CN102952812B - Ginseng amino acid transport protein gene PgLHT and encoding protein and application thereof - Google Patents

Abstract

The invention discloses a ginseng amino acid transport protein gene PgLHT and an encoding protein and an application thereof. The amino acid transport related gene PgLHT has a sequence as shown in SEQ ID NO.1, and the encoding protein has the sequence as shown in SEQ ID NO.2. Researches find that PgLHT genetic expression has the tissue specificity and is closely related to ginseng stress response. The PgLHT gene has obvious effect in enhancing the ginseng plant resistance and the like. Ginseng and other plants can be screened or improved through the gene or derivatives thereof, so that excellent plant varieties with strong environment adaptivity can be obtained. The organic nutrition metabolism of plants of ginseng and the like can also be promoted through adopting a tissue culture or transgene mode.

Description

Ginseng amino acid transporter gene PgLHT and proteins encoded and application

Technical field

The invention belongs to technical field of biological genetic engineering, relate to improvement plant trait, specifically promote genes involved and albumen and the application of the plant stress responses such as amino acid transport, participation ginseng.The present invention is having important using value aspect the cultivation of plants qualities such as the metabolism of regulating plant organotrophy, defensive raction and raising ginseng.

Background technology

Ginseng (P.ginseng C.A.Meyer) is Araliaceae Panax per nnial herb, is rare traditional Chinese medicine.But ginseng is a kind of plant of avoiding continuous cropping, and very harsh to requirements such as the nutrition of soil, and wherein nitrogen is extremely important to the growth of ginseng.Many important compound in ginseng body all contain nitrogen, as protein, nucleic acid, amino acid, pigment and some tethelin etc.In the cultivation and plant tissue and cell culture process of ginseng, inorganic nitrogen is the limiting factor of its growth normally, and unreasonable the using not only of nitrogenous fertilizer caused bad impact to the quality and yield of ginseng, but also causes environmental degradation.Thereby nitrogen use and plant comes into one's own day by day to the Absorption And Metabolism research of nitrogen.Along with the improvement of research means and going deep into of research, there is increasing evidence to show that plant also can absorb organic nitrogen.The long-term low temperature of ginseng planting's Climate, the soil organism is suppressed by the mineralization of microorganism, thereby soil organic matter content is high, and content of inorganic nitrogen in is usually lower; Organonitrogen content is often higher than inorganic nitrogen, thereby organonitrogen is extremely important to being grown in plant and the microorganism in this area.

Plant plasma membrane serves is being controlled the turnover of nutrient, there are some researches show, cell can be by the idiosyncratic carrier albumen active absorption amino acid on plasma membrane.More to the amino acid transporter research of castor-oil plant, Arabidopis thaliana plasma membrane in recent years.In addition, salt stress also can induce some plants to produce amino acid transporter, as Salt Stress-induced Arabidopis thaliana produces two proline transport protein (Rentsch, 1996), but Igarashi etc. (2000) report, the proline transport protein in rice plant body (OsProT) is not subject to the induction of salt stress.Boorer (1997) is cloned into the Arabidopis thaliana amino acid transporter AAPs mainly expressing in root in xenopus leavis oocytes and expresses and study it to amino acid whose absorption, finds that AAPs can transport the multiple amino acids such as neutral, acid and alkaline.

The cloning and identification of first amino acid transporter gene (Arabidopis thaliana AAP1/NAT2) of plant adopts exactly and makes plant cDNA on a certain amino acid transporter yeast mutants of disappearance, express and have complementary functions.Because lacking the yeast of a certain amino acid transporter, can not absorb this amino acid, and this yeast after expressing in this transporter gene importing yeast of plant just can be absorbed to this amino acid.Arabidopis thaliana AAP1/NAT2 Transshipment Permitted multiple amino acids, but be more inclined to running neutral amino acids, it contains 486 amino-acid residues, has 11 membrane spaning domains.Wherein N-terminal is in tenuigenin, and outside C-terminal Cell-oriented, known His337 is crucial amino-acid residue to the structure and function of AAP1/NAT2, and its change will affect this translocator to amino acid whose running (Bush etc., 1996; Chang and Bush, 1997).AAP1/NAT2 is wide expression in plant tissue, and it may participate in nitrogen reallocation in vivo.Two proline transport protein gene ProT1, ProT2 of Arabidopis thaliana are separated, ProT1 expresses in all organs, especially root, stem, spend middle expression amount large, ProT2 spreads all over whole plant, water or salt stress can be induced ProT2 strong expression, explanation ProT2 under stress conditions play an important role aspect nitrogen partition (Rentsch etc., 1996).From Arabidopis thaliana clone Methionin, LHT1 express in a organized way, strong expression in root, spire, flower, pollen and the long fruit of seedling, illustrate that LHT1 participation is transported to Methionin in heterotrophism tissue.Bick etc. (1998) obtain castor-oil plant two amino acid transporter gene RcAAP1, RcAAP2, in the cotyledon of seedling and root, expression amount is large, RcAAP1 mainly expresses in root, its transcript is distributed in tip of a root broad variety cell, comprise epidermis, cortex, in the middle pillar cell, also there is more expression amount, show that these two translocators may participate in seed germination and from soil, absorb amino acid, also may participate in the amino acid transportation of lateral root generation or xylem.Research also finds that castor-oil plant RcAAP3 amino acid transporter can participate in accumulating amino acid for protein synthesis, and not only cold zone plant can absorb amino acid whose plant, and farm crop are absorbable amino acid also.Liu etc. (2010) research finds that Arabidopis thaliana translocator AtLHT1 is by regulating the amino acid whose running balance such as glutamine to participate in the defensive raction of SA mediation.These discoveries are of great significance organic nutrition of plant metabolism and defensive raction aspect tool thereof.

