CN102586280A - Yunnan red pear delta PyTTG1 gene, prokaryotic expression vector thereof and application of prokaryotic expression vector - Google Patents

Abstract

The invention discloses a Yunnan red pear pigment synthesis and trichome development regulatory protein PyTTG1 gene delta PyTTG1 and a prokaryotic expression vector thereof. The Yunnan red pear delta PyTTG1 purified protein is obtained by cloning the specific segment delta PyTTG1 of the PyTTG1 gene from the Yunnan red pear, constructing the prokaryotic expression vector of the gene and expressing the gene in the Escherichia coli. The Yunnan red pear delta PyTTG1 purified protein is applied to preparation of a PyTTG1 specific antibody, detection on PyTTG1 protein expression, immunoprecipitation, and chromatin immunoprecipitation. The antibody has high specificity and can accurately detect the subcellular localization of the PyTTG1 protein, efficiently purify the PtTTG1 protein, efficiently separate the protein bound with the PyTTG1 protein from the DNA segment, and detect the expression condition of the the PtTTG1 protein in transgenic plants.

Description

YUNNANHONG pears Δ PyTTG1 gene and prokaryotic expression carrier and application

Technical field

The invention belongs to the genetically engineered field, be specifically related to a kind of YUNNANHONG pears pigment regulation and control and trichome genesis and development regulatory protein gene Δ PyTTG1And prokaryotic expression carrier, and the application of prokaryotic expression carrier in preparation Δ PyTTG1 albumen and specific antibody.

Background technology

Red epidermis is the important character index of pears molecular breeding.Yunnan Province has more red skin pears germ plasm resource because of its unique geography and weather condition, has successively selected early whitish honey, crisp, all over the sky red and No. 1 four Cultivars of the red pears of cloud of beauty since 1986.But remove red pears 1 extra of cloud in producing, other 3 kinds all can cause painted relatively poor even not paintedly because of climate change, therefore need the coloring mechanism of the red the operatic circle skin of research.Existing research shows that the synthetic of plant cyanidin(e) regulated and control by transcription complex MYB/bHLH/ WD40.Forefathers are more to MYB and bHLH research, and less to the research of the WD40 albumen in the plant.WD40 albumen contains WD40 motif (about 40 amino acid whose conserved sequences are with glycocoll-Histidine (GH) beginning, with tryptophan-aspartic acid (WD) ending).WD40 albumen contains 1-10 placed in-line WD40 motif, it is generally acknowledged that WD40 motif more than at least 4 could form senior and the structure of function is arranged.Research shows that WD40 albumen is a protein family that structure is various, function is different; Usually they have two common traits: the one, and structural domain is folded into β-propeller arrangement; The one, form the reversible assembling of polyprotein but the platform of any catalytic activity of tool not, they mainly are in Eukaryotic protein-protein complex forms, to work.Many researchs show that WD40 albumen has the support effect in the protein complex forming process.In plant, the proteic effect of WD40 mainly be as the plant specificity grow incident as bloom, flower development, meristematic tissue formation, pollen development and gamete form crucial regulatory factor.WD40 albumen mainly passes through its WD structural domain and other interactions between protein, the regulation and control of numerous metabolic reactions in the involved in plant body.People such as Morita find to have recessive gene in morning glory Ca(coding InWDR1 albumen) in the two mutants, except that CHS, the expression of the structure gene of cyanidin(e) route of synthesis all reduces synergistically.People such as Wallker find the synthetic of TTG1 protein regulation cyanidin(e) and trichome differentiation through positional cloning and complementary assay first.Arabidopis thaliana TTG1 albumen contains 4 WD40 motifs, its Ttg1The characteristics of two mutants are that shortage epidermal hair, kind skin are transparent, seed lacks cyanidin(e) fully, has unusual mutant phenotypes such as root development pattern with regard to direct germination, plant without dormancy, and this shows that the function of TTG1 is a multi-purpose.Cotton GhTTG1 and GhTTG3 have the similar functions of Arabidopis thaliana AtTTG1: not only can recover Arabidopis thaliana Ttg1Two mutants forms trichome and cyanidin(e), and can also complementary VT Ttg1Two mutants produces purple dot at petal.People such as De find WD40 albumen PhAN11 first in petunia, it is positioned tenuigenin, can connect signal transduction and transcriptional activation, can regulate and control the function of PhAN2 (MYB).Cyanidin(e) modulin AN11 in PAC1 and petunia and the Arabidopis thaliana in the corn, TTG1 are very similar, and 35S-PAC1 can be complementary TtglTwo mutants.People such as Dressel find that the sudden change (W158R) of single amino acids in the WD motif just can suppress the DFR expression of gene, and then cause spending in vain phenotype, and 54.1% high GC content of MiTTG1 is the signal of evolving.People such as Matus are the ectopic expression grape in Arabidopis thaliana WDR1Cause the synthetic of cyanidin(e) in Arabidopis thaliana over-ground part and the lotus throne leaf, and be positioned tenuigenin and nucleus through GFP fusion protein technology proof WDR1.People such as Zhang through yeast two-hybrid and plant cross expression technology find EGL3, GL3 can with TTG1 (WD40) and MYB albumen (GL1; PAP1, PAP2, CPC, TRY) combination, form the multi-functional regulation and control that the MYB/bHLH/WD40 mixture comprises that cyanidin(e) biosynthesizing and trichome form.People such as Brueggemann have confirmed that through complementary assay apple MdTTG1 has the function of similar Arabidopis thaliana AtTTG1.People such as Baudry find that through the method for heredity and molecule TT2 (MYB), TT8 (bHLH) and TTG1 (WDR) can form the expression of tetraplex direct regulation and control BAN in plant.Payne etc. have confirmed that through yeast two-hybrid the GL3/GL1/TTG1 mixture is regulated and control ciliary growth in Arabidopis thaliana.Bouyer catches-exhaust mechanism through what clonal analysis, ectopic expression and microinjection test had proposed that trichome forms: the trichome in high density promotes in the Protein G L3 cell; GL3 exhausts the TTG1 that closes in the cell through combining trichome to form albumen TTG1; Thereby cause trichome to form, and cell on every side can not form trichome because of the minimizing of TTG1.And people such as Zhao through the ion bombardment evidence TTG1; GL3; GL1 and GL2 do not shift between adjacent cells, and R3-MYB, CPC then can shift between adjacent cells; Propose the TTG1 mixture and directly regulate activation and suppress son, and ciliary type on the motion effects Arabidopis thaliana leaf of inhibition.TTG1 is many effects gene, and people such as Gonzalez find that also TTG1 can form the differentiation of mixture control exosper with MYB5 and TT2 (MYB).

