CN110256544A - NsNHX1 protein and its relevant biological material are cultivating the application in resistance to inverse type poplar - Google Patents

Abstract

The invention discloses NsNHX1 protein and its relevant biological material to cultivate the application in resistance to inverse type poplar.The present invention is with Nitraria sibirica Na⁺/H⁺Antiporter gene NsNHX1 is research object, is transferred to Chinese white poplar 84K, and acquisition turns NsNHX1 Chinese white poplar.It chooses wherein tri- transformants of N1, N2 and N3 and carries out resistance to reverse function identification.It is control with wild type Chinese white poplar, research turns saline-alkali tolerant and oxidation resistance of the NsNHX1 Chinese white poplar under environment stress.As a result, it has been found that survival rate, biomass, chlorophyll content in leaf blades and water content, plant height and the oxidation resistance for turning NsNHX1 Chinese white poplar are all remarkably higher than wild type Chinese white poplar under environment stress treatment conditions.Show that NsNHX1 can significantly improve the resistance to inverse ability and oxidation resistance of transgenic poplar, NsNHX1 can be applied to cultivate resistance of reverse Poplar Varieties as resistant gene of salt.

Description

Application of NsNHX1 protein and its related biomaterials in the cultivation of stress-tolerant poplar

technical field

The invention belongs to the field of biotechnology, in particular to the application of NsNHX1 protein and related biological materials in cultivating stress-tolerant poplars.

Background technique

Plant roots are the organs that directly contact Na ⁺ in the soil, so the Na ⁺ efflux from roots and the compartmentalization and concentration of Na ⁺ in shoot cells are important mechanisms that determine plant salt tolerance. The Na + /H ⁺ antiporter (NHX1) on the tonoplast membrane transports Na ⁺ in the cytoplasm against the concentration gradient to the vacuole for compartmentalization; the Na ⁺ /H ⁺ antiporter (SOS1) on the cytoplasmic membrane can transport the cytoplasmic Na ⁺ Na ⁺ in the reverse transport to the extracellular high Na ⁺ environment. Up to now, most of the Na ⁺ /H ⁺ antiporter genes developed and applied come from sweet soil plants such as Arabidopsis thaliana and tomato, while for woody plants, especially for halophyte Na ⁺ /H ⁺ The development and research of antiporter genes are relatively scarce. Previous studies have found that the expression level of ThSOS1 in the salt mustard is about 8-10 times that of Arabidopsis AtSOS1, and similar SOS signaling pathways have been found in the sweet soil plant rice and the halophyte salt mustard, indicating that higher plants may share a common A set of salt tolerance regulation mechanisms. Therefore, it is of great significance to develop and utilize the Na ⁺ /H ⁺ antiporter genes of halophytes.

Nirtaria L. is a small deciduous shrub belonging to the family Zygophyllceae and is a halophyte, mainly distributed in Inner Mongolia, Ningxia, Gansu, Qinghai and Xinjiang from northern to northwestern my country. One of the constructive species in the arid saline-alkali zone. The stems and leaves of the genus Alternaria have thick cuticles, the leaves are covered with waxy epidermal hairs, the stomata are invaginated, the palisade tissue and vascular bundles are developed, and the cell contents are filled with waxy crystals. These typical structural characteristics of xerophytes make It plays a role in resisting salt damage. The strong membrane protection ability and membrane repair ability under salt stress conditions, as well as the efficient intracellular ion compartmentalization and accumulation of osmotic regulating substances, endow the plants of the genus Alba with strong salt tolerance. There are four main species in Inner Mongolia, namely N.sibilica Pall, N.tangutorum Bobr, N.sphaerocarpa Maxim and N.roborowskii Kom , among which Siberian white thorn showed stronger salt tolerance. Siberian white thorn has extremely important ecological value. Due to the triple stress of salt, alkali and drought in its natural habitat, it has strong resistance to salt and alkali and drought, and contains abundant genetic resources for stress resistance. Therefore, it is of great significance to study the mechanism of its salt tolerance, identify the genes related to salt tolerance and apply it to the genetic improvement of the salt tolerance of pastures, agricultural and forestry crops.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is how to regulate plant stress tolerance.

In order to solve the above technical problems, the present invention first provides a new use of NsNHX1 protein.

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

1) Regulate plant stress tolerance;

2) Regulate plant growth and development;

3) Regulation of plant biomass;

4) Regulate plant height;

5) Regulate the chlorophyll content of plants;

6) Regulate the water content of plants;

7) Regulate the antioxidant capacity of plants;

8) regulating plant superoxide dismutase and/or peroxidase and/or catalase activity;

9) regulating plant proline content;

10) regulating the content of malondialdehyde in plants;

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

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

b) a fusion protein obtained by linking a tag to the N-terminal and/or C-terminal of the protein shown in SEQ ID NO: 2;

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

d) A protein having 75% or more homology with the amino acid sequence shown in SEQ ID NO: 2 and having the same function.

In order to facilitate purification of the protein in a), a tag as shown in Table 1 can be attached to the amino terminus or carboxyl terminus of the protein shown in SEQ ID NO: 2 in the sequence listing.

Table 1. Sequence of tags

Label Residues sequence 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

For the protein in the above c), the substitution and/or deletion and/or addition of one or several amino acid residues are substitutions and/or deletions and/or additions of no more than 10 amino acid residues.

The protein in the above c) can be obtained by artificial synthesis, or by first synthesizing its encoding gene and then biologically expressing it.

The gene encoding the protein in the above c) can be obtained by deleting the codons of one or several amino acid residues in the DNA sequence shown in the 149-1783 position of SEQ ID NO: 1, and/or making one or several base pair errors. A sense mutation, and/or the coding sequence of the tag shown in Table 1 is attached to its 5' end and/or 3' end.

In the above d), "homology" includes 75% or more, or 80% or more, or 85% or more, or 90% or more with the amino acid sequence shown in SEQ ID NO: 2 of the present invention, or amino acid sequences with 95% or higher homology.

In order to solve the above technical problems, the present invention also provides a new use of biological materials related to NsNHX1 protein.

