CN110205337A

CN110205337A - Application of the plant as host expresses source of people Telomerase

Info

Publication number: CN110205337A
Application number: CN201910549715.5A
Authority: CN
Inventors: 王跃驹; 陈书元; 唐辉
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-06-24
Filing date: 2019-06-24
Publication date: 2019-09-06

Abstract

The present invention relates to field of biotechnology, in particular to plant significantly extends bio-longevity as host expresses source of people Telomerase.The present invention expresses source of people Telomerase using simple and effective mediated by agriculture bacillus vacuum infiltration methods using plant such as romaine lettuce as the effective expression platform of recombinant protein production.The expression system determines that plant foreign protein can be collected after Agrobacterium is infected 4 days.Telomerase successful expression is determined using Western Blot protein hybridization method, goes out Telomerase with AKTA protein purification system successful purification.The life test of Caenorhabditis elegans is the result shows that significantly extend the service life using the Telomerase that the platform technology produces.

Description

Application of the plant as host expresses source of people Telomerase

Technical field

The present invention relates to field of biotechnology, in particular to plant significantly extends biology as host expresses source of people Telomerase Service life.

Background technique

The Nobel Prize committee, Royal Swedish Academy of Sciences on October 5th, 2009 announce 2009 annual Nobel's physiology or Medicine authorizes 3 American scientist Erie Sha fragrant plant Blackburns (Elizabeth H.Blackburn), Ka Luoer Gray moral (CarolW.Greider) it continues Stark (Jack W.Szostak) with Jack, to commend them " it was found that telomere and Telomerase are How chromosome is protected ".Their discovery illustrates the effect of Telomerase -- and it is stabilized the length of telomere and structure, from And chromosome, cell aging with shortening for telomere are protected, and when the activity of Telomerase is enough to safeguard the length of telomere, carefully Born of the same parents will delay senility, cancer cell obtain it is permanent this during, the activation of Telomerase has served very important.Phase Instead, some hereditary diseases eventually lead to the damage of cell just because of the active defects of Telomerase, just because of three scientists' Starting sex work, to allow people to instruct telomere and Telomerase not only closely related with the speciality of chromosome and stability, but also designs The aging and damage, the generation of cancer etc. of cell.

Telomerase (Telomerase) is the reverse transcription DNA synzyme for extending telomere.It is to be made of RNA and protein Ribose nucleoprotein.Its RNA group is divided into template, and protein component has catalytic activity, is to draw with the end telomere 5' Object synthesizes telomere repeat sequence.The activity of Telomerase can be detected in eukaryocyte, and function is synthesis end of chromosome Telomere is compensated the telomere length being gradually shortened by each cell division, and then stablizes telomere length.It is mainly characterized by With own carry RNA make template, using dNTP as raw material, after being catalyzed and synthesized by reverse transcription extend chain 5 ' end DNA fragmentation or Additional recurring unit.Principal biological function of the Telomerase in cell is to be replicated by its reverse transcriptase activity and extended end Grain DNA stablizes the length of chromosome telomere DNA.

In recent years the progress in relation to Telomerase and relation between tumor shows also to take part in tumour cell telomerase to swollen The stable regulation process of the apoptosis and genome of oncocyte.It is corresponding with the multi-biological of Telomerase activity, in tumour cell There is also complicated Telomerase regulated and control networks.It is horizontal to telomerase activation upon translation by protein-protein interaction And function is regulated and controled, then is one of the hot spot of current research Telomerase regulatory mechanism.But time-consuming for current expression system, pure Change complex process, there are gene contamination, the problems such as infecting the potential pest and disease damage of human body, and production cost is higher, there are product peaces The hidden danger of full property.

Summary of the invention

In view of this, the application the present invention provides plant as host in expression Telomerase.The present invention utilizes plant The especially efficient platform technology that produces as recombinant protein of romaine lettuce, expresses Telomerase.And succeed under mild conditions Isolate active foreign protein, it was demonstrated that plant especially romaine lettuce expression platform can successfully be used to produce telomerase protein. Time is short (4d), and purifying is simple, and it is convenient to produce.Gene contamination is eliminated, the potential pest and disease damage etc. of infection human body is eliminated.It substantially reduces Production cost improves Product Safety.

In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:

Application the present invention provides plant as host in expression Telomerase；The plant be selected from romaine lettuce, spinach, kind Eggplant, radish, Chinese cabbage, corn and soybean, wheat or tobacco；The organ of the plant is selected from seed, leaf, rhizome or whole plant.

