CN108250281A - Sodium, hydrogen reverse transport protein PbrNHX2 and its application in plant salt tolerance ability is improved in birch-leaf pear - Google Patents

Abstract

The present invention provides sodium, hydrogen reverse transport protein PbrNHX2 and its applications in raising plant salt tolerance ability in birch-leaf pear, belong to gene engineering technology field.The present invention provides sodium in birch-leaf pear, hydrogen reverse transport protein, encoding gene and primer pairs for cloning the encoding gene cDNA sequence.Sodium, hydrogen reverse transport protein, the encoding gene or the encoding gene of primer pair clone are to the application in plant salt tolerance ability is improved in the birch-leaf pear.Build tobacco transformation vector and Ussurian pear conversion carrier, the positive plant seedling of acquisition carries out salt treatment, it was found that the conductivity of positive plant seedling is significantly reduced compared with wild type, chlorophyll content is significantly increased compared with wild type, survival rate is significantly improved than wild type, while positive plant seedling has the ability of stronger anti-ROS than wild type.The encoding gene of overexpression can effectively enhance the oxygen scavenging activity ability of transfer-gen plant, so as to improve the salt tolerance of plant.

Description

Sodium in birch-leaf pear, hydrogen reverse transport protein PbrNHX2 and its improving plant salt tolerance ability In application

Technical field

The invention belongs to gene engineering technology fields, and in particular to sodium in birch-leaf pear, hydrogen reverse transport protein PbrNHX2 and Its application in plant salt tolerance ability is improved.

Background technology

Pears are one of fruit tree species of main cultivation in the world.Pear tree is very extensive in the plantation distribution of China, pears major production areas It is laid out as " three 4 points of areas ", from northeast to Guangxi, there is the plantation of pear tree from Yunnan to Shandong.The layout in pears advantage producing region with Planning has greatly facilitated the development of China's pears industry, exactly because but it is widely distributed, so it is highly susceptible to various environment The influence of factor, such as arid, damage or crop failure caused by waterlogging, saline and alkaline etc..Therefore, if excellent degeneration-resistant new varieties can be cultivated and become for pears The most vital factor of industry development.Because the breeding of pears exists：Genetic background is complicated, and juvenile phase is long, selfing is not affine, educates Plant the target not reasons such as confirmability so that conventional breeding method far can not meet the kind need of modern pears plantation It asks.So how to cultivate the degeneration-resistant new varieties of pears that can be used for production in a short time, become to put in breeding scholar face Preceding a difficult problem.With biotechnology and the rapid advances of tissue culture technique, the breeding of plant has new approach again, Such as tissue culture technique breeding, the methods of genetic transformation breeding, somatic hybridization.Using plant gene engineering technology to plant Character improved so that the cultivation of the efficiently and accuratelies of the degeneration-resistant new varieties of pears becomes possibility.

Salt stress is injured caused by plant most starts to behave as osmotic stress, and can continue the growth week in plant Phase exists；Followed by because poisoning the missing with nutrient caused by ion imbalance；It finally results in oxidative stress and causes film The change of permeability and the disorder of physiological metabolism and the accumulation of noxious material, finally cause the growth and development of plant hindered and It influences or even dead (Flowers, 2004).Plant mainly passes through the reconstruction of ionic equilibrium, osmotic adjustment, reactive oxygen species Removing improve the salt tolerant of plant.

According to existing research it is found that the salt tolerance of the NHX genes and plant in plant has close relationship.NHX genes Function be responsible for coding Na⁺/H⁺Reverse transport protein can regulate and control plant by changing the expression quantity of NHX family genes Internal Na⁺/H⁺The synthesis of reverse transport protein, so as to change salt tolerance of plant etc..Research shows that in higher plant, Na⁺/H⁺ The function of reverse transport protein is mainly： Na⁺Outer row and limitation Na⁺To aerial part transport (Shi et al., 2000； Shi et al.,2000).When plant cell is by Salt Strees Condition, it is easy to because of osmotic potential difference, and lead to cell dehydration. In order to maintain the ionic equilibrium in cell, the osmotic potential of cell is reduced, plant is enable normally to be inhaled from extraneous hypersaline environment Water, the Na on plasma membrane and tonoplast⁺/H⁺At this moment reverse transport protein will be activated, Na⁺/H⁺Reverse transport protein pass through by Na⁺Inverse concentration gradient is transported, by intracellular Na⁺In outer row or separating to vacuole, water stress (Padan is avoided and Schuldiner,1987).In addition to Na⁺Outer row and separating, some researches show that the Na being positioned on tonoplast recently⁺/H⁺Reverse transport protein also has K⁺Effect in separating to vacuole.When plant cell is by salt stress, the NHX1 of plant The expression quantity of gene can raise, so as to enhance K⁺Effect in separating to vacuole, by by K⁺Separating arrives vacuole In, cell is made to generate in cytoplasm and generates K⁺The signal of reduction, so as to activate high affine K on plasma membrane⁺Transporter.It is activated High-affinity K⁺Transporter can be specifically from low K⁺Environment is by more K⁺It is transported into the cell, so as to remain intracellular steady Fixed Na⁺/H⁺.At present from the point of view of research, salt stress can effectively activate NHX1 genes, so as to enhance Na⁺/H⁺Reverse transport protein Expression, improve plant salt tolerance (Ohta et al., 2002；Jiang et al.,2010；Leidi et al., 2010).Intracellular pH value is mainly the H generated by proton pump and metabolism⁺, OH^-It determines, NHX albumen passes through H in the cell⁺Transhipment, can change the ion concentration in vacuole play the role of adjust vacuolar pH.

Na⁺/H⁺Reverse transport protein is the discovery that in mammal for the first time, is then the matter in barley in plant (Ratner and Jacoby, 1976) is found on film for the first time.It is found from existing research, Na in the membranous system of plant⁺/ H⁺Reverse transport protein generally existing has had more in-depth study, in arabidopsis in common species OsNHX1 reverse transport proteins (Fukuda et in AtNHX1 reverse transport proteins (Apse et al., 1999), rice Al., 1999), the TtNHX1 reverse transport proteins (Lv Huiying, 2003) of New Zealand spinach, corn ZmNHX1 reverse transport proteins (Zorb Et al., 2005), citrus cNHX1 reverse transport proteins (Porat et al., 2002) etc..Saier etc. the study found that sun from Son, proton reverse transport protein can be divided into CPA1 and CPA2 Liang Ge families (Saier et al., 1999).With increasingly More Na⁺/H⁺It is found in animals and plants and fungi to transport protein, slowly constitutes the transport protein family of a NHX. According to its sequence homology, this family can be divided into two groups of class1 and class2, and sequence homology is respectively 20% to arrive 30%.From the point of view of the distribution of the transport protein family of NHX, class1 families are proved to be primarily present in terrestrial plant, NHX hypotypes are primarily present on tonoplast, this is also one of its key property.And class2 families are then to be present in quilt plant Object dragon spruce and bryophyte are primarily present and in gymnosperm in all kinds of vesicles in plant cell.According to existing It studies, the Na isolated in plant⁺/H⁺Reverse transport protein positions difference according to it and is divided into two classes, and one kind is positioned at cytoplasm It is another kind of on film, it is on cell liquid vacuolar membrane (Lu et al., 2005).

