CN110256544A

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

Info

Publication number: CN110256544A
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: 2019-09-20
Anticipated expiration: 2039-05-30
Also published as: CN110256544B

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

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

Technical field

The invention belongs to field of biotechnology, and in particular to NsNHX1 protein and its relevant biological material are resistance to inverse in cultivation Application in type poplar.

Background technique

Plant root is Na in direct contact soil⁺Organ, therefore the Na of root⁺Outlet and aerial part cell are to Na⁺ Separating concentrate be determine plant salt endurance important mechanisms.Na on tonoplast⁺/H⁺Reverse transport protein (NHX1) will be thin Na in cytoplasm⁺Inverse concentration gradient transports separating in vacuole；Na on cytoplasma membrane⁺/H⁺Reverse transport protein (SOS1) energy Enough by the Na in cytoplasm⁺Inversely it is transported to extracellular high Na⁺In environment.Up to now, the Na for developing and applying⁺/H⁺It is inverse To transporter gene, mostly from glycophytes such as arabidopsis, tomatoes, and for xylophyta, particularly to the raw wood of salt This plant Na⁺/H⁺The exploitation and research of antiporter gene are more deficient.Existing research discovery, the expression of salt mustard ThSOS1 Amount is about 8~10 times of arabidopsis AtSOS1, and has found similar SOS in glycophyte rice and halophytes salt mustard Signal path shows that higher plant may share a set of salt tolerant regulatory mechanism.Therefore, the Na of halophytes is developed and used⁺/H⁺It is inverse It is had a very important significance to transporter gene.

White thorn (Nirtaria L.) is a kind of machaka of zygophyllaceae (Zygophyllceae) Nitraria, raw for salt Plant is mainly distributed on the ground such as the Inner Mongol, Ningxia, Gansu, Qinghai and the Xinjiang of China the north to the northwestward, is arid salt-soda soil One of constructive species of band.The cauline leaf of Nitraria plant has thicker cuticula, blade by wax epidermal hair, stomata invagination, grid Column tissue and vascular bundle prosperity, cell inclusion fill wax crystal, these typical xerophyte structure features are supporting it It plays a role in imperial salt damage.Stronger film protective capability and film repair ability under condition of salt stress, and efficient ion intracellular The accumulation ability of separating and osmotic adjustment assigns the stronger salt tolerance of Nitraria plant.There are four Inner Mongolia is main Kind, i.e., Nitraria sibirica (N.sibilica Pall), Nitraria tangutorum (N.tangutorum Bobr), bubble pierce The white thorn (N.roborowskii Kom) of (N.sphaerocarpa Maxim) and tingia, wherein Nitraria sibirica is shown more Strong salt resistance ability.Nitraria sibirica have the extremely important ecological value, due to its in natural habitat by salt, alkali, Triple stress of drought, cause it with stronger saline-alkali tolerant and Drought resistance, contain adversity gene resource abundant.Cause This, studies its Mechanism of Salt-tolerant, identifies salt-resistant related gene and is applied to the salt tolerance genetic improvement of herbage, farm-forestry crop, has Highly important meaning.

Summary of the invention

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

In order to solve the above-mentioned technical problem, present invention firstly provides the new applications of NsNHX1 protein.

The present invention provides NsNHX1 protein following 1) -10) in it is any in application:

1) regulate and control plant stress tolerance；

2) regulating growth of plants；

3) regulate and control phytomass；

4) regulate and control plant plant height；

5) regulate and control chlorophyll content of plant；

6) regulate and control plant water content；

7) regulate and control plant anti-oxidation ability；

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

9) regulate and control plant proline content；

10) regulate and control plant mda content；

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

A) amino acid sequence is protein shown in sequence 2；

B) fused protein that the N-terminal of the protein shown in sequence 2 and/or C-terminal connection label obtain；

C) by amino acid sequence shown in sequence 2 by one or several amino acid residues substitution and/or missing and/or Add obtained protein with the same function；

D) homology with amino acid sequence shown in sequence 2 with 75% or 75% or more and egg with the same function White matter.

In order to make protein in a) convenient for purifying, can in sequence table the amino terminal of protein shown in sequence 2 or Carboxyl terminal connects upper label as shown in Table 1.

The sequence of table 1, label

Label	Residue	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

It is above-mentioned c) in protein, the substitutions of one or several amino acid residues and/or deletion and/or addition is not More than the substitution and/or deletion and/or addition of 10 amino acid residues.

It is above-mentioned c) in protein can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression and obtain.

It is above-mentioned c) in the encoding gene of protein can be by will be lacked in DNA sequence dna shown in sequence 1 149-1783 The codon of one or several amino acid residues is lost, and/or carries out the missense mutation of one or several base-pairs, and/or at it The coded sequence that 5 ' ends and/or 3 ' ends connect label shown in table 1 obtains.

It is above-mentioned d) in, " homology " include with amino acid sequence shown in sequence of the invention 2 have 75% or higher, or 80% or higher or 85% or higher or 90% or higher or 95% or more high homology amino acid sequence.

In order to solve the above-mentioned technical problem, invention further provides the new use of biomaterial relevant to NsNHX1 protein On the way.

The present invention provides biomaterials relevant to NsNHX1 protein following 1) -12) in it is any in application:

1) regulate and control plant stress tolerance；

2) regulating growth of plants；

3) regulate and control phytomass；

4) regulate and control plant plant height；

5) regulate and control chlorophyll content of plant；

6) regulate and control plant water content；

7) regulate and control plant anti-oxidation ability；

9) regulate and control plant proline content；

10) regulate and control plant mda content；

11) genetically modified plants that resistance of reverse improves are cultivated；

12) plant breeding.

In above-mentioned application, the biomaterial is following A 1) any one of to A12):

A1 the nucleic acid molecules of NsNHX1 protein) are encoded；

A2) contain A1) expression cassettes of the nucleic acid molecules；

A3) contain A1) recombinant vectors of the nucleic acid molecules；

A4) contain A2) recombinant vector of the expression cassette；

A5) contain A1) recombinant microorganisms of the nucleic acid molecules；

A6) contain A2) recombinant microorganism of the expression cassette；

A7) contain A3) recombinant microorganism of the recombinant vector；

A8) contain A4) recombinant microorganism of the recombinant vector；

A9) contain A1) the transgenic plant cells systems of the nucleic acid molecules；

A10) contain A2) the transgenic plant cells system of the expression cassette；

A11) contain A3) the transgenic plant cells system of the recombinant vector；

A12) contain A4) the transgenic plant cells system of the recombinant vector.

