CN106929492A

CN106929492A - Nitraria tangutorum tonoplast H+PPase GFPs NtVP1, its encoding proteins, cloning process

Info

Publication number: CN106929492A
Application number: CN201710014268.4A
Authority: CN
Inventors: 杨秀艳; 唐欣; 李焕勇; 刘正祥; 张华新; 朱建峰; 成铁龙; 常二梅; 段晓波; 王文军
Original assignee: Beijing Qiji Biotechnology Co Ltd; CHINESE ACADEMY OF FORESTRY
Current assignee: Beijing Qiji Biotechnology Co Ltd; CHINESE ACADEMY OF FORESTRY
Priority date: 2016-01-15
Filing date: 2017-01-09
Publication date: 2017-07-07
Anticipated expiration: 2037-01-09
Also published as: CN106929492B

Abstract

The invention belongs to biology field, there is provided Nitraria tangutorum tonoplast H⁺PPase GFPs NtVP1 and its encoding proteins, and there is provided the method for cloning the gene and the method for building the carrier comprising the gene.The albumen of gene of the invention and its coding is played an important role in Nitraria tangutorum resists salt stress, and new selection is provided to cultivate new variety of plant.

Description

Nitraria tangutorum tonoplast H+- PPase GFPs NtVP1, its encoding proteins, clone Method

Technical field

The invention belongs to biology field, it is related to the clone of gene and vector construction.

Background technology

Tonoplast H⁺Transhipment inorganic pyrophosphatase (V-H⁺- PPase) it is a kind of proton pump of unique acidifying vacuole, exist In terrestrial plant and a small number of algae, protozoan and bacterium.The major function of the enzyme is as follows：(1) proton-pump：Can be by Free energy and H that inorganic pyrophosphate (PPi) hydrolysis is produced⁺Proton transmembrane transport body phase is coupled, and PPi is hydrolyzed into the same of 2 Pi When, by the H in cytoplasm⁺Pumped into vacuole through tonoplast, play proton pump, with tonoplast H⁺- ATPase forms H together⁺ Across tonoplast electrochemical gradient, is active of various solutes (amino acid, carbohydrate, the cation and anion etc.) molecule across tonoplast Transport provides driving force.(2) ion compartment function：Under salt stress, plant is by intracytoplasmic a large amount of separating of inorganic ions It is that plant resists one of main policies of salinity to vacuole.In the active transport processes of various ions, tonoplast and plasma membrane turn Fortune protein exhibits important function, and the driving force of this transport protein, then come from the H that tonoplast proton pump is set up⁺Across Tonoplast electrochemical gradient.Can be tonoplast Na this imply that increasing the expression of tonoplast proton pump gene⁺/H⁺Antiport egg Stronger driving force is provided in vain, so as to further strengthen the salt tolerance of plant.(3) effect in growth and development of plants：Liquid Vacuolar membrane H⁺- PPase has substantial connection with the transport of auximone, the generation of growing that the interaction of the two can be to plant Influence.(4) biosynthesis of sucrose is participated in：The effect of sucrose in plant in sucrose synthase and UDPG pyrophosphorylases Under, glucose and pbosphohexose salt are converted into, and more PPi is had during this and is produced, and these PPi are generally by VHP Remove.Comprehensive these results of study are visible, and tonoplast VHP plays an important role in plant stress-resistance.

Nitraria tangutorum (Nitraria tangutorum) is under the jurisdiction of zygophyllaceae (Zygophyllaceae) Nitraria (Nitraria) it is, Chinese distinctive halophytes, is distributed mainly on northwest salination desert.Nitraria tangutorum well developed root system, Leaf is small and carnification, with anti-adversity abilities such as anti-blown sand, drought-resistant and Salt And Alkali Tolerances, be typical polysalt type (by salt ion gather in In vacuole) halophytes can be that the important of NORTHWEST CHINA deserta is built in region normal growth of the soil salt content up to 2% One of group's kind.Numerous research discoveries, Na⁺Separating in vacuole is the necessary links of plant salt tolerant under high-salt stress, this Applicant's early stage is to the tonoplast Na in Nitraria tangutorum⁺/H⁺Antiporter gene NtNHX1 successful clones and to its table Up to being studied, as a result find NtNHX1 genes be present in the Different Organs of Nitraria tangutorum and its receive NaCl induce and The salt treatment initial stage, its transcriptional level substantially increased, and these results imply that NtNHX1 genes are played in the salt tolerant of Nitraria tangutorum Important effect.Understand as previously described, tonoplast H⁺- PPase proton pumps are tonoplast Na⁺/H⁺Reverse transport protein provides driving Power, therefore to Nitraria tangutorum tonoplast H⁺- PPase GFPs are studied has important work for production salt-tolerant plant With.But in the prior art also without Nitraria tangutorum tonoplast H⁺- PPase GFPs and relevant report.

The content of the invention

It is an object of the invention to provide Nitraria tangutorum tonoplast H⁺- PPase GFPs and its cloning process, to solve Above mentioned problem of the prior art.

The first aspect of the invention is to provide Nitraria tangutorum tonoplast H⁺- PPase GFPs NtVP1, above-mentioned base The full length cDNA sequence of cause such as SEQ ID NO:Shown in 6.

Further, the coding region sequence of the gene such as SEQ ID NO:Shown in 106-2409 of 6.

The second aspect of the invention is to provide Nitraria tangutorum tonoplast H⁺- PPase albumen, the amino acid of the albumen Sequence such as SEQ ID NO:Shown in 7.

The third aspect of the invention is to provide a kind of clone's Nitraria tangutorum tonoplast H⁺- PPase GFPs NtVP1 Method, including step：

1) according to the tonoplast H of the plant for having Characterization with Nitraria tangutorum⁺The conserved region of-PPase GFPs sets Meter degenerate primer pair, it is preferable that the degenerate primer is to being NtVP1-F1 and NtVP1-R1：

NtVP1-F1：CATTCGCCATTCAGG(SEQ ID NO:1),

NtVP1-R1：ACATAGCAGCCAAAGT(SEQ ID NO:2)；

2) total serum IgE with Nitraria tangutorum blade is as template, using step 1) degenerate primer to the side by RT-PCR Method obtains cDNA fragments；Preferably, the cDNA fragment lengths are 1156bp (SEQ ID NO:3)；

3) according to step 2) the cDNA fragments that are obtained, design UPM primers, 3 ' RACE and 5 ' RACE primers, by nido Amplification obtains full length cDNA sequence, the nucleotide sequence such as SEQ ID NO of the full-length cDNA:Shown in 6；Preferably, it is described to draw Thing sequence is as follows：

UPM(SEQ ID NO:10)：

CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT；

3’RACE：TGCCAATCCAGTGGCAATAGTGCTGA(SEQ ID NO:4)；

5’RACE：ACAACTGCTGCCATCGGAAAGGGATT(SEQ ID NO:5).

Preferably, the plant for having Characterization with Nitraria tangutorum is overlord.

The fourth aspect of the invention is to provide a kind of expression vector or expression bacterial strain, the expression vector or expression bacterial strain Comprising SEQ ID NO:Nitraria tangutorum tonoplast H shown in 6⁺The cDNA sequence of-PPase GFPs NtVP1 or its coding Region sequence.Preferably, the expression vector is the carrier with GFP genes, and the expression bacterial strain is Agrobacterium.