Plant transient expression technology is will to contain mesh using a variety of different technical approach in plant growth to certain phase The plasmid of mark albumen is transferred in plant cell, and efficient, controllable expression system is established in plant cell, it is short to obtain the gene The technology of temporary controlled expression.It is short the time required to transient expression compared with stablizing expression, it does not need exogenous origin gene integrator to place In main plant chromosome, it is only necessary to which several days time can take experimental result.Compared with bacterial expression system, plant expression system Expressed albumen can correctly be folded, process, modify, protein active produced is higher than bacterial expression system； Compared with animal cell expression system, the early investment cost of plant expression system is very low, only its 1 percent arrive percentage Five.This research also found that recombination human source Telomerase significantly extends the service life of Caenorhabditis elegans.

On the basis of the studies above, the present invention also provides a kind of expression vectors, sequence including Telomerase and double First plant vector.

In some specific embodiments of the invention, the Telomerase is people's telomerase.

In some specific embodiments of the invention, the sequence of the Telomerase is to be by the codon optimization of Telomerase The codon of favorite plant, the sequence of the optimization Telomerase of acquisition.

In some specific embodiments of the invention, the sequence nucleotide sequence such as SEQ of the Telomerase of the optimization Shown in ID No.1；Amino acid sequence is as shown in SEQ ID No.2 in the sequence of the Telomerase of the optimization.

In some specific embodiments of the invention, the construction method of the expression vector includes the following steps:

Step 1: being the codon of favorite plant by the codon optimization of Telomerase, obtain the sequence of the Telomerase of optimization；

Step 2: Xbal restriction enzyme site is added in 5 ' ends of the sequence of the Telomerase of the optimization, in 3 ' ends Sac I site is added；It is cloned into pUC57 carrier, obtains pTelo cloning vector；

Step 3: genetic fragment being obtained from the resulting cloning vector of step 2 by Xbal/Sacl, is cloned into binary plant Carrier pCam35S obtains expression vector p35S-Telo.

The present invention also provides application of the expression vector in expression Telomerase.

On the basis of the studies above, a kind of method the present invention also provides plant as host expresses Telomerase will The expression vector is transformed into Agrobacterium, after entering plant tissue by mediated by agriculture bacillus vacuum infiltration, extracting and developing albumen Matter obtains Telomerase.

In some specific embodiments of the invention, the plant is selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage, jade Rice, soybean, wheat or tobacco；The organ of the plant is selected from seed, leaf, rhizome or whole plant.

In some specific embodiments of the invention, the mediated by agriculture bacillus vacuum infiltration includes the following steps:

Step 1: vacuumizing 25~45s；

Step 2: keeping 30~60s of vacuum (- 95kPa) pressure；

Step 3: releasing stress so that penetrating fluid penetrates into the plant tissue；

It repeats the above steps 2~3 times, is protected from light processing 4d.

The present invention is using plant such as romaine lettuce as the effective expression platform of recombinant protein production, using simple and have The mediated by agriculture bacillus vacuum infiltration methods of effect express source of people Telomerase.The expression system determines plant foreign protein in Agrobacterium It can be collected after infecting 4 days.Telomerase successful expression is determined using Western Blot protein hybridization method, it is pure with AKTA albumen Change system successful purification goes out Telomerase.The life test of Caenorhabditis elegans is the result shows that the telomere produced using the platform technology Enzyme significantly extends the service life.

Detailed description of the invention

In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described.

Fig. 1 shows cloning vector pTelo schematic diagram；

Fig. 2 shows that Telomerase plant binary expression vector p35S-Telo constructs process；Utilize restriction enzyme (Xbal/ SacI) double digestion cuts Telomerase from Fig. 1 cloning vector, connects the site Xbal/SacI into pCam35S, generates plant binary Expression vector p35S-Telo；

LB and RB:Ti plasmid right boundary；35S, the CaMV 35S with tobacco mosaic virus (TMV) (TMV) 5 ' UTR start Son；NPT II, the expression of the coding nptII gene for kalamycin resistance；Nos3 ', terminator；

Fig. 3 shows PAGE gel electrophoresis result；Swimming lane 1: non-to infect romaine lettuce；Swimming lane 2: the Telomerase of romaine lettuce expression；Swimming Road 3: Telomerase commercially produced product.