Apse etc. by the way that arabidopsis AtNHX1 genes are transferred in arabidopsis, obtains the Arabidopsis plant of overexpression, The salt tolerance of the strain improves a lot (Apse et al, 1999).Etc., it is found after AtNHX1 genes are transferred to tomato, mistake The tomato plant of expression can in 200mM NaCl normal growth (Zhang, 2001).Ohta etc. is by the NHX genes in Atriplex It is transferred in rice, also obtains the transgenic rice plant (Ohta et al., 2002) with salt tolerance.Pass through overexpression The plant of NHX genes can not only change the salt tolerance of plant, He etc., be found in the overexpression AtNHX1 gene plants of cotton, Under the NaCl treatment conditions of 200mmol/L, the output of cotton of transfer-gen plant is not only improved, and can be generated more Better cotton fiber (He et al., 2005).Some NHX genes have in can also spending it is specific expressed, in morning glory TvNHX1 genes, it is mainly specific expressed in spending, pattern (Ohnishi can be changed by adjusting the acid-base value of vacuole et al.,2005).NHX genes also have certain production-increasing function, and the overexpression of ZmNHX1 genes is resistance in enhancing plant in corn While salt, the yield (Li et al., 2010) of corn is also improved.NHX genes in most of species are in non-stress Under can also express, part NHX gene expressions increase under salt stress in the tissues such as root, stem and leaf, also have been reported that show gene by ABA, dehydration, arid, KCl, induction of chilling stress expression (Rodriguez-Rosales et al., 2009).It shows according to the study, It is isolated in the plants such as arabidopsis, tomato, rice, barley, soybean, the fluffy, salicornia europaeal of alkali.

Birch-leaf pear is that a kind of wide stock is applied in pears industry, and pole salt tolerant is research xylophyta salt tolerance and clone Ideal material in relation to salt resistant gene.Therefore, clone's birch-leaf pear salt resistance related gene is the key that salt resistant gene engineering and basis.

Invention content

In view of this, it the purpose of the present invention is to provide sodium, hydrogen reverse transport protein PbrNHX2 in birch-leaf pear and its is carrying The application of high plant salt tolerance ability, the PbrNHX2 have the function of salt resistance, effectively improve the salt-resistance of genetically modified plants.

In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：

The present invention provides sodium, hydrogen reverse transport protein PbrNHX2 in birch-leaf pear, have such as SEQ ID NO.1 in sequence table Shown amino acid sequence.

The present invention provides the encoding genes of sodium, hydrogen reverse transport protein PbrNHX2 in the birch-leaf pear, have such as sequence Nucleotide sequence in list shown in SEQ ID NO.2.

The present invention provides a kind of for cloning the primer pair of the encoding gene cDNA sequence, including forward primer and Primer is reacted, the forward primer has the nucleotide sequence as shown in SEQ ID NO.3 in sequence table；The reverse primer With the nucleotide sequence as shown in SEQ ID NO.4 in sequence table.

The present invention provides sodium, hydrogen reverse transport protein PbrNHX2, the encoding gene or institutes in the birch-leaf pear Application of the primer pair stated in plant salt tolerance ability is improved.

Preferably, the application includes the following steps：By sodium in the birch-leaf pear, hydrogen reverse transport protein PbrNHX2 or The encoding gene or the encoding gene of primer pair clone recombinantly express in plant, obtained recombinant plant tool There is salt tolerance.

Preferably, the program of the clone is 94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 58 DEG C of annealing 90s, 72 DEG C are prolonged 90s is stretched, 35 recycle, 72 DEG C of extension 10min after the completion of cycle.

Preferably, the plant includes tobacco or Ussurian pear.

Preferably, the salinity of the plant salt tolerance is not higher than 1000mmol/L.

Preferably, the salt of the plant salt tolerance includes Na⁺Or K⁺。

The present invention provides sodium, hydrogen reverse transport protein PbrNHX2 in birch-leaf pear, have such as SEQ ID NO.1 in sequence table Shown amino acid sequence.Table during function covering analysis is tested in salt density value yeast mutants AXT3 in the PbrNHX2 It is bright, PbrNHX2 in salt density value yeast mutants AXT3 is recombinantly expressed, shows that the PbrNHX2 can partly cover salt density value The salt resistance ability of yeast mutants AXT3；The saliferous ability in yeast is determined simultaneously, is as a result shown：Recombination yeast salt content Substantially less than blank control group yeast, containing K⁺Amount is higher than control group yeast, illustrates that the PbrNHX2 has and inhales K⁺Arrange Na⁺'s Function, so as to illustrate that albumen PbrNHX2 has the function of salt resistance.

The present invention provides the encoding genes of sodium, hydrogen reverse transport protein PbrNHX2 in the birch-leaf pear, have such as sequence Nucleotide sequence in list shown in SEQ ID NO.2.By birch-leaf pear seedling under the processing of 200mM NaCl solutions, corresponding Point in time sampling, using the relative expression quantity of the Real-time PCR Analysis encoding gene provided by the invention, as a result, it has been found that, As NaCl solution handles the extension of experiment in 12h, the relative expression quantity of the encoding gene gradually increases, this shows institute Encoding gene response salt stress processing is stated, there is salt resistance.

The present invention provides sodium, hydrogen reverse transport protein PbrNHX2, the encoding gene or institutes in the birch-leaf pear The encoding gene of primer pair clone stated is to the application in plant salt tolerance ability is improved.It is carried by building Transformation of tobacco respectively Body and Ussurian pear conversion carrier obtain positive plant seedling and carry out salt treatment respectively, as a result, it has been found that：The conductivity of positive plant seedling It is significantly reduced compared with wild type (WT), chlorophyll content is significantly increased compared with wild type, and survival rate improves 116% than wild type ~129%, show the ability that positive plant seedling has stronger anti-ROS than wild type.Hydrogen peroxide in transgene tobacco (H₂O₂) and superoxide anion (O^2-) content activity be intended to it is lower than wild type, plant activity in vivo oxygen residual it is lower, carefully Cellular damage smaller.These results indicate that the PbrNHX2 genes being overexpressed can effectively enhance the active oxygen of transfer-gen plant Scavenging activity, so as to improve the salt tolerance of plant.