Summary of the invention

The object of this invention is to provide a kind of can promote gene order that amino acid transport is relevant with participating in the plant stress responses such as ginseng as shown in SEQ ID NO.1 and the protein sequence of coding as shown in SEQ ID NO.2, for Future Development gene engineering product lays the foundation.

In order to achieve the above object, technical scheme provided by the invention is:

The invention provides a kind of ginseng amino acid transporter gene PgLHT, its gene order is as shown in SEQ ID NO.1.

The present invention also provides a kind of degenerated primer pair for the PgPDR1 gene that increases, and described primer pair is as shown in SEQ ID NO.5 and SEQ ID NO.6.

The present invention also provides the recombinant vectors that contains PgLHT gene, specifically comprises: plant expression vector pBI121, in empty carrier pBI121, obtains recombinant vectors pBI121-PgLHT by PgLHT gene clone that is; Prokaryotic expression carrier pET28a, in empty carrier pET28a, obtains recombinant vectors pET28a-PgLHT by PgPDR2 gene clone that is.PgLHT gene also can be cloned in other empty carriers, or is prepared into transgenic cell and recombinant bacterium.

The present invention also provides PgLHT gene promoting ginseng amino acid transport and coercing the application in reaction; The application of PgLHT gene in ginseng breeding.

The present invention also provides a kind of ginseng amino acid transporter by PgLHT genes encoding shown in SEQ ID NO.1, and the sequence of this albumen is as shown in SEQ ID NO.2.

The present invention also provides albumen shown in SEQ ID NO.2 promoting ginseng amino acid transport and coercing the application in reaction, and the application of this albumen in ginseng breeding.

The present invention utilizes existing plant gene engineering technology, utilize plant LHT DNA homolog design degenerated primer, adopt the separated gene order with identifying that the plant stress responses such as amino acid transport and participation ginseng are relevant of the methods such as degenerated primer round pcr and RACE, and by agrobacterium tumefaciens-mediated transformation, gene is proceeded to tobacco, through the rotating function of heterogenous expression assay certificate PgLHT gene.Research finds that PgLHT genetic expression has tissue specificity and coerces and react closely related with ginseng.There is significant effect the aspects such as resistance that described PgLHT gene strengthens panax ginseng plant.Can or improve even other plant of ginseng by this gene or derivatives thereof screening, obtain the strong good plant kind of adaptive capacity to environment.Can also adopt tissue culture or transgenosis mode to promote the organotrophy metabolism of the plants such as ginseng.

Accompanying drawing explanation

Fig. 1 is pcr amplification PgLHT gene diagram of the present invention; In figure, 1 represents degenerated primer pcr amplification product, and M represents Marker;

Fig. 2 is PgLHT albumen membrane spaning domain prognostic chart of the present invention;

Fig. 3 be PgLHT gene coding amino acid sequence of the present invention and with other plant LHT DNA homolog comparison diagram;

Fig. 4 is PgLHT gene coding amino acid sequence phylogenetic analysis figure of the present invention;

Fig. 5 is the expression level figure of PgLHT gene of the present invention in ginseng different tissues;

Fig. 6 is PgLHT gene of the present invention expression level figure in the ginseng-cell of (50mM NaCl processing) under condition of salt stress

Fig. 7 is PgLHT gene of the present invention expression level figure in the ginseng-cell of (20 μ M ABA process) under ABA inductive condition

Fig. 8 is PgLHT gene of the present invention expression level figure in the ginseng-cell of (100 μ M MeJA process) under MeJA inductive condition

Fig. 9 is PgLHT gene of the present invention expression level figure in the ginseng-cell of (200 μ M SA process) under SA inductive condition

Embodiment

The acquisition of embodiment 1 PgLHT gene

1, ginseng suspension cell culture

(1) get the 4 years raw ginsengs in Changbai mountain, Jilin ginseng producing region, tap water soaks 45min post-flush 2 times, first uses 75% alcohol immersion 30 seconds (sec), and aseptic water washing 2 times, uses 0.1%HgCl on sterilisable chamber Bechtop ₂sterilization, constantly stirs, and sterile distilled water rinses 4-5 time.