Specific embodiment

Application the invention discloses plant as host in expression Telomerase, those skilled in the art can use for reference this Literary content, is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to art technology It is it will be apparent that they are considered as being included in the present invention for personnel.Method and application of the invention has passed through preferably Embodiment is described, related personnel obviously can not depart from the content of present invention, in spirit and scope to side as described herein Method and application are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.

Application the present invention provides plant as host in expression Telomerase.Preferably, the plant be selected from romaine lettuce, Spinach, tomato, radish, Chinese cabbage, corn and soybean, wheat or tobacco；The organ of the plant is selected from seed, leaf, rhizome or whole strain Plant.

The present invention also provides a kind of expression vectors, sequence and carrier including Telomerase.

In some specific embodiments of the invention, the sequence of the Telomerase is to be by the codon optimization of Telomerase The codon of favorite plant, the telomeric sequences of the optimization of acquisition.

In some specific embodiments of the invention, the carrier is binary plant carrier.

Step 1: being the codon of favorite plant by the codon optimization of Telomerase, obtain: the sequence of the Telomerase of optimization；

Step 2: Xbal restriction enzyme site is added in 5 ' ends of the sequence of the Telomerase of the optimization, in 3 ' ends Sac I site is added；

It is grand into pUC57 carrier by golden Stryker, obtain pTelo cloning vector；

Specifically, in order to provide high efficient expression of the foreign protein in plant, the present invention is by human telomerase reverse amino acid sequence Column obtain nucleosides using anti-translation software (https: //www.ebi.ac.uk/Tools/st/emboss_backtranseq/) Acid sequence, and be the codon of favorite plant by its codon optimization, by Jin Sirui company (Nanjing, China) synthesis.Optimizing 5 ' end of telomeric sequences be added Xbal restriction enzyme site, 3 ' ends be added the site Sacl.And it is grand by golden Stryker Into pUC57 carrier, obtain pTelo cloning vector (Fig. 1).Genetic fragment is separated from cloning vector by XbaI/Sacl, and It is cloned into binary plant carrier pCam35S, is generated plant expression vector p35S-Telo (Fig. 2).

In addition, a kind of method the present invention also provides plant as host expresses Telomerase, by table provided by the invention It is transformed into Agrobacterium up to carrier, after entering plant tissue by mediated by agriculture bacillus vacuum infiltration, extracting and developing protein is obtained Telomerase.

Specifically, by plant expression vector p35S-Telo by with Multiporator (Eppendorf, Hamburg, Germany) Electroporation Transformation is into Agrobacterium tumefaciens GV3101.Obtained strains are equably spread over containing kanamycin anti- On the selective LB plate of raw element (50mg/L).In the dark after 28 DEG C of incubation 2d, picking single colonie is inoculated into 0.5L YEB (ferment Female extract meat soup, 5g/L sucrose, 5g/L tryptone, 6g/L yeast extract, 0.24g/L MgSO₄, pH7.2) and supplement Antibiotic liquid culture medium (50mg/L kanamycins).The culture of inoculation is incubated in oscillator (220rpm) with 25~28 DEG C Educate 72h.OD600 value is measured by addition YEB culture medium and is adjusted to 3.5~4.5.Then culture solution is collected, is centrifuged (4500 turns Speed) 10min.It is 0.5 that agrobatcerium cell, which is resuspended in osmotic medium (10mMMES, 10mMMgSO4) to O.D.600,.

It will prepare that mix containing p35S-Telo Agrobacterium equivalent to O.D.600 be 0.5；Culture suspension is placed in 2L beaker, is placed in drier.The romaine lettuce that this laboratory saves is inverted (core is upward) and lightly rotates on bacterium and is hanged In supernatant liquid, drier is sealed.Vacuum pump (Welch Vacuum, Niles, IL, USA) is opened to evacuate, and visible infiltration Transparent liquid is in leaf tissue.Keep 30~60s of pressure state.It opens the system quickly to release stress, makes penetrating fluid infiltration group Knit interior space.The process repeats 2~3 times, until high-visible penetrating fluid is spread obviously in romaine lettuce tissue.Then by romaine lettuce Tissue gently takes out from penetrating fluid, and three times with distilled water continuous flushing, is then transferred into the container of plastic foil covering.It will The sample of processing keeps 4d in the dark.