Description of the drawings

Fig. 1 is a kind of techniqueflow schematic diagram of the present invention；

Fig. 2 is expression schematic diagram of the PbNHX2 encoding genes under salt, dehydration and low temperature stress in embodiment 2；Wherein, Fig. 2-A be the encoding gene in birch-leaf pear seedling (non-transgenosis) under 200mM NaCl solution treatments, corresponding time point Sampling, using the relative expression quantity of encoding gene described in Real-time PCR Analysis；Fig. 2 B are that the seedling of birch-leaf pear takes off at room temperature The expression pattern of water different time points；Fig. 2-C be birch-leaf pear seedling 4 DEG C processing under, corresponding point in time sampling, using reality When the quantitative PCR analysis present invention gene relative expression quantity；

Fig. 3 is PbrNHX2 encoding gene subcellular localizations in embodiment 3, and wherein Fig. 3-A are GFP genes in light field, purple Imaging under outside line, light；Fig. 3-B are PbrNHX2 encoding genes in light field, ultraviolet, under light imaging；

Fig. 4 is that PbrNHX2 functions in salt density value yeast mutants AXT3 cover analysis result in embodiment 4；

Fig. 5 is the acquisition of 5 transfer PbrNHX2 tobacco plants of embodiment, and Fig. 5-a are vector construction, and Fig. 5-b are PbrNHX2 Turn the whole flow process figure of tobacco, Fig. 5-c identify transfer-gen plant for PCR, and Fig. 5-d are that RT-PCR identifies the super of genetically modified plants Expression analysis, Fig. 5-e are the protein expression level of Western blotting analysis overexpression plant；

Fig. 6 is that PbrNHX2 converts Ussurian pear and plant regeneration process schematic in embodiment 6, and wherein Fig. 6-A are conversions Photo afterwards；Fig. 6-B are that the material of 30 days is grown on screening and culturing medium；Fig. 6-C are regeneration bud root inductions；Fig. 6-D are to turn Gene plant shifting grows the photo of 30 days in soil；Fig. 6-E are to utilize T0 generations after gene specific primer progress PCR identification tobaccos Transfer-gen plant, wherein M：Marker ,+：Plasmid ,-：WT lines, 1-8：Transgenic line；Fig. 6-F are real-time quantitative The expression quantity of the encoding gene of PbrNHX2 in PCR analysis Ussurian pear difference transgenic lines；

Fig. 7 is the table of the encoding gene strain and wild type (WT) sodium chloride of 7 transfer PbrNHX2 of embodiment before and after the processing Type and physiological index determining, wherein Fig. 7-A are phenotype of 45 -day-old of the tobacco plant before 300 mM sodium chloride are handled 20 days；Figure 7-B is phenotype of 45 -day-old of the tobacco plant after 300mM sodium chloride is handled 20 days；Fig. 7-C are that 30 -day-old of tobacco plant exists 200mM salting liquids handle 4 days before phenotype；Fig. 7-D are that 30 -day-old of tobacco plant handles the table of 4 days in 200mM salting liquids Type；Fig. 7-E are that 30 -day-old of tobacco plant handles the survival rate statistical result of 4 days in 200mM salting liquids, and Fig. 7-F are 30 -day-old Tobacco plant handle the conductance measurement of 4 days as a result, the tobacco plant that Fig. 7-G are 45 -day-old exists in 200mM salting liquids 300mM sodium chloride handle 25 days after phenotype, Fig. 7-H be 45 -day-old tobacco plant 300mM sodium chloride handle 25 days after Chlorophyll measuring, Fig. 7-I are that 30 -day-old of tobacco plant handles the chlorophyll extraction of 4 days in 200mM salting liquids；

Fig. 8 is PbrNHX2 transgenosis Ussurian pear strains (OE2, OE6 and OE8) and WT lines (WT) in embodiment 8 500mM sodium chloride pour 15 days phenotype and physiological index determining as a result, wherein Fig. 8-A are the tables that 500mM sodium chloride pours 15 days Type, Fig. 8-B are that 500mM sodium chloride pours conductance measurement in 15 days, and Fig. 8-C~Fig. 8-D are that 500mM sodium chloride pours 15 days chlorophyll Measure (Fig. 8-C) and chlorophyll extraction (Fig. 8-D)；

The encoding gene tobacco and Ussurian pear histochemical stain that Fig. 9 is 9 transfer PbrNHX2 of embodiment analyze H₂O₂With O^2-Accumulation, Fig. 9-A and Fig. 9-B are 45 -day-old of the tobacco plant unconverted plant and two before and after 300mM sodium chloride handles 15d A transgenic line active oxygen histochemical stain is right respectively using nitro tetrazole (NBT) and diaminobenzidine (DAB) H₂O₂(Fig. 9-A) and O^2-(Fig. 9-B) is dyed, and Fig. 9-C are transgenosis cigarette

Specific embodiment

The present invention provides sodium, hydrogen reverse transport protein PbrNHX2 in birch-leaf pear, have such as SEQ ID NO.1 in sequence table Shown amino acid sequence.The PbrNHX2 encodes 542 amino acid, isoelectric point 8.49, molecular weight 60.09KDa.Institute Stating PbrNHX2 has suction K⁺Arrange Na⁺Function, while the ability of the anti-ROS of carrier plant can be increased, so as to illustrate this base Because having the function of salt resistance.In the present invention, the PbrNHX2 by the encoding gene of the PbrNHX2 by carrying out recombination table It reaches.The method of the recombinant expression is not particularly limited, using recombinant expression mode known in the art.

The present invention provides the encoding genes of sodium, hydrogen reverse transport protein PbrNHX2 in the birch-leaf pear, have such as sequence Nucleotide sequence in list shown in SEQ ID NO.2.The source of the encoding gene is preferably obtained using the method for clone.

The present invention provides a kind of for cloning the primer pair of the encoding gene cDNA sequence, including forward primer and Primer is reacted, the forward primer has the nucleotide sequence as shown in SEQ ID NO.3 in sequence table；The reverse primer With the nucleotide sequence as shown in SEQ ID NO.4 in sequence table.The source of the primer pair is not particularly limited, and is used Synbiotics AB's synthesis known in the art.In embodiments of the present invention, the primer pair commission Nanjing is raw together Object Science and Technology Ltd. synthesizes.

The present invention provides sodium, hydrogen reverse transport protein PbrNHX2, the encoding gene or institutes in the birch-leaf pear The encoding gene of primer pair clone stated is to the application in plant salt tolerance ability is improved.

In the present invention, the application preferably includes following steps：By sodium, hydrogen reverse transport protein in the birch-leaf pear PbrNHX2 or the encoding gene or the encoding gene of primer pair clone recombinantly express in plant, obtained weight Group plant has salt resistance ability (see Fig. 1).

In the present invention, the program of the clone is preferably 94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 58 DEG C of annealing 90s, 72 DEG C of extension 90s, 35 recycle, 72 DEG C of extension 10min after the completion of cycle.The encoding gene is preferably with pMV- The form of PbrNHX2 recombinant vectors is transformed into plant.The present invention is to the construction method of the pMV-PbrNHX2 recombinant vectors It is not particularly limited, using construction method known in the art.

The method that the encoding gene or the encoding gene of primer pair clone recombinantly express in plant is preferred Using the method for Agrobacterium tumefaciens-mediated Transformation.The present invention does not have special limit to the method for the Agrobacterium tumefaciens-mediated Transformation System, using conventional method known in the art.The plant preferably includes tobacco or Ussurian pear.