The biosynthesizing of YUNNANHONG the operatic circle peel anthocyanidin possibly regulated and control by MYB/bHLH/WD40.At present the PyTTG1 gene in YUNNANHONG pears and the apple is by clone and transgenic, but also is not specifically designed to the proteic Subcellular Localization of the accurate PyTTG1 of detection, carry out the proteic purifying of PyTTG1, high efficiency separation PyTTG1 albumen institute bonded albumen and dna fragmentation and detect the antibody of its expression in transgenic plant efficiently.The present invention selects painted best and stable ' No. 1, the red pears of cloud ' as test materials; Carry out YUNNANHONG pears ' No. 1, the red pears of cloud ' clone, sequential analysis and the prokaryotic expression of PyTTG1 transcription factor gene, the work of protein purification Antibody Preparation; With the blank that remedies this respect, also will act on the instrument that provides in the red colored mechanism of YUNNANHONG the operatic circle skin and in plant trichome genesis and development mechanism for studying YUNNANHONG pears and apple WD40 transcription factor TTG1.

Summary of the invention

The object of the present invention is to provide a kind of YUNNANHONG pears Δ PyTTG1Gene, this gene are the synthetic and trichome development regulatory proteins of YUNNANHONG pears pigment PyThe specific fragment of TTG1 gene has the protein of shown in SEQ ID NO.3 nucleotide sequence or coding aminoacid sequence shown in SEQ ID NO.4; This gene source is in the YUNNANHONG pears.