In above-mentioned application, A1) nucleic acid molecules be it is following 1) or 2) or 3) shown in gene:

1) its coded sequence is genomic DNA shown in cDNA molecule shown in sequence 1 149-1783 or sequence 1 point Son；

2) there is 75% or 75% or more identity with the nucleotide sequence 1) limited, and encodes NsNHX1 protein CDNA molecule or genomic DNA molecule；

1) or 2) 3) and cDNA points of NsNHX1 protein are encoded with the nucleotide sequence hybridization that limits under strict conditions Son or genomic DNA molecule.

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

Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation Method is mutated the nucleotide sequence of coding NsNHX1 protein of the invention.Those are by manually modified, with coding The nucleotide sequence 75% of NsNHX1 protein or the nucleotide of higher identity, as long as encoding NsNHX1 protein and having Identical function is derived from nucleotide sequence of the invention and to be equal to sequence of the invention.

Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair Amino acid sequence shown in bright coded sequence 2 composition protein nucleotide sequence have 75% or higher or 85% or Higher or 90% or higher or 95% or higher identity nucleotide sequence.Identity can with the naked eye or computer software It is evaluated.Using computer software, identity between two or more sequences can be indicated with percentage (%), can be with For evaluating the identity between correlated series.

Above-mentioned 75% or 75% or more identity can be 80%, 85%, 90% or 95% or more identity.

In above-mentioned application, the stringent condition is to hybridize at 68 DEG C in 2 × SSC, the solution of 0.1%SDS and wash film 2 times, each 5min, but in 0.5 × SSC, the solution of 0.1%SDS, hybridize at 68 DEG C and washes film 2 times, each 15min； Or, hybridizing under the conditions of 65 DEG C in the solution of 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS and washing film.

In above-mentioned application, A2) described in the expression cassettes of the nucleic acid molecules containing coding NsNHX1 protein be to refer to The DNA of NsNHX1 protein is expressed in host cell, which may include not only the promoter for starting NsNHX1 transcription, can also wrap Include the terminator for terminating NsNHX1 transcription.Further, the expression cassette may also include enhancer sequence.It is for use in the present invention Promoter includes but is not limited to: constitutive promoter；It organizes, the promoter and inducible promoter that organ and development are special.

The recombinant vector of the NsNHX1 expression casette can be contained with existing expression vector establishment.The plant expression Carrier includes double base agrobacterium vector and the carrier etc. that can be used for plant micropellet bombardment.As pAHC25, pBin438, PCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or PCAMBIA1391-Xb (CAMBIA company) etc..The plant expression vector also may include 3 ' end non-translational regions of foreign gene Domain, i.e., comprising polyadenylation signals and any other DNA fragmentation for participating in mRNA processing or gene expression.The polyadenylic acid letter Number bootable polyadenylic acid is added to 3 ' ends of mRNA precursor, as Agrobacterium crown gall nodule induces (Ti) plasmid gene (such as nopaline Synthase gene Nos), plant gene (such as soybean storage protein genes) 3 ' end transcription non-translational region all have similar functions. When using gene constructed plant expression vector of the invention, enhancer, including translational enhancer or transcriptional enhancer also can be used, These enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but must read with coded sequence Frame is identical, to guarantee the correct translation of entire sequence.The source of the translation control signal and initiation codon be it is extensive, Can be it is natural, be also possible to synthesis.Translation initiation region can come from transcription initiation region or structural gene.In order to just In transgenic plant cells or plant are identified and screened, plant expression vector used can be processed, it can as being added The coding expressed in plant can produce the enzyme of color change or gene (gus gene, luciferase genes of luminophor Deng), the marker gene of antibiotic (if assigned the nptII gene to kanamycins and associated antibiotic resistance, assigns to herbicide The bar gene of phosphinothricin resistance assigns the hph gene to antibiotic hygromycin resistance, and assigns to methotrexate resistance Dhfr gene is assigned to the EPSPS gene of glyphosate) or (such as anti-herbicide base such as anti-chemical reagent marker gene Cause), provide metabolism mannose ability mannose-6-phosphate isomerase gene.It, can not from the security consideration of genetically modified plants Add any selected marker, transformed plant is directly screened with adverse circumstance.

In above-mentioned application, the carrier can be plasmid, sticking grain, bacteriophage or viral vectors.The plasmid can be pBI101- 35::Gus-Hm。

In above-mentioned application, the microorganism can be yeast, bacterium, algae or fungi, such as Agrobacterium.The Agrobacterium can be GV3101。

In above-mentioned application, the transgenic plant cells system, Transgenic plant tissue and genetically modified plants organ are not wrapped Include propagation material.

In above-mentioned application, the resistance of reverse can be salt tolerance and/or alkali resistance.

The regulation plant stress tolerance is to improve plant stress tolerance.It is embodied in: under salt or alkaline stress, being planted with receptor Object is compared, and the chlorophyll content turned in survival rate raising, biomass or the raising of root biomass, blade of NsNHX1 plant mentions Water content high, in blade improves, plant height improves, oxidation resistance improves, hepatocuprein and/or peroxide in blade Compound enzyme and/or catalase activity improve, the proline content in blade improves, the mda content in blade reduces. The salt is specially NaCl, the NaCl concentration concretely 50mM or 100mM or 150mM；The alkali concretely NaHCO₃； The NaHCO₃Concentration concretely 100mM or 200mM or 300mM.

The regulating growth of plants is to promote plant growth and development or plant root growth is promoted to develop.

The regulation phytomass is to improve phytomass；The biomass can be root biomass or plant biology Amount.

The a height of raising plant plant height of regulation plant strain.

The regulation chlorophyll content of plant is the chlorophyll content improved in plant leaf blade.

The regulation plant water content is the water content improved in plant leaf blade.The water content is relative water content.

The regulation plant anti-oxidation ability is to improve plant anti-oxidation ability.It is embodied in: under condition of salt stress, Compared with recipient plant, turn hepatocuprein in NsNHX1 plant leaf blade and/or peroxidase and/or hydrogen peroxide Enzymatic activity improves, and proline content increases, and mda content is reduced.

The regulation plant superoxide dismutase and/or peroxidase and/or catalase activity are to improve to plant Hepatocuprein and/or peroxidase and/or catalase activity in object blade.