The fifth aspect of the invention is to provide a kind of construction method of expression vector, and the expression vector includes SEQ ID NO:Nitraria tangutorum tonoplast H shown in 6⁺The cDNA sequence or its coding region sequence of-PPase GFPs NtVP1, including： 1) Xba I and Kpn I restriction enzyme sites are added respectively in the cDNA sequence of NtVP1 genes or the two ends of its coding region sequence, and even Carrier T is connected to, T-NtVP1 carriers are formed；2) by T-NtVP1 carriers, and pCG-GFP and pSN1301 respectively with Xba I and Kpn I carry out digestion, and reclaim；3) by the genetic fragment got off from digestion on T-NtVP1 carriers and pCG-GFP and pSN1301 The endonuclease bamhi connection of carrier.

The sixth aspect of the invention is to provide a kind of by Nitraria tangutorum tonoplast H of the invention⁺- PPase albumen bases Transfected because of NtVP1 to the method for plant, including will be comprising SEQ ID NO:Nitraria tangutorum tonoplast H shown in 6⁺- PPase eggs The cDNA sequence of white gene NtVP1 or the carrier of its coding region sequence are converted into arabidopsis.

The seventh aspect of the invention is to provide Nitraria tangutorum tonoplast H of the invention⁺- PPase GFPs NtVP1 Application in preparing or cultivating genetically modified plants.

The present invention utilizes the tonoplast H of the different plant species nearer with Nitraria tangutorum affiliation⁺- PPase GFPs Conserved regions design degenerate primer, using RT-PCR and RACE methods, Nitraria tangutorum tonoplast H is cloned into first⁺-PPase GFP NtVP1, and functional analysis has been carried out to the gene, and it is right in the case of non-saline environment with high-salt stress respectively Its transcriptional level is studied, so that the zymoprotein of the clearly gene and its coding is resisted in Nitraria tangutorum on a molecular scale Effect in salt stress, new selection is provided to cultivate new variety of plant.

Brief description of the drawings

Fig. 1 shows Nitraria tangutorum tonoplast H⁺The cDNA sequence and amino acid sequence of-PPase (VP1).

Fig. 2 shows the tonoplast H in different plant species source⁺- PPase (VP1) Phylogenetic analysis figure.

Fig. 3 shows Nitraria tangutorum tonoplast H⁺- PPase (NtVP1) bioinformatic analysis figure：A, VP1 albumen knot Structure domain analysis；B, VP1 albumen hydrophobicity analysis；Analyze in C, VP1 protein transmembrane area；D, VP1 Protein secondary structure are predicted.

Fig. 4 shows Nitraria tangutorum tonoplast H⁺QRT-PCR of-PPase the GFPs (NtVP1) in Different Organs Expression analysis figure；The relative expression quantity of NtVP1 genes in A, Different Organs；In the lower Nitraria tangutorum blade of B, NaCl stress The relative expression quantity of NtVP1 genes.

Fig. 5 shows pCG-GFP carrier structure schematic diagrames.

Fig. 6 shows pSN1301 carrier structure schematic diagrames.

Fig. 7 shows the electrophoretogram that Nitraria tangutorum NtVP1 genes have been connected in carrier T；Wherein, Marker is DL2000plus；1-6 is 6 escherichia coli clonings of detection, wherein 2,4,5,6 is 4 positive colonies.

Fig. 8 shows the electricity of the genetic fragment for carrying out that NtVP1 is obtained after Xba I and Kpn I double digestions to T-NtVP1 carriers Swimming figure.

Fig. 9 shows and obtains open circular plasmid after Xba I and Kpn I double digestions are carried out to pCG-GFP and pSN1301 carriers Electrophoretogram.

Figure 10 is shown with the special primer T1 and T2 of NtVP1 genes, to pCG-35S::NtVP1-GFP and pSN1301-35S::The electrophoretogram that the positive spot of NtVP1 conversions is detected.M:Represent DL2000plus；Left side VP1-pCG is pCG-35S::NtVP1-GFP positive colony testing results；The right VP1-pSN1301 is pSN1301-35S::Positive gram of NtVP1 Grand testing result.

Figure 11 shows that after 30mmol/L NaCl stress T3 is for Arabidopsis plant and the leaf of wild-type Arabidopsis plants The comparison diagram of green element, proline and mda content and SOD activity；A, chlorophyll content comparison diagram；B, proline content contrast Figure；C, mda content comparison diagram；D, SOD activity comparison diagram.

Specific embodiment

The present invention is further illustrated by specific examples below, but the scope of the present invention not limited to this.Such as without special theory Bright, reagent, instrument, consumptive material used in following embodiments etc. are this area general reagent, instrument and consumptive material, can be from normal Obtained at the chemical article shop of rule or supplier.Unless otherwise specified, the method used in following examples is this area In conventional method；Technical staff can clearly know how to carry out the experiment according to experiment description.

In this application, NtVP1 represents Nitraria tangutorum tonoplast H⁺- PPase GFPs, NtVP1 represents Tang Gute White thorn tonoplast H⁺- PPase albumen.

Embodiment one：Nitraria tangutorum tonoplast H⁺The clone of-PPase GFPs (NtVP1)

According to 10 kinds of species (upland cotton (Gossypium hirsutum ADN96173.1), grape to having cloned (Vitis vinifera XP_002273207.1), comospore poplar (Populus trichocarpa XP_006381091.1), plan Southern mustard (NP_173021.1), salicornia europaeal (AEI17666.1), foliated kalidium (Kalidium foliatum ABK91685.1), water Rice (BAA31523.1), silique alkali fluffy (Suaeda corniculata ADQ00196.1), caspian halostachyses (Halostachys Caspica ABO45933.1), overlord (ABU92563.1)) the gene carry out Multiple Sequence Alignment, set in conservative area Meter a pair of degenerate primers NtVP1-F1 (SEQ ID NO:1) with NtVP1-R1 (SEQ ID NO:2).Extract Nitraria tangutorum blade Total serum IgE (the RNAiso Plus of Takara companies), expanded from the total serum IgE of Nitraria tangutorum by the method for RT-PCR and obtained Conserved region cDNA fragments (1156bp) (SEQ ID NO:3).The method of the RT-PCR is as follows：

Take total serum IgE, 1 μ l, 50 μM of oligomerizations (Dt) of 8 μ l₂₀, 1 μ l 10mM dNTP mixtures (being carried out on ice), add Into the centrifuge tube without RNase of 0.2ml, gently mix, 65 DEG C of denaturation 5min, place on ice at least immediately after at once 1min, and then according to the form below carry out the mixing of following each component.

The mixture of 10 μ l by more than, adds in the centrifuge tube of the mixture containing RNA/ primers, gently beats after mixing, 50 DEG C of 50min, 85 DEG C of 5min at once.After procedure above terminates, centrifuge tube is placed 1 minute on ice immediately, after being slightly centrifuged, will The RNase H of 1 μ l is added in centrifuge tube, 37 DEG C of incubation 20min.Synthesize the first chain cDNA.The cDNA that will synthesize is sharp as masterplate With primer NtVP1-F1 (SEQ ID NO:1) with NtVP1-R1 (SEQ ID NO:2) conservative region PCR amplifications and sequencing are carried out.

Based on having cloned the conserved region cDNA fragments that obtain, design UPM primers (SEQ ID NO:10), RLM-RACE primer 5’RACE(SEQ ID NO:4) with 3 ' RACE (SEQ ID NO:5), by cDNA ends rapid amplification (RACE) (the SMARTer of Clontech companies^TMRACE cDNA Amplification Kit kits), it is total from Nitraria tangutorum blade Amplification obtains Nitraria tangutorum tonoplast H in RNA⁺The full length cDNA sequence of-PPase GFPs.Through sequencing, the full-length cDNA Length be 2810bp (SEQ ID NO:6).