Step 1: vacuumizing 25~45s；

Step 2: keeping 30~60s of vacuum (- 95kPa) pressure；

It repeats the above steps 2~3 times, is protected from light processing 4d.

In some specific embodiments of the invention, Agrobacterium is specially Agrobacterium tumefaciens GV3101.

Clone's pTelo genetic fragment of the present invention, and binary plant expression vector p35S-Telo (Fig. 2) is constructed, After completing construct, being digested with specific restriction enzyme confirms that genetic fragment is complete.After infiltration, most romaine lettuce groups are woven in It is flooded during vacuum immersion, other than firm middle rib region, rest part shows after vacuum infiltration 4 days khaki Region.

Extracting and developing protein specifically: the romaine lettuce sample through Agrobacterium vacuum infiltration is stirred with blender, and uses body Product is than Extraction buffer (100mMKPi, the pH7.8 for 1:1 ratio；5mM EDTA；10mM beta -mercaptoethanol) blender high speed It is homogenized 1~2min.Homogenate is adjusted to pH8.0, with filtered through gauze, filtrate is centrifuged 15min with 10,000g at 4 DEG C to remove Remove cell fragment.Supernatant is collected, is mixed with ammonium sulfate (50%), and shakes be incubated for 60min on ice.By centrifuge (10, 000g) 15min is separated again at 4 DEG C.Obtained supernatant is subjected to the second wheel ammonium sulfate (70%) precipitating, is shaken on ice outstanding Floating 60min, is centrifuged 15min at 4 DEG C again with 10,000g.Then, liquid is discarded supernatant, processing sample pellet protein is molten In 5mL buffer (20mMKPi, pH 7.8；2mMEDTA；10m M beta -mercaptoethanol) in and store at 4 DEG C.

PAGE gel electrophoresis specifically: collect the protein purification extracted from Agrobacterium vacuum infiltration romaine lettuce, sampling (95 DEG C) load buffers (Biorad, Hercules, CA, USA) of product (5 μ L) thermal denaturation are in 4-12%Bis-Tris Plus SDS- denaturant gel (ThermoFisher Scientific, Waltham, MA, USA) runs electrophoresis.Equally, non denatured The affine degree of antibody is detected in gel electrophoresis.Then gel is clapped again after being dyed with Coomassie blue G250 (Biorad) According to.

The Downstream processing of the recombinant protein of plant origin is typically difficult to and valuableness, because cellulose cell wall is difficult to split Solution and secondary plant metabolites.We are stirred with blender and are homogenized, and greatly save homogenate cost and technique.Recombinate telomere Enzyme separates us by denaturant gel SDS-PAGE and observes that estimation molecular weight is about the item of 126.9kDa (Fig. 3) in swimming lane Band meets telomerase protein molecular weight.Egg based on Bradford measuring method and spectrodensitometry control group measurement purification of samples Bai Hanliang is about 1.81mg/g.

The present invention is using romaine lettuce come transient expression Telomerase, and (4d) can produce the albumen of high-content in a relatively short period of time Matter.Romaine lettuce is higher plant, can carry out posttranslational modification process, that is, the albumen expressed is automatically active.And this side Method reduces bio-safety problem to the maximum extent, because processed romaine lettuce tissue is usually in completely enclosed facility or appearance It is developed in device, biological pollution problem is not present.Romaine lettuce does not contain plant noxious material, and itself protein content is few, is conducive to The protein purification in downstream.Using romaine lettuce system production Telomerase, production cycle and production cost can be greatly shortened.

The present invention is found through experiments that, botanical system especially romaine lettuce system be it is more economical, efficiently express platform, be A kind of method of quick transient expression recombinant protein.The vacuum Agrobacterium permeating method that the present invention describes is simple, quickly, and And recombinant protein yield can be improved.Romaine lettuce can increase protein output by bearing vacuum pressure, and allow every leaf The more complete infiltration of son.Due to romaine lettuce be easy to grow and can commercial mass production, than other transient expression plants, such as Tobacco etc. is easier to obtain and cheaper, and due to not needing complicated special producing equipment, cost can be significantly reduced.It is comprehensive Upper described, the present invention, which can use, is mass produced Telomerase in the romaine lettuce system short time.

Plant provided by the invention raw materials used and city reagent Jun Keyou in the application in expression Telomerase as host Field is bought.