(2), under aseptic condition, the explant of sterilizing is inoculated in the MS substratum that contains 2,4-D (2.0mg/L) and KT (0.5mg/L).The complete dedifferentiation of explant after 3 succeeding transfer culture, after being completed into callus, 6 explants of every 100ml triangular flask inoculation, culture condition is as aforementioned, cultivate after 45 days (d) by the capable succeeding transfer culture of callus, to supplement the required nutritive substance of culture and other requirement.After inoculation 30d, carry out succeeding transfer culture for the first time, later every 3-4 week subculture once.

(3), after obtaining callus, be transferred in MS liquid nutrient medium.Callus lines diameter should be less than 3mm.If the large available aseptic scalper of tissue block is divided into fritter.Liquid nutrient medium is divided in the Erlenmeyer flask of 50ml, every 10ml liquid nutrient medium inoculation 1.5-2.0g callus, and after inoculation, concussion is cultivated, and 120rpm, secretly cultivates by 24 ℃.Every 4-7d subculture once.

2, the total RNA of ginseng suspension cell extracts

The suspension cell that the centrifugal 5min of (1) 3000 * g collects, in mortar with the abundant grind into powder of liquid nitrogen.The powder that every 40mg grinds is placed in 1.5ml centrifuge tube, adds 1ml TRIzol reagent, and 40 μ L beta-mercaptoethanols, with suction pipette head re-suspended cell precipitation, incubated at room 5-10min.

(2) add 0.2ml chloroform, jolting l5sec, incubated at room 10-15min.

(3) 4 ℃, the centrifugal 15min of 12000 * g, collect the colourless water in upper strata in 1.5ml centrifuge tube, do not inhale moving middle layer, abandons precipitation.

(4) add 0.4ml3mol/L ammonium acetate (pH5.2), 0.6ml Virahol, soft vibration mixes, incubated at room 5-10min (RNA precipitation).4 ℃, the centrifugal 10min of 12000 * g, abandon supernatant.

(5) add 1ml75% ethanol vibration; 4 ℃, the centrifugal 5min of 7500 * g, abandons supernatant, repeats this step.

(6) air drying 10min, adds 20 μ L DEPC water dissolution RNA.

(7) the non-sex change agarose gel electrophoresis of RNA carries out in 0.5 * TBE, agarose concentration is 1% (the 10mg/ml EB that adds 2.5 μ L in every 100ml gel), 4 μ L applied sample amounts, 8v/cm constant voltage 40min, the then integrity of observation post's extraction RNA in ultraviolet gel imaging system.

3, cDNA is synthetic

After purified mRNA, the o1igo (dT) of take is primer, the total mRNA of M-MuLV ThermoScript II reverse transcription.

(1) reaction system is as follows:

(2) soft stirring and evenly mixing; Room temperature is placed after 10min, moves to constant water bath box; 42 ℃, 1h; After end, cooled on ice 2min.

4, degenerated primer pcr amplification

(1) degenerated primer design is with synthetic

Utilize Bioedit software to 7 kind of plant LHT gene order soybean (the Glycine max that reported, GmLHT, XM003547627), castor-oil plant (Ricinus communis, RcLHT, XM002509522), grape (Vitis vinifera, VvLHT, XM_002265272), Root or stem of Littleleaf Indianmulberry (Lotus japonicus, LjLHT, JF705953), two fringe false bromegrass (Brachypodium distachyon, BdLHT, XM003573278), Arabidopis thaliana (Arabidopsis thaliana, AtLHT1, NM180778), comospore poplar (Populus trichocarpa, PtrLHT1, XM002321645) carry out sequence analysis, find out the region of high conservative, adopt Primer5.0 software design degenerated primer as follows:

P1：5′-TGGCAAATGGTKGADATGCATGA-3′；(SEQ ID NO.3)

P2：5′-WATCTGGTADCTWCCDATDACATG-3′。(SEQ ID NO.4)

Y=C/T wherein; D=A/G/T; R=A/G; I=inosinic acid.Primer is synthetic by the biological company limited of Shanghai English fine horse.

(2) pcr amplification

Take cDNA as template, with degenerated primer P1, P2, carry out " touchdown " pcr amplification.