Gained full length cDNA sequence is committed into NCBI to compare, the cDNA sequence and upland cotton (Gossypium is found Hirsutum), foliated kalidium (Kalidium foliatum), grape (Vitis vinifera) and salt mustard (Thellungiella The I types H of plant such as salsuginea)⁺The equal ＞ 80% of homology of-PPase GFPs, by full length gene cDNA sequence life Entitled NtVP1.

The cDNA sequence is translated into amino acid using NCBI, Nitraria tangutorum tonoplast H is obtained⁺- PPase albumen, life Entitled NtVP1, its amino acid sequence such as SEQ ID NO:Shown in 7.

Embodiment two：The tonoplast H of different plant species⁺- PPase protein sequences compare

10 tonoplast H from different plants are chosen from GenBank⁺The amino acid sequence of-PPase albumen, point Wei not upland cotton (Gossypium hirsutum ADN96173.1), grape (Vitis vinifera XP_ 002273207.1), comospore poplar (Populus trichocarpa XP_006381091.1), arabidopsis (NP_173021.1), Salicornia europaeal (AEI17666.1), foliated kalidium (Kalidium foliatum ABK91685.1), paddy rice (BAA31523.1), silique Alkali fluffy (Suaeda corniculata ADQ00196.1), caspian halostachyses (Halostachys caspica ABO45933.1), despot King (ABU92563.1), using DNAMAN softwares, Multiple range test is carried out by this 10 albumen and NtVP1 albumen of the invention.Knot Fruit display, NtVP1 amino acid sequences and the upland cotton H for having cloned⁺- PPase albumen homologies up to 93%, with studies of Arabidopsis thaliana vacuolar H⁺- PPase albumin A tVP1 homologys be 89.29%, and with studies of Arabidopsis thaliana vacuolar H⁺- PPase albumin A tVP2 homologys only have 33.53% (AAF31163.1).In addition, NtVP1 and other vegetation water vacuolar membranes H⁺- PPase albumen also has homology (figure higher 1).It will be seen from figure 1 that the albumen contains 3 conserved regions (CS1, CS2 and CS3), wherein contain in CS1 conserved regions DVGADLVGKVE sequences, with (E/X) (X)₇KXE configurations are consistent.The configuration is in many vegetation water vacuolar membrane H⁺Transport inorganic burnt phosphorus It is all highly conserved in sour enzyme, thereby increases and it is possible to be existed as the substrate binding site of pyrophosphoric acid (PPi) hydrolytic process.In addition, from Fig. 1 In it has also been found that the albumen contain ' EYYTS ' motif, the motif is related to the hydrolysis of pyrophosphoric acid and H⁺The coupling of transport, and it is present in In most of other vacuolar proton pump pyrophosphohydrolases (V-PPases).

Embodiment three：The tonoplast H in different plant species source⁺- PPase protein system chadograms

In tonoplast H⁺On the basis of the comparison of-PPase protein sequences, above-mentioned 10 are derived from using ClustalX softwares Tonoplast H in different plants⁺- PPase albumen (respectively upland cotton (Gossypium hirsutum ADN96173.1), Portugal Grape (Vitis vinifera XP_002273207.1) and (CAO41672.1), comospore poplar (Populus trichocarpa XP_006381091.1), arabidopsis (NP_173021.1) and (AAF31163.1), salicornia europaeal (AEI17666.1), foliated kalidium (Kalidium foliatum ABK91685.1), paddy rice (BAA31523.1), fluffy (the Suaeda corniculata of silique alkali ADQ00196.1), caspian halostachyses (Halostachys caspica ABO45933.1), overlord (ABU92563.1)) and this hair The amino acid sequence of bright NtVP1 albumen carries out multisequencing matching arrangement, then carries out systematic evolution tree using MEGA5.1 softwares Structure, the method is according to Neighbor-joining methods, by Poisson distance methods by 1000 times Bootstrap is analyzed.As a result (Fig. 2) show, Nitraria tangutorum tonoplast H⁺- PPase GFPs NtVP1 with belong to I type Upland cotton GhVP1, arabidopsis AtVP1, grape VvVP1 etc. genetic distance it is nearer, and with belong to the arabidopsis of II type AtVP2, grape VvVP2 genetic distance relatively far away from.

Example IV：Nitraria tangutorum tonoplast H⁺The sequence analysis of-PPase GFPs NtVP1

The protein sequence of NtVP1cDNA sequences coding is 768 albumen for amino acid.Speculated according to sequence, Its molecular weight is 80.5kDa, and isoelectric point is 5.33；Using hydrophobicity analysis software (http://web.expasy.org/cgi- Bin/protscale/protscale.pl) understand, hydrophobic amino acid is evenly distributed in the whole peptide chain of the albumen, and More than hydrophilic amino acid (A in Fig. 3).Therefore, whole polypeptide chain shows as hydrophobicity, illustrates that NtVP1 albumen meets film egg White feature, belongs to hydrophobic proteins (B in Fig. 3).Cross-film analysis is carried out using TMHMM softwares, shows that NtVP1 contains 14 Individual transmembrane region (C in Fig. 3), this and tonoplast H in other species⁺The membrane spaning domain of-PPase albumen is consistent.Use Swiss- Model(http://swissmodel.expasy.org/) NtVP1 protein structures are analyzed, it is seen that itself and 4a01.1.A The similitude of the sequence of template is up to 90.21%, belongs to proton pyrophosphatase model (D in Fig. 3).

Embodiment five：Expression analysis of the Nitraria tangutorum NtVP1 under NaCl high-salt stress

Total serum IgE (the post of Beijing day bounties gene Co., Ltd is extracted from Nitraria tangutorum spire, young stem and young root respectively Formula plant RNA out kits), it is template and reverse transcription synthesis cDNA the 1st chain (SuperScript III that 500ng is taken respectively Reverse Transcriptase).Based on NtVP1cDNA full length sequences, 1 couple of special primer NtVP1-F8 (SEQ NO is designed 8), NtVP1-R8 (SEQ NO 9), amplification obtains fragment (the SEQ ID NO that length is 207bp:13).With nitraria schoberi Actin Gene (Genbank：AB617805.1 it is) internal reference, using the method for quantitative fluorescent PCR to Tang Gute under the conditions of normal growth The expression of NtVP1 is detected in white thorn Different Organs.The experiment condition of quantitative fluorescent PCR is：95 DEG C of 10min predegenerations, so 95 DEG C of 15s, 60 DEG C of 1min afterwards, 40 circulations.Fluorescent dye is SYBR Green I.

Result shows that NtVP1 has expression, and the expression quantity in blade in Different Organs>Expression quantity in root>Table in stem Up to amount (A in Fig. 4).Whether to probe into Nitraria tangutorum VP1 genes by Salt treatment and regulation, further have detected 200mmol·L^-1NaCl stress 1,3,6,12,24,48h when Nitraria tangutorum blade in NtVP1 expression.Specific method For：The seed of Nitraria tangutorum is sowed in vermiculite：Turf is 3：In the blending agent of 1 (V/V), hot-house culture is placed in.Selection 3 monthly age seedling originally Aquaponic normal and of the same size is grown, water was changed 1 time every 4 days during culture.Water planting 1 week or so, adopts With 0 (CK) and 200mmol 〃 L^-1NaCl processes seedling 1,3,6,12,24,48h, each 3 repetition for the treatment of, and each repeats 3 plants, altogether 108 plants of meter.In the morning 10:00~11:Young leaf tissue is taken in 00 unification.Result shows that, with the extension of salt stress time, Tang is ancient Extra white thorn NtVP1 genes expression quantity in blade continues to increase, expression quantity highest during stress 12h, then gradually weakens (in Fig. 4 B), these results explanation NtVP1 genes may be played a significant role in Nitraria tangutorum Mechanisms of Salt Resistance.