The regulation plant proline content is the proline content improved in plant leaf blade.

The regulation plant mda content is the mda content reduced in plant leaf blade.

In order to solve the above-mentioned technical problem, the present invention finally provides a kind of genetically modified plants of cultivation resistance of reverse raising Method.

The method provided by the invention for cultivating the genetically modified plants that resistance of reverse improves includes improving NsNHX1 in recipient plant The expression quantity and/or activity of protein, the step of obtaining genetically modified plants；The resistance of reverse of the genetically modified plants be higher than it is described by Body plant.

Further, the resistance of reverse is salt tolerance and/or alkali resistance.

The resistance of reverse of the genetically modified plants is higher than the recipient plant and is embodied in: under salt or alkaline stress, turning base Because the survival rate of plant is higher than recipient plant；And/or the biomass or root biomass of genetically modified plants are higher than recipient plant； And/or the chlorophyll content in Transgenic plant leaf is higher than recipient plant；Water content in Transgenic plant leaf is (opposite Water content) it is greater than recipient plant；The plant height of genetically modified plants is higher than recipient plant；And/or the oxidation resistance of genetically modified plants Higher than recipient plant；And/or hepatocuprein and/or peroxidase and/or peroxidating in Transgenic plant leaf Hydrogenase activity is higher than recipient plant；And/or the proline content in Transgenic plant leaf is higher than recipient plant；And/or turn Mda content in gene plant blade is lower than recipient plant.The salt is specially NaCl, and the NaCl concentration is concretely 50mM or 100mM or 150mM；The alkali concretely NaHCO₃；The NaHCO₃Concentration concretely 100mM or 200mM or 300mM。

Further, it is described improve recipient plant in NsNHX1 protein expression quantity and/or active method be NsNHX1 protein is overexpressed in recipient plant；

The method of the overexpression is that the encoding gene of NsNHX1 protein is imported recipient plant.The NsNHX1 albumen The encoding gene of matter specifically can be by using Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity It leads, the conventional biology methods such as mediated by agriculture bacillus conversion plant cell or tissue, and the plant tissue of conversion is cultivated into plant.

The nucleotide sequence of the encoding gene of the NsNHX1 protein is DNA shown in sequence 1 149-1783 points Son.

In a specific embodiment of the present invention, the encoding gene of the NsNHX1 protein passes through recombinant vector pBI101- NsNHX1 is imported in recipient plant, and the recombinant vector pBI101-NsNHX1 is by the XbaI of pBI101-35::Gus-Hm carrier Segment between SacI restriction enzyme site replaces with DNA fragmentation shown in sequence 1 149-1783, and keeps pBI101-35:: The carrier obtained after the other sequences of Gus-Hm carrier are constant.

In the above method, the genetically modified plants are interpreted as not only comprising obtaining the NsNHX1 genetic transformation recipient plant The first generation genetically modified plants arrived also include its filial generation.For genetically modified plants, the gene can be bred in the species, The gene transfer can be entered to other kinds of same species with traditional breeding techniques, particularly including in commercial variety.Described turn Gene plant includes seed, callus, intact plant and cell.

In the above method or application, the plant is monocotyledon or dicotyledon.Further, the unifacial leaf Plant can be poplar；Further, the poplar can be Chinese white poplar.In one particular embodiment of the present invention, the hair White poplar can be Chinese white poplar 84K (Populus alba × Populus glandulose).

The present invention is with Nitraria sibirica Na⁺/H⁺Antiporter gene NsNHX1 is research object, is transferred to hair White poplar 84K (Populus alba × Populus glandulose), acquisition turn NsNHX1 Chinese white poplar.Choose wherein N1, N2 and Tri- transformants of N3 carry out resistance to reverse function identification.It is control with wild type Chinese white poplar, research turns NsNHX1 Chinese white poplar coerces in adverse circumstance Saline-alkali tolerant and oxidation resistance under compeling.As a result, it has been found that turning survival rate, the biology of NsNHX1 Chinese white poplar under the conditions of environment stress Amount, chlorophyll content in leaf blades and water content, plant height and oxidation resistance are all remarkably higher than wild type Chinese white poplar.Show NsNHX1 The resistance to inverse ability and oxidation resistance of transgenic poplar can be significantly improved, NsNHX1 can be applied to cultivate resistance of reverse poplar product Kind.

Detailed description of the invention

Fig. 1 is the amplification of NsNHX1 ORF.

The double digestion that Fig. 2 is recombinant vector pBI101-NsNHX1 is identified.

Fig. 3 is the genetic transformation of Chinese white poplar.(A) cutting and preculture of explant；(B) it disseminates；(C) differentiation culture；(D) Screening；(E) culture of rootage；(F) it transplants.

Fig. 4 is the PCR identification for turning NsNHX1 Chinese white poplar.M:200bp DNA Ladder；+: recombinant plasmid pBI101- NsNHX1；: wild type Chinese white poplar；N1, N2, N3: turn NsNHX1 Chinese white poplar.

Fig. 5 is the RT-PCR analysis for turning NsNHX1 Chinese white poplar.WT: wild type Chinese white poplar；N1, N2, N3: turn NsNHX1 maos it is white Poplar.

Fig. 6 is that wild type Chinese white poplar and three turn growth of the NsNHX1 Chinese white poplar strain under condition of salt stress and compare.(A) 2 weeks plant are cultivated in the P5 culture medium containing 0mM, 50mM, 100mM, 150mM and 200mM NaCl；(B) the growth feelings of root Condition compares；(C) 2 weeks plant are cultivated in the P5 culture medium containing 0mM, 50mM, 100mM, 150mM and 200mM NaCl；(D) wild Raw type Chinese white poplar and the biomass for turning NsNHX1 Chinese white poplar strain root；(E) wild type Chinese white poplar and turn NsNHX1 Chinese white poplar Survival rate.WT: wild type Chinese white poplar；N1, N2, N3: turn NsNHX1 Chinese white poplar.Error line represents standard deviation (SD), from Three independent biology repeat to test；The different lowercases of label respectively represent the significant difference (P between every group of sample <0.05)

Fig. 7 is that the expansion of Chinese white poplar is numerous.(A) by seedling cuttage into culture medium；It (B) will be in seedling replanting to soil；(C) it covers Preservative film；(D) it is ready for the seedling of salt stress.