The present invention provides the application of the biological material related to NsNHX1 protein in any one of the following 1)-12):

1) Regulate plant stress tolerance;

2) Regulate plant growth and development;

3) Regulation of plant biomass;

4) Regulate plant height;

5) Regulate the chlorophyll content of plants;

6) Regulate the water content of plants;

7) Regulate the antioxidant capacity of plants;

8) regulating plant superoxide dismutase and/or peroxidase and/or catalase activity;

9) regulating plant proline content;

10) regulating the content of malondialdehyde in plants;

11) Cultivate transgenic plants with improved stress tolerance;

12) Plant breeding.

In the above application, the biological material is any one of the following A1) to A12):

A1) a nucleic acid molecule encoding the NsNHX1 protein;

A2) an expression cassette containing the nucleic acid molecule of A1);

A3) a recombinant vector containing the nucleic acid molecule of A1);

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

A5) a recombinant microorganism containing the nucleic acid molecule of A1);

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

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

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

A9) a transgenic plant cell line containing the nucleic acid molecule of A1);

A10) a transgenic plant cell line containing the expression cassette of A2);

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

A12) A transgenic plant cell line containing the recombinant vector described in A4).

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

1) Its coding sequence is the cDNA molecule shown in position 149-1783 of sequence 1 or the genomic DNA molecule shown in sequence 1;

2) A cDNA molecule or genomic DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) and encodes the NsNHX1 protein;

3) A cDNA molecule or a genomic DNA molecule that hybridizes to the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the NsNHX1 protein.

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

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

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or higher, or 85% or higher, or 90% or higher, or 95% or Nucleotide sequences of higher identity. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The above-mentioned 75% or more identity may be 80%, 85%, 90% or more than 95% identity.

In the above application, the stringent conditions were hybridized and washed twice at 68°C in a solution of 2×SSC, 0.1% SDS for 5 min each, and then in a solution of 0.5×SSC, 0.1% SDS, at 68°C. Hybridize and wash the membrane twice at 68°C for 15 min each; or, in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS, hybridize and wash the membrane at 65°C.

In the above application, the expression cassette containing the nucleic acid molecule encoding the NsNHX1 protein described in A2) refers to the DNA capable of expressing the NsNHX1 protein in the host cell. terminator. Further, the expression cassette may also include enhancer sequences. Promoters useful in the present invention include, but are not limited to: constitutive promoters; tissue, organ and development specific promoters and inducible promoters.

The recombinant vector containing the NsNHX1 gene expression cassette can be constructed by using the existing expression vector. The plant expression vectors include binary Agrobacterium vectors and vectors that 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 company) and so on. The plant expression vector may also contain the 3' untranslated region of the foreign gene, ie, containing the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The poly(A) signal can guide the addition of poly(A) to the 3' end of the mRNA precursor, such as Agrobacterium crown gall-inducing (Ti) plasmid genes (such as nopaline synthase gene Nos), plant genes (such as soybean The untranslated regions transcribed from the 3' end of the storage protein gene) have similar functions. When using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG initiation codons or adjacent region initiation codons, etc., but must be associated with the coding. The reading frames of the sequences are identical to ensure correct translation of the entire sequence. The translation control signals and initiation codons can be derived from a wide variety of sources, either natural or synthetic. The translation initiation region can be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding a gene (GUS gene, luciferase gene, luciferase gene) that can be expressed in plants encoding an enzyme that can produce a color change or a luminescent compound. Gene, etc.), marker genes for antibiotics (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, the hph gene that confers resistance to the antibiotic hygromycin , and the dhfr gene conferring resistance to methotrexate, the EPSPS gene conferring resistance to glyphosate) or marker genes for chemical resistance (such as herbicide resistance genes), mannose-6- which provides the ability to metabolize mannose Phosphoisomerase gene. Considering the safety of transgenic plants, the transformed plants can be directly screened under 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 applications, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium. The Agrobacterium can be GV3101.

In the above application, the transgenic plant cell line, transgenic plant tissue and transgenic plant organ do not include propagation material.

In the above applications, the stress resistance may be salt resistance and/or alkali resistance.

The regulation of plant stress tolerance is to improve plant stress tolerance. Specifically, under salt or alkali stress, compared with recipient plants, trans-NsNHX1 plants have improved survival rate, increased biomass or root biomass, increased chlorophyll content in leaves, increased water content in leaves, and plant height. Increased, 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 . The salt is specifically NaCl, and the NaCl concentration may be specifically 50 mM or 100 mM or 150 mM; the base may specifically be NaHCO ₃ ; the NaHCO ₃ concentration may specifically be 100 mM or 200 mM or 300 mM.

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

The regulation of plant biomass is to increase plant biomass; the biomass can be root biomass or plant biomass.

The regulating plant height is increasing the plant height.

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

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

The regulation of plant antioxidant capacity is to improve plant antioxidant capacity. Specifically, under salt stress conditions, compared with recipient plants, the activities of superoxide dismutase and/or peroxidase and/or catalase in leaves of NsNHX1 transgenic plants increased, and the proline content increased, and the content of malondialdehyde decreased.

Described regulation of plant superoxide dismutase and/or peroxidase and/or catalase activity is to improve superoxide dismutase and/or peroxidase and/or hydrogen peroxide in plant leaves enzymatic activity.

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

The regulating and controlling plant malondialdehyde content is reducing the malondialdehyde content in plant leaves.

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

The method for cultivating transgenic plants with improved stress tolerance provided by the present invention includes the steps of increasing the expression and/or activity of NsNHX1 protein in recipient plants to obtain transgenic plants; the transgenic plants have higher stress tolerance than the recipient plants. plant.

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

The stress tolerance of the transgenic plant is higher than that of the recipient plant, which is embodied in: under salt or alkali stress, the survival rate of the transgenic plant is higher than that of the recipient plant; and/or, the biomass or root biomass of the transgenic plant higher than the recipient plant; and/or, the chlorophyll content in the 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 higher than that of the recipient plant; the plant height of the transgenic plant is higher than that of the recipient plant and/or, the antioxidant capacity of the transgenic plant is higher than that of the recipient plant; and/or, the superoxide dismutase and/or peroxidase and/or catalase activity in the leaves of the transgenic plant is higher than that of the recipient plant and/or, the proline content in the leaves of the transgenic plant is higher than that of the recipient plant; and/or the content of malondialdehyde in the leaves of the transgenic plant is lower than that of the recipient plant. The salt is specifically NaCl, and the NaCl concentration may be specifically 50 mM or 100 mM or 150 mM; the base may specifically be NaHCO ₃ ; the NaHCO ₃ concentration may specifically be 100 mM or 200 mM or 300 mM.