Another purpose of the present invention provides the YUNNANHONG pears Δ PyTTG1Prokaryotic Expression carrier pET32a- Δ PyTTG1, this carrier contains PyTTG1Gene, PyTTG1There are T7 promotor and bacterial ribosome binding site RBS in the upper reaches of gene, and the downstream of T7 promotor have can be by IPTG inductive operator sequence, PyTTG1The N end and the C end of gene all have 6 * His label.

Another purpose of the present invention is prokaryotic expression carrier to be applied in the synthetic trichome genesis and development that reaches of preparation YUNNANHONG pears pigment regulate and control among the specific proteins Δ PyTTG1.

Another purpose of the present invention is that prokaryotic expression carrier is applied in the preparation PyTTG1 specific antibody, the PyTTG1 specific antibody be applied in PyTTG1 and the proteic Subcellular Localization detection of MdTTG1, the proteic purifying of PyTTG1, PyTTG1 albumen institute bonded albumen and dna fragmentation high efficiency separation, detect in its expression in transgenic plant, the chromatin co-immunoprecipitation (ChIP).

Above-mentioned purpose of the present invention is to be achieved through following technical scheme:

1, construction of prokaryotic expression vector

(1) according to the YUNNANHONG pear PyTTG1In gene (the GenBank accession number is HQ641374) encoder block and apple, Arabidopis thaliana, corn, purple perilla, the petunia WD40Gene and prokaryotic expression carrier pET-32a polyclone restriction enzyme site design 1 pair of special primer PyTTG1-F:5 '- CCG CCATGGAGAACTCTACGCAAGAATCG-3 ', PyTTG1-R:5 '- CCG CTCGAGGTTCGGCTTTATTGAAAGGGTATCC-3 ' adds NcoI and XhoI restriction enzyme site and protection base (underscore partly is restriction enzyme site, and italicized item is the protection base) respectively at 5 of upstream and downstream primer ' end.With the total RNA of YUNNANHONG pears is template, with using behind the synthetic cDNA of M-MLV ThermoScript II PyTTG1-F with PyTTG1-R increases, and obtains specificity PyTTG1Fragment;

(2) reclaim PyTTG1Full length fragment;

(3) use NcoI with XhoI is right PyTTG1Fragment and carrier pET32a carry out double digestion, reclaim respectively, connect, and obtain prokaryotic expression carrier pET32a- PyTTG1

2, PyTTG1Prokaryotic Expression

Use the thermal stimulus method with pET32a- PyTTG1Change intestinal bacteria over to Rosetta (DE3)In, induce screening optimum expression condition down at IPTG, under optimum condition, carry out great expression.

3, the proteic purifying of PyTTG1

Collect thalline, carry out ultrasonication, cross the nickel post and carry out purifying, the albumen behind the collection purifying.

4, use Δ PyTTG1 purifying protein to prepare PyTTG1 antibody, use Δ PyTTG1 antibody can detect PyTTG1 albumen.

Recombinant vectors pET32a-of the present invention PyTTG1Can express the acquisition purifying protein, the antibody of preparation can be used for detecting PyTTG1 albumen, the present invention's preparation PyTTG1Antibody, can remedy do not have at present special detection PyTTG1 with MdTTG1, separate the antibody of PyTTG1 institute bonded protein, the proteic Subcellular Localization of PyTTG1, achievement of the present invention will lay the foundation for the molecular breeding of fruit tree and flowers.

Description of drawings

The total RNA electrophoresis synoptic diagram of Fig. 1 for extracting among the present invention.

Fig. 2 is the amplified production electrophoresis synoptic diagram after the gene amplification of the present invention.

Fig. 3 is a prokaryotic expression carrier pET32a-Δ PyTTG1 electrophoresis synoptic diagram of the present invention, and 1 is control plasmid pET32a-PyMT among the figure; The 2nd, pET32a-Δ PyTTG1.