Reaction system is: 10 * PCR damping fluid, 5 μ L, MgCl ₂(25mmol/L) 3 μ L, dNTP (2.5mmol/L) 8 μ L, P1 (20 μ mol/L) 2 μ L, P2 (20 μ mol/L) 2 μ L, cDNA2 μ L, dd H ₂o27.5 μ L and LA Taq DNA polymerase (5U/ μ L) 0.5 μ L.

Reaction conditions is: 94 ℃ of denaturation 3min; 94 ℃ of sex change 30s, 62 ℃ of annealing 2min, 72 ℃ of extension 2min, 2 circulations; 94 ℃ of sex change 30s, 60 ℃ of annealing 2min, 72 ℃ of extension 2min, 2 circulations; 94 ℃ of sex change 30s, 58 ℃ of annealing 2min, 72 ℃ of extension 2min, 2 circulations; 94 ℃ of sex change 30s, 56 ℃ of annealing 2min, 72 ℃ of extension 2min, 26 circulations; 72 ℃ are extended 10min.1.0% agarose gel electrophoresis is analyzed PCR product.

(3) amplified fragments glue reclaims, connects, checks order and analyzes

After PCR product glue is reclaimed, be connected on pGEM-T Easy carrier, and transform bacillus coli DH 5 alpha.After PCR preliminary evaluation is correct, recombinant plasmid pGEM-LHT is served to the order-checking of Hai Yingjun company.After order-checking, obtain 1 gene fragment that sequence length is 729bp, sequencing result utilizes Blast instrument in NCBI and dbEST database and the albumen database of GenBank to carry out sequence analysis, with other plant LHT gene height homology.

4, cDNA total length amplification

729 bp LHT gene order design 5 ' ends and 3 ' end amplimer that according to degenerated primer, amplification obtains, 5 '-RACE primer (GSP5) and 3 '-RACE primer (GSP3) is respectively:

GSP5：5′-CTCGGGTGTTGAAGGGATTGTTGC-3′；(SEQ ID NO.5)

GSP3：5′-GTCCACTTTGTCCTCTCCCATCTCC-3′。(SEQ ID NO.6)

1. RNA extracts and reverse transcription

RNA extracts and adopts aforesaid method to carry out, and reverse transcription adopts ThermoScript II and primer in SMART RACE cDNA Amplification Kit.

5 2. ' end amplification reaction system

5 3. ' end amplification reaction condition

94℃ 2min，1cycles:

94℃ 30sec，72℃ 1min，5cycles；

94℃ 30sec，70℃ 30sec，72℃ 1min，5cycles；

94℃ 30sec，68℃ 30sec，72℃ 1min，20cycles

3 4. ' end amplification system

3 5. ' end amplification reaction condition

94℃ 2min，1cycles:

94℃ 30sec，72℃1min30sec，5cycles；

94℃ 30sec，70℃ 30sec，72℃ 1min30sec，5cycles；

94℃ 30sec，68℃ 30sec，72℃ 1min30sec，20cycles

6. electrophoresis and order-checking thereof

PCR product is after 1.0% agarose gel electrophoresis detects, and glue reclaims and is connected with pGEM-T Easy carrier, transforms bacillus coli DH 5 alpha, extracts single bacterium colony plasmid.Plasmid is carried out to double digestion is identified and PCR detects, detect and serve Hai Yingjun company after errorless and check order.After order-checking, obtaining 5 ' end cDNA length is 909bp, and 3 ' end cDNA length is 1250bp, and the sequence (PgLHT) that the total length that obtains comprising complete encoder block after splicing is 1350bp, is submitted to Genbank, and accession number is JX896444.

5, sequential analysis

The cDNA sequence that successfully to have obtained length be 1889bp through checking order, utilizing in NCBI on-line analysis tools BLAST and ORF Finder (http://blast.ncbi.nlm.nih.gov/Blast.cgi and http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi) to compare with ORF searches and finds that in this sequence, CDS sequence length is 1350bp, the aminoacid sequence of its coding is adopted to the comparison of CLUSTAL X software analysis, with other plant LHT gene as soybean (GmLHT), castor-oil plant (RcLHT, ), grape (VvLHT), Root or stem of Littleleaf Indianmulberry (LjLH), two fringe false bromegrasses (BdLHT), Arabidopis thaliana (AtLHT1), the homology of comospore poplar (PtrLHT1) is greater than 75%.Utilize TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/) on-line analysis software analysis membrane spaning domain, result shows that this albumen is transmembrane protein.And use MEGA4 software to set with adjacent method (Neighbor-Joining, NJ) constructing system.In genealogical tree, for the confidence level of branch, bootstrap value (Bootstrap test) is estimated, repeat 1000 times, result shows that the LHT gene affinity of this gene and grape, castor-oil plant and soybean is nearest.