In the present invention, before the salt resistance ability for examining recombinant plant, the sieve of positive transgenic plant is preferably further included Choosing.The screening technique of the positive transgenic plant is preferably carried out using the method for PCR amplification.During the screening, PCR amplification There is the nucleotide sequence as shown in SEQ ID NO.3 in sequence table with the forward primer of primer；The PCR amplification primer Reverse primer has the nucleotide sequence as shown in SEQ ID NO.4 in sequence table.The response procedures of the PCR amplification are as follows：

Step	94℃	58℃	Elongating temperature	4℃	Recurring number
						Step 1	3min				1
Step 2	30s	30s	55s(58℃)		35
									90s(60℃)		1
Step 3			10min(72℃)
						Step 4				30min

The PCR amplification is as follows with PCR reaction systems：

Reactive component	Dosage (μ l)
		Template DNA	1
PCR buffer solutions	2
		dNTDMix(2.5mmol/L)	1.6
Forward primer	1
		Reverse primer	1
Taq DNA polymerase (5U)	0.2
		Nuclease free water	13.2

After the PCR amplification, if plant to be detected system can amplify the segment (1626bp) of expected size, this It is positive transgenic strain to show them.

In the present invention, obtained positive transgenic strain is subjected to salt treatment, the positive obtained after measure processing turns base Because of the phenotype, physical signs and active oxygen of strain, to verify its salt resistant function.The assay method of the active oxygen preferably uses DAB With NBT histochemical staining methods to plant leaf H₂O₂And O^2-Accumulation is analyzed (according to the depth and degree of dyeing), It detects by an unaided eye and claps.The active oxygen of measure includes hydrogen peroxide (H respectively₂O₂) and superoxide anion (O^2-).It is lived by measuring Property oxygen content variation find：Before salt stress processing is done, do not have between positive transgenic strain and control group coloured aobvious Difference is write, after salt stress 30d, the blade that is dyed with DAB, the leaf area that brown occurs in wild strain type significantly compares transgenosis Strain is big, and darker, the blade dyed with NBT, and WT strain is deeper than the blue of transgenic line, and area is more Greatly, this shows that positive transgenic strain has lower ROS (H under salt stress compared with wild type₂O₂And O^2-) accumulation, so as to ensure Cellular damage smaller.

In the present invention, conductance measurement and the extraction of chlorophyll and assay method are not particularly limited, using this field Known extraction and assay method.The measurement result for analyzing conductivity and chlorophyll is found：The conductance of positive plant seedling Rate is significantly reduced compared with wild type (WT), and chlorophyll content is significantly increased compared with wild type, and survival rate is improved than wild type 116%~129%, show the ability that positive plant seedling has stronger anti-ROS than wild type.

In the present invention, the salinity of the plant salt tolerance is preferably not higher than 1000mmol/L.The plant salt tolerance Salt preferably includes Na⁺Or K⁺.The plant preferably includes tobacco or Ussurian pear.

With reference to embodiment to sodium in birch-leaf pear provided by the invention, potassium, hydrogen reverse transport protein PbrNHX2 and its The application for improving plant salt tolerance ability is described in detail, but they cannot be interpreted as to the scope of the present invention It limits.

Embodiment 1

The clone of birch-leaf pear PbNHX2 full length genes cDNA

This seminar early period using efficient yeast expression system, screens a sodium hydrogen antiport egg from birch-leaf pear White PbrNHX2 designs primer according to the sequence of PbrNHX2 genes and primer premier 5.0, with RT-PCR method from Du Its overall length is expanded on pears.Detailed step is as follows：1 μ g of birch-leaf pear RNA is taken to be put immediately after 37 DEG C of processing 30min of DNase I of 1U Enter on ice, be immediately placed on ice after adding in 1 μ l 50mM EDTA, 65 DEG C of processing 10min.The synthesis reference of the first chains of cDNA The operation manual of TOYOBO reverse transcription reagent box carries out.First chain cDNA of gained is used for the amplification of PbrNHX2 genes. PCR It is completed by following procedure：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 58 DEG C of annealing 90s, 72 DEG C of extension 90s, 35 recycle, 72 DEG C of extension 10min after the completion of cycle.The PCR product of single band is generated after the completion of amplification, through 1% Ago-Gel electricity After swimming, with plastic recovery kit according to operation instruction extraction step, special purpose band is recycled.

The solution of recovery purifying is attached with pMD19-T carriers, and the molar ratio of gene and carrier is 3 in linked system: 1 connection.It is 10 μ l to react total volume, wherein the product of the PCR purifying of 5 μ l buffer, 4.5 μ l, 0.5 μ l carriers.16 DEG C of companies Night is taken over, is transformed into E. coli competent DH5 α using thermal shock method, PCR verifications are carried out simultaneously with objective gene sequence primer It is sequenced (by Shanghai, Invitrogen Corp. completes).

Bioinformatic analysis cDNA sequence shows, PbNHX2 full length gene 1626bp, including encoding reading frame codes 542 amino acid, isoelectric point 8.49, the molecular weight of prediction is 60.09KDa.BLASTX analyzes the sequence and known (institute Have the document and database delivered) plant sequence very high homology.Amino acid sequences alignment shows, the PbrNHX2 bases of derivation Diversiform-leaved poplar PeNHX2, Mains Zumi MzNHX1, apple MdNHX2 and MdNHX1, the corn of the amino acid sequence of cause and report ZmNHX2, arabidopsis AtNHX1 and AtNHX2 sequence very high homology.ExPASy is analysis shows the amino acid PbrNHX2 of coding has One vacuole membrane positioning signal.From the point of view of the chadogram built according to the Multiple Sequence Alignment of amino acid, pears PbrNHX2 family proteins Nearest with apple MdNHX2 affiliations, MdNHX1 takes second place, with furthest away in arabidopsis AtNHX5 and AtNHX6 evolution.

Embodiment 2

The qRT-PCR analyses of the different lower PbrNHX2 genes of adverse environmental factor processing and the subcellular localization of PbrNHX2 genes

In order to analyze PbrNHX2 gene pairs low temperature in birch-leaf pear, with high salt and dehydration response modes use Real-time Round pcr analyzes the expression pattern of PbrNHX2 genes.RNA, the synthesis reference of the first chains of DNA are extracted using CTAB methods The operation manual of TOYOBO reverse transcription reagent box carries out.Have in the reaction system of 20 μ l：10 μ l 2 × Mix, 0.1 μ l CDNA, 5 μ l primers (using ubiqutin as internal control primer (SEQ ID NO.5 and SEQ ID NO.6), length 208), 4.9 μ l Water.The program of quantitative PCR is as shown in table 1：

1 quantitative PCR program of table

As a result see Fig. 2.A kind of expression of the Fig. 2 for PbNHX2 encoding genes of the present invention under salt, dehydration and low temperature stress Schematic diagram, wherein, Fig. 2-A are the encoding genes in birch-leaf pear seedling (non-transgenosis) under 200mM NaCl processing, accordingly Point in time sampling, using the relative expression quantity of Real-time PCR Analysis gene of the present invention；Fig. 2-B are the seedling rooms that grows directly from seeds of birch-leaf pear The expression pattern of the lower dehydration different time points of temperature；Fig. 2-C be birch-leaf pear seedling 4 DEG C processing under, corresponding point in time sampling, The relative expression quantity of the encoding gene is analyzed using real-time quantitative PCR.The prolonging with processing time it can be seen from Fig. 2-A It is long, incremental trend is presented in the expression quantity of 12h interior coding genes, expression quantity is begun to decline after 12 hours.This shows institute State encoding gene is influenced by salt stress, so as to respond Salt-resistance mechanism, birch-leaf pear is made to realize salt resistant function.