Embodiment six：The structure of Nitraria tangutorum NtVP1 expression vectors

PCG-GFP carriers (Fig. 5) (brightness fine horse is biological) and pSN1301 carriers (Fig. 6) (brightness fine horse is biological) are determined first, in gene Two ends add Xba I and Kpn I restriction enzyme sites respectively, and are connected to carrier T (Transgen, CT11-01, http:// Www.transgen.com.cn/products/63.html on)；Insertion point fragment is carried out with gene specific primer T1 and T2 Amplification, is checked to obtain by PCR and contains NtVP1 gene bands, confirms that NtVP1 genes have been connected in carrier T (Fig. 7).Wherein, The primer sequence of T1 and T2 is respectively：T1:5’-GTTTGCCTTCGTCCTTCA-3’(SEQ ID NO:11)；T2:5’- CATTGTCCCATGCACCTC-3’(SEQ ID NO:12)。

T-NtVP1 carriers, and pCG-GFP and pSN1301 are carried out into digestion with Xba I and Kpn I respectively, is obtained NtVP1 genetic fragments (Fig. 8) and pSN1301 and pCG-GFP open loops carrier (Fig. 9), by NtVP1 genes connection pSN1301 and PCG-GFP, converts Escherichia coli, then chooses bacterium PCR detections, obtains detection figure (Figure 10), it was demonstrated that vector construction is completed, and obtains sun Property bacterium clone.Sequencing analysis are carried out to NtVP1 genes connection pSN1301 carriers, the gene flanking sequence for being measured all is carrier Sequence.Prove that NtVP1 genes are had been coupled on expression vector.

Embodiment seven：Nitraria tangutorum NtVP1 genetic transformation

Two kinds of binary vectors that embodiment six builds are transformed into Agrobacterium C58 respectively, are cultivated, be inoculated into 5ml and contain In the LB fluid nutrient mediums of antibiotic (rifampin 20mg/L, kanamycins 50mg/L), concussion and cultivate 2 days at 28 DEG C.1ml is trained Foster Agrobacterium is transferred in the LB fluid nutrient mediums that 100ml contains antibiotic, and 28 DEG C are continued concussion and cultivate 24 hours.By bacterium solution Pour into centrifuge tube, 6000rpm/min, under room temperature condition, be centrifuged 10 minutes, then pour out supernatant.Precipitation is contaminated with 200ml Liquid (10% sucrose, containing 0.02%silwet) is resuspended, forms uniform agrobacterium suspension (OD₆₀₀=0.8), and by Agrobacterium Suspension is transferred in a vessel for opening (500ml beakers).The healthy and strong arabidopsis of just fruiting period is chosen, band basin alms bowl back-off is in Sheng There is the container top of agrobacterium suspension, only immerse about 20-30 seconds in above-mentioned agrobacterium suspension whole inflorescence, basin alms bowl is taken Under, traverse is about 24 hours in camera bellows.Treated Arabidopsis plant is put under 22~25 DEG C of illumination condition after 24 hours Normal growth, mature seed is collected after three weeks.

Embodiment eight：The checking analysis of arabidopsis salt stress

The arabidopsis seed harvested in embodiment seven is seeded into the 1/2MS culture mediums containing 25mg/L hygromycin to be carried out Screening, is transplanted to (turfy soil in the culture medium containing turfy soil and vermiculite when two panels true leaf is grown：Perlite=1：1), will Arabidopsis plant is put in normal growth under 22~25 DEG C of illumination condition, and mature seed is collected after three weeks.Aforesaid operations are repeated, directly To collecting T3 for seed (TR).T3 is sowed into culture respectively for arabidopsis seed (TR) and wildtype Arabidopsis thaliana seed (WT), Two weeks after to be transplanted, carry out NaCl Stress treatments.Control group (0) and treatment group (1) are respectively provided with, three repetitions are respectively set, wherein It is processed as 30mmol/L NaCl.Blade is collected after Stress treatment 4d, immediately liquid nitrogen frozen, chlorophyll content, dried meat ammonia are measured respectively Acid content, mda content and SOD activity changes.

Result shows that T3 is 0.0334mg/g when (TR0) is compareed for the chlorophyll content of plant, after salt stress treatment (TR1) it is 0.0338mg/g, there was no significant difference (p>0.05)；Mda content is 0.0025umol/g when (TR0) is compareed, It is changed into 0.0031umol/g after salt stress treatment (TR1), also there was no significant difference (p>0.05)；And T3 contains for plant proline Amount is respectively 33.38ug/gFw (TR0) and 105.47ug/gFw (TR1) before and after salt stress, and content is significantly raised after salt stress, There is significant difference (p<0.05)；SOD activity is 7.445U/gFW when (TR0) is compareed, and is changed into after salt stress treatment (TR1) 17.056U/gFW, SOD activity are significantly raised after being processed through salt stress, there is significant difference (p<0.05) (Figure 11).

After salt stress, chlorophyll content is changed into 0.029mg/g (WT1) to WT lines from 0.039mg/g (WT0), deposits In significant difference (p<0.05)；Mda content rises to 0.0048umol/g (WT1) by 0.0034umol/g (WT0), There is significant difference (p<0.05)；Proline content is changed into 68.52ug/gFw (WT1), difference from 55.43ug/gFw (WT0) Not notable (p>0.05)；SOD activity is 5.42U/gFW (WT0) in control group, is 6.77U/gFW after salt stress treatment (WT1), same there was no significant difference (p>0.05) (Figure 11).

This result explanation salt stress promotes synthesis of the T3 for plant osmotic adjustment proline, and mda content increases Not significantly, synthesis and the degraded of chlorophyll are not affected by significantly affecting, and show that membranous system is more stablized；SOD activity significantly increases simultaneously Plus demonstrate its removing Superoxide radical anion ability enhancing.More than experiment prove, NtVP1 genes in Arabidopsis plant into Work(conversion makes T3 be significantly improved for plant salt tolerance, i.e., NtVP1 genes have actively work in terms of plant salt endurance is improved With.