Below with reference to embodiment, the present invention is further explained:

The building of 1 plant transient expression vector of embodiment

For the high efficient expression by foreign protein in plant, telomere enzyme amino acid sequence is utilized into anti-translation software (https: //www.ebi.ac.uk/Tools/st/emboss_backtranseq/) obtains nucleotide sequence, and by its password Son is optimized for the codon of favorite plant, by Jin Sirui company (Nanjing, China) synthesis.In the end telomeric sequences 5' of optimization Xbal restriction enzyme site is added, the site Sacl is added in the end 3'.And be cloned into pUC57 carrier by Jin Sirui company, It obtains pTelo cloning vector (Fig. 1), genetic fragment is separated from cloning vector by Xbal/Sacl, and is cloned into binary plant Carrier, pCam35S are generated plant expression vector p35S-Telo (Fig. 2).By plant expression vector by using Multiporator (Eppendorf, Hamburg, Germany) Electroporation Transformation is into Agrobacterium tumefaciens GV3101.Equably by obtained strains It spreads on the selective LB plate of antibiotic containing kanamycin (50mg/L).In the dark after 28 DEG C of incubation 2d, picking list Colony inoculation is to 0.5L YEB (yeast extract meat soup, 5g/L sucrose, 5g/L tryptone, 6g/L yeast extract, 0.24g/ L MgSO4, pH7.2) and supplement antibiotic liquid culture medium (50mg/L kanamycins).By the culture of inoculation in oscillator With 25~28 DEG C of incubation 72h in (220rpm).OD600 value is measured by addition YEB culture medium and is adjusted to 3.5~4.5.Then Culture solution is collected, (4500 revolving speed) 10min is centrifuged.By agrobatcerium cell be resuspended in osmotic medium (10mM MES, 10mMMgSO₄) in O.D.600 be 0.5.

The vacuum infiltration of 2 mediated by agriculture bacillus of embodiment

It will prepare that mix containing p35S-Telo Agrobacterium equivalent to O.D.600 be 0.5.Culture suspension is placed in 2L beaker, is placed in drier.The romaine lettuce that this laboratory saves is inverted (core is upward) and lightly rotates on bacterium and is hanged In supernatant liquid, drier is sealed.Vacuum pump (Welch Vacuum, Niles, IL, USA) is opened to evacuate, and visible infiltration Transparent liquid is in leaf tissue.It is kept for pressure state 30~60 seconds.It opens the system quickly to release stress, makes penetrating fluid infiltration group Knit interior space.The process repeats 2 to 3 times, until high-visible penetrating fluid is spread obviously in romaine lettuce tissue.Then by romaine lettuce Tissue gently takes out from penetrating fluid, and three times with distilled water continuous flushing, is then transferred into the container of plastic foil covering.It will The sample of processing is kept 4 days in the dark.

3 Protein Extraction of embodiment and separation

Romaine lettuce sample through Agrobacterium vacuum infiltration is stirred with blender, and is buffered with the extraction that volume ratio is 1:1 ratio Liquid (100mM KPi, pH7.8；5mM EDTA；10m M beta -mercaptoethanol) homogenate of blender high speed 1-2 minutes.By homogenate It is adjusted to pH8.0, with filtered through gauze, filtrate is centrifuged 15 minutes with 10,000g at 4 DEG C to remove cell fragment.Collect supernatant Liquid is mixed with ammonium sulfate (50%), and shakes be incubated for 60 minutes on ice.Divided again at 4 DEG C by centrifuge (10,000g) From 15 minutes.Obtained supernatant is subjected to the second wheel ammonium sulfate (70%) precipitating, shakes suspend 60 minutes on ice, again 4 With 10,000g centrifugation 15 minutes at DEG C.Then, liquid is discarded supernatant, processing sample pellet protein is dissolved in 5mL buffer (20mM KPi, pH 7.8；2mM EDTA；10mM beta -mercaptoethanol) in and store at 4 DEG C.

The Downstream processing of the recombinant protein of plant origin is typically difficult to and valuableness, because cellulose cell wall is difficult to split Solution and secondary plant metabolites.We are stirred with blender and are homogenized, and greatly save homogenate cost and technique.