Fig. 8 is the resistance of reverse analysis for turning NsNHX1 Chinese white poplar.(A) Stress treatment；(B) turn in NsNHX1 leaves of Populus Tomentosa Chlorophyll content；Error line represents standard deviation (SD), repeats to test from three independent biology；The difference of label is small The mother that writes respectively represents the significant difference (P < 0.05) between every group of sample.WT: wild type Chinese white poplar；N1, N2, N3: turn NsNHX1 Chinese white poplar.

Fig. 9 is that NaCl coerces lower wild type Chinese white poplar and turns the phenotypic analysis of NsNHX1 Chinese white poplar.(A) salt stress is to wild Type Chinese white poplar and the influence (scale bar=10cm) for turning NsNHX1 Chinese white poplar complete stool；(B) salt stress to wild type Chinese white poplar and turns The influence of NsNHX1 leaves of Populus Tomentosa.

Figure 10 is the resistance of reverse analysis for turning NsNHX1 Chinese white poplar.(A) biomass；(B) plant height；(C) leaf chlorophyll Content；(D) leaf r elative water content；(E) SOD activity；(F) POD activity；(G) CAT activity；(H) proline content；(I)MDA Content.WT: wild type Chinese white poplar；N1, N2, N3: turn NsNHX1 Chinese white poplar.Error line represents standard deviation (SD), from three A independent biology repeats to test；The different lowercases of label respectively represent between every group of sample significant difference (P < 0.05)。

Specific embodiment

Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even Mean value.

Vegetable material Chinese white poplar 84K (Populus alba × Populus involved in following embodiments Glandulose) the condition of culture of sterile tissue-cultured seedling: temperature is 24 ± 2 DEG C, and intensity of illumination is 200 μm of olm^-2·s^-1, illumination Period is 16h illumination/8h dark.

PBI101-35::Gus-Hm carrier is recorded in document " WANG L, MA Y K, LI N N, et in following embodiments al.Isolation and characterization of a tonoplast Na⁺/H⁺ antiporter from the Halophyte Nitraria sibirica [J] .BIOLOGIA PLANTARUM, 2016,60 (1): 113-122, in 2016 ", The public can obtain from applicant, which only attaches most importance to used in the related experiment of duplicate invention, not can be used as other purposes It uses.

Culture medium prescription involved in following embodiments is as follows:

Precultivation 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 (200 μm of ol/mL).

Differential 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 and culturing 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).

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

Huo Gelan culture medium (Huo Gelan nutrient solution) and MS culture medium are Phytotechnology Laboratories The product of (https: //phytotechlab.com).

Nitraria sibirica Na in following embodiments⁺/H⁺Sequence is such as antiporter gene (abbreviation NsNHX1 gene) Shown in sequence 1, the open reading frame (sequence 1 149-1783) comprising a 1635bp, the west primary of NsNHX1 gene coding The white thorn Na of Leah⁺/H⁺The amino acid sequence of reverse transport protein (abbreviation NsNHX1 albumen) is as shown in sequence 2.

Embodiment 1, the acquisition for turning NsNHX1 Chinese white poplar and its resistance of reverse analysis

One, turn the acquisition of NsNHX1 Chinese white poplar

1, the building of recombinant vector

(1) amplification of target gene

According to Nitraria sibirica Na⁺/H⁺Antiporter gene (abbreviation NsNHX1) sequence (the gene order number of logging in: AB859847 pair of primers (NHX-pBI-F:5 '-GC) is designedTCTAGAATGGATCAATTAAGTT-3 ', 5 ' ends include that XbaI knows Other sequence；NHX-pBI-R:5 '-CGAGCTCTCACTGCCATTGGGGGAT-3 ', 5 ' ends include that SacI identifies sequence), Yi Xibai The RNA of the white thorn Nitraria sibirica Pall of Leah is template, using NHX-pBI-F and NHX-pBI-R primer, is utilized TaKaRa RNA PCR Kit (AMV) Ver.3.0 kit expands the region NsNHX1 ORF, obtains PCR product.Reaction condition Are as follows: 94 DEG C of 3min；94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 1min, 35cycles；72℃5min.The electrophoresis result of PCR product such as Fig. 1 It is shown.

(2) target gene is connect with expression vector

Binary expression vector pBI101-35::Gus-Hm and above-mentioned PCR product carried out respectively using XbaI and SacI double Digestion obtains recombinant vector pBI101-NsNHX1 after being then attached using T4 ligase.And to recombinant vector pBI101- NsNHX1 carries out sequencing and digestion identification.

Sequencing result shows: recombinant vector pBI101-NsNHX1 be by the XbaI of pBI101-35::Gus-Hm carrier and Segment between SacI restriction enzyme site replaces with DNA fragmentation shown in sequence 1 149-1783, and keeps pBI101-35:: The carrier obtained after the other sequences of Gus-Hm carrier are constant.

The digestion qualification result of recombinant vector pBI101-NsNHX1 is as shown in Figure 2.Weight after double digestion and sequencing identification Group carrier pBI101-NsNHX1 is used for the genetic transformation of poplar.

2, the screening and identification of the genetic transformation of Chinese white poplar and genetically modified plants

Recombinant vector pBI101-NsNHX1 is converted into Chinese white poplar with the leaf disk method that agrobacterium strains GV3101 is mediated, is obtained Transgenosis Chinese white poplar.The process of Chinese white poplar genetic transformation is as shown in Figure 3, the specific steps are as follows:

(1) the sterile tissue culture of eugonic Chinese white poplar 84K (Populus alba × Populus glandulose) is chosen Seedling leaf, vertical vane main lobe arteries and veins cut 3-4 knife, then vacuum side of blade are laid in upward in P1 culture medium, and preculture 4 days.

(2) recombinant vector pBI101-NsNHX1 is imported in GV3101 Agrobacterium, is obtained containing pBI101-NsNHX1's GV3101 Agrobacterium.

(3) the GV3101 Agrobacterium single colonie containing pBI101-NsNHX1 that picking is activated through coated plate, is added to and contains In the LB liquid medium of 20mg/L kanamycins, 50mg/L rifampin and 200 μm of ol/L acetosyringones, 28 DEG C of constant temperature oscillations About 8h is cultivated, until OD₆₀₀For 0.6-0.8,0.01% Silwet L-77 is added as dip dyeing liquid for shell.

(4) blade of preculture is placed in dip dyeing liquid for shell and impregnates 10min, blot blade surface with filter paper after taking-up blade Vacuum side of blade is laid in P2 culture medium by bacterium solution upward, 24 DEG C dark culture 2-3 days.