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

The overexpression method is to introduce the gene encoding the NsNHX1 protein into the recipient plant. The encoding gene of the NsNHX1 protein can specifically transform plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrical conductance, and Agrobacterium-mediated transformation, and transform the transformed plant cells or tissues. Plant tissue is grown into plants.

The nucleotide sequence of the gene encoding the NsNHX1 protein is the DNA molecule shown at positions 149-1783 of SEQ ID NO: 1.

In a specific embodiment of the present invention, the encoding gene of the NsNHX1 protein is introduced into the recipient plant through the recombinant vector pBI101-NsNHX1, and the recombinant vector pBI101-NsNHX1 is a combination of XbaI and SacI of the pBI101-35::Gus-Hm vector. The fragment between the restriction sites was replaced with the DNA fragment shown in 149-1783 of sequence 1, and the vector obtained by keeping the 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 its progeny. For transgenic plants, the gene can be propagated in that species, and conventional breeding techniques can be used to transfer the gene into other varieties of the same species, including in particular commercial varieties. The transgenic plants include seeds, callus, whole plants and cells.

In the above method or application, the plant is a monocotyledonous plant or a dicotyledonous plant. Further, the monocotyledonous plant can be poplar; further, the poplar can be Populus tomentosa. In a specific embodiment of the present invention, the Populus tomentosa may be Populus tomentosa 84K (Populus alba×Populus glandulose).

The present invention takes the Siberian white thorn Na ⁺ /H ⁺ anti-transporter gene NsNHX1 as the research object, and transforms it into Populus tomentosa 84K (Populus alba×Populus glandulose) to obtain the NsNHX1-transformed Populus tomentosa. Three transformants, N1, N2 and N3, were selected for the identification of reverse-tolerant function. Taking wild-type Populus tomentosa as a control, the salinity-tolerance and antioxidant capacity of NsNHX1-transformed Populus tomentosa under adversity stress were studied. The results showed that the survival rate, biomass, leaf chlorophyll content and water content, plant height and antioxidant capacity of NsNHX1-transformed Populus tomentosa were significantly higher than those of wild-type Populus tomentosa under stress conditions. The results indicated that NsNHX1 could significantly improve the stress tolerance and antioxidant capacity of transgenic poplars, and NsNHX1 could be used to breed stress-tolerant poplar varieties.

Description of drawings

Figure 1. Amplification of NsNHX1 ORF.

Figure 2 is the identification of double restriction digestion of the recombinant vector pBI101-NsNHX1.

Figure 3 shows the genetic transformation of Populus tomentosa. (A) explant cutting and pre-culture; (B) infiltration; (C) differentiation culture; (D) screening; (E) rooting culture; (F) transplantation.

Figure 4 shows the PCR identification of NsNHX1 transgenic Populus tomentosa. M: 200bp DNA Ladder; +: recombinant plasmid pBI101-NsNHX1; -: wild-type Populus tomentosa; N1, N2, N3: transgenic NsNHX1 Populus tomentosa.

Figure 5 is the RT-PCR analysis of NsNHX1 transgenic poplar tomentosa. WT: wild-type Populus tomentosa; N1, N2, N3: NsNHX1-transformed Populus tomentosa.

Figure 6 is a comparison of the growth of wild-type P. tomentosa and three NsNHX1-transformed P. tomentosa lines under salt stress conditions. (A) Plants grown for 2 weeks in P5 medium containing 0 mM, 50 mM, 100 mM, 150 mM and 200 mM NaCl; (B) Comparison of root growth; (C) 0 mM, 50 mM, 100 mM, 150 mM and 200 mM NaCl Plants cultured in P5 medium for 2 weeks; (D) biomass of wild-type and NsNHX1-transformed P. tomentosa lines; (E) survival rate of wild-type and NsNHX1-transformed P. tomentosa. WT: wild-type Populus tomentosa; N1, N2, N3: NsNHX1-transformed Populus tomentosa. Error bars represent standard deviation (SD) from three independent biological replicate experiments; different lowercase letters marked represent significant differences between each group of samples (P<0.05)

Figure 7 shows the expansion of Populus tomentosa. (A) The seedlings were cut into the medium; (B) The seedlings were transplanted into the soil; (C) Covered with plastic wrap; (D) The seedlings were prepared for salt stress.

Figure 8 shows the stress tolerance analysis of trans-NsNHX1 poplar tomentosa. (A) Stress treatment; (B) Chlorophyll content in NsNHX1 transgenic P. tomentosa leaves; error bars represent standard deviation (SD), from three independent biological replicate experiments; different lowercase letters marked represent the difference between samples in each group, respectively There was a significant difference between them (P<0.05). WT: wild-type Populus tomentosa; N1, N2, N3: NsNHX1-transformed Populus tomentosa.

Figure 9 shows the phenotypic analysis of wild-type Populus tomentosa and NsNHX1-transformed Populus tomentosa under NaCl stress. (A) The effect of salt stress on the whole plant of wild-type P. tomentosa and trans-NsNHX1-transformed P. tomentosa (scale bar = 10 cm); (B) the effect of salt-stress on the leaves of wild-type P. tomentosa and trans-NsNHX1-transformed P. tomentosa.

Figure 10 shows the stress tolerance analysis of trans-NsNHX1 poplar tomentosa. (A) biomass; (B) plant height; (C) leaf chlorophyll content; (D) leaf relative water content; (E) SOD activity; (F) POD activity; (G) CAT activity; (H) proline Acid content; (I) MDA content. WT: wild-type Populus tomentosa; N1, N2, N3: NsNHX1-transformed Populus tomentosa. Error bars represent standard deviation (SD) from three independent biological replicates; different lowercase letters marked represent significant differences between each group of samples (P<0.05).

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.

The cultivation conditions of the plant material Populus tomentosa 84K (Populus alba×Populusglandulose) sterile tissue culture seedlings involved in the following examples: the temperature is 24±2°C, the light intensity is 200 μmol·m ^-2 ·s ^-1 , the light cycle 16h light/8h dark.

In the following examples, pBI101-35::Gus-Hm carrier is recorded in the document " WANG L, MA YK, LI NN, etal.Isolation and characterization of a tonoplast Na ⁺ /H ⁺ antiporter from thehalophyte Nitraria sibirica [J].BIOLOGIA PLANTARUM, 2016, 60(1): 113-122, 2016", the public can obtain from the applicant, the biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.