Sequence table

SEQ ID NO:1：CATTCGCCATTCAGG

SEQ ID NO:2：ACATAGCAGCCAAACT

SEQ ID NO:3：

GATGATTGGGAGGGTCTTTTGAGGCTATCCRAGCATTTGGGCTGACGGGATCGACTACGCATGCTATGG GACGACCGGGCGGCGGTCTCTATCCGATTGCTGCTGATGTTGGTGCTGATCTTGTGGGCAAGGTCGAGAGAAACATT CCAGAAGACGAGCCAAGAAACCCTGCTGTCATTGCTGACAACGTTGGTGACAATGTTAGGGACGTTGCTCGCATGGG CTCAGATCTTTTCGGTTCATATGCCGGGTCATCTTGTGCTGCGCTCGTAGTTGCATCCATTTCATCCTTTGGAATCA ACCATGACTTCACTGCCATGTTGTATCCTCTGCTCATCAGTTCGATGGGTATCCTTGTTTGTTTGATCACAACTCTC TTTGCCACTGATATCTTTGAGATCAAGGCTGTTAAAGAGATCGAGCCAGCATTGAAGAAGCAGCTTATCATCTCTAC TATTCTTATGACTGTTGGAATTGCAATTGTTTCATGGGTTGGTTTGCCTTCGTCCTTCACAATCTACAATTTTGGGA CTCAGAAGGTTGTCAAGAACTGGGAACTTTTCTTGTGTGTGGGTGTTGGTCTTTGGGCTGGACTCATCATTGGATTT GTGACCGAGTACTATACTAGCAACGCATACAGCCCTGTACAAGATGTTGCTGACTCCTGCAGAACAGGAGCTGCCAC CAATGTTATCTTTGGTCTTGCTTTGGGATACAAATCTGTCATCATTCCAATTTTTGCCATTGCTATCAGTATTTTTG TCAGTTTTAGTTTGGCTGCTATGTATGGCATTGCAGTGGCTGCCCTTGGTATGCTCAGCACTATTGCCACTGGATTG GCAATTGATGCCTACGGTCCCATCAGTGACAATGCTGGAGGCATTGCTGAGATGGCTGGCATGAGTCACCGCATCCG TGAGAGGACTGATGCCCTTGATGCAGCTGGAAACACAACTGCTGCCATCGGAAAGGGATTTGCAATTGGATCAGCAG CCCTTGTCTCTTTGGCTCTGTTTGGTGCTTTTGTTAGCCGTGCAGCAATCTCAACAGTTGATGTCTTGACTCCAAAG GTGTTCATCGGTTTGATTGTTGGTGCTATGCTCCCGTACTGGTTCTCGGCCACATGACCATCGGAAGAGTGTNGGAA GTGCAATCT

SEQ ID NO:4：TGCCAATCCAGTGGCAATAGTGCTGA

SEQ ID NO:5：ACAACTGCTGCCATCGGAAAGGGATT

SEQ ID NO:6：

ACATGGGGAAACATCTTCACTTGAAAATACTTCCCTCTCCTCTGTCTTCTGCCTCTTCTCTTTTTCTTT TTTTTTTTTTTCGTTGTTTTTCTGGTTTTGGTAGGTATGGGAGTGGCGTTGCTGTCCGAGCTGGCGACGGAGATACT GGTTCCGGTCTGTGCCGTGATCGGTATCGTGTTCTCGCTCGTTCAGTGGTACCTCGTCTCGCGCGTGTCGCTCACGC ACGACCGGTCGGCCGGGAACAACAACAACAACAAGAAGAATGGATTCAACGATTATTTGATCGAGGAAGAGGAAGGA ATTAATGACCAGAGCGTCGTGACCAAGTGTGCTGAAATTCAGAACGCTATTTCTGAAGGTGCAACATCCTTTCTTTT CACTGAATATCAGTATGTTGGGATCTTCATGGTTGCTTTTGCAATCTTGATTTTCCTCTTCCTGGGTTCTGTGGAGG GCTTCAGCACAAAGAGCCAGCAATGTACTTACGATAAAACAAGGACGTGCAAGCCTGCACTTGCCACTGCTATCTTC AGCACAGTAGCATTTGTGCTTGGTGGCGTCACATCTGTCCTTTCTGGCTTCCTTGGGATGAAAATTGCTACTTATGC AAATGCCAGAACTACCCTGGAAGCAAGAAGGGGTGTCGGAAAGGCTTTTATTACTGCATTTAGGTCTGGTGCAGTAA TGGGCTTCCTCCTTGCAGCAAATGGTCTCTTGGTGCTTTACATTGCTATCAATCTCTTTAAGTTGTACTATGGTGAT GACTGGGAAGGCCTATTTGAGGCTATTACTGGATACGGTCTTGGGGGTTCTTCAATGGCTCTCTTTGGACGAGTGGG TGGTGGTATCTATACCAAGGCTGCTGATGTTGGTGCTGATCTTGTGGGCAAGGTCGAGAGAAACATTCCAGAAGACG ATCCAAGAAACCCTGCTGTCATTGCTGACAACGTTGGTGACAATGTTGGGGACATTGCTGGCATGGGCTCGGATCTT TTTGGTTCTTATGCTGAGTCATCCTGTGCTGCGCTTGTTGTTGCATCCATTTCATCCTTTGGAATCAACCATGACTT CACTGCCATGTTGTATCCTCTGCTCATCAGTTCGATGGGTATCCTTGTTTGTTTGATCACAACTCTCTTTGCCACTG ATATCTTTGAGATCAAGGCTGTTAAAGAGATCGAGCCAGCATTGAAGAAGCAGCTTATCATCTCTACTATTCTTATG ACTGTTGGAATTGCAATTGTTTCATGGGTTGGTTTGCCTTCGTCCTTCACAACCTACAATTTTGGGACTCAGAAGGT TGTCAAGAACTGGGAACTTTTCTTGTGTGTGGGTGTTGGTCTTTGGGCTGGACTCATCATTGGATTTGTGACCGAGT ACTATACTAGCAATGCATACAGCCCTGTACAAGATGTTGCTGACTCCTGCAGAACAGGAGCTGCCACCAATGTTATC TTTGGTCTTGCTTTGGGATACAAATCTGTTATCATTCCAATTTTTGCCATTGCTATCAGTATTTTTGTCAGTTTTAG TTTGGCTGCTATGTATGGCATTGCAGTGGCTGCCCTTGGTATGCTCAGCACTATTGCCACTGGATTGGCAATTGATG CCTACGGTCCCATCAGTGACAATGCTGGAGGCATTGCTGAGATGGCTGGCATGAGTCATCGCATCCGTGAGAGGACT GATGCCCTTGATGCAGCTGGAAACACAACTGCTGCCATCGGAAAGGGATTTGCAATTGGATCAGCAGCCCTTGTCTC TTTGGCACTGTTTGGTGCTTTTGTTAGCCGTGCAGCAATCTCAACAGTTGATGTCTTGACTCCAAAGGTGTTCATCG GTTTGATCGTTGGTGCTATGCTTCCGTACTGGTTCTCTGCCATGACCATGAAGAGTGCAGGAAGTGCTGCATTGAAA ATGGTTGAGGAGGTTCGCAGGCAGTTCAACACCATTCCTGGCCTCATGGAGGGCACTGCCAAGCCTGATTACGCTAA CTGTGTCAAGATCTCTACTGATGCTTCCATCAAGGAGATGATTCCTCCTGGTGCTCTTGTCATGCTCACACCCCTCA TCGTCGGAACCTTCTTCGGTGTGGAAACCCTCTCTGGTGTTCTTGCTGGCTCTCTTGTATCTGGTGTTCAGATCGCA ATATCTGCATCAAACACTGGAGGTGCATGGGACAATGCCAAGAAGTACATTGAGGCAGGTGCTTCTGAGCACGCAAG GACCCTTGGACCCAAAGGGTCAGAGCCACACAAGGCAGCTGTGATCGGTGACACCATCGGAGACCCACTCAAGGACA CGTCGGGGCCATCACTGAACATCCTTATCAAGCTTATGGCCGTAGAATCGCTTGTTTTTGCTCCCTTTTTCGCCACC CACGGTGCCTTTCTTTTCAAAATTTTTTGAAGAAAGTTAAATAATGACCAGAAGAAGGGGGGGAAGGGAGGGGGTTT GTTAAGCTAGTTTTATTTAGGGAAGTTAAAATACTATTTTTGATTTTGAGGAGGGATGAGGAGGAGGATTTGGACAC CAATGGAGATTGGGGTAAAACAAAAATTTGATGGGATGGGATGTGTGAACCAAAATTGGAGGCATAAATATGGGGCA TCAGGACTTGGGTGGGTTGGGGGTATGTTCTGGTCCTTTTTAATTTTAATTTTTGGTTTTTTTTTTTTTTGTTTCAT GTAGGGGGTAGAACAATTTTCCCTTTTTTATTTTATTTTAACATTTGTACTGTTCATCGTTTAATCAAGAAAAGACC ATTTGGATTTTTTACCAAAAAAAAAAAAAAAAAAAAAAAAGTACTT