Fig. 4 is expression synoptic diagram 0.5 mM condition induce under at 16 ℃ with 37 ℃, IPTG final concentration for prokaryotic expression carrier pET32a-Δ PyTTG1 of the present invention, and wherein 1 is the total protein of conversion pET-32a empty carrier Rosetta (DE3) thalline; The 2nd, the total protein of prokaryotic expression carrier before IPTG induces; The expression that 3-5 is a prokaryotic expression carrier after 16 ℃, IPTG final concentration are to induce 2,4,6 h under the 0.5 mM condition; The expression that 6-8 is a prokaryotic expression carrier after 37 ℃, IPTG final concentration are to induce 2,4,6 h under the 0.5 mM condition; The 9th, protein molecular weight standard SM0431.

Fig. 5 is the optimization electrophoresis synoptic diagram of Δ PyTTG1 protein purification condition among the present invention, and wherein 1 is Rosetta (DE3) total bacterial protein of expressing Δ PyTTG1; The 2nd, the stream behind the hanging column is worn liquid; 3-5 is respectively the result behind 30,150, the 500 mM imidazoles wash-outs; 6 is results of second pipe behind the 500 mM wash-outs; The 7th, molecular weight of albumen standard Marker0431.

Fig. 6 is the optimization electrophoresis synoptic diagram of Δ PyTTG1 protein purification condition among the present invention, and wherein 1 is the total protein that Rosetta (DE3) the bacterium 0.5 mM IPTG of expressing protein Δ PyTTG1 induces 16 h; The 2nd, Rosetta (DE3) total bacterial protein before IPTG induces; The 3rd, molecular weight of albumen standard Marker0431; 4-7 is the result who is respectively behind 150,200, the 300 and 500 mM imidazoles wash-outs.

Fig. 7 is the proteic purifying synoptic diagram of Δ PyTTG1 among the present invention, and wherein 1 is 0.1% BSA of 10 μ l; The 2nd, molecular weight of albumen standard Marker0441; 3-10 is the result of Δ PyTTG1 albumen under 300 mM imidazoles wash-outs.

Fig. 8 is for using Δ PyTTG1 antibody test 35S::PyTTG1 transgene tobacco blade PyTTG1 protein expression synoptic diagram among the present invention, wherein 1,2,3,5 and 6 is transgenic lines, and the 5th, the wild-type contrast.

Fig. 9 is the construction strategy synoptic diagram of prokaryotic expression carrier pET32a-Δ PyTTG1 of the present invention.

 

Embodiment

Below in conjunction with accompanying drawing and embodiment the present invention is done further explain, but protection domain of the present invention is not limited to said content.

The reagent that uses among the embodiment is mainly molecular biology test reagent; Various restriction enzymes, Pfu archaeal dna polymerase, RNA enzyme inhibitors, dNTP etc. are Dalian precious biotechnology ltd product; ThermoScript II is a Promaga company; PCR product purification test kit, plasmid extraction kit are given birth to worker Bioisystech Co., Ltd available from Shanghai Rosetta (DE3)Bacterial strain with DH5 αCompetent cell is Beijing Transgene Company products, and all the other reagent are homemade analytical pure.

His Trap pillar (GE Health), Other Instruments are molecular biology and genetically engineered laboratory common instrument equipment.All primer sequences are synthetic, and all life worker biotech company carries out in Shanghai with gene sequencing.

Method therefor is ordinary method if no special instructions among the present invention.

Embodiment 1: prokaryotic expression carrier pET32a- PyTTG1Structure

(1) design of primers

According to the YUNNANHONG pears PyTTG1In gene (the GenBank accession number is HQ641374) encoder block and apple, Arabidopis thaliana, corn, purple perilla, the petunia WD40Gene and prokaryotic expression carrier pET-32a polyclone restriction enzyme site design 1 pair of special primer PyTTG1-F:5 '- CCG CCATGGAGAACTCTACGCAAGAATCG-3 ', PyTTG1-R:5 '- CCG CTCGAGGTTCGGCTTTATTGAAAGGGTATCC-3 ' adds respectively at 5 of upstream and downstream primer ' end NcoI with XhoI restriction enzyme site and protection base (underscore partly is a restriction enzyme site, and italicized item is the protection base).

(2) extraction of total RNA

Use guanidine isothiocyanate method to extract total RNA, electrophoresis detection result such as Fig. 1 of red the operatic circle skin.

（3）RT-PCR

With the total RNA of YUNNANHONG pears is template, with using behind the synthetic cDNA of M-MLV ThermoScript II PyTTG1-F with PyTTG1-R increases, and obtains PyTTG1Fragment.