(5) to be seen to after there is Agrobacterium bacterium colony on P2 culture medium, with ceftiofur sodium containing 300mg/L and 200mg/L The sterilizing ddH of Ticarcillin/Clavulanate Acid₂Blade is transferred in P3 culture medium and continues to cultivate by O cleaning blade, and suck dry moisture, about after two weeks, Occurs the adventitious bud broken up on blade.

(6) long to 1-2cm to adventitious bud, it is cut in the P4 culture medium for be transferred to the kanamycins containing 50mg/L and is sieved Choosing selects resistance seedling and carries out PCR identification after three weeks.

(7) NsNHX1 Chinese white poplar plant will be turned by the positive of tissue PCR identification and is transferred to ceftiofur sodium containing 100mg/L P5 culture medium in carry out culture of rootage, the transgenic plant after taking root is transplanted to Nutrition Soil, be placed in a greenhouse cultivate and be used for Resistance of reverse detection.

3, turn the PCR identification of NsNHX1 Chinese white poplar

Using the TransDirect Plant Tissue PCR Kit of Quan Shi King Company, primer 35S-F:5 '-is used It is white that AGGAAGGTGGCTCCTACAAATG-3 ' and NHX1-pBI-R:5 '-CGAGCTCTCACTGCCATTGGGGGA-3 ' carries out hair The PCR of poplar resistant plant is identified.

Reaction condition are as follows: 94 DEG C of 5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min30s, 35cycles；72℃5min；12 ℃Forever。

PCR product finds through detected through gel electrophoresis, three resistant plants N1, N2, N3 and recombinant vector pBI101-NsNHX1 Amplifiable size out is about 1.9kb (the NsNHX ORF sequence+6bp digestion sequence of the 35S promoter sequence+1635bp of 252bp Column) NsNHX1 specific band, and not no band of wild type Chinese white poplar shows foreign gene NsNHX1 by successfully It is transferred to Chinese white poplar.

4, turn the RT-PCR analysis of NsNHX1 Chinese white poplar

The plant for being confirmed as turning NsNHX1 Chinese white poplar by tissue PCR identification is transferred to ceftiofur sodium containing 100mg/L P5 culture medium in carry out culture of rootage, after plant leaf is grown up carry out RT-PCR detection.With TaKaRa MiniBEST Plant RNA Extraction Kit kit (the program Protocol-I that specification is shown in operating process) extracts positive strain RNA, useFirst-Strand cDNA Synthesis SuperMix Kit synthesizes the first chain of cDNA, The expression of NHX-pBI-F and NHX1-pBI-R primer detection NsNHX1 gene.Using Chinese white poplar Actin gene as internal reference, primer is Actin (Populus)-F:5 '-AAACTGTAATGGTCCTCCCTCCG-3 ', Actin (Populus)-R:5 '- GCATCATCACAATCACTCTCCGA-3 ', reaction condition: 94 DEG C of 3min；94 DEG C of 30s, 63 DEG C of 30s, 72 DEG C of 20s, 30cycles；72℃5min.

As a result as shown in Figure 5.The result shows that: turn amplify in NsNHX1 Chinese white poplar strain N1, N2, N3 at three NsNHX1 expression product.Illustrate that NsNHX1 gene stablizes expression in turning NsNHX1 Chinese white poplar.

Two, turn the resistance of reverse detection of NsNHX1 Chinese white poplar

1, turn resistance of reverse detection of NsNHX1 Chinese white poplar under the conditions of sterile culture

It chooses eugonic wild type Chinese white poplar and turns the sterile tissue-cultured seedling of NsNHX1 Chinese white poplar, intercept the cuttings of 5~6cm (retaining terminal bud and three pieces leaf).Wild type Chinese white poplar and three turn NsNHX1 Chinese white poplar strain N1, N2, N3 and respectively choose 20 plants, Cuttage carries out salt stress processing into the P5 culture medium containing various concentration NaCl (0,50,100,150 and 200mM) respectively.Place Reason after two weeks, measures the survival rate of plant and the biomass (the fresh amount of root) of root.

As a result as shown in Figure 6A.The result shows that: under the conditions of 0mM, 50mM and 100mM NaCl, wild type Chinese white poplar and three A phenotype for turning NsNHX1 Chinese white poplar strain is without marked difference (Fig. 6 A), and survival rate is 100%, but three turn NsNHX1 maos The biomass of white poplar strain root is all remarkably higher than wild type Chinese white poplar (Fig. 6 B, D)；Under the conditions of 150mM NaCl, wild type Chinese white poplar and three turn NsNHX1 Chinese white poplar strain and phenomena such as yellow, necrosis, growth inhibition occur, but three turn NsNHX1 The survival rate (N1-83.33%, N2-50%, N3-66.67%) of Chinese white poplar strain is significantly higher than the survival rate of wild type Chinese white poplar (25%) (Fig. 6 E)；Under the conditions of 200mM NaCl, wild type Chinese white poplar and three turned NsNHX1 Chinese white poplar strain by two weeks Salt stress is all dead, shows that the transgenic poplar for overexpressing NsNHX1 can be in culture of the NaCl concentration not higher than 150mM (Fig. 6 A) is survived in base.On the other hand, the root system development for overexpressing NsNHX1 transgenosis Chinese white poplar is good compared with wild type Chinese white poplar, nothing By being under normal growth or condition of salt stress, three biomass for turning NsNHX1 Chinese white poplar strain root are all remarkably higher than open country Raw type Chinese white poplar (Fig. 6 B, C, D).The above result shows that the overexpression of NsNHX1 not only increases the salt tolerant of transgenic poplar Property, and promote the growth and development (Fig. 6) of root system.

2, the resistance of reverse detection for turning NsNHX1 Chinese white poplar being transplanted in soil

It chooses eugonic wild type Chinese white poplar and turns the sterile tissue-cultured seedling of NsNHX1 Chinese white poplar, 5~6cm's of interception inserts Fringe, retains terminal bud and three pieces leaf, cuttage carry out culture of rootage into P5 culture medium.It cultivates in illumination box and turns after two weeks It moves on on illumination cultivation frame and continues to cultivate 3 days (plant has sent out roots at this time), plant is taken out from culture medium, it is residual to clean root Remaining culture medium is simultaneously dried.Then it is transplanted in soil (ratio of peat soil and vermiculite is 5:1) hardening, pours Huo Gelan nutrition Liquid covers preservative film moisturizing, sooner or later respectively primary with Huo Gelan nutrient solution sprinkling blade daily.Remove preservative film after a week, obtains The almost the same plant of growing way.