SEQ ID NO:7：

MGVALLSELATEILVPVCAVIGIVFSLVQWYLVSRVSLTHDRSAGNNNNNKKNGFNDYLIEEEEGINDQ SVVTKCAEIQNAISEGATSFLFTEYQYVGIFMVAFAILIFLFLGSVEGFSTKSQQCTYDKTRTCKPALATAIFSTVA FVLGGVTSVLSGFLGMKIATYANARTTLEARRGVGKAFITAFRSGAVMGFLLAANGLLVLYIAINLFKLYYGDDWEG LFEAITGYGLGGSSMALFGRVGGGIYTKAADVGADLVGKVERNIPEDDPRNPAVIADNVGDNVGDIAGMGSDLFGSY AESSCAALVVASISSFGINHDFTAMLYPLLISSMGILVCLITTLFATDIFEIKAVKEIEPALKKQLIISTILMTVGI AIVSWVGLPSSFTTYNFGTQKVVKNWELFLCVGVGLWAGLIIGFVTEYYTSNAYSPVQDVADSCRTGAATNVIFGLA LGYKSVIIPIFAIAISIFVSFSLAAMYGIAVAALGMLSTIATGLAIDAYGPISDNAGGIAEMAGMSHRIRERTDALD AAGNTTAAIGKGFAIGSAALVSLALFGAFVSRAAISTVDVLTPKVFIGLIVGAMLPYWFSAMTMKSAGSAALKMVEE VRRQFNTIPGLMEGTAKPDYANCVKISTDASIKEMIPPGALVMLTPLIVGTFFGVETLSGVLAGSLVSGVQIAISAS NTGGAWDNAKKYIEAGASEHARTLGPKGSEPHKAAVIGDTIGDPLKDTSGPSLNILIKLMAVESLVFAPFFATHGAF LFKIF

SEQ ID NO:8：GTTCGCAGGCAGTTCAACACCATTC

SEQ ID NO:9：ACCAGATACAAGAGAGCCAGCAAGA

SEQ ID NO:10：

CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT

SEQ ID NO:11：GTTTGCCTTCGTCCTTCA

SEQ ID NO:12：CATTGTCCCATGCACCTC

SEQ ID NO:13：

GTTCGCAGGCAGTTCAACACCATTCCTGGCCTCATGGAGGGCACTGCCAAGCCTGATTACGCTAACTGT GTCAAGATCTCTACTGATGCTTCCATCAAGGAGATGATTCCTCCTGGTGCTCTTGTCATGCTCACACCCCTCATCGT CGGAACCTTCTTCGGTGTGGAAACCCTCTCTGGTGTTCTTGCTGGCTCTCTTGTATCTGGTGTTCAGATCGCAATAT CTGCATCAAACACTGGAGGTGCATGGGACAATGCCAAGAAGTACATTGAGGCAGGTGCTTCTGAGCACGCAAGGACC CTTGGACCCAAAGGGTCAGAGCCACACAAGGCAGCTGTGATCGGTGACACCATCGGAGACCCACTCAAGGACACGTC GGGGCCATCACTGAACATCCTTATCAAGCTTATGGCCGTAGAATCGCTTGTTTTTGCTCCCTTTTTCGCCACCCACG GTGCCTTTCTTTTCAAAATTTTTTGAAGA

Sequence table

<110>China Forestry Science Research Institute

Beijing fine horse Bioisystech Co., Ltd

<120>Nitraria tangutorum Vacuolar H~ +-PPase GFP NtVP1, its encoding proteins, cloning process