The media formulations involved in the following examples are as follows:

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

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

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

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

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

Both Hoagland medium (Hoagland nutrient solution) and MS medium are products of Phytotechnology Laboratories (https://phytotechlab.com).

The sequence of the Siberian white thorn Na ⁺ /H ⁺ antiporter gene (NsNHX1 gene for short) in the following example is shown in sequence 1, which contains an open reading frame of 1635bp (position 149-1783 of sequence 1), and the NsNHX1 gene encodes The amino acid sequence of the Siberian white thorn Na ⁺ /H ⁺ antiporter (abbreviated as NsNHX1 protein) is shown in sequence 2.

Example 1. Acquisition of NsNHX1-transformed Populus tomentosa and analysis of its stress tolerance

1. Obtainment of NsNHX1-transformed Populus tomentosa

1. Construction of recombinant vector

(1) Amplification of the target gene

A pair of primers (NHX-pBI-F: 5'-GC TCTAGA ATGGATCAATTAAGTT-3', 5' end was designed according to the sequence of Siberian white thorn Na ⁺ /H ⁺ antiporter gene (NsNHX1 for short) (gene sequence accession number: AB859847) Including the XbaI recognition sequence; NHX-pBI-R: 5'-C GAGCTC TCACTGCCATTGGGGGAT-3', the 5' end includes the SacI recognition sequence), using the RNA of Nitraria sibirica Pall as a template, using NHX-pBI-F and NHX -pBI-R primer, using TaKaRa RNA PCR Kit (AMV) Ver.3.0 kit to amplify the NsNHX1 ORF region to obtain PCR product. The reaction conditions were: 94°C for 3 min; 94°C for 30 s, 52°C for 30 s, 72°C for 1 min, 35 cycles; 72°C for 5 min. The electrophoresis results of PCR products are shown in Figure 1.

(2) Linking the target gene to the expression vector

The binary expression vector pBI101-35::Gus-Hm and the above PCR product were respectively digested with XbaI and SacI, and then ligated with T4 ligase to obtain the recombinant vector pBI101-NsNHX1. The recombinant vector pBI101-NsNHX1 was sequenced and identified by restriction enzyme digestion.

The sequencing results show that: the recombinant vector pBI101-NsNHX1 is a DNA fragment shown at positions 149-1783 of sequence 1 by replacing the fragment between the XbaI and SacI restriction sites of the pBI101-35::Gus-Hm vector, and the pBI101- The vector obtained after the other sequences of the 35::Gus-Hm vector remain unchanged.

Figure 2 shows the results of restriction digestion and identification of the recombinant vector pBI101-NsNHX1. The recombinant vector pBI101-NsNHX1 identified by double enzyme digestion and sequencing was used for the genetic transformation of poplar.

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

The recombinant vector pBI101-NsNHX1 was transformed into Populus tomentosa by leaf disc method mediated by Agrobacterium strain GV3101 to obtain transgenic Populus tomentosa. The process flow of the genetic transformation of Populus tomentosa is shown in Figure 3, and the specific steps are as follows:

(1) Select vigorously growing Populus tomentosa 84K (Populus alba×Populus glandulose) aseptic tissue culture seedling leaves, cut 3-4 cuts vertically to the main leaf veins, and then flatten the back of the leaves in P1 medium for pre-cultivation 4 days.

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

(3) Pick a single colony of GV3101 Agrobacterium containing pBI101-NsNHX1 activated by plating, and add it to LB liquid medium containing 20 mg/L kanamycin, 50 mg/L rifampicin and 200 μmol/L acetosyringone, Incubate with constant shaking at 28°C for about 8 hours until the OD ₆₀₀ is 0.6-0.8, and add 0.01% Silwet L-77 as a dip solution.

(4) Immerse the pre-cultured leaves in the dip solution for 10 minutes, take out the leaves and dry the bacterial liquid on the surface of the leaves with filter paper, lay the back of the leaves up in the P2 medium, and cultivate in the dark at 24°C for 2-3 days .

(5) After Agrobacterium colonies were observed on the P2 medium, the leaves were washed with sterilized ddH ₂ O containing 300 mg/L ceftiofur sodium and 200 mg/L Timentin, and the water was absorbed, and the leaves were transferred to The culture was continued in P3 medium, and after about two weeks, differentiated adventitious buds appeared on the leaves.

(6) When adventitious buds grow to 1-2 cm, they are cut and transferred to P4 medium containing 50 mg/L kanamycin for screening. After three weeks, resistant seedlings are selected for PCR identification.

(7) The positive transgenic NsNHX1 poplar tomentosa plants identified by tissue PCR were transferred to the P5 medium containing 100 mg/L ceftiofur sodium for rooting culture, and the rooted transgenic plants were transplanted to nutrient soil and placed in a greenhouse for cultivation And used for reversal resistance testing.

3. PCR identification of NsNHX1 transgenic Populus tomentosa

The PCR identification of P. tomentosa resistant plants was carried out using the TransDirect Plant Tissue PCR Kit from Quanzhou Gold Company, using primers 35S-F: 5'-AGGAAGGTGGCTCCTACAAATG-3' and NHX1-pBI-R: 5'-CGAGCTCTCACTGCCATTGGGGGGA-3'.

The reaction conditions are: 94°C for 5 min; 94°C for 30s, 55°C for 30s, 72°C for 1min30s, 35cycles; 72°C for 5min; 12°C Forever.

The PCR products were detected by gel electrophoresis, and it was found that the three resistant plants N1, N2, N3 and the recombinant vector pBI101-NsNHX1 could amplify about 1.9kb (252bp 35S promoter sequence + 1635bp NsNHX ORF sequence + 6bp) The specific band of NsNHX1 in the enzyme cleavage sequence), while the wild-type Populus tomentosa does not have this band, indicating that the exogenous gene NsNHX1 has been successfully transferred into Populus tomentosa.

4. RT-PCR analysis of NsNHX1 transgenic Populus tomentosa

The plants identified as NsNHX1 transgenic Populus tomentosa identified by tissue PCR were transferred to P5 medium containing 100 mg/L ceftiofur sodium for rooting culture, and RT-PCR detection was performed after the leaves of the plants grew. Use the TaKaRa MiniBESTPlant RNA Extraction Kit (see the procedure Protocol-I in the manual for the operation process) to extract the RNA of the positive strains, and use The First-Strand cDNA Synthesis SuperMix Kit was used to synthesize the first strand of cDNA, and the NHX-pBI-F and NHX1-pBI-R primers were used to detect the expression of NsNHX1 gene. Taking the Actin gene of Populus tomentosa as the internal reference, the primers were 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; 72℃ for 5min.