(1) leaf disk method detection turns the saline-alkali tolerant and oxidation resistance of NsNHX1 Chinese white poplar

By cuttage culture three weeks wild type Chinese white poplars and turns the sterile tissue-cultured seedling of NsNHX1 Chinese white poplar and is transplanted in peat soil, Hot-house culture after two months, choose the blade unfolded of same position stalwartness, avoid main lobe arteries and veins with punch and break into blade directly The leaf disc of diameter 1cm.Leaf disc is put into respectively NaCl solution (0mM, 50mM, 100mM and 150mM) containing various concentration, NaHCO₃Solution (0mM, 100mM, 200mM and 300mM) and H₂O₂Solution (0%, 1.0%, 1.5% and 2.0%), is placed in illumination Period is 16h illumination/8h dark environment, utilizes Suzhou Ke Ming Bioisystech Co., Ltd plant leaf green after 24 DEG C of incubation 72h Chlorophyll content in cellulose content kit measurement blade.

As a result as shown in Figure 8, the results showed that: under conditions of 0mM and 50mM NaCl, wild type Chinese white poplar and turn The leaf disc of NsNHX1 Chinese white poplar does not have significant difference；But under conditions of 100mM and 150mM NaCl, wild type hair is white Most leaf discs strong penetration caused by high level salt solution of poplar acts on due to makes cell dehydration that shrinkage occur, and causes leaf disc Softening.Meanwhile under conditions of 100mM and 150mM NaCl, the chlorophyll of three leaf discs for turning NsNHX1 Chinese white poplar strain Content is higher than wild type Chinese white poplar.Illustrate that the leaf disc for overexpressing NsNHX1 transgenosis Chinese white poplar has stronger salt tolerance. In 100mM, 200mM and 300mM NaHCO₃Under conditions of, although wild type Chinese white poplar and turning NsNHX1 Chinese white poplar leaf disc Chlorophyll content does not have significant difference；But the leaf disc of wild type Chinese white poplar shows more serious damage, illustrates to turn The leaf disc of NsNHX1 Chinese white poplar has stronger alkali resistance.In 1.0% and 1.5%H₂O₂Under conditions of, turn NsNHX1 Chinese white poplar Strain N2 and N3 is compared with wild type and N1, and show stronger inoxidizability: the quantity of leaf disc albefaction is less, and chlorophyll contains It measures higher.In 2.0%H₂O₂Under conditions of, wild type Chinese white poplar and the leaf disc for turning NsNHX1 Chinese white poplar are seriously damaged. The above result shows that the overexpression of NsNHX1 gene can improve the salt tolerance, alkaline-resisting of transgenosis Chinese white poplar to a certain extent Property and oxidation resistance.

(2) turn the salt tolerance and phenotypic analysis of NsNHX1 Chinese white poplar

The pretreatment seedling that wild type Chinese white poplar and three turn NsNHX1 Chinese white poplar strain is chosen, is divided into two groups, one group with suddenly Glan nutrient solution pours, as control；Another group is poured with the Huo Gelan nutrient solution containing NaCl, progress salt stress processing: first Week, the Huo Gelan nutrient solution of the NaCl containing 25mM was poured once every three days；The concentration of second week, NaCl increases every three days 25mM is gradually increased to 150mM；It in third week, is poured once, is continued every three days with the Huo Gelan nutrient solution of the NaCl containing 150mM Stress one week.Every plant of plant pours the Huo Gelan nutrient solution of same volume or the Huo Gelan nutrient solution containing NaCl every time, and puts It sets in a pallet, to keep soil salt content.After 3 weeks, measuring its upgrowth situation, (biomass M, plant height h, leaf chlorophyll contain Amount and leaf r elative water content)；The content of proline (Proline, PRO) and malonaldehyde (Malondialdehyde, MDA)；It is super Oxide dismutase (Superoxide dismutase, SOD), peroxidase (Peroxidase, POD) and catalase The activity of (Catalase, CAT).

1) measurement of upgrowth situation

A, biomass: the biomass M of plant before and after the processing is measured respectively with electronic balance.

B, plant height: with the plant height h after ruler measurement processing.

C, chlorophyll content in leaf blades: after salt stress processing, choosing the blade of plant same position, portable using SPAD-502 Formula Chlorophyll meter measures chlorophyll content.

D, leaf r elative water content: after salt stress processing, the blade of plant same position is chosen, measures fresh weight Wf.Then Blade is impregnated for 24 hours with distilled water under room temperature dark condition, measures imbibition weight Wt.Finally sample is dried under the conditions of 80 DEG C Dry 48h, measures its dry weight Wd.The calculation formula of leaf r elative water content (RWC) are as follows: RWC=(Wf-Wd)/(Wt-Wd) × 100%.

2) proline content measures

About 0.1g sample (blade) is weighed, 1mL extracting solution is added, carries out ice bath homogenate；It is placed in mechanical shaking extraction in boiling water bath 10min；10000g is centrifuged 10min under room temperature, takes supernatant, to be measured after cooling.Sequentially added into EP pipe 0.25mL sample, 0.25mL reagent one and 0.25mL reagent two, cover tightly nozzle, are placed in boiling water bath and keep the temperature 30min, primary every 10min oscillation. After cooling, 0.5mL reagent three is added, vibrates 30s, stands a moment so that pigment is gone in reagent three；Draw 0.2mL upper solution In 96 orifice plates, with the light absorption value A at microplate reader (preheating 30min or more) measurement 520nm wavelength₅₂₀, calculation formula are as follows:

Pro content (μ g/g fresh weight)=38.4 × (A₅₂₀+0.0021)÷W_{Fresh weight}。

Wherein W_{Fresh weight}: sample fresh weight (g).

3) malonaldehyde (MDA) assay

About 0.1g sample (blade) is weighed, 1mL extracting solution is added, carries out ice bath homogenate, 8000g, 4 DEG C of centrifugation 10min take Supernatant is placed in be measured on ice.0.3mL reagent one and 0.1mL sample are added into 1.5mL centrifuge tube, mixes, in 90 DEG C of water-baths Keeping the temperature 30min (covering tightly nozzle), ice bath is cooling afterwards, and room temperature 10000g is centrifuged 10min.200 μ L supernatants are added into 96 orifice plates, With the absorbance A 532 and A600 at microplate reader (preheating 30min or more) measurement 532nm and 600nm, calculation formula are as follows:

MDA content (nmol/g fresh weight)==51.6 × Δ A ÷ W_{Fresh weight}。

Wherein Δ A=A532-A600；W_{Fresh weight}: sample fresh weight (g).