6, expression analysis

Root, adventive root, bud, seed and the root of hair of getting fresh ginseng, extract respectively RNA; Again with 20 μ MABA, 200 μ M SA, 100 μ M MeJA and 50mM NaCl process respectively ginseng 0,1,3,6,12 and 24h, extract the total RNA of each time point ginseng.Oligod (T) 18 of take is primer, and AMV ThermoScript II is synthesized cDNA, and take β-actin as internal reference, utilizes real-time pcr amplification PgLHT gene, and PgLHT and β-actin amplimer are respectively:

PgLHT：F：5′-CTGGGAGATGTAGCGTTTGC-3′；(SEQ ID NO.7)

R：5′-GGTGTTGAAGGGATTGTTGC-3′；(SEQ ID NO.8)

β-actin：F：5′-CGTGATCTTACAGATAGCTTCATGA-3′；(SEQ ID NO.9)

R：5′-AGAGAAGCTAAGATTGATCCTCC-3′。(SEQ ID NO.10)

Reaction system is:

Adopt two-step approach pcr amplification standard program: 95 ℃ of 30sec; 95 ℃ of 40sec, 60 ℃ of 30sec totally 40 circulations; 95 ℃ of 15sec, 60 ℃ of 1min, 95 ℃ of 15sec.Each sample repeats for three times.Reaction finishes rear confirmation amplification curve and solubility curve, adopts 2 ^{-Δ Δ Ct}method is calculated the difference of PgLHT gene expression level under different tissues and different treatment condition.Result shows that it expresses the highlyest in gemma, is secondly adventive root and root, expresses minimum in seed.MeJA and NaCl can significantly lure PgLHT genetic expression and be time-dependent manner and change.

Structure and the conversion of embodiment 2 expression vectors

1, the structure of plant over-express vector and transfer-gen plant screening

(1) use respectively BamH I and Sac I double digestion pBI121 and PgLHT, reclaim pBI121 carrier and PgLHT gene fragment.Two fragments connected and transform DH5 α, filtering out recon, recombinant plasmid called after

pBI121-PgLHT。The upstream and downstream primer of PgLHT gene of wherein increasing is respectively:

PgLHT：F1:5′-CG GGATCCCGCACCATGGTTTCCCAAGCTCAGAC-3′；(SEQ ID NO.11)

R1:5′-CGC GAGCTCGGCTCCTTTGCTGCTTATGAGAA-3′。(SEQ ID NO.12)

Wherein line part is BamH I and Sac I restriction enzyme site.

(2) preparation of agrobacterium tumefaciens competent cell

1. streak culture Agrobacterium LBA4404 on the YEB flat board that contains 100 μ g/ml rifomycins, secretly cultivates 2d for 28 ℃.

2. picking list colony inoculation is in the YEB substratum that contains 100 μ g/ml rifomycins, and 28 ℃ of shaking culture are spent the night.

In the YEB substratum of the kanamycin that 3. Agrobacterium of the activation of spending the night is contained to 100 μ g/ml by the dilution proportion of l:50 to 50ml, 28 ℃ of shaking culture are about 0.4-0.6 to OD600, ice bath 30min.

4. in 4 ℃, the centrifugal 10min of 5000rpm, abandons supernatant liquor, with 10ml0.15mM NaCl suspension thalline.

5. in 4 ℃, the centrifugal 10min of 5000rpm, abandons supernatant liquor, uses 2ml20mM CaCl ₂suspension thalline.

6. every pipe 200 μ L packing, liquid nitrogen flash freezer ,-70 ℃ of preservations.

(3) conversion of agrobacterium tumefaciens competent cell

1. agrobacterium tumefaciens competent cell is placed on ice, slowly thaws.

2. add 0.5 μ g pBI121-PgLHT plasmid DNA, mix gently ice bath 30min.

3. put freezing 2min in liquid nitrogen, move into rapidly heat shock 5min in 37 ℃ of water-baths, rapider ice bath 2min, add afterwards 800 μ L YEB liquid nutrient mediums, in 28 ℃ of shaking culture 3-4h.

4. the centrifugal 5min of 5000rpm precipitation thalline, discards Eddy diffusion thalline after 800 μ L supernatants, evenly coats on the YEB substratum that contains 100 μ g/ml rifomycins and 50 μ g/ml kantlex, cultivates 2-3d for 28 ℃.

(4) agrobacterium tumefaciens plasmid extraction and evaluation

1. PCR identifies

The single colony inoculation of Agrobacterium that picking transforms containing 37 ℃ of shaking culture in the LB liquid nutrient medium of kantlex, is used PgLHT gene-specific primer in 1.5ml:

F：5′-CTGGGAGATGTAGCGTTTGC-3′；(SEQ ID NO.7)

R：5′-GGTGTTGAAGGGATTGTTGC-3′；(SEQ ID NO.8)

After pcr amplification, agarose gel electrophoresis detects whether contain expection fragment.PCR response procedures is as follows: 94 ℃ of 30sec, 55 ℃ of 45sec, 72 ℃ of 30sec, 35 circulations; 72 ℃ of 2min.