(4) PyTTG1Segmental recovery

The PCR product is carried out (see figure 2) behind the 1 % agarose gel electrophoresis, downcut the purpose fragment; Use glue to reclaim test kit, the purpose fragment is reclaimed according to the test kit specification sheets.

(5) construction of prokaryotic expression vector

Use NcoI with XhoI is to the PCR purified product PyTTG1Carry out double digestion to specifications with carrier pET32a, obtain 5 ' end respectively and have NcoI and 3 ' end have XhoI's PyTTG1Carry out glue behind fragment and the pET32a carrier, leakage of electricity swimming respectively and reclaim, according to the mol ratio goal gene: carrier=3:1 application of sample, add then and connect liquid I, 16 ℃ connect 16 h (see figure 3)s.With 100 μ l competence intestinal bacteria E.coliDH5 α adds mixing in the 6 μ l linked systems; Mixed solution ice bath 30 min, behind 42 ℃ of thermal stimulus 45 s, ice bath 2 min; Add 900 μ l liquid nutrient medium SOC, 37 ℃, 200 rpm shaking tables are cultivated 60 min and are made the thalline recovery; After cultivating end, centrifugal 1 min of normal temperature 8000 rpm collects thalline; On super clean bench, inhale and remove supernatant, when remaining about 0.1 ml, use the liquid-transfering gun mixing, insert and have on the LB solid plate of Amp resistance, even with the coating of aseptic triangle rod; 37 ℃ of incubated overnight; The picking white colony is inoculated in the substratum of LB liquid+Amp, after 37 ℃, 180 rpm are cultivated 12 h, extracts plasmid; Carry out after enzyme cuts checking, deliver to Shanghai again and give birth to the order-checking of worker bio-engineering corporation and carry out the exactness that sequence verification is inserted gene, obtain prokaryotic expression carrier pET32a-at last PyTTG1

Embodiment 2: prokaryotic expression carrier pET32a- PyTTG1Prokaryotic expression

Use the thermal stimulus method with recombinant plasmid pET32a-Δ PyTTG1Be transformed into intestinal bacteria Rosetta(DE3) in the competent cell, coat on LB+Amp solid plate picking pET32a-Δ PyTTG1The reorganization bacterium colony in LB+Amp liquid nutrient medium, 37 ℃, 200 rpm shaking table overnight cultures are inoculated on the identical LB substratum in 1: 100 ratio and are cultured to OD ₆₀₀Be 0.6-0.8, adding IPTG is 0.5 mM to final concentration, and collection bacterium liquid is used for analyzing total albumen after cultivating 0,2,4,6 h under 16 ℃ and 37 ℃ respectively; 4 ℃ of bacterium liquid after the collection, centrifugal 1 min of 12 000 rpm abandon supernatant, the deposition with 100 μ l sds gel sample loading buffers (Tris-HCl 50 mM, pH 6.8; SDS 2 %; DTT 100 mM; Tetrabromophenol sulfonphthalein 0.1 %; Glycerine 10 %) resuspended, boil 5 min after, centrifugal 1 min of 12 000 rpm gets 20 μ l supernatants and carries out SDS-PAGE and detect; With Xylene Brilliant Cyanine G R-250 staining fluid (0.1 % Xylene Brilliant Cyanine G R-250,40 % methyl alcohol, 10 % glacial acetic acids) dyeing, after destainer (25 % methyl alcohol, 6 % glacial acetic acids) decolours, use gel imaging system to take pictures.The result shows the recombinant plasmid pET32a-Δ that is inserted with exogenous segment PyTTG1After IPTG induces, about about 29 kD of molecular weight of albumen (comprising TRX albumen on the carrier) of expection, 1 protein band is arranged, and inductive does not transform recombinant plasmid and this protein band (see figure 4) does not appear in the pET-32a control plasmid.Be illustrated in 16 ℃ and 37 ℃, the IPTG final concentration is a recombinant plasmid pET32a-Δ under 0.5 mM PyTTG1Intestinal bacteria Rosetta(DE3) in abduction delivering Δ PyTTG1 albumen.Because low temperature induction more helps the formation of soluble proteins, thus follow-up test to select 16 ℃, IPTG final concentration for use be to carry out abduction delivering under 0.5 mM.