<130> TD1612546F

<160> 13

<170> PatentIn version 3.5

<210> 1

<211> 15

<212> DNA

<213>Artificial sequence

<220>

<223>Degenerate primer NtVP1-F1

<400> 1

cattcgccat tcagg 15

<210> 2

<211> 16

<212> DNA

<213>Artificial sequence

<220>

<223>Degenerate primer NtVP1-R1

<400> 2

acatagcagc caaagt 16

<210> 3

<211> 1156

<212> DNA

<213>Nitraria tangutorum

<400> 3

gatgattggg agggtctttt gaggctatcc ragcatttgg gctgacggga tcgactacgc 60

atgctatggg acgaccgggc ggcggtctct atccgattgc tgctgatgtt ggtgctgatc 120

ttgtgggcaa ggtcgagaga aacattccag aagacgagcc aagaaaccct gctgtcattg 180

ctgacaacgt tggtgacaat gttagggacg ttgctcgcat gggctcagat cttttcggtt 240

catatgccgg gtcatcttgt gctgcgctcg tagttgcatc catttcatcc tttggaatca 300

accatgactt cactgccatg ttgtatcctc tgctcatcag ttcgatgggt atccttgttt 360

gtttgatcac aactctcttt gccactgata tctttgagat caaggctgtt aaagagatcg 420

agccagcatt gaagaagcag cttatcatct ctactattct tatgactgtt ggaattgcaa 480

ttgtttcatg ggttggtttg ccttcgtcct tcacaatcta caattttggg actcagaagg 540

ttgtcaagaa ctgggaactt ttcttgtgtg tgggtgttgg tctttgggct ggactcatca 600

ttggatttgt gaccgagtac tatactagca acgcatacag ccctgtacaa gatgttgctg 660

actcctgcag aacaggagct gccaccaatg ttatctttgg tcttgctttg ggatacaaat 720

ctgtcatcat tccaattttt gccattgcta tcagtatttt tgtcagtttt agtttggctg 780

ctatgtatgg cattgcagtg gctgcccttg gtatgctcag cactattgcc actggattgg 840

caattgatgc ctacggtccc atcagtgaca atgctggagg cattgctgag atggctggca 900

tgagtcaccg catccgtgag aggactgatg cccttgatgc agctggaaac acaactgctg 960

ccatcggaaa gggatttgca attggatcag cagcccttgt ctctttggct ctgtttggtg 1020

cttttgttag ccgtgcagca atctcaacag ttgatgtctt gactccaaag gtgttcatcg 1080

gtttgattgt tggtgctatg ctcccgtact ggttctcggc cacatgacca tcggaagagt 1140

gtnggaagtg caatct 1156

<210> 4

<211> 26

<212> DNA

<213>Artificial sequence

<220>

<223>3'RACE primers

<400> 4

tgccaatcca gtggcaatag tgctga 26

<210> 5

<211> 26

<212> DNA

<213>Artificial sequence

<220>

<223>5'RACE primers

<400> 5

acaactgctg ccatcggaaa gggatt 26

<210> 6

<211> 2810

<212> DNA

<213>Nitraria tangutorum

<400> 6

acatggggaa acatcttcac ttgaaaatac ttccctctcc tctgtcttct gcctcttctc 60

tttttctttt tttttttttt cgttgttttt ctggttttgg taggtatggg agtggcgttg 120

ctgtccgagc tggcgacgga gatactggtt ccggtctgtg ccgtgatcgg tatcgtgttc 180

tcgctcgttc agtggtacct cgtctcgcgc gtgtcgctca cgcacgaccg gtcggccggg 240

aacaacaaca acaacaagaa gaatggattc aacgattatt tgatcgagga agaggaagga 300

attaatgacc agagcgtcgt gaccaagtgt gctgaaattc agaacgctat ttctgaaggt 360

gcaacatcct ttcttttcac tgaatatcag tatgttggga tcttcatggt tgcttttgca 420

atcttgattt tcctcttcct gggttctgtg gagggcttca gcacaaagag ccagcaatgt 480

acttacgata aaacaaggac gtgcaagcct gcacttgcca ctgctatctt cagcacagta 540

gcatttgtgc ttggtggcgt cacatctgtc ctttctggct tccttgggat gaaaattgct 600

acttatgcaa atgccagaac taccctggaa gcaagaaggg gtgtcggaaa ggcttttatt 660

actgcattta ggtctggtgc agtaatgggc ttcctccttg cagcaaatgg tctcttggtg 720

ctttacattg ctatcaatct ctttaagttg tactatggtg atgactggga aggcctattt 780

gaggctatta ctggatacgg tcttgggggt tcttcaatgg ctctctttgg acgagtgggt 840

ggtggtatct ataccaaggc tgctgatgtt ggtgctgatc ttgtgggcaa ggtcgagaga 900

aacattccag aagacgatcc aagaaaccct gctgtcattg ctgacaacgt tggtgacaat 960

gttggggaca ttgctggcat gggctcggat ctttttggtt cttatgctga gtcatcctgt 1020

gctgcgcttg ttgttgcatc catttcatcc tttggaatca accatgactt cactgccatg 1080

ttgtatcctc tgctcatcag ttcgatgggt atccttgttt gtttgatcac aactctcttt 1140

gccactgata tctttgagat caaggctgtt aaagagatcg agccagcatt gaagaagcag 1200

cttatcatct ctactattct tatgactgtt ggaattgcaa ttgtttcatg ggttggtttg 1260

ccttcgtcct tcacaaccta caattttggg actcagaagg ttgtcaagaa ctgggaactt 1320

ttcttgtgtg tgggtgttgg tctttgggct ggactcatca ttggatttgt gaccgagtac 1380

tatactagca atgcatacag ccctgtacaa gatgttgctg actcctgcag aacaggagct 1440

gccaccaatg ttatctttgg tcttgctttg ggatacaaat ctgttatcat tccaattttt 1500

gccattgcta tcagtatttt tgtcagtttt agtttggctg ctatgtatgg cattgcagtg 1560

gctgcccttg gtatgctcag cactattgcc actggattgg caattgatgc ctacggtccc 1620

atcagtgaca atgctggagg cattgctgag atggctggca tgagtcatcg catccgtgag 1680

aggactgatg cccttgatgc agctggaaac acaactgctg ccatcggaaa gggatttgca 1740

attggatcag cagcccttgt ctctttggca ctgtttggtg cttttgttag ccgtgcagca 1800

atctcaacag ttgatgtctt gactccaaag gtgttcatcg gtttgatcgt tggtgctatg 1860

cttccgtact ggttctctgc catgaccatg aagagtgcag gaagtgctgc attgaaaatg 1920

gttgaggagg ttcgcaggca gttcaacacc attcctggcc tcatggaggg cactgccaag 1980

cctgattacg ctaactgtgt caagatctct actgatgctt ccatcaagga gatgattcct 2040

cctggtgctc ttgtcatgct cacacccctc atcgtcggaa ccttcttcgg tgtggaaacc 2100

ctctctggtg ttcttgctgg ctctcttgta tctggtgttc agatcgcaat atctgcatca 2160

aacactggag gtgcatggga caatgccaag aagtacattg aggcaggtgc ttctgagcac 2220

gcaaggaccc ttggacccaa agggtcagag ccacacaagg cagctgtgat cggtgacacc 2280

atcggagacc cactcaagga cacgtcgggg ccatcactga acatccttat caagcttatg 2340

gccgtagaat cgcttgtttt tgctcccttt ttcgccaccc acggtgcctt tcttttcaaa 2400

attttttgaa gaaagttaaa taatgaccag aagaaggggg ggaagggagg gggtttgtta 2460

agctagtttt atttagggaa gttaaaatac tatttttgat tttgaggagg gatgaggagg 2520

aggatttgga caccaatgga gattggggta aaacaaaaat ttgatgggat gggatgtgtg 2580

aaccaaaatt ggaggcataa atatggggca tcaggacttg ggtgggttgg gggtatgttc 2640

tggtcctttt taattttaat ttttggtttt tttttttttt gtttcatgta gggggtagaa 2700

caattttccc ttttttattt tattttaaca tttgtactgt tcatcgttta atcaagaaaa 2760

gaccatttgg attttttacc aaaaaaaaaa aaaaaaaaaa aaaagtactt 2810

<210> 7

<211> 767

<212> PRT

<213>Nitraria tangutorum

<400> 7

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

1 5 10 15

Val Cys Ala Val Ile Gly Ile Val Phe Ser Leu Val Gln Trp Tyr Leu

20 25 30

Val Ser Arg Val Ser Leu Thr His Asp Arg Ser Ala Gly Asn Asn Asn

35 40 45

Asn Asn Lys Lys Asn Gly Phe Asn Asp Tyr Leu Ile Glu Glu Glu Glu

50 55 60

Gly Ile Asn Asp Gln Ser Val Val Thr Lys Cys Ala Glu Ile Gln Asn

65 70 75 80

Ala Ile Ser Glu Gly Ala Thr Ser Phe Leu Phe Thr Glu Tyr Gln Tyr

85 90 95

Val Gly Ile Phe Met Val Ala Phe Ala Ile Leu Ile Phe Leu Phe Leu

100 105 110

Gly Ser Val Glu Gly Phe Ser Thr Lys Ser Gln Gln Cys Thr Tyr Asp

115 120 125

Lys Thr Arg Thr Cys Lys Pro Ala Leu Ala Thr Ala Ile Phe Ser Thr

130 135 140

Val Ala Phe Val Leu Gly Gly Val Thr Ser Val Leu Ser Gly Phe Leu

145 150 155 160

Gly Met Lys Ile Ala Thr Tyr Ala Asn Ala Arg Thr Thr Leu Glu Ala

165 170 175

Arg Arg Gly Val Gly Lys Ala Phe Ile Thr Ala Phe Arg Ser Gly Ala

180 185 190

Val Met Gly Phe Leu Leu Ala Ala Asn Gly Leu Leu Val Leu Tyr Ile

195 200 205

Ala Ile Asn Leu Phe Lys Leu Tyr Tyr Gly Asp Asp Trp Glu Gly Leu

210 215 220

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

225 230 235 240

Phe Gly Arg Val Gly Gly Gly Ile Tyr Thr Lys Ala Ala Asp Val Gly

245 250 255

Ala Asp Leu Val Gly Lys Val Glu Arg Asn Ile Pro Glu Asp Asp Pro