2. enzyme is cut evaluation

The single colony inoculation of picking Agrobacterium is in the YEB substratum of the kantlex of the rifomycin that contains 100 μ g/ml and 50 μ g/ml, 28 ℃ of overnight incubation are cultivated, extract recombinant plasmid pBI121-PgLHT, adopt aforesaid method to cut the plasmid of extraction with BamH I and Sac I enzyme.

(5) containing the agrobacterium tumefaciens of pBI121-PgLHT plasmid, cultivate

28 ℃ of about 2d of the streak culture Agrobacterium that contains pBI121-PgLHT plasmid.Picking list colony inoculation is in the YEB liquid nutrient medium that contains 100mg/L rifomycin and 50mg/L kantlex, and 28 ℃ of shaking culture are spent the night, and to OD600, be about at 0.6 o'clock and collect bacterium liquid, the centrifugal 10min of 4000rpm, precipitation is resuspended in 1/2MS liquid nutrient medium.

(6) leaf dish method infects tobacco

Tobacco spire is placed in to tap water and rinses 3-4 time, then in 70% ethanol, soak 30-60sec, the 10-20min that sterilizes in 10% clorox, more fully rinse with sterilized water, be then placed in sterile petri dish, blade is cut into 0.5-l cm ²small pieces, the blade shearing is put into respectively to the Agrobacterium bacterium liquid that contains pBI121-PgLHT plasmid and infects 5min, the bacterium liquid on blade is blotted with aseptic filter paper.

(7) cultivate altogether

The leaf dish that infected and blotted surperficial bacterium liquid, pore faces up, and nestles up and is placed on (MS+6-BA1.0mg/L+IAA0.1mg/L) on common substratum, and 2d is cultivated at dark place.

(8) screening and differentiation culture

Dark cultivation after 2d, be transformed on screening division culture medium (MS+6-BA 1.0mg/L+IAA 0.lmg/L+Kan 100mg/L+Cef 300mg/L), every 15d changes primary screening division culture medium, when the tobacco differentiating grows to 3-5cm, goes in root media.

(9) root culture

The tobacco breaking up to 3-5cm is cut respectively, be transferred to root media (1/2MS+IBA 2.0mg/L).

(10) transplant

When the root of regeneration plant grows to 5-8cm, open sealed membrane hardening 2-3d, by seedling replanting to flowerpot, the initial upper transparent plastics of 5-l0d cover after transplanting, the humidity of maintenance 90%-100%, and beat a little apertures be beneficial to gaseous interchange on covering.The matrix of transplanting and flowerpot be sterilization in advance all.

(7) transfer-gen plant PCR identifies

The genomic dna of transfer-gen plant of take is template, adopts above-mentioned recombinant plasmid to identify that PgLHT gene-specific primer used carries out pcr amplification, and PCR reaction system and reaction conditions are the same.Through identifying the T that obtains seedling differentiation ₀for positive transfer-gen plant.

2, the anti-evaluation of coercing ability of seedling

Get form growing way basically identical wild-type plant and transfer-gen plant, be divided into two parts, respectively to containing 20 μ MABA, 200 μ M SA, on the MS solid medium of 100 μ M MeJA and 50mM NaCl, in (25 scholar 2) ℃, relative humidity 70%, every day 16h illumination/8h dark illumination box cultivate in growth, each processes at least 10 young plants.Continue to cultivate after 10 days, observe the growing state of transgene tobacco and wild-type tobacco seedling, found that transfer-gen plant has the stronger anti-ability of coercing.

3, prokaryotic expression analysis

(1) pcr amplification

The PgLHT gene having increased of take is template, and the primer that adds respectively BamH I and Sac I restriction enzyme site sequence with upstream and downstream increases, its primer used that increases following (line part is respectively BamH I and Sac I restriction enzyme site):

5′-CGG GAATTCATGGTTTCCCAAGCTCAGAC-3′(SEQ ID NO.13)

5′-CGG GAATTCTCCTTTGCTGCTTATGAGAA-3′(SEQ ID NO.14)

Reaction system is the same, and PCR response procedures is as follows: 94 ℃ of 30sec, 55 ℃ of 45sec, 72 ℃ of 4min 30sec, 35 circulations; 72 ℃ of 10min.

(2) enzyme is cut, is connected and transforms and screening

Use respectively BamH I and Sac I double digestion pET28a and PCR product, reclaim pET28a and PCR product fragment.Two fragments connected and transform BL21, by kantlex screening, pcr amplification and enzyme, cutting and identify recon, recombinant plasmid called after pET28a-PgLHT.

