hoagland nutrient solution or with NaCl and placed in a tray to maintain the salt content of the soil. After 3 weeks, the growth conditions (biomass M, plant height h, leaf chlorophyll content and leaf relative water content) were determined; proline (PRO) and Malondialdehyde (MDA) content; superoxide dismutase (Superoxide dismutase, SOD), peroxidase (POD), and Catalase (CAT) activity.

1) Measurement of growth conditions

A. Biomass: biomass M was measured separately before and after plant treatment with an electronic balance.

B. Plant height: the treated plant height h was measured with a ruler.

C. Leaf chlorophyll content: after salt stress treatment, leaves at the same position of the plant are selected, and the chlorophyll content is measured by using a SPAD-502 portable chlorophyll meter.

D. Blade relative water content: after salt stress treatment, selecting leaves at the same position of the plant, and measuring fresh weight Wf. Then, the leaf was immersed in distilled water for 24 hours under a dark condition at room temperature, and the swelling weight Wt was measured. Finally, the sample was dried at 80℃for 48 hours and its dry weight Wd was measured. The calculation formula of the Relative Water Content (RWC) of the blade is as follows: rwc= (Wf-Wd)/(Wt-Wd) ×100%.

2) Proline content determination

About 0.1g of the sample (leaf) was weighed, 1mL of the extract was added, and ice-bath homogenization was performed; placing in boiling water bath, and extracting for 10min; centrifuging 10000g for 10min at normal temperature, collecting supernatant, cooling, and testing. To the EP tube, 0.25mL of sample, 0.25mL of reagent I and 0.25mL of reagent II were added sequentially, the tube mouth was closed, and the mixture was placed in a boiling water bath for 30min with shaking every 10 min. After cooling, 0.5mL of reagent three was added, and the mixture was shaken for 30s and allowed to stand for a moment to transfer the dye into the reagent three; absorbing 0.2mL of the upper layer solution into a 96-well plate, and measuring the absorbance A at 520nm wavelength by using an enzyme-labeled instrument (preheated for more than 30 min) ₅₂₀ The calculation formula is as follows:

pro content (μg/g fresh weight) =38.4× (A ₅₂₀ +0.0021)÷W _{Fresh weight} 。

Wherein W is _{Fresh weight} : sample fresh weight (g).

3) Malondialdehyde (MDA) content determination

About 0.1g of sample (leaf) is weighed, 1mL of extract is added, ice bath homogenization is carried out, 8000g of sample is centrifuged at 4 ℃ for 10min, and the supernatant is taken and placed on ice for measurement. 0.3mL of the first reagent and 0.1mL of the sample are added into a 1.5mL centrifuge tube, the mixture is uniformly mixed, the mixture is kept in a water bath at 90 ℃ for 30min (a pipe orifice is tightly covered), then the mixture is cooled in an ice bath, and 10000g of the mixture is centrifuged at room temperature for 10min. 200. Mu.L of the supernatant was added to a 96-well plate, and absorbance A532 and A600 at 532nm and 600nm were measured by an enzyme-labeled instrument (preheated for 30min or more) as follows:

MDA content (nmol/g fresh weight) = 51.6XΔA/W _{Fresh weight} 。

Wherein Δa=a532-a 600; w (W) _{Fresh weight} : sample fresh weight (g).

4) SOD activity detection

Sequentially adding 45 mu L of the first reagent, 100 mu L of the second reagent, 2 mu L of the third reagent, 18 mu L of a sample (leaf) or 18 mu L of distilled water and 35 mu L of the fourth reagent into a 96-well plate, fully mixing uniformly, standing at room temperature for 30min, and measuring the absorbance A560 at 560nm by using an enzyme-labeled instrument (preheating for more than 30 min), wherein the calculation formula is as follows:

SOD Activity (U/g fresh weight) =11.11×A _{Percent inhibition} ÷(1-A _{Percent inhibition} )÷W _{Fresh weight} X sample dilution.

Wherein A is _{Percent inhibition} ＝(A _{Control tube} -A _{Measuring tube} )÷A _{Control tube} ×100％；W _{Fresh weight} : sample fresh weight (g).