Embodiment 3

The subcellular localization of the encoding gene of PbrNHX2

According to the nucleotide sequence of the encoding gene of PbrNHX2 and pJIT166-GFP carrier figures, before and after gene order It is separately added into SalI and BamHI restriction enzyme sites.The correct target gene of sequencing result is extracted into plasmid as template, with addition Primer (the SEQ ID NO.7 and SEQ ID NO.8) amplification of restriction enzyme site, PCR programs used are：94 DEG C of pre-degeneration 3min；94 DEG C denaturation 30s, 58 DEG C of annealing 1min, 72 DEG C of extension 1min 30s, 35 cycles；72 DEG C of extension 10min.Gene 3 ' eliminates Terminator codon TAG, it is therefore an objective to gene be allowed to be merged with GFP.PCR product is tried after 1% agarose gel electrophoresis using gel Agent box recycles purpose band.The amplified fragments of recovery purifying are cloned into pMD19-T carriers, are transformed into E. coli competent In DH5 α.Bacterium solution after conversion is detected with PCR, the bacterium solution that PCR identifications are positive sends to sequencing, and extraction sequencing result is correct The plasmid of bacterium solution and pJIT166-GFP carriers.To double digestion both be carried out with SalI and BamHI, respectively purifying recycling digestion Later product PbrNHX2 genes and pJIT166-GFP carriers.The two is connected through T4-DNA ligases, and 16 DEG C overnight, conversion Obtained recombinant vector is named as pJIT166-GFP- PbrNHX2 by E. coli competent DH5 α.

Using the reagent and method of protoplast transformation, agents useful for same：Protoplasts of Arabidopsis thaliana broken by ultrasonic enzymolysis liquid (carries the previous day It prepares at night, 4 DEG C of preservations)：0.4M mannitol；1% cellulase；0.1% macerozyme； 5mM MES；0.1% pectase.

Method：10min is heated in 55 DEG C of water-baths, inactivates various protease, while the dissolving of reinforcing fiber element enzyme etc. Property, it is then cooled to room temperature, adds following two reagents：0.15%BSA and 8 mM CaCl₂；MaMg solution：0.4M sweet dews Alcohol；0.1%MgCl₂；4mM MES；W5 solution (now with the current, when culture pays attention to plus carboxylic benzyl antibiotic)：154mMNaCl； 125mM CaCl₂； 5mM KCl；2mM MES；PEG solution (40%, v/v)：4g PEG 4000 (sigma-Fluka, It #81240) is dissolved in 4mL water；0.72868g mannitol is added in 3mL water, adds in 1mL 1M CaCl₂Dissolving, it is such as insoluble If, 65 DEG C of hydrotropies can be put into.The two is mixed together, after complete mixing, constant volume to 10mL is finally arrived with KOH tune pH 7.5–8.0。

As a result see Fig. 3.Fig. 3 is the encoding gene subcellular localization of the PbrNHX2, wherein Fig. 3-A, and GFP genes are (right According to) imaging under light field (figure right), ultraviolet light (UV) light (figure is left), figure (in) for both imaging after superposition；Fig. 3-B, Imaging of the PbrNHX2 genes under light field (right side), UV light (left side), figure (in) for both superposition after imaging.According to cellular localization Figure can obtain albumen PbrNHX2 and be located in tonoplast position.

Embodiment 4

Yeast strain AXT3 (ean1::HIS3::ena4,nha1::LEU2,nhx1::TRP1)(Na⁺Transport protein is endogenous Gene is knocked) research NHX gene pairs salt tolerant effects covering effect.Expression carrier used thereof is pYES2 (Invitrigen). It is used after the method for empty pYES2 carriers and the carrier chemical conversion for carrying PbrNHX2 genes is converted into mutant strain respectively The solid medium screening of SC/-Ura.Selection positive transformant is cultivated in liquid SC/-Ura culture mediums, will be in logarithm and be given birth to Long-term turns pYES2, pYES2::PbrNHX2 and pYES2::The yeast transformant of PbrNHX2 is adjusted to OD with distilled water₆₀₀= 1.0, after take respectively 3 μ L yeast stock and dilution 10¹、10²And 10³Times yeast culture get ready to respectively contain 0, 90th, 120 the growing state of different transformants, is observed on the SC/-Ura Selective agar mediums of 150mMNaCl concentration.As a result it shows： The yeast transformant growing state for turning PbrNHX2 encoding genes is significantly better than the yeast transformant for turning sky pYES2 carriers, shows The encoding gene of PbrNHX2 can partly cover the salt resistance ability of salt density value yeast mutants AXT3.Them have also been surveyed in ferment simultaneously Saliferous ability in mother, the yeast salt content that as a result display turns the encoding gene of PbrNHX2 are substantially less than a turn sky pYES2 carriers Yeast, containing K⁺Amount is higher than zero load, illustrates that this gene has and inhales K⁺Arrange Na⁺Function (see Fig. 4), so as to demonstrate The encoding gene of PbrNHX2 has the function of salt resistance.

Embodiment 5

The genetic transformation of tobacco

1. plant conversion carrier is built

According to the multiple cloning sites of the pMV carriers plant binary transformation vector pBI121 of gus gene (excision in) and The coding region sequence of PbrNHX2 genes goes out to expand according to the principle primer primier5.0 Software for Design for being typically designed primer (primer pair is exactly to expand the primer of PbrNHX2 gene cDNA sequences to the upstream and downstream PCR primer of the entire code area of increasing gene It is right).PCR amplification is carried out by template of the clone of PbrNHX2 genes.The annealing temperature of PCR amplification is 58 DEG C, PCR reactants System and amplification program are identical with PbrNHX2 gene clonings.Double digestion is carried out later in amplification, and double digestion system total volume is 20 μ l, wherein：10 μ l, 10 × G buffer solution of the purified product 2 each 1 μ l of μ l, KpnI and SalI, 6 μ l of distilled water of PCR.Digestion is 37 It is carried out in DEG C, glue purification recycling is done after digestion overnight.PMV carrier double digestions reaction volume is 20 μ l, wherein：There is the carrier of pMV 8 μ l, 10 × M buffer solution of plasmid, 2 each 1 μ l of μ l, KpnI and XhoI, add 8 μ l of distilled water.After being equally positioned over 37 DEG C of digestions overnight Purifying recycling.The molar ratio that PbrNHX2 genes and carrier pMV are added in coupled reaction system is 3:1, reacting total volume is 10μl.Wherein contain：10 × buffer, 1 μ l, T4DNA ligases, 1 μ l, the PbrNHX2 genes 4ul of double digestion recycling, double enzymes The 2 μ l of pMV support products cut back to close, 2 μ l of distilled water react 14-16h at 16 DEG C and obtain connection product.Connection product conversion is big Enterobacteria DH5 α are cultivated in the LB solid plates of the kanamycins containing 50mg/L.The positive colony that will be filtered out adjusts point After shake bacterium, extracting plasmid carries out digestion and PCR identifications, and sequencing determines no encoder block mutation, obtains containing being inserted into target fragment Recombinant clone, be named as pMV-PbrNHX2 recombinant vectors, using freeze-thaw method (with reference to Pehanorm Brooker, Huang Peitang is translated, 《Molecular Cloning:A Laboratory guide》The third edition, Science Press, 2002) recombinant vector pMV-PbrNHX2 imported into Agrobacterium In GV3101.