The results are shown in Figure 5. The results showed that the NsNHX1 expression products could be amplified in the three NsNHX1-transformed Populus tomentosa lines N1, N2 and N3. This indicated that NsNHX1 gene was stably expressed in NsNHX1 transgenic poplar tomentosa.

2. Stress tolerance test of NsNHX1-transformed Populus tomentosa

1. Stress tolerance test of NsNHX1-transformed Populus tomentosa under sterile culture conditions

Select vigorously growing wild-type Populus tomentosa and trans-NsNHX1 aseptic tissue culture seedlings, and cut 5-6 cm cuttings (retain the terminal bud and three leaves). Wild-type Populus tomentosa and three NsNHX1-transformed Populus tomentosa strains N1, N2, and N3 were selected for each of 20 strains, and were cut into P5 medium containing different concentrations of NaCl (0, 50, 100, 150, and 200 mM) for salt stress treatment. . After two weeks of treatment, plant survival and root biomass (fresh root mass) were determined.

The results are shown in Figure 6A. The results showed that under the conditions of 0 mM, 50 mM and 100 mM NaCl, the phenotypes of the wild-type P. tomentosa and the three NsNHX1-transformed P. tomentosa lines were not significantly different (Fig. 6A), and the survival rates were all 100%, but the three NsNHX1-transformed P. tomentosa lines had no significant difference (Fig. 6A). The root biomass of poplar lines was significantly higher than that of wild-type poplar (Fig. 6B, D); under 150 mM NaCl conditions, both wild-type poplar and three NsNHX1-transformed poplar lines showed yellowing, necrosis, and growth inhibition. etc., but the survival rates of the three NsNHX1-transformed P. tomentosa lines (N1-83.33%, N2-50%, N3-66.67%) were significantly higher than those of the wild-type P. tomentosa (25%) (Fig. 6E); Under the condition of 200 mM NaCl, the wild-type P. tomentosa and the three transgenic NsNHX1 P. tomentosa lines died after two weeks of salt stress, indicating that the transgenic poplars overexpressing NsNHX1 could survive in the medium with NaCl concentration not higher than 150 mM ( Figure 6A). On the other hand, the root development of NsNHX1-overexpressing P. tomentosa was better than that of the wild-type P. tomentosa, and the root biomass of the three NsNHX1-transgenic P. tomentosa lines was significantly higher than that of the wild-type P. tomentosa under normal growth and salt stress conditions. Poplar (Figure 6B,C,D). The above results indicated that overexpression of NsNHX1 not only improved the salt tolerance of transgenic poplars, but also promoted root growth and development (Fig. 6).

2. Stress tolerance test of NsNHX1 tomentosa poplar transplanted into soil

Select vigorously growing wild-type Populus tomentosa and trans-NsNHX1 aseptic tissue culture seedlings, cut 5-6 cm cuttings, retain terminal buds and three leaves, and cut them into P5 medium for rooting culture. After culturing in a light incubator for two weeks, it was transferred to a light incubator for 3 days (the plants had grown roots at this time), the plants were taken out from the medium, and the residual medium at the roots was washed and wiped dry. Then transplanted into the soil (the ratio of peat soil and vermiculite is 5:1) to refine the seedlings, watered with Hoagland nutrient solution, covered with plastic wrap to keep moisture, and sprayed the leaves with Hoagland nutrient solution once every morning and evening. After one week, the plastic wrap was removed to obtain plants with basically the same growth.

(1) Detection of salinity-tolerance and antioxidant capacity of NsNHX1-transformed Populus tomentosa by leaf disk method

The wild-type poplar tomentosa and trans-NsNHX1 aseptic tissue culture seedlings of Populus tomentosa cultured for three weeks were transplanted into peat soil, and after two months of cultivation in the greenhouse, the robust and stretched leaves in the same position were selected, and a hole punch was used to avoid the main plant. Leaf Vein Punch the leaves into leaf discs with a diameter of 1 cm. The leaf discs were put into different concentrations of NaCl solutions (0 mM, 50 mM, 100 mM and 150 mM), NaHCO ₃ solutions (0 mM, 100 mM, 200 mM and 300 mM) and H ₂ O ₂ solutions (0%, 1.0%, 1.5%) and 2.0%), placed in an environment with a light cycle of 16h light/8h dark, incubated at 24°C for 72h, and used the plant chlorophyll content kit of Suzhou Keming Biotechnology Co., Ltd. to determine the chlorophyll content in leaves.

The results are shown in Figure 8. The results show that: under the conditions of 0 mM and 50 mM NaCl, there is no significant difference between the leaf discs of wild-type P. tomentosa and NsNHX1 transgenic P. tomentosa; but under the conditions of 100 mM and 150 mM NaCl, the wild-type P. tomentosa Most of the leaf discs of poplar are shrunken due to the strong osmosis caused by the high salt solution, resulting in the softening of the leaf discs. Meanwhile, under the conditions of 100 mM and 150 mM NaCl, the chlorophyll content of the leaf discs of the three NsNHX1 transgenic P. tomentosa lines was higher than that of the wild-type P. tomentosa. This indicated that the leaf discs of NsNHX1 transgenic Populus tomentosa had stronger salt tolerance. Under the conditions of 100 mM, 200 mM and 300 mM NaHCO ₃ , although there was no significant difference in chlorophyll content between wild-type and NsNHX1-transformed P. tomentosa leaf discs, the leaf discs of wild-type P. tomentosa showed severe damage, indicating that Leaf discs of P. tomentosa transfected with NsNHX1 had stronger alkali resistance. Under the conditions of 1.0% and 1.5% H ₂ O ₂ , the NsNHX1-transformed Populus tomentosa lines N2 and N3 showed stronger antioxidant activity compared with the wild type and N1: the number of leaf disc albino was less, the chlorophyll higher content. Under the condition of 2.0% H ₂ O ₂ , the leaf discs of both wild-type and NsNHX1-transformed Populus tomentosa were severely damaged. The above results indicated that the overexpression of NsNHX1 gene could improve the salt tolerance, alkali tolerance and antioxidant capacity of transgenic poplar to a certain extent.