4) SOD Activity determination

45 μ L reagents one, 100 μ L reagents two, 2 μ L reagents three, 18 μ L samples (blade) or 18 are sequentially added into 96 orifice plates μ L distilled water, 35 μ L reagents four, mixes well after being placed at room temperature for 30min, with microplate reader (preheating 30min or more) measurement Light absorption value A560 at 560nm, calculation formula are as follows:

SOD activity (U/g fresh weight)=11.11 × A_{Inhibit percentage}÷(1-A_{Inhibit percentage})÷W_{Fresh weight}× Sample Dilution multiple.

Wherein A_{Inhibit percentage}=(A_{Control tube}-A_{Measurement pipe})÷A_{Control tube}× 100%；W_{Fresh weight}: sample fresh weight (g).

5) POD Activity determination

10 μ L samples (blade), 60 μ L distilled water, 120 μ L reagents one, 30 μ L reagents two, 30 are sequentially added into 96 orifice plates μ L reagent three mixes well simultaneously timing immediately, with the light absorption value A of 30s at microplate reader (preheating 30min or more) measurement 470nm₁With Light absorption value A after 1min30s₂, calculation formula are as follows:

POD (U/g fresh weight)=5000 × Δ A ÷ W_{Fresh weight}

Wherein Δ A=A₂-A₁；W_{Fresh weight}: sample fresh weight (g).

6) CAT Activity determination

25mL reagent is added into reagent two to mix well together, 10 μ L samples (blade) and 190 μ L are added in UV plate Working solution mixes simultaneously timing immediately, with initial light absorption value A under microplate reader (preheating 30min or more) measurement 240nm₁After 1min Light absorption value A₂, calculation formula are as follows:

CAT (U/g fresh weight)=918 × Δ A ÷ W_{Fresh weight}

Wherein Δ A=A₁-A₂；W_{Fresh weight}: sample fresh weight (g).

It is handled by three weeks salt stress, wild type Chinese white poplar and three turn the phenotype and physical signs of NsNHX1 Chinese white poplar (biomass, plant height, chlorophyll content in leaf blades and leaf r elative water content) testing result is as shown in Figure 9 and Figure 10.The result shows that: Compared with the wild type Chinese white poplar grown under normal operation and turning NsNHX1 Chinese white poplar, the plant under salt stress is shorter and smaller, raw Length is slower, but either under normal growing conditions or under condition of salt stress, turns NsNHX1 Chinese white poplar strain N1 and N3 Growing way go with N2 than wild type, the growing way of N2 is slightly better than wild type Chinese white poplar (Fig. 9 A).Under paired observation normal condition and The 7th leaf (from terminal bud number) of plant under condition of salt stress finds wild type Chinese white poplar under condition of salt stress and turns There is yellow, withered, phenomena such as wilting in the blade of NsNHX1 Chinese white poplar, and wild type Chinese white poplar and turns NsNHX1 Chinese white poplar strain Even there is the withered spot of gangrenosum acne in the blade of N2, but the damage that wild type leaves of Populus Tomentosa is subject to is more serious (Fig. 9 B).

By measurement wild type Chinese white poplar and turn the biomass discovery of NsNHX1 Chinese white poplar, under normal growing conditions and salt Under stress conditions, the biomass for turning NsNHX1 Chinese white poplar is all remarkably higher than wild type Chinese white poplar (Figure 10 A).It is white to measure wild type hair Poplar and the plant height discovery for turning NsNHX1 Chinese white poplar turn NsNHX1 either under normal growing conditions or under condition of salt stress The plant height of Chinese white poplar strain N1 and N3 are all remarkably higher than wild type and N2, and there was no significant difference between wild type Chinese white poplar and N2 (Figure 10 B).Measurement wild type Chinese white poplar and the chlorophyll content hair for turning the 5th leaf (from terminal bud number) of NsNHX1 Chinese white poplar Existing, wild type Chinese white poplar and the chlorophyll content turned in NsNHX1 leaves of Populus Tomentosa are poor without conspicuousness under normal growing conditions It is different；And under condition of salt stress, turn that chlorophyll content in NsNHX1 leaves of Populus Tomentosa increases and to be significantly higher than wild type hair white Poplar (Figure 10 C).Measurement wild type Chinese white poplar and the relative water content hair for turning the 4th piece of blade (from terminal bud number) of NsNHX1 Chinese white poplar Existing, there was no significant difference for wild type Chinese white poplar and the relative water content turned in NsNHX1 leaves of Populus Tomentosa under normal operation；And Under condition of salt stress, wild type Chinese white poplar and the relative water content turned in NsNHX1 leaves of Populus Tomentosa decline, but turn Relative water content in NsNHX1 Chinese white poplar strain blade is slightly above wild type Chinese white poplar (Figure 10 D).The above result shows that super table Transgenosis Chinese white poplar is not only improved up to NsNHX1 to the tolerance of salt stress, but also promotes its growth.

Since salt stress can cause oxidative stress to plant, make to generate active oxygen in plant, therefore, in order to determine that salt is coerced Whether NsNHX1 functions in Chinese white poplar antioxidant system under the conditions of compeling, and also has detected wild type Chinese white poplar and turns NsNHX1 The content of the activity of SOD, POD, CAT in leaves of Populus Tomentosa, proline and malonaldehyde.

The results are shown in Figure 10.The result shows that: under condition of salt stress, three SOD for turning NsNHX1 Chinese white poplar strain, POD and CAT activity significantly increases, and noticeably greater than wild type Chinese white poplar (Figure 10 E, F, G)；It is wild under condition of salt stress Type Chinese white poplar and the proline content for turning NsNHX1 Chinese white poplar significantly increase, and turn the dried meat ammonia of NsNHX1 Chinese white poplar strain N1 and N3 Acid content is significantly higher than wild type Chinese white poplar and N2 (Figure 10 H)；Wild type Chinese white poplar and turn the MDA in NsNHX1 leaves of Populus Tomentosa Content significantly increases under salt stress, but the MDA content in wild type leaves of Populus Tomentosa is significantly higher than and turns the strain of NsNHX1 Chinese white poplar It is (Figure 10 I).The above result shows that the overexpression of NsNHX1 can be improved the activity of transgenosis Chinese white poplar antioxidase, increase The content of proline reduces the accumulation of active oxygen, reduces the damage of biomembrane, and then improve the anti-oxidant energy of transgenosis Chinese white poplar Power.