2. agriculture bacillus mediated tobacco genetic transformation step is as follows：

(1) Agrobacterium is cultivated：The Agrobacterium tumefaciems bacterium solution preserved in ultra low temperature freezer is taken, is being added to kanamycins The flat lining out of the LB of 50mg/L, scraping scribing line bacterial plaque, adds in liquid MS minimal mediums, and 28C ° turns min oscillation trainings It supports, is disseminated when bacterial concentration reaches OD=0.3-0.8.

(2) it disseminates：The tobacco leaf of non-transgenosis is taken, is cut into 0.5 × 0.5cm sizes, is then placed in the crown gall prepared In Agrobacterium bacterium solution, 8-10min is impregnated, is during which constantly vibrated.

(3) it co-cultures：The tobacco leaf after dip dyeing is taken, aseptic filter paper blots bacterium solution above, is then inoculated in common training It supports on culture medium (blade back is downwards), 25 DEG C of light culture 3d.

(4) screening and culturing：Tobacco leaf after co-culturing 3d washes one with the cephalosporin solution of 500mg/L concentration Time, then aseptic water washing time 3-5 times, retransfers into the hoof for being added to 100mg/L kanamycins and 500mg/L cephalosporins It selects in culture medium.

(5) culture of rootage：When hoof selects the adventitious bud on culture medium to grow to 1cm or so, cut and be transferred to and be added to On the Cef root medias of 100mg/LKm and 500mg/L.

(6) tobacco seedling is transferred to earth culture：Transformation seedlings after under growth root cover with culture bottle, by being taken out in root media, with certainly Water cleans the culture medium on transformation seedlings, and transplants in the Nutrition Soil of sterilizing.Transformation of tobacco seedling used medium is shown in Table 2.

2 Transformation of tobacco seedling used medium formula of table

3. the screening of transgenic positive seedling

It obtains turning PbrNHX2 genetic tobaccos according to the method described above, every plant of tobacco is extracted DNA, designs and draws in primer gene Object carries out the positive seedling of PCR amplification identification.

3.1 tobacco leaf DNA are extracted

(1) suitable tobacco young leaflet tablet is taken to be put into 1.5mL centrifuge tubes, liquid feeding nitrogen is fully ground into powdered；So DNA Extraction buffers cetyltriethylammonium bromide (abbreviation CTAB, the formula of 700 μ l, 65 DEG C of preheatings are added in afterwards：100mM Tris-HCl (pH8.0), 1.5MNaCl, 50mM EDTA (pH=8.0) solution add 1% polyvinylpyrrolidone, 2% (volume) CTAB, 65 DEG C of water-baths are fully dissolved spare, with preceding after 65 DEG C of water-baths preheat, are added in 1~4% (volume) mercaptoethanol, are mixed It is even),

(2) 65 DEG C of warm bath 60-90min take out every 15min and gently overturn mixing up and down；10000g room temperature centrifuges 10min； Supernatant (can directly pour into centrifuge tube) is taken, adds 600 μ l chloroform isoamyl alcohol (chloroforms：Isoamyl alcohol volume ratio is 24：1) it, overturns mixed Even extracting 3min；

(3) 10000g centrifuges 15min；450 μ l of supernatant (albumin layer is not drawn in attention, leads to protein contamination) are taken, are added in Absolute ethyl alcohol is pre-chilled in 900 μ l-20 DEG C, and 34 μ l 5MNaCl, after overturning mixing, -20 DEG C freeze 30min.

(4) 10000g centrifuges 10min；After abandoning supernatant, after washing 3 times with the ethyl alcohol of 1mL 75%, blank pipe centrifuges one point Clock is put into superclean bench and blows half an hour, until DNA is in colorless and transparent, adds appropriate distilled water, is positioned over 37 DEG C of incubators Middle dissolving 40min, does glue detection.

3.2 positive transgenic plant detect

PCR amplification is carried out using primer gene specific primer.Response procedures and system are shown in Table 3, table 4 respectively.Using 35S Gene right inner primer (SEQ ID NO.9 and SEQ ID NO.10) carries out PCR, in the transgenic line of selection, there is transgenosis Strain can amplify the segment of expected size, and showing this, they are positive transgenic strain.

3 PCR response procedures of table

Step	94℃	58℃	72℃	4℃	Recurring number
						Step 1	3min				1
Step 2	30s	30s	55s		35
						Step 3			90s		1
Step 4			10min
						Step 5				30min

4 PCR reaction systems of table

Reactive component	Dosage (μ l)
		Template DNA	1
PCRBuffer	2
		dNTDMix(2.5mmol/L)	1.6
Right side forward primer	1
		Left side reverse primer	1
Taq DNA polymerase (5U)	0.2
		Nuclease free water	13.2

PbrNHX2 is expressed in tobacco by agriculture bacillus mediated.It is analyzed by molecular genetic, identification has obtained single copy Homozygosis is inserted into, stablizes the transgene tobacco for expressing PbrNHX2 from T1-T2 generations, therefore phenotypic character can stablize heredity.It inserts The character mutation that angle of striking analytical proof turns PbrNHX2 transgenic lines respectively is not to influence other genes by transgeneic procedure Caused by.Therefore the transgenic line provides material guarantee for this item purpose research.Vector construction such as Fig. 5-a, transgenosis Tobacco whole flow process is shown in Fig. 5-b, utilizes RT-PCR identification genetically modified plants such as Fig. 5-c (35S primers+downstream of gene primer), profit Further identify that TG17 and TG20 is two overexpression systems (Fig. 5-d and Fig. 5-e) with RT-PCR and Western blotting.

Embodiment 6

The overexpression analysis of transgenosis Ussurian pear plant

Transgenosis Ussurian pear plant is built according to the method for embodiment 5.Extract 8 plants of transgenic positive seedlings of transplant survival RNA, detect its structural integrity, such as Fig. 6 by running glue, measure after its concentration that (concentration is in 200- using nanodrop 600ng/ul), RNA total amounts are adjusted to carry out after 3ug reverse transcription into cDNA, then expanded as internal reference by the use of the Tublin of pears Increase.Tublin primer nucleotide sequences are：

Tublin forward primers：5’-TGGGCTTTGCTCCTCTTAC-3’(SEQ ID NO.11)

Tublin reverse primers：5’-CCTTCGTGCTCATCTTACC-3’(SEQ ID NO.12)

With Tublinn amplify come band brightness it is consistent, illustrate that the cDNA concentration of reverse transcription is identical, after use again PbrNHX2 special primers are as template amplification purpose band, PbrNHX2 primer nucleotide sequences：

Forward primer：5’-ATGGCTGTTGCACATTTGAGCATGATG-3’(SEQ ID NO.3)

Reverse primer：5’-TTGCCACTGAACGTTGTTGTCCCGTTC-3’(SEQ ID NO.4).