260 265 270

Arg Asn Pro Ala Val Ile Ala Asp Asn Val Gly Asp Asn Val Gly Asp

275 280 285

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

290 295 300

Cys Ala Ala Leu Val Val Ala Ser Ile Ser Ser Phe Gly Ile Asn His

305 310 315 320

Asp Phe Thr Ala Met Leu Tyr Pro Leu Leu Ile Ser Ser Met Gly Ile

325 330 335

Leu Val Cys Leu Ile Thr Thr Leu Phe Ala Thr Asp Ile Phe Glu Ile

340 345 350

Lys Ala Val Lys Glu Ile Glu Pro Ala Leu Lys Lys Gln Leu Ile Ile

355 360 365

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

370 375 380

Leu Pro Ser Ser Phe Thr Thr Tyr Asn Phe Gly Thr Gln Lys Val Val

385 390 395 400

Lys Asn Trp Glu Leu Phe Leu Cys Val Gly Val Gly Leu Trp Ala Gly

405 410 415

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

420 425 430

Pro Val Gln Asp Val Ala Asp Ser Cys Arg Thr Gly Ala Ala Thr Asn

435 440 445

Val Ile Phe Gly Leu Ala Leu Gly Tyr Lys Ser Val Ile Ile Pro Ile

450 455 460

Phe Ala Ile Ala Ile Ser Ile Phe Val Ser Phe Ser Leu Ala Ala Met

465 470 475 480

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

485 490 495

Gly Leu Ala Ile Asp Ala Tyr Gly Pro Ile Ser Asp Asn Ala Gly Gly

500 505 510

Ile Ala Glu Met Ala Gly Met Ser His Arg Ile Arg Glu Arg Thr Asp

515 520 525

Ala Leu Asp Ala Ala Gly Asn Thr Thr Ala Ala Ile Gly Lys Gly Phe

530 535 540

Ala Ile Gly Ser Ala Ala Leu Val Ser Leu Ala Leu Phe Gly Ala Phe

545 550 555 560

Val Ser Arg Ala Ala Ile Ser Thr Val Asp Val Leu Thr Pro Lys Val

565 570 575

Phe Ile Gly Leu Ile Val Gly Ala Met Leu Pro Tyr Trp Phe Ser Ala

580 585 590

Met Thr Met Lys Ser Ala Gly Ser Ala Ala Leu Lys Met Val Glu Glu

595 600 605

Val Arg Arg Gln Phe Asn Thr Ile Pro Gly Leu Met Glu Gly Thr Ala

610 615 620

Lys Pro Asp Tyr Ala Asn Cys Val Lys Ile Ser Thr Asp Ala Ser Ile

625 630 635 640

Lys Glu Met Ile Pro Pro Gly Ala Leu Val Met Leu Thr Pro Leu Ile

645 650 655

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

660 665 670

Ser Leu Val Ser Gly Val Gln Ile Ala Ile Ser Ala Ser Asn Thr Gly

675 680 685

Gly Ala Trp Asp Asn Ala Lys Lys Tyr Ile Glu Ala Gly Ala Ser Glu

690 695 700

His Ala Arg Thr Leu Gly Pro Lys Gly Ser Glu Pro His Lys Ala Ala

705 710 715 720

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

725 730 735

Ser Leu Asn Ile Leu Ile Lys Leu Met Ala Val Glu Ser Leu Val Phe

740 745 750

Ala Pro Phe Phe Ala Thr His Gly Ala Phe Leu Phe Lys Ile Phe

755 760 765

<210> 8

<211> 25

<212> DNA

<213>Artificial sequence

<220>

<223>Special primer NtVP1-F8

<400> 8

gttcgcaggc agttcaacac cattc 25

<210> 9

<211> 25

<212> DNA

<213>Artificial sequence

<220>

<223>Special primer NtVP1-R8

<400> 9

accagataca agagagccag caaga 25

<210> 10

<211> 45

<212> DNA

<213>Artificial sequence

<220>

<223>UPM primers

<400> 10

ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45

<210> 11

<211> 18

<212> DNA

<213>Artificial sequence

<220>

<223>T1 primers

<400> 11

gtttgccttc gtccttca 18

<210> 12

<211> 18

<212> DNA

<213>Artificial sequence

<220>

<223>T2 primers

<400> 12

cattgtccca tgcacctc 18

<210> 13

<211> 483

<212> DNA

<213>Artificial sequence

<220>

<223>The fragment of 207bp

<400> 13

gttcgcaggc agttcaacac cattcctggc ctcatggagg gcactgccaa gcctgattac 60

gctaactgtg tcaagatctc tactgatgct tccatcaagg agatgattcc tcctggtgct 120

cttgtcatgc tcacacccct catcgtcgga accttcttcg gtgtggaaac cctctctggt 180

gttcttgctg gctctcttgt atctggtgtt cagatcgcaa tatctgcatc aaacactgga 240

ggtgcatggg acaatgccaa gaagtacatt gaggcaggtg cttctgagca cgcaaggacc 300

cttggaccca aagggtcaga gccacacaag gcagctgtga tcggtgacac catcggagac 360

ccactcaagg acacgtcggg gccatcactg aacatcctta tcaagcttat ggccgtagaa 420

tcgcttgttt ttgctccctt tttcgccacc cacggtgcct ttcttttcaa aattttttga 480

aga 483

Claims

1. a kind of Nitraria tangutorum tonoplast H⁺- PPase GFPs NtVP1, the full length cDNA sequence such as SEQ ID of the gene NO:Shown in 6.

2. Nitraria tangutorum tonoplast H according to claim 1⁺- PPase GFP NtVP1, wherein, the gene Coding region sequence such as SEQ ID NO:Shown in 106-2409 of 6.

3. a kind of Nitraria tangutorum tonoplast H⁺- PPase albumen, the amino acid sequence such as SEQ ID NO of the albumen:Shown in 7.

4. it is a kind of to clone Nitraria tangutorum tonoplast H⁺The method of-PPase GFPs NtVP1, including step：

1) according to the tonoplast H of the plant for having Characterization with Nitraria tangutorum⁺The conserved regions design degeneracy of-PPase GFPs Primer pair；

2) total serum IgE with Nitraria tangutorum blade is as template, using step 1) degenerate primer the method by RT-PCR is obtained To cDNA fragments；

3) according to step 2) the cDNA fragments that are obtained, 3 ' RACE and 5 ' RACE primers are designed, total length is obtained by nested amplification CDNA sequence.

5. method according to claim 4, wherein the degenerate primer is to being NtVP1-F1 and NtVP1-R1：

NtVP1-F1：CATTCGCCATTCAGG(SEQ ID NO:1),

NtVP1-R1：ACATAGCAGCCAAAGT(SEQ ID NO:2)；

Wherein, step 2) obtain the cDNA fragments length be 1156bp (SEQ ID NO:3)；

Wherein, step 3) in, the primer sequence is as follows：

UPM(SEQ ID NO:10)：

CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT；

3’RACE：TGCCAATCCAGTGGCAATAGTGCTGA(SEQ ID NO:4)；

5’RACE：ACAACTGCTGCCATCGGAAAGGGATT(SEQ ID NO:5)；

Wherein, the nucleotide sequence of the full-length cDNA of acquisition such as SEQ ID NO:Shown in 6.

6. a kind of expression vector or expression bacterial strain, the expression vector or expression bacterial strain include Tang as claimed in claim 1 or 2 Gu Tebaici tonoplasts H⁺- PPase GFPs NtVP1.

7. expression vector according to claim 6 or expression bacterial strain, wherein, the expression vector is the carrier with GFP, The expression bacterial strain is Agrobacterium.

8. a kind of construction method of expression vector, the expression vector includes Nitraria tangutorum liquid as claimed in claim 1 or 2 Vacuolar membrane H⁺- PPase GFP NtVP1, including：1) cDNA sequence in NtVP1 genes or the two ends point of its coding region sequence Not Jia Shang Xba I and Kpn I restriction enzyme sites, and be connected to carrier T, form T-NtVP1 carriers；2) by T-NtVP1 carriers, and PCG-GFP and pSN1301 carry out digestion with Xba I and Kpn I respectively, and reclaim；3) will get off from digestion on T-NtVP1 carriers Genetic fragment be connected with the endonuclease bamhi of pCG-GFP and pSN1301 carriers.

9. one kind is by Nitraria tangutorum tonoplast H⁺- PPase GFPs NtVP1 is transfected to the method for plant, including will by right The expression vector described in 6 or expression bacterial strain is asked to convert into arabidopsis.

10. Nitraria tangutorum tonoplast H as claimed in claim 1 or 2⁺- PPase GFPs NtVP1 turns in preparation or cultivation Application in gene plant.