The present invention is had been described in detail above.To those skilled in the art, do not depart from spirit of the invention and Range, and without carrying out under unnecessary experimental conditions, can synchronization parameters, concentration and under the conditions of, it is real in a wider range Apply the present invention.Although The present invention gives particular embodiments, it is understood that, the present invention can be improved further. In short, pressing the principle of the present invention, the application is intended to include any change, purposes or improvement of the present invention, including departing from this Shen Please in the open scope, and the change carried out with routine techniques known in the art.By the range of following attached claims, It can carry out the application of some essential characteristics.

Sequence table

<110>Agricultural University of the Inner Mongol, University of the Inner Mongol

<120>NsNHX1 protein and its relevant biological material are cultivating the application in resistance to inverse type poplar

<160>2

<170>PatentIn version 3.5

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aattatattt aatgcaggat tccaggtgaa aaagaagcaa tttttccgta 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

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caatgtgaca gagagttcaa gaatcactac caagcatgct tttgcaacct tatcatttgt 1080

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gatgttggga agagcagctt ttgtttttcc tttatccttc gtctccaatt tgatgaagaa 1260

atcacctacg gataaagtgg gcttcaaaca gcagattgtg atatggtggg ctgggctcat 1320

gagaggtgct gtgtctatgg ctcttgctta caatcagttt acaaggtcag ggcacactca 1380

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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 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

1.NsNHX1 protein is following 1) -10) in it is any in application:

1) regulate and control plant stress tolerance；

2) regulating growth of plants；

3) regulate and control phytomass；

4) regulate and control plant plant height；

5) regulate and control chlorophyll content of plant；

6) regulate and control plant water content；

7) regulate and control plant anti-oxidation ability；

9) regulate and control plant proline content；

10) regulate and control plant mda content.

2. biomaterial relevant to NsNHX1 protein is following 1) -12) in it is any in application:

1) regulate and control plant stress tolerance；

2) regulating growth of plants；

3) regulate and control phytomass；

4) regulate and control plant plant height；

5) regulate and control chlorophyll content of plant；

6) regulate and control plant water content；

7) regulate and control plant anti-oxidation ability；

9) regulate and control plant proline content；

10) regulate and control plant mda content；

11) genetically modified plants or cultivate resistance of reverse plant variety that resistance of reverse improves are cultivated；

12) plant breeding.

3. application according to claim 1 or 2, it is characterised in that: the NsNHX1 protein be it is following a) or b) or c) Or d) shown in protein:

A) amino acid sequence is protein shown in sequence 2；

C) amino acid sequence shown in sequence 2 is passed through to the substitution and/or deletion and/or addition of one or several amino acid residues Obtained protein with the same function；

D) homology with amino acid sequence shown in sequence 2 with 75% or 75% or more and albumen with the same function Matter.

4. application according to claim 2 or 3, it is characterised in that: the biomaterial is following A 1) appointing into A12) It is a kind of:

A1 the nucleic acid molecules of NsNHX1 protein) are encoded；

A2) contain A1) expression cassettes of the nucleic acid molecules；

A3) contain A1) recombinant vectors of the nucleic acid molecules；

A4) contain A2) recombinant vector of the expression cassette；

A5) contain A1) recombinant microorganisms of the nucleic acid molecules；

A6) contain A2) recombinant microorganism of the expression cassette；

A7) contain A3) recombinant microorganism of the recombinant vector；

A8) contain A4) recombinant microorganism of the recombinant vector；

A11) contain A3) the transgenic plant cells system of the recombinant vector；

A12) contain A4) the transgenic plant cells system of the recombinant vector.

5. application according to claim 4, it is characterised in that: A1) nucleic acid molecules be it is following 1) or 2) or 3) shown in Gene:

1) its coded sequence is genomic DNA molecule shown in cDNA molecule shown in sequence 1 149-1783 or sequence 1；

2) there is 75% or 75% or more identity with the nucleotide sequence 1) limited, and encodes described in claim 1 The cDNA molecule or genomic DNA molecule of NsNHX1 protein；

1) or 2) 3) and NsNHX1 described in claim 1 is encoded with the nucleotide sequence hybridization that limits under strict conditions The cDNA molecule or genomic DNA molecule of protein.

6. -5 any application according to claim 1, it is characterised in that: the resistance of reverse is salt tolerance and/or alkali resistance.

7. a kind of method for cultivating the genetically modified plants that resistance of reverse improves, the table including improving NsNHX1 protein in recipient plant Up to amount and/or activity, the step of obtaining genetically modified plants；The resistance of reverse of the genetically modified plants is higher than the recipient plant.

8. according to the method described in claim 7, it is characterized by: the resistance of reverse is salt tolerance and/or alkali resistance；

Or, the resistance of reverse of the genetically modified plants be higher than the recipient plant be embodied in it is any in following (1)-(9):

(1) survival rate of genetically modified plants is higher than recipient plant；

(2) biomass of genetically modified plants or root biomass are higher than recipient plant；

(3) chlorophyll content in Transgenic plant leaf is higher than recipient plant；

(4) water content in Transgenic plant leaf is greater than recipient plant.

(5) plant height of genetically modified plants is higher than recipient plant；

(6) oxidation resistance of genetically modified plants is higher than recipient plant；

(7) hepatocuprein in Transgenic plant leaf and/or peroxidase and/or catalase activity are higher than Recipient plant；

(8) proline content in Transgenic plant leaf is higher than recipient plant；

(9) mda content in Transgenic plant leaf is lower than recipient plant.

9. method according to claim 7 or 8, it is characterised in that: described to improve NsNHX1 protein in recipient plant Expression quantity and/or active method are that NsNHX1 protein is overexpressed in recipient plant；

Or, the method for the overexpression is that the encoding gene of NsNHX1 protein is imported recipient plant；

Or, the nucleotide sequence of the encoding gene of the NsNHX1 protein is DNA shown in sequence 1 149-1783 points Son.

10. -6 any any method of application or claim 7-9 according to claim 1, it is characterised in that: institute Stating plant is monocotyledon or dicotyledon；Or, the monocotyledon is poplar.