Embodiment 3: the proteic purifying of Δ PyTTG1, and concrete steps are following:

A, bacterial cell disruption: will be at the thalline behind a large amount of abduction delivering 1 L under 16 ℃, 0.5 mM IPTG, through ultrasonication thalline (3 s that work, 6 s have a rest) 5 min;

Cleer and peaceful deposition in b, the collection:, keep respectively and go up cleer and peaceful deposition with 4 ℃ of bacterial cell disruption liquid, centrifugal 20 min of 12 000 rpm;

C, the broken supernatant of albumen use 0.22 μ m filter to filter, and remove impurity;

The pre-treatment of d, His-Trap HP post: use 5 times of column volume pure water to wash post; 5 times of column volume binding buffer liquid (sodium phosphate buffer (pH7.4) 20 mM, NaCl 0.5M, imidazoles 30 mM) balance pillar, flow velocity is 1 ml/min;

E, protein sample upper prop: flow velocity is 1 ml/min, collects effluent;

F, wash post: use 5 times of column volume binding buffer liquid (20 mM sodium phosphate buffer pH7.4, NaCl 0.5M, imidazoles 30 mM) wash-out;

G, wash-out: use 5 times of column volume elution buffers (20 mM sodium phosphate buffer pH7.4, NaCl 0.5M, imidazoles 30,150,500mM) wash-out successively respectively, collect elutriant;

The aftertreatment of h, His-Trap HP post: 5 column volume elution buffers (20 mM sodium phosphate buffer pH7.4, NaCl 0.5 M, imidazoles 500 mM) all albumen of flush away; 5 times of column volume pure water are washed post; 5 times of column volume 20 % ethanol are washed post; Seal before and after the pillar, in 4 ℃, the ethanol of 20 %, preserve;

I, SDS-PAGE detect: get each imidazoles gradient current fluid of 50 μ l respectively, add 5 * albumen sample-loading buffer of 5 μ l, behind the mixing, boil 5 min, behind 4 ℃, centrifugal 5 min of 13 000 rpm, respectively get appearance 20 μ l on, carry out the SDS-PAGE analysis.Result such as Fig. 5, behind 150 mM imidazoles wash-outs, 500 mM imidazoles obtain purifying protein.For further optimizing imidazoles wash-out concentration, adopt the imidazoles gradient elution of 150,200,300 and 500 mM, result such as Fig. 6 show that 300 mM imidazoles are best albumen wash-out concentration.Subsequently, adopt 300 mM imidazoles to carry out large-scale purification, result such as Fig. 7 have obtained high purity protein, after use 1 * phosphoric acid buffer (pH7.4) dialysis, send the cloud mcroorganism to prepare the polyclonal antibody of rabbit the 7th swimming lane purified proteins.

The Western of embodiment 4:PyTTG1 protein specific antibody detects

Be to detect the validity of PyTTG1 protein specific antibody, use Δ PyTTG1 protein antibodies to 35S:: PyTTG1Transgene tobacco detects.Detailed process is following:

The reagent and the instrument that use in the experiment are following:

Vertical protein electrophoresis appearance, pvdf membrane (millipore), Semi-Dry Transfer Cell (BIO-RAD) changes the film system;

Acrylic amide, two methene acrylic amides, 10 %APS, TEMED, 1 * glycine buffer, 1 * phosphoric acid buffer, 1*PBT, skim-milk, SDS-PAGE and Western reagent such as Whitman 3MM filter paper.

The protein sample preparation: protein sample and 1/5 volume, 5 * albumen sample-loading buffer (Tris-HCl (pH6.8) 250 mM, SDS 10%, and BPB 0.5%, glycerine 50%, beta-mercaptoethanol: 5%), 95 ℃ of heating 5 min are placed on 5 min on ice.Normal temperature 13 000 rpm, centrifugal 1min.

1, SDS-PAGE detects

(1) preparation SDS-PAGE gel;

(2) electrophoresis: need constant current: concentrate glue 30 mA, separation gel 40 mA.