(2) Salt tolerance and phenotype analysis of NsNHX1 transgenic poplar tomentosa

The wild-type Populus tomentosa and the pretreated seedlings of three transgenic NsNHX1 Populus tomentosa lines were selected and divided into two groups, one group was irrigated with Hoagland nutrient solution as a control; the other group was irrigated with Hoagland nutrient solution containing NaCl , salt stress treatment: in the first week, the Hoagland nutrient solution containing 25 mM NaCl was watered every two days; in the second week, the concentration of NaCl was increased by 25 mM every two days and gradually increased to 150 mM; in the third week, with Hoagland nutrient solution containing 150 mM NaCl was watered every two days for a further week of stress. Each plant was watered with the same volume of Hoagland nutrient solution or NaCl-containing Hoagland nutrient solution and placed in a tray to maintain soil salinity. After 3 weeks, the growth status (biomass M, plant height h, leaf chlorophyll content and leaf relative water content); content of proline (PRO) and malondialdehyde (MDA); superoxide Dismutase (Superoxide dismutase, SOD), peroxidase (Peroxidase, POD) and catalase (Catalase, CAT) activities.

1) Measurement of growth status

A. Biomass: use an electronic balance to measure the biomass M of the plants before and after treatment.

B. Plant height: use a ruler to measure the plant height h after treatment.

C. Leaf chlorophyll content: After the salt stress treatment, select leaves in the same position of the plant, and use the SPAD-502 portable chlorophyll analyzer to measure the chlorophyll content.

D. Relative water content of leaves: After the salt stress treatment, the leaves at the same position of the plant were selected, and the fresh weight Wf was measured. Then, the leaves were soaked in distilled water for 24 h at room temperature in the dark, and the imbibed weight Wt was measured. Finally, the samples were dried at 80 °C for 48 h, and their dry weight Wd was measured. The calculation formula of relative water content (RWC) of leaves is: RWC=(Wf-Wd)/(Wt-Wd)×100%.

2) Determination of proline content

Weigh about 0.1 g of the sample (leaf), add 1 mL of extract, and homogenize it in an ice bath; place it in a boiling water bath to shake and extract for 10 min; centrifuge at 10,000 g for 10 min at room temperature, take the supernatant, and cool it for testing. Add 0.25 mL of sample, 0.25 mL of reagent 1 and 0.25 mL of reagent 2 to the EP tube in sequence, cover the mouth of the tube tightly, place it in a boiling water bath for 30 min, and shake it every 10 min. After cooling, add 0.5mL of reagent three, shake for 30s, and let stand for a while to transfer the pigment to reagent three; pipette 0.2mL of the upper layer solution into a 96-well plate, and use a microplate reader (preheated for more than 30min) to measure the chromatin at 520nm wavelength. Absorbance value A ₅₂₀ , the calculation formula is:

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

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

3) Determination of malondialdehyde (MDA) content

Weigh about 0.1 g of the sample (leaf), add 1 mL of extract, homogenize in an ice bath, centrifuge at 8000 g at 4°C for 10 min, take the supernatant, and place it on ice for testing. Add 0.3 mL of reagent 1 and 0.1 mL of sample to a 1.5 mL centrifuge tube, mix well, incubate in a 90°C water bath for 30 minutes (close the mouth of the tube), cool in an ice bath, and centrifuge at 10,000 g at room temperature for 10 minutes. Add 200 μL of supernatant to the 96-well plate, and measure the absorbance A532 and A600 at 532 nm and 600 nm with a microplate reader (preheated for more than 30 min). The calculation formula is:

MDA content (nmol/g fresh weight)＝51.6×ΔA÷W _{fresh weight} .

Wherein ΔA=A532-A600; W _{fresh weight} : sample fresh weight (g).

4) SOD activity detection

Add 45 μL of reagent 1, 100 μL of reagent 2, 2 μL of reagent 3, 18 μL of sample (leaf) or 18 μL of distilled water, and 35 μL of reagent 4 to the 96-well plate in sequence. ) measure the absorbance value A560 at 560nm, the calculation formula is:

SOD activity (U/g fresh weight)=11.11×A _{inhibition percentage} ÷(1-A _{inhibition percentage} )÷W _{fresh weight} ×sample dilution ratio.

Wherein A _{inhibition percentage} =(A _{control tube} -A _{measurement tube} )÷A _{control tube} ×100%; W _{fresh weight} : fresh weight of the sample (g).

5) POD activity detection

Add 10 μL of sample (leaf), 60 μL of distilled water, 120 μL of reagent 1, 30 μL of reagent 2, and 30 μL of reagent 3 in sequence to the 96-well plate, mix thoroughly and time immediately, and use a microplate reader (preheated for more than 30 min) to measure the 30s at 470 nm. The absorbance value A ₁ and the absorbance value A ₂ after 1min30s, the calculation formula is:

POD(U/g fresh weight)=5000×ΔA÷W _{fresh weight}

Wherein ΔA=A ₂ -A ₁ ; W _{fresh weight} : fresh weight of the sample (g).

6) CAT activity detection

Add 25mL of reagent to Reagent 2 and mix thoroughly, add 10μL of sample (leaf) and 190μL of working solution to the UV plate, mix immediately and time, measure the initial absorbance value at 240nm with a microplate reader (preheated for more than 30min). The absorbance value A ₂ after A ₁ and 1min, the calculation formula is:

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

Wherein ΔA=A ₁ -A ₂ ; W _{fresh weight} : fresh weight of the sample (g).

After three weeks of salt stress treatment, the phenotypic and physiological indicators (biomass, plant height, leaf chlorophyll content and leaf relative water content) of wild-type and three NsNHX1-transformed Populus tomentosa are shown in Figures 9 and 10. Show. The results showed that compared with wild-type Populus tomentosa and trans-NsNHX1-transformed Populus tomentosa grown under normal conditions, the plants under salt stress were shorter and grew more slowly, but under normal growth conditions or under salt stress conditions, the transgenic plants were shorter. The growth vigor of NsNHX1 Populus tomentosa lines N1 and N3 were better than wild type and N2, and the growth vigor of N2 was slightly stronger than that of wild type Populus tomentosa (Fig. 9A). The seventh leaf (counting from the terminal bud) of the plant under normal conditions and under salt stress conditions was compared and observed, and it was found that under salt stress conditions, the leaves of wild-type Populus tomentosa and NsNHX1-transformed Populus tomentosa had yellowing, withering, wilting and other phenomena. , the leaves of the wild-type P. tomentosa and the NsNHX1-transformed P. tomentosa line N2 even appeared necrotic withered spots, but the leaves of the wild-type P. tomentosa suffered more severe damage (Fig. 9B).