According to PbrNHX2 special primers amplify come purpose band brightness size, it can be determined that go out PbrNHX2 bases Because of expression quantity in positive transgenic pears, select that brightness is high 2,6 and 8, i.e. three high overexpression strains names of expression quantity Then OE2, OE6 and OE8 expand numerous respectively as individual transgenic line.

As a result as shown in Figure 6.Fig. 6 converts Ussurian pear and plant regeneration process schematic, wherein Fig. 6-A for PbrNHX2 Photo after conversion；Fig. 6-B are that the material of 30 days is grown on screening and culturing medium；Fig. 6-C are regeneration bud root inductions；Fig. 6-D It is the photo that genetically modified plants shifting grows 30 days in soil；Fig. 6-E are after carrying out PCR identification tobaccos using gene specific primer T0 is for transfer-gen plant, wherein M：Marker ,+：Plasmid ,-：WT lines, 1-8：Transgenic line；Fig. 6-F are fixed in real time Measure the expression quantity of the PbrNHX2 encoding genes in PCR analysis Ussurian pear difference transgenic lines.Fig. 6 illustrates that pears turn The PbrNHX2 genes overexpression of PbrNHX2 gene plants.

Embodiment 7

PbrNHX2 transgenic resistance plant Resistance Identifications

1. the salt resistance analysis of transgenic tobacco plant

In order to identify whether PbrNHX2 transgene tobaccos are related with salt stress-resistant, control series and transgenosis system are carried out short The salt stress of time and the processing of long-term salt adverse circumstance.PbrNHX2 transgenosis systems (TG17, TG20) tobacco that same batch receives It is sowed at respectively on MS screening and culturing mediums and common MS nonreactives culture medium after seed and the processing of wild type (WT) Seed sterilization, etc. It is transplanted in soil within about 3 days after germination and is cultivated.The transfer-gen plant of different seedling ages is taken to carry out salt treatment, is observed at its Phenotype after reason counts survival rate, measures conductivity and chlorophyll etc..

Fig. 7 is to turn the encoding gene strain and wild type (WT) sodium chloride of PbrNHX2 phenotype before and after the processing and physiology refers to Mapping is determined, and wherein Fig. 7-A are phenotype of 45 -day-old of the tobacco plant before 300mM sodium chloride is handled 20 days；Fig. 7-B are 45 days Phenotype of the big tobacco plant after 300mM sodium chloride is handled 20 days；Fig. 7-C are that 30 -day-old of tobacco plant is molten in 200mM salt Liquid handle 4 days before phenotype；Fig. 7-D are that 30 -day-old of tobacco plant handles the phenotype of 4 days in 200mM salting liquids；Fig. 7-E are 30 -day-old of tobacco plant handles the survival rate statistical result of 4 days in 200mM salting liquids, and Fig. 7-F are 30 -day-old of tobacco plants The conductance measurement of 4 days is handled in 200mM salting liquids as a result, Fig. 7-G are 45 -day-old of tobacco plants at 300mM sodium chloride Phenotype of the reason after 25 days, Fig. 7-H are chlorophyll measuring of 45 -day-old of the tobacco plant after 300mM sodium chloride is handled 25 days, are schemed 7-I is that 30 -day-old of tobacco plant handles the chlorophyll extraction of 4 days in 200mM salting liquids.The measure of These parameters is to comment The important measurement index of its salt-resistance of valency can obtain the salt resistance of transfer-gen plant of PbrNHX2 this genes amplification in Fig. 7 Property.

2. the salt resistance analysis of transgenosis Ussurian pear

In order to identify whether PbrNHX2 transgenosis Ussurian pear is related with salt stress-resistant, control series and transgenosis system are carried out The processing of salt adverse circumstance.By PbrNHX2 transgenosis Ussurian pear strains (OE2, OE6 and OE8) and WT lines (WT) 500mM chlorinations Sodium pours phenotype and the physiological index determining of 15 days.

The results are shown in Figure 8.Fig. 8 be embodiment 8 in PbrNHX2 transgenosis Ussurian pear strains (OE2, OE6 and OE8) and WT lines (WT) 500mM sodium chloride pours 15

3rd, histochemical stain analysis H₂O₂And O^2-Accumulation

In transgenic line (tobacco/Ussurian pear), conductivity is relatively low and survival rate height shows that they may have than WT There is the ability of stronger anti-ROS.So by identifying that the accumulation of ROS in plant just necessitates.It is texturized with DAB and NBT It learns decoration method to dye plant leaf, is respectively intended to detection hydrogen peroxide (H₂O₂) and superoxide anion (O^2-) content.

As a result enter shown in Fig. 9.Fig. 9 analyzes H to turn PbrNHX2 genetic tobaccos and Ussurian pear histochemical stain₂O₂And O^2- Accumulation, Fig. 9-A and Fig. 9-B are 45 -day-old of tobacco plant unconverted plant and two before and after 300mM sodium chloride handles 15d The histochemical stain of transgenic line active oxygen is using nitro tetrazole (NBT) and diaminobenzidine (DAB) respectively to H₂O₂ (Fig. 9-A) and O^2-(Fig. 9-B) is dyed, cell death chromosome after Fig. 9-C are handled for transgene tobacco salt stress, Fig. 9-D With Fig. 9-E be 30 -day-old Ussurian pear plant 500mM sodium chloride handle 15d before and after unconverted plant and two transgenic lines It is that active oxygen histochemical stain uses nitro tetrazole (NBT) and diaminobenzidine (DAB) respectively to H₂O₂(Fig. 9-D) and O^2-(Fig. 9-E) is dyed, and Fig. 9-F are cell death chromosome after the processing of transgenosis Ussurian pear salt stress.Such as Fig. 9-A and figure 9-B), before salt stress processing is done, do not have coloured significant difference between transgenosis system and control.Such as Fig. 9-A and Fig. 9-B After salt stress 30d, there is the leaf area of brown significantly than transgenic line in the blade that is dyed with DAB, wild strain type Greatly, blade that is and darker, being dyed with NBT, WT strain are deeper than the blue of transgenic line, and area is with big.These Evidence shows that transgenic line ties up to and has lower ROS (H under salt stress compared with wild type₂O₂And O^2-) accumulation.Same result exists Similar result is obtained in transgenosis Ussurian pear plant (as shown in 9-D and Fig. 9-E).

4th, comprehensive analysis shows to identify its function after the gene is transferred in tobacco and Ussurian pear, it is found that transgenosis surpasses table Up to strain, saline-alkaline tolerance has very big promotion compared with compareing wild type.Hydrogen peroxide (H in transgene tobacco₂O₂) and it is super Oxygen anion (O^2-) content activity be intended to it is lower than wild type, plant activity in vivo oxygen remain lower, cellular damage smaller. These results indicate that the PbrNHX2 genes being overexpressed can effectively enhance the oxygen scavenging activity ability of transfer-gen plant, from And improve the salt tolerance of plant.