2, Western operation

(1) change film: setting electric current is 2.0 mA/cm ², change film 40 min;

(2) sealing (5% skimmed milk);

(3) add one anti-(Δ PyTTG1 antibody);

(4) add two anti-(the commercialization antibody of goat-anti rabbit);

(5) result observes

Abandon two and resist, after the washing, add 1 ml working fluid; Soak into whole pvdf membrane, use imaging system Chemidoc XRS (BIO-RAD) to be carried out to picture and observe result such as Fig. 8; Swimming lane 1,2,3,5,6 is the transgene tobacco total protein; 4 is the total protein of wild-type tobacco, in transgene tobacco, detects the proteic expression of PyTTG1, and in the wild-type contrast, does not detect PyTTG albumen; This polyclonal antibody specificity that shows that the present invention prepares is strong, is fit to carry out the proteic detection of expression of PyTTG1, detection and localization etc.

SEQUENCE?LISTING

 

< 110>Kunming University of Science and Technology

 

< 120>YUNNANHONG pears Δ PyTTG1 gene and prokaryotic expression carrier and application

 

<160> 4

 

<170> PatentIn?version?3.5

 

<210> 1

<211> 29

<212> DNA

< 213>artificial sequence

 

<400> 1

ccgccatgga?gaactctacg?caagaatcg 29

 

<210> 2

<211> 34

<212> DNA

< 213>artificial sequence

 

<400> 2

ccgctcgagg?ttcggcttta?ttgaaagggt?atcc 34

 

<210> 3

<211> 213

<212> DNA

<213> Pyrus?Pyrifolia

 

<400> 3

atggagaact?ctacgcaaga?atcgcatctc?aaggcggaga?actcggtgac?gtacgagtcg 60

 

ccgtacccgc?tgtacgcctt?ggcgtttgtc?tcgccccaaa?cccgaacccg?ccaccagcac 120

 

caccgaatcg?ccgtcggcag?ctttatcgag?gagtactcga?accgggtcga?catcctttcc 180

 

ttcgacccgg?ataccctttc?aataaagccg?aac 213

 

<210> 4

<211> 71

<212> PRT

<213> Pyrus?Pyrifolia

 

<400> 4

 

Met?Glu?Asn?Ser?Thr?Gln?Glu?Ser?His?Leu?Lys?Ala?Glu?Asn?Ser?Val

1 5 10 15

 

Thr?Tyr?Glu?Ser?Pro?Tyr?Pro?Leu?Tyr?Ala?Leu?Ala?Phe?Val?Ser?Pro

20 25 30

 

Gln?Thr?Arg?Thr?Arg?His?Gln?His?His?Arg?Ile?Ala?Val?Gly?Ser?Phe

35 40 45

 

Ile?Glu?Glu?Tyr?Ser?Asn?Arg?Val?Asp?Ile?Leu?Ser?Phe?Asp?Pro?Asp

50 55 60

 

Thr?Leu?Ser?Ile?Lys?Pro?Asn

65 70

Claims (5)

1. YUNNANHONG pears PyTTG1Gene is characterized in that: PyTTG1Gene has the protein of shown in SEQ ID NO.3 nucleotide sequence or coding aminoacid sequence shown in SEQ ID NO.4.

2. YUNNANHONG pears according to claim 1 PyTTG1Gene is characterized in that: gene source is in the YUNNANHONG pears.

3. said YUNNANHONG pears of claim 1 PyTTG1Prokaryotic Expression carrier pET32a- Δ PyTTG1, it is characterized in that: this carrier contains PyTTG1Gene, PyTTG1There are T7 promotor and bacterial ribosome binding site RBS in the upper reaches of gene, and the downstream of T7 promotor have can be by IPTG inductive operator sequence, PyTTG1The N end and the C end of gene all have 6 * His label.

4. the described prokaryotic expression carrier of claim 3 is at the synthetic and trichome genesis and development modulin of preparation YUNNANHONG pears pigment

Application among the Δ PyTTG1.

5. the application of the described prokaryotic expression carrier of claim 3 in preparation PyTTG1 specific antibody.

Images