By measuring the biomass of wild-type P. tomentosa and trans-NsNHX1 P. tomentosa, it was found that under normal growth conditions and under salt stress conditions, the biomass of NsNHX1-transformed P. tomentosa was significantly higher than that of wild-type P. tomentosa (Figure 10A). The plant heights of wild-type and NsNHX1-transformed Populus tomentosa were measured, and it was found that the plant heights of NsNHX1-transformed Populus tomentosa lines N1 and N3 were significantly higher than those of wild-type and N2 under normal growth conditions or under salt stress conditions, while There was no significant difference between wild-type P. tomentosa and N2 (Fig. 10B). Measured the chlorophyll content of the fifth leaf (counting from the terminal bud) of the wild-type and NsNHX1-transformed Populus tomentosa, and found that there was no significant difference in the chlorophyll content in the leaves of the wild-type and NsNHX1-transformed Poplar tomentosa under normal growth conditions; However, under salt stress conditions, the chlorophyll content in the leaves of trans-NsNHX1 P. tomentosa increased and was significantly higher than that of the wild-type P. tomentosa (Fig. 10C). The relative water content of the 4th leaf (counting from the terminal bud) of the wild-type and NsNHX1-transformed Populus tomentosa was measured, and it was found that there was no significant difference in the relative water content in the leaves of the wild-type and NsNHX1-transformed Populus tomentosa under normal conditions. Under the condition of salt stress, the relative water content in the leaves of both the wild-type and NsNHX1-transformed P. tomentosa decreased, but the relative water content in the leaves of the NsNHX1-transformed P. tomentosa line was slightly higher than that of the wild-type P. tomentosa (Fig. 10D). . The above results indicated that overexpression of NsNHX1 not only improved the tolerance to salt stress of transgenic poplar tomentosa, but also promoted its growth.

Since salt stress will cause oxidative stress to plants and generate reactive oxygen species in plants, in order to determine whether NsNHX1 functions in the antioxidant system of Populus tomentosa under salt stress conditions, the leaves of wild-type Populus tomentosa and trans-NsNHX1-transformed Populus tomentosa were also tested. The activity of SOD, POD and CAT, the content of proline and malondialdehyde.

The results are shown in Figure 10. The results showed that the SOD, POD and CAT activities of the three NsNHX1-transformed P. tomentosa lines were significantly increased under salt stress, and were significantly greater than those of the wild-type P. tomentosa (Figure 10E, F, G). , the proline content of both wild-type and NsNHX1-transformed Populus tomentosa was significantly increased, and the proline contents of N1 and N3 of the NsNHX1-transformed Populus tomentosa lines were significantly higher than those of wild-type tomentosa and N2 (Fig. 10H); The MDA content in the leaves of P. tomentosa and trans-NsNHX1-transformed P. tomentosa was significantly increased under salt stress, but the MDA content in the leaves of the wild-type P. tomentosa was significantly higher than that of the trans-NsNHX1-transformed P. tomentosa line (Fig. 10I). The above results indicated that the overexpression of NsNHX1 could increase the activity of antioxidant enzymes in transgenic poplar tomentosa, increase the content of proline, reduce the accumulation of reactive oxygen species, reduce the damage of biofilm, and then improve the antioxidant capacity of transgenic poplar tomentosa.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. While the invention has been given particular embodiments, it should be understood that the invention can be further modified. In conclusion, in accordance with the principles of the present invention, this application is intended to cover any alterations, uses or improvements of the invention, including changes made using conventional techniques known in the art, departing from the scope disclosed in this application. The application of some of the essential features can be made within the scope of the following appended claims.

sequence listing

<110> Inner Mongolia University Inner Mongolia Agricultural University

Application of <120>NsNHX1 protein and its related biomaterials in the cultivation of stress-tolerant poplar

<160>2

<170>PatentIn version 3.5

<210>1

<211>2182bp

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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aattgcttga ttgattaaga ggttcgagat ggatcaatta agttcggttg tatcgagatt 180