4 PAGE gel electrophoresis of embodiment

The protein purification extracted from Agrobacterium vacuum infiltration romaine lettuce is collected, (95 DEG C) of thermal denaturation of sample (5 μ L) loads are taken Buffer (Biorad, Hercules, CA, USA) is 4~12%Bis-Tris Plus SDS- denaturant gel (ThermoFisher Scientific, Waltham, MA, USA) runs electrophoresis.Equally, it is detected in native gel electrophoresis anti- The affine degree of body.Then Coomassie blue G250 is used

(Biorad) it takes pictures again to gel after dyeing.Recombinant granzyme separates me by denaturant gel SDS-PAGE Observed in swimming lane estimation molecular weight be about 126.9kDa (Fig. 3) band, meet telomerase protein molecular weight.It is based on The protein content of Bradford measuring method and spectrodensitometry control group measurement purification of samples is about 1.81mg/g.

5 telomerase protein Activity determination of embodiment

20 Caenorhabditis elegans ovum are put into the culture dish containing purifying plant source recombinant granzyme (0.1mg/ml), The culture dish of no any addition Telomerase is as negative control.It is cultivated at 20 DEG C, observes the life cycle of nematode.Life cycle day Number is average value ± SD.The results show that the life of the Caenorhabditis elegans in the culture dish containing purifying plant source recombination telomere Period greatly prolongs (P≤0.001), is 30 ± 3 days；And the nematode service life in the culture dish without containing plant source recombinant granzyme It is 20 ± 2 days.

Embodiment 6

Control group: Telomerase is produced using zooblast；

Experimental group 1: plant production Telomerase provided by the invention；

Experimental group 2: leaf tobacco production Telomerase is utilized；

1 recombinant granzyme of table

^*Show P≤0.05 compared with the control group；^**Show P≤0.01 compared with the control group；

^#Show P≤0.05 compared with experimental group 2；^##Show P≤0.01 compared with experimental group 2；

As shown in Table 1, experimental group 1 is compared with the animal system of control group, romaine lettuce transient expression telomere provided by the invention Enzyme, extremely significant (P≤0.01) shorten the production cycle, and extremely significant (P≤0.01) improves protein content, simplifies protein purification Complexity, extremely significant (P≤0.01) reduces production cost.

Experimental group 1 is compared with the tobacco leaf system of experimental group 2, romaine lettuce transient expression Telomerase, and significant (P≤0.05) shortens Production cycle, significant (P≤0.05) improves protein content, simplifies the complexity of protein purification, extremely significant (P≤0.01) Reduce production cost.

Compared with the control group, tobacco leaf transient expression Telomerase (P≤0.05) more significant than animal system shortens life to experimental group 2 The period is produced, the complexity of protein purification is simplified, significant (P≤0.05) reduces production cost.In summary test result Show botanical system especially romaine lettuce system be it is more economical, efficiently express platform.It being capable of quick transient expression recombinant protein Telomerase can be mass produced in matter in a short time.

The present invention is using romaine lettuce come transient expression antibody, and (4d) can produce the protein of high-content in a relatively short period of time. Romaine lettuce is higher plant, can carry out posttranslational modification process, that is, the albumen expressed is automatically active.And this method is most Reduce to limits bio-safety problem, because processed romaine lettuce tissue is usually in completely enclosed facility or container Biological pollution problem is not present in exploitation.Romaine lettuce does not contain plant noxious material, and itself protein content is few, is conducive to downstream Protein purification.Using romaine lettuce system production, production cycle and production cost can be greatly shortened.Nematode result of study also table Bright, Telomerase greatly prolongs the service life of nematode.

The above is only a 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 are also answered It is considered as protection scope of the present invention.

Sequence table

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caaatgccag ctcatggact ttttccatgg tgtggacttc ttcttgatac tagaactctt 2820

gaagttcaat ctgattattc ttcttatgct agaacttcta ttagagcttc tcttactttt 2880

aatagaggat ttaaggctgg aagaaatatg agaagaaagc tttttggagt tcttagactt 2940

aagtgtcatt ctctttttct tgatcttcaa gttaattctc ttcaaactgt ttgtactaat 3000

atttataaga ttcttcttct tcaagcttat agatttcatg cttgtgttct tcaacttcca 3060

tttcatcaac aagtttggaa gaatccaact ttttttctta gagttatttc tgatactgct 3120

tctctttgtt attctattct taaggctaag aatgctggaa tgtctcttgg agctaaggga 3180

gctgctggac cacttccatc tgaagctgtt caatggcttt gtcatcaagc ttttcttctt 3240

aagcttacta gacatagagt tacttatgtt ccacttcttg gatctcttag aactgctcaa 3300

actcaacttt caagaaagct tccaggaact actcttactg ctcttgaagc tgctgctaat 3360

ccagctcttc catctgattt taagactatt cttgat 3396

<210> 2

<211> 1132

<212> PRT

<213>Telomerase (Telomerase)

