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 companiesTMRACE 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