gcaaatggtg tcgacgtcgg atcattcttc cgtagtttcg atgaatctat ttgtggccct 240

tctctgcgcg tgtattgtga ttggtcatct tcttgaagag aataggtgga tgaatgagtc 300

gattactgct cttttgattg gtgtttgtac tggtgtcatc atcttgctgg ttagtgggggg 360

aaaaagctcg catctattag tgtttagtga agatctgttc tttatatatc tgctaccgcc 420

aattatattt aatgcaggat tccaggtgaa aaagaagcaa ttttttccgta acttcatcac 480

aatcatattg tttggtgcca tcggtacttt aataagctgt accatcatat ctctaggtgc 540

tatgcaggct tttaagagat tggacattgg ttctctggat ttgggggatt ttctagcaat 600

cggtgctata tttgctgcaa cagattctgt ttgcacgttg caggttctta accaggatga 660

gacaccttta ctttacagtc tggtgttcgg tgagggtgtt gttaatgatg ctacatctgt 720

ggtgcttttc aatgcaatcc agagctttga tctctctaat ttgaacacca gctctgcttt 780

tcagcttctt ggcaacttct tatatttatt tttcgcaagt actatgcttg gcgtcattac 840

tggactactt agcgcttata ttatcaaaaa gctatatttt gccaggcact caacggaccg 900

tgaggttgca ctgatgatgc ttatggcata cctctcatac atgctggctg aactctttga 960

catgagtgga attctcacag tatttttctg tgggattgtg atgtcccatt acacctggca 1020

caatgtgaca gagagttcaa gaatcactac caagcatgct tttgcaacct tatcatttgt 1080

tgccgagatc tttctctttc tctatgttgg tatggatgcc ctggacattg agaagtggag 1140

gtttgtaagc gacagccctg gaacatcagt tgcagtaagt tcgatactga tgggtttggt 1200

gatgttggga agagcagctt ttgtttttcc tttatccttc gtctccaatt tgatgaagaa 1260

atcacctacg gataaagtgg gcttcaaaca gcagattgtg atatggtggg ctgggctcat 1320

gagaggtgct gtgtctatgg ctcttgctta caatcagttt acaaggtcag ggcacactca 1380

attgcggggg aatgcggtaa tgatcacaag cacaataact gttgttcttt tcagcacagt 1440

ggtctttggt ttgatgacta aacctctcat aaggttactt ctacctcagc aaaaagccgc 1500

aagaagcatg tcactatcgg atccagaaaa ccaaaaatca gtgaccacac cgcttctcgg 1560

acaatcacaa gactctgagg ctgaccttgg tagcacacca cttggcaacg gtatccatcg 1620

gccaggtagc ttacgtgcac ttctaaatgc tcctacacac acggtccact actattggcg 1680

taaatttgat gatgccttta tgcggcctgc ctttggtggc cgaggcttta cccccttcgt 1740

tccgggctca ccaacagaac ggagcgtccc ccaatggcag tgagagaaga aaactaatcg 1800

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aaaaaaaaaa aaaaaaaaaa aa 2182

<210>2

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Met Asp Gln Leu Ser Ser Val Val Ser Arg Leu Gln Met Val Ser Thr

1 5 10 15

Ser Asp His Ser Ser Val Val Ser Met Asn Leu Phe Val Ala Leu Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Phe Gln Val Lys 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

210 215 220

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

225 230 235 240

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

500 505 510

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

515 520 525

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

530 535 540

Claims (10)

1. Use of NsNHX1 protein in any of the following 1)-10): 1) Regulate plant stress tolerance; 2) Regulate plant growth and development; 3) Regulation of plant biomass; 4) Regulate plant height; 5) Regulate the chlorophyll content of plants; 6) Regulate the water content of plants; 7) Regulate the antioxidant capacity of plants; 8) regulating plant superoxide dismutase and/or peroxidase and/or catalase activity; 9) regulating plant proline content; 10) Regulate the content of malondialdehyde in plants. 2. Use of a biological material related to NsNHX1 protein in any of the following 1)-12): 1) Regulate plant stress tolerance; 2) Regulate plant growth and development; 3) Regulation of plant biomass; 4) Regulate plant height; 5) Regulate the chlorophyll content of plants; 6) Regulate the water content of plants; 7) Regulate the antioxidant capacity of plants; 8) regulating plant superoxide dismutase and/or peroxidase and/or catalase activity; 9) regulating plant proline content; 10) regulating the content of malondialdehyde in plants; 11) Cultivate transgenic plants with improved stress tolerance or cultivate stress-tolerant plant varieties; 12) Plant breeding. 3. The application according to claim 1 or 2, wherein the NsNHX1 protein is the protein shown in the following a) or b) or c) or d): a) the amino acid sequence is the protein shown in sequence 2; b) a fusion protein obtained by linking a tag to the N-terminal and/or C-terminal of the protein shown in SEQ ID NO: 2; c) a protein with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in Sequence 2; d) A protein having 75% or more homology with the amino acid sequence shown in SEQ ID NO: 2 and having the same function. 4. The application according to claim 2 or 3, wherein the biological material is any one of the following A1) to A12): A1) a nucleic acid molecule encoding the NsNHX1 protein; A2) an expression cassette containing the nucleic acid molecule of A1); A3) a recombinant vector containing the nucleic acid molecule of A1); A4) a recombinant vector containing the expression cassette described in A2); A5) a recombinant microorganism containing the nucleic acid molecule of A1); A6) a recombinant microorganism containing the expression cassette described in A2); A7) a recombinant microorganism containing the recombinant vector described in A3); A8) a recombinant microorganism containing the recombinant vector described in A4); A9) a transgenic plant cell line containing the nucleic acid molecule of A1); A10) a transgenic plant cell line containing the expression cassette of A2); A11) a transgenic plant cell line containing the recombinant vector described in A3); A12) A transgenic plant cell line containing the recombinant vector described in A4). 5. application according to claim 4 is characterized in that: A1) described nucleic acid molecule is the gene shown in following 1) or 2) or 3): 1) Its coding sequence is the cDNA molecule shown in position 149-1783 of sequence 1 or the genomic DNA molecule shown in sequence 1; 2) a cDNA molecule or a genomic DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) and encodes the NsNHX1 protein described in claim 1; 3) A cDNA molecule or a genomic DNA molecule that hybridizes to the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the NsNHX1 protein described in claim 1. 6. The application according to any one of claims 1-5, wherein the stress resistance is salt resistance and/or alkali resistance. 7. A method for cultivating a transgenic plant with improved stress tolerance, comprising the steps of increasing the expression and/or activity of NsNHX1 protein in a recipient plant to obtain a transgenic plant; the stress tolerance of the transgenic plant is higher than that of the recipient plant. body plants. 8. The method according to claim 7, wherein the stress resistance is salt resistance and/or alkali resistance; Or, the stress tolerance of the transgenic plant is higher than that of the recipient plant as shown in any one of the following (1)-(9): (1) The survival rate of transgenic plants is higher than that of recipient plants; (2) The biomass or root biomass of the transgenic plant is higher than that of the recipient plant; (3) The chlorophyll content in the leaves of the transgenic plants is higher than that of the recipient plants; (4) The water content in the leaves of the transgenic plants is greater than that of the recipient plants. (5) The plant height of the transgenic plant is higher than that of the recipient plant; (6) The antioxidant capacity of transgenic plants is higher than that of recipient plants; (7) The superoxide dismutase and/or peroxidase and/or catalase activity in the leaves of the transgenic plant is higher than that of the recipient plant; (8) The proline content in the leaves of the transgenic plants is higher than that of the recipient plants; (9) The content of malondialdehyde in leaves of transgenic plants was lower than that of recipient plants. 9. The method according to claim 7 or 8, wherein the method for increasing the expression and/or activity of NsNHX1 protein in recipient plants is to overexpress NsNHX1 protein in recipient plants; Or, the method for overexpression is to introduce the gene encoding the NsNHX1 protein into the recipient plant; Or, the nucleotide sequence of the gene encoding the NsNHX1 protein is the DNA molecule shown at positions 149-1783 of SEQ ID NO: 1. 10. The application according to any one of claims 1-6 or the method according to any one of claims 7-9, wherein the plant is a monocotyledonous plant or a dicotyledonous plant; or, the monocotyledonous plant for poplar.