5) POD Activity detection

Sequentially adding 10 μl sample (leaf), 60 μl distilled water, 120 μl first reagent, 30 μl second reagent and 30 μl third reagent into 96 well plate, immediately mixing, timing, and measuring absorbance A at 470nm for 30s with enzyme-labeled instrument (preheated for more than 30 min) ₁ And absorbance A after 1min30s ₂ The calculation formula is as follows:

POD (U/g fresh weight) =5000×Δa++w _{Fresh weight}

Wherein Δa=a ₂ -A ₁ ；W _{Fresh weight} : sample fresh weight (g).

6) CAT Activity assay

Adding 25mL of the reagent into the reagent II, fully mixing, adding 10 mu L of the sample (blade) and 190 mu L of the working solution into a UV plate, immediately mixing, timing, and measuring an initial absorbance A at 240nm by using an enzyme-labeling instrument (preheated for more than 30 min) ₁ And absorbance A after 1min ₂ The calculation formula is as follows:

CAT (U/g fresh weight) =918×ΔA/W _{Fresh weight}

Wherein Δa=a ₁ -A ₂ ；W _{Fresh weight} : sample fresh weight (g).

The results of the detection of the phenotype and physiological index (biomass, plant height, leaf chlorophyll content and leaf relative water content) of the wild type aspen and the three transformed NsNHX1 aspen after three weeks of salt stress treatment are shown in fig. 9 and 10. The results show that: plants grown under salt stress were shorter and grew slower than wild-type aspen and ns nhx 1-transformed aspen grown under normal conditions, but N1 and N3-transformed NsNHX1 aspen lines grew slightly stronger than wild-type aspen grown under normal growth conditions or under salt stress conditions (fig. 9A). The seventh leaves (counted from the top buds) of the plants under normal conditions and under salt stress conditions were compared, and the leaves of the wild type aspen and the transgenic NsNHX1 aspen under salt stress conditions were found to have the phenomena of yellowing, withering, wilting and the like, and the leaves of the wild type aspen and the transgenic NsNHX1 aspen strain N2 even had necrotic withered spots, but the leaves of the wild type aspen were more severely damaged (FIG. 9B).

The biomass of the transformed NsNHX1 aspen was found to be significantly higher than that of the wild-type aspen under both normal growth conditions and salt stress conditions by measuring the biomass of the wild-type aspen and the transformed NsNHX1 aspen (fig. 10A). The plant heights of wild-type aspen and ns nhx 1-transformed aspen were measured and found to be significantly higher than that of wild-type aspen and N2, with no significant difference between wild-type aspen and N2, both under normal growth conditions and under salt stress conditions (fig. 10B). Measurement of chlorophyll content of fifth leaves (counted from terminal buds) of wild type aspen and ns nhx 1-transformed aspen shows that there is no significant difference in chlorophyll content in the leaves of wild type aspen and ns nhx 1-transformed aspen under normal growth conditions; while under salt stress conditions, chlorophyll content in leaves of transformed NsNHX1 aspen was increased and significantly higher than that of wild-type aspen (fig. 10C). Measuring the relative water content of the 4 th leaf (counted from the top bud) of the wild type aspen and the NsNHX 1-transformed aspen, and finding that the relative water content in the wild type aspen and the NsNHX 1-transformed aspen leaves has no significant difference under normal conditions; while the relative water content in both wild-type aspen and ns nhx 1-transformed aspen leaves decreased under salt stress conditions, the relative water content in ns nhx 1-transformed aspen strain leaves was slightly higher than that of wild-type aspen (fig. 10D). The above results indicate that the overexpression of NSNHX1 not only improves the tolerance of transgenic aspen to salt stress, but also promotes the growth thereof.

Since salt stress causes oxidative stress to plants and active oxygen is generated in the plants, in order to determine whether or not NsNHX1 functions in the antioxidation system of populus tomentosa under the condition of salt stress, the activities of SOD, POD, CAT in leaves of wild populus tomentosa and trans-NsNHX 1 populus tomentosa, and the contents of proline and malondialdehyde are also detected.