The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications It should be regarded as protection scope of the present invention.

Sequence table

<110>Agricultural University Of Nanjing

<120>Sodium, hydrogen reverse transport protein PbrNHX2 and its application in plant salt tolerance ability is improved in birch-leaf pear

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<170> SIPOSequenceListing 1.0

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Met Ala Val Ala His Leu Ser Met Met Ile Ser Lys Leu Gln Asn Val

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

His Ser Pro Lys Gln Thr Thr Ser Met Leu Ser Ser Glu Pro Thr Ser

450 455 460

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

465 470 475 480

Leu Val Gly Gln Asp Ile Arg Arg Pro Ala Ser Leu Arg Asp Leu Leu

485 490 495

Thr Thr Pro Thr His Thr Val His Arg Tyr Trp Arg Lys Phe Asp Asn

500 505 510

Ala Phe Met Arg Pro Val Phe Gly Gly Arg Gly Phe Val Pro Phe Val

515 520 525

Pro Gly Ser Pro Thr Glu Arg Asp Asn Asn Val Gln Trp Gln

530 535 540

<210> 2

<211> 1629

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 2

atggctgttg cacatttgag catgatgatc tcaaagttac aaaatgtatc cacttcggac 60

cactcctccg tggtttcgat gaacctattt gtggcgctac ttttagcttg tattgtgatc 120

ggacatcttc tcgaggagaa tcgatgggtg aatgagtcga tcaccgccct tttgattggg 180

ctttgcaccg gagtagttat tcttctgatc agtagaggaa gaagctcgca tcttttggtt 240

ttcagtgaag atcttttctt tatatacctc ttgccgccta ttatttttaa tgccgggttc 300

caggtgaaaa agaagcagtt ctttgttaac ttcatgacca ttgtaatgtt tggtgctatt 360

ggtacattag tatcctgcac tatcatatca ttaggtgcta cacaattctt taagaagttg 420

gacattggaa ctctggagtt gggggacttc cttgcaattg gtgcaatatt tgctgcaacg 480

gattctgttt gcacgttgca ggtgctcaat caggatgaga cacctttact ctacagtctt 540

gtatttggag agggtgtcgt taacgatgcg acatctgtgg tccttttcaa tgctattcag 600

agctttgatc tcacccacat tgattccagt attgctttgc actttatggg caacttctta 660

tatttgtttt tcgcaagcac tatgctagga gtgtttgcag ggctgcttag tgcttacatt 720

atcaaaaagc tttattttgg aagccactct acggatcgtg aggttgctct tatgatgctc 780

atggcatact tgtcatatat actggctgaa ttattctatt tgagtggcat tctcactgtt 840

ttcttttgtg ggatcgtgat gtcgcattac acttggcaca atgtgactga gagttcaaga 900

gttacgacca agcatgcttt cgcaaccttg tcatttgttg ccgaaacatt tatcttcctt 960

tatgttggta tggatgcctt ggacattgaa aagtggcgat ttgtaagtga cagtcctgga 1020

acatcagtgg cagtgagttc aatactgcta ggtcttgtta tgcttggaag agcagctttc 1080

gtcttcccct tatcattctt gtcgaactta acaaagaaaa accaacgtga taaaattagc 1140

ctccggcagc aagttataat atggtgggct ggtctcatga gaggtgctgt gtctatggca 1200

cttgcttaca atcagtttac aaggtcaggc cacacgcaat tgcgagcaaa tgcaatcatg 1260

atcactagca cgataactgt tgttcttgtc agcacagtgg ttttcggatt gatgacgaaa 1320

cctcttataa ggttcttgct gcctcattca ccaaaacaaa caaccagcat gttgtcgtca 1380

gaaccaacct ctccaaaatc agtcattgtt ccacttctag ggcaggattc tgtagatgat 1440

cttgttggcc aagatattcg acggccggcc agcttacgcg atcttctgac aactccaacg 1500

cacacagtcc atcgctattg gcgtaagttt gacaacgcat tcatgcgtcc agtgtttgga 1560

ggccggggtt ttgttccctt tgttcccggg tcaccaactg aacgggacaa caacgttcag 1620

tggcaatga 1629

<210> 3

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<213>Artificial sequence (Artificial Sequence)

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atggctgttg cacatttgag catgatg 27

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<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

ttgccactga acgttgttgt cccgttc 27

<210> 5

<211> 19

<212> DNA

<213>Artificial sequence (Artificial Sequence)

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tgggctttgc tcctcttac 19

<210> 6

<211> 19

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

ccttcgtgct catcttacc 19

<210> 7

<211> 33

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 7

gtcgacatgg ctgttgcaca tttgagcatg atg 33

<210> 8

<211> 33

<212> DNA

<213>Artificial sequence (Artificial Sequence)

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ggatccttgc cactgaacgt tgttgtcccg ttc 33

<210> 9

<211> 27

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 9

cgccgtaaag actggcgaac agttcat 27

<210> 10

<211> 27

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 10

ttgccactga acgttgttgt cccgttc 27

<210> 11

<211> 19

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 11

tgggctttgc tcctcttac 19

<210> 12

<211> 19

<212> DNA

<213>Artificial sequence (Artificial Sequence)

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ccttcgtgct catcttacc 19

Claims (9)

1. sodium, hydrogen reverse transport protein PbrNHX2 in birch-leaf pear, which is characterized in that have as shown in SEQ ID NO.1 in sequence table Amino acid sequence.

2. the encoding gene of sodium, hydrogen reverse transport protein PbrNHX2 in birch-leaf pear described in claim 1, which is characterized in that have Nucleotide sequence as shown in SEQ ID NO.2 in sequence table.

3. it is a kind of for cloning the primer pair of encoding gene cDNA sequence described in claim 2, draw including forward primer and reaction Object, which is characterized in that the forward primer has the nucleotide sequence as shown in SEQ ID NO.3 in sequence table；It is described reversed Primer has the nucleotide sequence as shown in SEQ ID NO.4 in sequence table.

4. encoding gene described in sodium in birch-leaf pear described in claim 1, hydrogen reverse transport protein PbrNHX2, claim 2 or Application of the primer pair in plant salt tolerance ability is improved described in claim 3.

5. application according to claim 4, which is characterized in that the application includes the following steps：It will be described in claim 1 Birch-leaf pear in encoding gene described in sodium, hydrogen reverse transport protein PbrNHX2 or claim 2 or drawing described in claim 3 Object recombinantly expresses the encoding gene of clone in plant, obtains the recombinant plant with salt tolerance.

6. application according to claim 5, which is characterized in that the program of the clone is 94 DEG C of pre-degeneration 3min；94℃ 30s is denaturalized, 58 DEG C of annealing 90s, 72 DEG C of extension 90s, 35 recycle, 72 DEG C of extension 10min after the completion of cycle.

7. application according to claim 4 or 5, which is characterized in that the plant includes tobacco or Ussurian pear.

8. application according to claim 4, which is characterized in that the salt resistant character of the recombinant plant represents with salinity, The salinity is not higher than 1000mmol/L.

9. the application according to claim 4 or 8, which is characterized in that the salt includes Na⁺Or K⁺。