(3) the protein induced expression of PgLHT and functional analysis thereof

The bacterium colony BL21 (DE3) that picking contains restructuring pET28a-PgLHT plasmid, to 37 ℃ of incubated overnight in l0ml LB (containing Kan 50 μ g/ml).Get l ml bacterium liquid and join containing 100m1LB substratum (containing Kan50 μ g/ml), 37 ℃ of concussions are cultured to OD600 and are about 0.4-0.6.Adding IPTG is that 1mM induces to final concentration, every 1h, collects a bacterium liquid, the centrifugal 10min of 5000 * g, results precipitation, adds the ratio of 4ml PBS damping fluid resuspended with 1g thalline, adds 5 * SDS-PAGE sample-loading buffer, mix, boiling water bath 5min gets supernatant and carries out SDS-PAGE analysis.

Choose the time point that PgLHT expressing quantity is high, add the substrates such as different sorts amino acid, analyze the function of PgLHT albumen, and in conjunction with His-tag, carry out protein purification, its structure of external parsing.

SEQUENCE LISTING

<110> Central South University

<120> ginseng amino acid transporter gene PgLHT and proteins encoded and application

<160> 14

<170> PatentIn version 3.3

<210> 1

<211> 1350

<212> DNA

<213> homo sapiens

<400> 1

atggtttccc aagctcagac ggatcaccat gacaactcca acaacgttga tcgaagaact 60

gcgaaagaga aagccattga tgactggctt cctataacgt cttcaaggca tgcaaaatgg 120

tggtattcag cattccacaa tgtcactgcc atggttggag ccggagttct cagtctcccc 180

tatgccatgt cagaactcgg atggggccct ggtgtagctg tactggtgat atcatgggtt 240

gtgactttat acacattgtg gcaaatggtg gagatgcatg agatggtacc tgggagacgt 300

ttcgacagat accatgagct aggccagtat gcttttgggg agaagctcgg cctctacatc 360

gtggtgcccc agcagctaat tgtagaagtt gggatggaca tagtctacat ggtcaccggt 420

ggcaaatctc tgaaaaaatt ccacgattta atctgcaaaa aggattgcaa agatatcaaa 480

ttaacattct ttattatgat ctttgcctct gtccactttg tcctctccca tctccccaac 540

ttcaattcca tctcttgggt atctttggtt gcagcaatca tgtccttgag ttactctact 600

attgcttggt cagcttctgt taagaagggt gttcaaccag atgttgaata tggctacaaa 660

gccaagtcta cagccggaac agtttttaac tttttcagtg cgctgggaga tgtagcgttt 720

gcatatgcag gtcataatgt ggttttggag attcaagcaa caatcccttc aacacccgag 780

aagccttcaa agggtcctat gtggaaggga gtcgtcgtgg cctacatagt tgtcgccttg 840

tgctacttcc ccgttgctct gataggatat tggatgtttg gaaatgcagt ttccgacaat 900

attctgatca ccttagagaa acctacttgg ctcattgcaa tggctaacat ttttgttgtt 960

tttcatgtca tcggaagcta tcagatttat gcaatgccgg tatttgatat gattgaaact 1020

gtgctggtaa agaaattaca tttcgcacca agtataacac ttcgtttcgt ttcaaggaac 1080

ctatacgtgg ctttcacaat gtttgttgcc atttgtttcc ccttttttgg gggtcttctt 1140

ggattctttg gaggattcgc cctcgcccca actacatatt ttctcccttg cgtcatgtgg 1200

cttgctatct acaaaccgag gagatacagc ctatcttggt ttactaactg gatttgcatc 1260

tttttcggag tttgtttgat gattttatct cctattggag gactaaggca gatcataatt 1320

caagccaaaa cctacgaatt tttctcataa 1350

<210> 2

<211> 449

<212> PRT

<213> homo sapiens

<400> 2

Met Val Ser Gln Ala Gln Thr Asp His His Asp Asn Ser Asn Asn Val

1 5 10 15

Asp Arg Arg Thr Ala Lys Glu Lys Ala Ile Asp Asp Trp Leu Pro Ile

20 25 30

Thr Ser Ser Arg His Ala Lys Trp Trp Tyr Ser Ala Phe His Asn Val

35 40 45

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

50 55 60

Glu Leu Gly Trp Gly Pro Gly Val Ala Val Leu Val Ile Ser Trp Val

65 70 75 80

Val Thr Leu Tyr Thr Leu Trp Gln Met Val Glu Met His Glu Met Val