The results are shown in FIG. 10. The results show that: under salt stress conditions, SOD, POD and CAT activity of the three transformed NsNHX1 aspen lines were all significantly increased and significantly greater than that of wild-type aspen (fig. 10E, F, G); under the condition of salt stress, the proline content of the wild aspen and the trans-NSNHX 1 aspen is obviously increased, and the proline content of the trans-NSNHX 1 aspen strains N1 and N3 is obviously higher than that of the wild aspen and N2 (figure 10H); the MDA content in the wild-type aspen and the ns nhx1 transformed aspen leaves was significantly increased under salt stress, but the MDA content in the wild-type aspen leaves was significantly higher than the ns nhx1 transformed aspen line (fig. 10I). The result shows that the overexpression of the NsNHX1 can improve the activity of the transgenic populus tomentosa antioxidant enzyme, increase the content of proline, reduce the accumulation of active oxygen, reduce the damage of a biological film and further improve the antioxidant capacity of the transgenic populus tomentosa.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

<110> university of inner Mongolia agricultural university

<120> NsNHX1 protein and application of related biological material in cultivation of stress-tolerant poplar

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<170>PatentIn version 3.5

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Cys Ala Cys Ile Val Ile Gly His Leu Leu Glu Glu Asn Arg Trp Met

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

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Ile Leu Leu Val Ser Gly Gly Lys Ser Ser His Leu Leu Val Phe Ser

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Glu Asp Leu Phe Phe Ile Tyr Leu Leu Pro Pro Ile Ile Phe Asn Ala

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Gly Phe Gln Val Lys Lys Lys Gln Phe Phe Arg Asn Phe Ile Thr Ile

100 105 110

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

115 120 125

Leu Gly Ala Met Gln Ala Phe Lys Arg Leu Asp Ile Gly Ser Leu Asp

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Ser Ala Phe Gln Leu Leu Gly Asn Phe Leu Tyr Leu Phe Phe Ala Ser

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

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

245 250 255

Met Leu Met Ala Tyr Leu Ser Tyr Met Leu Ala Glu Leu Phe Asp Met

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

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Thr Trp His Asn Val Thr Glu Ser Ser Arg Ile Thr Thr Lys His Ala

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

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

325 330 335

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

340 345 350

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

355 360 365

Met Lys Lys Ser Pro Thr Asp Lys Val Gly Phe Lys Gln Gln Ile Val

370 375 380

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

385 390 395 400

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

405 410 415

Val Met Ile Thr Ser Thr Ile Thr Val Val Leu Phe Ser Thr Val Val

420 425 430

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

435 440 445

Lys Ala Ala Arg Ser Met Ser Leu Ser Asp Pro Glu Asn Gln Lys Ser

450 455 460

Val Thr Thr Pro Leu Leu Gly Gln Ser Gln Asp Ser Glu Ala Asp Leu

465 470 475 480

Gly Ser Thr Pro Leu Gly Asn Gly Ile His Arg Pro Gly Ser Leu Arg

485 490 495

Ala Leu Leu Asn Ala Pro Thr His Thr Val His Tyr Tyr Trp Arg Lys

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Phe Asp Asp Ala Phe Met Arg Pro Ala Phe Gly Gly Arg Gly Phe Thr

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Pro Phe Val Pro Gly Ser Pro Thr Glu Arg Ser Val Pro Gln Trp Gln

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Claims

Use of an nsnhx1 protein in any one of the following 1) -9):

1) Promoting the growth and development of the root of the populus tomentosa;

2) Increasing the plant height of the populus tomentosa;

3) Improving the survival rate of the populus tomentosa under the stress of salt;

4) In salt stress or H ₂ O ₂ Increasing chlorophyll content in aspen leaves under stress;

5) Reducing the damage degree of the populus tomentosa under the alkali stress;

6) Increasing the water content in the aspen leaves under salt stress;

7) Increasing superoxide dismutase and/or peroxidase and/or catalase activity in the aspen leaf under salt stress;

8) Increasing the proline content in the aspen leaves under salt stress;