<400> 2

Met Pro Arg Thr Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser

1 5 10 15

His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly

20 25 30

Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Ser

35 40 45

Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro

50 55 60

Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu

65 70 75 80

Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val

85 90 95

Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro

100 105 110

Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr

115 120 125

Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val

130 135 140

Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val

145 150 155 160

Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr

165 170 175

Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly

180 185 190

Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg

195 200 205

Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg

210 215 220

Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg

225 230 235 240

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

245 250 255

Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val

260 265 270

Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala

275 280 285

Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His

290 295 300

Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro

305 310 315 320

Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly

325 330 335

Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro

340 345 350

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

355 360 365

Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln

370 375 380

Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His

385 390 395 400

Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg

405 410 415

Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln

420 425 430

Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu

435 440 445

Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe

450 455 460

Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser

465 470 475 480

Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser

485 490 495

Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met

500 505 510

Ser Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys

515 520 525

Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe

530 535 540

Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe

545 550 555 560

Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr

565 570 575

Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His

580 585 590

Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln

595 600 605

His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile

610 615 620

Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val

625 630 635 640

Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser

645 650 655

Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg

660 665 670

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

675 680 685

Ala Trp Arg Thr Phe Val Leu Arg Val Ser Ala Gln Asp Pro Pro Pro

690 695 700

Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile

705 710 715 720

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

725 730 735

Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His

740 745 750

Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp

755 760 765

Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser

770 775 780

Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu

785 790 795 800

Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His

805 810 815

Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro

820 825 830

Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp

835 840 845

Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu

850 855 860

Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala

865 870 875 880

Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys

885 890 895

Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu

900 905 910

Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe

915 920 925

Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser

930 935 940

Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe

945 950 955 960

Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly

965 970 975

Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn

980 985 990

Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln

995 1000 1005

Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln Gln

1010 1015 1020

Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr Ala

1025 1030 1035 1040

Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser Leu

1045 1050 1055

Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu Ala Val Gln Trp

1060 1065 1070

Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg Val Thr

1075 1080 1085

Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr Gln Leu Ser

1090 1095 1100

Arg Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu Ala Ala Ala Asn

1105 1110 1115 1120

Pro Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu Asp

1125 1130

Claims

1. application of the plant as host in expression Telomerase；The plant be selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage, Corn and soybean, wheat or tobacco；The organ of the plant is selected from seed, leaf, rhizome or whole plant.

2. a kind of expression vector, which is characterized in that sequence and binary plant carrier including Telomerase.

3. expression vector as claimed in claim 2, which is characterized in that the Telomerase is people's telomerase.

4. expression vector according to claim 2 or 3, which is characterized in that the sequence of the Telomerase is by Telomerase Codon optimization is the codon of favorite plant, the sequence of the optimization Telomerase of acquisition.

5. expression vector according to claim 4, which is characterized in that the sequence nucleotide sequence of the Telomerase of the optimization Column are as shown in SEQ ID No.1；Amino acid sequence is as shown in SEQ ID No.2 in the sequence of the Telomerase of the optimization.

6. expression vector according to any one of claims 2 to 5, which is characterized in that its construction method includes the following steps:

Step 2: Xbal restriction enzyme site is added in 5 ' ends of the sequence of the Telomerase of the optimization, is added in 3 ' ends Sac I site；It is cloned into pUC57 carrier, obtains pTelo cloning vector；

7. according to application of the described in any item expression vectors of claim 2 to 6 in expression Telomerase.

8. a kind of method of plant as host expresses Telomerase, which is characterized in that will be as described in any one of claim 2 to 6 Expression vector be transformed into Agrobacterium, after entering plant tissue by mediated by agriculture bacillus vacuum infiltration, extracting and developing protein, Obtain Telomerase.

9. method according to claim 8, which is characterized in that the plant be selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage, Corn and soybean, wheat or tobacco；The organ of the plant is selected from seed, leaf, rhizome or whole plant.

10. method according to claim 8 or claim 9, which is characterized in that the mediated by agriculture bacillus vacuum infiltration includes following step It is rapid:

Step 1: vacuumizing 25~45s；

Step 2: keeping 30~60s of vacuum (- 95kPa) pressure；

It repeats the above steps 2~3 times, is protected from light processing 4d.