85 90 95

Pro Gly Arg Arg Phe Asp Arg Tyr His Glu Leu Gly Gln Tyr Ala Phe

100 105 110

Gly Glu Lys Leu Gly Leu Tyr Ile Val Val Pro Gln Gln Leu Ile Val

115 120 125

Glu Val Gly Met Asp Ile Val Tyr Met Val Thr Gly Gly Lys Ser Leu

130 135 140

Lys Lys Phe His Asp Leu Ile Cys Lys Lys Asp Cys Lys Asp Ile Lys

145 150 155 160

Leu Thr Phe Phe Ile Met Ile Phe Ala Ser Val His Phe Val Leu Ser

165 170 175

His Leu Pro Asn Phe Asn Ser Ile Ser Trp Val Ser Leu Val Ala Ala

180 185 190

Ile Met Ser Leu Ser Tyr Ser Thr Ile Ala Trp Ser Ala Ser Val Lys

195 200 205

Lys Gly Val Gln Pro Asp Val Glu Tyr Gly Tyr Lys Ala Lys Ser Thr

210 215 220

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

225 230 235 240

Ala Tyr Ala Gly His Asn Val Val Leu Glu Ile Gln Ala Thr Ile Pro

245 250 255

Ser Thr Pro Glu Lys Pro Ser Lys Gly Pro Met Trp Lys Gly Val Val

260 265 270

Val Ala Tyr Ile Val Val Ala Leu Cys Tyr Phe Pro Val Ala Leu Ile

275 280 285

Gly Tyr Trp Met Phe Gly Asn Ala Val Ser Asp Asn Ile Leu Ile Thr

290 295 300

Leu Glu Lys Pro Thr Trp Leu Ile Ala Met Ala Asn Ile Phe Val Val

305 310 315 320

Phe His Val Ile Gly Ser Tyr Gln Ile Tyr Ala Met Pro Val Phe Asp

325 330 335

Met Ile Glu Thr Val Leu Val Lys Lys Leu His Phe Ala Pro Ser Ile

340 345 350

Thr Leu Arg Phe Val Ser Arg Asn Leu Tyr Val Ala Phe Thr Met Phe

355 360 365

Val Ala Ile Cys Phe Pro Phe Phe Gly Gly Leu Leu Gly Phe Phe Gly

370 375 380

Gly Phe Ala Leu Ala Pro Thr Thr Tyr Phe Leu Pro Cys Val Met Trp

385 390 395 400

Leu Ala Ile Tyr Lys Pro Arg Arg Tyr Ser Leu Ser Trp Phe Thr Asn

405 410 415

Trp Ile Cys Ile Phe Phe Gly Val Cys Leu Met Ile Leu Ser Pro Ile

420 425 430

Gly Gly Leu Arg Gln Ile Ile Ile Gln Ala Lys Thr Tyr Glu Phe Phe

435 440 445

Ser

<210> 3

<211> 23

<212> DNA

<213> homo sapiens

<400> 3

tggcaaatgg tkgadatgca tga 23

<210> 4

<211> 24

<212> DNA

<213> homo sapiens

<400> 4

watctggtad ctwccdatda catg 24

<210> 5

<211> 24

<212> DNA

<213> homo sapiens

<400> 5

ctcgggtgtt gaagggattg ttgc 24

<210> 6

<211> 25

<212> DNA

<213> homo sapiens

<400> 6

gtccactttg tcctctccca tctcc 25

<210> 7

<211> 20

<212> DNA

<213> homo sapiens

<400> 7

ctgggagatg tagcgtttgc 20

<210> 8

<211> 20

<212> DNA

<213> homo sapiens

<400> 8

ggtgttgaag ggattgttgc 20

<210> 9

<211> 25

<212> DNA

<213> homo sapiens

<400> 9

cgtgatctta cagatagctt catga 25

<210> 10

<211> 23

<212> DNA

<213> homo sapiens

<400> 10

agagaagcta agattgatcc tcc 23

<210> 11

<211> 34

<212> DNA

<213> homo sapiens

<400> 11

cgggatcccg caccatggtt tcccaagctc agac 34

<210> 12

<211> 32

<212> DNA

<213> homo sapiens

<400> 12

cgcgagctcg gctcctttgc tgcttatgag aa 32

<210> 13

<211> 29

<212> DNA

<213> homo sapiens

<400> 13

cgggaattca tggtttccca agctcagac 29

<210> 14

<211> 29

<212> DNA

<213> homo sapiens

<400> 14

cgggaattct cctttgctgc ttatgagaa 29

Claims (3)

1. a ginseng amino acid transporter gene PgLHT, is characterized in that, described PgLHT gene order is as shown in SEQ ID NO.1.

2. a recombinant vectors, is comprised of empty carrier and the goal gene that inserts this empty carrier, it is characterized in that, described goal gene is PgLHT gene claimed in claim 1.

3. recombinant vectors according to claim 2, is characterized in that, described empty carrier is pBI121 or pET28a.