9) Reducing malondialdehyde content in aspen leaves under salt stress;

the NsNHX1 protein is a protein shown in the following a) or b):

a) The amino acid sequence is a protein shown in the sequence 2;

b) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein represented by the sequence 2.
2. Use of a biological material associated with NsNHX1 protein in any one of the following 1) -10):

1) Promoting the growth and development of the root of the populus tomentosa;

2) Increasing the plant height of the populus tomentosa;

3) Improving the survival rate of the populus tomentosa under the stress of salt;

4) In salt stress or H ₂ O ₂ Increasing chlorophyll content in aspen leaves under stress;

5) Reducing the damage degree of the populus tomentosa under the alkali stress;

6) Increasing the water content in the aspen leaves under salt stress;

7) Increasing superoxide dismutase and/or peroxidase and/or catalase activity in the aspen leaf under salt stress;

8) Increasing the proline content in the aspen leaves under salt stress;

9) Reducing malondialdehyde content in aspen leaves under salt stress;

10 Breeding aspen;

the NsNHX1 protein is a protein shown in the following a) or b):

a) The amino acid sequence is a protein shown in the sequence 2;

b) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein represented by the sequence 2.
3. The use according to claim 2, characterized in that: the biomaterial is any one of the following A1) to A8):

a1 Nucleic acid molecules encoding NsNHX1 protein;

a2 An expression cassette comprising A1) said nucleic acid molecule;

a3 A) a recombinant vector comprising the nucleic acid molecule of A1);

a4 A recombinant vector comprising the expression cassette of A2);

a5 A) a recombinant microorganism comprising the nucleic acid molecule of A1);

a6 A) a recombinant microorganism comprising the expression cassette of A2);

a7 A) a recombinant microorganism comprising the recombinant vector of A3);

a8 A recombinant microorganism comprising the recombinant vector of A4).
4. A use according to claim 3, characterized in that: a1 The nucleic acid molecule is a gene as shown in the following 1) or 2):

1) The coding sequence is DNA molecule shown in 149-1783 of sequence 1;

2) A DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) and encoding the NsNHX1 protein of claim 1.
5. A method for cultivating transgenic aspen with improved stress tolerance comprises the steps of improving the expression level of NsNHX1 protein in receptor aspen to obtain transgenic aspen; the stress tolerance of the transgenic aspen is higher than that of the receptor aspen; the stress resistance is salt resistance and/or alkali resistance;

the NsNHX1 protein is a protein shown in the following a) or b):

a) The amino acid sequence is a protein shown in the sequence 2;

b) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein represented by the sequence 2.
6. The method according to claim 5, wherein: the stress tolerance of the transgenic aspen is higher than that of the receptor aspen, and the stress tolerance is represented by any one of the following (1) - (9):

(1) The survival rate of the transgenic aspen is higher than that of the receptor aspen under the stress of salt;

(2) Under salt stress, the biomass or root biomass of the transgenic aspen is higher than that of the receptor aspen;

(3) In salt stress or H ₂ O ₂ Under stress, chlorophyll content in leaves of transgenic aspen is higher than that of receptor aspen;

(4) Under salt stress, the water content in the leaves of transgenic aspen is greater than that of receptor aspen.

(5) Under the stress of salt, the plant height Yu Shouti of the transgenic aspen is higher than that of aspen;

(6) Under the alkali stress, the damage degree of the transgenic aspen is smaller than that of the receptor aspen;

(7) Under salt stress, the activity of superoxide dismutase and/or peroxidase and/or catalase in the transgenic aspen leaves is higher than that of receptor aspen;

(8) Under salt stress, the proline content in the leaves of the transgenic aspen is higher than that of the receptor aspen;

(9) The malondialdehyde content in transgenic aspen leaves is lower than that of receptor aspen under salt stress.
7. The method according to claim 5, wherein: the method for improving the expression level of the NsNHX1 protein in the receptor white poplar is to over-express the NsNHX1 protein in the receptor white poplar.
8. The method according to claim 7, wherein: the over-expression method is to introduce the coding gene of the NsNHX1 protein into a receptor populus tomentosa.
9. The method according to any one of claims 5-8, wherein: the nucleotide sequence of the coding gene of the NsNHX1 protein is a DNA molecule shown in 149-1783 of the sequence 1.